Immune modulating agents

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Laura Williams Cheever, RachelAnn, Jennifer Lynn, and Rebecca Marie

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In recent years,the fie1 of immunology has expanded significantly with the identification of numerous bi ogical response modifiers. Elucidation of these molecules in the systemic responses of the immune system has rapidly followed. Thus, networks or immuneresponsepathwayshave been identified and consistentlydepictedin immunological texts and slide presentations as a complex array of arrows, inscripI ~ ~ u n e ns, and pathways ti context inr the to ancient lendars. d u ~ ~ t i nAgents g a es the complexity of the sys une response and providesacomprehendescription of immunemodulatingagentsinthecontext of disease and therapy. The initial chapters deal with the identification of biological response modifiers and the interactions of these molecules with cells, tissues, and organs. The first eight chapters deal with the immune mediators as individual components of immunologically bas d systemic pathways. Initially,the general rulesof the immune system are outlined. n this context, specificversus nonspecific immunity is presentedalong with cognateandnoncognat neinteractions.Cytokinesand immunomodulatory interventions are listed. ody regulation, cellular signal transduction,andcytokinepathwaysareint d in Chapter 2. A serological property of antibodiesand cellularreceptors,idiotypy,anditsroleinimmune regulation pathways are presented in Chapter 3. Chapter 4 presents the pathways for stem cells to differentiate into cells of the immune system. This chapter introduces theimportance of regulatorygrowthfactorsforcellulardifferentiation. etween the neuroendocrine system Chapter 5 introduces the complex interaction andthecomponents of theimmune system. diators betweenthese systems include growthand lactogen hormone family, hormones and factors of the hypothalaus-pituitary-adrenal axis, thyroid hormones, steroid sex hormones, and vitamin . Chapter 6 completes the picture of growth hormones and immune modulation by describing the immune modulatory aspectsof transforming growth factors. Chapters 7 and 8 create a transition to immune modulation in disease states. Chapter 7 presents aspects of a growing field based on ourpresent-day lifestyle. The role of psychological stress and immune competenceis addressed. This is followed

i

by the use of an immunological mediator, interleukin 12, as an adjuvant to modulate the systemicimmuneresponses, i.e., the use of an immune mediator as a vaccine to boost normal immuneresponses. This topic progresses in Chapter 9 into the description of cytokine pathways induced in infectious pathogens. Chapter 9 initiates a series of 10 chapters focused on immune modulation in disease states. Following a description of the ability of the host to defend against infectious pathogens with either T helperl or 2 cytokine profile and how these immune response profiles could also be deleterious to the host, cellular immune responses to infectious pathogens are described. Chapter 10 details antigen presentation through the class I pathway and cytolytic T cells in the context of viral infection. Chapter 1 1 focuses this discussion on the changes in the cellular immune T cell repertoire on infection with the human immunodeficiency virus, These changes are eloquently portrayed by flow cytometry analysis. Additional regulation of T cells is presented in Chapter 12: idiotypic networks are described that modulate T cell-mediatedhepatic granulomatous inflammation induced by an infectious pathogen. The discussion of immunoregulation of disease then broadens to include immune modulation through human monoclonal antibodies (Chapter 13), immune modulation as a function of nutritional states (Chapter 14), immune modulation in gastrointestinal disease (Chapter 15), sepsis (Chapter 16), cancer (Chapter 17), and immune modulation and the neuroendocrine system (Chapter 18). Each of these chapters provides a unique focus on the immune system in the context of systemic infections and disease. The remaining eight chapters present immune modulationin the form of both accepted and experimental therapies. Thus, the book concludes with an emphasis on immune modulation in the therapy of disease. An important aspect of new experimental therapies is the reduction of side effects through a reduction in systemic interactions. Thus, a review of targeted antigen delivery systems for experimental therapies is presented in Chapter 19. Chapter 20 presents immune modulation in gene therapy. In this exciting new field, cytokine gene therapy and cellular adoptive therapy is described. Chapters 21 and 22 return to the human immunodeficiency virus and present apoptosis pathways and immune-based therapies for human immunodeficiency virus infection. Chapter 23 provides a descri~tionof the novel observation of microchimerism in immune-mediated allograft acceptance in organ transplantation. As thelastdisease-based chapter, Chapter 24 providesa review of immune modulation and therapy in arthritis, followed by a presentation of vaccination via mucosal- and oral-induced immune modulation (Chapter 25). The book closes with the use of immune modulating agents in the performance of human clinical trials. t i ~ gis acomprehensive,currentmanualof Thus, Z ~ ~ ~ uo ~ uel ~Agents pathways and individual agents that describe and affect the immune response. As such, this book is a unique referencethat summarizes current state-of-the-art immunological paradigms related to clinically relevant immune modulation. Thomas F. Kresina

Preface Contributors

1. The Immune System and Immune Modulation Fran~oisHirsch and Guido Kroemer 2.

Molecular Mechanisms Controlling ImmunoglobulinE Responses Rachel L. Miller and PaulB. Rothman

3. Idiotypes and T Cell Selection Bjarne Bogen, Zlatko DembiC, andSiegfried Weiss

4. In Vivo Modulation of Lymphohemopoietic Stem Cell Populations with Cytokines Gerald de Haan and Gary Van Zant 5.

Hormones as Immune Modulating Agents Istvan Berczi and Eva Nagy

6 . Transforming Growth Factor-@: A Cytokine Paradigm ~ichelle FrazierR, Jessen, Nancy McCartney-Francis, and Sharon M. Wahl

V X

1

21

35

55

75

121

~ i i

iii

IlS

7. Psychological Stress and Immune Competence ~lizabeth A. Bachen, Anna L. ~arsland, Stephen and Sheldon Cohen

8.

Interleukin 12: A Potent Vaccine Adjuvant for Promoting Cellular Immunity and Modulating HumoralImmunity Stanley F. Wolf

9. Cytokine Immunomodulation of InfectiousDiseases Joseph F. Urban, Jr., Fred Douglass Finkelman, ?'ere2 Shea-Donohue, and William C. Cause 10.

lating Cytolytic Responses to Infectious Pathogens ca Pogue Caley and Jeffrey A . Frelinger

11.

I m ~ u n o l o g yof 1:Cells in AIDS: Dynamics Revealed by * ht-Color Flow Cytometry rio Roederer, S t e ~ h e nC. De Rosa, Leonore A . Herzenberg, and Leonard A . Herzenberg

12. Immunomodulation of Schistosomal-Induced Inflammation Thomas F. Kresina 13.

noclonal Antibodies and Modulation e in Schistosomiasis, itis, Infection and T h o ~ aF: s Kresina, Carry A . Neil, and Steven K. H. Foung

145

161

169

187

209

22 1

237

14. Nutrition and Immunity Srinivas ~ e n d u l u rand i ~ a n j iKurnar t ~handra

255

15. Immunomodulating Agents in Gastrointestinal Disease Samir A. Shah, Athos Bousvaros, and A. Christopher Stevens

267

16. Immune Modulation in Sepsis Janet M . J. Hammond and Peter D. Potgieter

301

17.

Int~rleukin6: Role in the Pathogenesis of Cancer Ofoniel ~ a r t ~ n e z - ~ a z a

345

i

l$, Neuroendocrine-Induced Immune Modulation and Autoimmunity Terence Smith and Adrian K. Hewson

363

rt 19. Antigen Delivery Systems Used to Induce Immunomodulation . ~ a h i r uI.l Khan, Ian G. Tucker, and Joan P. Opdebeeck 20.

Immunomodulation in Gene Therapeutics lock, Susan S. Rich, Shu-Hsia Chen, and Savio L. C. Woo

21. Cytokines, Apoptosis, and Immune Therapyin HIV Infection Jer6me Estaquier and Jean-Claude Ameisen 22.

ost-Directed and Immune-Based Therapies for Human deficiency Virus Infection Valdez, ~ i c h a e l MLederman, . Bharat Ramratnam, and ~ i m ~ t P. h yFlani~an

385

42 1

439

457

23. Transplantation Tolerance, Microchimerism, and the Two-way Paradigm 483 Thomas E. Starzl,Anthony J. Dernetris, ~ o r i k o ~ u r a s e , S. Rao, and John J. Fung Massimo Trucco, Angus W . ~homson, Abdul 24.

25.

Immunomodulation of Cytokines and TCells by Biologicals in ~avinderN.Maini and arc Feldmann

507

Immunomodulation at Mucosal Surfaces: Prospects for the Development Antiinfectious of and Antiin~ammatoryVaccines Cecil Czerkinsky and Jan Ho~mgren

529

26. New Statistical Designs for Clinical Trials of Immunomodulating Agents 539 R~chardSimon

Index

551

.

eam-Clau~emei is em, Bernard, Paris, France

INSERM U13, Groupe Hospitalier Bichat-Claude

E ~ i ~ a ~ A. e t hBachen, 1Ph.E). Postdoctoral Fellow, Center for Social and Behavioral Sciences, University of California-San Francisco, San Francisco, California

.,

Ph.D. Professor, Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada .D. Clinical Associate,Departmentof Medicine, University Hospital Eppendorf, University of Hamburg, Hamburg, Germany Professor, Department of Medicine, Institute of Immunology and Rheumatology, University of Oslo, Oslo, Norway

.

AttendingPhysician,DepartmentofGastroenterology, Children’s Hospital, Boston, Massachusetts

.

Postdoctoral Fellow, Department of Microbiology and Immunology,University of North Carolina, ChapelHill, North Carolina

umar Chan~ra,M.B.B.S., .C. Professor, Department of Pediatrics,Memorial University of Newfoundland,Health Sciences Centre,St. John’s, Newfoundland, Canada

.

Assistant Professor, Institute for Gene Therapy and lecular Medicine, Mount Sinai School of Medicine, New York, New York

.

Sbel~onCohem, Ph. Professor,Department University, Pittsburgh, Pennsylvania

of Psychology,Carnegie

No-

Mellon X

.

Cecil C ~ e r ~ i n s ~ y , Hospital, Lyon, France

Research Director, INSERM

U80, Herriot

.

Blood and Bone Marrow Transplant Program, University of Kentucky Medical Center, Lexington, Kentucky Senior Scientist, Department of Medicine, Institute of Immunology and Rheumatology, University of Oslo, Oslo, Norway Professor, Department of Pathology, Pittsburgh Medical Center, Pittsburgh, Pennsylvania

University of

.

Postdoctoral Fellow, Department of Immunology, Memorial University of Newfoundland, Health Sciences Centre, St. John’s, Newfoundland, Canada Postdoctoral Scholar, Department of Genetics, Stanford University, Stanford, California

.

~ e r ~ m e E s t a ~ ~ i e r , INSERM 1113, Groupe Hospitalier Bichat-Claude nard, Paris, France

Ber-

ath. Professor, The Kennedy Institute of Rheumatology, Hammersmith? London, England

.

McDonaldProfessorandDirector, Division of Immunology, Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio

.

Division of Geographic Medicine and Clinical Immunology, The Miriam Hospital, Brown University, Providence, Rhode Island

.

~teven Associate Professor, Department ford University School of Medicine, Stanford, California

of Pathology, Stan-

.

Intramural Research Fellow, Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland

.

Jeffrey A. F r ~ ~ i n ~ e r , SarahGraham KenanProfessor, University ofNorth Carolina, Chapel Hill, North Carolina AssociateProfessor,Department of Surgery, and Chief, Division of Transplantation Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania e

illiam C. ~ a ~ sAssociate e Professor, Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland

xii

.

Postdoctoral Fellow, Divisions of Clinical Pharmacology and InfectiousDisease, Johns HopkinsUniversity, Baltimore, Maryland

.

Professor, Department of Genetics, Stanford Uni-

epartment of Genetics, Stanford University, Stanford, California Multiple Sclerosis Laboratory, Miriam Marks Department of Neurochemistry, Institute of Neurology, London, England Research Director, Equipe d’Immunologie Cellulaireet de Transplantation, Centre National de la Recherche Scientifique, Villejuif, France

.

09 Professor,Departmentof Medical Microbiology and Immunology, University of Goteborg, Goteborg, Sweden

.

09 Project Manager, Drugs velopment Department, Research Institute, PLIVA d.d., Zagreb, Croatia

De-

Division of Digestive Diseases and Nutrition, National Digestive and Kidney Diseases, NationalInstitutesof ealth, Bethesda, Maryland

.

UPR 420, Centre National de la Recherche Scienti-

fique, Villejuif, France

.

Professor, Department of Medicine, CaseWestern Reserve University, University Hospitals of Cleveland, Cleveland, Ohio *9

and Director, The England

09

Kennedy Institute of Rheumatology, Hammersmith, London,

.

Professor, Department of Psychology, Pittsburgh, Pittsburgh, Pennsylvania

University of

.S. DoctoralCandidate, Psychology Department, University of Pittsburgh, Pittsburgh, Pennsylvania

.

Associate Professor,epartmentsof Obstetrics and Gynecology, and Microbiology and Immunology,University of California-Los Angeles School of Medicine, Los Angeles, California

.

Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland

rck,

.

ae Instructor, Department sity, New York, New York

of Medicine, Columbia Univer-

.

Associate Professor,DepartmentofSurgery,ThomasE. Starzl Transplantation Institute, University of Pittsburgh Medical Center, burgh, Pennsylvania hAssociate,DepartmentofImmunology,Facultyof itoba, Winnipeg, Manitoba, Canada

arry epar

Executive Director,

C.,

of

, Brisbane, Australia

Director of Critical Care, Intensive Care ital, Riyadh, Saudi Arabia

.

Division of Geographic Medicine and Clinical Immuital, Brown University, Providence, Rhode Island

.

AssistantProfessor,DepartmentsofSurgeryand ion of Cellular Transplantationand Medical I tics, Thomas E. Starzl Transplantation Institute, University of Pittsburgh Center, Pittsburgh, Pennsylvania

.

Professor, Departments of Microbiology and Immunology, aylor College of Medicine,

.

Research Associate,Department University, Stanford, California

.

of Genetics, Stanford

AssistantProfessor,Departmentsof crobiology, Columbia University, New York, New York

Medicine and

.

Department of Medicine, The Miriam Hospital, and Division of Gastroenterology,rown University Schoolof Medicine, Providence, Rhode Island

.

Associate Professor, Department of Medicine, Uniformed Services University of the HealthSciences, Bethesda, Maryland hief, Biometric Research Branch, National Cancer Insti-

.

Research Fellow,Multiple Sclerosis Laboratory, Marks Department of Neurochemistry, Institute of Neurology, London, England

omas E. Starzl, Professor of Surgery and Director, Thomas E. Starzl Institute,University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

.

r i s t o ~ ~ Stevens, er AssistantProfessor,Department ofMedicine, Harvard Medical School, Boston, Massachusetts ngus W. T~omson, ath. Research Professor, Department of Surgery, Universityof Pittsburgh Medical Center, Pittsburgh, Pennsylvania

.

Hillman Professor of Pediatric Immunology, Department of Pediatrics, Children’s Hospital, and Head, Division of Immunogenetics, Director,HistocompatibilityLaboratoryforTissueTyping,University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

.

Professor, Department of Pharmaceutical Sciences, School of Pharmacy, University of Otago, Dunedin, New Zealand

.

SupervisoryMicrobiologist,Immunologyand Disease Resistance Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland

.

AIDS Clinical Trials Unit, Case Western Reserve University, University Hospitals of Cleveland, Cleveland, Ohio Professor, Department of Blood and Marrow Transplant Program, University of Kentucky MedicalCenter, Lexington, Kentucky e

.

ActingChief, OralInfectionand ntalResearch, NationalInstitutesof

.

Senior Scientist, Gesellschaft fur Biotechnologische Forschung, Braunschweig, Germany

.

Senior Scientist, Preclinical Molecular and Cellular Biology, Genetics Institute, Inc., Cambridge, Massachusetts

.

Professor and Director, Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, New York

Centre ~ ~ t ~ odenlaaRecherche l ~ c i e n t ~ q ~ e , France Villej~~

It is generally agreed that the concept of immune modulation emerged in 1796, when Edward ~ e n n e succeeded r in protecting people against smallpox by infecting them with cowpox virus. Since then, many attempts have been undertaken to help the immune system to face external attacks such as bacteriaor viruses and internal attacks such as cancers or autoimmune disorders. It is particularly during the past two decades that information on how to manipulate immune responses has been accumulating. The first breakthrough came from the elucidation of the primary structure of the antigen receptor (antibodies and Tcell receptors) and the way their enerated. The description of how immune reactions can be elicited or insight mechanisms allowing self-tolerization (in particular deletion and anergy), and the discovery of classes of response (for instance, the T helper I [Thl] and Th2 responses) have provided the conceptual bases of interventions on the immune system. Otherinnovations in immune modulation became possible when the conceptof cellular “cross-talk” arose. Thediscovery of cytokines, capable of influencing the fate of immune cells over some distance, was a major breakthrough to theu n d e r s t a n ~ i nof~ how immune responses could be coordinated in the body. oreo over, it is now well established that immune cells, in order to function properly, must cooperatevia direct cell-to-cell contacts. For the purpose of immune modulation, it is also important that some open-minded scientists conceived that the immune system does not function in a strictly autarchic manner but rather as an open integrated system. mmune cells, for instance, continuallyreceive inputs from the neuroendocrine system and in turn produce factors that can modulate neural and endocrineresponses. In this chapte~,wewill outline some general rules governing the immune response. We then will provide an overview on communication within the immune system and between the immune system and other communication systems.On the basis of these premises, we will discuss the possible levels of immunomodulatory interventions. As a cautionary note, this survey does not pretend to be exhaustive, but rather to appeal to the reader’s curiosity, which will, it is hoped, be satisfied by the subsequent chapters of this handbook.

city

. 1 l'

S (peptide/

concerns a minor fraction of the overall imm cytes receive signals via monomorphic recept Such noncogna~esignals are nonspecific ina ~ a j o r i t yor a large fraction of ly interventions on monomorphic rec cules that are designed to interact hus, for instance, antibodies lymphocytes bearing a@-type antigenrec complex will have nonspecific effects, w specific effects.Onanintermediatest portion of the i m m ~ n erepertoir products froni V genes of the TC semispeci~icmod~lators(Table 1).

lymphocyte^ are constantly challenged wit rsus death, (2) response versus anergy, a response (Figure 1). At a first level, T and lymphocytes are notoriously pr

Classification of Immunopharmacological Interventions According to the Degree ofSpecific~ty of

Degree " "

.

-

Immunization with pepodulation monoclonal of tides or proteins; applicaor oligoclonal response tion of~odifiedantigens to a determined antigen SemispecificinterventionInterventiononafraction of the immune repertoire bodies, and immunotoxdetermined bythe expresins. 'I' cell vaccination sion ofdetermin~dV genes Ap~licationof cytokines, ~ o n s p ~ c i fintervention ic Application of substances hormones; ablation of'I' that alter immune recell subsets sponses independently from the antigenor antigen receptor@) involved

Specific interve~tion

a

~ T cell~

receptor. ~

;

XL-l, IL-6, TNF-a

.g:::%.: ....

C

g:;:.:.’

costi

Schematic representation of the immune system showing the different levels of mmune modulation. Naive peripheralT cells can be activated eitherto di intoa T helperprecursor or to undergoprogrammedcelldeath(apoptosis). activationbyantigenpresentingcellsdoesnotimplyproductiveresponsesby T cellcanbecomeanergic or Dependingonthesimultanousisionofcostimuli,the respond productive a in fashion. T cells “decide” between at least two T This decision process determined is options, namely, to acquire a by costimuli type the of provided dominant he cytokine profile the in environment of the T cell. Thl and Th2 cells are mutually antagonistic. Thus, for example, Thl cytokines (especially IFN-y) favorthe differentiation from the Tho to the but inhibit that of Tho to Th2 cells. In contrast, theTh2 product IL-4 hasan opposite effect. It wouldbe an oversi~plification toassumethat T cells are sequentiallyexposed to the survival/apo~tosis,anergy/response, and class of response dilemmas. Instead, it is conceivable that T cells can be induced to undergo apoptosis or to enter a state of anergy at any differentiation stage. In the upper part of the figure, one particular type of neuroendocrine immune control is depicted. APC can produce inflammatory cytokines (IL-l, IL-6, TNT;) that will causean endocrine arousal reaction and ultimately an increase in adrenal glucocorticoidrelease.Glucocorticoidsthenfunctionasendogenousimmunosuppressorsandexert multipleinhibitoryeffects, at thelevelsofboth the APC and the T cell. A presentingcell;GC,glucocorticoids;xis,hypothalamic-pituitary-adrenal axis; IFN-y, TGIF-@,transforminterferon-y;IL,interleukin;Thp,Chelperprecursorlymphocyte; ing growth factorp; TNF, tumor necrosis factor.

during their differentiation in the thymus or bone marrow, respectively, and later of lymphoc~esresults during theirlifeinperipherallymphoidorgans.eletion from programmed cell death (apoptosis) and can achieved by numerous adverse conditions:ligation of “deathreceptors” (Fas/apoptosis 1-1/CD95, CD30, tumor necrosis factorreceptor(TNF-R),cytotoxicTlymphantigen(CTLA)-4 on T cells; F a s / A ~ O - l / C ~ 9on 5 B cells) via cell-to cell contact, presence of soluble death-promoting factors (glucocorticoids tumor necrosis factor [TNF], transforming growth factor-@ [ T ~ F - @ ] unfavorable ), combinations of signals, or simply abs T andB cells can sence of obligatory trophic factors[ 1,2]. It appears n o ~ a d a y that be driven into apoptosis at any differentiation stage. Thus, the dilemma of death versus survival constitutes one primary level of en~ogenousim At a secondlevel, surviving lymphoc~es that are activat can respond in two opposite fashions. On the one hand, lymphocytes can receive the entire set of signals required to mount a productive immune response and thus exert cytotoxic functions to destroy the antigen-bearing target or secrete soluble effector molecules (cytokines in the case of T cells, anti odies in the case of On the other hand, failure to provide certain signals, the so-called costimuli, can abort the immune response and cause anergy, n a m e l ~ ,a reversible partial loss of cellular functions [3,4]. Anergic cells thus, instead of becoming deleted, will fail to exert effector functions, although they may conserve other functions such as self-tolerization. At a thirdlevel, surviving nonanergic lymphocytes can mount different classes of responses. Thus, it is established that undiffere~tiated 4’ T cells can become either Thl cells (which are specialized in facilitatingtoxicresponses, e.g., against tumors orgrafts) or Th2cells (which produce a different setof l y ~ p h o ~ i n e s important for humoral immunereactions), that, by the way, are mutuallysuppressive [5,6]. It appears that CDS’ T cells also can acquire Thl-like or Th2-like phenooreover, it has long been ~ n o w nthat tion of different Ig classes,a choice thatis determined by the or Th2 cytokines. It may be important to note that an inap sponse can be pathogenic. As an example, the elicitation of i antibodies (with Th2 help) against pollen is pathogenic and thus will cause allergy, whereas a preponderant IgG response (with Thl help) against the same antigen will have no (or little) pathologica~ conse~uence.imilarly, the development of an inappropriate Thl (instead of Th2) response gain st or an-specific autoantigen can have deleterious consequences[S]. The choices discussed (surviv 1 versus deletion, response versus anergy, response classes) are disposed in a fail-safe hierarchy. Thus, to mount a productive immune response, a l y m p h o c ~ emust avoid deletion, escape anergy induction, and choose the correct class of response. In accord with this postulate, autoaggressive responses only occur after accumulation of molecular or cellulardefectsin the sense thatall self-tolerizing mechanisms (deletionof autoreactive cells, induction of anergy, deviation to an innocuousclass or response) must fail simultaneously[3,4]. oreover, infectious microoganisms and parasites have developed multiple different strategies to subvert the immune response and to induce deletion, anergy, or choice of the “wrong” classof response. of the three Therapeutic immune modulation also aims at influencing one different levels of immune regulation. Immunostimulatory regimes can aim at the

e/

prevention of clonal deletion, the prevention(or reversal) or anergy induction, and the stimulation of the appropriate class of response. In contrast, immunosuppressive therapies can be designed to delete specific lymphocyte subsets, to provoke anergy, orto deviate the immuneresponse from a noxious to aninoffensive class of response (Table 2).

Lymphocytes continuously receive signals via multiple functionally distinct receptors. First of all, T or cells possess surface antigen receptors (T cell receptors and surface immunoglobulins,respectively) that detect thepresence of antigen. Ligation of the antigen receptor can trigger the T cell receptor (TCR) or the l3 cell receptor CR) complex to transmit a complex signal transduction cascade. Thus, the affinity/avidity of antigen receptor/antigen, as well as thetime courseof this interaction, will influence the lymphocyteresponse. At least in the case of the TCR, it appears possible that the overall sha eof the M C-peptide complex not only influences the quantitativeoutcome of triggering butcan influence thequalitativeoutcome of a T cell response (aner In addition to the clonotypically distributed receptors, lymphocytes possess invariantcoreceptorsthat detect the presenceofcostimuliprovided by antigen presenting cells (APC~).Thus, T cells can perceive costimulivia a 5 , CD$, CD28, CTLA-4, CD4OL, CD4

Classification of Immunomodulatory Interventions Accordingto the Level of Interventiona Level Examples of intervention Survival versus apoptosis Survival/growth factors: cytokines, linomide (apoptosis inhibitor) Apoptosis inducers: glucocorticoids, superantigens, antibodies, i~mmunotoxins AnergyversusresponseInducers of anergy:blockade of costimuli by neutralizing antibodies or soluble decoy receptors; applicationof antigen-pulsed APC that fail to provide costimuli; injection of F(ab ')2 fragment of anti-CD3 or anti-CD4; application of antagonistic peptides; Inhibitors of anergy: IL-2 and possibly other cytokines Response class Thl/Th2 shift: application of Thl/ThlL-like cytokines Ig class switch: interventionon Th 1 /Th2 cells aAPC, antigen presenting cell; CD, cluster designation; IL, interleukin; Th, T helper; Ig, immunoglobulin.

etc.) that interactwith defined counterreceptors onspecialize the activation and differentiation stage, a determined minimum required to mount a productive immune response and to preven anergy or apoptosis in the T cell. ~nteractionwith nonspecializ cause abortive immuneresponses. L y m p h o c ~ e salso possess receptors for a panoply of soluble factors. They thus receive signals via lymphokines (c~tokines elaboratedby lymphoc~es monokines (cytokines produced by monocytes/macrophages), growth factors el by different tissues, local factors such as prostaglandins and leukotrienes, hormones (steroids and protein hormones), and neurotransmitters (catecholamines, endorphins, etc.). The outcome of an immune response depen S on the av~ilabilityof cognate signals as well as on the context of such cognate signals9 namely, the simultaneous deliv~ryof nonspecific contact-dependent or factor-~ediatedsignals. Thus, it is mainly the context which determines which of the three existential choices of lymphocytes (apoptosis versus survival, anergy versus response, class of response) is made.

In order to become activated, T cells must recognize an appropriate peptidecomplex via theTCR(signal 1). Signal 1 has to be providedduringa minimum interval and thus critically depends on overcoming char~e-dependentrepulsion between the T cell and the APC via adhesion molecule-dependent interactions (e.g., leukocyte function antigen l/intracellular adhesionmolecule 1) [g]. This first signal, however, is not sufficient to elicit a productive respon§e, and additional signals (signal 2) are required to cause full activation. These ~‘costimuli’~ mostly depend on thedirect physical interaction between the T cell and the that this interaction requires direct cell contact rather than soluble advantage, in teleological terms, of preventing the unwarranted bystander activaoreover, itguaranteesthatonlythe“right” will deliver the required for elicitation of immune response costimuli can cause deletion or anergy of the T cell [ 10,111, agreed not only that thereis one signal 2, but that several costimuli (signals 2a, b,c)

of the cosignals [14,151. molecules, which neutralize

vation [ 181. €37-2 initiates of the immune response

cells, respectively [20,21). Anotherligand of stimulation9was discovered on activated T cells. It appears cells functions as an endogenous “death uses apoptosis of activated T cells [22]. Thus, in specified circumstances9 costimuli can abort immune responses.

tosis of the activated T cell. Thus,mutualcontact-

~ytokines are soluble, monomor hie (g1yco)proteins that are released by living cells in a highly regulatedfashion to regulate cell functions via specific 7). They ~ a r t i c i ~ a tine the 01 of all immunologically relevant hertheyconcerntheactivatifferentiation,maturation9proliferation, a~optosis, or ~cquisition of effector functions. Cytokines influencethe quantitative, as well as thequalitative, outcome of the immune response. ~ y t o k i n e sare rather leiotropic in nature. They can promote different func-

ncester. The pleiotropy of cytokines is also illustrated by the fact that certain mediators are functioning in very

disparate tissue contexts; e.g., IL-6, which is involved in immune activation, inflammation, tissue damage, etc.[35], may also serve asa neurotrophic factor[36]. As shown in Table 3, another characteristic of cytokines is their redundancy, as defined by the ability of different cytokines to achieve the same functions. dundancy is observed onseveral levels of the cytokinesystem: (1) on thelevel of the molecules that may belong to a familyof gene products with o v e r l a ~ ~ i norg identiCytokines Involved in the Immune Responsea Principal

Cytokines IL-la, p

IL-1ra IL-2

IL-5 IL-6 IL-7 IL-l0 IL-12 IL-14 IL-15 IL-16 IL-17 TNF TGF-p

APC, lymphocytes,NK cells

Lymphocyte proliferation, NK cell activation, induction of lymphokine gene expression,promotion of inflammatory reactions, endogenous pyrogen Monocytes/macrophages, ker- Inhibition of IL-l functions atinocytes T lymphocytes T andB lymphocyte proliferation, Ig production, NK cell activation Lymphocytes, mast cells, baso- Proliferation and differentiationof B phils cells, productionof IgE, promotion of Th2 cells T lymphocytes, mast cells, eo- Promotion of Th2 cells, differentiation sinophils of B cells T lymphocytes, APCs, astroProliferation and differentiation of lymcytes, mast cells phocytes, endogenous pyrogen Bone marrow stroma cells, Proliferation and differentiationof cythymic epithelial cells, totoxic lymphocytes,sti~ulation of APCs macrophages Lymphocytes, macrophages Promotion of Th2 cells, inhibition of macropha~e-produced proinflammatory cytokines B lymphocytes, macrophages Promotion of Thl cells, proliferationof activated-T lymphocytes T lymphocytes Proliferation of activated-B lymphocytes T lymphocytes, monocytes, T lymphocyte proliferation bone marrow stroma cells T lymphocytes CD4' T lymphocyte growth factor Memory CD4 + T lymphoCostimulation of T cell proliferation, promotion of IL-6 secretion cytes T lymphocytes, APCs, astro- Modulation of cytokine gene exprescytes sion, promotionof in~ammatoryreactions Promotion of Thl cells, inhibitionof T lymphocytes, monocytes lymphocyte proliferation, promotion of IgA production

aIL, interleukin;APC, antigen presenting cell; NI(,natural killer; Th, T helper;CD4+, cluster designation 4'; TNF, tumor necrosis factor; TCF-@, transf~rminggrowth factor-@. Sources: References may be found in the review in Ref. 99; for IL-16 in Ref. 100 for IL-17 Refs. 101, 102.

rece~tor§that may share a c ~ m ~

ine manner an

a slower kinetic mechani§~.

topographical restri cytokine transport. tion by c ~ o k i n erec rapid elimination intervene at the re

tor agonists.

ioneeringworkofesedovsky andSorkiopened igations showing an extensive interrelatio r o e ~ ~ o c r i systems. ne In determined contexts, imm amounts of cytokines to provoke systemic effects cells from the immune system, cytokines can act which in t ~ carries ~ n out immunore~ulatory functi

roen~ocrineimmune control [46]. In particular,t

a new area of intensive

basis, lamic-pituitary-adrenal ( )-activating potential. IL-l is farmorepotentthan IL-6 TNF-a [49]. ~uboptimalamounts of IL- 1a and IL-6 synergize to induce glucocorticoid responses [50]. ther cytokines, such as -y,have delayed a (> -stimulating potential and proba~ly former cytokines. In this context, it is interesting that a physiological neuromodulator/neurotrans~itter whose sent in the circulation to brain only [53]. Thus, circulating docrine effectsat thelevel of cortico-

d that IL-6-deficient mice fail to oxins or IL-lP. This suggests the -6 in temperature control at the level of the preoptic areaof

y of immunecells to respond

u~lishedresults). The lterations in nuclear

lsoinduceapoptosis of thymocytes,pre cells, andactivated cells; inhibit the secretion of T h l lympho es; induce transcription of the immunosuppressivecytokine TCFP; favor a shift from Thl to Th2 responses; and inhibit both APC and lymphocyte effector functions [46]. In other terms, gl~cocorticoids affect ‘‘existential the choices” of lymphocytes (Table2) at all hey thus serve as major endogenous inhi~itorsof specific i m m ~ n eand fic in~ammatoryresponses [69] art from the obvious impact of glucocorticoids on the immu present the exact impact of hormones and neurotransmitters the in vivo p~ysiological characteristics of immune cells is poorlyunderstoodowever,itappears clear thatinterventionsontheneuroendocrine systemmaye majoreffectson immune functions, a fact that may explain ~nwarrante proautoimmune effects of certain phenothiazianes, as well as of a- e t h y l ~ o ~ 1701. a It may be future immune interventions may be based on the modulation of such neuroendocrine interactions.

~mmunopharmacologicalinterventions maybe classified a g to several independent criteria: degree the antigen of speci (Table onthe l), functio~allevel interventions of with rega e “existential choices” of cyte physiological processes (in~uctionof apoptosis versus survival, inducruption of a n e r ~ y changes , in the cytokine profile [Table 2]), the target S of an intervention (antigen receptors, contact-dependent costimuli,cyto-

re presently in use are small molecules(< 1 This applies to many different areasof ~harmacolical examples of thisclass of compoun~s c application in nons~ecificimmunosup-

nists [97], monoclonal orpolyclonal anti-

into p~tients elicit to immune reactions. It would be beyond the scope of this review to discuss all these types of immune intervention. In the following paragraphs, we will discuss one particularly ~romisingtype of immune intervention, namely, gene t h e r ~ p .y

Classification of Immunomodulatory Interventions According to the Chemical of

Class Small molecules( C 1

Linomide ercuric salts ~zathioprine Schiff base-forming molecules Glucocorticoids

Peptides Proteins Cytokines

Inhibits depletion of peripheral T cells Induce T cell apoptosis Cytotoxic for proliferating lymphocytes romote Th1 response

Induce T-cell apoptosis, inhibit cytokine production Promotes Th2 response Progesterone C-binding peptides Inhibits antigen presentation T cell receptor peptides Prevent autoimmunity

Promotes antitumor immunity Immunosuppression IL-lRa, TNF-a, etc. Passive immunization Specific antisera Antibodies Treatment of autoimolyclonal, mixed Igs mune diseases onoclonal antibodies Immunosuppression against lymphocyte subsets and immunotoxins ~eutralizingantibodies Immunosuppression against cytokines Prevents autoimmunity Soluble inhibitors CD4 Immunosuppression CTLA4 Ig Promotes immunosupAntisense oligonucleo- Cytokine antisense pression tides Enco~ingT-cell receptor Prevents autoimmunity NA Vaccination Encoding IL-2, IL-12, IL-4, etc.

cobacteriu~tuberculosis antigen

Cells munity

Peptide-pulsed dendritic Promote antitumor imcells clones Prevent cell autoimmunity T

aTh, T helper; NHC,major histocompatibility complex; IL, interleukin; TNT;, tumor necrosis factor; CD, cluster designation; CTLA, cytotoxic T lymphocyte antigen.

the simpliest systems of gene therapy would be the applicationof antisense oligonucleotides to prevent thetranslation of specific species. This approach has been applied mostly in vitro, to modify cytokine production [77]. remising results were thus accumulated showing the ability of small length sequences ranging from15 to 30 nucleotides to inhibit the productionof in~ammatory cyto~ines ( ~ L -TNF-a), ~ ~ , cytokinesinvolved in tumorgrowth (IL-6), or cytokiolvedinother imm~noolling IgE synthesis). es have been used in els for vaccination. Iff et al. showed gene tr ained by simple intramuscular injectio~sof naked plasmid S of cytokine genes injecte~ mostconclusive nstrations of the so-called genetic vac tituberculosisvaccineswereobtain encoding a single antigenic molecule of shock protein). ~ u r ~ r i s i n g l ydespite , the nique (little plasmid molecule uptake by cells, low persistence of the transgene), mice respondedto antigenic challengeup to6 months fter the lastplasmid injection [SO]. Gene therapy has also been successfully applie to the re vent ion of experimental autoimmuneencephalomyelitis (EA ). This disease is indue tion of susceptible ro yelin basic protein ( Th 1 cells expressing repertoire and res sclerosis. Genetic vaccination was obtained b intramuscular injections of a lasmid iablesequence of a specifically used by anti AE was accompani shift a from the cytokine profile [$l]. These findings suggest that future protocolsof immunomodulation may take advantage of genetic vaccination.

Various pathological conditions are now approached with manipulated cells as vectors of immunomodulation. This kind of manipulation has been thoroughly researched in several cancers, including malignous melanomas. he simplest method consists in injecting irradiated tumorcells [82]. The scientific rationale of this treatment is that melanoma cells bear specific tumor antigens that may elicit cytototic T cell responses in vivo. Improving the immunogencity of tumor cells by genetic As anexample,tumor cells can nsfected manipulationsmaybeattempted. withimmunostimulatorycytokinegenesorbemodified to express class I1 costimulatory andmolecules tumor alternative Sto an cells, normal dendritic cells vitro in with tumor-related p e p t i ~ eantigenscanbeused to elicit ant es, This was documented by the rejection cell expressing the chicken egg ovalbumin S [86]. This antitumor therapy is m latory signals and participation of Transfection of APC with costimulatory molecules or cytokines favoring Thl reL-12) [SS] may enhance the antitumor response. Another experjmental method of tumor vaccination is based on the use of transfected insect cells. An

~chematicrepresentation of the principle ofa ~ t ~ e c t iAn o ~antibody . directedto is chemically cQmplex~d to a plasmid DNA. an internalizable cell surface epitope ' ulates through theendo~ytic pathway, be transcribed, leadingto the producleic acid;Ab, antibody.

Hinrch and Kroemer

16

of the specific antibody (Figure 2), the plasmid can be expressed in a transitory fashion. Depending on the specificity of the monoclonal antibody, reporter genes have thus been specifically targeted to B cells (anti-IgD antibody) or to T cells (anti-CD3e antibody) [91,92]. Moreover, the IL-3 gene has been transferred into hemopoietic progenitor CD34+ cells in vitro [93]. The technique of “antifection” thus may facilitate the expression of genes in discrete subsets of immune cells. Since both partners of the hybrid molecule used for “antifection,” the antibody and the plasmid, can be easily exchanged, this technique may provide a near-inexhaustible playground of experimental immunomodulation. VI. CONCLUSIONS

In clinical and outpatient routine, immunomodulatory interventions nowadays are still limited to rather few approaches. However, medical practice will soon be enriched by the availability of an increasing amount of sophisticated tools for immunomodulation. On the one hand, recombinant gene technology allows the generation of recombinant proteins. On the other hand, gene therapy may facilitate novel, previously inconceivable types of immunomodulation. As a caveat, it should be noted that both increasing costs and practical obstacles will limit the use of these novel types of immunomodulation to severe diseases of the economically privileged classes in Western countries. Therefore, the search for simple chemicals for immune therapy should not be abandoned at the expense of recombinant proteins or DNAbased molecules. Whatever the future will bring to us, it appears clear, however, that any kind of immunomodulation will have to take into account the three existential choices made by B and T cells, as defined earlier: survival versus death, response versus anergy, and different classes of response. Therefore, the succesful development of immunomodulatory regimens will depend critically on the fundamental understanding of the immune system. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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,

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I.,’Y.*;I.IS~x.MIA

_...:

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ac Colum~ia~niversity,New York,New York

ImmunoglobulinE(1gE)-mediated allergic reactionscomprisethe classic typeI immediate hypersensitivity reactions that occur within seconds to minutes of exposure to allergens or antigens. These immune responses can result in systemic anaphylaxis, wheal and flare reactions,allergic rhinitis, food allergies, or bronchial asthma. The underlying mechanism that links these reactions is the binding of IgE to highaffinity Fc receptor (FceRI) on the surface of mast cells or basophils and crosslinking of FcdWIgE complex by antigens. These events trigger the release of several different inflammatory mediators such as vasoactive amines, chemotactic factors, andcytokinesthatinturnareresponsibleforthevariedsymptomsof allergic disease. The route of allergen exposure (e.g., inhaled, intravenous, ingested), the dose, and individual genetic predispositions all can modulate the type or site of reaction that an individual may experience. The ability of €3 lymphocytes to produce IgE in responseto antigens relies on a complex set of molecular events, several of whichhavebeendelineatedonly recently. One key advance to theunderstanding of this pathway was theidentification of distinct phenotypic subsets of CD4' T helper cells, termed Thl and Th2 cells. It appears that Th2-"type" T cells secrete cytokines that are responsible for stimulating anIgE response. Another advance was learning how the cytokines these cells secrete are able to initiate and regulate signals required for B cells to undergo immunoglobulin (Ig) heavy-chain class switching to IgE. In this chapter wewill review the molecularpathway that leads to IgE synthesis, beginningwithTh2secreted cytokines that bind to their cell surface receptors on resting B cells and trigger tyrosine phosphorylation of the receptor complex, activating intracellular signal transducing proteins. These proteins in turn stimulate specific transcriptional events that lead to Ig heavy-chain class switch recombination, resulting in an Ig heavy-chain locus that can produce messenger ribonucleic acid (mRNA) encoding IgE heavy-chains and ultimately allergic immune response.

In 1986, Mosmann, Coffman, and colleagues [l] reported that murine C clone lines can be classified into subsets defined by unique patterns of cytokine synthesis and secretion. Type 1 T helper (Thl) cells secrete interleukin-2 (IL-2), tumor necrosis factor (TNF)-P, andinterferon-gamma (I , whereastype2T helper (Th2) cells can secrete IL-4, IL-5, IL-10, and IL-13 of producing IL-3, IL-6, granulocytemonocytecolony CSF), and TNF-a!. A third subset, referredto as ThOcells, exhibit combined type 1 and type 2 patterns of cytokine secretion and comprises the predominant subset isolated fromhumanperipheralblood lymphocytes [2,3]. Type1cytokines are important for delayed-type hypersensitivity immune responses, contact sensitivity, macrophage activation, and heavy-chain Ig class switch to IgG2a and IgG3. In contrast, type 2 secreted cytokines provide essential helper function for humoral immune responses and can induceIg class switch to IgCl andIgE [4] (Figure l). Both Thl and Th2cells are believed to arise froma common progenitor Tcell population that only secretes IL-2 on antigen stimulation. These naive progenitor Th cells are termed Thp. The generationof a polarized Thl or Th2response from naive Thp cells is influenced by the nature of the cytokines present at the time of initial antigen stimulation. For instance, several groups have shown that the presence of IL-4 at the time of antigen stimulation increases the frequency of IL-4(Th2)-producing cells generated [reviewed in Refs. 5-71. The presence of IL-4 also inhibits the development of IFN-7-producing Thl cells[8]. In addition to IL-4, another Th2 cytokine, IL-10, can decrease the production of IFN-7 in antigenstimulated Th culture systems. In contrast to IL-4, which appears to mediate its effects on Th development by directly influencing T cell differentiation¶ IL-l0 appears to decrease priming for IFN-7 production by its activity on antigen presenting cells [g]. Several studies have demonstratedthat thecytokines IL-12and IFN-7increase the production of Thl cytokines and diminish the production of Th2 cytokines (reviewed in Ref. 5). Interleukin-l2 has clearly been shown to increase the priming

TH 1 IFNy IL-2 TNF-B

Naive CD4e THP

IIL-2

1 T helper subsets and their cytokines. DTH, delayed-type hypersensitivity.

S

for IFN-y (Thl) production at the of time antigen stimulation[lo, l l]. Thereis good evidence, both in vitro and in vivo, that IFN-y can decrease the production of IL-4 during an immune response. heth her this effect is secondary to an inhibition of differentiation of hp cells into Th2 cells, or to an inhibitionof the growth of Th2 cells, is not clear. In addition, conflicting data exist as to whether IFN-y by itself ming for IFN-y production [6,7,12]. Thus, the role IFN-y plays in fferentiation remainsless certain. In ad~ition the to developmental effects, Th cell cytokines can alter the growth and proliferation of differentiated T cells. These effectsare thought to be important in the cross-regulation of different Th cell populations. For example, IFN-y inhibits the proliferation of Th2 cells but has no effect onThl cellular proliferation [13,14]. It has been propo hat this selective antiproliferative activity may be the mechay present atthetimeofantigenstimulation to inhibit the nism thatallows outgrowth of any Th2 cells that were generated in response to antigen stimulation cells that produce IFN-y (which include Thl cells, natural killer [NK] subsets) to suppress the development of a Th2 response. Alternately, IFN-y producing cells may inhibit an ongoing secondary Th2 response through the antiproliferativeeffects of IFN-y. A second example of the selective effects of cytokines on Th proliferation is the finding that IL-4 can support the growth of Th2, but notThl, cells [3,15]. This relationship raises the possibility that the defect in Th2 responses reported in IL-4 knockout mice [16,17] may, in part, be due to the re~uirementof this cytokine for Th2 cellular proliferation. In effect, through self-amplification and cross-regulation, Th subsets can modifyand skew an immune response toward a specific and polarized pathway. Finally, costimulatory signals have been shown to help regulate Th differentiation both in vitro [l81 and in vivo [ 191. For example, in experimental allergic encephalomyelitis (EAE), anti-B7-l antised the incidenceof disease inthe setting of increased IL-4production, 2 antibodies exacerbated the diseaseseverity [20]. Anti-B7-2 antibody treatment also suppressed the disease progression in the nonobese diabetic (NOD) model of autoimmune diabetes [21], supporting a role for B7-2 costimulation in the initiation of autoimmune disease. One important example of the relevance of the polarized Th response occurs in the regulation of immunoglobulin isotypes. The IFN-y produced by Thl cells promotesthe selective production of IgG2aandIgG3,theprincipalantibodies involved in opsonization and phagocytosisof microbes, and suppresses the production of IgE and IgGl. In contrast, type 2 cytokines promote selective the production of IgGl [22,23], whichcontributes to the neutralization of toxinsproduced by extracellular bacteria and of IgE, which is critical to mast cell degranulation (reence, the ability of the immune system to produce isotypespecific humoral antibodies relies on the differential productionof cytokines and is a key factor in the developmentof diverse biological responses.

The Th type 2 cytokines IL-4 and IL-13 are the key mediators in Ig class switching to IgE and therefore are important for IgE-dependent mast cell degranulation. uch of the early evidence for this was derived from finding that the presence of

IL-4 was necessary for class switchingto IgE. by l3 lymphocytes, observedin several in vitro [22,23] and in vivo (reviewed in Ref. 24) models. In addition, mice homozyous for a mutation in the IL-4 gene were unable to produce substantial IgE[16,171, re recently, it has been demonstrated that a second cytokine produced by human cells can stimulate IgE production. This cytokine, IL-13, shares both considerable sequence homology and biological activity with XL-4, including IgE synthesis [25,27]. Although the human IL-13 shares si~nificanthomology with the murine equivalent [28], themurineprotein is notimportantforIgE ~roduction,likely because of the lack of response of murine l3 cells to IL-13. Further, considerableevidence has accumulated supporting the model that the pathogenesis of IgE-mediated allergic diseases, such as rhinitisand extrinsic asthma, involves heightened Th2 mediated immune responses. For example, the great majority of allergen-specifi~CD4’ T cell clones derived from peripheral blood lymphocytes of patients with allergies express aTh2 and T h o phenotype consisting of high levels of IL-4 and IL-5, and little or no production of IFN-y [29,30]. Interleukin-4 and IL-5, but not IFN-y, levels are elevated in sera of patients with allergic asthma [31,32]. Also, allergic asthmatics’ peripheral blood possesses an increased number of B cells bearing the IgE receptor CD23, which is evidence of cell activation and IL-4 and IL-13 up-regulation[33]. The presence of activated Th2 cells in target organs during human allergic disease has been well documented. As examples, increased numbers of activated Th2 lymphocytes and mast cells expressing IL 4 mRNA or producing IL-4 have been assayed from bronchoalveolar fluid [34,35] and nasal mucosa [36,37] of patients with symptomatic allergic asthma and rhinitis, respectively. These results can be replicated experimentally after allergen inhalation challenge, but not after diluent challenge in atopic patients [38], Further, these results are specific to allergic disease, as exemplifiedby one reportwhich measured increasedlevels of IL-4 and IL-5 in the bronchoalveolar lavage fluid of patients with allergic asthma, in contrast to increased IL-2 and IL-5 measured in bronchoalveolar lavage fluid of patients with nonallergic or intrinsic asthma [39], Interestingly, a close correlation between the number of activated T cells and severity of atopic asthma hasbeen demonstrated in both blood and bronchialfluid [40). More recently, IL-13 hasbeen detected in target organs during allergicdisease. Although it has been difficult to measure IL-13 in the peripheral blood during allergic exacerbations[41], IL-13 mRNA and IL-13 transcripts have been measured in the bronchoalveolar lavage cells of atopic patients after segmental bronchial allergen challenge E421 and in the nasal mucosa of patients with perennial allergic rhinitis [41], The roleof IL-13 expression in vivo is not yetwell defined.This expression of IL-13 predominantly in the target tissues may be a manifestation of a more limited local roleof 1L-l3 in IgE regulation rather than a sy~temicone. some circumstances, the presence of IL-13 may substitute for IL-4 in promoting IgE synthesis, In support of this latter model is one study which described that “naive” (~D4’45Ro-) human T cells can be primed in vitro, to secrete large amounts of IL-5, IL- 13, and IFN-y, but not IL 4, and these cells can induce IgE production efficiently. This IgE response was blocked completely by neutralizing anti-IL-l3 antibodies, but not by anti-IL-4 [43]. Whereas IL-4 is critical to IgE production, another type 2 cytokine, IL-5, acts as a differentiation and growth factor for eosinophils during allergic disease.

Interleukin-5, IL-3, and CM-CSFall induce eosinophil production, as demonstrated in both invitro and in vivo experiments [44], but only IL-5 is specific for the eosinophil lineage. The IL-5 stimulates eosinophilopoiesis in the bone marrow [45] and promotes the terminal differentiation of myeloid precursors into eosinophils [46]. Eosinophils play an essential role during allergic diseases and parasitic infections by releasing eosinophil-derived proteins such as major basic protein ( and the eosinophilic cationic protein (ECP). In helminthic and parasitic infections, the eosinophilie-derived granules are believed to be toxic to the infesting cells. In asthma, epithelial sheddingis believed to result from therelease of eosinophil major basic protein 2471, and mucus hypersecretion and airway hy~ereactivity believed are to be consequences of the release of the eosinophil and mastcell-derived leukotriene C4, which is cleaved to the active products leukotriene D4 and E4 [48]. In addition, IL-5 promotes the migrationof eosinophils from the blood to the tissues in response to antigen challenge [49,50] and increases eosino~hil, butnot neutrophil, adhesion to vascular endot~elium[Sl]. Although IL-5 does not contribute to IgE production, the Th2 immune response that promates IL4, IL-13, and IL-5 secretion, all contribute to the in~ammation-related symptoms in complementary ways.

Many of the biological activities regulated by cytokines arise from their ability to by induce thetrans~riptionof target genes rapidly. Gytokines initiate this regulation binding to their cell surface receptors and activating intracellular signalin cules that then can induce transcription. The JAK-STAT pathway is one important signalingpathwayresponsible for transcription of specific genes in response to cytokines and growth factors. Bindingof cytokines to their receptors results in the oligomerization of the receptor proteins. These oligomers can be dimers hetero), trimers, or tetramers, depending on the cytokine. This oligomer the receptors results in the activation, likely by transphosphorylation, of the Janus kinase (JAK) family of tyrosine kinases, which are physically associated with the cytoplasmic tails of the cytokine receptors and exert a low basal level of kinase activity constitutively. To date, the JAK familyis known to comprise four kinases, referred to as JAK1, JAK2, JAK3, and Tyk2. They are expressed ubiquito except JAK3, which is primarily expressed in hematopoietic cells (reviewed in 52). The activation of the receptor-associated JAKs then leads to the phosph tion of tyrosine residues within the cytoplasmic tailof the receptor proteins. These phosphorylated tyrosines in turn serve as docking sites for the binding signaling proteins, the signal transducer and activatorof transcription (S teins, via interactions betweenthe receptor phosphQtyrosy1 motifs and src homQlogy 2 (SH2) domains of the STATs. The STAT proteins, once recruitedto complexes, become activated via tyrosine phosphorylation by the J in the receptor complex. Subsequently, the STATs form homo- or heterodimers, dissociate from thereceptor complexes, and translocate into the nucleus, wher activate transcription of genes whose promoters contain STAT bindingsites. of these STAT binding sites were initially characterized as elements important for the activation of gene expression by IFN-7 and were therefore termed gammaacti-

vation site (GAS) elements (reviewed in Refs. 52-54). It is now clear that many of these elements can act as response elements for other cytokines. TheJAK-STATsignalingpathwayfortheinterferons was the first to be studied extensively. The cell surface receptor for IFN-y is composed of a heterodimer of an CY chain, whichis associatedwithJAK1, and a (l chain, which is associated with JAK2. Althoughthe presence of only theCY component allows a cell to bind IFN-y it is not sufficient to generate a biological response (as measured either by IFN-y-stimulatedtranscriptionorantiviralresponses) [55,56]. Experiments in which the (l subunit was transfecte~ intocells that only expressed the CY subunit demonstrated "that the p subunit was necessary for these cells to activate -STAT signaling pathway and generate antiviral responses 155,561. This subunit, also referred to as accessory factor-l (AF-l), is absent in Thl cells, but not Th2 cells, and is believed to be the reason underlying the inability of Thl cells to activate the JAK-STAT signaling pathway in response to IFN-y [57]. After binding to its cell surface receptor, IFN-y stimulates the activation of JAK1 and JA leading to the phosphorylation of the receptor proteins. The phosphorylated tyrosines of the CY chain act as a docking site for the binding of Statl . Activated Stat l then homodimerizes and translocates into the nucleus, where it acts as a transcriptional activator for several IFN-induciblegenes [54,58,59]. It is now recognized that other cytokines share this general model of signal transduction, and that at least seven members of the STAT family exist (Stats l , 2, 3, 4, Sa, 5b, and 6). These proteins share significant homology over several fu tiona1,domains. The greatest homology is observed within the regions of the S 3 motifs and the deoxyribonucleic acid (DNA) binding domain (reviewed in 60) (Figure 2). Nonetheless, different cytokines/ligands are able to activate rent STATs with great specificity. Specificity of signaling is achieved at the level of the cytokine receptor/STAT interaction and not regulated by the particular volved. This specificity results from the interaction between the S main of the STAT and motifs on the receptor chains that, after activation by ligand, contain phosphorylated tyrosine residues [6l]. This interaction allows the recruitment of different STATs into differentreceptor complexes. The signal transduction pathway activated by IL-4 and L 1 3 both utilize the Stat 6 molecule. Originally Stat 6was purified and cloned from nuclear extractsof IL-4-stimulated human myeloid cells. These nuclear extracts contained DNA binding activity capable of specific interaction with a CAS element. hen this activity was' purified, it was found to be a specific l00 kD polypeptide that functions in a dimeric state and reacts with an antibody thatrecognizes phosphotyrosine, confirming its re~uirement fortyrosine phosphorylation 1621. In hematopoeitic cells, IL-4

SW3

Signaltransducerandactivator residue.

SW2

of transcription (STAT) protein, Y, tyrosine

binds to acell surface receptor complexthat consists of a heterodimer of the ligandspecific cy chain(termed IL-4Rcx) and the common y (yC) chain that is also a component of the receptors for IL-2, IL-7, IL-9, and IL-15. Dimerization leads to the activation of JAKl, which is associated with the IL-4Ra, and JAK3, which is bound t o y e . The activation of these JAKs results in phosphorylation of several tyrosine residues contained within the cytoplasmic tail of the IL-4Rcy. Then Stat 6 can be drawn into the receptor complexby interaction between the S Stat 6 and one of several phosphotyrosyl motifs of the IL4Rcr. S tyrosine-phosphorylated, allowing it to form homodimers using two r phosphotyrosyl interactions. omodimers of Stat 6 can then translocate into the nucleus, where they bind to two distinct types of elements. The Stat 6 can bind to, but not activate transcription from GAS elements that contain the sequence TTC N(2-3) GAA. In addition, Stat 6 can bind to the sequence TTCN(4) GAA 163,641. This sequence, which does not bind other STATs, is found in the promoter of IL-4-inducible genes, such as the CD23 gene and theIg heavy-chain germline epsilon ( E ) gene (discussed later) [65,66] (Figure 3). Proof of the importance of Stat 6 to IL-4-mediated signal transduction came from mice which lack expression of Stat 6 as a result of gene targeting of the Stat 6 gene. Mice deficient in Stat 6 by gene targeting are unable to activate several immune events normally induced b IL-4 tivation of a Th2 proliferative response induction of CD23 and cells, nor can they produce significant IgE and IgGl response helminth infection 167,691.

nucleus

IL-4-mediated Stat 6 signal transduction pathway. IL4RE, interleukin-4 response element; PY, phosphorylated tyrosine.

It was believed that the IL-13 receptor was composed of the a chain that is “common to the IL-4 receptor (IL-4Ra) and associated with JA component that is not completely defined but differs from the yC chain andis not associated with JAK3 (reviewed in Refs, 70-71). It has since been learned that a lower-affinityreceptor component, termed IL-13 receptor a, alsoiscapable of binding to IL-13 but not IL-4 [72]. It also has been proposed that coexpressionof the IL-13 a receptor subunit and IL-4Ra can act ashigh-affinity a receptor complex [72], Because the IL-4Ra chain is a c o ~ p o n e n tof the IL-13 receptor, the downstream signaling initiated by IL-13, including the activation of Stat 6 , overlaps with the signaling activated byIL-4.

Immunoglobulin heavy-chain classswitching is the process whereby an activated cell changes the heavy-chain constant region at the carboxy t e r ~ i n u s(CH) of t antibody it produces to alter its effector function while still retaining its antigenic specificity. In general, Ig class switching occurs by a deletional recom~inationevent between regions of repetitive DNA, called switch ( S ) regions, that are present upstream of each heavy-chain constant region, except C& This recombination juxtaposes the fully assembled variable region (composed of rearranged VDJ gene segments) directly upstreamof different CH genes that encode for different Ig isotypes. For example, in the case of Ig class switching from IgM to IgE, the C@locus and downstream genes are deleted so that the CE gene becomes juxtaposed directly3’ to the VDJ region and VDJ-CE transcripts are encoded 1241. High levels of Ig class switching require two types of signals. One is a B cell activation signal that follows contact between the B cell surface molecule CD40 and itsligand CD4OL expressed by activated Th cells[73]. Recognition of the anti~en-specific MHC class I1 complex by the T cell receptor leads to the expression of CD40L on the Thcells. This interaction occurs in germinal centersof secondary lymphoid tissues and in part prevents the B cells from undergoing apoptosis [74]. Of note is that the X-linked hyperimmunoglobulinM syndrome results from muta* in the genes encodingfor CD4OL and is characterized by impairedCD40/ L interactions, defective class switching, and absence of all immunoglobulin isotype classes except IgM [75]. The second typeof signal responsib~e forIg class switching involves cytokineinduced expressionof particular germline CH transcriptsvia cytokine activation of the corresponding germline promoter, Immunoglobulin class switching has been shown to be preceded by the expressionof germline transcriptsin the area surrounding the switch region. Also, certain cytokines have been shown to regulate specific Ig class switching differentially through the induction of particular germline transcripts. For example, it hasbeen demonstrated that the addition of IL-4 to lipopolysaccharide (LPS) cultures of B cells induces high levels of germline E and y l transcripts that correlate with the amount of Igl? andIgG1induction, respectively ~ 7 ~ , 7 8More ]. recently, IL-13 has been shown to induce germline IgE heavy-chain gene transcription in purified B cells, even in the presence of neutralizing anti-IL-4

antibody, confirmingits independent role in IgE production [25]. In contrast, IFN-y has been shownto suppress theIL-4-induced germlineE transcription. Furthermore, regulation of IgE switching seems to occur through these cytokines9 antagonistic effects on the germline E promoter [79]. Theseobservationsseem to support a model in which specific heavy-chain germline transcription always precedes switch recombination and in which type 1 versus type 2 cytokines differentiallyregulate the switching processto CE. Althoughtheassociation betweenheavy-chaingermlinetranscription and switch recombinationis highly correlative, direct proofof a causal relationship has, until recently, been lacking. Germline transcription initiates at exon an (I exon) that is 5 of the corresponding S region and initially proceeds through the S region and the CH gene. The transcript is processed to a stable form which is relatively conserved among different CH transcripts. This conservation raises the possibility that the transcripts themselves may play an important functional role switch in recombination. Germline transcripts cannot encode large proteins because they contain stop codons in the open reading frame of the CH gene but are believed essential for making the DNA accessible to the recombination machinery (reviewed in Ref.24). The transcripts may either act as RNA molecules or encode important short polypeptides [go]. An alternative model is that the germline transcripts themselves are not essential but that transcription per se (or perhapsassociatedtopoisomerase activity or chromatin change) is essential for targeting switch recombination to specific heavy-chain loci. A third possibility is that germline transcription is an epiphenomenon of other cytokine-in~uced events (e.g., switch region recombinases) that areessential for specific switching events. Theimportance of germlinetranscription to class switchinghasbeen addressed by gene targeting experiments in which the I region promoters and exons have been removed or replaced. For instance, removal of the Iyl element from the heavy-chain locusby homologous recombination markedly inhibitedIg switching to IgG1. Also, heterozygous mice with one wild-type allele and one mutated allele switched to y l on the wild-type allele but not the mutated one [81]. In another study, mice lacking the Iy2b germline promoter could not undergo switching to the IgC2b isotype [82]. These studies suggest that sequences within the Iyl or Ig2b locus, including genes encoding promoter activity, are required for switching to IgGl and IgG2b, respectively. In comparison, mutated B lymphoid cells in which enhancer and the Ig heavy-chain E (IE)locus was replaced by the Ig heavy-chain EIA, Vh promoter to allow constitutive transcription through the E switch region still transcribed the E locus independentlyof the presence of IL-4. These cells can switch to E in the absence of IL-4, but at levels 10-fold lower than that of nonmutated cells cultured with IL-4[83]. These findings have been confirmed in transgenic mice with the same mutation [77]. These studies suggest that optimal efficiency of the switching process requires the presence of an intact IE region, implicating factors besides the transcription through theSEregion in the regulationof class switching. Similar studies in which the Iyl region was replaced by the metallothioneine promoter also illustrate thatproviding germline transcriptsto a heavy-chain locuscan supplant the requirement for cytokine in the switching process and supports a model in which cytokine (e.g., IL-4) targets Ig class switching simply by the induction of these germline transcripts [84].

Y

Y

IgE switch recombination. IL-4 binds to its cell surface receptor on the resting IgM' B cell. After tyrosine phosphorylation of the receptor complex, Stat 6 is activated, rendering it competent for translocation into the nucleus and binding to specific DNA sequences found in the promoter region, thereby stimulating germlineE transcription. A deletional recombination event occurs between § regions, juxtaposingthe fully assembled variable region directly beside theC E ,and VDJ-CEtranscripts are encoded.

In summary, IgE production is the end result of highly coordinated and specific immune responses beginning with exposureto allergens in a genetically predisposed individual. Activated Th2 cells secrete IL-4 and IL-13 that bind to their cell surface receptor on the resting IgM’ cells. After tyrosine phosphorylation of the receptor complex, Stat 6 is activated, ndering it competent for translocation into the nucleus and binding to specific A sequences found in the promoter region, cell-Bcell contact involving stimulatinggermline E transion.AfterT a deletional r~combinationevent occurs, resulting in mRNA that enhe IgE heavy-chain polypeptide (Figure 4). Immunoglobulin E binds to ast cells and basophils, allowingcross-linking of FC€RI/IgE complex by antigens and release of a variety of inflammatory mediators that cause allergic symptoms. What remains unclear at this point is precisely how this pathway is modified during human allergic disease or after exposure to certain antigens and what dictates anindividual’s genetic predisposition to a heightened IgE production. The 5q3 1-q33 chromosome locus containing genes encoding for IL-4, IL-5, IL-l3 has been associated with clinical atopy, bronchialhyperresponsiveness, and elevated IgE levels [$5,$7], but delineating howinteractionsbetweenenvironmentaland genetic influences modify complex immune responses remains a great challenge.

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Previous work has demonstrated that immunoglobulins are processed by antigen presenting cells (APCs) and thatidiotypic peptidesderived from thevariable regions are presented on major histocompatibility complex (MHC) molecules to T cells. This has raised the questionof whether idiotypic peptides could influence the Tcell repertoire in the thymus andin the periphery, be it by positive or negative selection. Some answers have come from studies in mice made transgenic for a T cell receptor which recognizes an idiotypicpeptide from a U 3 " immunoglobulin light chain, presented on class I1 (I-Ed) molecules. Deletion of idiotope-specific T cells, both double positive (CD4'8') thymocytes and peripheral single CD4' T cells, is observed when the serum Xz3l5 immunoglobulin concentration increases beyond 50 pg/ml. On the basis of serum i~munoglobulin(Ig) concentration considerations, the results suggest that germline-encoded idiotypic peptides induce clonal deletion. y contrast, the highly diversified idiotypic peptides spanning the complementary determining region 3, as well as the unique idiotypic peptides generated by somatic mutations, probably do notcause deletion because their concentrationsare toolow. In addition to negative selection, idiotypic peptides may constitute a rich source of antagonist and agonist self-peptides thought to be importantin positive selection of thymocytes.

ical Besides their well-known function in elimination of foreign antigen (Ag), Ig molecules have attracted the attentionof immunologists because they may have a role in the internal regulation of the immune system of an individual. The basis for this idea is that billions of differentIgs, which have highly diversified variable (V) 3

regions [l], exist. Because of the low concentration of each antibody (Ab), the immune system is thought not tobe tolerant [2] to unique antigenic determinants in the V regions, called idiotopes (Ids) [3,4]. Therefore, immunization with an Ig may elicit anti-Id antibodies in syngenic animals [5] and even in the autologous animal itself [6]. Thus, the B cell part of the immune system may to a large extent be composed of Ig molecules which mutually bind each other. Such Id-anti-Id interactions may regulate the immune system, as hypothesized in the network theory of Jerne [2].

Jerne’s network theory only dealt with B cells and antibodies. owever, it became evident that Tcells wereneeded for anIg to induce an anti-Id response because anti-Id ab responses could not be elicited in athymic nude or neonatally thymectomized mice owever, the mechanism by which T cells recognize Id remained unclear. In fact, a large bodyof work performed in the seventies and early eighties on Id-specific T cells of suppressor andhelper type, claimed to recognize complete Ig in the absence of MHC molecules, has not been substantiated by later experiments. There were, however, reports suggesting that T cells can distinguish Ids and recognize Id as a “carrier” in in vivo adoptive transfer experiments [g, lo]. Work in KristianHannestad’slaboratoryshowedthatsuchT cells could recognize Idcontaining fra~mentsof Ig and thus seemed to be independent of Ig conformation f10-13). Furthermore, the Id-specific T cells were shown to recognize epitopes that depended on somatic mutations for their expression [14,15]. The response of these polyclonal Tcells wasunder ~ H C - l i n ~ immune ed response (Ir) gene control [12,16181. Collectively, these studies suggested that Id-specific T cells behave like conventional Tcells, but since only polyclonalin vivo responses wereaccessible for studies, further progresswas hampered.

ic, The emo on strati on that CD4’ Id-specific T cells could be cloned facilitated further studies [19]. Employment of these clones unambiguously showed that Id~specific T cells were MHC-restricted and that they recognize an idiotypic fragment of an Ig light (L) chain presented on class I1 molecules [19,20]. Furthermore, complete Ig molecules wereshown to be endocytosedandpd by professional A subse~uentlypresented Id peptides on their class 11 molecules [19,21]. Thus, the way T cells recognize Id seems to be identical to the way they recognize conventional antigens. The studies discussed were performed with an Id epitope which comprises residues 91-101 of the U 3 I 5 Ig light (L) chain of the BAL (IgA,The Id (X2”’) peptide is presentedonthe I-Ed class I1 molecule to B A L ~ / cCD4’ T cell clones [ 19,20,22] (Figure 1). ~ecognitionof X2315 by Id-specific T cells strongly depends on residues Phe94, Argg5,and wit hi^ the 91-101 sequence [19,201 (Figure 1). These three residues are nongermline as a result of somatic mutations in the VX2 gene segment of the ~ ~ P C 3 1plasmacytoma 5 cell line [23]. We have suggested thatthe positively

98 99 100

~

1 The Id ( U 3 ’ ’ ) model system: processing of endocytosed Ig and presentation of an Id peptide on class I1 molecules to Id-specific CD4’ T cells. The length of the naturally processed Id (u~”)is not known, but a synthetic peptide covering amino acid positions 91101 isthe minimal length of a maximal stimulatory synthetic peptide. Residues 94-96, which influence or are essential for recognition by Id-specific CD4” cells, are shown in bold in the inserted table [19-221. 11, major histocompatibility complex (MHC) class I1 molecule; T-cell receptor, heterodimeric symbol, opposed to the Id peptide/class I1 MHC symbols. Id, idiotypic; CD, cluster designation.

charged Arg” residue fits into negatively a charged pocket in the I-Ed molecule [22], a suggestionwhich is supported by a recent study of Schild et al. [24] on the peptide binding motif for I-Ed. Furthermore, since some h23’5-specificT cell clones do not distinguish between Phe94 (U3*’)and Tyr94 (germlineh2), while others require the Phe94 amino acid, position94 appears to be a T cell receptor (TCR) contact residue [20,25]. Interestingly, all clones which cross-reactbetween Phe94,and Tyr94 peptides use a very conserved T cell receptor (Va3, Jal; V069 JOl.l), while those clones which only react to Phe94use more diversely composed TCRs [25]. The narrow specificity for the 94-96 residues, as well as the conserved nature of the Id (u~”)specific TCR, may be caused by T cell tolerance to germline X2 Ig; i.e., Id-specific T cells focus exclusively on determinants dependent on somatic mutations (or the CDR3 regions) [14,15,19,26]. The demonstration of Id-specific T cell clones that recognize Id peptides presented on MHCclass I1 molecules has since been confirmedby work from a number of laboratories [26-321. Moreover, peptides derived from the constant parts of Ig (allotypic peptides) have alsobeen shown to be presented by class I1 molecules to T cells [33,34]. These functional studies have been biochemically confirmed by the elution of Ig V region [35,36] and C region[35-371 peptides from class I1 molecules.

1.

3

The fact that the efficiency of presentation of Id peptides depends on the conformation of the moleculeitself appears to be of physiological importance. For example,the successfulIdpresentationrequireshigh concentration of the complete M3 15 molecule and the Fab3I5 fragment, but the presentation isvery efficient even with low concentrations of VXt3”, Fv315,and U 3 I 5 fragments of 1191. A likely explanation is that complete Ig and Fab fragments are relatively resistant to processing and do not readily yield Id peptides [ 191. This finding has been confirmed for otherIgs [29,30].

The study of Yamoto et al. [38] demonstrated that cloned Tcells specific for the Id 104E plasmacytomawere class I ( -2Dd)-restricted. ecent studies have extended these findings [38] and conclusively shown that Id peptides [39,40] and a C K peptide [41] can be presented on class I molecules to cloned Id-specific CD$’ cells. Interestingly, in the study of Cao et al. [40], the CD8 clone was raised against an influenza hemagglutinin epitope and fortui usly discovered to cross-r an Id peptide (residues49-58) of the ~ O P C 2 1 chain in association with

In addition to the conventional class I1 pathway utilized for processing larly derived 1g and presentation of Id, newly synthesized U 3 * ’ Ig in cells probably is processed in the endoplasmic reticulum(ER) or a pre-Golgi appa-

Transfection of 3’5 gene

h2 (BALBlc B cell l y ~ p ~ o ~ a )

Transfectant Proliferation

Blymphomacellsprocess and present Id peptides derived from their endogenously produced Ig[21]. By extending these experimentsto KDEL-tagged h23’5,it was demonstrated that neither secretion nor surface expression was needed for processing and presentation. In fact, expression of the “mutated”X23’5chain in the ER/Golgi seems to suffice for processingandpresentationofIdpeptides [42]. Id, idiotypic;Ig,immunoglobulin; ER, endoplasmic reticulum.

ratuscompartment [21,42]. The DEL motifattachedto was thoughttocause retention of proteins priorto cis-Golgi. owever,it is nowknownt tagged proteins, although usually foundthe ER, maytravel until before being retrieved [43]. Since signals involvedin cellular traffic are oftenleaky, transport of minor amounts of ? d 3 I 5 into an endocytic compartment cannot be excluded so far. In this endogenous pathway for Id presentation, neither display on the cell surface nor secretion of X2315Ig is required [42]. Others have also demonstrated processing of endogenous antigen in or close to the ER [44,45] and an ER proteolytic degradation pathway has been described [46,47]. Furthermore, binding of synthetic peptides to nascent class I1 molecules has been demonstrated in vitro in he presence of invariant chain, which is supposed to block the class I1 in the ER [48,49]. Moreover, peptides derived from proteins in the secretorypathway, and in particular Ig peptides, are frequently eluted from class I1 molecules [35-37] (however, such elution studies do not identify where inthe cell the processing occurs). The endogenous Id peptides found on class I MHC molecules could be generated by proteolytic degradation in or close to the ER. These peptides would be available either to class I or to class I1 molecules, dependingon length and complementarity tothepeptidebindinggrooves of the particular MHC molecules. It of the Id peptide to class I1 should be emphasized, however, that the actual binding molecules could occur more distally in the secretory pathway (e.g., Golgi apparatus or even endosomes)wherethepH is lowermorefavorableforbinding. Sirnilarly,alternativepres tationalpathways fo C class I have been described [SO]. cellis likely to predpeptidesderivedfromthe Ig it producesonboth itsclassI and class I olecules andthereforebe subject to the same token, B cells could regulate regulation by both D4' and CD8' cells. selection of both C ' and CD8' cells.

ecause Id peptides are spontaneously and continuously generatedby the two processing pathways of Ig (Sections 1I.Cand II.E), it would be of interest to investigate what influence Id peptides might have on positive and negative selection of the T cell repertoires within an individual, both in the thymus andin the periphery.

, Jerne'snetworktheory er of antigenic structures the B cell repertoire for foreign antigens and thus render theindividual immunodeficient. Thisis a relevant argument for antibody recognition idiotypes of becauseanti-Id antibodies recognize conformationally dependent Id on completeIg such as a combinationof heavy (H) and light (L) chains. owever, as dicussed earlier [Sl], the argument is less relevant for Tcell recognition of Id peptides because Id-specific T cells recognize short linear sequences (i.e., an l1 amino acid long synthetic peptide for A2315).

4

For the simplicity of the argument? let us assume that there are l00 equally expressed variable heavy (V,) germline gene segments (Figure 3). On the protein level, each VHmay be associated withan equal number of germline-encoded variable light (V,) sequences, yielding 100 X 100 = lo4combinations. Importantly, antigen processing will undo this combinatorial complexity, making the ~ermline-encoded T cell-de~in~dId peptides far less diverse than serologically defined Id on complete -t- L) Ig molecules [5 l] (Figure 3). Thus, a given VH germline-encoded amino acid sequence will be present on every hundredth Igmoleculeinserum,i.e., 100 pg/ml (m0.7 PM), whichis a rather high concentration. Processing of serum may Ig thus yield considerable numbers of identical Id peptides, even though most of these Id peptides actually are derived from different antibodies with different L chains, produced by different plasma cells. Because Ig molecules permeate most tissues, APC-like dendritic cells ( ~ ~throughout s ) the body, including the thymus, are expected to process all Ig molecules circulating in the individual continuously and present a collection of Id

Serological (VH+VL) Id ‘(m14

-

4

10 Abs with

100 VH

VLI

vL2

VLn

X

=

100 VI,

....,.,.

equally many conforma~onaIIy dependent serological Id

200 iinear 100 VH

Processing and presentation of Id on class I1 MHC

“+e=.

+

loo vL

=

sequen~esfrom which Tcell defined Id peptides are derived

0

Antigen processing simplifies combinatorial idiotypic (Id) complexity of antibodnd A are ID peptides encoded by germline VH1 and VL1 gene segments, respectively. These Id peptidesare shown integrated ina complete Ig molecule. Processing ofthe Ig liberates short Idpeptides,which are presented to Tcellsiftheyhaveanchorresidues matchingthepeptide-bindingmotifsoftheclassI1moleculesof the APC.Serologically defined Id usually require H + L assembly and their heterogeneity is high as a resultof do not combinatorial association of H and L chains. Because T cell-defined Id peptides require H + L association, their complexity should be far less than that of the serologically defined Id. For the sake of simplicity, we have deliberately excluded N, I), and J region sequences that may greatly contribute to both conformational and linear diversity of the idiotypes. Id, idiotypic; Ig, immunoglobulin; APC, antigen presenting cell.

I

7

peptides on theirclass I1 molecules. It is important to note, however, that the collection of Id peptides being presented by an APC is likely to be much smaller than the numberof different Id peptides generatedby processing, This is so because only those Id peptides containing sequences matching the peptide-binding motifs of C molecules of the individual will be presented [19,22,52]. addition, because B cells spontaneously processand present Id on boththeir and class I molecules, one would expect that 1070 of polyclonal B resent identical Id peptides derived from the same germline V, (alcells have different V, sequences). Importantly, a B cell is expected ndogenous Id peptides derived from its homogeneous Ig more efficiently than exogenous Id peptides derivedfrom endocytosed heterogeneous Ig [52]. This is so because endogenous monoclonal Ig is present in higher concentrations inside the cell than each of the diversified Igs from the extracellular origin. This should yield a lot of identical endogenously derived peptides which w highnumberofclass I1 molecules.Ongoingexperimentsutilizing ~2315-transgenic mice support such a notion (unpublished data). This statementmay also hold true for peptides generated from processing of anti-Id antibodies binding to Id' serum Ig (sIg) because anti-Id antibodies are polyclonal, The relative resistance of extracellular Ig to processing also contributes to inefficient Id presentation of serum Ig (see Ref. 52 for anextensive discussion). Taken together, APC-like dendritic cells and macrophages are expect any time point, to present a multitude of Id peptides derived from serumIg. molecules will be charged with identical Id ted to present Id peptides efficiently from 4). the particular Ig which they produce themselves (Figure

In order to study the influence of Id peptides on T cell selection, we establis T cell clone 4 mouse strain transgenic for thea@T C of the Id (X2315)-specific (Val, Jal9, V~8.2,J61.2) [53]. Expression of the transgenic TCR on T cells was monitored by use of a clonotype-specificmousemonoclonalantibody(mAb)

ic, Thymocytes acquire their clonally distributed TCR by stochastic genetic processes. Thymocytes which recognize endogenousp e p t i d e / ~ H Ccomplexes on thymic cortical epithelial cells with low avidity seem to receive a survival signal; this phenomenon is called positive selection. Such positively selected t h y m o c ~ e smay later, as mature T cells in the periphery, bind with high avidityto foreign peptide presented HC molecules on APCs; binding may be followedby T cell activation [55-591. When compared to other TCR-transgenic strains [60], our X23'5-specificTCRtransgenic strain shows weak positive selection of transgenic thymocytes: The peal lymphoid compartment is lymphopenic and there is a light skewing toward cells [53]. This is surprising because a preponderance of CD4' cells is to be I

I

Difference of Id presentation between€3 cells and other APCs. Dendritic cells and macrophages can present many different Id peptides derived from extracellular (serum) Ig, but each at a low Id/class I1 concentration. The heterogeneity of serum Ig and the resistance of extracellular complete Ig to processing 1191 may both contribute to this. By contrast, l3 cells preferentially and efficiently present their own idiotypic peptides because they have a high concentrationof intracellular endogenous, homogeneous Ig available for processing. In addition, APC and €3 cellsprocess and presentamultitude of non-Igself-antigens(not shown). Id, idiotypic; APC, antigen presenting cell; Ig, immunoglobulin. expected in transgenic mice with a TCR (cYTPT) derived from a class 11-restricted T cell clone [60]. The frequent CD$' cells virtually all express both the transgenic as well as endogenous TCRa chains [53,61]. In addition, only ,= 70% of CD4' cells are while = 30% are negative for t Moreover, even among thoseCD4' cells which are in addition endogenous TCR, chains [til], while chains [63]. Expression of endo enou shutdown of recombination activation gene AC) expression and is a sign of poor positive selection of the transgenic TCR[64]. The size of the thymus of TCR transgenic mice is not much changed, being somewhat larger than normal in young, but slightly smaller than normal in older mice [[53], unpublished data]. As in most TCR-transgenic mice, the intensity of expression is prematurely increased in double positive thymocytes [53], perhaps as a result of early transcription of the already rearrangeda and P transgenes. Again indicative of a weak positive selection is a reduced frequency of both single 8' thymocytes [53]. Furthermore,double positive thymocytes develop into single positive cells, endogenous TC chainsarein~reasingly expressed [61].

Even though the findings in the periphery andin the thymus collectively indicate a poor positive selection, it should be positive selection was quency of peripheral --2b) mice were intermediate [53]. A further disclosure of positive selection was obtainedwhen making the transgenic mice deficient for rearrangement of endogenous TCR by back-crossing them "transgenic scid/scid mice an almost corned in the periphery, while e the expected skewing, th ith only about 5% of the normal absolute

ecently it has been shown that thepeptides involved in positive selectionof thymocytes could be either antagoni§tic peptides [SS] or agonistic peptides at low concentration [57,59] or partial agonists (for review see Refs. 65 and 66). Antagonistic or partial agonistic peptides are similar to the nominal peptide but contain amino acid substitution§ which affect the binding TC to C Antagonistic peptides inhibit the stimulation of mature T cells in an antigen-specific way and at low concentrations; i.e., their activity is not due to competition for MHC molecules. Partial agonists only elicit some effector functions compared to the agonistic (nominal) peptide and are inhibitory at low concentrations. Because of their diversity, Id peptides could represent a particularly rich source for agonist/partial agonist/ antagonist peptides involved in positive selection (Figure 5 ) . Presumably, certain endogenouspeptidepeptidespresented by I-Edcan se positive selectio the h2315-specificTC transgenic double positive (DP) 4'8' thymocytes. peptide could be either an antagonist/agonist peptide at low concentration or a agonist. Apeptideagonist will probablynot ariseearly in the life of ansgenic mice because for thisto occur, specific mutations in codons 94,95, and 96 of the Vh2 gene segment are required (Figure 1). Thus, it is not likely that such agonistic peptide has an influence on positive selection of h23'5-specificTC transgenic thyrnocytes. Partial agonist or antagonist peptides that can positive select the h2315-specificTC could conceivablybe other Ig-derived peptides, because icular X chains) are most likely to contain sequences similarto that scheme outlined, it has been demonstrated that processed 15 produced in the pery of amousebyatu ells withinthethymus ( gen etal.,unpublished) ever, it may be arguedthat a demonstrationof Id sensitization of this context because positive selection is thought mainly to occur on cortical epithelial cells [6'7,68], and it remains to be demonstrated that Ig peptides are presented onthatkind of stromal cell. Infact,theputativeblood-thymus barriercould erurn-derived Ig from being processed and presented by cortical wever, circumventing a putative blood-thymus barrier, complete Ig can enter the thymus by a transcapsular route and can bind antigens within the cortex [69].

~ypothetica1influenceofIdpeptidesderivedfromantibodies on selection of thymocytes: a, Id priming of thymic epithelial cells (TECs) in the cortex may result from Ig diffusion into the thymus by the transcapsular route 1691; doubly positive(DP) CD4'8' cells may bind partial agonist, antagonist, and agonist peptides withthe indicated outcomes. The reality is more complex because a single DP thymocyte may bind all these peptides at the same time, or sequentially, even on different TECs, and possibly integrate these signals to byNegative selection of thymocytes by Id-primed dendritic result in one of the three destinies. cells (DCs) at the corticomedullary border. The DCs are presumably Id-sensitized by serum Ig originating from vessels in the medulla. Id-sensitizedDCs may kill corticalDP thymocytes close to the medulla, and perhaps even single CD4+ cells in the medulla (GF Lauritzsen, PO ~ofgaard,K Schenck, B Bogen, submitted). Id, idiotypic; CD, cluster designation; Ig, immunoglobulin. If cortical epithelial cells really process and present Ig derived from serum, they may display a bewildering arrayof diverse Id peptides. Some of these may be antagonists, while others may be partial agonists or even agonists for a given Idspecific TCR, Further adding to the complexity, the absolute number of each peptide isp played on a cortical epithelial cell could vary for different Id peptides. The developing double positive thymocyte would have to deal with all these signals,and the outcome could be a survivalsignal(positiveselection), no signal(death by neglect), or a deletional signal (negative selection, although that is thought mainly to be effected by dendritic cells) (Figure sa). We hypothesi~e that d ~ v e l o ~thymoin~ cytes can integrate TCR signals during the selection process in the cortex; similarly the neurons can integrate electrical potentials in order to fire the action potential. For example, a DP cell may integrate signals from the interaction involving low concentration of an antagonist or anagonist with additional signals stemming from

5 \

ex

\ \

\

\

\ \

\ \ \ \ \ \ \ \

\

positive selectlon

\ \ 1

I \

l I I

l I l l l I i

I I I I I

I I I I I

I

Continued.

single or multiple TCR connections with other antagonistic, partial agonistic, or even agonistic peptide/MHC complexes. The outcome of such integration would depend on two hypothetical thresholdswhich constitute the bordersbetween neglect and positive selection, on the one side, andbetween positive and negative selection, on the other. Each of these thresholds would be a function of the affinity and concentration of each molecular constituent: TCR with coreceptors, peptide, and the complementaryMHC molecule. The poor positive selection observed in h23'5-specific TCR-transgenic mice may be related to polyclonal serum Ig's having an effect on positive selection of , as a result of sequence similarities. Indeed, the finding of better skewing toward C 4' T cell lineage in TCR-transgenicSCIDmice(which lack serum Ig) may be explained accordingly, but may also be due to a lack of endogenousTCR,chains. To explorethese possibilities, we are establishing TCRtransgenic micedeficient for Ig by breeding them homozygous fora pMT knock out construct 1701: If such mice show altered positive selection, Id peptides are highly likely to be the cause. Some circumstantial evidence supports the idea expressed previously because maternalanti-Id S107 injectionsuppressed Id S107 expression in offspring and reduced T cell responses to a S107 GDR2 peptide [71]. Although the evidence is indirect and of a functional nature, that study[71] suggests that the polymorphisms of self Ig may contribute to the mature cell T repertoire by positive selection mechanisms. Id peptides from the CDR3regions provide an extremely diversified source of self-peptides 1721; such CDR3 Id peptides may therefore serve as agonists, partial

agonists, or antagonists for peptides derived from ordinary foreignantigens. Thus, 3 peptides presented by cortical epithelial cells may be important to positive S. This hypothesis may be selection of thymocytes specific for conventional anti tested by studying postive selection in various other T transgenic trains of mice (e.g., hemagglutinin-specific) made Ig-deficient (see previous discussion).

A major mechanism for T cell tolerance to self-antigens is clonal deletionof thymocytes (1731, reviewed in Ref. 74). Id peptides from Ig are an extremely diverse source of self-peptides and mayextensively influence he T cell repertoire. W ectioncaused by Idpeptides,gtransgenicmice cells and in serum Ig have been established [75]. Suchheterozygous ~2315-transgenic mice have been crossed with heterozygous TC 5 , yielding four types of offspring: TG) mice specific fo hIL3I5-TG, anddoubly - and~2315-transgenicfigure 6). In o forbothTCRand X2 structs,apronounced deletion of tra is observed; the remaining few thymocytes are mainly C 1). In the periphery, lackof both CD4’ (xT& and CD8’ (Table l), consistent with deletion at the C stage of thymocyte development [76,77]. As would be expected from such staining results, lymph node T ce doubly transgenic mice had ~rasticallyreduced in vitro responses to Xz3l5. trast, X2315-expressing cells were present and could respond normally to cloned Id (~2315)-specificcells T [5 l ] (Table l). Thus, when Id-specific T cells and Id-expr~ssing cells are forced by transgenes to be expressed in the same individual, the animal does not “explode” by B cells mutuallystimulatingeachother.Rather,IdmultiplicationofTand expressing B cells persist while Id-specific T cells become deleted [S l]. Such a result couldhave several explanations.First,serum Ig with L chainsmayenterthe thymus, be processed, be presented, and induce deletion of thymocytes (Figure 5b). T

x X2315-Transgenic

Deletions Obtained in Offspring from TCR-Transgenic No exposure to maternal IgG u 3 1 5 :

[Offspring from (TCR-TGV X23’5o*)F,]

TCR-TG

Id-specific thymocytes Id-specific peripheral T cells Deletion B cells

TCR

X

Exposure to maternal IgG Xz3l5: [Offspring from 0Q3”-TGO X TCR-TC o)FJ

+ ~ 2 3 1 5 ~ ~ aTCR-TG

TCR

+x

(-> ( 4

1 5-

( 4 ( 4

5-

(->

(”4

(->

(->

~ ~ ~ ~ T G

l

al,deletion; (-), no changes; TCR, T cell receptor; IgG, immunoglobu~in G;TG, transgenic; Id, idiotypic.

Second, U 3 1 5 produced within the thymus (e.g., by thymic cells or plasma cells) may be endoc~osed andpresented by local A cytes. Third, in addition to thymocyte deletion [sl], mature C deleted in the periphery. To address these questions we have performed several experiments.

m (U” 5 - Q ~x ~ should have bee t themselves produce U 3 1 5 -TG mice have IgG a maternally transmitted IgG hus, the “effective” I GX1315 concentration may be around 125 prisingly, such TC X2315showed no e thymocyte deletion at birth [51]. Thus, a U 3 1 5 Ig serum concentrationof about 100 pg/ml was evidently not enough to delete Id-specific T thymocytes in utero (Table l). To obtain an even higher serum concentration of IgU315,we injected newborn TC genic mice i.p. with larg ounts of IgG2bhZ3l5mAbobtainedfrom cell omas of ~ z ~ ’ ~ -mice. TG ion was obtained at 111-410 pg/ml, wh tested 3 days later [5 l]. ~ i m i l a rinjection ex~erimentswere performedusingadult TC

tra~splacentall~ transferred

~xperimentswithmicedoublytransgenic for TCR andtransgenes. gous female U315-TGwere mated with male TCR-TG mice. In the opposite cross, mitted maternal IgG from the TCR-TG female does not contain the u 3 1 5 L, chai cell receptor; TG, transgenic; IgG, immunoglobulinG; L, light.

mice. In that case, 51-525 pg/ml serum JgC3h2315, and 180-270 pg/ml serum Ig62bh23'5 was required to obtain deletion. Deletion was observed within 24-48 hr after injection. Astonishingly, the thymus of such mice had already regained a normal appearance 7 days after injection, at which time the serum still contained 180 pg/ml of theX23'5Ig [ S ] . This surprising finding atteststo thehigh proliferative capacity of immature (progenitor) thymocytes, which enables them to repopulate thymic epithelial structures rapidly. In another experimental approach,we have injected the TC with such a high number of M ~ P C 3 1 5 p l a s m a c ~ o mcells a that their previously reported resistance to a MOPC315 plasmacytoma cell challenge 162,783 was overwhelmed. Such TCR-transgenic mice are continuo~sly exposed to increasingly high serum M315 concentrations as the tumor grows. A partial deletion of CD4'8' t h y m o c ~ e sis observed when serum M3 15 reaches 50 pg/ml, and the deletion is complete whenM315 reaches 200 pg/ml[79]. The deletion is not causedby metastasis to the thymus, because outgrowthof M ~ P C 3 1 5cells from thymus cultures has never been observed. Definitely excluding metastasis as an explanation, d obtained by i,p. injection of transgenic mice with purified myeloma prote but then a slightly higherM3 15 serum concentration(1 13- 176pg/ml) appears tobe required [79]. BALWc mice with large MOPC3 15 tumors contain dendritic cells in their thymuses which can present Id X231J.Thymic DC from MOPC315-bearing mice induces in vitro proliferation and IL-2 production by peripheral T cells derived from TCR-transgenic animals. Moreover, X2315-sensitizedthymic DC induce apoptosisof transgenic CD4'8' thymocytes during 39 hr of coculture.It is most likely that serumIg molecules enter thethymusandsensitizedendritic cells, whichpredominantly reside at the corticomedullary junction (Figure 5b); itis less likely that dendritic cells become X23'5-sensitizedin the periphery (e.g., within the tumor) and subseq~entlyrelocalize to the thymus. Regardlessof the mechanism, dendritic cells at the corticomedullary junction may sequentially delete the cortical CD4'8' thymocytes, starting with those nearest the corticomedullary junction. Taken together, double positive TCR-transgenic thymocytes are deleted in newborns as well as in adults by serum Ig of various isotypes, in a concentration range of 50-270 pg/ml. Thymic dendritic cells are probably being charged by circulatory Ig entering the thymus. The presented Id peptides then can induce clonal deletion. The high concentrationof Ig (50-270 pg/ml) needed for deletion is surprising, especially since the transgenic TCR is expressed in higher than normal amounts on double positive thymocytes which therefore should be more sensitive to deletion than normal thymocytes. Mostlikely, the high Concentration requirement couldbe due to resistance of complete Ig to processing by thymic APC, as previously observed in vitro for splenic APC [19,29,30]. Alternatively, those isotypes already investigated (IgC2b, IgG3, IgA) require a high concentration for deletion, while isotypes not yet tested (i.e., IgN, IgD, IgC1, IgC2a, IgE) may be moreefficient at inducing deletion. Finally, TCRs may differ in their concentration requirement for deletion: Perhaps the transgenic TCR used in the present experiments requires a particularly high IgU3I5 concentration fordeletion to occur. In another model, in which the transgenic TCR recognizes an IgC2ab allotype, <0.003 pg/ml failed to induce thymocyte deletion (higher serum concentrations were not tested 1801). IgC

may have a peculiar high concentration requirement for inducing deletion [51,80] because in a CS-specific TCR-transgenic model, 50 pg/ml of C5 sufficed for deletion of single positive(but not doublepositive) thymocytes(lower concentrations of C5 were not tested [Sl]). Taken collectively, we would like to suggest [51], thymocytes with TC cific for~ermline-encoded Idpept(present in serum Ig ataconcentrationof l00 pg/ml)are clonallydeleted. contrast,thymocytes with TCR specific for somatically mutated Id peptides or CDR3 Id peptides are not negatively selected because this category of Id peptides is present at too low concentration in serum [72] (Table 2).

ly X23’5-and TCR-transgenic offspring from a (TCR-TG9X h 2 3 1 5 T G cross ~) notbeen exposed to Ig in uterobecausethemotherdoesnotproduce Xz3l5. Such doubletransgenic neonates have undetectable U 3 ” IgM in serumbecause they have hardly started their own antibody production [51]. Even so, such offspring already have at birth a pronounced deletion of TCR-transgenic thymocytes [Table 21. Thymic XZ3l5 B cells in such neonates are few but may conceivably cause deletion. Another possibility is that plasma cells within the thymus may locally secreteenough Ig to sensitize thymicDCwith u 3 I 5 efficiently.Such amechanism may operate in in~uctionof tolerance to IgE in newborn mice [82]. Finally, the H chain enhancer used in the Xz3l5construct [75] possibly also causes transcription of the transgene in T cells [51]. If such messenger RNA is translated, ? d 3 1 5 secreted by thymocytes may locally sensitize DC. Alternatively, intrace~lular? d 3 1 5 may be released from double positive thymocytes that die by apoptosis. The latter possibility is especially interesting because it may represent a mechanism to obtain T cell tolerance to most intracellular housekeeping proteins shared by T cells and other cells of the body ( ifi

uring the lastfew years, it has become increasingly clear that continuous exposure to antigen can induceclonal deletion of peripheral T cells (reviewed in Ref. 74). To test whether peripheral Id-specific T cells could be deleted by continuous exposure Effects of Various Typesof Processed Id Peptides Id-dependent Positive Negative Negative selection,

Type Frequency of Id mus us ery Ig serum in peptidea Germline “mutated” CDR3

0.5-1 .O p M

Very rare Very rare

Yes Unlikely Unlikely

selection, Yes Unlikely Unlikely

selection,

Possibly Possibly Possibly

T-B cooperation Unlikely Likely Likely

aCermline Id peptides are encoded by V, or VL gene segments; CDR3 Id peptides span V-D-J or V-J junctions; “mutated” Id peptides depend on somatic mutations. Id, idiotypic; Ig, immunoglobulin; CD, cluster designation.

15, we have performed the following experiment: TC ctomized and injected with a sufficiently large numbe overcome their tumor resistance. Such mice started to delet cells when theserum 15 concentrationsrhed 50 pg/ml(tumor weight m 0.5 5 mm). The deletio rogressed with incr were enriched for expr on of endogenous TCR. The deletionwas found in lymph nodeof all localizai.e., they expressedaEPT tions, as well as in the spleen and in the blood. C omitant with the peripheral deletion, T cells became unresponsive to C in vitro. They were also y had retainedtheir expected of cells expressing aEPT e Id-responsive [til], are not de

+

1 have a reduced express recognize Id on class I1 (I-Ed)withouttheh most likely are too insensitive [61] to be del peculiarity of class IItion of conventional c terns [83]. The mechanism of deletion of the experimental setting used to de are only weakly sensitized with X be sufficient fora continous lowis possible that the peripheraldel differentiation9” or “clonal ex igen [83], virus [84] r 1, native myelin basic protein [87], all0 and various synthetic pep situ in lymphoid organs or travel to the gut[90] or the liver [91] is not yet clear. An alternateexplanationwouldbethat Id-specific T cells are lost fromperipheral lymphoid organs because they relocalize to the plasma CD4’ cells are present and activated in small tumors. larges, deletion of Id-specific CD4’ T cells is seen the [79]. Therefore, at least in later tumor stages, sequestr the tumor does not suffice to explain the peripheral deletion.

eletion of both double positive Id-specific thymocytes and peri heral 4’ cells caused by serum Ig is first observed when serum ut 50 pg/ml.Thisfindingdoesnot necessarily argueag thymocytes are intrinsically more sensitive to antigen thanperi because in our whole animal model, the local concentration of Ig in the thymus is not known. There are some differences between central and perip~eral deletion which S faster because it is compl ed out: (1) Central deletion proc 15 level reaches about 200 pg/ml. this tumor stage, some peri

era1 Id-specific C 4+ cells are still present. (2) Injection of urified Id subcutane24-48 hr.contrast,peripheral ously (s.c.) resultsinthymocytedeletionwithin deletion is observed after a longer interval (3-17 days) [?!l]. CD4'8' thymocyte CD4' and 8' cells (through deeletion results in deletionofbothperipheral creased output of bot types of cells fromthethymus).contrast,peripheral deletion only affects C ' 4 and not Id-specific CD8' cells. ' cells appearsto be Insummary,thereduction in peripheral Id-specific caused by both thymic and peripheral deletions. The deletions starta concentration and mayrepresent redundant mechanisms to obtain tolerance.

peptides may be presented on references). The processing viously outlined for Ig. The ell selection, asalready -derived peptides (T-cell Ids) taken upby thymic epithelial cells or dendritic cells from apoptotic thymocytes may conceivably contribute to both positive and negative selection in the thymus. Indeed, there are functional data which indicate that thymocytesspecific for germline TCR Id peptides are functionally silenced [95].

. As outlined, T cells may be rendered tolerant to germline-encoded Id but probably can respond to Id peptides covering the third hypervariable region and Id peptides generated as a result of somatic mutations (Table 2). What happens when such mature Id-specific T cells fortuitously encounter cells expressing the complementary,rare Idpeptide?Which ty of cell, if any, dies? Whichtypeof cell, if collaboration has been theoretically discussed eprocess of reconstituting mice with mature TG LN cells, hoping to answer these questions. atter would be to look for peptide binding motifs orrnal mice: Do they match(Le., they areselected for) or not match(i.e. they are counterselected) the peptide binding motifs of the I3 cell's class I1 and class I molecules?

The data obtaine by injection of Ig, or establishment of plasmacytomas, in Id-transgenic mice can probably be extrapolated to normal mice. The e~perimentalevidence suggests that Id peptides derived from Ig negatively select T cells by clonal deletion. Clonal deletion affects not only double positive (CD4'8') thymocytes, but also single CD4' cells in the periphery;in both instances, deletions require the same serum Ig concentration in order to occur ( 2 5 0 pg/ml). On the

52

basis of concentration considerations, only Id peptides encoded by germline V-gene segments may negatively select T cells. By contrast, the highly deversified GDR3 Id peptides (covering the third hypervariable region), as well as Id peptides expressing somatic mutations, may have concentrations too low to select T cells negatively. The effect of Id peptides on positive selection less is well studied, but on t~eoretical grounds it may be suggested that Id peptides may constitute a particularly rich source of diversified self-peptides,thus representing an important supplyof (partial) agonistic and antagonistic peptides involved in positive selection of thymocytes. The TCR-derived Id peptides may have similar effects on negative and positive selection, but this possibility hasbeen far less extensively studied.

We would like to acknowledge all those individuals who have contributed over many years to this long-term project. Anne Grete Olsen and Suzanne Garman-Vik helped in typing the manuscript. Ludvig A. Munthe expertly prepared someof the figures. This workis supported by the NorwegianResearch Council and the Norwegian Cancer Society.

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3 26. MC Eyerman, LJ Wysocki. Immunology 152:1569, 1996. V Teeuwsen, AJ 27. F UytdeHaag, I Claassen, H Bunschoten, H Loggen, T Ottenhoff, Osterhaus. Mol Cell Immunol3:145, 1987, 28. SJ Waters, CA Bona. Cell Immunol 11 1957, 1988. 29. Y Saeki, JJ Chen, L Shi, Y Okuda, HJ Kohler. Immunology 144:1625, 1990. 30. A Wilson, AJT George, CA King,FK Stevenson. J Immunol 145:3937, 1990. 31. AY Rudensky, SM Mazel, JM Blechman, VL Yurin. EurJ Immuno120:1691, 1990. 32. A Rademaekers, E Kolsch. Eur J Immunol25:623, 1995. 33 E Bikoff, BK Birshtein. J Immunol 137:28, 1986. 34. AY Rudensky, VL Yurin. Eur J Immunol 19:1677, 1989. 35. AY Rudensky, P Preston-Hubert, BK Al-Ramadi, J Rothbard, CA Janeway. Nature 359:429, 1992. 36. RM Cicz, RC Urban, JC Corga, AAV Dario, WS Lane, JL Strominger. J Exp Med 178:27, 1993. 37. JR Newcomb, PJ Cresswell. Immunology 150:499, 1993. 38. H Yamamoto, K Araki, S Bitoh, M Takata, S Takahashi, S. Fujimoto. Eur J Immunol 17:719, 1987. 39. D Chakrabarti, SK Ghosh. Cell Immunol 144:455, 1992. 40. W Cao, BA Myers-Powell, TJ Braciale. J Exp Med 179:195, 1994. 41. D Rueff-Juy, P Sanches, M Faure, AM Drapier, PA Cazenave. Eur J Immunol 25: 2752, 1995. 42. S Weiss, B Bogen. Cell 64:767, 1991. 43 MJ Lewis, HRB Pelham. Cell 68:353, 1992. 44. A Brooks, S Hartley, L Kjer-Nielsen, J Perera, CC Goodnow, A Basten, J McCluskey. Proc Natl Acad Sci USA 88:3290, 1991. 45. DJ Kittlesen, LR Brown, VL Braciale, JB Sambrook, MJ Gething, TJ Braciale. J Exp Med 177:1021, 1993. 46. RD Klausner, R Sitia. Cell 62:611, 1990. 47. MR Knittler, S Dirks, IG Haas.Proc Natl Acad Sci USA 92:1764, 1995. 48. MJJE Bijlmakers, P Benaroch,HL Ploegh. EMBO J 13:2699, 1994. 49. ML Hedley, RG Urban, JL Strominger. Proc Natl Acad Sci USA 91:10479, 1994. 50. R Germain. Immunol Rev 1515, 1996. 51. B Bogen, Z Dembic,S Weiss. EMBO J 12:357, 1993. 52. B Bogen,S. Weiss. Intern Rev Immunol 10:337, 1993. 53. B Bogen, L Gleditsch,S Weiss, Z Dembic. Eur J Immunol22:703, 1992. 54. B Bogen,GF Lauritzen, S Weiss. Eur J Immunol20:2359, 1990. 55. MJ Bevan. Nature 269:417, 1977. 56. RM Zinkernagel, GN Calahan, A Althage, S Cooper, PA Klein, JJ Klein.ExpMed 147:882, 1978. 57* PG Ashton-Richardt, A Bandeira, JR Delaney,LV Kaer, HP Pircher, RM Zinkernagel, S Tonegawa. Cell 76:651, 1994. 58. KA Hogquist, SC Jameson, WR Heath, JL Howard, MJ Bevan, FR Carbone. Cell76: 17,1994. 59. E Sebzda,VA Wallace, J Mayer, RSM Yeung, TW Mak, PS Ohashi. Science 263: 1615,1994. 60. H von Boehmer. Annu Rev Immunol8:531, 1990. 61. L Munthe, A Sollien, Z Dembic, B Bogen. Scand J Immunol42:651, 1995. 62. B Bogen, L Munthe, A Sollien, P Hofgaard, H Omholt, F Dagnaes, Z Dembic, GF Lauritzsen. Eur J Immunol25:3079, 1995. 63. L Munthe, E Blichfeldt, A Sollien, Z Dembic, B Bogen. Cell Immunol 170:283, 1996. 64. P Borgulya, H .Kishi,Y Uematsu, H VonBoehmer. Cell69529, 1992. 65. BD Evavold, J Sloan-Lancaster, PM Allen. Immunol Today 14:602, 1993. 66. A Franco, GY Ishioka, L Adorini, J Alexander, J Ruppert, K Snoke, HM Grey, A Sette. Immunologist 2:97, 1994. *

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67. C Benoist, D Mathis. Cell 58: 1027, 1989. 68. J Bill, E Palmer. Nature 341:649, 1989. 69. P Nieuwenhuis, RJM Stet, JPA Wagenaar, AS Wubbena, J Kampinga, A Immunol Today 9:372, 1988. 70. D Kitamura, J Roes, R Kuhn,K Rajewsky. Nature 350:423, 1991. 71. JJ Chen, SV Kaveri, H Kohler. EurJ Immunol22:3077, 1992. 72, IJ Sanz. Immunology 147:1720, 1991. 73* JW Kappler, N Roehm, P Marrack. Cell 49:273, 1987. 74. J Sprent, S Webb. Curr Opin Immunol7:196, 1995. 75. B Bogen,S Weiss. Eur J Immunol21:2391, 1991. 335: 174, 1988. 76. HR MacDonald, H Hengartner, T Pedrazzini. Nature 77.

78. 79. B Bogen. Eur J Immunol26:267 1, 1996. 80. 203, 1996. 81. 82. 83. B Rocha, H VonBoehmer. Science25 1: 1225 1991. 84. D Moskophidis, F Lechner,H Pircher, R 85. H Wang, RF Gill, D Lichlyter, A Iglesias, 2950, 1994. 86. L Forster,RHirose,JMArbeit, BE Clausen,anahan.Immunity Critchfield, MK Racke, JK ~uniga-P~ucker, B Cannella, CS 87. 88. 89. 90. 91. 92. 93* 94. 95.

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~ n i v e r ~ iof t y~ e n t u c k y

~ e Center, d i c a ~~ e x i n ~ t o n * ~ e n t u c k y

The varioustypes of white blood cells that regulate an arrayof host defense mechanisms are all derived from a very small population of primitive lymphohemopoietic n of mature cells from these primitive ancestors is strictly the i n ~ u e n c eof en~ogenouscytokines. There are numerwhich interference with immune cell production may be attractive. The manipulation and modulation of the production of committed mactor cells are, therefore, extensively discussed in various chapters this chapter, an overview of our current understanding of the structure of the primitive hopoietic cell compartment is presented.Datathat focus on howexogenously inistered growthfactorsmayperturbthisearly cell compartment and es such a modulation may have in clinical situations will be discusse eason,onlydataand perspectives onin vivo use of growth factors will ecause b of the cumbersome methods of measuring primitive stem cell cycling activity, manipulation of these cell compartments in vivo has until now received little attention in the literature. t perturbation of this critical cell population may have p r o f o u n ~ neral immune status and thus may be a factor in the process of aging. It can be expected, given the current availability and continuing discoveryof new growth factors, that in the near future assessment and subse~uentmodulation of the fre~uencyand behavior of primitive stem cells will prove to be significant areas of research.

. efore an overview can be given on how., in vivo, various growth factors affect hemopoietic stemcell kinetics, it is important torealize that ourbasic understanding of this population of cells is limited. This lack of knowledge is predominantly due

to the extremely low frequency of these cells (the frequency of true long-term repopulating stem cells is estimated to be about 1 per lo5bone marrow cells) as well as the distinctively poor morphological and antigenic characteristics of stem cells [l]. From a cell biological point of view, the most apparent feature of the stem cell population that has emerged during the last decade is that these cells are not a homogenous pool. Stem cell populations consist of various distinct subcompartments [2,3]. These subcompartments are hierarchically linked. As such, there is a continuous differentiation from a small pool of totipotent lym~hohemopoietic stem cells to successively larger pools of more lineage-restricted pluripotent progenitor cells. During this differentiation program, cells fromconsecutivecompartments gradually increase their rateof proliferation fromvirtual quiescenceto a highly and continuously cycling state [4-71. In contrast, little progress has been made with respect to how the highly organized, rapidly proliferatingh~mopoieticcell compartments maintain themselves in vivo. Ironically, it is exactly in this respect that hemopoietic growth factors may 'play prominent roles. Two classes of questions in this context in particular stand out: 1.

How does the hemopoieticcell system control the frequencyof component cells? What mechanisms regulate the pool size of the various stem cell subsets? ow does the hemopoietic system regulate the various rates at which its cellular ~omponentsproliferate? hat mechanisms control the characteristic cell cycling activity during steady-state hemopoiesis of primitive cell subsets?

The ability to manipulate in vivo both the poolsize and the cycling activity of stem cells and their subsets is of significant importance. For example, theability to increase stem cell numbers would have a clear benefitin bone marrow transplantation in the context of increasing the yield of harvested stem cells. In this setting, patients who are scheduled to undergo autologous transplantation could be pretreated with myeloablative agents. Second, increasing the stem cell pool prior to chemotherapy may be accompanied by a reduced cycling status of primitive cells [7,8], This relative quiescence makes them less sensitive to S-phase-specific cytotoxic drugs, and thus a prophylacticin vivo stem cell expansion offers a potential method to reduce hemotoxicity. The ability to modulate the cycling of primitive cells would also beof considerable interest. Decreasing the cycling activity by administration of inhibitory cytokines has been shown to result in resistance against cytotoxic agents [g]. Increasing the cycling activity in vivo also has clear applications. Obviously it may enhance recovery of peripheral blood cell numbers in classic chemotherapy-growth factor protocols. Cycling of primitive cells is also essential for many gene therapy protocols to be effective [lo]. In addition, if it were possible to increase the cycling activity of primitive cells selectively, this population would become sensitized to relatively mild cytotoxic drugs like ~ - ~ u o r o u r a c iCombination l. therapy of cytokines and cytotoxic drugs may be used for moreeffective eradication of endogenous stern cells in transplantations [1 1- 1 31. Cytokines are obviously attractive candidates be to used in all of these settings. Several growth factors with assumed lineage-restricted potential are at present administered to patients, mainly to increase the recovery rate of peripheral blood

cells. Several novel cytokines appear to have the potential to affect more primitive, undifferentiatedhemopoietic cells. In this chapter data on the effects of these growth factors on the primitive cell compartments will be discussed. It is likely that the manipulation of pluripotent stem cells in vivo will be more complicated than that of mature blood cells. efore further substantial progress in this field can be realized, additional informationis needed on thebiological characteristicsof several fundamental features of the stem cell compartment; discussion of some of these aspects follows.

It is likely that the size of each of the primitive cell compartments during steadystate hemopoiesis is carefully regulated, in much the sameway that mature, peripheral blood cell counts are controlled. The frequency of peripheral blood cell numbers is determined by “demand,” an by regulatory loops involving cytokines. Asan example, the erythrocyte poolhas to be adequate to supplyall tissues with oxygen, and leukocytes have to be sufficiently numerous to prevent widespread invasion of microorganisms. Thus, by analogy, the stem cell pool has to be large enough to the number of mature blood cells required to replenish these circulating e, it would seem likely that stem cell equency is indirectly regulatedby ofmature cells inthecirculation.owever,variousconditionshave been reported in which the number of stem cells is either substantially lower or higher than normal, and yet peripheral blood cell numbers are normal. ~ o n g - t e r m and irreversible deficits of stemcells occur in miceas a result of exposure to certain treatments9suchasbusulfan, or total bodyirradiation [14-1?1. been treated with these agents have permanent stemc peripheral blood cell numbers are often normal [17]. cell-philia,” has been observed by us recently. When mice age from 6 weeks to 1 year, approximately f their normal lifespan, their peripheral blood counts do not changesignificantly. wever, duringthesameperiodthefrequency of the most primitive stem cells increases about fourfold [18]! It is interesting to combine these data in aged mice with data recently presented on stem cell frequencies in fetal liver, an early site of hemopoiesis. It was demonstrated that the absolute number of stem cells in young adult mice was approximately 13-fold higher than in the fetal liver [ 191. Thus, whereas one may expect that during life the number of stem cells may remain constant or decrease gradually(i.e., the self-renewal probability of the stem cell pool would be 0.5 or slightly less), surprisingly, the opposite appears to occur. Figure 1 shows that there is a constant increase of the number of stem cells during development (i.e., the self-renewal probability is slightly higherthan 0.5). Nevertheless, perip~eralblood cell numbers remain unchanged during this period, indicating that the number of mature cells produced per stem cell gradually declines during aging. An additional interesting observation is that the size of the stem cell pool varies from mouse strain to mouse strain, whereas, again, peripheral blood cell countsinthesestrains do not differ appreciably[18,20,21].Thus,someinbred strains manage to maintain normal blood cell counts with only a fraction of the stem cells that other strainsseem to use, suggesting that undiscovered genetic deter-

60 50 40

30

20 10

liver

fetal

young marrow

old marrow

~ e v e / o ~ ~ stage e~ta/

The pool of primitive, long-term repopulating stem cells increases during aging. Shown are the number of stem cells, normalized for fetal liver values. The total stem cell pool present in fetal liver increases approximately 13-fold compared to that in marrow of 6 week old mice. (Data from Ref.19. In addition, the total stem cell pool increases on average 4-fold from 6-week-old mice to1-year-old mice. (Data from Ref,18.) minants may be im~ortant.Taken together, these ata argue against a tionshipbetweenthenumber o heral blood cells andt tive stem cells during steady-sta opoiesis. If stem cell frequency is not regulated by fee back loops involving the sizes of mature peri~heralbloo cell com~artments,what then is it t of this pool ofcrucial c Is? At present, any answer can only alternative options may be considered. First, the stem cell its own size by some intrinsic, progr regulation could conceivably be dete tion factors, or other regulatory elements. overexpression of

mal blood cell counts, but primitive cell compartments were [22]. This is the first studyto our knowledge that has demon permanent stem cell ex~ansion,without n ously, if itturnsoutthat stem cell fre~uencyis determi~edby intracellular controlmechanisms,it will not beveryeasy to late these compartments in vivo, and the role forexogenously applied

stem cell pool is and also determines how large it may grow. Such a sensor may be

stem cell-specific growth factor concentrations.If growth factor levels are high, the stem cell frequency increases; if they are low the stem cell pool shrinks. Interestingly, ithas recently eendemonstratedthatserumconcentrations of Flk2-Flt3 ligand (FL), a growth factor considered to act primarily on primitive cells 124,251, are high in many patients with Fanconi anemia and acquired aplastic anemia. These are clinical conditions in which stemcell numbers arereduced [26]. Anotheroptionfor externl size control wouldbe thatthefrequency of stem cells is determinedbythen of availablemicroenvironmental “niches.” A niche could be consideredas an anatomical site necessary for stem cell viability and 1. The existence of niches has never been unambiguouslydemonstrated9 evidence that strQma1cell elements, at least in vitro, are necessary for long-term survival of stem cells and hemo~oiesis.Similarly, in vivo stem cell infusion in nonmyeloablhostsdoesnot result in rapidengraftment, presumably because of a lack of hes. Only when massive numbers cells of are infused is engraftment o this does not result fromanincrement of the cells expel host cells and the total number of mechanism suggests that a maximum size of the stem cell compartment may exist, determined by the numberof available niches. It is of great importance toassess which of the hypotheses mentioned applies. If the size of the stem cell pool is determined directly by growth factor concentrations, then it is likely that altering those concentrations by exogenous administration of growth factors will have an impact on stem cell frequency. However, if it is the number of niches that determines stem cell frequency, then our efforts should be directed at modulating the numberof niches. Niche modulation could be performed by manipulations which in theory arepossible by exogenous growth factor administration. If, in contrast, stem cell pool size is determined by intracellular regulatory factors, it is unlikely that exogenously administered growth factors will havea sustained effect on stem cell frequency. In the following section a brief overview will be given by published data indicating that the administration of growth factors can atleast temporarily affect stemcell frequency in vivo.

ost growth factors that are currently available were identified by their ability to support in vitro proliferation of relatively committed hemopoietic cell stages in clonogenic assays. Therefore, the effect of these proteins on more primitive cell compartments has, until now, not received much attention. In addition, it is likely that this experimental approach has resulted in a bias to select for relatively lineagespecific factors, leaving growth factors that act preferentially primitive on stem cells as yet un~iscovered. Theprevailing concept that stem cells were “null” for specific surface markers and cytokine receptors discouraged such searches until relatively recently. Nevertheless, growth factors that act predominantly on lineage-restricted cells may also have an indirect, but substantial, effect on stem cells. It can easily be envisioned that irect stimulationof ~ a t u r eblood cell production by lineagerestricted, late-acting growth factors may indirectly perturb the stem cell compartment that replenishes the committed compartments. It may therefore be appropriate

nt

to distinguish here between such direct stem cells.

and indirect effects of growth factors on

Few growth factorshave been discoveredthat appear to have ( s ~ m e selective ) sensitivity for primitive cells. Of these, stem cell factor (SCF, alsocalled mast cell growth factor, steel factor, c-kit ligand) was the first tobe identified and is by far the best characterized. Primitive cells have receptors for SCF (c-kit) and have been shownin numerous studies to respond in vitro to SCF stimulation [29,30]. More convincingly, naturally occurring SCF knock-outmice (SISld mice) show profound hematological abnormalities, in particular a deficiency in pluri~otent and erythroid cell lineages, which are reversed on administration of SCF [3l]. When SCF was administered to normal mice for 7 days, the numberof pluripotent hemopoietic stem cells decreased significantly in marrow. Concomitantly, however, the number of these cells in the peripheral blood and the spleen rose strongly [32,33]. Bodine et al. reported that this resulted in a net expansion of the stem cell pool (about threefold)1321, whereas Fleming et al., evaluating Sca-l' cells (an antigen thatis presumably exclusively expressed on primitive hemopoietic cells), reported a mere relocalization of the stem cell pool from the marrow to thespleen [33]. In a morerecent study, we have also evaluated the effect of SCF,in combination with interleu~in-ll (IL-l l , o nin vivo stem cell expansion [7]. A powerful assay to quantify stem cell frequency that we used in this experiment is the so-called cobblestone area formingcell (CAFC) assay. This assay, developed and validated in an array of different experimental protocols by Ploemacher et al., is a limiting dilution type long-term bone marrow culture 1341. The presence of myeloidcolonies growing underneath a stromal cell layer is evaluated at various time points subsequent to theinitiation of thecell culture. The number of colonies appearing early in culture (7 t o l 4 days,formurine cells) is a reflection of the size of relatively committed cell compartments(colony-formingunit-granuloid,erythroid,monocyte, m e g a ~ a r y o c ~ e [ C F U - ~ ~ M colony-forming M], unit spleen [CFU-S]) present in the originalcell suspension. In contrast, murinecolonies appearing only after28 or 35 daysare derived from primitivestem cells (long-termrepopulating cells, competitive repopulating units) [35]. When mice were treated for 7 days with high doses of SCF and IL-l l, a fivefold expansion of the number of CAFC day 7 was observed in marrow. More primitive cell stages that have been reported to be decreased by using SCF alone [32,33] were not affected in the femur by adding IL-1 l to SCF[7]. In addition, where SCF aloneincreased primitive stemcell subsets in the spleen 4- to 15-fold, SCF IL-11 increased splenic values of all stern cell subsets 100-fold [7]. When the total number of stem cells per animal was calculated in our study, significant increases of all primitive cell compartments were observed. owever, the expansion of the CAFC day 7 poolwas more pronounced than that of more primitive stages. Thus SCF in combination with IL-l1 appears to be one of the most potent growth factor combinationsto induce in vivo stem cell expansion. This in vivo effect has been confirmedin several in vitro studies [30,36] and apparently is due to unique synergistic intracellular signaling through c-kit (the SCF receptor) and gp130 (part of the IL-l1 receptor)[37]. Another cytokine thatmay havethe capabilityto expand stem cell numbers is the more recently discovered Flk-2/Flt-3 ligand (FL). Surpris-

+

ell

ingly, at present, detailed reports on the effects of in vivo administration of this cytokine on stem cells are lacking. It has been demonstratedin vitro, however, that FL preferentially stimulates primitive cells. Thus, it may be speculated that FL may be at least as potent as SCF [24,38]. Interestingly, patients with aplastic anemia, characterized by a serious deficit in the numberof stem cells in their bone marrow, show increased FL serum levels, whereas their SCF concentration is normal [26]. This finding obviously supports the hypothesis thatFL is a physiological regulator of stem cell frequency. ~e have recently begun studies on the effects of FL on the frequency andcycling activity of stem cell populations in the mouse. But becauseof the time scale that we chose (effects within 24 hr) no changes of cell frequencies could be expected, nor were they observed. owever, striking effects oncell cycling (discussed later) were seen.

A prolonged stimulation of mature cells is likely to affect the kinetics of stem cells indirectly, since it can be anticipated that feedback loops which regulate cell production operate within and between the various primitive cell compartments. Surprisingly, although growth factors that stimulate the production of mature peripheralblood cells are widely used in the clinic ry little is knownof indirect effectsof these cytokines on stem cell kinetics. haveassessed theeffects of long-term (3 weeks)granulocytecolony-stimulatictor(G-CSF)administration to normal mice on the behavior of the various stem cell subsets [39]. In marrow, where normally 99% of the total number of stem cells per mouse can be found, G-CSF induced a rapid and severe depletion of the most primitive stemcell subsets to 5% to loolo of normal values. Simultaneously, the number of these cells in the spleen rose, an effect (extramedullary hemopoiesis) that can be observed mice in on administration of almost any cytokine. This increase of cell numbers in thespleen is very likely due to a migration of stem cells via peripheral blood. This possibility is substantiated by theobservationthat splenectoedmicehavehigherperipheral ntotal stem cell numbersper bloodstem cell levels thannormal mice [40], mouse were calculated after 3 weeks of G-CSF administration, committedprogenitors were increased threefold, intermediate primitivecell stages were increased twofold, but the number of the most primitive cell subset evaluated was not different er, 9 5 ~ of 0 the total cell pool was located in the spleen. Four reatment, this situation was reversed and normalized again. In em cells found in bone marrow atthis time was twofold higher than in the marrowof untreated control mice. This observation indicateda rebound effect common in cell po~ulationstightly regulated by complex feedback loops. Thus, even though G-CSF is generally regarded as a lineage-specific, late-acti~g growth factor, these data de~onstrate that the entire stem cell compartment is seriously perturbed during, and after, its administration. This perturbationis most likely due toindirect regulatory loops between distinctcell populations that control in vivo blood cell production. irect stimulation of relatively committed progenitors has indirect effects on more primitive cell stages that are required to supply the increased mature bloodcell production. n even more strikin demonstration of the existence of these indirect loops was recently reportedbydineetal. [41]. In thisstudyG-CSF, in combination

with SCF, was administered for 5 days to splenectomized mice. growth factor treatment thecompetitive repopulating ability (aCO ment of the number and quality of the stem cells present) of bone marrow cells harvested from treatedmice was low comparedto thatof untreated controls. Simultaneously, however, the repopulatingability was high in blood from growth factortreated mice. These results are in full agreement with the data that we obtained previously (discussed earlier),althourstudy G-CSF as single cytokine was administered.More interestingly, et al.observed that1 days after cessation of this 5 day G-CSF SCF treatment, a converse effect occurred: the repopulating ability of bone marrow from growth factortreated mice was estimatedto be 10-fold higher than that of normal bone marrow. This finding indicates that the number of stem cells in the marrow, and therefore in the total mouse,was expanded considerably afterthisgrowthfactor regimen.This effect was not o period of growth factor administration, rather, it was present after treatment. Thus, itseems to be an indirecteffect of the growth factor stimulation. The stem cell population of the treated mice was never depleted (a condition that would result fromtreatmentwithcytotoxic drugs). Themarrow depletion caused by growth factor treatment apparentlyresults in a strong amplification of stem cells when the treatment is stopped. This amplification not only restores marrow stem cell populations to their normal pool size but also results in a significant overshoot (a 10-fold expansion of primitive stem cell numbers). These findings reveal fundamental regulatory loops in various clinical protocols. donors may be pretreated with growth factors, and marrow cells ca vested at the point of greatest rebound, thus maximizing stem cell yields. In addition, it is likely that this in vivo amplification is the result of transiently increased cellcycling of these primitive cell populations.This is an are interest since the efficiency of many gene transfection protocols cycling status of the cells to be transfected [IO]. ~ o r m a l l ythese very primitive lymphohemopoietic cells are deeply quiescent(see later discussion). Taken together, the available data on the effects of growth factors on stem cells in vivohaveshown that these primitive cell compcananddo react in response toat least somegrowthfactors.Theprimitivepartmentsappear to be very flexible yet resilient in nature, quickly reestablishing their normally quiescent behavior. At present, it appears to be difficult stem cell numbers permanently. This inability may priate growth factors. ~ d d i t i o n a lstem cell-specifi FL is capabl remain as yet undiscovered. hether in vivo over the short and/ indirect stimulation of stem cells has demons quency can be obtained by depleting the marrow pool size bymobilizanwith growth factors. The exact mechanism of this e earlier, it is not unlikely that thestem cell population size during steady-state hemopoiesis is at least partly determined by the number of available microenvironmental niches. Growth factorshave proved to be very efficient stemcell mobilizers, and since mobilization occurs at the expense of marrow cell frequencies, it is possible that growth factors empty these hypothetical niches. Some time after the mobilizing regimen is stopped, the number of stem cells in marrow exceeds by far the number

+

of cells present normally l]. This line of reasoning then leads to the conclusion that the number of availa~leniches can be increased above normal values. If this proposition is correct, the implication would bethat a direct stimulation of stem cell e achieved by growth factors that increase the numberof niches. Since the exact nature of the stem cell niche remains elusive, identifying growth factors that increase the number of niches will prove to be difficult, perhaps more difficult than identifying stem cell-specific growth factors. derstanding of how stem cell pool size is regulated that has ajor manipulations of stem cell frequency with cytokines. made in the manipulation withcytokinesof the other acteristic that we have described earlier: stem cell cycling. f o l ~ o w i nsection. ~

There are multiple mechanisms by which growth factors can affect proliferationof stem cells, depending on the physiological status of these cells. Stem cells can be either quiescent, in active cell cycle, or dying an apoptotic death (Figure2). Growth factors can haveeffects on all these three processes.

It is well k n o ~ nthat during steady-state hemopoiesis most stem cells having longterm repopulating ability are deeply quiescent, i.e., are in the Go phase of the cell igure 2). This is revealed by their resistance to cell cycle-specific cytotoxic

Lymphohe~opoieticstem cellsare of three types: a, quiescent and thus maintaining the population size, also referred to as being in the Go phase of the cell cycle; b, cycling and expanding the population size, positioned in either the G,, S , or G2M phase of the cell cycle; c, dying bythe process of apoptosis, thus leadingto a decreaseof population size.

drugs,likehydroxyurea ( ) or5-~uorouracil (5-FU) [4,’5,2~,4~].Inaddition, from a clinical perspective, se cells are very difficult to transduce retrovirally with foreign genetic material.Th6 manygene therapy efforts demonstrate the absence of deoxyribonucleic acid (DNA) replication [lo].It is unknown ow long an individual stem cell is able to remain in this ~uiescentstate. Nor has it been assessed whether during this apparently inert phase qualitative changes of the proliferative capacity of stern cells take place.

Although the majority of primitive stem cells are ~uiescent,it has been well established that these very cells canrapidlyenter the cellcycle. ample, if the cell cycle-specific c ~ o t o x i cdrug 5-FU is administered to nor surviving long-term repopulating cells is close to 100% second dose of 5-FU is administered to these same animals 3 days after a first injection, almost all stem cells are killed, ing the high cycling status of the cells at that time [43]. It has independently be ermined that the fraction of primitive stem cells in the S phase of the cell cy reases from 0% to 3 period of 3 days after5-FU administrat

Although apoptosisis normally associated with more lineage-restricted hemopoietic cells (e.g., erythroid precursors, lymphocytes [45- 81) there is no reason to believe that this process would be reserved exclusively to regulate pool size of committed cells. It is probable that the low frequency of stem cells, in conjunction with the rapid disappearance of apoptotic cells in vivo, has precluded the demonstration of apoptotic hemopoietic stem cells during hemopoiesis, either steady-state or perturbed. An apoptotic stemcell would irreversibly have left the cell cycle (Figure 2), but the prevention of apoptosis would simultaneously increase the numberof cells in the cell cycle. Thus, apoptosis andcell cycling are tightly linked. It hasbeen well documented in vitro thatcells with characteristics of stem cells do indeed undergo apoptosis [49,50]. Employing a mathematical approach, Necas etal. recently suggested that it is very likely that apoptosis occurs in primitive cell compartments [Sl]. They compared the maximal proliferative capacity of an individual mouse stem cell (CFU-S), the total number of ~ F U present, - ~ and the steady-state production of blood cells in the mouse. Theyconclud~d that the proliferative potential of CFU-S is several-thousand-fold higher than actually observed, and thus needed to maintain homeostasis. This quantification of extremely inefficient hemopoietic cell production may provide an estimate of the extent to which apoptosis regulates primitive stem cell pool size. It underscores the possibility that in vivo substantial, possibly massive, apoptosis normally takes place in the primitive oiesis eachdistinctstem cell compartments [S l].Duringnormal steady-stateh cell subset has a characteristic proliferation rate. ng differentiation cells “travel” through the successive compartments in which the fraction of cells in cycle gra~uallyincreases (Figure 3). It is interesting to note that in mice the level of cycling for a specific stem cell subset varies widely from mouse strain to mouse

C3H/He mice (6 weeks oh

0 14

21 35

28

7

Stern cell subset (CAFC day type)

The various distinctive stem cell compartments have characteristic cycling activity during steady-state hemopoiesis. Bone marrow cells obtained from young C3H/He mice were incubated for 1 hr in the presence of hydroxyurea, after which a regular cobblestone area forming cell (CAFC) stem cell assay was performed. CAFC scored after 35 days in culture, which contained cells with long-term repopulating ability [35], were completely resistant to hydroxyurea, indicating the absence of cells inS phase. In contrast, CAFC-day7, containing 18.) clonogenic progenitors, were highly cycling. (Data from Ref.

strain. In addition, thelevel of cycling is highly influenced by the age of the animal. We have recently quantified the cycling of stem cells in young and old mice of five different inbred strains. §tem cells in short-lived mouse strains, like C3 A/J, had far higher fractions of §-phase cells than their counterparts in longlived strains like BALB/c and C57BL/6 [18,20]. In addition, stem cells from shortlived mice showed a pronounced decrease in cycling activity during aging, whereas this decrease was less obvious or not detectable in long-lived strains [ 181. These observations imply that stem cell kinetics, at least during steady-state hemopoiesis, is under strict genetic control. In these strains of mice, hemopoiesisis not dependent on peripheral blood cell counts, which are virtually the same among the strains. Lord et al. have suggested that the intramedullary “concentration” of progenitor cells (i.e., the number of stem cells per lo5bone marrow cells, and not the absolute pool size per femur) determines the rate of stem cell cycling [ 6 ] . Thus, stem cells would have the abilityto “sense” the frequency of the progenitors. Our datasuggest that such steady-statecell cycling isgenetically predetermined. Overall, itis likely that the gradualincrease in cycling activity from aquiescent long-term repopulating cell population to a highly cycling progenitor pool is caused by the expression of an array of cytokine receptors on the stem cell surface and the availability of theirligands. The rate andextent to which this occurs would be under genetic control. Later, in the discussion of the general impact of growth factors on stem cell cycling, an experimentalexample will be given of the importanceof genetic background and responsivenessto cytokines.

ate a ~ the othree ~ ~ n. ~trictlys ~ e a ~ i n ~ ast, is defined by an irreversi~le ce p r e v e ~ t i oof~ apo~tosispermits of the cell cycle, these processes

l

C57BU6 mice (12 weeks ok

U5-FU SCF+5-F-U

7

14

21

28

35

Stem cell subset (CAFC day t y ~ e ) SCE; increasesthecycliivity of committedstemcells,buthas no effect on L/6 miceweretreatedwith SCF, threeinjections of itivecellsubsets.Young 2.0 pg within 24 hr. Two hours after last SCE; injection, 5-FIJ (150 mg/kg) was administered, and again24 hr later marrow cells were harvested to assess the surviving fraction of the various stem cell subsets.5-FU, 5-~uorouracil.(Data from

eclines until C57

cytokine a ~ ~ i n i s t r a

with ~ r o w t hfactors.

-l

7

14

21

35

28

Stem ce/lsubset (GAFG day type)

~

Flt-3 ligand (FL) increases the cycling activity of all hemopoietic stem cell subsets, but only in DBA/2 mice. Young DBA/2 and C57BL/6 mice were injected with FL, three injectionsof 2.0 kgwithin24 hr.Twohours after thelastinjectionmarrowcellswere harvested and incubatedfor 1 hrin the presenceofhydroxyurea,afterwhicharegular FL increasedthe cobblestone area formingcell(CAFC)stemcellassaywasperformed. percentage of CAFC day 7 Sinphase of both strains butwas only effectiveon more primitive cells in DBA12 mice. (Data from Ref. 57.)

IlS

As opposed to long-term repopulating stemcells, committed pluripotential progeninot have theability to remain in a quiescent Gophase for prolonged periods. , changes in cell cycle activities observed within these cell compartments are more likely to be caused by a lenghtening or shorteningof the total cell cycle time; i.e., a shortened GI phase (see Figure 2). Many in vivo effects of cytokines on progenitor cell cycling have been reported. But, basically, cytokines acting on the proliferative activity of cycling stem cells can be separated in those having inhibitory, as opposed to those having stimulatory, activity. In vivo studies have clearly demonstrated the potency of various inhibitory cytokines that reduce the percentage of cells in S phase. Treatment of mice with macrophage infla~matory protein-la( M ~ ~ -orl transforming ~ ) growth factor+ (TGF-P) before the injection of a cytotoxic drug has resulted in improved hematological recovery and survival [60,61]. Obviously, these inhibitors may provide a potential prophylactic treatment to prepare patients receiving chemotherapy, but, at present, the impact of these inhibitors in such clinical conditions has yet to be established. The effectiveness of inhibitory cytokines is dependent on the cycling activity of stem cells present in cancer patients. It is not unlikely that the proliferation of stem cells in these patients, particularlyin those with malignant bonem a r r o ~ involvement, is substantially different from that in normal, hea~thyindividuals,

Limited information on this subjectis available. However, data of Broxmeyer et al. show that the cycling activity of various hemopoietic progenitors in patients with sarcoma is significantly reduced as compared to that of normal bone marrow [62]. If this situationproves to be a general finding, then pretreatmentof cancer patients with inhibitory cytokines may not be very effective, since their stem cells are slowly cycling. The list of cytokines with stimulatoryactivity is much longer than the one for inhibitors. To what extent thisis due to a biased search for, and resulting discovery of, these stimulatory cytokines, comparedto a relative neglect of inhibitors, remains to be seen. Many of the stimulatory cytokines have remarkably quickly found their way into clinical trials of all sorts. These trials, in general, assess the ability of hematopoietic growth factorsto accelerate endogenous recovery of peripheral blood cell values after cytotoxic therapies of cancer patients. Although only few clinical studies have addressed the issue of how increased blood cell production is actually achieved after administration of cytokines, it is likely that most effects can be attributed to the ability of cytokines to reduce the cellcycle time of stem and progenitor cells. For example, administration of GM-CSF or G-CSF to patients with various malignancies increased the percentage of progenitor cells in S phase from 10% to 50% [62,63]. Also, IL-11 administration to mice induced a marked increase in progenitor cell cycling within 3 hr, suggesting a direct effect of IL-11 on these cells [64]. As reported previously, SCF and FL administrationincreases the in vivo 5-FU sensitivity of progenitor cells within 24 hr, suggesting a rapid shortening of the total cellcycle time by these cytokines [12,57]. Although no systematic attempt has been made to evaluate the effects of all known cytokines on progenitor cell cycling, the picture emerges, particularly from data from in vitro studies, that the ability to reduce the GIphase of the cell cycle is a characteristic of many,if not all, stimulatory cytokines. Taken together witha massive amount of data obtained from in vitro studies, which show synergistic effects of almost all cytokines on colony growth and size, it appears as if relatively committed progenitors are sensitive to many cytokines. Thus, they have acquired the respective receptors for those cytokines during their differentiation from the quiescent totipotent compartments to the cycling pluripotent compartments. At present, it is not understood how this seemingly unspecific process results in very specific and coordinated blood cell production. Although progenitor cells are responsive to many administered cytokines in vivo, it is unclear to what extent this can be exploited optimally in various clinical settings. One can only speculate about whether the administrationof exogenous recombinant cytokines that is currently employed has reached its maximal utility.

~Q~tQs wit i shi^ the Stern The concept of apoptosis in regulating pool size of lineage committed and mature blood cells is substantiated by experimental data [46]. In stark contrast, it is unknown to what extent this process may alsoaffect primitive stem cells in vivo. Since an apoptotic-prone stem cell population would be actively and irreversibly leaving the cell cycle (Figure 2), the prevention of apoptosis would consequently have an impact on the cycling activity of the stem cell compartment as a whole. As stated

namic nature of the process of apoptosis, in conjunction with the fre~uencyof hemopoietic stem cells in the bone It to detect, let alone ~uantify,apoptotic stem cells in the a with isolated candidate stemcells have shown that U factors were with rawnfromtheculture,these cells doundergo his processcanbeented,orat leastdelayed, by various andSCF [50,65~. ever, as always,itisunclearwhether echanisms actually also apply to invivo situations. e possible existence of a~optosiswithin the primitivecell compartan a c a d e ~ i issue, c it is of high clinical relevance. It would necessarany stem cells are lost during normal hemopoiesis. These could inistration of cytokines that then act as the appropriate survival factors.

ur current understandingof the invivo regulation of primitive lympho~emo~oietic l compartments is limited. ccordingly, at present it is difficult to predict ication of cytokines in future clinical therapies aimed at modulating stem the one hand, it is difficult to believe that cytokines, which have been numerous in vitro studies to have a major impact on the production and ctionality of stem cells, cannot be used to stimulate these same aspects in vivo. theotherhand,however,most in vivo studiesuntil now have onlyshown moderate effects of the capacity of cytokines to perturb stem cell numbers and difficulties that are encountered in modifying stem cell rimarily duetotheabsence o ufficient fundamental cell biological characteristics. ost importantly in this experiments should be aimed at identifying the biological laws that define and regulate stem cell population size and cycling activity. Only when these aspects have been unraveled can successful" manipulation of the endogenous system be achieved by the use of an ever-expanding arrayof cytokines.

rtment of InterCancer Center. 1s a recipient of a

Spangrude,feld, L Smit J Friedman, IL Weissman. use hematopoieticS Jones, JE ~ a g ~ e r Separation arkis. pluripotent of haematopoietic stem cells from spleen colony-forming cells. Nature 347:188, 1990.

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Cells 12563 1 94. 11.

inducedbypegy 349 1 , 1994. 12.

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a

RH Wiltrout. Recombinant transforming growth factor beta 1 and beta 2 protect mice from acutely lethal doses of 5-fluorouracil an S Cooper, DEWilliams, G 62.HEBroxmeyer, Growth characteristics of marrow hematopoietic progenitor/precursor cells from patientsonaphase I clinicaltrialwithpurifiedrecombinanthumangranulocytelony-stimulating factor. Exp Benninger, , LPatel, SR RS aj. Kinetic response of ~ u m a nmarrow myeloid progenitor cells to in vivo treatment of patients with granulocyte colony-stimulating factoris different from the responseto treatment with granulocyte-macrophage colony-stimulatingfactor. Exp Hematol22: 100, 1994. 64. G Wangoc, T Yin, S Cooper, P Schendel, YC Yang, HE Broxmeyer. In vivo effects of 8 1:965, 1993. recombinant interleukin-l1 on myelopoiesis in mice. Blood 65. JE Rasko,DMetcalf, MT Rossner, CG Begley,NA Nicola.Theflt3/flk-2ligand: Receptor distribution and action on murine haemopoietic cell survival and proliferation. Leukemia 9:2058, 1995.

University of ~ a n ~ ~ o~bian, n i ~~ea~n ,i t o bCanada ~,

It has long been suspected that there is interaction between the neuroendocrine and immune systems [l]. The suppressive effect of the adrenocorticotropic hormoneinfluence of sex hormones on immune organs were [2-41. Selye [5] also bserved that the inflammatory corticoids.In 1949 ench andcoworkers described and glucocorticoids for the treatment of rheumatoid arthritis. These authors proposed the first hypothesis for the roleof pituitary hormones in the pathogenesis of rheumatoid disease [6]. These observations led to numerous studies related to the effect of horm~nes on the immune system. The animal studies yielded contradictory results, and most ~ a t i e n t swithrheumatoid disease showed “normal”glucocorticoidserum levels. oreover, the long-term use of glucocorticoid therapy in inflammatory disease led to serious com~lications. was It concluded that the immunosuppressive and antiinflammatory effect of these drugs is pharmacological rather than physiological [7]. evelopments in immunobiology gave rise to the concept that the immune system is self-regulatory and is capable of functioning even outside the body in culture systems. everth he less, evidence has been accumulating slowly to support the role of hormones in immunoregulation and the neural regulation of inflammatory reactions. Thus brain lesions were shown to inhibit the anaphylactic reaction [8], and neurogenic infla~mationhas been discovered [S]. It has been established that hypophysectomized raimmunodeficient [lo] andthatimmunereactionscan beconditioned in the viansense [1 1,121. It was also revealed thatlymphoid organsareinnervated 1, that lymphocytes arecapable ofproducing classical h o r ~ o n e sas well as neuropeptides [ 141, and that cytokines which were originally thought to be immune-derived are also producedin the centralnervous system [IS]. These shared mediators are viewed today as the basis of neuroendocrine immune interaction. Evidence is rapidly increasing that neuroimmune mechanisms play an important role in basic physiolo~icalprocesses, and that abnor~alities of this regulatory network contribute to causation of diseases with underlying i m ~ u n abnore rospects for the hormonal treatment of inflammatory and autoimmune diseases are ever more encouraging withrecent significant advances. 7

f

ere we provide a brief overview of the immunomodulating effects of hormones, with some discussion on the mechanism of action and with some reference to disease causes and management. For detailed information the reader is referred to Goetzl [16], Guillemin et al. [17], Berczi [18], Plotnikoff et al. [19], Berczi and Kovacs 1201, Jankovic et al. [21], Ader et al. [22], Fabris et al. [23], Stead et al. 1241, Berczi and Szelenyi [25], Grossman 1261, RothwellandBerkenbosch [27], Hoghe-Peters and Hoghe 1281, Chikanza 1291, Kellen 1301, Friedman et al. [3l].

Prolactin (PRL), growth hormone (GH), and placental lactogen (PL) diverted from a common ancestral gene during evolution.All of these hormones show heterogeneity and currently are referred to as the growth and lactogen hormone (GLH) family. There is evidence to indicate that the variant hormones also differ in biological activity [32-371. The PRL receptors have been classified as short, intermediate, and long, and GH receptors as short or longin various species. These receptors share features with cytokine receptors and may be designated as the ~LH-cytokinereceptor family. The receptors for erythropoietin (EPO), for granulocyte colony-stimulating factor ( G - ~ S ~ granuloc~e-macrophage ), colony-stimulating factor (~M-CSF),and the beta chain of interleukin (1L)-2, -3, -4, -5, -6, and -7 belong to this family, The glycoprotein gp130, which is associated with the IL-6 receptor,is also a member of this family [38,39]. In the thymus, spleen, lymph. nodes, and bone marrow of rats and mice, both the short and the long forms of PRL receptor were present [40]. There is evidence to indicate that immunization [41] and autoimmune disease [42] lead to the increased expression of PRL receptorby lymphocytes. ~ r o w t hhormone induces insulinlike growth factor-I (IGF-I) in the liver and in many other tissues and organs; IGF-I plays a role in the mediation of the biologi. It is structurallyrelated to insulin and cross-reacts with it at [43]. Prolactin stimulates similar intermediate hormones, named synlactin by Nicoll and coworkers[44]. Receptor cross-linking (dimerization or oligomerization) by a single hormone molecule is required for signal transduction by the growth hormone receptor. Because of this require~ent, athigh hormone concentrations when each receptor is occupied by a single hormone molecule, cross-linking becomes impossible and therefore the hormoneserves as its own antagonist (Figure 1) 1451. The signaling pathway for PRL and related hormones is not fully resolved. G proteins, thyrosine kinase, and protein kinase C have allbeen suggested to play a role[39,46,47]. Direct nuclear signaling by PRL and GH has also been proposed [48-501. Recent information suggests that phosphorylation of transcription factors by protein kinases plays a A ) and transmajor role in signal transduction. ~eoxyribonucleic acid ( ~ ~ binding activation can be effected both positively and negatively by these factors. Comand cytokines use signal transducer and activator of transcription (STAT) f a ~ i l yfactors in the early phase of induction" The use of other signaling systems, such as Src-family kinases, phospholipase CYprotein kinase C, and mito-

Figure 1 A possible mechanism of cell activation by the growth and lactogenic hormone (GLH)-cytokine receptor family, using human growth hormone (hGH) as an example. In order to generate an active signal, two receptor molecules are cross-linked by a single molecule of ligand hormone/cytokine (oligo~erization).At high concentration, the ligand serves as its own antagonist.(Source: Ref. 45).

gen-activated protein (MAP) kinase, is variable. Although initially there are shared protein kinases and STATs, the combination of transacting factors working at a particular promoter in a given celltype is distinct, specifying a very different biological effect on hormonal stimulation. Thus,specificity of hormone action dependsto a large extent on the target cells' repertoire of transacting factors [Sl]. The IGF-I receptor belongs to the transmembrane thyrosine kinase receptor family. Signal transduction involves the phosphorylation of certain enzymes, which include phospholipase C, phosphatydil-inositol-3 'kinase, ras, guanosi~etriphosphatase activating protein, andSre and Src-like thyrosine kinases. Phospholipase C activation generates inositol triphosphate and diacylglycerol, which act as second messengers mobilizing intracellularCa+ and activating protein kinaseC [38]. +

1. ~

~ ~~ e ~ v e l oo ~ ~ n e ni ~ ~ The immune system develops normally in fetuses lacking the pituitary gland [52]. This suggests that during embryonic development placental and perhaps maternal growth and lactogenic hormones (CLHs) control thedevelopment of the hemopoietic and immune systems. Bone marrow, thymus, and immune function can be hypophysectomiz~d( ypox) ratsbyhumanplacentallactogen(PL) man PL alsoexerted itogenic effectontheNb2ratlymphoma cell line which is dependent on lactogenic hormones for proliferation [33]. Studies on chicken embryos revealedthat PRL and pituitary grafts placed onto the chorioallantoicmembranestimulatedtheearlymaturation of thymocytes in decapitated chicken embryos [ S ] . Neonatal rats treated with anti-growth hormone (anti serum for 8 weeks showed a significant decrease in thymus and spleen W and cellularities and a diminished antibody response. All these abnormalities were corrected by treatment with bovine G

rmones have long been suspected of playing a role in bone marrow function [57]. e erythropoietic effectof PRL was first demonstratedby Jepson and Lowenstein [ S S ] . Anemia develops in Hypox rats, which show grossly impaired DNA and ribonucleic acid (RNA) synthesis in the bone marrow, which is associated frequently, but not always, with leu~ocytopenia andthrombocytopenia. All these deficiencies can be restored to normal levels by syngeneic pituitary afts (SPCs) placed under the kidney capsule or by treatment with purified PRL, or human PL [53,54,59611. Human hemopoietic progenitorcells formed granul te and erythroid colonies if stimulated with PRL in the presence of IL-3, CSF, and erythropoietin (EPO) [62]. Insulinlike gr th factor-I mediates the ac hormone on the bone marrow, includin ymphopoiesis [63-651. Prolactin was shown to regulate the functionof bursa of Fabricius inbirds 166,671.

3. ~~e~ h y ~ u s The stimulatory effect of C H on thymus growth haslong been recognized [68-701. It induces thymus growth in hormone-deprived and old animals, which parallels with an increase in immunocompetence, Crowth hormone has a direct mitogenic effect on thymocytes in vitro and stimulates the production of thymic hormones [ 18,7€-731. At least some of the effects of C on the thymus are mediated by Hormone producti and rat thymic epithelial cells was stimulated and ICF-I [74-761 promoted the engraftment of human thymocytes in micewithseverecombinedimmudeficiency [77]. Theimpaired synthesis andinvolution of thethymus in poxrats, which are associatedwith profound immunodeficiency, can be reversedby syngeneic pituitary graft (SPC) or by treatment with GH or PRL: thymocyte proliferation resumes and immunocompetence returns to normal [73]. Pituitarygraftsinducedan increase in thymus weight and in the number of thymocytesin Ames dwarf mice[78]. Prolactin induced the expressionof theThy-l, LT-34 ( C ~ 4 ) ,andTLantigensby thymocytes

ormone production by thymic epithelial cells was stimulated by Placental lactogen increased selectively the growth of the thymus in Snell pituitary dwarf mice ['75,81].

promotes the antibody response [ls]. The imypox rats and of rats immunosuppressedby bromoreatment can be restored by either G enhanced the antibody response acc in both young and old mice [83]. iated the antibody responseof cockerels [67,84]. In pituitary dwarf children the mitogenic response of per c tes and immu

tivated withS t ~ ~ h -

nificant amounts of IGF-I after stimulationwi

response to tetanus toxoid [93].

Contact sensitivity reactions are impaired and survivalof allogeneic skin grafts are rats [lo]. Contact sensitivit reactions are restored if G or treated with , or PL. Treatment of rats with

1 The Immune Effects of Peptide Hormones and Neuropeptides

Effects on" Bone

tibody Thymus marrow NEP ACTH AVP CGRP CRF @-END ENK

CM1

1(DEN)

In~ammation

1 (DENI)

t 1(DNI) t 1(DNI) t (D)

GW

INS LW MEL CY-MSH NGF PL PRL SOM SP VIP

IL-2, -4 1(D)

"D, direct effect on immunocytes; E, effect through endocrine mediation; N, effect on and through the nervous system; I, in vivo effect, mechanism not clear; t, increased response/activity; 4, decreased response/activity; t 4, variable effect; 0 = no effect. NEP, neuropeptides; CMI, cell-mediated immunity; ACTH, adrenocQrticotropichormone; AVP, arginine vasopressin; CGRP, calcitonin gene-related peptide; CRF, corticotropin releasing factor; @-END,beta-endorphin; ENK, enkephalin; GH, growth hormone; INS, insulin; LH, luteinizing hormone; MEL, melatonin; a-MSH, a~pha-melanocytestimulating hormone; NGF, nerve growth factor; PL, placental lactogen; PRL, prolactin; SOM, somatostatin; SP, substance P; VIP, vasoactive intestinal peptide.

bromocriptine (BRC) or pergolide, which inhibit pituitary PRL secretion, prevents the development of contact sensitivity reactions to dinitrochlorobenzene. Reactivity can be restored by additionaltreatment with PRLorGH.storation may be antagonized if ACTH treatment is also given [53,94]. Treatm of rats with BRC prolonged the survival of allogeneic skin grafts [53]. ~ o m b i n e dtreatme 3 ) cyclosporine with dopaminergic agents (e.g., BRC or C ~ P - ~ 0 1 - 4 0 and showed synergism in the suppression of mixed lymphocyte reaction, the local graft versus host reaction, and therejection of kidney and cardiac allografts[95-971. In mice given allogeneic skin grafts, pituitary PRL messenger RNA (mRNA) was significantly increased and serum PRL bioactivity was elevated [98]. Bromoof macrophage tumoricriptine treatment prevented the T cell-dependent induction cidal activity in mice by Listeria ~ o n o c y t ~ g e nor e s ~ y c ~ b a c t e $bovis, i u ~ Treat[99]. SerumPRL levels increased ment with ovinePRL reversed thiseffect significantly in patients with cardiac allografts during the primary rejection episode, but such an increase became irregularduring recurrent rejections [loo]. Prolactin enhanced the proliferation of human T cells stimulated by IL-2 or

Hormones as Immune Modulating Agents

81

phytohemagglutinin (PHA); it was stimulatory for B, T, and natural killer (NK) cells when applied together with mitogens at physiological concentrations, whereas 5- to 10-fold higher levels inhibited the response of T cells to IL-2 [101,102]. Bovine PRL, but not bovine GH, administered to mice increased the expression of IL-2 receptors by CD4’ and CD8’ lymphocytes in the peripheral lymph nodes. The expression of IL-2 receptors in popliteal lymph nodes of dwarf mice was only 50% . of normal after the injection of concanavalin A (ConA). Treatment with PRL or 6.H corrected this deficiency [103]. Human T cell lines derived by transformation with the human T lymphotropic virus responded to GH in vitro by proliferation and IGF-I production, which in turn stimulated colony formation by these cells. T cell lines derived from individuals with Laron-type dwarfism did m t respond to GH by proliferation and IGF secretion [104]. Murine spleen cells also produced IGF-I after exposure to GH [1051. Acute rejection episodes of renal allografts were observed in two children after treatment with GH [106]. Myositis developed in two boys while on growth hormone therapy. The disease resolved in both patients 2 months after the cessation of therapy, but returned within 3 months when therapy was resumed [107]. In 22 adults with growth hormone deficiency the percentage and absolute number of NK cells were significantly decreased [108]. In 14 girls with Turner syndrome, GH treatment decreased the CD4/CD8 T cell ratio and increased the number of NK cells [log]. Treatment with GH of female patients with impaired GH secretion and of healthy adults increased NK cell-mediated cytotoxicity [l 10,1111. The effect of PRL on NK cell-mediated cytotoxicity is controversial. Matera et al. [112] showed that PRL stimulates the growth and cytotoxic activity of purified NK cells but has no effect on NK activity when unseparated peripheral blood lymphocytes (PBLs) are treated. This lack of reactivity of PBL was due to the activation of suppressor cells by PRL. Treatment of PBL or NK cells for 4 days with PRL in serum free medium increased target cell killing, but novel cytotoxicity was not induced when NK susceptible target cells (K562 and U937) were used. In contrast, cytotoxicity against lymphokine-activated killer (LAK) target cells (HL60, Jurkat, Daudi, and Sutt-1) was induced in both the NK and T cell populations. Prolactin had a biphasic effect on NK cells with peaks at either 25 or 200 ng/ml, whereas LAK activation occurred only at 200 ng/ml. Physiological concentrations of PRL supported significantly the generation of NK and LAK activities with low doses of IL-2. Pathological concentrations of PRL reversibly inhibited the generation of LAK cells, whereas IL-2-activated NK cells were stimulated [113]. 1.

The Effect of GLH on Phagocytic Cells

Human GH, at picomolar concentrations, exerted a chemotactic effect on human monocytes. Somatostatin and its long-acting analogue, octreotide, had a similar effect. However, micromolar concentrations were required. Somatostatin antagonized the chemotactic effect of GH on monocytes in a dose-dependent manner. Interferon and substance P were also inhibitory [114]. Phagocytic functions of monocytes and polymorphonuclear leukocytes were increased significantly in children treated with GH for 6 months. This was also true for long-term GH replacement therapy [115]. Growth hormone, PRL, and growth hormone-releasing hormone at very high doses enhanced hydrogen peroxide production in human monocytes stimulated with phorbol myristate acetate (PMA);

..

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...... . _. ..._.

- . . ~ ~ ~ . . ~ ~ . ~ . - , ~ . . . . . , ~.,.

.

...

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. ...

I

82

Berczi and Nagy

IGF-I was without effect, Monocytes expressed mRNA for GH and PRL receptors and bound specifically radiolabeled GH [116]. Treatment with GH, and to a lesser extent with PRL, increased significantly the number and size of testicular macrophages in long-term Hypox rats. When testicular Leydig cells were killed selectively with ethylenedimethanesulfonate, the clearance of dead cells was largely increased in GH- or GH plus PRL-treated rats [ 1171. Growth hormone stimulated lysosomal enzyme production, oxidative metabolism, adhesiveness, and modulated chemotaxis and priming for superoxide prodW' tion in neutrophilic leukocytes [118,119]. The reduction in superoxide anion secretion and in bactericidal activity of neutrophilic leukocytes from aged rats could be corrected in vitro by treatment with y-interferon (IFN-y) or GH. Neutrophils from aged rats that were grafted with the syngeneic GH secreting pituitary tumor GH3, but not neutrophils of control aged rats, could be primed in vitro for superoxide secretion by IFN-y [120]. Neutrophils from patients with acromegaly and hyperprolactinemia showed decreased chemotactic activity [ 1211. The Effect of GLH on Cytokine Production Production of IFN-y by murine spleen cells and by human peripheral blood mononuclear cells is enhanced by prolactin [85,99,122]. Interferon-regulatory factor-I gene expression and IFN-y production were induced by PRL in the Nb2 rat lymphoma cell line and in T lymphocytes. Growth hormone enhanced the production of IL-2 by human lymphocytes and IL-1, tumor necrosis factor-a (TNFa), and superoxide anion production by monocytes [51,72,123] (Table 2). Others found that GH activated monocytes for superoxide production, but not for TNF production, cell adherence, or killing of Mycobacterium tuberculosis [ 1161. Growth hormone increased the release of IFN-y from murine splenocytes stimulated with enterotoxin A, whereas the release of IL-la was inhibited. Prolactin also decreased the release of IL-la but did not affect IFN-y release under these conditions [124]. Growth hormone treatment of cows reduced the plasma levels of:TNFa, cortisol, thromboxane B2, and thromboxane/prostacyclin ratios in response to endotoxin injection [ 1251, Considerable information is available with regard to lymphocyte-derived PRL, which is produced by B and T lymphocytes, some B lymphoblast cell lines, and some lymphoid tumors [ 126-1291, The molecular weights of lymphocytederived PRL varied from 11 to 48 kDa, indicating that monomeric, dimeric, and cleaved forms of PRL all are produced within the immune system [128-1321. The regulatory sequences of the PRL gene in lymphocytes are analogous to the placental and not the pituitary regulatory sequences. Pituitary regulators of PRL secretion had no effect on hormone production by the IM-9 human B cell line, but dexamethasone was inhibitory [133-1361. 2.

E. lnsulfn (INS)

Insulin shows a regulatory interaction with GH and also with IGF-I. Insulin receptors are barely detectable on resting T and B lymphocytes, but high-affinity specific receptors appear on both cell types after activation [137]. Insulin has a variable effect on lymphocyte mitogenesis; it was required for the human mixed lymphocyte reaction in serum free medium. Both B and T lymphocytes need INS for normal

The Effect of Peptide Hormones and Neuropeptideson Cytokine Secretiona HPGF ACTH CGRP CRF @-END ENK FSH GH INS LH

PRL SOM SP TRH TSH VIP

IL-1

TNFcY

IL-6

t l

t 71 f

TI t

t t t t

t

IL-4

l tl

t t

l

t

t l

l

1

1

t 1

t l

l IL-3 t, GMCSF t

IL-2 01

l tl

Tl t t tl l t t

IFN?

t

t t T

l

l

HPGF, hypothalamic pituitary growth factor; IL-l, interleukin-l; TNFa, tumor necrosis factor a; IL-6, interleukin-G; IFNy, interferon y; IL-2, interleukin-2; IL-4, interleukin-4; ACTH, adrenocorticotropic hormone; CGRP, calcitonin gene-related peptide; CRF, corticotropin releasing factor; @-END, betaendorphin; ENK, enkephalin; FSH, follicle stimulating hormone; GH, growth hormone; INS, insulin; LH, luteinizing hormone;MEL,melatonin; a-MSH, alpha-melanocyte stimulating hormone; PRL, prolactin; SOM, somatostatin; SP, substance P; TRH, thyrotropin releasing hormone; TSH, thyroid stimulating hormone;VIP, vasoactive intestinal peptide; GM-CSF, granulocyte macrophage-colony stimulating factor.

development and function. Activated human T cells respond to INS with chemotaxis [ 18,1381. Insulin potentiates anaphylaxis, induces an anaphylactic inflammatory promoting factor, enhances fibrinolysis and phagocytosis. On the other hand, Fcreceptorsofguinea pig macrophagesweredown-regulatedandantibodydependent cytotoxicity by human lymphoid cells was inhibited by IN§ treatment. Insulin suppressed the release of IL-l and IFN-y by murine spleen cells in response to stimulation with staphylococcal endotoxin-A. Glucagon and somatostatin antagonized INS action on lymphoidtissue [ 18,124,1391.

7. ~ Q ~ ~ C Q t f Q ~ i ~ Corticotropin releasing factorstimulatesthe release ACTHfromthepituitary glandand mediates, in part,cyto~ine-inducedACT release fromthepituitary gland. It is a major stress response that integrates peptide of the central nervous system and acts centrally as an immunosuppressive agent, which is not mediated by glucocorticoids but ratherby the stimulation of sympathetic outflow. Receptors for F are present in the immunesystem and immunocytes produceCRF [ 140,1411. ~ i t h i nthe immune system CRF production increases during inflammation,

Y

There is evidence to suggest that CRF is an important antiinflammatory hormone. owever, some forms of inflammationactually are enhanced by C cotropin releasing factor reducedlipopolysaccharide-(LPS)-inducedpulmonary vascular leak in mice, which was mediated by the stimulation of glucocorticoids. Leukocyte infiltration was significantly repressed and a modest survival occurred in CRF-treated mice. Corticotropin releasing factor anta~onized the stimulation of human granulocytes by tumor necrosis factor (TNF) [30,143, 1441. Corticotropin releasing factor treatment of rats led to a decrease in various parameters of immune cell function [145]. Elevated levels of synovial fluids of rats with experimental arthritis and of pa rheumatoid arthritis [146]. In vitro studies on the effect of reactions have produced c tradictory findings. T lymphocy IL-2, and IL-6 secretion; cell activity have all been influenced

d r e ~ o c o ~ i c o t r o~~ ioc r ~ ~~C~~~ o ~ e In various species of mammals and birds, ACTH treatment has an antiinflammatoryeffectand influences leukocyterecirculation.These effects are due to the stimulation of glucocorticoid production by the adrenal gland.~ n t i b o d yformation, antibody-mediated reactions (anaphylaxis, arthus type hypersensitivity)? and cellmediatedimmunity(graftrejection,tuberculinresponse)were all inhibited by treatment of animals [181. The antagonistic effect of ACTH and growth hormone on t sponse was first described by Hayashida and Li in 1957 [1471. In restoration of immunocompetence by either PRL or C treatment was antagonized by replacement doses ofACTH 1591. Immunocompetent cells synthesize proopiomelanocortin ( cies and generate POMC-related peptides, incl Immunocytes express specific receptors these for peptides [ the of tes epidermis proimmunoregulatory duce cytokines, A yte-stimulating hormone SH) [15 l]. Lymphocyte-derived ACTH was shown to beinsufficient to stimulate adrenalsteroidogenesis in Hypox rats [ 15121. Adrenocorticotropic hormone has a direct effect on lymphocyte proliferation [153,154] and modulates Ig and IgE synthesis in vitro. Its effect on Ig ated by accessory cells [155,1561. It suppressed the Ca"-dependent and "independent phagocytosis of latex beads by murine peritoneal macrophages. This suppression was not mediated by cyclic 3' ,5'-adenosine monophosphate (CA Adrenocorticotropic hormone exerts an antipyretic effect on the central nervous system [1581. 3. ~ e t ~ - ~ n d o and r ~O~t ~i n e r O ~ i~ o ied ~ t i ~ e s Opioid receptors of K , 6, and p types are present on lymphocytes, on monocytes/ macrophages, and on polymorphonuclear leukocytes [l 59-1621. Opioid receptors consist of about 400 amino acids and have the characteristics of 7 transmembrane domain structure of C-protein-coupled receptors. The amino acid homology between the different opioid receptor types is about 60%, and there is about 9 0 ~ 0 identity of the same type of receptors cloned from different animal species. On agonist binding, opioid receptors mediate inhibition of adenylyl cyclase 11631. eta-endorphin (&END) is derived in the pituitary gland and in other tissues from the POMC peptide by enzymatic cleavage. The production and secretion of

@-ENDin the pituitary gland areregulated by CRF and immune-derived cytokines, such as IL-1@and TNFcY[ 164,1651. eta-endorphin is also produced in lymphoid cells, especially those infiltrating inflamed tissues. There is evidence to suggest that opioids in general and &END in particular exert an antiinflammatory effect and down-regulate the immune response [166,167], In vitro, bothp and K opioid agonists exhibit immunosuppressive activity for antibody responses [1681. The K opioid agonist agent U5094$$ suppressed the production of IL-l and TNFcYby mouse peritoconcentrations. macrophages neal l at suppressive This effect could be completely reversed the byK-selective antagonist norbinaltorphimine[ 1693. In general, the effectsof endogenous opioid peptideson immune function are any of the findings are contradictory. Antibody production; NK cell ell activity; cytotoxic T lymphocytes; the mixed lymphocyte reaction; of IL-1, IL-2, IL-4, IL-6, IFN-y,andprostaglandins;mast cell degranulation;andthe activity of neutrophilicleukocytesare all influenced by opioid peptides. ~ p i o i d sare also capableof immunomodulation by acting through the central nervous system [ 19,170- 1781. f f o ~ y ~ e~ i ~ f f ~ l ~o r~ ~i of ffCY-^^^) f ~e is a significant regulatorof fever and inflammation. It antagonizes the pyrogenic and proinflammatory effects of IL-1, IL-6, IFN-y. It actswithin the brainto inhibit fever and peripheral inflammation. clearly also exerts an antiinflammatory effect on peripheral targets [179]. Alpha-melanocyte stimulating hormone is produced by spleen cells and by' keratinocytes and is present in the aqueous humourof the eye [ 150,180,181~. It has an inhibitory effect on thymocyte proliferation9 neutrophilia, the production of se proteins by the liver, TNF and IFN-y production, inductionof prostain fibroblasts, and contact sensitivity reactions. The immunosuppressive effect of IL-l@administered by the intracerebroventricular route could be blocked of a[ 182- 189). Whenadministered to mice by thesimultaneousinfusion either systemically or locally at the si treatment with contact sensitizing agents, led to the induction of hapten-specific tolerance. Lymphocytes from regional lymph nodes of CY failed to produce IL-2 5 days after sensitization. Tolerance abrogated could be i in vivo by the administration of antibodies to IL-l0 at the site ofsensitizati The antiinflammatory effectof centrally administered hibited in mice by the &adrenergic receptor blocker propra 62-adrener ic receptor antagonist. A marked antiinflammatory effect was exerted by hen injected intrape allyanimals into spinal with cord tra involvement e of ashown was in adjuvant arthritis, adultrespiratory distress syndrome9and septic shock.Plasma levels of CYare elevated in patients suffering from acquired immunodeficiency syndrome and in the early stages of myocardial infection. Alpha-melanocyte stimulating hormone found in the synovial fluid of joints affected by rheumatoid arthritis did not correlate with plasma levels [ 1791.

1

I

ukocytes express glucocorticoid Expression r is highest in thymic epithelial cells and bone marrow cells. numbers r increase in stimulated lymphocytes and are regulated by plasma corticosteroid levels, hormones, cytokines, and genetic factors [ 1931.

SteroidandthyroidhormonesandvitaminsAandandtheirmetabolites have intracellular (cytoplasmic or nuclear) receptors that b ong to the same receptor family and directly regulate gene expression in thenucleus. Steroid and thyroid hormones and vitamins A and D are small lipophylic molecules which diffuse into the cells and bind intracellularly to their receptors. The glucocorticoid r located in the cytoplasm, whereit is bound to heat shock protein 90 Glucocorticoid causes the dissociation of the receptor from receptor translocation into the nucleus. In the case of thyroid h acid the receptors are located in the nucleus. The mode by which steroid hormones and vitamins A and D regulate gene expression is not entirely understood. The regulation of the transcriptional activator (AP-l), which is composed of homo- and heterodimers of .Tun and Fos proteins, is one pathway to gene regulation [194]. The immunosuppressive activity of glucocorticoids has been shownrecently to be dueto the transcriptional activation ofI K B ~which , is the Cytoplasmic inhibitor of nuclear regulatory factorNF-KB,which activates many immunoregulatory genes in response toinflammatorystimuli;inhibitor of NFK a)trapsactivated NF-K dersit inactive inthecytoplasm.Theinon of NF-KBmarkedlydecreases cytokine secretion and plays a major role in the antiinflammatoryactivity of glucowhich corticoids [195,196]. A thirdnucleartranscriptionfactor glucocorticoids is the CAMP-responsive element binding protein(C A glucocorticoid-induced protein, lipocortin 1 , inhibited ne mation. Lipocortin 1 plays a role in the inhibitory effect of glucocorticoids on the e of the hypothalamic-pituitary-adr~nal axis to cytokines [ 198,1991. esistance may develop to the antiinflammatory effectsof glucocorticoids in asthma and in other inflammatory and immunological diseases. Under these conditions there is a marked reduction in the binding of glucocorticoid receptorto which appears tobe the consequence of increased binding of glucocorticoid re GC-R) to transcriptional activator A -1. The release ofc tolcines that activate -1 may induce secondary steroid resistance in concentrations h of "adrenoreceptor agonists activate another GCanscription factor,

ow concentrations of corticoidsweresho to inducethe ~roductionof ) frommacrophagesigrationinhibitoryfactor is migrationinhibitoryfactor ( capable of overriding the glucocorticoid-mediated inhibition of cytokine secretion by lipopolysaccharide (LP§), activates monocytes, and antagonizes the protective effect of glucocorticoid in lethal endotoxemia[201]. There is overwhelming evidenceto indicate that glucocorticoids are fundamenof thehypothalamic-pituitary-adrenal theimmunosuppressivefunction ) axis (Table 3). This conclusion gains further support from studies in transenic mice expressing glucocorticoid receptor antisense RNA. Endo~enousGCwere markedly reduced in such mice, and the sex dimorphism was gh plasma corticosteronelevels were present from early postnatal ages through adulthood. There was a marked increase in CD4+ C 8 double positive cells in the thymus, and the CD4/CD8 ratio was incr by 4 0 ~ 0 - 6 0 ~as 0 ,a result of the increase of CD4' cells and the decreaseof thesubset.Transgenic mice failed to show the postpubertal reduction in thymocytes. A significant increase in immunoreactivity was observed[202]. The expression of the glucocorticoid receptor antisense transgene was targeted +

The Immune Effectsof Steroid Hormones Hormone

ona one Antibody Thymus marrow

l

GC

E2

l

TS

t

DHEA PS

VD3

l l

CM1

Inflammation

l t

tl l

l

l T l l

l

t l

t

aCMI, cell-mediated immunity; G C , glucocorticoid; E2, estradiol; TS, testosterone; DHEA, dehydroepiandrosterone; PS, progesterone; VD3, 1,25-dihydroxyvitamin D3.

to the thymusby using the proximallck promoter, which is predominantly active in immature thymocytes. The embryonic and postpartum development of thymocytes was significantly altered in such mice. There was a decrease in thymic size which was due primarily to the de se in the number of C 4+ CD8 + cells. There was an enhanced susceptibility to ell receptor-(TCR)-mediatedapoptosis,although thymocyte loss was also de before the expression of T lymphocytes bearing a@ typeantigenreceptor (T . Theauthors concluded that glucocorticoids are necessary forthetransiti ymocytes fromtheCD4- CD8" stage tothe C 8' stage and that glucocorticoids support the survivalof the latter cell PO tion in the thymus [203]. These findings are compatible with the observation that physiological concentrations of corticosterone (CON), which is the principal glucocorticoid of rat,accelerated the anti-T cell receptor-induced growth of rat lymphocytes after 2-3 days in culture; inhibited cell growth followed after 5-7 days. Cortine had to be present within 60 min after the initiation of T cell receptor activation in order to produce maximal enhancement. An increased expression of the IL-2 receptor a chain on CON-treated TCRa@+ andC observed after 48-72 hr in culture[204]. Apparently, glucocorticoids also exert some immunoregulatory effect through the mineralocorticoid receptor (also called type I corticosteroid receptor). This repated in the cortisol-induced inhibition of interleukin-l receptor antagstimulated monocytes. The type I receptor antagonist spironolactone I1 receptor antagonist RU38486 both partially reversed this inhibitory terone was also inhibitory; itwas blocked by spironolactone [205]. uman mature T cells are considered to be resistant to the apoptotic effectof glucocorticoids. Such T cells show, however, apoptosis if exposed to GC soon after onandarealso sensitive to apopto induced by growthfactor 8' cells weremore sensitive than C cells. Apoptosis was increased by macromol ular synthesis inhibitors (cyclohexamine and puromycin) and antagonized by IL-2, -4, IL-10 [206]. It was also observed that cells from patients with sepsis or septic shock were more sensitive to the antiproliferative effect of CCs in the mito~en-stimulated lymphocyte proliferation assay when compared with normal controls. Suppression could be antagonized by additional treatment with +

IL-2, and to a lesser extent with the combination of IL-l, IL-6, and TNFa. The authors suggested that the general hypersensitivity to GCis counteracted at thesite of in~ammationby locallyproducedcytokines [20'7], Cortisol (200 mg/kg)increased IL-4 and IgE serum levels significantly in Balb/c mice which were also treated with monoclonal antibodies to IgD to induce polyclonal [208]. Glucocorticoidscausedneutrophiliawithaconcomitantdecrease of other leukocyte species in the circulation. This effect is attributed to theaction of GC on the vascular endothelium rather than directly on lymphocytes 12091. Glucocorticoids increase the rate of apoptosis in thymocytes, leading to thymus involution [210], The CC sensitivity of thymocytes is influenced by thymic hormones and genes of themajor histocompatibilitycomplex ( C) [l 81. Corticosteroidssuppress the productio~of prostaglandin E2 ( ~ G E 2 and ) 6-keto~ Gby phagocytic ~ * cells ~ in the thymus [21l]. In cells of the monocyte-macrophage series metabolism; chemotaxis; phagocytosis;cytotoxicreactions;capacity to presentantigen;secretion of IL-1, IL-1 receptor antagonist, and IL-6; and ability to respond to lymphokines are all inhibited by glucocorticoids. Glucocorticoids suppress the prod~ctionof collagenase, elastase,plasminogen-activated TNFa, superoxide,andnitricoxide by macrophages [18,205,212,213] (Table 4). n monocytes glucocorticoids increased the expression of h u ~ a n l e u k o c ~ e antigen ( H L ~ and ) IFN-y receptors [214,215] and induced high-affinity IL-l receptors on human blood lymphocytes 12161, up-regulated high-affinity IL-6 receptors on human epithelial cells [217], and potentiated the ind~ctionof Fc-y receptors in human monocytesby IFN-y [218]. The functionof dendritic cells, which are capable of presenting antigen to naive T cells, was impaired in mice after treatment with dexamethasone (Dex) [219]. The helper, suppressor, and killer functions of T lymphocyt GC. The production of IL-2, IFN-y, colony-stimulatin~ factor( phage activating factor ( ~ ~byFT )lymphocyteswas inhibited b be abrogated by treatment with IL-2 /220,221], Serum Ig level is decreased in CCtreated animals and patients. This effect was followed by an overshoot reaction

The Effect of Steroid Hormoneson Cytokine Secretiona Hormone HPGF

IL-1

TNFa

IL-6

IFN-y IL-4 IL-2

GC E2 DHEA AET PS VD3 *

"HPGF, hypothalamic pituitary growth factor; IL-l, interleukin-l; TNFcu, tumor necrosis factor a; IL-6, interleukin-6; IFN-y, interferon y; IL-2,interleukin-2;IL-4,interleukin-4; GC, glucocorticoid; E2, estradiol; DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; AET, androstentriol; PS, progesterone; VD3, 1,2S-dihydroxyvitamin D3.

afterthetreatment was terminated.InC-treatedanimalsthenumberofbone marrow-derived lymphoc~esin spleens and lympodes is decreased, butit is ell activationare notaffectedinthebonearrow.The early stages o by GC,but preactivated cells arenotaffected.Thetionof IgA and mucosal surfaces is inhibited by GC, whereas the production of secretory component (§C), which is responsible for the tra~sportationof IgE and tissue fluid onto mucosal surfaces, is stimulated by GC in rat hepatocytes and inhibitedby estradiol [222], Glucocorticoidsinhibit N -cell-mediated cytotoxicity andantibody-dependent cellular cytotoxicity ( A ~ C ~This ) . inhibitory effect is potentiated by PGE2 and abrogatedby IFN-.)/or IL-2 [18,2231. The GC treatment of rats induced mast cell destruction. Local GC treatment led to the dramatic depletion of cutaneous mast cells in humans [224]; inhibited mediator release from mast cells and basophilic leukocytes 12251 methasone down-regulated IgE receptors and inhibited the IgE-d~pendent release of hexosaminidase and the synthesis and release of leukotrienes LT murine bone marrow-derived mast cells, whereas the release of significantly [226]. In vivo treatment or in vitro exposure of human eosinophils to GC inhibits the chemotactic response. The functionof human neutrophilic granulocytes is impaired by GC as Fc receptors, complement receptors, and chemotactic receptorsarerenderednonfunctionaland@-adrenergicreceptorsare unc~upled from adenylatecyclase [181. Systemic GC treatment exerts an immunosuppressive effect in a number of species, including human, mouse, rat, guinea pig, rabbit, chicken, lizard, and frog. Serum immunoglobulin levels and humoral and cell-mediatedimmune reactions are depressed in chronically treated animals. Immunological memory and the cells responsible for the maintenance of graft versus host reaction are resistant to the suppressive effect of GC [181. Glucocorticoids have a powerful antiinflammatory effect. This is the result of glucocorticoid action on avariety of pathophysiological mechanisms that play a role in inflammation, including the inhibitionof cytokine and other mediatorsecretion, inhibition of leukocyte priming, reduction of vascular permeability, and synergism with other antiinflammatory mediators such as catecholamines, @-EN etc. This multifaceted effect on the inflammatory response explains the unparalleled inhibitory effect of glucocorticoids on inflammatory disease 1[227,228]. Glucocorticoids are also capable of inhibiting neurogenic inflammation, which is initiated by substance P and other inflammatory mediators released from sensory nerve fibers [198,229]. Recent observations showed that glucocorticoids play a role in develthe opment of T cell anergy during graft versus host reaction in mice 12301 and in the inhibition of immune and inflammatory reactions in the aqueous humor .of the anterior chamberof the eye, which is an immunologically privileged site. §ignificant concentrations of glucocorticoids and little or no cortisol binding globulin were found in aqueous humor of mouse, rat and human 123 l]. Glucocorticoids also play a role in strain-specific variations in the immune response and, along with other steroid hormones, in the age-related abnormalities of immune inflammatory reacis capable of synthesiztions [232]. Moreover, it appears that the thymic epithelium ing GC, which plays a si~nificantrole in the normal development of thymocytes [203,2331. ~ifferenceshave been observed in the activation of adrenal steroid receptors 9

in the brain and in immune tissues of Sprague- awley, Fischer 344, and Lewis rats. F344 rats exhibited the greater magnitude of type I1 adrenal receptor activation in brain tissues, whereas Lew rats showed the lowest magnitude in immune tissues during stress 12341. In the spleen and thymus of a~renalectomized rats, type I J adrenal steroid receptor binding was significantly increased; it could be bloc~edby aldosterone administration. This indicates that typeI receptors may play a tonic inhibitory role in type I1 receptor expression in immune cells which expressboth receptor subtypes12351. The effect of selective type I and typeI1 adrenal steroid receptoragonists on lymphocyte distribution in the peripheral blood and spleen of young adult rats has been stu~ied. The type I1 receptor agonist ~ 4 $ 3 6 2decreased T and cell and natural killer cell numbers to a very low absolute level. Aldosterone, which is a type I receptor ago' significantly reduced the number of lymphocytes and monocytes and, $3629 also decreased the number of ~eutrophils.T helper cells and N were decreased by aldosterone, but therewas no effect on cytotoxic cells. Corticosterone at physiological doses beh agonist in these experiments 12361. The response of sple mized animals to concanaval was stimulated by ment for 10-13 min to low concentrations (3-30 aldosterone of if 2 was also present. ~orticosterone had a similar effect. These effects became evident in the presence of glucocorticoi~ receptor antagonist and after short corticosteroid exposure times. After prQlonged preincubation with high concentrations of corticosteroids9 an immunosuppressive effect was observed 12371.

teinizing hormone (L ) modify immune roid hormone production and through a nificantly increased the proliferation of murine lymphocytes in response to mitogens and stimulated the secretion of IL-1 and IL-2. stimulated also It econdary antibody responses [238]. ~ ~ y s i o l o g i c aconcentrations l enhanced ofresponse the human of peripheral blood mononucle cantly decreased the percent enhanced the percentage of

cells stimulated by leased IL-6 from

various immune reactions in rats by treatment with hCG led to negative results. In guinea pigs hCG inhibited the development of ~elayed-type hypersensitivity reac-

tions. The hCG-treated guinea pigs showed depressed lymphocyte responses to P and to purified protein derivative in vitro. Several investigators pointed out that commercial preparations of hCG contain impurities which are responsible for the suppressive effect on lymphocyte reactionsin vitro [ 181.

Estrogen and androgen receptors are present in the thymus and in the bursa of Fabricius; however, classical estrogen receptors (detectable by the dextran-coated charcoal assay) in secondary lymphoid tissue are not present, with the exception of lymphocytes, which expressreceptors for estradiol. Progesteroneis capable of acting throughGC receptors, although theexistence of specific receptors forthis hormone in lymphoid cells has also been described. At high concentrations estrogens and androgens also act on GC receptors [26,240-2421. There are indications tence of as yet unidentified steroid receptors that affect lymphocyte or instance, estradiol and triphenylethylene antiestrogens(e.g., tamoxifen or toremifene) both sensitize lymphoid and nonlymphoid target cells for cellme~iated ~illing in the absence ofclassical estrogen receptors. This findingsuggests the existence of a novel steroid receptor on which both estradiol and antiestrogens act synergistically ratherthanantagonistica 31. ~embrane-boundsteroid receptorswhichincludethepolyglycoproteinpumphavealsobeen described. Triphenylethylene-derived antiestrogens are capable of inhibiting lymphocyte proliferation under conditions in. which estradiol has no effect. A membrane-bound antiestrogenbindingsiteforwhichthenaturalligand is unknownseems to be involved in this phenomeno~[243]. stradiol(E2)suppressesbonemarrowfunction;causesthymicinvolution;and Inhibits the helper, suppressor, and effector (delayed-type hypersensitivity) functions of T lymphocytes. The function of natural killer cells, neutrophils, and mast cell degranulationarealso inhibited by E2.Incontrast, phagocytosis,humoral immune reactions, and certain autoimmune diseases are enhanced by E2 in laboratperitonealmacrophagesexposed toE2at lom2and ng/ml in vitro ignificantly greater amounts of TNFa than untreated macrophages. In contrast, macrophages treated with lom4ng/ml of E2 showed significan~lyreduced TNF secretion [246]. ~imilarly,the release of superoxide by rat peritoneal phages was not affected by E2 at concentrations between 10"' and stimulation occurred at concentrations above or below that range. Estradiolsignifihibited nitrite release by macrophages at most concentrations used, except at which release was not affected. In the presence of anti-TNF antibody, superoxide and hydrogen peroxide release was moderately reduced, but nitrite release was inhibited dramatically[212]. Antigen presentation by uterine epithelial cells was stimulated in ovariectomized rats by E2, and antigen presentationby stromal uterine cells was inhibited at 1. Estradiolincreasedthe production ofimmunoglobulin by phocytes stimulated by IL-2 in culture. A significant incr * S was also induced by E2 alone. Phytohemagglutinin- (

induced DNA synthesis by T cells was enhancedby pharmacological concentrations of E2 [248]. The treatmentof various mouse strains with estradiol for 31 weeks, but not for 4 weeks, significantly increased the frequency of IgC- and Ig cells [249~. stradiol reduced NK cell cytotoxicity in a number ofmousestrains in a dose-dependent manner. C3H/N, DBA/l, and NZB/W mice displayed high (more than 5 0 ~ 0 degree ) of suppress ion^ whereas C57BL/6 and ~ ~ L / l ~ rmice / l ~disr played low susceptibility (less than 30910). Repeated injections of mice with antiasialo C ~ l - a n t i b o d i e swhich 9 affect NK cells, for 4 weeks raised the frequency of IgC-producing cells several-fold. The authors hypothesized that estrogen-mediated suppression of NK cells, which inhibits cells, led to the increasedfrequencyof Ig-producing cells [249]. The in vitro cytotoxic activity of murine CD4' T cells was dependent on estrogen.ThisestrogendependencycouldalsobeshowninP2-microglobulinient mice infected withlymphocytic chori eningitis virus. In suchmice, cytotoxi~T cells are not fun~tional andC ' CTL can only exert antiviral immunity, resulting in fatalimmune meningitis if orchidectomizedanimals are treated with estrogen[250]. ~hysiologicalconcentrations of estradiol (10"' significantly reduced the chemotacticresponse of humanpolymorphonuclearleukocytes to wformyl-Lmethionyl-leucyl-phenylalanine( F ~ ~ reinc ~ )cub. at ion with the estrogen receptor antagonist drugs clomiphene and tamoxifen eliminated this inhibitory effect [25 l]. l ~ P - ~ s t r a d i and o l myelin basic protein, which is a major autoantigen from brain tissue, had a synergistic effect on inducing degranulation of rat mast cells. This effect was more pronouncedin Lewis rats, which are susceptible to thedevelopment of experimental allergic encephalitis, than in Sprague-Dawley rats, which are resistant [252].

In general, testosterone has a suppressive or moderating effect on immune reactions. The development of the bursa of Fabricius can be prevented by testosterone of treatment in chicken embryos. Testosterone antagonizes the enhancing effect estrogens in a number of autoimmunedisease models. It has been proposedto favor selectively the differentiation of suppressor T lymphoc~esin the thymus [18,253]. The effect of androgens on the immunesystem is influenced by ~ ~ C - l i n k genes ed S exert a hemopoietic effect [255]. of adult male mice led to the fall of transforming growth factor-@ ( ~ C F - P , )level in the thymus, it increased 2.3-fold after replacement therapy with testosterone.Bioactive TCF-P, levelfell approximately to 50% aftercastration and was normalized l week after testosterone treatment. ~roductionof TCF-P by thymocytes was modulated by testosterone 12561. In aging rats the thymus can be regenerated by castration and the regeneration inhibitedby testosterone t r e a t ~ e n t . Testosterone is converted to estradiol by aromatase, and estradiolis known to be a powerful inducer of thymiG involution. When young adult and aging (18 months old) orchidectomized male rats were treated with testosterone and with aromatase ors (l ,4,6-androstatriene-3,17-dione[ATD]; or 4-hydroxyandrostenedione ), the effects of testosterone were blocked by ATD in both the thymus and 0th inhibitors restored the thymusin aging rats with normal histologi-

on

cal appearance, and ATD enlarged the thymus in young intact animals. In old rats the effects of testosterone were blocked by ATD in both thymus and spleen. Cytosolic estrogen receptors were not detectable in the thymus of old rats but were measurable in orchidectomized or ATD-treated old animals [257]. Treatment of rat peritoneal macrophages with testosterone in vitro inhibited nitrite release and stimulated release of reactive oxygen intermediates. The addition of a n t i - T N ~antibody to the system moderately reduced superoxide an peroxide release and profoundly inhibited nitrite release [212]. Treatment of murine T lymphocytes in vitro by dihydrotestosterone reduced the amount of IL-4, IL-5, and IFN-y in response to activation wit CD3 antibodies; the production of IL-2 was not affected. Androstenedione or testosterone (metabolic precursors of DHT) had no effect in thi tem. The presence of theenzyme S~-reductase,which convertstestosterone to has been found in macrophages. of this of mice with D conversion bl production. IL-4 tment d S to produce lymphokines. altered the capacity of T cells in the Treatment of aged dehydroepiandrosterone mice com d with a restored the capacity of T cells to produce IL-2, IL-4, and IFN-y to a level e ~ u i v a lent to thatof younger mice[263]. Treatment of neonatal ratswith testosterone (TS) T increased the reactivity of lymphocytes from males and females in the mixed lymphocyte reaction; it was further increased by additional gonadectomy. Serum immunoglobulin levels were increased by this hormonal treatment in males, but suppressed in females[258]. Recent studies revealed thatthe weak androgendehydroepiandrosterone ( ~ H E A and ) its metabolites play a major role in the regulation of the immune system. In the adrenal gland, which serves as a precursor for regarded as a “mother steroid” from which 0th ity, such as testosterone (TS), Recent observations revealed t spleen and that “I-hydroxy derivatives of more active in stimulating the antibody response in mice than are the recurso or hormones [260]. Androstendiol and androstentriol (AET), which are also D metabolites, have both been shown to be morepotentimmunoregulators EA. Dehydroepiandrosterone suppressed Con A- and LPS-driven l y m p h o c ~ e proliferation, whereas AED had little influence, and AET potentiated with both mitogens. Similarly, the secretion of IL-2 and IL-3 by Con lymphocytes was depressed by D E and potentiated by AET. The suppression of Con A-induced 1 ocyte proliferation drocortisone was strongly antagonized by AET; had no effect; and showed a weak antiglucocorticoid effect [261]. thymocyte proliferation assay, calc’ -related both inhibi~ed proliferation. When antagothe proliferative response was ations.Inaddition,the in this system. None of he absence of Con type helper cell, which is

stimulated for proliferation and IL-2 secretion. ehydroepiandrosterone is capable of overcoming the suppression of IL-2 secretion glucocorticoids. The production of IFN-y is also promoted by DHEA, whereas production of IL-4 and inhibited. The deficient prod n of IL-2, IL-4,and IFN-y oflymphocytes from old mice in response to anti- antibodies could berestored to the level oflymphocytes from young donors by combinationtreatmentwith drotestosterone [259,263,264]. In mice high dose tolerance of delayed-type hypersensitivity to sheep red bloodcells could be abolishedby a single subcutaneous (s.c.) EA. The treatment of spleen cells from tolerant mice in vitro with A prevented the transfer of tolerance to naive recipients. A-treated tolerant mice produced more IFN-y and less I -4 and IL-6 than those of untreated tolerant animals12651. D receptors in humanmonocyteshasbeendemonThe presenceof strated.Theinductionof xicity, IL-lsecretion, reactive nitrogenintermediate release, and c o m p l e ~ e nreceptor-l t and TNFa protein expression wereall enhanced animal model systems HEA hasbeenshown to increase host resistance against viral, bacterial,andparasiticinfectionsandntiatevaccination,includingmucosalimmunity [232,259]. Treatmentwithprotected mice against thermalinjury,whereasprednisonetreatment increas rate of mortality 12671. The blood level of DHEA declines progressively after the second decade by approximately 20% per decade and will decrease to 10%-15% of maximum by age 85-90. In aging animals and humans the immune system is gradually disregulated; the process is referred to as immune senescence. The proportion of C increased with age; frequently the cells produce autoantibodies. Thl-type helper activity which produces IL-2declines, whereas activity of Th2 cells and production of IL-4, IL-5, and IL-6 increases. Treatment of ex~erimental animalswith stored the reactivity of old sulfate led to the reversal of immunosenesc animals to the level of young ones. Curren being investigated for the potentiation vaccination of in elderly peo 2701, Treatment of post-' menopausalwomen wit hphysiologicaldoses for 3 weeks in aprospective randomized double-blind cross-over study, S y a 2 week w a s h o ~ period, t led to the modulation of immune function. There was a decrease in C and increase in C a1 killer) cells. T cell mitogenic and IL-6 responses inhi were cell cytotoxicityincreased was dramatically EA levels decrease during chron ness. ~ r e a t m e n of t huma~s alsosuppressesplasma levels ofEA-sulfate,androstendiol, and testosterone. These hormonal alterations may be corrected by AC [259]. Levels of D EA-sulfate are depressed in postmenopausal women with rheumatoid arthritis [272].

uman progesterone receptors have two isoforms: the 120 k and the endterminally truncated 94 k reside in the cytoplasm, where they ted with heat shoc of 70 and90kDa molecular weight binding leads tothe dissoc S 70 and 90 from the receptors; they becomephos~horylated and dimerized and

bind to inthe nucleus. Threedimeric species, namely A/A, A/ bind to progesteroneresponseelements ( ) and regulatetranscripti dimerization is necessary for binding to but is notsufficient to ormone. In pure heterodimers A receptors are dominant receptors [273,274]. an important role in the harmonization of immune function with reproduction. Its overall effect on systemic immunity is suppressive. cent studies in infertile women treated with the fertility drug clomiphene citrate revealed that only where theimmunosuppressiveeffect was present did women conceive, whereas W nwho experienced no immunosuppressiveeffectdidnot achieve ~regnancy. esterone levels were significantlydifferent in these two groups of women,asthoseofothersteroids were notdifferent [275]. In women suffering from primary habitual abortions theincidence of pregnancy after ' unotherapycould be increased with additionaltreatment with progesterone , suggesting a synergistic effect [276]. rogesterone is known to decrease host resistance against viral and fungal infections [277,278]. In vitro lymphocyte proliferation in responseto Candida a cans was reduced by approximately 5 0 ~ in 0 the presence of luteal phase levels of , as opposed to proliferative phase levels (0.15 ng/ml). This inhibitory ted when monocytes were removed from the culture [279]. concentration suppressed the anti-Candida activityof neutrophils from mice, whereas testosterone, estradiol, and estriol did not [280]. Progesterone significantly inhibited nitriterelease from rat macrophages and stimulat release of reactive oxygen intermediates, with the exception of 10"' and concentrations which had no effect [212]. "2 ng/ml) stimulated the release of TNF from rat peritoneal ripheral blood mononuclear cells from healthy donors stimuless IL-1 on exposure to PS, whereas IL-6 secretion was failed to inhibit IL-1 secretion of peripheral blood monom male donors with rheumatoid arthritis [281]. Progesterone deficiency is present infemalepatientssuffering from thyroid and ovarian autoimmune disease and from rheumatoid arthritis [282]. The Severe premenstrual exacerbationofasthma was reportedinthreepatientswhodidnotrespond to treatment with high-dose glucocorticoids, but intramuscular (i.m.) treatment with PS eliminated the premenstrual dips in peak airflow and led to the reduction of daily doses of prednisone [283]. The secretion of IgA by mucous membranes and exocrine glands is regulated by sex hormones. Thus, testosterone promotes the secretion of IgA and the synthesis of secretory component (SG) in the lacrimal gland; estradiol, progesterone, and prolactin stimulate the synthesis of IgA and SC in the mammary gland,where testosterone is inhibitory. Estradiol stimulates SC production and Ig secretion in the uterus, sterone promotes, however, cervical which is antagonized by progesterone. IgA and SC progesterone estradiol and prod seem ,to inhibit Ig secretio nd progesterone-treated ewes inhibited the and mouse. The injection of these proteins

to mice reduced NK activity and prevented the increase in fetal loss caused by the injection of poly(1) poly(C) [285]. Progesterone treatment of ewes (100 mg/day i.m.), particularly after60 days, causeda reduction in the number of CD45' cells in e glandular epit~elium and associated subepithelial stroma and MHC class I1 positive cells in all regions of the intercaruncular endometrium.These results indicate that progesteroneregulates the migration or proliferationof endometrial lymphocyte popul~tions[286].

The precursor of 3, cholecalciferol, is taken up in diet or synthesized in the skin under the influence of ultraviolet (UV) radiation, 25- ydroxyvitamin D3 is generated in the liver and is further processed by l-hydroxylase in the kidney. This latter enzyme is also present in monocytes/macrophages, keratinocytes, bone marrow, placenta, glial cells, and pneumocytes. The vitamin D receptor (VDR) is of 50 kDa molecular weight and is a member of the superfamily of steroi receptors. It interacts with vitamin D-responsive elements (V also with the retinoic S receptor. 0th ositive (stimulation) and negative (inhibissible through VDVitamin D regulates theDNA binding oteinkinaseC is olved in receptor-mediated signaling by mune system, monocytes/ agent and prodifferentiation mediator for macrophages, lymphocytes, a cyte/macro~hageadherence, chemotaxis, p ~ a g o c ~ o s icytotoxi s, radical, and heat shock protein production are all hand, antigen presenta production of IL-l, IL-6, IL-12, and TNFa are ' * ited. The function S is also inhibited and the pro~uctionof IL-2, -y, andTNF is dec 3. Suppressor T cell fun~tionis also inhibited, whereas the effect of otoxiq lymphocytes T is not clear. Natural killer cell cytotoxicity is stimulated. B lymphocyte proliferation and im~unoglobulinsecretion are inhibited[287,288,290]. deficiency in mice suppressed the induction of contact sensitivity reaction to dinitrofluorobenzene. The ingestion of a vitamin -sufficient diet for 8 weeks corrected this immunodeficiency [291]. Tuberculin sk reactivity developed more slowly in V 3-deficient guinea pigs immunized with purified protein derivative, buteventuallyachieved normal levels. The proliferation ofsplenocytes in response to the antigenwas suppressed by both deficient and excess dietary vitamin D3. everth he less, vitaminstatusdidnotaffecttheability of naiveanimals to control primary pulmonary tuberculosis or to develop protective immunity after bacillus Calmette-Guerin (BCG) vaccination[292]. Lymph node cells ofmiceexposed in situ to V 3 produced less IL-2 and IFN-y andfarmore IL-4, IL-5, and XL 0 than did lymp~ocytes from control animals. When vaccination with hepatitis surface antigen was coupled with VD3 treatment, both systemic and common mucosal immune responses were induced. The humoral and mucosal immune responses were augmented if added to the immunizationregime as an immunomodulator[293]. treatment prevents experimental autoimmune disease, including nephritis, experimental allergic encephalitis, diabetes, thyroiditis, and systemic lu-

pus erythematosus. If therapy is attempted, however, the disease will only be ded. In humans, the treatment of psoriasis with ever, the natural vitamin is toxic, and curren structural analogues withmuchrd toxicity are employed. Combination of the 6 , orrapamycinimprovesthe efficiency of treatment withcyclosporine A, treatment.Insarcoidosistheh lcemia andsofttissuegranulomatosisand calcification are due to the extrarenal production of V

Immune cells express receptors for cantlyincreased theproliferative res Inneonatallythymectomi~ed

ofspleen cells to Con A [296]. ) mice, the developmentofintestinalin-

tigen receptors (TC

Thyroid stimulating hormon cells and on human T and hormone moderately increased immunoglobulin secretion by activat ffect on lymphocyte proliferati -activated h ~ m a nblood lymphoc~es[298], enhanced the proliferati~eresponse of murine spleen cells to IL-2, and significantly increased IL-2-induc cytotoxicity [301]. Thyroid stimulating hormone enhanced the express C-11 surface molecules by human thyroid epithelial cells [302]. The stimulation of nocytes h in vitro wit by the administratio zed the p s u ~ u n iot egulated by th~romimetic compounds [304].

L y m p ~ o c ~and e s monocytes express nuclear receptorsfor both T3 and T4 lymphocytes convert 154 to T3, which is biologically active. Triiodothyroni encedsodiumexchangeandglucoseuptake in lymphocytes,stimulatedthymus owth and hormone production, and promoted erythroid burst forming clones and cell maturation in the bone marrow. The role of thyroid hormonesin immunoregulation remains controversial. iverse effects were reported repeatedly in various species on mitogen-induced lymp~ocyteproliferation, antibody formation to various antigens, and various forms of cell-mediated immunity, including graft rejec-

tion, delayed-type hypersensitivity reaction, created thyroid deficiency was usually, but n ciency, which could be restored by treatmen immunodeficiency. associated with alsowas of normal animals with T3yielded mostly n Lytic activity by splenic diet but was normal if thediet

ntal t r e a t ~ e n t

uman peripheral blood mononuclearcells expres autologous mixed lymphocyte culture was enhanc stimulated the productionof prostaglandin E2 by human and the production of endorphin by human peripheral nonuclear cells. It is an antipyretic hormone andis capable of attenuating fever after central administration [18,313,314]. Argininevasopressinwasdetected in rat thymocytes and splenic l y m p h o c ~ e s[3 1 5,3 16J.

In primitive vertebrates the pineal gland is a direct photosensory organ withs i ~ i l a r structure to the eye. During evolution the light regressed and in mammals the secretes the hormone melatonin. derived from serotoninby ~-acetyltransferase. The pineal plays an importantrole in the regulation of seasonal breedingin various vertebratespecies and in the circadian rhythm of vertebrate physiological processes. It in~uencesthermal regulation9electrolyte metabolism, hemopoiesis, immune reactions, an tion is suppressed by light and increased in darkness. involved in visible light-induced neuroendocri hormones e that show majorcircadianrhythmaregrowthhormone,thyroidhormones,prolactin, on theretina is conducted andplasmacortisol. A small part of light enering to the suprachiasmatic nucleus (SCN), which is located in the hy~othalamus. The SCN is thought to direct the circadian rhythm of the body and a1 events, such as temperature, reproductive cycle, appetite, and mood. amus is directly connectedwiththe pineal andpituitaryglandst pathways.Through these pathways visible light falling ontoretina induces the production ofneurotransmittersandneuropeptides in the S pituitary,and pineal gland [317,318]. ~ i t h i the n immunesystem melatoninrece~torshave been d e ~ o ~ s t r a t e pleen cells, in CD4' T cells, and in the bursa of Fabricius i latonin receptors in primary lymphoid organs, but not those in the spleen, demonstrated diurnal variationin density. organs may be coupled to G proteins 13221. In cytotoxicity, the secretion of IL-l and the produc ates. It exerteda synergistic effect with L

monocyte-associated IL-la andIL-16 activities with an activation thresholdof elatoninenhancedantigenpresentation by murine enic macrophages hich was concomitant with increased oppression o cules and enhanced production of IL-1 and TNFa [324]. A er T cell. At physiological concentrations ne marrow IL-4 induced the release , from stromal cells [320]. man peripheral blood mononuclear in 22% of cases [325]. dependent cellular cytotoxicityin in winter. This seasonal effect is due toa difference e course of the year [326]. age. Melatonin treatment of old mice or pineal graft placed into the thymus increased thymus weight and cellularity; that effect was associated with the recovery of peripheral blood lymphocyte numberand spleen cell subsets and increased mitogen responsiveness. Zinc turnover was also restored in aged mice to a rate similar to that of young mice. Zinc plasma levels re the reduced immunologand zinc supplementation is able to is due, at least in part, to mmunorestoration of old mice by the correction of zinc metabolism [327].

Nerve growth factor was discovered in murine submandibular glands as a growth factorfor sensory and sympatheticganglia [328]. Low-affinity panneurotropic y receptors, thyrosine kinases, desigimarily for brain-derived neurotropic pin 3, are distinguished. These recepthe tumor necrosis factor receptor superfamily and also include the S on lymphoid tissue. trkA are expressed in the thymus and in spleen and are localized primarily to the stromaof these or ans. Some expression was also found, however, in splenocytes and thymocytes. phocytes and antigen presenting cells (follicular dendritic cells) also express receptors [329-3331. Nerve growth factor has a mitogenic effect on human lymphoblastoid cell lines which is associatedthyrosinephosphorylation and activation of mitogenactivatedproteinkinase 1. Itpromotedproliferationin mixed lymphocyte cultures [335], response of leen cells to T and cell mitogens [336], and growth phocytes 13371. The production of IgM and IgC4 by 1337,3381; it inhibited the induction. of IgE by IL-4 od mononuclear cells. On the other hand IL-4 prevented increaseinIgA and I production [339]. ratperitonealmastc synthesize biologi factor treatment increased the number and size of mast stimulated histamine release from mast cells, both in vivo stimulated the development of hemopoietic colonies and the chemotactic and phagocytic activity of neutrophilic leukocytes [347,348]. The stim~latory effect of NGF on mast cells and neutrophilic leukocytes sug-

7

Y

gests a proinflammatory role. Yet the suppression of inflammation has been observed in vivo by NCF in several experimental models[ 3 ~ ~ , 3 5 0 ]he . reason forthis paradox is not well understood. Itis possible that in vivo NCF activates the ACT adrenal axis through the stimulationof cytokines.

lymphocytes,macrophages,granulocytes,andmast cells laminereceptors,whereasplatelets have a-type receptors [181. adrenergic agents inhibit allergicand asthmatic reactions [35 1,3521.In general, catecholamines inhibit various immune phenomena, including lymphocyteresponses to mitogens and antigen, histamine release from ~eukocytes and mast cells, and skin reaction to antigen orhistamine. at echo la mines have a variable effecton antibody pro~uction,theyinhibitneutrophilactivation by for block the stimulatory effect of IFN-y on macropha~ecytotoxicit teredsystemically,adrenalineelicitsleukocytosis and eosinoph lowed by eosinopenia. These effects on l e u ~ o c y part, to glucocorticoid release through the ACT ~ i b i t smast cell degranu~ation and histamine release from leukocytes. It has a variable effect on antibody formation[353-3561.

Acetylcholine receptors of the muscarinic and nicotinic types have been demonstrated on lymphoidcells. In general, choliner~icagents enhance immune includelymphocytemito~enesis,c oxic reactions, and relea othermediatorsfrommast cells scarinicreceptorsmediatethese effects. Acetylcholine or carbamylcholine stimulated th com~onentsby human m o n o c ~ e sthrough the nicotinic show an increased sensitivity to cholinergic stimulatio are involved in exercise- induce^ anaphylaxis [l 8,3

Substance P receptors are expresse and T lymphoc~es,macrophages, mast cells, and astrocytes. Substance P plays a major role in the mediation of neuro~enic inflammation and is capable of inducing mast cell degranulation, plasma extravasation, and bronchoconstriction. Uterine mast cells of rats did not release ~istamine or cytokines in responseto substance P at proestrus oron day 4 of pregnancy, but preincubationof these cells with SP signi€icantly enhanced~ e d i a t o r release in response to anti-IgE antibodies. In contrast, during thediestrus phase S amine and cytokinerelease from purified mast cells [360]. also acts directlyon lymphocytes, macrophages, and neutrophils. ~ymphocyteproliferationandlymphokineproduction whereas the effect on immunog~obulinsecretion is v a r i a ~ l E receptors and decreased C3b on eosinophils. ~ubstanc

macrophages and modifies macrophage function during stress [361-3631. In polymorphonuclear leukocytes stimulated the respiratory burst and chemotactic and phagocyticactivities [3643. It stimulated t&e release of PGE2 and collagenase thromboxane rheumatoid and from 2 from astrocytes [370]. Substance F secretionmarrow by bone secretion was ulation pa IL-6 of andIL-l by marrow cells [371]. Substance P is capable of activating platelets for cytotoxicity n i [372]. against ~ c ~ i s ~ o ~s ao n~~aolarvae

hocytes, macrophages, and bone marrow cells. Calcitonin gene-related peptide receptors are functionally coupled to adenylate cyclase [373,374]. Calcitonin gene-related peptide inhibited the proliferative responseof murine thymuscells to ConA. ~ihydroepiandrosteronealso had an inhibitory effect, which couldbe prevented by treatment with the CGRP antagonist CGRP-[80-37] [262]. Calcitonin gene-related peptide can trigger mastcell discharge and induce slow-onsetintense erythema and vasodilatation in the skin[365]. It has an inhibitory effect on immune phenomena, including interference with antigen nhibition of lymphocyte proliferation, IL-2 production, and interferNAsynthesis for TNFcx, TNF-P, and IFN-y. Interferon-y induced 202 production by human mononuclear phagocytes is also suppressed by CG [375-3771. Calcitonin gene-related peptide-containing nerve fibers were shown to be assoangerhans cells in the human epidermis. In three different functional d antigen presentation by human skin Langerhans cells [378]. P inhibited the induction of cutaneous hypersensitivity reaction to haptensin mice [379]. Topically applied CGRP increased cutaneous inflammation toallergens and irritants and also boosted the sensitization process [380].

Somatostatin receptors are present on T and lymphocytes and mast cells possess receptors for SO It inhi~itstheIgE-dependentmediator release fromhuman basophilsandmast cells. Lymphocyteproliferation,enn-inducedleukocyto, and it hasavariable sis,IgAsecretion, and 1FN.y production are inhibited b effect onantibody-dependentcytotoxicity.Somatostatinalsoregulatesmacrophages [364,365,381-3831.

onocytes and lymphocytes express VIP receptors. Vasoactive intestinal peptide protects murine and human thymocytes against the cytolytic effectof prednisolone [384]. It plays a role in the regulation of T cell homing to gut-associated lymphoid tissue. Vasoactiveintestinalpeptidestimulatedmacrophagechemotaxis through protein kinase C activation and inhibited lymphocyte chemotaxis through activation of adenylate cyclase [385]. It enhanced phagocytosis by mouse peritoneal macrophages in a dose-dependent manner [386]. It inhibits lymphocyte mitogenesis and

has a variable effect on immunoglobulin secretion and N cell activity [3$7,3$$1. The addition of VIP to cultures of human colonic lamina pria mononuclear cells increased IgA production andsignificantly reduc peptide induced IgA production by isotype SW production of IL-2 and IL-4 by murine thymoc was found in rat thymus, spleen, and lymph nodes in both lymph~idand nonlymphoid cells E3911 In conjunction with acontributes to the immunosuppressive properties of aqueous humor in the eye. Antigen presentation in the eye is deviated towardantibodyproduction, whereascell-mediated immune respons pressed. Substance P acted in the aqueous humor as an antagonist Vof or TNFa induced VI release into the aqueous h the reexposureof dark-reared mice to light [181,

Growth hormone, PRL, and possibly also placental lactogenic hormones during embryonic development are required for the normal ~ e v e l o ~ m e and n t function of theimmune system.Lymphocytesdeprivedofgrowth and lactogenic hormone ) are incapable of proliferation and thus are unable to mount an immune response. Therefore, these hormones have been termed competence hormones for the immune system [393]. Even though it has been shown recently that lymphoid ypox animals lose t cells are capable of producing P petence, indicating that pituitary the maintenanceof the immune system. Immunocompetent lymphocytes respond to antigen with cell proliferation. The antigen needs to be presented by specialized antigen presenting cells ( that display partially digested (processed) antigen in association with the histocompatibility type I and 11 antigens on their surface to C .t he1 er T lymphocytes and also to lymp T S of the killer/suppressor subset recognizing antigen in thecontextof -I molecules). The antigen recepto lymphocytes C antigens, belongs to the immuno(immunoglobulin) and of T cells, as well as globulin family of adhesion molecules. Inaddition to antigenrecog latory signals mediated by additional adhesion molecules between phocytes and betweenT and lymphocytes are required fortheinduction of lymphocyte proliferation. Integrins and selectins represent two addition of adhesion molecules, and some of them also appear on lymphocytes. signals rank second in lymphocyte activation. Thesesignals also govern lymphocyte recirculation, homing to tissues and organs, and response to in~ammatorystimuli [393]. The third categoryof lymphocyte activating signals is locally acting hormone molecules, called cytokines. Cytokine signals are required for the completionof the cell cycle. They play a role in differentiation, functional activation and regulation, survival, and even activation of thesuicide p a t h ~ a y Immune-derive .

serve as regulatory signals towar the endocrine

system and toward other tissues

hat the activation of the growth hormone receptor requires rization) by a single hormone molecule ( ~ i g u r e1) makes it mes its own antagonist at high concentrations, as discussed and to somecytokines and hemopoietic growth ing to the same family. Indeed, it is well recogose res onse to P L constitutes a bell-shaped curve. The S best observed in hormone-deprived animals C1-treated), whereas PRL exerts little, if any, effect on immune f ~ n c t i o nif administered to normal animals. Evidence that P influences immune reacti rding to bell-shaped a dose-response fromtheobservations of o et al. [S21 on antibodyformation, the mitogenic response of T cells to IL-2, and activation of lymphokine-activated 11s was stimulated by high levels of at the inhibitory effect of hyperprolactinemia on immune

he evidencereviewed indicat~s that the hypQthalamus-pituitary-adrenal axis exerts a powerful suppressive effect on immune and inflammatory reactions. Corticotro-

nizes the immunostimulatory effects of ng cells in the pituervations show that

also in the periphery. a-

o implicated in hapten-specific tolerare the most important immuno-

te theexpression ofthe IL-2 receptor and the expression of

cytes by IFN-v. Through these effects, glucocorticoids promote general leukocyte activity, which is an important featureof the acute-phase response. are alsonecessary, in additionto IL-6, for the production of acute-phase proteins in the liver during febrile illness. ones were catego ecade ago as immunomodulators [181. ~ l t h o u g hthe immune systemis capable of functioning without the presence ofsex steroids, these hormones are necessary for normal immune function. Sex steroids have the capacity to influence certain immune functions selectively and to modulate immune reactivity primarily in the interest of reprod Sex hormones also ern, to a large extent, mucosal immune function. In cells and cell-mediated immunity but promote hum androsterone boosts both cell-mediated and humoral i testosterone function as immunosuppr~ssive ~ormones. the inductionof immunological tolerancetoward the promotes differentiation, enhances N activity, and suppresses immune reactions. Several steroid hormones are converted to biologically active metabolites by immune cells, allowing for local immunoregulatory function, as required. Thus, testosterone is converted to dehydrotestosterone and also to estradiol; 7-a-hydroxy derivatives; -a-hydroxy derivativ androstenetriol; 25-hy 3 is metabolized to l Sex hormones are in interaction with luteinizin~ ho follicleand stimulating L ho exert immunoregulatory effects also through the modification of the serum level of these hormones. Evide~ceis increasing that the hypothalamus-pituitary-thyroid axis important rolein immune function. Thyroid stimulating hormone (TS tropin releasing hormone (TR ) have a direct immunomodulatory effect, and thyroid hormones are required for immunocompetence. The mechanism of action of thyroid ~ormones on immunocytes is not fully understood at thepresent time.

m

europ peptides are secreted by nerve terminals in tissues and are also produce lymphoi~cells. They are important local regulators o reactions aswell as neuralactivity in thetissues. Local peptides has both physiological and pathological si~nificance. ological function is the control of immune reactions in the anterior chamberof the eye b erful regulatory effect on inflammatory reactions. S , are released from sensory ~roinflammatoryneuropeptides, such as C nerve fibers in responseto a variety of irritants. These mediators induce mastcell discharge, increase the erm me ability of blood vessels, and also act on smooth muscle elements; resulting in neurogenicinflammation.Neuraleffectorjunctionsare formed between mast cells and C fibers for c e regulatory interaction. ~eurogenic inflammation may be regarded as an instant st defence mechanism in response to potentially noxious agents, which can be activated rapidly and does not require

specific immune products to initiate. owever, neurogenic inflammation may contribute to the pathogenesis of numerous immune diseases, which include allergy, rhinitis, arthritis, and gastrointestinalhypersensitivity [365,394,395]. ucosal surfaces are constantly bombarded with antigen, infectious agents, toxins, many of which have the capacity to stimulate the immune r to elicit inflammation. An exaggerated mucosal response to a mild irritant (e.g., asthma) is just as dangerous as an inadequate response to a pathogenic agent. Therefore, the immune/inflammatory response must strictly be controlled on mucosal surfaces at a level which assures adequate responses against pathogenic agentsanddetoxificationandclearance of harmfulnts by inflammatory cells, inflammatory response must not be excessive. ause mucosal surfaces fall the body, the capacity of the immune system t tinguish self from nonself is virtually useless in thissituation.e suggestedearlier thatimmunoregulatory substances produced by salivary glands, especially in the submandi~ulargland in laboratory rodents, are fundamental to immunoregulation in the gastrointestinal f these substa~ces,nerve growth factor, transforming growth factor-@, epidermal growth factor, and glandular kallikrien are most important. The sympatheticnervoussystem regulates the secretion of these substances.Throughthis neuroim~u~oregulatory function the submandibular gland is suggested to play a key role in the regulation of mucosal immune and inflammatory responses [396]. This hypothesis is supported by clinical observations that the failure of the major salivary glands (Sjogrendisease) in humans is frequently associated with rheumatoid ,indicating a systemic disturbance of immunoregulation[396]. any neuropeptides and neurotransmitters, including opioids, substance P, VIP, and catec~olamines,have variable effects on immune reactions. mediators &ay be classified as immunomodulatory agents with the capacity of increasing or suppressingimmune reactions. Althoughmanyquestionsremain unanswered regarding the immunoregulatory function of these mediators, it seems clear that they are designed to act locally in specific areas of the body and are capable of delivering opposite signals to the immune system, such as adrenergic versus cholinergic stimuli,SP,CG versus somatostatin,orstimulatory versus inhi~itoryopioid signals, which may be dependenton receptor class. Many of these agents function assignal modulators having receptors coupled with G proteins instance, catecholamines regulate cyclic 3 ' ,S '-adenosine monophosphate ( and C a + + influx.Theretwouldbeexpectedthattarget cells with low cytoplasmic Ca+ or cycliclevel wouldreactstrongly to stimulationwiththe respective catecholamin reas cells with a maximum level of these signalmodulating factors may at all to such stimulation. The observation that would selectively stimulate the development of intestinalTlymphocytes es the possibility thatcertainneuropeptideswould selective ate lymphocytes in a given compartment, allowing for organ-tissuespecific Another on. example is immunoregulation in the eye by glucocorticoids, a, SP,andCGRP. +

ost peptide hormones and immunoregulatory neuropeptides are produced within the immune system, as pointed out repeatedly in this chapter. For a number of these regulatory peptides, good evidence exists that they are identical with their

counterpart produced in the endocrine or nervous system, includingbiological activity. These regulatory molecules fulfilllocal autocrine/paracrine immunoregulatory functions and also act as messengers between nerve terminals an tissues. They are produced at low conc is reason do not exert a systemic effect. conclusion This isobservations sup that pituitary function is essential for the maintenance of immunocompetence as well as by some deliberate experiments designedto elucidate the function of immune derived neuropeptides [l 5 1,3971.

annemacherandcoworkers 13981 discovered theleukocyteendogenousor fever, whichwas thefirstindicationthatimmune-derivedfeedback sign he central nervous system exist. for the existence of immune d n for a long time ring the early 1980s the leukocyte endogenous i n t e r l e ~ ~ i n -From l. these factsitwaspredictedthatIL-lfunctionsas derivedsignal to the pituitarygland 1181. S number of laboratories that, indeed, IL-lis ca the secretion of a number of other pituitar voluminous literature has accumulated on th inflammatory cytokines on pituitary hormone secretion [l 8,139,140,4 Although many of the findings remain controversial and much remainsto be elucidated, it is quite clear that cytokines (e.g., IL-l, IL-6, T N F ~ alter ) pituitary hormone secretion during systemic immune and inflammatory reactions. For instance, during acute-phase responses levels of glucocorticoids, catecholaon, arginine vasopressin, and a1 PRL, estrogens, androgens, ins either elevated or suppressed, depending on the sever condition e [355]. Interleukin-6 activates the genes of acute-phase protein the DNA binding protein named NF-IL-6. Some other leukemiainhibitoryfactor, TGFP, oncostatin , and ciliary neurotropicfactor, also increase acute-phase protein pr liver. Catecholamines and glu corticoidscaninduce at least someandgreatlyamplifytheinduction ofA by IL-6. Acute-phase proteins are also producedin the placenta and in the choroid plexus [355]. During acute-phase reactionsnew proteins (acute-phase reactants) synthesized in the liver rise to extremelyhigh levels in the serum at the e of other serum proteins.Someof these proteins,suchasC reactive proteinandendotoxin binding protein (EBP), represent ancient defense molecules capable of recognizing surface moieties that commonly occur on pathogenic microbes. After combining with their specific ligand, they activate humoral and cell-mediated immune defense reactions of the host nonspecifically. Other acute-phase proteins influence blood clotting (fibrinogen),exert an antiinflammatory effect,or function asenzyme inhibitors. All ofthese functions arelikely to be essential for thesurvival of the host. The specific immuneresponse is profoundly suppressed andcytokineproduction is

tightly controlled, rimarily by the PA axis, during acute-phase reactions. Acutephase reaction (A ) is characterized by catabolism, with the exception of bone marrow and leukocyte metabolism, which is greatly enhanced. One may suggest that the acute-phase response is an emergency reaction to fight infectious disease, and other harmful insults, in cases where the specific immune response failed to control the situation. Therefore, specific immune responses are switched off and the body resorts to more ancient responses which are less specific but can be mounted within 24-48 hr and provide a wide spectrum of defense against numerous microbes and other harmfulagents [355].

~ e u r o i m m u n emechanisms are involved in normal physiological processes, which include tissue turnover, involution and atrophy, intestinal function, regulation of endocrine glands, and reproduction. These mechanisms are also fundamental to host defense against infection, sepsis,trauma, and shock. The number of pathological conditions where neuroimmune abnormalities have been detected is increasing steadily and includes abnormalaging, acquiredimmunodeficiency,allergy and asthma, anemia, ~ l z h e i m e rdisease, autoimmune disease, cancer, chronic inflammatory disease, connective tissue disease, gastrointestinal disease, endotoxin shock, toxic shock syndrome, fatigue, infectious disease, kidney and liver failure, pain, psychological disorder, regeneration and healing, rheumatoid disease, fever, and recovery from disease [7,18,25,402-411].

A detailed review of therapeutic experiments in animals and therapeutic trials in humans with hormones, neurotransmitters, and neuropeptides and their agonists and antagonists is beyond the scope of this chapter. owever, by now it should be clear to the reader that the neuroendocrine system the highest regulator of immune reactions and has the capacity to regulate immunocompetence; to increase, suppress, and balance immune reactions; to promoteselectively either cell-mediated or humoral immunity; to initiate immunoglobulin class switch;to induce or abolish immunological tolerance; to maintain immunological responsiveness in privileged sites; and tocorrect the age-related declineof immune function. Not so long ago all ts were attributed to the internal regulatory pathways of the immune wever, the internal regulatoryn e t w o r ~of the immunesystem is subjected to neuroendocrine regulation which has the power to set thresholds to immune reactions and to alter immune and inflammatory reactivity profoundly in order to maintainhomeostasiswithin the body. On this basis, it should be obvious that hormones, neurotransmitters, neurop~ptides, andtheir analogues do and will have major application in medicine. Thus, glucocorticoids have been indispensable for the treatment of immune and inflammatory diseases for several decades. The search is on for better and moreefficient compounds with less severe side effects, criptine, which suppresses serum PRL levels, is currently being tested in the clinics

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for the treatment of systemic lupus erythematosus, rheumatoid arthritis, and some other inflammatory conditions. Opioid receptor agonists and antagonists arebeing studied at present in acute-phase-type responses, where someof them show activity in the preventionoflethaloutcome. a-M 1-13] hasproved to bean effective antiinflammatoryagent.Thetriphenylethyantiestrogenicagentstamoxifenand toremifene were shown in our laboratory to amplify target cell destruction by natural killer, lymphokine-activated killer, and cytotoxic T lymphocytes. These agents are effectivein the potentiation of immunotherapy of murine tumors by killer cells. Androgens and antiandrog ave been tested in various situations, including autoimmunedisease.AtpresentA is being tested forthe nationandcorrection ofage-re mmune decline.Vitamin 100% efficiencyfor the therapyof psoriasis. This finding may indicate that psoriasis is a disease of abnormal vitamin D metabolism in the skin. Analogues of VD3 are also effective in the prevention of a number of autoimmune conditions in animals. Melatonin is being testedin patients for cancer immunotherapy and also for boosting of immunity. Beta-adrenergic agents have been used for a long time for the suppression of asthmatic and allergic symptoms. Substance P antagonists are effective in the experimental treatment of gastroenteritis; somatostatin analo~uesmay also have an antiinflammatory effect.

Theimmune system is regulated by theneuroendocrine system. growth hormone are required for the normal development and immune system and serve as the hormones of immunocompetence. The hypothalamus-pituitary-adrenal PA) axis,whichincludescorticotropinreleasing factor produced by hypothalamic neurons; adrenocorticotropic hormone, a-melanocyte stimulatinghormone,and endorphin producedand released by thepituitary gland; and glucocorticoids secreted by the adrenal cortex, performs immunosuppressive, antiin~ammatory, andantipyretic functions. This axis is fundamental to normal immune function andto recovery from febrile illness. The major role in the metabolic conversion during acute-phase react coids and catecholamines potentiate the inductionby cytokines (e. phase proteins in the liver during febrile illness. Sex hormones have thecapacity to stimulate, suppress, orselectively modify immune reactions and to re~ulatemucosal immune function. Sex hormones coordinate immune function with reproduction. Thyroid hormones are necessary for normal immune function, but the mechanism of action of thyroxinon immune phenomena has not been elucidated in full detail. Neuropeptides which may be secreted by nerveterminalsin the tissues but are also released by cells of the immune system, have local regulatory functions of physiolo~ical andpathophysiological significance. They regulate immune, inflammatory, and neuralactivity in the tissues. The centralnervous system receives feedback signals from the immune system via c ines and nerve impulses, thisregulatory loopmay lead to disease.tipleendocrine abnorm been detected in a number of diseases with underlying immune/inflammatory conditions. The pharmacologica~ correction of endocrine abnormalities in immune/in-

flammatory diseases holds promiseof more effective management and even cure in some cases (e.g., psoriasis).

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~ a t ~ o nInstitute al of Dental Research, ~ a t ~ o nInstitutes al of Health, Bethesda,~ a r y l a n d

Unraveling the complex mechanisms whereby immune cells modulate the host response to injury, inflammation, and/or infection has revealed the involvement of small protein molecules, released by activated cells, which function as chemical messengers. These messenger molecules, called cytokines and/or growth factors, provide an essential means of communication between cells within the body and mediate cellular, physiological, and immunological processes that arecritical to the host response to injury or antigenic insult. The balanceof these activities is critical not only to maintain normal homeostasis but also to allow for a controlled biologiof cytokine expression cal response that is not injurious to the host. regulation is often observed in disease and may provide sis for therapeutic intervention in certain situations. Transforming growth factor-beta (TGF-@) is such a cytokine/ growth factor that has a wide range of biolo a1 activities and has been implicated in immune and in~ammatorydisorders [l]. anipulation of TGF-@expression in these disorders may provide insight into its mechanism of action as well as its therapeutic usefulness.

-P Of the growth factors discovered to date, one that has been extensively studied for its wide spectrum of actions is TG -p. Originally defined as a factor that induced a transformed phenotype in mesenchymal cells, allowing for anchorage-independent growth [2], TGF-@exhibits multiple, often opposing, activities in the regulation of immune function. It is instrumental in both the initiation and the resolution of inflammatory events [ 1,3,4]. Transforming growth factor-@ is a homodimeric protein of which there are five known isoforms. Of these, only TGF-@l,-62, and -p3 have been emo on st rated in mammalian tissues. The TCF-@S belong to a su structurally related regulatory proteins that include activins, inhibins, hibiting substance,

l

and the bone morphogenetic proteins [ 5 ] . by unique genes on separate chromosomes9 acid sequence homology. E ~ ~ r e s s i oand n activity of lated at transcriptional and translational levels, invol moter interactions9 message"stability,processi biolo~icalactivities of these isoforms are nearl and mechanisms of synthesis and in vivo local three isoforms bind to the same receptors a them to some degree interchangeabl gest that the individual isoforms are

amino acids with

, which is transcribe

-P ligand, where TO on the cell surfa kinases at multip lating activity is not regulated by ligand S TGF-P bound to 11 butnotfreein the medium orboundtomembraneproteins [ 151. The table complex wit which propagates the signal to substrates downstream of ex, ~onsistentwith this model, the nature of the biologiily by the type I receptor isoform that is that either lack the receptor or have a S has been observed in some tumor cell effects of TGF-P, which could provide receptors, the TGF-P type 111 receptor nsmembrane $53-amino-acid proteoglying both heparan sulfate and chondroiane segment and a ~~-amino-acid cytoines [16]. Thetransmembraneand ave significant homology to endoglin, othelial cells and mesangium of the TGF-P isoforms with equal affinity, GF-P2 [ 17,181. Thus, the purported or of ligand access to the signaling receptors [19], since it is not directly involved in signal transduction.

-P

TGF-P can have both inhibitory ndsti and withoutcome the most likely dependent status ation and tor expression [3].

T lymphocytes, of

~

iferate and undergo immunoglob11s expressing cytoplasmicp heavy er successfully rearranging their oncert with light ch making an antigen binding complex. After antigenic stimulation, matur switchheavychainisoty andsubsequentlydifferentiateintomemory cells or Ig-secreting plasma cells. ,potentially such as autoreactive clones, are selectively eliminated cell maturation [20], and TGF-P is likely a key mediator in the deletio information is available abo

has been localized in most cells within the bone marrow, including those of B cell lineage, suggestingan as yet unelucidated role@) in this process. ore em~hasishas been placed on characterizing the impact of TGF-/3 on later stages in tion, particularly in the regulation of Ig synthesis [21]. At high concentrations( 20.1 ng/ml), TGF-01 inhibits productionof secreted Ig of all classes,and this effect couldbe mostly dueto inhibition of light chain gene transcription. In contrast, atlow concentrations, TCF-P works as a costimulator to of class switching induce IgG secretion in vitro. TGF-P also increases the frequency to IgA [21]. Peripheral blood Bcells from patients with IgA deficiency (an autoimmune disorder) do not express Iga messenger ribonucleic acid (mRNA) in vivo, but are capable of generatinga germline transcripts onin vitro stimulation with active TGF-/3 [22]. Although B cells from these patients express TGF-P m ~ N Aa, defect in activation of latent TCF-/3 hasbeen suggested t o contribute to thedefective regulatory controlof these cells. In addition, TGF-(31 is a potent inhibitor of lymphocyte proliferation,effectively blocking progression of the cell cycle in the mid-G, phase. Not only does TCF-/3 reduce c-myc mRNA levels; it also appears to block the phosphorylation of Rb, an anti-oncogene that regulates cell cycle progression and is differentially phosphorylated during thecell cycle 1231. ~ypophosphorylated Rb reversibly arrests cell cycle progression in G,. In addition, TGF-Preduces the levels of E2F mRNA, a downstream transcription factor for Rb [2 t can inhibit proliferation via inactiassociation,such as cyclin E/CDK2 vation of cyclin/cyclindependentkinase ( via the inhibitory protein ~ 2 7 ~ [25,26] ' ~ ' and cyclin D/CDK4 via the inhibitor ~ 1 5 ' 1271. " ~ ~ In ~addition toinhibiting cell growth, TGF-P1 can promoteapoptosis, particularly in resting I3 lymphocytes [28], which also could provide a mechanism for deletion of sei€-reactive clones at all stagesof I3 lymphocyte maturation.

While TGF-/3 was originally shown to inhibit both T cell growth [29] and generation of cytotoxic T cells [30], TGF-Pl also supports the generationof effector T cells in both human and murinesystems [31-331. TGF-/3 can regulate the cell cycle progression and differentiationof murine CD4-/CD81° precursor T cells into CD4+/CD8' t h y m o c ~ e s[34]. In addition, TGF-/31 can costimulate the growth of a n t i - 0 3 activated murine CD8 T cells, resulting in induction of a memory phenotype,loss of lytic function, and expressionof a mixed cytokine pattern (interleukin-2 [IL-21, interferon-? [IFN-?I, IL-10, and TGF-61) [35]. Naive CD4'/CD45RBhi/CD44'ow T cells, on stimulation with TCF-P, become CD45RB'ow/CD~4hi, a phenotype characteristic of memory T cells [32]. These effector cells are distinguished by a high rate of cell proliferation and IL-2 production on restimulation. Individual cells in this population areable to switch to either the Thl orTh2 cytokine secretion phenotype in the presence of TGF-/3 or IL-4, respectively [32]. Furthermore, human T cells activated in thepresence of TGF-/3 display increased viability on restimulation, as a result of reduced apoptosis sensitivity in response not only to CD2 but also to Fas triggering [36]. Thus, the differentiation state of thecellT appears to beparticularly critical in determiningthe responseto TGF-/3 [3].

+

Immunological tolerance is a basic property of the immune system that allows for protection of the host from external pathogens without reacting against self. Autoimmune disease results when there is a breakdown in tolerance, leading to self-reactivity and subsequent destructionof tissues. Characteristic clinical presentations of autoimmune disorders include increased lymphocyte activation, increased major histocompatibility complex (MHC) expression, production of inflammatory cytokines, and presence of both polyclonal and autoreactive antibodies, resulting in a chronic inflammatory state.Since TGF-6 plays a critical role in the maintenance of normal immune function, dysregulationof this growth factor mayplay a role in autoimmune pathogenesis, as has been observed in transgenic mice in which the TGF-61 gene has been functionally deleted or is overexpressed 137-401. TGF-6 appears tobe such a pivotal cytokine in the regulation of immune and autoimmune diseases,many approaches have been undertaken to modulate immune pathogenesisby manipulating TGF-6. These approaches include neutralization of excess TGF-6 activity by neutralizing antibodies E411 and use of binding proteins such as decorin 142,433. Alternatively, to promote immune suppression, increased TCF-6 is required; that increase can be achieved through systemic exogenous administrationof TCF-6 1441, as well as augmentationof endogenous production through agents such as tamoxifen [45] or through inductionof oral tolerance. hile tolerance can be induced by several mechanisms, early studies found feeding a protein and then subse~uentlychallenging with that protein, a state of systemic hyporesponsivenessto the proteinis created [46]. In an attempt to provide a more focused, and ideally less a toxic, treatment than is presently offered by current i~munosuppressivedrugs, tolerance induction via oral ingestion has been extensively exploited in the last decade. While the actual mechanism(s) behind oral tolerance induction is not completely understood, TGF-6 is considered to play a key role [46]. After ingestion, antigenis taken up anddelivered to thegut-associated lymphoid tissue (GALT), which includes eyer’s patches and intraepithelial lymphocytes, for antigen processing. Antigenic stimulation leads to a changein the cytokine secretion profile by GALT lymphocyte populations. Production of Thl cytokine such as IL-2 and IFN-7, is down-regulated, whereas Th2 cytokines, such as ILand IL-10 aswell as TGF-6, are up-regulated(Figure 1) [47]. Release of TGF-61 by T cells is dependent upon antigen~specifictriggering by the oral toleragen. As previously mentioned9 local secretion of TGF-61 within the GALT aids in IgA class switchingbyresident cells. Im~unoglobulinA is essential for the clearance of orally ingested antigens (food), preventing the development of deleterious inflammatory responses within the gut.

Much of our current understanding of the ~echanism(s)underlying oral tolerance has been gained from animal models of autoimmune disease that involve immunization of self-proteins, such as experimental autoimmune encephalomyelitis ( which parallelsthe ~athological changes observed in human multiple sclerosis

7

Role of TGF-P in the induction of oral tolerance. On administration of antigen ral route, processing occurs in Peyer’s patches by resident lymphocytes. A switch from Thl cytokines (IL-2, IFN-y)to Th2 cytokines andTGF-P occurs. GALT are induced to secrete IgA. GALT T lymphocytes migrate to the peripheral site of inflammation, where they continue secreting TGF-P, thereby inhibiting local inflammatory cell populations in a nonspecific fashion. TGF-P, transforming growth factor beta; Th, T helper; IL-2, interleukin 2; IFN-y, interferon gamma; GALT, gut-associated lymphoid tissue; Ig, immunoglobulin.

Characteristic central nervous system (CNS) lesionsin both broader context in the majority of chronic in~ammatorydisorders) are i n ~ a m m a tion, demyelination of nerves by infiltrating macrophages (tissue destruction), and astrocyte proliferation/scarring, which lead to impaired electrical conductio~(loss of function)within theCNS.Low-doseoralan of myelin basicprotein ive cellular suppressionof fic T cell proliferation via 8’ T cells and dramaticall the severity and duration of EAE [48]. This suppression can be adoptively transferred via splenic T cells and lasts for several months. Furthermore, chronic can be suppressed after diseaseonset by thistherapy.This ap effective in the suppression of other experimental autoimmune diseases [49,50], arent cytotoxicities. T cell clones isolated from the mesenteric lymph nodes of mice orally ot ically identical to Thl encephalit restriction,and e * fectively suppress types may represent a different subset of T cells (Th3) [S11 and are thought to migrate to the periphery, where they continue to secrete TGF-61, resulting in nonspecific suppression at the site of tissue i n ~ a m m a t i o a~ phenomenon , known as bystander suppression 1461.

-P Elevated numbers of mononuclearcells expressing IL-4, IL-10, and TGF-6 are lood and cerebrospinal fluid from untreate

in comparison to

deficiency in immunosuppressive cytokine expression extendsto the afsues since immunohistochemical analysisrevealed perivascular infiltration with mononuclear cells expressing in~ammatorycytokines in brains of rats with inhibitorycytokineswereabsent. In -tolerizedanimals,there -regulation of inflammather inhibitory cytokines 1561. while elevated in comparison to rison to other inflammatorycytoS

ration and cytokine synthesis,

endothelial activation [ B ] . astrocyte cell lines E601 as well ible form of nitric oxide synt

*

*

t of complement) in human 'de (NO) throughin~ibition of theinducby infiltrating macrophages, suggesting

production of cytocidal factors [61]. S have been made with regard to tolerance induction in nonobese ~iabetic( mice, inwhich insulin-depend en^ diabetesmellitus spontaneously develops after mononuclear cell infiltration of the pancreaticislets of Langerhans and destruction of insulin-producing beta cells. Oral administrationof insuof diabetesin the NOD mouseand is linsuppressesinsulitisanddevelopment associated with increase^ production of TGF-61, IL-4, IL-10, and prostaglandin E ear cells, whereas mononuclear infiltrates from untreated ytokine profile [62]. S in animal models, trials of oral tolerization to out in individuals with early relapsing-remitting

[63]. Fewer myelin-treated patients had major exacerbations of disease than e~o-treatedpatients. Although the overall change in disa~ilityscores was not greater with myelin thanwithplacebo,genderand C phenotypealsoaffected treatment outcome. No toxicities were associated with treatment, and, in fact, several patients in the placebo group subsequently treated with myelin experienced disease stabilization in open-label treatment [63]. T cell lines generated from these S patients hada marked increase in therelative secretion as compared to those of nontreated antigen-specific TGF-0 l-secreting Th3 cells are elieved to localize to the target organ and then suppress inflammation in the local microenvironment. Oral tolerization with self-antigens may provide a therapeutic approach for the treatment of cell-mediated autoimmune disease which does not necessarily depend upon knowledge of the antigenspecificity of the original T cell clone triggeringthe autoimmune cascade [64]. The studies discussed have clearlydemonstrated that TGF-0plays a role inthe establishment of oral tolerance. This r rthersupported by theobservations thataddition of anti-TGF-01antibody-activatedlymphnode cells enhances theT cell proliferativeresponsein viv vitroand injections of anti-TGF-01 oreover, systemic antibody worsen both incidence and severity of EA administration also is effective since long-term of chronic relapsE with either TGF-01 or TGF-02 reduces the clinical severity of the disease, as evidenced by bothdecreased in~ammationand demyelwithinthe CNS. No abnormality or deleterious side are erapy EAEin organs other observed in [66], trials ical prov involving S to 1l patients with administratio the d progressi chronic of 0.2, 0.6, and 2.0 y are currently being . Calabresi, personal communication).

S , autoimmune processes can lead to destruction matoid arthritis (RA) is a prototypic autoimmun terized by chronic inflammation of the synovial joints and infiltratio~by activated leukocytes, primarily lymphocytesand m a c r o p h a ~ ~ [4]. s This chronicin~ammatory state can, in most instances, lead to progressive destruction of cartilage and bone, which occurs after invasion of these tissues by the cellular synovial tissue and is believed to be mainly mediated by cytokine induction of free radical species and destructive enzymes such as matrix metalloproteinases. In experimental acute and chronic arth~itis, systemicad mini st ratio^ of TGF-0, which may impair l e u k o c ~ e recruitment to the inflamed joint(^)^ nearlyobliterates the disease process [44], whereas local administration,resulting in increased leukocyte recruitment and subation of immature leukocyte populations, exacerbatesit [67]. AE, oral administ~ation of antigen, in this case native type11 collagen, nimal models of arthritis induced by type I1 coll~gen or complete Freund’s a d j ~ v a n [49]. t In addition, the cond ients receiving oral type 11 collagen stabilizes or improves and this ef last for several months an after cessation of therapy [68]. T lymphoc~tesexpressing the~EO~-integrin, adhesion molecul~associated with cells of intestinal origin, and its ligand,

are also present in synovial tissues of patients with RA [69]. In vitro stimulation with TGF-61upregulates aEP7expressionon PIBMCs fromhealthy volunteers. These results indicate that T cells of mucosal origin may migrate to the inflamed E-cadherin-express in^ joints, but once in the synovium they down-regulate their aE6,-integrin as a result of some as yet unidentified inhibitory stimuli. The exact function of these T lymphocytes in the jointis unknown, but it is possible that this migratory pattern from the gut to the periphery is associated with the cell populations involved in orally induced tolerance. Certainly, these observations warrant further investigation of the cytokine profile (Thl, Th2, or Th3) which these gutderived Tcells elicit on their arrival inthe synovium. Abundant TGF-@l, LTIBP, and TGF-PR11 mRNA is synthesized in the most actively proliferating synovial intimal cells, with peak expression during the active phase of the disease [70]. In addition, RAsynovial tissues (STs) have a significantly higher degreeof inflammation, macrophages, bloodvessels, and TGF-P immunoreactive cells whencompared with STs from patients with osteoarthritis (OA) or normal ST [71,72]. These observations suggest that TGF-P is involved in regulation of the synovial inflammatory process and that endogenous TGF-P may function as promoter, self-regulator, and suppressorof the arthritic lesions [l]. Furthermore, in a panelof monoclonal antiendoglin antibodies, increased staining was demonstrated in RA ST versus OA or normal ST. The increased staining was localized to subsynovialmacrophagesandSTlining cell layers,implicatingendoglin, whichis a capture receptor for TGF-P1 and$3, in RA pathogenesis[72]. Punalysis of gene expression in synovial fluid MNC and PBMC from by Northern [71] and ~uantitative RT-PCRrevealed an increase in several inflammatory cytokines as well as IL-10 and TGF-0, but adecreased TGF-P/P 2 microglobulin ratio [73]. The presence of TGF-P mRNA in RA indicates that immunosuppressive cytokines may also operate in the inflamed joint, although their level ofe~pressionmay not be sufficient for down-~odulationof immune activation.

In order to determine the specific regulatory role@)of TGF-61 in vivo, transgenic mice homozygous for a mutated allele (TGF-01 knockout mice)were generated by gene targeting in embryonic stem cells [9,74]. The TGF-01 knockout mice are indistinguishable from their normal littermates at birth, but by 2-3 weeks of age pathological changes develop, characterized by multifocal tissue infiltration of inflammatory cells and a severe wasting syndrome which becomes more pronounced at the timeof weaning. Death usually occurs between 3 and 4 weeks o f age, mainly as aresult of cardiopulmonary failure. The progressive inflammatory process found in TGF-61 knockout mice is associated with several manifestations of autoimmunity, including circulating antibodiesto nuclear antigens, immune complex deposition, increased expression of both class I and class I1 MHC antigens, and tissue destruction (see Table 1). In these animals, pre- and postnatal health may be sustained by maternally transferred TGF-P1[75]. However, as early as 7 days post partum, leukocyte adherenceandextravasationthroughendotheliumareobserved in TGF-Plknockout mice. The absenceof TGF-@l, orpossibly the low levels of maternal TGF-01which couldestablishachemotactic gradient, mayberesponsible for recruitment and

Characteristic Changes Observed in Immune Cell ____

.~

Cell acrophag~

TAdh~sion C classI and I1 expression TCytokine production

lymphocyte lymphocyte 4IgA synthesis TAutoantibody production aTGF-/3,transforming growth factor beta;MHC, major histocompat~bility class; IL-2, interleukin 2; IL-2R, interluekin 2 receptor; Th,T helper; Ig, immunoglob-

ulin.

ockout thy~ocyte§ the tis§ue§, expres-

n § t r u ~ e n t ain l

P

levels are elevated in these mice [38], consistent with increa§ed elae, consistent wit

II-defici~nt back~round.

strated an u ~ - r e ~ u l a t i oof n tissue as early as postnatal day

cytokine n e t ~ o r k , absence, aberrant i

.

-P

It is becomin~ increa§in~ly clear that cytokines suchas T role ini n ~ a m ~ a t oand r y immuneevents, are de~endentup0

7

for their proper functioning. The context in which a particular cytokineis found is the key to its function [84]. Not the least of these determining factors is concentration,butthemicroenvironment,extracellularmatrix,target cell differentiation influences must alsobe considered, state, presenceof antagonists, and myriad other The role of TGF-@ in host defense is, like its role in other biological processes, bifunctional, both promotional and suppressive, depending upon context. In this regard, augmentation of TGF-#3 levels isindeed beneficial to the controlof inflammatory disorders, but conversely, this immunosuppression may be deleterious in that it will increase susceptibilityto infectious pathogens [l]. It is evident that growth factors and cytokines play an important role in the immune response to pathogens. Susceptibility to infection by a particular pathogen and the host’s ability to resolve the resultantinfection are dependent upon the cytokine profile elicited, Pathogenic organisms may promote their own survival by alteration of host cytokine production thatis conducive to their survival, a phenomenon that has also been observedintumor cell evasion from detection by the immune system [85].

. In bacterial and parasitic infection/diseases, such as those causedby ~ o x o p l ~ s ~ o s ~ s g o n ~ iand i Trypanoso~a cruzii, leishmaniasis, and schistosomiasis, TGF-P hasbeen recognized as an important immunoregulating factor. Active TGF-#3is produced during infec~ion of murine peritoneal macrophagesand human macrophage cultures with the intracellular parasite ~ e i ~ h ~[86,87]. a ~ i aIn murine models, addition of exogenous TGF-#3increases parasite numbers, whereas neutralizing anti-TGF-@antiserum has a reductive effect [86]. Similarly, TGF-#3 added to cultures of human macrophages infectedwith L. ~razi~iensis led to a 50% increase in parasite numbers [87]. ~oncomitantadministration of parasites and TCF-@increases lesion size in mice [86]. In human leishmaniasis lesionsTGF-#3has also been detected immunohistochemically,suggesting that itmay contribute to localized immunosuppression [87]. Thus, TCF-#3appears toincrease susceptibilityto leishmaniasis. This increased susceptibility could be due to the inhibitory effect of TGF-#3 on iNOS, one of the enzymes responsible for NO production and a potent cytotoxic mediator of pathogens. Pronounced increased NO production through up-regulation of iNOS has been observed in mouse strains resistant t o L e i ~ h ~ a ninfection, ia whereas Leish~ ~ ~ i a - s u s c e p t imouse b l e strains have increased TGF-@ immunoreactivity in combination with decreased iNOS immunoreactivity and NO production [88]. Infections withcommon bacterial pat~ogensare a significant causeof morbidity and mortality in immunocompromised individuals. Transforming growth factor-@,which occurs at elevated levels in some of these immunosuppressive conditions, has been implicated in susceptibility to pathogens, in part, by suppression of normal cyto~ine-stimulated cytotoxicactivity,mostnotably from macropha~es. However,Lowranceetal. [89] haveshownthatpolymorphonuclearneutrophil leukocytes (PMNs) from ~ ~ Lmice/ (a ~murine r model of systemic lupus erythematosus [SLE]) are unable toextravasate adequately into theperitoneal cavity after intraperitoneal (i.p.) bacterial challenge, resulting in an increased susceptibility to lethality. This defect was attributed to an increased production of TCF-#3 by ~ ~ L / Zpr mice, as anti-TGF-@ antiserum improved PMN migration and survival of in-

L/lpr mice. Likewise, intravenous ( i x ) injection of TGF-6 into normal mice caused a mimicking of the effects observedin ~ ~mice. ~ / ~ In contrast, Nakane and colleagues [go] recently demonstrated that TGF-61 administered to mice 2 hr before infection with the intracellular bacterium Listeria monocytogenes significantly decreased spleenand liver bacterial load, whereas antiTGF-61 antibody had the reverse effect. In addition, 80% of mice administered TGF-61 after a lethal dose of L. monocytogenes survived. Thus, it appears that TGF-/3 augments antilisterial resistance, perhaps by decreasing the initial bacterial load.

In addition tointracellular parasites, TGF-6 mayalso influencethe host responseto extracellular organisms. Schistosomiasis is a chronic helminthic disease that affects more than 200 million people worldwide, and the morbidity in schistosome infections is primarily due to fibrosisresulting from the granulomatous response to parasite eggs in tissues 1911. In Sc~istosoma mansoni-infected mice, egg granuloma formation has been characterized as a CD4+ T-cell-dependent delayed-type hypersensitivity reaction, and egg deposition is associated primarily with increases inTh2 cytokine responses [92]. In spleen cell supernatants from mice that failed to elicit protective immunity against S. m a ~ s o n iTGF-6 has been detected [93]. Furthermore, TGF-61 gene expression is increased concomitantly with gene expression of extracellular matrix (ECM) proteinsduring murine S. mansoni infection. Treatment with the chemotherapeutic agent praziquantel decreases mRNA expressionof both TGF-01 and ECMs, suggesting that TGF-61 also contributes to S. mansoni pathogenesis by increasing the fibrosis associated with this disease [94]. Preliminary studies from our labsuggest that liver granuloma size and collagen content are increased in transgenic mice that overexpress the TGF-61 gene, whereas mice heterozygous for a null mutation in the TGF-61 gene have decreased liver granuloma volume and collagen content [9S]. Thus, our preliminary studies suggest that TGF-6 influences S. mansoni infection and the fibrotic granulomatous response.

Because of its potent immunosuppressive actions on mononuclear cells, TGF-6 may contribute t o the propagation and patho~enicity of a variety of infectious diseases. Much evidence to date suggests that it plays an important role in the immunosuppressive effects attributed to human immunodeficiency virus-l (HIV-l) [96-981. The HIV-1 protein Tat, which contains an RGD sequence allowing it to interact with ECM components, can induce TGF-6 production in human monocytes in vitro. Both exogenous TGF-fl and Tat can influence viral replication in HIV-l-infected monocytes, depending on concentration and other parameters[96]. The number of PBMCsfromHIV-l-infectedpatientsexpressingTGF-6mRNA is significantly higher than thatof normal volunteers [99], possibly contributing tosystemic immunosuppression. Thus, TGF-6 is likely an important pathogenic mediator in HIV-1 infection in vivo, as its overexpression could result in increased viral replication. Recently,Zauli andcoworkers[loo]foundthat in vitrostimulation of human CD34' hematopoieticprogenitor cells with eitherheat-inactivated HIV-IIIBor

r

t extravasion into the

lesion [106].

tracell~lar~ a t r i x

-P

forma~ion.

of gene expression as well as localization of protein via immunostaining) has been observed to be up-regulatedin animal models of kidney fibrosis as well as in human biopsy results from patients with fibrotic kidneydisease [l 161. Although renal allografts are designed to replace failing kidneys, rejection often occurs, leading to gradualprogressive deterioration inrenal function, proteinuria, and hypertension. Fibrosisbegins in the graftarteries and arterioles, as well as the glomeruli and interstitium. The accumulatio~of ECM leads to tissue destruction graft and loss of function. In biopsy specimens from patients un~ergoing rejection, elevated expression of all three TGF-P isoforms was observed [1171, demonstrating the many similarities between matrix accumulation in chronic rejection and that in models of tissue repair. Furthermore, immunosuppressive agents such as cyclosporine A (CSA) administered not onlyto graft recipients but also to patients with chronic autoimmune disorders may invoke renal toxicity, resulting in kidney fibrosis. One of the multiple mechanisms for CSA’s potent immunosuppressive efficacy is theprevention of IL-2production.owever, recent in vitrostudies suggest that besides inhibiting IL-2 mRNA expression and protein production in activated humanTlymphocytes,CSAstronglyup-regulatesTCF-P pro~uction [l l S]. These results implicate a link among TGF-6, renal toxicity, and fibrosis and suggest that the ability to limit TGF-P production during CSA treatment might improve graftsurvival as well as renal toxicity. These observations are further substantiated by the development of transgenic mice in which the TCF-P1 gene has been linked to the murine albumin promoter, allowing for expression of the protein exclusively within the liver [ l 191. In these animals, circulating levels of TGF-P maybe transiently elevated eightfold over those of normal age-matched mice. Concurrent with therise in circulatin~ TCF-Plis the development of glomerular disease, characterized by deposition of Ig, fibrinogenrelated molecules, increased extracellular matrix, and moderate cellular infiltrates. Mortality in these mice is due to nephrotic syndrome and end-stage renal disease [no]. While treatment with neutralizing antibodies to TCF-P might be one effective method for improvingkidney fibrosis in these instances, itis likelynot cost-effective and may lead to other side effects. Therefore, treatment with TGF-P antagonists or even antisense therapy might prove more efficacious. Isaka and colleagues have recently demonstrated an alternative form of drug delivery in an animal model of chronic renal glomerulosclerosis [43]. In this approach, rat muscle cells were transfected with a vector containing the gene for human decorin, a proteoglycan that is associated with the extracellular matrix and is able to bind and neutralize TGF-P via its core protein. In rats receiving muscle-based gene therapy, accumulation of pathological matrix is not observed, proteinuria (a measure of kidney function) issignificantlyreduced, andglomerularTGF-P1expression is alsosubstantially reduced. Another potential therapeutic treatment targeting TGF-P involves the use of antisenseoligodeoxynucleotides (ODNs) directedagainstTGF-P.Although the mechanism whereby antisense ODNs function is complex, it has been postulated that they interfere with transcription, RNA processing, mRNA stability, mRNA , and/or mRNA translation. Imai and coworkersrecently introduced N for TGF-/3 selectively into the left kidney 2 days after the induction of mesangial proliferative glomerulonephritis by anti-Thy-lantibody [121). A single

introduction of TGF-01 antisense ODNs dramatically suppressed the increase in mesangial matrix expansion and mesangial cell proliferation. Inhibition of TGF-P expression by antisense ODNs might open up new venues for therapy forglomerulosclerosis.

. Although excess TGF-0 is often blamed for tissue abnormality, interestingnew data implicate inadequate levels of TGF-0 as a contributing factor in the evolution of atherosclerotic lesions. The atherosclerotic lesion is thought to begin as a typical protective responseto vascular endothelialcell injury due to thrombosis, mechanical stress, hyperlipidemia, and/or immune reaction [1221. Circulating blood m o n o c ~ e s are attracted to the damaged vessel wall,where the subsequent interaction and activation of these immune cells result in VSMC migration and proliferation, as well as synthesis of extracellularmatrixproteins. The resultant lesiontakes on the characteristics of a chronic inflammatory reaction, with extensive fibrosis and permanent tissue damage, in this case, occlusion of the artery. Decreased localTCF-P production may bea risk factor for plaque formation, as TGF-01 gene expression is reduced in high-stress regionsof arteries [ 1233. While total TGF-P protein levels are not significantly altered in patients with advanced atherosclerotic disease, the level of active TGF-0 is depressed approximately fivefold below that of age-matched control patients [124], correlating with increased serum lipoprotein (a) (Lp(a)) and plasminogen activator inhibitor-l (PAI-1) levels. While VSMCs derived fromatheroscleroticlesionsexpressdecreased levels of TpR-I1 [125], it is possible that the decrease in active TCF-6 results from clearance of TGF-P by circulatingTGF-6 receptors [1261. Epidemiological studies have demonstrated that elevated levels of Lp(a) are associated with an increased risk for coronary artery disease. Lipoprotein (a) is localized in blood vessel walls and atheroscleroticplaques, both intra- andextracellularly, colocalizing with fibrinogen/fibrin [12’71. The underlying mechanism for Lp(a)’s role in atherosclerotic lesionsis not completely understood. However, plasminogen shares homology with apoprotein (a) (apo(a)), a glycoprotein component of Lp(a) [l281and, when converted to plasmin, may compete with Lp(a)for binding sites on fibrin. Since plasmin is felt to be the main activator of latent TGF-/3 in vivo [14], competition between apo(a) and plasminogen could effectively limit the availability of biologically active TCF-6. In transgenic apo(a) mice fed a lipid-rich diet,vascularlesionssimilar to thoseobservedinearly humanatherosclerotic plaques develop [1291. While total plasminogen content is not significantly altered in vessel walls of apo(a) mice and normalmice, plasmin content is threefold less in apo(a) mice than in controls. Likewise, while total TGF-6 levels are not altered, active TCF-P levels are decreased in both the serum and the vessel wall, where staining for active TGF-P is dramatically diminishedat sites of highest apo(a) accumulation withinvessel walls. Since an insufficient level of TGF-P may contribute to the development of atherosclerotic lesions, treatment of patients with TGF-&based therapies may be useful in diminishing lesionsize and/or formation. Tamoxifen, anestrogen agonist, to up-regulate is a potential candidate for this type of therapy, as it has been shown production of active TGF-@ in cancer studies [130,13l]. Furthermore, tamoxifen

1.

Current Therapeutic Strategies That Target TGF-p" ~__

_

_

_

_

_

_

~

~

chanism Condition Treatment Oral ingestion of protein

Systemic TGF-Phecombinant TGF-0 Antibodies directed against TGF-fi Tamoxifen

Antisense TGF-0 viatargeted gene therapy Decorin production viatargeted gene therapy

~ u t o i m ~ udisease ne (rheu- Oral tolerance induction; matoid arthritis, multiple up-regulation of TGF-6 sclerosis, uveitis, etc.) production by immune cells withinGALT Autoimmune disease; scar Suppression of autoimformation ~une-mediatedinflammation; decreased scarring with TGF-03 Scar formation; kidney fi- Decreased scarring with brosis; rheumatoidaranti-TGF-P 1, -62; dethritis creased fibrosis with kidney, joint Breast cancer Up-regulation of allTGF-6 isoforms; inhibition of cellular proliferation in susceptible tumors Kidney fibrosis Reduction of localTGF-/3 gene expression resulting in decreased fibrotic response Kidney fibrosis Binding/inactivation of TGF-6 resulting in decreased fibrosis

"TGF, transforming growth factor; GALT, gut-associated lymphoid tissue.

has already proved efficacious in reducing aortic lesion size and up-regulating circulating/aorticconcentrations of TGF-P [45] in ce fed a high-fat diet. overexpression of TGF-@may be detrimental a actually worsen lesion [132]. Therefore, the development of therapeutic agents that minimally modify the TGF-@system will likely induce fewer detrimentalside effects.

on many key Clearly TGF-@is an important moleculewithwidespreadeffects biological processes as well as a profound influence on immune function. the course of an inflammatory response (as elicited by pathogens or injury, oreven in response to tumorcells), the initialrelease of TGF-P createsa chemotactic gradient for mononuclear cells, causing their mi~rationfrom the peripheral circulation into the in~ammatory site. These cells differentiate andelicit other factorsnecessary for the resolution and repair of the injured site, in large part through the actions of TGF-P. While in most instances these processes are fairly well balanced, excessive or insufficient amounts of TGF-@can be deleterious,resulting in fibrosis or chronic, nonhealing wounds, respectively. Alternatively, cells may become refractory to the biological effects to TGF-P, as a result of loss or sheddingof TGF-@receptors.

In manycases, treatment with exogenous TGF-P or antibodies directed against of disease(Table 2) ver, administration of it mayamelioratetheoutcome TGF-Por even anti-TCF-P antibodiesmayhave untode effects and/or alter host defense in deleterious ways, especially if chronic administration is required. The development of TCF-P antagonists, agonists, and soluble receptors has potential merit in the treatment of certain conditions. Other current approaches include the developmentof therapeutic strategies that employ reintroduction of the gene of interest into the host,resulting in altered synthesis of the desired protein. tion of complex mediators, such as TGF-P, which already has comple regulatory mechanisms with regard to its synthesis, secretion, and activation, will present a challenge, assuchstrategiesmayhaveunpredictablesideeffects. dless, continued exploration of the biological characteristics of TGF-P is essential for the understanding of disease processes as well as the design of better treatment regimens for individuals suffering from a variety of inflammatory and immune disorders.

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rnla-S~nFrancisco, San Francisco, Cal~ornia ~niversityof Pi~tsburgh,~ittsburgh,Pennsylvania Carnegie ell on ~niversity,Pittsburgh, Pennsylvania

Psychoneuroimmunology (PNI) is an interdisciplinary science which attempts to elucidate the relations among behavioral factors; nervous, endocrine, and immune systems; and health. major hypothesis in this discipline is that stressful life events influencerisk for the onset or prssionofimmune-related illness. This chapter focuses on the human literature i and explores evidence linking stressful events to immune-related disease, as well as potential mechanisms that may mediate this relationship. Particular attention is given to the hypothesis that altered immune functioning during stress is one plausible biological pathway through which stress influences health outcomes.

Stressful life eventsare commonly believed to suppress host resistance. These events include major stressful experiences such as the loss of a spouse or a job, but also include the accumulation of minor daily hassles such as misplacing keys, missing a bus, or having an argument with a spouse or coworker. When demands imposedby events exceed a person’s ability to cope, a ps~chologicalstress response composed of negative cognitiveand emotional statesis elicited [l]. In turn,psychological stress is thought toinfluence immune function through autonomic innervates of lymphoid tissue or hormone-mediated alterations of immune cells. Stress may also alter immune response through coping behaviors such as increased smoking and alcohol consumption.

Stressful life events havebeen associated with a range of immune-relat including autoimmune diseases, infectious illness, and cancer l[%”]. most compelling evidence involves susceptibility to upper prospectiveepidemiologicalstudieshavefoundth , chronic family conflict, and disruptive daily events verified infectious illness [5,6]. Converging evidence comes from viral challeng control for exposure toinfectious agents. In these st and negative mood are assessed, vo and then monitored in quarantine Three recent viral-challenge studies havesuggested that PS factor for susce tibility to upper respiratory infe volunteers, reports of stres d negative affect all predict ment of a cold. This association o prechallenge antibody to the experimental virus and was independent of health practices (e.g., smoking and alcohol consumption) or numbers of various white blood cell populations or total (nonspecific) antibody level easured before viral challenge [7,8]. The relationships among stress, mood, an across five different upper respiratory viruses. Finally, in a S ors of disease severity, rather than episode onset, negative viral exposurewas related to more severe colds and influen amount of mucus produced over the course of the illness [ 10 In addition todisease outcomes, stressful of wounds. ~iecolt- laser and colleagues [l l] a severely ill family memberwas associated with heig~tened slower healing of a 3.5-mm-punch biopsy WO evidence and wound responseto hydrogen peroxide average of 9 days longer in care givers than in age ealth-related variables such as alcohol and caffeine mass, weight change, and smoking did not account served. As the authors note, such decrements in wound repair may have important clinical imPlications with respect to surgical recovery [l 11.

ow stress influences health outcomes is not entirely clear. people cope with stress by initiating coping behaviors, such and that these behaviors compromise health an ively, stressmayinfluencehealthbydirectly the activation of neuroendocrine pathways and rele neurotransmitters, such as cortisol and tomical links exist between the nervous sympathetic innervationof lymphoid org

addition, lymphocytes have receptors for a variety of hormones that are secreted ing catecholamines, hins/enkephalins, S ings,along with evidence i hormones can influence immune function in vitro, suggest that hormonal and neuroendocrine substances mayplay a role in altering cell function duringstress. The remainder of this chapter focuses on existing evidence that changes in immune function are associated with moo alterations evoked by life events and, sible pathway by which stress influences susceptibilityto is important to keep in mind, however, that while subin i ~ m u n efunction have been shown to index health status (e.g., * r declines in T helper lymphocyte counts in human immunodeficiency virus ] infection), it is not clear whether less pronounced alterations associated with psychological stress are sufficientto influence health. As will be discussed further, it is possible that the most pronounced health consequenceswill occur in individuals who have already wea~enedimmune systems (e.g., the elderly or individuals with IV infection) [l51.

uman studies in PNI are limited to quantitative and functional assessments of immune parameters sampled from peripheral blood and mucus. lude the absolute numbers or percentages of circulation, including T helper, T suppressor, T cytotoxic, and lymphocytes; natural killer (N ) cells; and phagocytes. functional assessments, lymphocyte proliferation assays are I research. These typically includethe T cell mitogens phytoand concavalinA (Con A), as well as the T and ). Natural killer cell cytotoxicity is alsofrequently lant pokeweedmitogen (P d.Insome cases, cells may be initiallyincubated with stimulatory es, such as interleukin2 (IL-2) or gamma-interferon (IFN-7). The effectiveness of these cytokines in enhancing N cell activity is then compared to cytotoxicity levels found in unstimulated samples. Finally, in vitro functional assays are also used to measure cytokines that are produced and secreted by lymphocytes and monocytes after stimulation by mitogens. Frequently levels of IFN-7 and various interleukins, such as IL-2, are measured. er functional assays measureantibody responsesto novel antigens in such as va~cinationW an influenza virus, recombinant hepatitis keyhold limpet hemocyanin (KL antibodies to a primarychallenge, investigators have measured the level of proliferase of blood lymphocytesto aspecific challenge virus in vitro. ough greater' antibody response is usually interpreted as better immunocompetence, elevated antibodylevels to latent herpesviruses may reflecta weakened ability of the immune systemto prevent such viruses from becoming active. Therefore,higherantibody levels to herpesviruses areoften interpretedasindicating poorer immunocompetence [15]. Studies frequently measure antibody titer to Ep-

V) because EBV is widespread, with seropositivity rates at approximately 90% in the adult population 116,171. Finally, levels of the pteridine compound neopterin, which is released from activated macrophages, can also be obtained from blood or urine and are considered an index ofcell-mediated immune activation.

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rvi

A growing literature in both humans and animals indicates that immunolo~ical changes are associated with psychological and physical forms of stress (for extensive reviews involving humans, see Refs. 18-19). Changes in the immune system have been found to accompany major and minor life events, such as exams, bereavement, divorce, work-related stress, and the ongoing uncertainty associate Three Mile Island (TMI) several years after the nuclearaccident.

ino or ~ t r e s s f ~ u~ a i l y vents inorstressfulevents(i.e.?dailyhassles,such as losing one’s associated with changes in salivary immunoglobulin A (sIgA) and serum antibody response to a novel antigen. In a study by Stone et al. [20], participants ingested a novel protein capsule daily for 12 weeks to stimulate an sIgA antibody response. Saliva samples were collected each day to assess sIgA levels to the protein, and participants recorded the occurrence of both desirable (e.g., accomplishing a goal) and undesirable (e.g., arguing with a spouse) daily events. ~ e s i r a b l eevents were related to greater sIgA antibody production, whereas undesirable events were related to lower levels. These findings were recently replicated by Stone et al. [21], who also demonstrated that undesirable events were associated with lowered antibody levels primarily when accompanied by increased negative mood. In contrast, desirable events were linked to increased a n t i ~ o d yproduction when accompanied by decreases in negative mood. Jabaaij et al. [22] also found that serum antibody responses to three recombinant hepatitis B vaccines covaried with the frequency and intensityof daily hassles and concurrent distress. In this prospective study, an index of combined hassles and distress scores measured early in the i ~ m u n i ~ a t i oseries n predicted less production of antibody in response to the vaccine. With respect to daily events and health outcomes, it is interesting that Stone, Reed, and Neale [231 found that ~ e f o r ethe onset of an infectious illness, individuals reported a peak number of undesirable events (e.g., difficulties with their boss or coworkers) and a diminished number of desirable events, Thissuggests that peoplemay become more susceptible to infection when negatively perceived events appreciablyexceed good ones.

I.

in at ions Perhaps the most commonly studied stressful events in relation to immunological status are examinations. Academic examinations have long been known to elicit stressfulreactions in students, including heightened anxiet elevated concentrations of cortisol and catec~olamines [2 examination stress and immune function compare blood samples collected on an

2.

exam day to those taken during a relatively stress-free period of the semester (e.g., just after a vacation). Overall, several indices of immunosuppression have been observed in medical students during important examinations. These include decrements in lymphocyte response to mitogenic stimulation, reduced N cell populations, decreased antibody responsesto vaccinations, and duction of cytoki S and neopterin [15,26-321. Increased levels of circulating antibodies to Epstein rr virus and other herpesviruses have also been ination periods, indicating reactivation of latent virus by either ne influencesor weakened immunocompetence[15,291. have found that certainpeople are more susceptibleto immune alterations during exams than others. For example, the largest exam-related immunologicalchangesndinstudentswhoreportedhigher levels ofoveralllife stress or lonelinessSimilarfindingswerereported by Claseretal. [31], who foundan associatienanxiety levels duringexamsandantibody response to a series of three hvaccinations.Althoughtherewas no effect of anxiety on theantibody leve afterthethirddose ofantigen,heightenedanxiety was related to a delay in seroconversion to hepatitis B. In a similar study, Bovbjerg et al. [32] vaccinated S entswithtrivalentinfluenzavaccineimmediately after an examination period. re, greater reported distress was related to lowerproliferase of blood lymphocytesto the influenzavirus in vitro. long do immunological changes persistafter exams? While immune alterations are thought to returnbaseline to shortlyafter exam periods, some individuals may showa delayed recovery lastingup to4 weeks after examinations end[33], This corresponds to periods of persistent stress hormone elevations in some students I241 and is interesting in light of anecdotal reports that students often become ill after, but not during, exams[2'7].

3. The loss of an intimate relationship through either death or divorce has also been immunity, including suppressionof lymphocyte responsesto reduced NK cell activity, and changes in T cell subpopulations. Early investigationsfound lowered lymphocyte proliferationamong bereaved loss of their spouse, as compared to that of both nonbereaved the prebereavement period [35]. In these studies, immunological alterations persisted from2 to 14 months after the loss. ecent studies indicate that the degree of immune change among bereaved varies greatly and may be related to the severity of depressed mood. Thus, Linn, Linn, and Jensen [36] reported reduced lymphocyte proliferation to P only among bereaved 'spouses with concomitant depressive symptoms. This is consistent with findings by Irwin, ~ a n i e l s , ~ m iBloom, th, and Weiner [37] that greater depression during bereavementis associated with lowerNK cell activity (r = - .89). In addition todepressed mood, the availability of a supportive social network may influence immunological responses to the anticipated or actualloss of a loved of patients with cancer, aron et al. [38] found that one. In a sample of spouses individuals reportin from their social ties had higher NK cytotoxicity and proliferativ A than those reporting less support. The presence of depressiontherlifeeventsdid not account for theseassociations. ecause individuals in the gay community are often subjected to multiple

losses attributable to acquired immunodeficiency syndrome have begun to examine the immunological correlates of b immunodeficiencyvirus- ( 1V)-seropositive and cently, Kemeny IV-sero~ositive et men who had lost an intimate partner to A showed ear decreases in proliferative reA and increases in serum neopterinlevels after theloss. Life-style and medicationfactors(recreationaldrusmoking, level of exercise, sleep 10s use of azidothymidine(zidovudine) I) didnotaccountforthe findings. ~ levels in ~ e a l t h yindividuals [ ~ 7 ] , stresshasbeenlinked to ~ e c r e a s eneo~terin IVis known to increase neopterin production, and hown to be a strong predictor of the development of owever, in the case of IV-negative men, no immunological changes were found after the deathof an intimate partner. Immunological alterations were also observed in IV-seropositive gay men by Goodkin et al. [41], who assessed immune function after the death of a lover or close friend. When th were compared to nonbereaved controls, loss was associatedwith dirninis cell cytotoxicitywithin 6 rnonthsafterthedeathand again 6 months later. mitogenesis d to A was also found at the later ,was related to elevations in plasmaCorti there were nodi rences in thenumber ed and nonbereav groups in either stud

ations. ~iecolt-Glaser,Glaser, and colleagues found t divorced women had lower percen creased proliferative responsesto P thanacomparisongroup of marriedwomen [42]. S ings were reported in ordivorcedforupto asubsequentinvestigation of 32 menwhohadbeen 3 years [43]. As in the previous study, separ body levels to latent viruses (here than matched married con between the two groups. marital quality was related to higher levels of distress, loneliness, and latent virus antibody response.Finally,in a recent investigation o decrements in immune functioning were similarly relate Newlyweds who demonstrated more negative or hostile interactions discussion of maritalproblems wed greaterdecrea and proliferativeresponses to P and Con A overa viduals also had higher antibody titers to latent hostile counterparts.

Immunological changes accompany other prolonged stressful events, such giving for a terminally ill patient, long-term unemployment, job stress, a dence near a damaged nuclear power plant. Several studies ha ocial consequences of caring for a family member with ). In general, such care givers are found to be at higher

health complaints, an logical se~uelaeof c

mer and current care givers did not differ from each other and IFN-7 and IL-2) than a gest that the psychological stressors may persist well responses were also found

cations, or substanceuse.

changes persist over prolonged periods?

events, such as the death of a loved one, may unfold a number of other stressful situations relating to finances, social relations, and other long-term concerns [SS]. Finally, cognitive influences, such as intrusive thoughts and imagery, may sustain stress reactions even when the objective event no longer exists [49]. For exam elevated anxietyand immunological changes in T o experience intrusive thoughts or imagery abou nt and its potential health effects.

Taken together, moststudies involving stress and immunity indicate that psychological stressors are as with changes in imm l numbers and functions [l8]. The most cons erations include reduce cell activity and lymphocyte ~roliferationtoandCon A; diminished dy response to viral antigens, such as hepa nd increase^ antibody levels to latent herpesviruses.

Decreases in percentage or absolute number of circulating cells, T cells, T helper cells, T suppressor/c~otoxiccells, and PJK cells are also equently reported immune responses to stress. Evidence suggests that such alterations may persist over protracted intervals during particularly intense or prolonged stress. Despite the general consistencyof findings, there are somelimitations associated with naturalistic studies of psychosocial influences on immune function. design, naturalistic studies are able to demonstrate CorreZatio~aZ,but not causal relations between stressand immunity. Furthermore, the potential mechanisms linking stressto immunity are difficultto delineate in such investigations. nat~ralisticstudies have examined health practices or neuroendocrine factors as mediators of changes in immunity, and at this point, it is not clear whether more modest changes in health practices during stress result in altered immune function [l 81

hile most of the literature on stress and immune relationships involves nat~rally occurringstressors,there are now anumber of controllederimentalinvestigamune function. Extionsexaminingtheimpact of acutepsychologicalstress o perimental manipulation, in which subjects are assigned ra ly either to a stressexposed condition or to an unstressed, control condition is required to evaluate whether stress causes alterations in immune function. Experimental studies also serve as a useful model for transient daily life stressors and provide a means to investigate potential endocrine mechanisms underlying associated immunological changes. Short-term laboratory studies have examined immunological responses to a range of behavioral stimuli perceived by subjects as aversive, deman~ing, orinterpersonally challenging. Such stimuliare typically presented to participants for brief intervals (e.g., 10 to 120 minutes) and include either computer tasks, mental arithmetic, electrical shocks,loud noise, unsolvable puzzles, graphic films depicting combat surgery, evaluative speeches, marital discussions involving conflict, or interviews eliciting the recollection of positive and negative experiences and mood states (for a f. 56)" Immunological measurements are obtained * of the stressor and are compared resting to valu prestress period of adaptation and baseline measurement; oc measureme~ts taken several minutes or hours after thetask are Exposure to suchchallengingtaskshasbeenshown to enumerative immune changes; the most consist~ntfindings i n c l ~ ~ ane increase in the numbers of circulating NK cells and T suppressor/c~otoxic lymphoc~es and a decrease in the ratio of T helper to T suppressor cells. ome (but not all) studies have shown alterations in the number of T helper and l ~ m p h o c ~ t as e s well [57611. respect With to functional unity, stress-induced decrea T cell proliferative responses are to freqently reported [53,5

iminished mitogenic responsesto PWM have also been shown, suggesting similar reductions inB lymphocyte function [58,68]. Finally, there are numerous reports of altered NK cell activity after exposure to brief psychological stress, and herestudieshavedemonstratedincreasesas well as decreasesinthisparameter [53,58,59,62,65,70-741. The onset and duration of immunological reactions to acute mental stress are not entirely known, but preliminary data suggest that such changes are rapid and transient, occuring as early as5 minutes from stressoronset [63]. In the case of cell subset redistribution, these changes returnto baseline within l 5 minutes of stressor termination [75]. In contrast to quantitative alterations, reductions in lymphocyte proliferationhave been found to persist for at least 90 minutesaferchallenge [67,69]. Elevations in NK cell activity may last at least 1 hour [S]; however, the decreases reported inone experiment persisted as long as 72 hours after exposureto laboratory stress [73]

There is a growing body of evidence that acute immuneresponses to psychological stress are mediated by activation of the sympathetic nervous system. First, the rapid appearance of such changes makes it unlikely that other, slower-responding hormones (e.g., cortisol) are contributing to the effects. Indeed, two studies found immune alterations in the absence of concomitant cortisol responses 164,691. Second, infusion of catecholamines leadsto functional and enumerative immune alterations that are similar to those seen during acute mental stress (e.g., elevated T suppressor/cytotoxic and NK cells, decreased ratio of T helper to T suppressor/ cytotoxic cells, and diminished proliferative response to mitogens) [76,77] Finally, only those subjects with the most pronounced sympathetic responsesto laboratory stressorsdisplaysuppressionofmitogen-stimulatedlymphocyteproliferation [63,64,69]. Additional and more direct evidence for sympathetic mediation derives from the observation that changes in cellular immune function under stress are ameliorated by the administration ofan adrenoceptor antagonist. In particular, the administration of labetalol (a nonselective alpha- and beta-adrenergic antagonist) was shown to prevent stress-induced increases in NK cell number andactivity, reductions in the ratioof T helper to T suppressorkytotoxiccells, and decreases in~roliferative responses to PHA and Con A during two cognitive tasks and a public speaking [70], who stressor [62]. Similarfindings were demonstrated by Benschopetal. found that the selective beta-:! antagonist propranolol blocked stress-induced rises in NK cell numbers andcytotoxicity.

n i s ~ of s Sy~pat~etic-lmmun~ ~ediation Precisely how the sympathetic nervous system mediates immune reactions to stress remains unclear. One potential mechanism underlying cell redistribution involves the extrusion of stored lymphocytes from spleento periphery after the contraction of sympathetically innervated smooth muscle in the spleen [78]. It is also possible that catecholamines may influence leukocyte redistribution between the marginating

pool of blood vessels and the bloodstreamby altering adhesion receptor expression on these cells and/or the vasculature. It is now known that blood-borne lymphocytes enter some lymphoid organs by first attaching, via adhesion molecules, to specialized endothelial cells lining the postcapillary venules of lymphatic tissue. In a recent experiment,Benschop,Oostveen,Heijnen,and ux [79] foundthat N cell adhesion to endothelial tissue was markedly reduce ) in vitro after incubation with epinephrine, and that this process was dep adenylate cyclase (cyclic adenosine monophosphate [c altered mitogenesis may, in part, involve sympathetically mediated impairments of IL-2 production by T helper lymphocytes [SO], altered IL-2 receptor expression [Sl], and decreased antigen presentation by macrophages[SO].

The accumulating evidence supporting sympathetic modulation of immune parameters during stress has implications for individual differencesin stress-immune intereople are known to vary markedly in the magnitudeof their sympatheticadrenal responsivity to stress, and these differencesdenotea relatively stable attribute of individuals [82,83]. Recent findings suggest that individuals differ substantially in both neuroendocrine (epinephrine, norepinephrine) and cardiovascular (heart rate, systolic and diastolic blood pressure) indices of sympathetic arousal, and that reactivity in these parameters covaries with cellular immune res psychological stress [64]. anuck et al. 1641 found that only individuals who were identified as “high sympathetic responders” (on the basis of relatively high catecholamineandcardiovascularresponses to mental stress) showedstress-induced insuppressor/cytotoxic cell numbers and a diminished mitogenic response ills et al. 1611 extended these findings by demo~strating thatindividual in the magnitude of plasma norepinephrine responses to mental stress and thesensitivity of beta-2 adrenergicreceptors on lymphocytes ~redictedconcomitant changes in circulating NK and T suppressor/cytotoxic graphic and healthvariables, such as ethnicity, gender, age, bo pressure status (i.e., hypertensive or normotensive blood pressure), and smoking, failed to predict these immune alterations. Individual differences in several immunological reactions to mental stress are moderately stable over time, Moreover, the propensity for individuals to react to one stressor with heightened sympathetic arousal predicts immunological re to other stressful situations [60,65,84]. For example, Sgoutas-Emch et al. that persons responding with high heart rates when required to give a speech showed greater increases in NK cytotoxicity when exposed to a mental arithmetic task on another day thanindividuals who demonstrated low heart rate reactions to the speech. Other investigations assessing the stability of cellular immune reactions to laboratory stressorsdelivered 2 to 6 weeks apart have found signi correlations for the magnitude of changein proliferative response t of T suppressor/cytotoxic and NK cells, and ratio of T helper cytotoxic cells (ranges of .40-.6O)[6O,84]. Taken together, these findings suggest that individuals differ in the magnitude of immune responses to brief mental stress, that changes in some parameters are moderately reproducible on retesting, and that the magnitude of change may therefore denote a stable dimension of individual differences [6O].

. ith the exception of increase NI( activity, functional immune cha stress usuallyresembloseaccompanying chronicnaturalistic stress though,thedirection~uantitative changes in somelymphocyte S will differ between acute and chronic stressors. hereas laboratory stressors elicit immediate elevations in N and T suppressor/c~otoxic lymphocytes,naturalistic stressors tend to be associ d with reductions inthese cell types [l 81. The reasons forthese discrepancies are notyet clear but may reflect differences in the hormonal environment surrounding immune cells. Long-termchangesin health habits, which influence both immune and neuroendocrine systems, may in art underlie differences that are observed in acute and chronic stress [12,85,86]. so, it is possible that homeostatic processes, such as receptor down-regulation d desensitization, may be involved. For example, Maisel et al. [87] found that oral administrationof the beta-2 agonist terbutaline for1 week caused a decrease in thenumberofbetareceptorsonTsuppressor/cyt , andT helper cells, and a substantialdropinisoproterenol-inducedcgeneration in NI( andT suppressor/cytotoxic cells in humans. Interestingly, T helper and showed change of in either beta-receptor density or lation. ion is indeed essential recruitment the in o the margin at in^ pool of blood vessels to the cir sting bloodstream [79], these adaptations maypartlyexplain why peripheral cell numberstend to increase acute stress ~onditions anddecrease durin onic forms of stress. inally, i m m u ~ o l o ~ i c differences al may be attributable to a more complex hormonal milieu associated with chronic than acute stress. As slower-responding hormonal systems become activatedand release substances such as cortisol, adrenocorticotropic hormone (ACT ), and ad endorphin, both independent and interactive hormonal effects on immune function are likely to occur. Although reports are few, there is preliminary evidence suggesting that cortisol may play an important role in modulating some immune reactions in chronic stress conditions. As indicated previously, ~ o o d k i net al. [ found associations between increased plasma c ative response to PHA during bereavement i , in a study assessing cortisol response to Antoni et al. [$$l found that anxiety levels and intrusive thoughts before serostatus ificantly to higher plasma cortisollevels and lower proliferaPlasma beta-endorphin levels were also related to reduced lymphocyte proliferation, although associations were less consistent than for cortisol.

There is now ample evidence suggesting that stressful life events are related to health outcomes,suchasinfectious illness, autoimmunedisorders,cancer,andwound efs. 2-11). Theliterature in PNI also clearly demonstratesthat immunological alterations occur in response to a variety of stressful events in hu-

7

mans, and it is through such changes thatstress may, in part, be linked to disease. Studies conducted in laboratory settings provide particularly strong evidence that the sympathetic nervous system mediates some immunological changes during mental stress. Because individuals differ in the magnitude of immune and sympathoadrenal responsivit~ to stress, it is conceivable thatthere is a meaningful distribution of response differences among people which, in turn, may influence their susceptibility to immune-related illness. Future research in PNI needs to determine whether the nature or magnitude of stress-induced immune alterations bears relevance to increased disease susceptibility and progression. I m ~ u n responses e of stressed persons generally fall within normal ranges [SS], and where reductions in antibody responses to vaccinations are observed, it is unclear whether these decrements are sufficientto influence protection against infection. Additional studies that employ longitudinal designs and measure immune parameters relevant to the disease under study and health outcomes are needed. It is also likely that stress-related immune alterations may have more pronounced health consequences for individuals with weakened immune systems. Itis well known, for example, that aging is associated with a decline in immunefunction, as indicated by lowering of the proliferative response to mitogens, natural killer cell activity, antibody production, and phagocytic activity 1901. Thus, the significance of stress-related immunealterations may begreater for theelderly or forindividuals infection or autoimmune disorders [l5]. With respect to individuals who nfected, Kemeny et al. [39] reported that elevated neopterin level during bereavement placed some individuals at levels previously shown to be associated with a higherrisk of development of AIDS[39,40]. Over the last 20 years, PNI research has established links!between psychological stressors and altered functioning in the immune system. This remains one of the most promising pathways through which stress may alter host 'resistance to disease onset or progression, Prospective studies that measure all aspects of the stressimmune disease modelare needed to understand these associations more fully.

1. RS Lazarus, S Folkman. Stress, Appraisal, and Coping.New York: Springer, 1984. 2. S Cohen, GM Williamson. Psychol Bull 1095, 1991. 3. I Grant, GW Brown, T Harris, W1 McDonald, T Patterson, MR. Trimble. J Neurol Neurosurg Psych 5223, 1989. 4. MR Jensen. J Pers 55:317, 1987. 5 . NMH Graham, RB Douglas,P Ryan. Am J Epidemiol 124:389, 1986. 6. RJ Meyer, RJ Haggerty. Pediatrics29539, 1962. 7. S Cohen, DAJ Tyrrell, AP Smith. N Eng J Med 325:606, 1991. 8. S Cohen, DAJ Tyrrell,AP Smith, J Person. Soc Psychol 64:131, 1993. 9. AA Stone, DH Bovbjerg, JM Neale, A Napoli, H Valdimarsdottir, D Cox, FG Hayden, JM Gwaltney. Behav Med 18: 115, 1992. 10. S Cohen, WJ Doyle,DP Skoner, P Fireman, JM GwaltneyJr., JT Newsorn. J Pers SOC Psychol 68:159, 1995. 11. JK Kiecolt-Glaser, PT Marucha, WB Malarkey, AM Mercado, R Glaser. Lancet 346: 1194, 1995. 12. JK Kiecolt-Glaser, R Glaser. Brain Behav Immun 2:67, 1988.

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S Livnat, SY Felten, SL Carlson, DL Bellinger, DL Felten. J Neuroimmunol 105, 1985. M Plaut. Annu Rev Immunol5:621, 1987. JK Kiecolt-Glaser, R Glaser. Ann Behav Med 9:16, 1987. DD Porter, I Wimberly, M Benyesh-Melnick. JAMA 208:1675, 1969. CV Sumaya. Pediatr Infect Dis5:337, 1986, TB Herbert, S Cohen. Psychosom Med 55:364, 1993. A O’Leary. Psychol Bull 108:363, 1990.

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AA Stone, BR Reed, JM Neale. J Hum Stress 13:70, 1987. KT Francis. Psychosom Med41:617, 1979. G Johansson, A Collins, VP Collins. Motiv Emotion 7:1, 1983. JP Dobbin, M Harth, GA McCain, RA Martin, K Cousin. Brain Behav Immun 5:339,

AAStone, JM Neale,DSCox,ANapoli,HValdimarsdottir,EKennedy-Moore. Health Psychol 13:440, 1994. 21. Cox, JM Neale. Int J Behav Med 3:1, 1996. 22. k, HJ Duivenvoorden, RE Ballieux, AJJM Ving-

1991. 27. PR Dunbar, J Hill, J, TJNeale. Aust NZ J Psychiatr27:495, 1993. 28. R Glaser, J Rice, CE Speicher, JC Stout, JK Kiecolt-Glaser. Behav Neurosci 100:675, 1986. 29. R Glaser, JK Kiecolt-Glaser, CE Speicher,JE Holliday. J Behav Med 8:249, 1985. 30. JK Kiecolt-Glaser, W Garner, C Speicher, GM Penn, J Holliday, R Glaser. Psychosom Med 46:7, 1984. laser, RH Bonneau, W Malarkey, S Kennedy, J Hughes. 31. 52:229, 1990. 32. DH Bovbjerg, SL Manne, PA Gross. Psychosom Med Jemmott, JZ Borysenko, M Borysenko, DC McClelland, R Chapman, D Meyer, H 33. nson. Lancet 1:1400, 1983. 1:834, 1977. 34. R Bartrop, L Lazarus, E Luckhurst, LG Kiloh, R Penny. Lancet JC Thornton, M Stein. JAMA250:374, 1983. 35. SJ Schleifer, SE Keller, M Camerino, 36. MW Linn, BS Linn, J Jensen. Psychol Rep54:219, 1984. 37. M Irwin, M Daniels,TL Smith, E Bloom, H Weiner. Brain Behav Immun1:98, 1987. 38. RS Baron, CE Cutrona, D Hicklin,DW Russell, DM Lubaroff. J Pers Soc Psychol59: 344, 1990. 39. ME Kemeny, H Weiner, R Duran, SE Taylor, B Visscher,JL Fahey. Psychosom Med 57547, 1995. 40. JL Fahey, JMG Taylor, R Detels, B Hofmann, R Melmed, P Nishanian, JV Giorgi. N Engl J Med322:166, 1990. 41. K Goodkin, DJ Feaster, R Tuttle, NT Blaney, M Kumar,MK Baum, P Shapshak, MA Fletcher. Clin Diagnost Lab Immunol3:109, 1996. 42. JK Kiecolt-Glaser, LD Fisher, P Ogrocki,JC Stout, CE Speicher, R Glaser. Psychosom Med 49: 13, 1987. 43. JK Kiecolt-Glaser,S Kennedy, S Malkoff, L Fisher, CE Speicher, R Glaser. Psychosom Med 50213, 1988. 44. JKKiecolt-Glaser, VVB Malarkey,MChee, MT Newton, JT Cacioppo,HMao,R Glaser. Psychosom Med 55:395, 1993. for 45 * E Light, BD Lebowitz, Alzheimer’s Disease Treatment, Family Stress: Directions Research. Rockville, MD: National Institute of Mental Health,1989. rr, JM Zarit. The Hidden Victims of Alzheimer’s Disease: Families 46. Under Stress. New York: New York University Press, 1985. CE Speicher. 47. JK ~iecolt-Glaser,RGlaser,ECShuttleworth,CSDyer,POgrocki, Psychosom Med 49523, 1987. 48. BA Esterling, JK Kiecolt-Glaser, JC Bodnar, R Glaser. Health Psychol13:291, 1994.

49. A Baum. Health Psychol 9:653, 1990. 50. cKinnon, CS Weisse, CP Reynolds, CA Bowles, A Baum. Health Psychol 8:389, 51.

rnetz, J Wasserman, B Petrini, SO Brenner, L Levi, P Eneroth, H Salovaara, R Hjelm, L Salovaara, T Theorell, IL Petterson. Psychosom 52. B Dorian, P Garfinkel, E Keystone, R Gorczyinski, P Darby, D Garner. Psychosom Med 47:77, 1985. 53. AD Futterman, ME Kemeny, D Shapiro,JL Fahey. Psychosom 54. PH Knapp, EM Levy,RG Giorgi, PH Black, BH Fox,TC Weer 133, 1992.

SVKasl. In: A Steptoe, AMathews,eds.HealthCare,HumanBehavior.London: Academic Press, 1984, p. 41. 56. JK Kiecolt-Glaser, JT Cacioppo, WBMalarkey,RGlaser.Psych 55.

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EABachen,SBManuck,ALMarsland, S Cohen, SI3 Malkoff, Rabin. Psychosom Med 54:673, 1992. 5 8 . AR Caggiula, CC McAllister, KA Matthews, SL Berga, JF Owens, roimmunol59: 103, 1995. 59. W Gerritsen, CJ Heijnen, VM Wiegant, B Bermond, NH Frijda. Psychosom Med 58:

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273, 1996. 60. AL Marsland, SB Manuck, TV Fazzari, CJ Stewart, BS Rabin. Psychosom 295, 1995. 61. PJ Mills, CC Berry, JE Dimsdale, MC Ziegler, RA Nelesen, BP Kennedy. Brain Behav Immun 9:61, 1995. 62. EABachen,SBManuck, S Cohen, MF uldoon, R Raible,TBHerbert,BSRabin. Psychosom Med 57:366, 1995. 63. TB Herbert,S Cohen, AL Marsland, EA Bachen, BS Rabin, Psychosom Med 56:337, 1994. 64 SB Manuck,S Cohen, BS Rabin, MF Muldoon, goutas-Emch, JT Cacioppo, BN Uchino, W 65. aser. Psychophysiology 31:264, 1994. 8:269, 66. AA Stone, HB Valdimarsdottir, ES Katkin, J Burns, DS Cox. Psychol Health 1993. 67. CS Weisse, CN Pato, CC McAllister, R Littman, A Breier, SM Paul, A Baum. Brain Behav Immun 4:339, 1990. A Baum. Int J Behav 68. SG Zakowski, L Cohen, LMH Hall, K Wollman, 1994. Zakowski, CG McAllister, M Deal, A Baum. Health Psychol11:223, 1992. 69. Benschop, EES Nieuwenhuis, EAM Tromp, GLR Godaert, RE Ballieux, LJP van 70. ornen. Circulation 89:762, 1994. D Naliboff, D Benton, GF Solomon, JE orley, JL Fahey, ET Bloom, T 71. Gilmore. Psychosom Med 53: 121, 1991. Naliboff, GF Solomon, SL Gilmore,D Benton, JE orley, JL Fahey. J Psychosom 72. 39:345, 1995. S Ortega,NCummings.BrainehavImmun 6:141, 73. VVJ Sieber,JRodin,LLarson, 1992. Uchino, JT Cacioppo, W Malarkey, R Glaser. 74. Brosschot,RJBenschop, GL Godaert, MBM 75. allieux. Psychosom Med 54:394, 1992. IL Kutz, C 76. B Crary , SL Hauser, M Borysen ko, enson, J Immunol 131:1178, 1983. JH vanTits,MCMichel,HGrosse-Wide,Happel,FWEigler,ASoliman, OE 77. Brodde. Am J Physiol258:E191, 1990.

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~enetics I~stitute, Inc., ~ a ~ b r~assachusetts i ~ ~ ~ ,

Interleukin 12 (IL-12) is a u n i ~ u ecyto ine produced by antigen presenting cells. As discussed in more detail in sev recent reviews [l-31, among the activities of IL-l2 are several whichbear on its entia1 asavaccine adjuvantforbothpromoting cellular immune responses and modulatin and enhancing humoral responses. 'vities of IL-l2 is i duction of y-interferon (IFN-7) from T cells. Enhancement of antigen presentation and suppression 11 development by IFN-y play key roles in p r o ~ o t i n gand directing the immune response developed in the presence of IL-12. Interleukin-l2 also specifically and uni~uelypromotes the differentiation of T helper progenitors to be come Thl cells. In both mice and humans atleast two classes of T helper cell subsets thatexert specific effects on immune development develop. T helper 1(Thl) cells express IFN-y and IL-2 cytokines that promotea cellular immune response by activating macrophage and enhancing development a cells. Th2 cells express IL-4 and IL-5 and lack expr on of IFN-y and IL-2. As a conse~uence, Th2 cells promote the differentiationo ells to express noncytophilic antibodies (e.g., immunoglobulin G1 IgGl] in mice) and IgA and recruit eosinophils, Although IL-12 has only moderate activity in promo cells, costimulationwithaccessorymoleculessuchashas been. showndramatically to enhanceproliferationof C '8 T cells induced by IL-12 141. Thus as an adjuvant,IL-12 has the potential to(1) promote differentiation of antigen-specific Thl cells capable o N-y response in to reexposure to differentiatio capable recognizing of endogeantigen, (2) promote nously expressed antigens encoded rus and tumor cells, (3) expand the population of antigen-specific cells alone and more orpotently combination in with other costim dulate the antibody response via IF - -dependentandIFN-y-independentmechanisms to express cytophilic antimportant in opsonizing targets and leading to complement activation. The

7

evidence for these activities and their functional conse~uences are §ummarized in the following discussion.

that immune mechanisms and IFN-y expression are necessary for initial cure and notti et al. [8] confirmed the role i exposure of tumor-bearing mice G26 melanoma growth as aresul t is cuss ion) protected naivemice from tumor gro cells. These findings suggest that I tumor-specific immunity, in effect a and thus support the notion that exogenous tumor antigen.

Immuni~ationwith cells ( t u ~ o or r fibroblasts) transfected wit cyto~ines or other factors has been tested for efficacy in many tumor models wit varying degrees of success 191. Local andsustained delivery ofcytokine at the tumor site and the

in atleast one case tumor cell expres vaccine than the combinationof turn al. [lo] inoculated mice with either

Zitvogel et al. treated mice beari

were treated with irradiated tumortransfected with control plasmid, In control experiments9 tumor bearing mice were treated with IL-12 by i.p. injection. Of mice therapeutically immunized with IL-12 transfected tumor 90% were cured and resistant to rechallenge with wild-type tumor. Of mice in the other groups 0 ~ 0 - 1 0 were ~ 0 cured.

In studies perhaps more directly relevant to clinical practice, several investigators have demonstrated the efficacy of IL-12 as an adjuvant in therapeutic antitumor ous for three missense point mutatide encoding one of these is capable cell responses [12]. Vaccination with this peptide in combination with QS21 given subcutaneously and IL-l2 given i.p. elicited a T cell response and cured mice of established tumor [13]. Immunization with any combination other than all threewas not effective. The importanceof both cells was demonstrated by depletion with antibodies. primary but not theboost immunization abrogated icells at either the primary immunization or the boost abrogated lly delivered IL-l2 asadjuvant in tumor models has also antigen delivered as complimentary deoxyribonucleic acid virus. Irvine et al. [l41 treated mice bearing established T26 melanoma cells transfected with p-galactosidase [ped with @-gal cDNA on day 2 and given IL-12 i.p. on days 3 through 7. The number of pulmonary metastases measured on day 12 was uced by the combination therapy relative to treatment lone. In subsequent studies with the sametumor model [ 151 used vaccinia virus expressing 1. Viral inoculation was on day 3 and IL-12 on days 3 through 5 for the cDNA immunogen, onlymice given L-12 showed signifi reduction in pulmonary metastases on day 12. The combination also prolonged survival of tumor bearing mice. Depletion of '8 cells, beginning immunization, e abrogatedthetherapeutic response. owever,depletion of cells onlypartially reduced the efficacy. hese results directly demonstrate the activity of IL-12 as an of established tumor in mice and establish a role for bot ells in the response. Nevertheless, the mechanism by which th cell populations cure established tumors, the optimal protocols for using IL-12 in combination with antigens, the optimal immunogens, and the means to suppress escape variants remain dauntingchallenges.

L-l2 as a key mediator in antigens and well-char-

If

I

acterized mechanisms have extended these in vitro studies to animal models and established the role and efficacy ofIL-12 in promoting Thl responses in vivo. Afonso et al. [l71 were first to establish that IL-12 given as an adjuvantin combinationwithsolubleantigenpromotedaprimary Thl response in vivo. Micewere inoculated subcutaneously (s.c.) with soluble leishmani~lantigen mixed with IL-12. Lymph node and spleen cells collected on day 10 after immunization and cultured in vitro in the presence of antigen expressed elevatedlevels of IFN-y anddiminished levels of IL-4, characteristic of a Thl response. Immune cells from mice immunized in the absence of IL-12 expressed the converse profile. Finkelman et al. [l81 made similar observations in mice immunized with the nematode parasite ~jppostrongyius ~r~sjijensjs. As adjuvant IL-12promotedenhancedexpression of IFN-y on challengewithparasite. In a more defin~dha~ten-proteinmodel syste ght et al. [l91 demonstrated that immunization with IL-l2 as adjuvant an enhancement of antigen-specific IFN-y expression from lymph node cells and suppression of IL-4 expression. Extensions of these studies by liss et al. ~20,211 established that L 1 2 in the absence of other adjuvants was sufficient to promote a primary Thl response to a soluble protein antigen. Moreover, in mice immunized with L 1 2 as adjuvant a Thl recall response thatwas retained for atleast 6 months developed. ~ p t i m a conditions l for the use of I -12 and antigen remain tobe established. Nevertheless, these findings indicate that, in mice, not only is I - 12 effective as an adjuvant in promoting a primary Thl response in vivo, but that the capacity to recall an antigen-specific Thl response promoted by IL-12 is long-lived. The characteristics of the memory cells induced by immunization with IL-12 as adjuvant remain anactive and interesting area of study [22-241.

y-Interferon mediated immune activation is a central mec anism in the cure of many parasitic diseases. In fact, the activity of IL-l2 in promoting cure of Leish~ ~ n j ~ - i n f ~mice c t e [25,26] d by induction of IFN-y was key in establishing the role of IL-12 as an immune modulator. Efficacy in several models has been demonstrated.Inconjunction with thestudies whic ished the activity of IL-12 as adjuvant in promoti~g t al. demonstrated the that igen and IL-l2 was sufficient sequent parasitechallen ~accination protoc effective pa other in ~accinationof mice with radiation attenuated cercariae of S c h j s t o s o ~ ~ ~ ~ n s o n j can promote protectionmediated by CD4' T cell and IFN-y. Nevertheless,standard immunization protocols elicit both Thl and Th2responses and only partial protection. Studies by ynn et al. [27] demonstrated that immunization with irradiated cercariae IL-12 enhanced parasite-induced IFN-y expression in lungs and significantly reduced parasite burden. Neither IL-12 nor cercariae alone had comparable ountford et al. [28] ext~ndedthese studi~s toshow that soluble S. ~ ~ n s antigen o ~ j IL-12 could also afford significant protection and that the protective response was associated with development of a Thl response. In addition to su~gesting the use of IL-12 in combination with some antigens

+

+

for IFN-y-sensitive infectious diseases such as leprosy, tuberculosis, leishmaniasis, and schistosomiasis, these findings emphasize the potency of IL-12 as an adjuvant in promoting immune memory associated with IFN-y production.

A consequence of the shift in cytokine expression induced by immunization with antigen and IL-12 is a shift inthe isotype of antigen-specific antibodies. y-Interferon promotes differentiation of B cells toward production of cytophilic antibodies in mice (IgG2a, IgC2b, Xg ). Absence of Th2-associated cytokines (IL-4 and IL-5) reducesdevelopment of ells expressing IgCl e,IgE,andIgA.These effects have been confirmedin era1 model systems. ightetal.[l91showedsignifilevels inaprimaryresponsein mice immucantreduction of hapten-spec nized incombinationwithILive tocontrols.Hapten-specificIgC2a levels were concomitantly elevated. al. [20] extended these findings and demonstratedenhancementofIgC2aexpressionmediated by a recall T cell response in mice immunizedwithantigen IL-12. Similarfindings were obtained by Buchanan et al. [29] using a second model antigen system. In a thorough examination of the activity of IL-12 in inducing complement-fixing antibody subclasses in mice, Germann et al. [30] demonstrated that IL-12 as adjuvant promotedincreased IgC2a, IgC2b, and IgC3 levels to phospholipase A2 (PLA,), keyhold limpet hemocyanin (KLH), and typeI1 collagen. Levels of IgCl were slightly enhanced and IgE was suppressed. upp press ion of a mucosal IgA response by the use of L 1 2 as adjuvant was used to advantage by Yang et al. [31] to allow repeated gene therapy with recombinant adenovirus. With no additional manipulation, mice infected intratracheally with adenovirus are resistant to reinfection. Resistance is antibody-dependent. Yang et al. [31] demonst~ated that treatment with IL-12 (intratracheally or i.p.) at the time of primary infection allowed animals to be reinfected and express a virally encoded reported gene. Depletion of CD4' cells before the primary infection and treatment with IFN-y were also effective. Concordant with the ability to reinfect el and a selective inhibition of

+

In addition to shifting the antibody isotype toward complement-fixing subclasses, IL-12 appears to enhance absolute levels of antibody expressed in serum. These findings were suggested in the data of Cermann et al. [30] and established more liss et al. [20]. In the latter study, hapten-specific IgG2a and IgGl levels were significantly enhanced after hapten-protein challenge of mice primed with hapten-protein IL-l2 relative to those of mice primed and challenged with hapten-protein alone. The mechanism for enhanced antibody production remains to be determined but may reflect expansion of T helper cells and enhanced cytokine expression effectedby IL-12. Thesefindings suggest that IL-12 may be effectivenotonly in promoting

+

7

humoral immune responses with greater efficacy in cases where complement fixation is important, but also in enhancing the magnitude and/or kinetics of the humoral response.

2 in modulating and enhancing humoral immunity has been demonstrated in several models. Schijns et al. [32] demonstrat~d thatIL-12 as adjuvant with an inactivat pseudorabies virus (PRV) vaccine enhancedprotection fromlethal challenge.sistancecorrelated with increased serum titers of PRV-specific IgC2a and was dependent o expression. In a second model, Wynn et al. 1331 demonstrated that multiple zations with irradiated S. ~~~~0~~cercariae I ~ - 1 2enhanced expression of antigen-specific IgG2a, Ig62b, and IgG1 isotypes and that the serum from these mice increased protection in naive recipients.

+

Overall, the studies summarized argue strongly thatIL-12 is a potent vaccine adjuvant with potential in many therapeutic and prophylactic indica~ions. Its abilityto enhance cellular immunity to specific tumor antigens (both described andyet to be identified) and demonstrated efficacy in therapeutic antitumor models suggest that prudent and creative extension to the clinical setting holds great promise. In addition the sustained memory response for antigen-specific IFN-y production offers opportunities for prophylactic immunization against many parasitic diseases. Finally, the observation that IL-12 shifts the humoral response to expressionof antibody subclasseswhich fix complement and the finding that it also increases antibody titers offers thepotential for IL-12 as a prophylacticvaccine in many viral andother infectious diseases.

1. G Trinchieri. Interleukin-12: A cytokine produced by antigen-presenting cells with immunoregulatoryfunctionsinthegenerationofT-helpercellsTypelandcytotoxic lymphocytes. Blood 84:4008-4027, 1994. 2. G Trinchieri.Interleukin-12: A pro-inflammatorycytokinewith muno no regulatory functions that bridge innate resistance and antigen-specific adaptive im~unity.Annu Rev Immunol13:251-276, 1995. 3. JA Hendrzak, MJ Brunda. Biologic activity, therapeutic utility, and role in disease. Lab Invest 72:619, 1995. 4. M Kubin, M Kamoun, G Trinchieri. Interleukin 12 synergizes in inducing efficient proliferation and cytokine production o Med 180:2 1 1 -222, 1994. H Edington, McKinney, T H Taha Brunda,Gately, M Nastala, 5 . CL SF Wolf, RD Schreiber, WJ Storku, MT Lotze. nterleukin 12 administra-

7

tion induces tumor regression in association with IFN-gamma production. J Immunol 153:1697-1706, 1994.

e is correlated with a striking reversalof suppressed 17~1135-1145, 1995. G FSpreafico,MWysocka, mune response against a murine colon carcinoma cells transduced with interleukin-l2

D Old. A mouse mutant p53 recognized byCD4' and CD8' T

2223, 1995. 14.

immu-of DNA enhancement Cytokine estifo. nization leads to effective treatment of established pulmonary metastases. J Immunol W Carrol, KR Irvine, B Moss, SA Rosenberg, NP Restifo. Interleukin-l2 inan effective adjuvant to recombinant vaccinia virus based tumor vaccines: Enhancement by simultaneous B7-1 expression. J Immunol 156:3357-

, KM Murphy. Development duced macrophages. Science 2603547-549,1993. 17. LCC rinchieri, Afonso,

adju-

The P Scott.

adden, AW Cheever, IM Katona, 12 on immune responses and host protection in mice infected with intestinal nematode parasites. J Exp

, GJ Zimmer, IFogelman, SF Wolf, AK Abbas.Effects of IL-12 on

vaccine adjuvant: Characteristics of primary, recall, and long-term responses. Ann NY AcadSei795:26-351996. A O'Garra. urphy, NA Hosken,ino,KDavis,KMurphy, racellularcytokine S S at the single-cell levelinpolarized T elper 1 and Thelper2populations. J Ex182~1357-1367,1995.

7

lf

KMMurphy.RolesofIFN23. CAWenner,MLGuoler,SEMacatonia,AO’Carra, gamma and IFN-alpha in IL-12-induced T helper cell-l development. Am Assoc Immuno1 156: 1442-1447, 1996. 24. E Murphy, K Shibuya, N Hosken, P Openshaw, V Maino, K Davis, K Murphy, A O’Garra. Reversibility of T helper 1 and 2 populations is lost after long-term stimulation. J Exp Med 183:901-913, 1996. 25. JP Sypek, CL Chung, SEH Mayor, JM Subramanyam, SJ Goldnam, DS Sieburth, SF Wolf, RC Schaub.Resolutionofcutaneousleishmaniasis:Interleukin-l2initiatesa protective T helper Type 1 immune response. J Exp Med 177:1797-1802, 1993. 26 FP Heinzel, DS Schoenhaut, RM Rerko, LE Rosser, MK Gately. Recombinant IL12 cures mice infected with leishmania major.J ExpMed 177:1505-1509, 1993. 27. TA Wynn, D Jankovic,S Hieny, AW Cheever, A Sher. XL-l2 enhances vaccine~induced immunity to Shistosoma mansoni in mice while decreasing Th2 cytokine expression, IgE production, and tissue eosinophilia. J Immunol154:4701-4709, 1995. 28, A Mountford, S Anderson, R Wilson. Induction of Thl cell-mediated responses and n imice, by coadministration of larval protective immunity to ~ c ~ i s f o s ~o a~ na ~ o in antigens and IL-12 asan adjuvant. J Immunol 156:4739-4745, 1996, 29. JM Buchanan,LAVogel,VHVanCleave,DWMetzger.Interleukin-l2altersthe isotype-restricted antibody response of miceto hen eggwhite lysozyme.Intl Immunol7: 1519-1528, 1995. 30. TGermann,MBongartz,HDlugonska,Hess,ESchmitt,LKolbe,EKolsch, FJ Podlaski, MK Cately, E Rude. Interleukin-l2 profoundly up-regulatesthe synthesis of antigen-specific complement-fixing IgG2a, IgG2b and IgG3 antibody subclasses in vivo. Eur J Immunol25:823-829, 1995. 31. Y Yang, G Trinchieri, JM Wilson. Recombinant IL-12 prevents formation of blocking IgA antibodies to recombinant adenovirus and allows repeated gene therapy to mouse lung. Nature Med 1:890-893, 1995. 32. VEC Schijns, BL Haagmans, M Horzinek. Interleukin-l2 stimulatesan antiviral type l cytokine response but lacks adjuvant activity in interferon-~-receptor-deficient mice. J Immunol155:2525-2532, 1995. 33. TA Wynn, A Reynolds, S James, AW Cheever, P Caspar, S Hieny, D Jankovic, M Strand, A Sher. Interleukin-l2 enhances vaccine-induced immunityto schistosomes by augmenti~gboth humoral and cell mediated immune responses againstthe parasite. J Immunol 157:4068-4078,1996,

~ n i v e r s i otf~ Cincinnati Collegeof ~ e d ~ c i~incinnati, ~e, Ohio

ines produced in r infectious agent is critical to the -being of the host. 4' T cell responses that express a distinct pattern of type 1 cytokines (interferon-~[I lymphotoxin [LT]) or type 2 c ~ o k i n e s(IL-4, IL-5, often suffici~nt to neutralize the infectivity of distinc their capacity to cause disease. subsets and cells of the innate i macrophages, and neutrophils) that init responses9 important in the control of i

the develop~entof T helper l omavariety of T cell subs omeostasis is maintained by tions of these cytokines acros counterregulatory cytokines host resistance that is an i e interactions with the host that minimize divert or protective an avenue for infection and convert acute disease to more rnatively, positive feedback induction of high concentrations of protective cytokines can limit superinfection by homologous parasites and reduce competing heterolo~ousinfections that debilitate the host or interfere with the further develop~entof the primary parasitic infection. Furthermore9unregulate~ pro-

mes chronic 161. imilar treatment at the time of a secondary challenge self-cure of the primary infection, does not block resistance. ear to engage directly in worm killing but promote velopment of ~ r i c ~ u r i s to ~ uegg-laying ris (fecund) adult worms in the cecum and proximal colon is strictly Treatmentofnormallyresistantmousestains withanti-Clocks resistance and allows larval development to mature egg-laying adults [cl] rus normal1 esta~lishesa chronicprimaryinfection in the small intestine of aracterized by pe nt worm burden and long-term egg excretion limiting development of new superinfections ting infective larval develo~mentto adult therapeutic drug cure of 4 enhances worm fecunS resistance to a challenge infection is]. T rongly induced by all of these parasites, tion of infection varies. Neutralization b has no conse~uencein any of these gastrointestinal ema at ode infections.

dependent because injectio~of neutralizing a n t i - ~ ~ mAb N - ~ restores normal

and a functional STAT6 molecule are needed to activate type 2- ependent expulsion of N. br~siZie~sis and (2) IL-4 is not required to induce worm e ulsion in immunocompetent mice suggest that another cytokine that binds to the IL-4 activatesSTAT6,most likely IL-13, can ce wormexpulsion.Thishypothesis is supported by the fact thatanti-IL-can effectivelyblock e~pulsionof ALWc bac~ground), butnot normal IL-4 intact oben-Trauth, and Finkelman, unpublished results). e been treated with exogenous IL-4cC provide insi mechanismsof c~okine-induced wormexpulsion,OnesuchIL-4-dependenteffector mechanism appears to change parasite feeding behavior in situ. N i ~ ~ o s t r o ~ g y br~silie~sis ~ ~ ~ not do have mouthparts that directly engage host e~ithelialtissue and blood but may damage microvilli and epithelialcells by the mechanical action of their cuticular ridges, with subsequent oral ingestion of cellular tents via a s u c ~ i n gaction created by their muscular esophagus[1S] injected intravenously into N. ~r~silie~sis-infected SCImice leaks into the lumen and is subse~uentlyingested by the worm and becomes visible in its intestine when explanted worms are viewed with fluorescence microscopy. Treatment of infected mice with IL-4C over a 3 day period results in an inhibition of dye uptake by the worm (Urban, Rinehart, & Finkelman, unpublished results). ~nte~leukin-4de~endentreduction in worm feeding lowers metabolism, reduces fecundity, and laces wormsfromtheirintestinal niche. The exactmechanism is may be related t o 1 ~ - 4 ~ - i n d u c e dcontraction of ga~trointestinal smooth muscle and changes in the neuroimmune regulationof epithelial ion transport in the host intestine [19,20].

Susceptible mousestrains eshibit chronic infectionof the cecum and proximal colon after ingestion of infective 7'. ~ ~ reggs, i s while resistant strains expel worms at different intervals before devel~pmentof egg-laying adults [21]. Changes in the in vivo cytokine environment, through reciprocal activation of T helper subsets, can interchangeably convert chronic and acute mouse nt in strains [22]. Chronic T. ~ ~ rinfection i ssusceptible in A mice induces a Thldominatedcytokineresponse;treatment of these mice with a n t i - 1 F ~ - mAbinand IL-5 synthesis and worms are expelled. In contrast, mice and have Th2-dominant a res~onse att tern; t r e a t ~ e ected 2 response and resultsin ce with anti-IL-4RmAbblocksthetype chronic in~ection. In addition,injection of IL-4cC cures chronically infected AKR mice, while treatment of normally resistant mice with IL-12, during the secondweek of infection, induces an I F ~ - ~ - d e p e n d e increase nt in host susceptibility (A. J. J. Else, &c R. K. Crencis, personal communication). Unlike obsermice infected withN. brusi~ie~sis, resistance to T. ~ ~ ris associi s ated with type 2 cytoki -dependent mechanisms because IL-4mice cannot expel 7:~ ~ rnorm i sall^ (A. .Crencis, personal communication). Conditions for resist ~ygyr~ are dependent sthealso on balance b e t ~ e e n T h l / T hcell~regulated 2 mechanisms. Some protective features are

with those regulating d mice. Injection of IL

evelopment of N. brasiliensis and T. ~ u $ i in s res primary H. polygyrus infection in immuno-

iensis. Role of K - 4 in Resistance to eligmosomoidespolygyrusTreatmentwith us feeding behavior in /candSCID mice with a amine dye injected intravenously intomice with a primary visible in the intestine of explanted worms within 45 min a similar injectionof dye into H. polygy$us-infected mice treated 18 hr earlier with a single intravenous injection of IL-4C greatly reduces worm uptake of dye compared to worm uptake from mice not given IL-4C. The nse is more rapid than that observed in chronicN. ~$asiliensisppressed mice or N. brasiliensis-infected SCID mice treated ; two injections of IL-4C over 3 days is required to alter dye uptake by N. ~rasiziensis. Thismay relate to the direct tissue ingestion by H. polygyrus and the intimate interaction of the worm with host blood-borne resistance factors [23] versus dilution of effectors into the ambient microenvironment of N. brasiliensis which feeds on cellular debris and lumen contents. Adult egg production (EPG) can drop substantially by 18 hr after IL-4C treatment of infectedmice,suggesting thatsubtlechangesinwormmetabolismmayrapidly follow host exposure to IL-4, but are manifested later as reduced egg output. The mechanismremainslargelynown, but IL-4induceschanges in hostintestinal physiologicalmechanismsi poly~yrus-infected mice,some of which are mediated by products of mucosal mast cells ( CS). Thesechangesinhostintestinal physiological characteristics may create an altered microenvironment that is inhospitable to the worm and may contribute to its expulsion. First, increased small intestinal smooth muscle contractility is observed during an immunologically enhanced secondary polygy~usinfection [24], but not during a primary infection, when immunity is S evident [25]. This effect is blocked by treatment with antimAb and induced in normal uninfected mice by injection of IL-4C over a 6-day period. It is not seen in mice with a primary infection after a single injection with IL-4Cy even though worm feeding behavior is altered under these conditions. Changesincontractilityinduced by prolongedIL-4Ctreatment are blocked by

an i n ~ i ~ i t oofr leu~otrien~

by treat~entwit^ anti-

mucosa derived fro

e ~ i t ~ e l icells. a l In

le site in the in r o i ~ t e s t i n ~nern l

mAb at the timeof inoculation with C. p with low-level oocyst output that is great [36].Neonatal ice represent a model of treatment with anti-IFN-7 mAb natural infection because they are initially susceptible, but age-related develops within a few weeks of birth; in C. p ~ r v # ~ - i n o c u l a t eneonatal d mice chronic infection slowly develops. The infection apid increase in IFN-y cytokine gene expression in esenteric lymph nodes without increases in IL-2 or [37]. Treatment with exogenous rIL-12 enhances IFN-7 gen tion of rIL-12 before inoculation with C. ~ ~ oocysts r c v ~ ~ opment of the pa~asite. This effect is immune system-ind activated in neonatal SCI

ut a regulatory role for endogenous production of these cytokines is indicated by the fact that disease is exacerbated and the number of intestinal epithelial cells increased ~ afterinjectionof C. ~ ~ r v ~ ~ - i n f e c t e d infectedwith C. p ~ r v # with mAbs that neutralize IL-12 and IFN-7 [371. Although a link betwken IL-1 an increase in ~FN-7-dependent NO productionhas not been made in this infection model,reductionsinNOproductihavebeenlinked to enhanc oocyst output in athymic mice [39). owever, the observation that mice and their normal litter mates are comparably resistantto infection with C. ~ ~ r v # ~ does not support a role for NO in blocking intracellular parasite development in intestinal epithelial cells (Urban, Trout, fink elm an^ & Fayer, unpublished results).

The IL-4~-in~uced amelioration of a n established adult p o ~ y g ~infection r~s in e indicates that adult worms are one target of an IL-4-dep ependent protective mechanism[13). Yet there is only a p of adults worms in this system and resistance mechanisms that destroy developing ~ o ~ y g y larvae ~ # s in the intestinal submucosal tissue [25] and shortly after erge from the tissue into the intes [40~may be r e ~ u i r efor ~ effective protection. Immunoglo~ulin ajor immunoglobulin isotype induced by H.p o ~ y g y r ~ s , apassive nd trans ~ u r i n gthe larval phase of develop Although treatment with anti-IL locks protective immu , it affect does not the polyresponse [43]. IL-4 It activates tis p cell types or chanisms that synergize with IgC, antibody to facilitate expulsion or that production of parasite-specific IgC, antibody is more IL- -dependent than the polyclonalresponse.Adults from a primaryinfectiongenerallyremaininplace

during elimination of larvae from a challenge inoculum (concomitant immunity) [25,44]. Concomitant immunity is IL-4-dependent because mice treated with antimAb andgiven three successive inoculations withH . ~oZygyr#sover a 21-day accumulate increasing numbers of adults, while control mice establish an adult worm burden derived principally from the initial primary infection that does not increase with additional inoculations with larvae[45]. The ability of the host to limit superinfection by successive larval infections protects the host from an ultimately lethal b u i l ~ u pof adult worms. Although parasite-specific IgG, antibodies were not measured experimen this in ~oZygy~#s-specific IgG, supported by the observation that IL-4 ed for a second time with , are more susceptible to a c fection and a significantly reduce ~oZygy~#s-specific IgG, antibody response dev Zygy~#s-infected, 1~-4-competentcontrol mice Finkelman, unpublished results). It is unlikely that a response to infection is relevant to protection because mice treated thro infection with anti-IgE mAb that blocks parasite-induced IgE synthesis Finkelman9unublishedresults)or mice with a deletedhi receptor for IgE (Fc 0 mice) (~ombrowicz,Urban, & results) are resistant hallenge infection with cell contribution to a protectiv 0 mice are more susceptible to than genetically similarcontrol an, unpublished results). The mice notonlyhave a defectinantibodyproduction, but express a twofold to threefold reduction i 2 cytokine gene expression (Gause & Urban, unpublis results) and a reduc C response to infectionthat suggest that a diminishe cell and/or antibody-enhanced presentationof parasite antigens in these mice limits T cell activation, Thus, afferent processing and presentation of parasite antigens site-specific effector mechanisms that are IL-4-dependent may syne resistance against several stages H. of ~ o Z y ~ y r # s more f o r efficient er studies have measured afferent activation of T cells during parasitic infection [46,47] and demonstrated that the injectionof the chimeric antibody CTLA4 Ig, which blocks the interaction between T cell C ~ 2 8 / C T L A 4with and B7-2 on antigen presenting cells (APCs), into BALB/c mice inoculated H. ~ o Z y g y ~ blocks us parasite-induced T cell activation that normally leads to the S treatment, or the alternative strategyof specific antibody 2 on APCs, also inhibits T cell-dependent parasite-induced ood eosinophils9 and mucosal mastcells. In contrast, mice given CTLA4 Ig only at th time of a secondary challenge infection with H. ~oZygyrushaveunalteredtypecytokinegeneexpression;IgE,IgG,,eosinophil,and MMC responses; and resist nce to infection, indicating thatparasite-induced memory T cells have a diminisuirement forcostimulatory molecules [48]. 4of course entire Ig the during ever, weekly treat primary the both a oZygyr#s resistance reducedid T cell activation to a challenge infection, indicating that continual interference with byblockingcostimulatorymoleculescanaffectprotectiveimmunity(Urban Cause, unpublished results). In marked contrast to the effect of treatment of H.

interaction is no he activation of

chal~en~e-ex~ose~ > 5 ~ red~ction ~ 0

N.~ r ~ s i Z i e ~larvae s i ~ can establish a chronic i~fection

ode antigens that has the lytic enzymes used di~estionof host tis likely ever, the nature of these common features would response specific could that resistance regulate a to of classically induced type 2 specific action the e production, can be largely nontheir response. There is rowing evidence that

expected to act in a very specificmanner.

~ o ~ y g yThere r ~ ~are . apparent advaninherent in the facts that (1) a single cing multiple effects, some or all of which (2) some parasites appear to be susceptible to that can be based on different cytokine-induced 1 sense for the host because(1) it is unlikely that in a particular parasite an adaptivestrategy to avoid multiple resistance mechanisms could simultaneously develop and (2) host responses to commonparasite features rather than specific charactersare auseful strategy to combatparasites that share similar life cycles and reside in sites in the host intestine that often maintain mixed infections with multiple generaand species. S

~ o r m a l l yacute infections with gastrointestinalnematodesinresistanthostscan become chronic if low-dose inocula are used to establish the infection [55,60]. In contrast, disease becomes self-limiting and immunity is established in the normally infections with the protozoan L. ~ ~ j o r tions in which low antigen or infective f a Th 1 response and the down nt with the classical observation of and antigen dose determine the ex ession of the generally ~ n t a g o ~ i s tconditions ic of a delayed-type hypersentitivity versus atory role for cytokines in determining e infected with L . ~ ~ j has o rbeen sh nd intervention with anticytokine neutral

shift susceptibility and resistance [63]. Footpad lesion size, after inoculation with r from l@to lo7 organisms, was compared increasing doses of L. ~ f f j ometacyclics for resistant C57B1/6 and susceptible /c mice. All infectiousdoses were selflesion size was doselimitingin the C57 116 mouse strain, although maximum dependent. In cont st, only the lowest infectious dose ( l e ) was self-limiting in /c mice inoculatedwith lo2 meta/c mice. Resolution of the lesion in cyclics was dependent on the in situ c ~ o k i n emilieu because treatment of infected mice with anti-IL-4 mAb considerably shortened the time of lesion resolution while treatment with either anti-IFN-7 or anti-IL-l2 markedly exacerbated and accelerated lesion size and development. This phenomenonwas immune system-dependent because lesions failed to resolve in SCID mice at any infectious dose and were refractory t o treatment with a n t i - I ~ - 4 , a n t i - I L - l ~ , o r a n t i -mllbs. I ~ ~ - 7It was of additional interest that infectious dose ra an parasiteantigen load appeared to becritical to modulation of lesion size. /c mice given lo2 rnetacyclics along with 106heat-killed metacyclics or lo6nonreplicating procyclics,to increase antigen nonreplicating parasite source, were still able to exhibit lesion size owever, in vitro parasite antigen-induced productionof IL-4 from the iteal lymph nodes of these mice taken at l wee and 7 weeks after inoculation revealed a transient burst of IL-4 production only frommice that were injected with the high antigen load inocula at 1 week after exposure. It would appear /c mice, a sustained stimulation of T cells by an optimal number of infectious particles is required to promote Th2 development and suppress Thlcell development. The balance between Thl and Th2 responsiveness may be ultimately dependent on the cytokine milieu [64,65], the availability of cell-to-cell costimulatory molecule interactions1661, or the natureof APCs [67] that engage thereplicating organism and influence subsequent cell T subset development. Infectious dose is also critical resolving in a mouse infectionwith a gastrointestinal nematode like T. ~ # r [68]. ~ s Adult worms fail to mature in BALB/K mice inoculated with a high dose of eggs (400 eggs), and resistance is associated with increases in parasiteantigen-inducedIL-5andIL-9,andlittleIFN-7synthesis. However, a low-dose infection results in a patent infection and sus challenge infection. The cytokine patternin the low-dose inoculated is more Thl-like, T cell subset development is also affected by diffe 7'. ~ # r i 1693 s and reflected in their potential to elicit protective immunity in differexpress agenetically determined ent strainsof mice [70]. Three inbred mouse strains responsephenotype to infection with 2". ~ ~ where r the ~ speed s of expulsion parasite is strain-d A mice high are responders, C57 sponders, and B10 mouse all hosts; in strains the isolate was expelled early, E/N isolate expelled later,and S isolate very late ornotat all.Susceptibilitywascorrelatedwith low parasiteantigen-induced release of IL-5 in vitro, high IFN-7 release, and a predominant parasite antigenspecific IgGzaantibody response; resistance correlated with high IL-5 release and a predominant IgG, antibody response to parasite antigens. The fact that these isolates invade similar sites in the host intestine with initially comparable infection densities would suggest that differences in parasite-derived products among the isolates are skewing the cytokine response pattern to favor resistance or susceptibility.

A polarized cytokine response would inherently be expected to down-regulate its reciprocal cytokine pattern 171-731. Therefore, the cytokine statusof the host could influence the process of active immunization or natural development of immunity that can control an infection.Inductionof a strong T h l responsewould likely interfere with successful control of gastrointestinal nematode infections that are sensitive to Th2-dependent protective mechanisms. In fact, treatment of BAL mice with exogenous rIL-l2 (12), rIFN-y or rIFN-a 1'741 down-regulates the type protective response to N. brasiliensis. The parasiticstage of N.brasiliensis migrates to the lungs and then to the small intestine after skin penetration or experimental subcutaneous injection of infective larvae, Larvae migrating through the lung induce an eosinophil-rich perivascular infiltration,and furthermigration and development in the intestine are accompanied by mucosal mastocytosis and systemic IgE production. The stereotypical type 2 cytokine gene expression pattern of high IL-4, IL-5, and IL-9 production is completely reversed by treatment with IL-12 via an IFN-y-dependent down-regulation of the response. As a result, there are anabsence of an inflammatory response he lungs and decreases in blood and tissue eosinophilia,IgEsynthesis, and M development,Inflammatory andimmune cell attack of developing larvae and adults is reduced and worms are more robust, are larger, and producesignificantly more eggs over an extended period pastthe time of self-cure. Once IL-12 treatment is terminated, expulsion of adult N. brasiliensis follows a period of increasing type 2 cytokine activity, indicated by increases in MMG and serum , probablyfrominduction of Th2 cells by productsderived of IFN from a metabolicenhancedwormpopulation.Endogenousactivators can also down-regulate N. brasiliensis-induced type 2 responses in rily inhibit parasite expulsion. For example, injection of mice with a b o r t ~ cells s [75] or inoculation with live oocysts of ~ i ~ e r i a ~ e r r i s i , zoan parasite ofmice that invades the intestinal mucosa, will temporarily block expulsion[54,74], This effect is IF~-y-dependentbecause itis reversible by neutralization of IFN with specific mAb. This type of interaction could be of epidemiologicalsignificance if h~lminth-infectedindividuals are exposed to microbes or intracellular parasites that stimulate strong type l responses leading to IFN-dependentsuppressionofwormresistancemechanisms that couldincrease worm fecundity and enhance the spreadof infective stages. One could alsoimagine exacerbated disease conse~uences frommore robust worm activity in a suppressed host, such as enhanced muscle larvae deposition by Tric~inellas~iralis,copious blood loss fromhookworm-infected individuals, or superinfection by pinworms and S t r o ~ g ~ l o ispp. ~es ~ o r m - i n d u c e dpolarization toward type 2 responses and down-regulation of T h l immunity may be significant t o ~ u m aand n animal populations witha propensity to acquire large worm burdens. For example, tetanus toxoid '(TT) vaccination n s o ~ i results in an in vitro T h ~ - l i ~ e of humans with a S c ~ i s t o s o ~ a ~ a infection response to TT by their peripheral blood mononuclear cells ( M[Gs), while TTa or Tho-like response [76]. vaccinated but uninfected individuals express Theamount of TT-specificIFN-yproduction by from S. ~ansoni-infected

c)

0 E: c)

I

S

2I

U

#

onse to an infection or fectious agent can also

ive responses that arepolarized have provided insight not t establish resistance to infections but into theway countilitate host s~sceptibility(T induced protection and IL ar events of processing and presentathat lead to para acrophage [ 1-33. how a role reversalfor IL-4 in which y can have an impact on immunologicallyS an also change the physiological characteri hese effectors can be turned off, ctive potential of a “c e storm” [4] that repr tokine-induced damageto the host djuvant andcurative [82,833 have not only been useful ensitization of the an L. ~ ~ j o r - r e s i s t a nphenotypes t but as an antipathology agent that modulates

7

the exacerbating effects of IL-4 during S, ~~~~0~~granuloma formation [84-861. heth her IL-4- or other type 2-related cytokines can act as adjuvants to facilitate responses to certain e~tracellularparasites remains to be seen, but the generally antiin~ammatoryproperties of type 2 cytokines couldbe important therapeutically in limiting disease [81,87,88]. However, there is a caveat: counter regulator^ cytokines, when inappropriately applied, can also limit protective responsesand directly or indirectly contribute todisease ~77,78,8~].

1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21 22. 23. 24. 25. 26.

, 1995, SL Reiner, RM Locksley. Annu Rev Immunol 13: 15 1 SL Reiner, ZE Wand,F Hatam, P Scott, RM Locksley. Science 259:1457, 1993. AE Wakil, Z Wang, RM Locksley. Exp Parasitol84:214, 1996. CA Biron, RT Gazzinelli. Cur Opin Immunol7:485, 1995. FD Finkelman, T Shea-Donohue, J Goldhill, CA Sullivan, SC Morris, KB Madden, WC Gause, JF Urban Jr. Annu Rev Immunol15:505, 1997. IM Katona, JF Urban Jr., FD Finkelman. J Immunol 140:3206, 1988. K1 Koyama, H Tamauchi, Y Ito. Parasite Immunol 17:161, 1995. JF Urban Jr., IM Katona, FD Finkelman. Exp Parasitol73:500, 1991. B Madden, JF Urban Jr., WJ Ziltener, JW Schrader, FD Fink~lman,IM Katona. J Immunol147:1387, 1991. RL Coffman, BWP Seymour,S Hudak, J Jackson, D Rennick. Science 245:308, 1989. RA Lawrence, CA Gray, J Osborne, RM Maizels. Exp Parasit FD Finkelman, KB Madden, AW Cheever, IM Katona, SC Hubbard, WC Gause, JF Urban Jr. J Exp Med 179:1563, 1994. JF Urban Jr., CR Maliszewski, KB Madden, IM Katona, FD Finkelman. J Immunol 154:4675, 1995. FD Finkelman, KB Madden, SC Morris, JM Holmes, N Boiani, I Katona, CR Maliszewski. J Immunol 151:1235, 1993. K Rajewsky,W RAMorawetz,LGabriele, LV Rizzo,NNoben-Trauth,RKuhn, ller, TM Doherty,F Finkelman, RL Coffman,WC Morse 111. J Exp Med 184:1651, 6. K Shimoda, J van Deursen,MY Sangster, SR Sarawar, RT Carson, RATripp, C Chu, RW Quelle,TNosaka,DAVignali, PC Doherty,GGrosveld,WE Paul, JN Ihle. Nature 380:630, 1996. KTakeda, TK Tanaka, W Shi, M Matsumoto, M Minami,S Kashiwamura, K Nakanishi, N Yoshida, T Kishimoto,S Akira. Nature 380:627, 1996. M Ogilvie, VE Jones. Exp Parasitol29:138, 1971. orris, CMaliszewski, JF Urban Jr., CDFunk,FDFinkelman,T Shea-Donohue. Gastrointest Liver Physiol in press. Sullivan, JM Goldhill, JF Urban Jr., FD Finkelman, SC Morris, CR Maliszewski, Pineiro-Carrero, T Shea-Donohue. Gastroenterology in press. Wakelin. Parasitol96543, 1988. KJ Else, FD Finkelman, CR Maliszewski,RK Grencis. J Exp Med 179:347, 1994. AD Bansemir, MV Sukhdeo. J Parasitol80:24, 1994. J Goldhill, SC Morris, C Maliszewski, JF Urban, FD Finkelman, T Shea-Donohue. Gastroenterology in press. V Chaicumpa, SJ Prowse,PL Ey, CR Jenkin. Aust J Exp Bioi ed Sci 55:393, 1977. PK Goyal.Parasite JM Behnke,FWahid, RK Grencis,KJElse,AWBen-Smith, Immunol 15:415, 1993.

27. 28 29, 30. 31. 32. 33. 34.

ay, M Cervone, A Bernstein, P ata, S Nishikawa, K Yoshinaga, S Kobayashi, K Nishi, S Nishikawa. Develop 116:369, 1992. L Puddington,S Olson, L Lefrancois. Immunity 1:733, 1994. T Kassai, C Takats, PRed1 J Parasitol73:345, 1987. EE Barth, WFH Jarrett, GM Urguhart. Immunology 10:459, 1966. arkos, JB Matthews, L D'Andrea, CS Awtry, AH Lichtman, C Delp, Immunol Rev 124:5, 1991. f, A Sher. Proc Natl Acad Sci USA 90:6115,

1993. obiol Rev50:458,1986. 35. RFayer ,BLPUngar . Irnmunoll47: 1014,1991. 36. BLPUngar,TKao,Jrris,FDFinkelman. K Gately, FD Finkelman.JImmunol 37 JF Urban Jr., RFayer, S Chen,WCGause, 156:263, 1996. uhls, DE Mosier, VL Abrams, L Crawford, RA Greenfield. J Parasitol 80:4$0, 38.

. Infect Immun625173, 1994. 39. Lucia, HA Dunsford,FJ Enriquez. J Helminth01 62:69, 1988. 40. 41. DJ Williams, JM Behnke. Immunology 48:37, 1983. 42. D1 Pritchard, DJ Williams, JM Behnke,TD Lee. Immmunology 49:353, 1983. Katona, JF Urban Jr., MP Beckmann, LS Park, KA 43* smann, WE Paul, Annu Rev Imrnunol8:303,1990. Parasitol47: 116, 1979. 44. E Paul, FL) Finkelman. Proc Natl Acad Sci USA 885513, 45. 1991. P Linsley, JF Urban 46. P Lu, X Zhou, SJ Chen, M Moorman, SC Morris, FD Finkelman, Jr., WC Gause. J Exp Med 180:693, 1994. J Halvorson, P Lu, R Greenwald, P Linsley, JF Urban Jr., FD Finkel47. man. Immunol Today 18: 115, 1997. 48. WC Gause, P Lu, X Zhou, SJ Chen, KB Madden, SC Morris, PS Linsley, FD Finkelman, JF Urban Jr. Exp Parasitol84:264, 1996. J 49. PLu, JF Urban Jr., X Zhou, S Chen,SCMorris,FDFinkelman,WCCause. Immunol 156:3327,1996. so. JF Urban Jr., HR Gamble, IM Katona. Am Zoo1 29:469, 1989. 51. NO Christensen, P Nansen, BO Fagbemi, J Monrad. Parasitol Res 73:387, 1987. 52. RG Bell, DD McGregor. Infect Immun 29:1$6, 1980. 53. JK Dineen, P Gregg, G Windon, AD Donald, JD Kelly. Int J Parasitol7:211, 1977. K Madden, SC Morris, 54. JF Urban Jr., R Fayer, C Sullivan, J Goldhill, T Shea-Donohue, Gause, M Ruff, LS Mansfield, FD Finkelman. Vet Immunol ImmunopaDC Jenkins, RF Phillipson. Parasitol62:457, 1971. 56. AJ Curry, KJ Else, F Jones, A Bancroft, K Grencis, DW Dunne. J Exp Med 181:769, 1995. 57. J Hermanek, PK Goyal, D Wakelin. Parasitol Immunol 16:111, 1994. 58. V Palanivel, C Posey,AM Horauf, W Solbach, WF Piessens, DA Harn. Exp Parasitol 84: 168, l 996. 59. Urban Jr. Parastiol Today 8:311, 1992. 60. Ali. Ann Trop Med Parasitol78:509, 1984. 61. PA Bretscher, G Wei,JN Menon, H Bielef-Eldt-Ohmann, Science 257539, 1992. 62. CR Parish, FY Liew. J Exp Med 135:298, 1972.

55.

63. 64. 1991. 65. 66. 67. 68. 69. 70. 71. 72. Sci USA 90:948, 1993. 73. 74. 75. 76. 77. 78.

Immunol Today 12346, 1991. en, AWCheever, PP Trotta, I A Svetic,YC Jian, P Lu,FD Finkelman, WC Gause. Int Immunol5:877, 1993. EA Sabin, MI Araujo,EM Carvalho, EJ Pearce LS Mansfield,JF Urban Jr. Vet Immunol Immu LS Mansfield, JF Urban Jr., RR Holley-Shanks, A Canals, W Gause, JK Lunney. Vet Immunol I

79. 80.

81. 82.

SeiUSA 923142, 83. CS Nabora,LCCAfonso, JP Farrell,PScott.ProcNatlAcad 1995 E Williams, TA Wynn, FD Fin 84. r. J Immunol 153:753, 1994. 85. TA Wynn, I Eltourn, I Oswald, AW Cheever, A Oswald, P Caspar,D Jankovic, TA Wynn, E 86. 1994. 87. EJ Pearce, JP Vasconcelos, LR Brunet, EA Sabin. ExpParasitol84295, 1996. 88. F Powrie, RL Coffman. Immunol Today 14270, 1993.

University of North Carolina, Chapel Hill,North Carolina

Survival in a worldofpathogens requires an effective immune response.Some pathogens, such asviruses and intracellular bacteria, invade the host’s cells and seize the cell’s machinery in order to replicate. It is critical for the host’s survival to recognize these assaults an to remove the affectedcells from the body. Theprocess* ndpresentation of endogenousproteinsonmajorhistocompatibilitycomplex C) class I molecules allow the immune system to detect intracellular changes. Antigen presentation involves the degradation ofcytosolic proteins into small peped into the endoplasmic reticulum (ER) throu eptides then bind within a cleft formed by the C/@,-microg1obu1in (@,M)complex is transported cell surface via the normal secretory pathway. This cell surface peptide/ complex is the ligand which the cellular immune system targets for recogni(CTLs), recognize C class I/peptide complexes tion.CytotoxicTlymphocytes and are important and major effector cells in thedestructionof virus-infected cells. The CTL recognition of a virus-encoded peptide complexed to the can result in the destructionof that infected cell, thereby stopping theviral replication cycle within the cell and limiting virus production in the host. The virus’s goal is to produce as many progeny as possible in order to increase the probability for transmission to the next host. Thus, if a viral population contains peptides which elicit strong immune responses, the CTL can easily destroy cells producing that strain of virus. As a result, the immune system selects against such strong immune response-inducing strains. Such selection results in the development of virus escape mutants and initiates the coevolution of virus and host. As a consequence of this warfare, many viruses have generated mechanisms to evade CTL immune response through interfering in one or moreof the steps in normal peptide presentation. For example,awild-typeimmunodominantpeptidecanbed in the virus such thatthepeptide is incapable of binding efficiently to the class Iheavychain. Alternatively, changes in the amino acid sequence can occurin the region surround-

r

7

ing the epitope. Such changes may leadto a diminished ability for the proper epitope to be generated from the whole protein through altered proteolysis (discussed later). ther viral mechanisms of immune system evasion exist, including viral latency; down-regulation of M C class I, or adhesion molecules; and rep~ication in “immune-privileged”sites [l]. Our focus in this reviewis on alteration of viral epitopes to induce stronger, more effective CTL responses while retaining activity toward the original virus-encoded peptide. In somecases, the coevolution of mammals with viruses and intracellular bacteria has resulted in pathogen genomes lacking strong immunodominant epitopes. The mammalian host is consequently hindered in its ability to mount a strong, specific, effective, and protective response quickly. A method to improve on these moderate to weak epitopes in viruses, intracellular bacteria, and tumors is imperative in order to generate epitopes which can induce enhanced CTL activity. Epitope improvement can lead to more direct and efficient immune responses in vaccine development and bypasses the higher costs, side effects, and lower immunogenicity associated with some attenuate^ or heat-killed virus-basedvaccines.

a

~ Strffcture ~ ~and eunction plex class I restricted antigen presentation primarily y chain binding peptides which have been generated 1. Several lines of evidence implicate the proteasome as involved in this degradation of cytosolic proteins for antigen presentation. First, S encoding for twoof the proteasome% subunits, low-molecular-weight protein P)-2 and LMP-7, are located within the MHC class I1 region [2,3]. Second, roteasome is involved in both ubiquitin-dependent and u~i~uitin-independent protein degradation pathwayswithin the cell. Third, inhibitors of proteasome function, such as dipeptide aldehydes, can block the presentation of model peptides [4]. The 20s and 26s forms of the proteasome produce a barrel-shaped complex of four rings, two inner and two outer rings [S]. In mammals, the outer rings of the 20s proteasome comprise seven different C\! subunits while the inner rings contain seven distinct p subunits. The p chains contain the catalytic sites for protein degra6s proteasome consists of the 20s subunits plus regulatory subunits, P-2 and LMP-7. This 26s proteasome is involved in the degradation odified proteins. ~ b i q u i t i nmodification has been shown to improve the generation of peptides for presentation on MHC class I molecules, thereby the causal linkage [6]. and LMP-7 subunits mayalterthetype of peptides that are d .by pro tea so^^ degradation, althoughthis hypothesis is still controversial. proteasomes from y-interferon IFN-y-induced cells have shown, in vitro, a ecrease in protein cleavage after acidic residues [7,8]. A number of studies have shown that IFN-y stimulation increases proteasome cleavage after basic residues [7,8]. These latter residues are pc many class I restricted peptides. Thus, the L

o~ses

1

peptide pool toward peptides which are able to bind efficiently to class I heavy chains. Further, alteration of the C-terminal-flanking residue of two mouse restricted epitopes resulted in dramatic differences in the generation of the proper epitope. Proline, cysteine, leucine, and isoleucine substitutions inhibited the efficient roduction of these epitopes [g]. In addition, knockout mi and -7 have demonstratedtheimportance The -2 mice showeddecreased levels of C mice have lower levels of class I expression on the cell surface [10,1l]. In spite of thisdefect, theL -2 knockoutmicecanstillmountsomeantiviralresponses, suggesting that th bunit is not necessary for the presentation of all peptides [lo]. 2 cells lack both the LM subunits and the transporter m result, they have a substantial decrease in the cell surface levels of expression [ 121. Cell surface levels can be restored to normal throug of thetransporter genes alon 14). Inconclusion,theL P subunitsarenot crucial forexpression of the molecules in mice or in vitrobut may likely enhance the presentationof certain viral epitopes.

Once the peptides are generatedin the cytosol, they are transported into the lumen of the endoplasmic reticulum (ER), where they are bound by the class I heavy chains (Figure 1). The major mechanism in the antigen presentation pathway for peptides to be transported is through the transporter complex. The transporter associated with antigen processing (TAP) consists of two subunits, TAP-l and TAP-2. Cells lines which lack functional TAP molecules show a marked decrease in their cell class surface levels of class I [12,151. Some human leukocyte antigen A2 (HLA-A2) I complexes which reach thecell surface in the mutantcell line T2 have bound signal [ 16,171. While the signal sequence pathway provides some sequence derived peptides of the peptidesbound to class I, the majority of peptidesentering the antigen presentation pathway enter via the TAP pathway. Transfection of the TA into the TAP-deficientcell lines restores antigen presentation and cell surface levels [18-20]. The subunits of T A P 4 and TAP-2 are76 and 70 kDa,resp and are noncovalently associated. They are thought to function as a dimer, although it is unclear whether high-order multimers occur, The TAP molecules belong to a family of transport proteinswhich containanadenosinetriphosphate-(ATP)binding cassette 1211. The TA -dependent peptide transportis ATP-dependent E221 complexcantransportpeptideswhichare 7-40 amino acidsin h the most efficient transport is seen with peptides of 8-13 amino acids [23]. This correlates well with the length of the majority peptides which have been eluted from cell surface class I molecules [24]. Efficient transport has shown both preference for peptide length and sequence specificity. As a result, different peptides can have dramatically different efficiencies in TAP-dependent transport. AhydrophobicC -terminal amino acid is preferred for peptidetransport by mouseTAP-2and AP-2" [25]. Thisbias is not TAP-2" [26]. Somepartiality for certain amino positions has been noted for peptide bindingt o human

transport subunits both and port, ecan cont

occur. also

7

Long peptides can be exported out of the ER for further “trimming” in the cytoplasm and reimport into the E through the TAP molecules. The proteins involved in this export and trimming h e not been identified; however, the protease inhibitor ~-acetyl-L-leucyl-L-leucyl-L-norleucinal appears toin [29]. For two human epitopes, influenza matrix 58-66 and TAP-independent transport has been noted. The former is highly hydrophobic and may simply diffuse acrossthe ER membrane [30]. The latter is thought to enter the pathway through the cotranslational translocation of the envelope protein into the lumen of the ER[31,32]. class I heavy chain( ) and ~2-~icroglobulin (p2 ) are cotranslationally d into the lumen of ER. In humans, the clas heavy chain interacts with a number of chaperone proteins, including not mice, calnexin appears to bind free heavy chain and not the dimer [33]. In humans, this heterodimer associateswith calreticulin (Figure 1). The role of calreticulin in the assembly of mouse class I is unknown. The calreticulin/

”... ”*....

Class I

Class I Calreiiculin

Protein

Schematic of human antigen processing and presentation: les, the heavy chain first interacts with calnexin. Binding of MHC heavy chain facilitates the interaction of the heterodimer with calreticulin and Tapasin. Binding of the complex to TAP via Tapasin positions the class I heavy chain/@,M heterodimer for peptide binding. Once the peptide/classI heavy chain/@, complex is formed, it is released fromTAP, Tapasin, and calreticulin andis transported to the cell surface. (Courtesy r.)MHC,majorhistocompatibilitycomplex;Tapasin,TAP-associated transporter associated with antigen processing.

alone to the calret complex whethercalreti the Tapasin/TAP mplex binds a pathwaycausestheeterodimer

ated glycoprotein (Tapasin, also known as tapasin interactions in human require p2 , Tapasin and calreticulin may bind to the site. It is unclear whether Tapasin binds and then binds the toTAP dimer or complex. tobelocated beside thepoint of e

own the importanceof binding to calreticulin and d calreticulin and enter the mutant, T134K, fails to lack of association results in r and cell surface. As a result, tional phenotype is similar to 1s for CTL recognition if the peptideis loaded o sensitize targets when infected with virus. binding of calreticulin is imperative for the A humanmutant lym blastoid cell line, 1353. As a result, the il to bind peptide 1371. decrease of cell surface class I complexes hese studies show the great importance of theTapasin molecule to bridging theheterodimerwiththe TAP complex. The Tapasin molecule appears to have two isoforms, as displayed on twolysis 1353. The Tapasinmolecules may be involvedin loading the cleft. Alternately, Tapasin may function in a manner analogous to that of the invariant chain in C class II assembly. In this model, the different a result of peptide loading and degradation of one isoforrn of Tapasin ess” form after peptideloading. Once the complete complexis formed, from the transporter, tapasin, andcalreticulin and enters the secretory pathway. In the Golgi network, the heavy chain is glycosylated and the complex continues along the pathwayto thecell surface for recognition asself or nonself by the imrnune system,

Some of the class I complexes which reach the cell surface are peptide-receptive. It is unclear whether these molecules lack peptide or contain low-affinity peptides

the same epitope is normally processed and presented from the cytosolic protein. The main effector function of the CTL is to be able torecognize changes that occur

7

within the cell, not peptides derived from sera. While bone marrow macrophages have been shown to present class I binding peptides after macropinoc~osis,this type of exogenous derived presentation appears to be an exception invivo [40].

Proper activation of CTL requires multiple interactions at the cell surface. These interactions, if above the required threshold9 trigger the cell signaling machinery to send the activation signal tothe nucleus. The specificity CTL-target cell recognitio~lies in the interaction of theT cell receptor ) with the class I complex. IfT the heavy chain bound with a particular signaling peptide, exassociated and serine/threonine its kinases is activated. The antigen specificity of this interaction provides a “proofreadin ” mechanism to ensure that thetarget cell is presenting nonself peptides. of recentstudieshave sugge econcept of “serialtriggering” of ontheT cell froma single / ~ e p t i d ecomplex 141,421. Ithas been shownthat 200 TCRs seriallyrecognizeasingle C/pepti~ecomplex,and that approximately 8 ~ TC 0 ed to trigger for proper T cell recognition if there 8. This findingcorrelates well with previousdata is notcostimulationthroug which suggested that 50-2 /peptide complexes are require face for proper T cell recognition 1431. Given that the l chain of t has been shown to associate with the c ~ o s k e l ~ t o it n 9is plausi to induce down-regulation of surface TC peptide complex can result incomplete signaling and a lack of f the ZAP-7Qmolecule [45- 3. This lack of signaling reduces the T cell’s ability to respond adequately to the pathogen. Such partial agonists and anta~onistsprovide a mechanismof viral evasion from thei m ~ u n response. e

mplex, additional ulatory molecules threonine kinases

from approximately 40 to fewer than 10. ~ ~ o k i n also e s interact with T cell surface receptors and aid in the activation and differentiationof the Tcell.

The genes of the MHC loci are uniquely polymorphic. Over l00 known, and the2 0 ~ 0 - 3 0 ~ diversity 0 among alleles is found within the peptide binding site. One explanation for this diversity is an increase in the host’s ability to present a large array of peptides to the immune system. Consequently, probabilthe it of the host’s presenting peptides derived fromnew infectious agents is improved. k sequencing of acid eluted peptides from purifiedcell surface MHCcomplexes shownapredominance of certain amino acids,termed anchor residues, at particular residues, which vary by class I protein [24,52], For HLA-~*0201,the most common human class I allele, the anchor residues are at position 2 and position 9, where sequence analysis showed a preponderance of the aminoacids leucine, methio~ine, andisoleucine at position 2 and hydrophobic residues, primarily valine, at position 9 [53]. This “primary” anchor residue pattern is termed a motif and is specific for each MHCclass I allele. Structural crystal analysis of MHC class I was used to examine the nature of the interaction between the peptide and the binding cleft [52,53]. The peptide was shown to bind within a cleft formed by the heavy chain that consists of two alpha helices over a floor of eightbetapleatedsheets [52,53] (Figure 2). Within this groove, the amino acid residues form multiple interaction sites, originally termed binding pockets [52]. The amino terminus of the peptide fits into the A pocket and maintains conserved hydrogen bond interactions with the heavy chain. The C terminus fits into the F pocket with similar conserved interactions. The anchor residues fit down into other pockets which line the cleft (Figure 2). The complete submersion of the anchor residue’s amino acid side chain in a pocket suggests these 66favored” amino acids significantly contribute to peptide/HC affinity. ever, mutational analysis of both peptides and heavy chain has clearly shown that the primary anchor residues are not the sole determinant of peptide affinity [54,55].

utations in any of the class I binding pockets can alter peptide binding and/or nctional CTL recognition. Thus, nonanchor residues are crucial not only for TCR contact, but also for MHC binding [54,56]. The recent crystallographic data of TCRinteraction W theMHC/peptidecomplex clearly showthat all theCDR involved in the interaction with both class I heavy regions of both TC hains are chain and nonanchor peptideresidues [57]. Binding of iodinated peptide librariesto heavy chain has also shown the importance of nonanchor residues in peptide binding [58]. Structural analysis of H2 Kb bound with two different peptides suggested that preferential nonanchor residues are necessary in order to maintain conserved water molecule interactions[59]. In order to determine whether a hierarchy of preferred amino acids existed at nonanchor positions, Ruppert et al. analyzed HLA-A2-restricted peptides for binding [60]. The peptides separated into three groups: high-, intermediate-, and lowaffinity binders. The amino acids in the nonanchor positions of the high-affinity binders were compared with intermediate- and low-affinitypeptides. There was

01 heavy chain cornplexed

*

A-AZrestricted

ticular no~anchor also been a ~ ~ lt i e ~

t on the amino acid

Schematic of proposed secondary anchors for (a) *35Ol-restricted peptides we compete with a radiolabeled high-affinity marker peptide for igh-, intermediate-, and low-affinity peptides were then analy nonanchor positions, The frequency of some amino acids was higher some nonanchor residues, suggesting their preferential use in high-affinity favoredresidueshavebeentermed“secondaryanchors.” In aminoacidsinapeptidepositioninnitypeptidessuggested that t ~ o s amino e acids detrimental may be to anchors indicated yare by an ,human leukocyte antigen. region. Adapted from

ary anchors to weak bindi specific. It is self-evi oes not resultin the 1 indirectly.

in the

ene era ti on of the

epitope. Because of the severe overlap of the two epitopes, the HLAepitope is cleaved when the HLA-B8-restricted epitopeis generated. This shows that not only is the sequence surrounding the epitope important, but the coexpression of different heavy chains can have a dramatic effect on which epitope is favored for presentation.

Amino acid substitutions within the epitope can resultin a decreased affinityof the peptide for theclass I molecule.If the virus alters epitopes which bindto frequently expressed class I alleles, viral fitness may improve. One nat strategy is Epstein-Barr virus (EBV) evolution in China and 1691. In both these populations, the HLA-A11 allele is highly prevalent. Previous studies of EBV-specific CTL responses showed that the HLA-All-restricted CTL response is particularly strong in Caucasian populations. Subsequent analysis defined an epitope in EBV-encoded nuclear antigen-4 gene (EBNA-4) which bindsto the HLA-A1 l molecule. In China and New Guinea, immune selection has apparently selected againstthispeptide. Instead,the EBNA-4proteinhasaltered an amino acid which normally serves as an anchor residue for peptide binding. This destruction of the anchor residue results in a peptide which poorly binds to the HLA-All molecule andelicits no detectable CTL through HLA-A11 results in nonresponsiveness of a T cell and is caused by the interaction with a peptide similar to, but different from, the eliciting peptide. These altered peptides cause T cell inactivation to result from a lack of complete signals [45-471. As a result, the T cell is rendered anergic, and TCR down-regulation has been shownin vitro to persist for 24 hr [41]. Given the extensive TCR contact region involved in MHC/peptide recognition, the ability of single amino acid substit~tions to affect recognition dramaticallyis not surprising 1571. Antagonist peptides tendto have a lower affinity for theclass I molecule. Twoelegant studies using C displayed the effect of antagonism using natural peptide variants from HBV epitopes [70,71]. The role in antagonistpeptides was furthera BV infection. In one HBV patient an oligoclonal response to the wildtype HBV peptide/MHC combination developed. Variationwithin this epitopegenerated an antagonist peptide. This altered epitope resulted in the complete loss of CTL recognition [72]. The authors suggest that the chronicHBV infection was the result of narrowly focused immune response which was readily antagoni~ed through viral mutation. Together theseresults suggest that intracellularpathogens have evolved to contain peptides which are poorly processed and/orfail to interact with either MHC class I or TCR.

+

II.

I

L

. Viruses are forced to generate mechanisms to evade the immune system in order to propa~ate. The mutation rate of ribonucleic acid (RNA) viral genomesis inherently high because of their replication mechanism. Even deoxyribonucleic acid (

viruses can rapidly accumulate advantageous mutations. Many viral epitopes are located in external viral proteins since these proteins are under constant immune surveillance and selection from both the humoral andcellular arms of the immune response. In the development of peptide-based immunotherapy or vaccine components, these highly variable epitopes are ineffective starting points. In contrast to these highly variable segments, conserved regions of viral genomes mayserve as the virus’s Achilles’ heel for peptide-based vaccines against hepatitis B virus (HBV), hepatitis C virus(HCV),andhumanimmunodeficiencyvirus(HIV).Twoapproaches for addressing the variant peptide binding specificity of different HLA alleles have been proposed. One strategy is simply to target high-frequenc alleles. The HLA-A2-restricted peptides are frequently targeted since this allele is fairly predominant, with 40% of Caucasians expressing HLA-A*0201 [73] garding uncommon subtypes, the majority of individuals can be targeted LA molecules. The second approach takes arecent observation into consideration. Groups of LA molecules have been assembled together on the basis for similar binding motifs. These groups, because of their similar binding specificity, then together display a supermotif (Figure 4). Single peptides which fit this supermotif may be able to target all the HLA molecules within that supermotif family [74]. Since the predominant MHC alleles varybetweenraces,various“peptide” cocktailsmay be produced, depending on the targeted area of the world being vaccinated. Alternatively, sequentialminigenes encoding the peptides may be placed in a viral vector, such as vaccinia. Such an approach has been shown to be effective using different strainsof mice without any apparent problemswith processing [75]. Whitton et al. have shown that linked minigenes of lymphocytic choriomeningitis virus (LCMV) epitopesin recombinant vaccinia virus can protect mice against lethal doses of LCMV. This linkage of several epitopes, termed a “string-of beads” approach, apparently did not adversely affect processing as CTL activity was detected against multiple epitopes [76]. Further, the combination of several epitopes in one viral vector acted synergistically to aid in host protection. Several studies in both mouse and human suggest that targeting conserved epitopes and common MHC alleles can be a reasonable approach forvaccine &vel-

Allele

Position 9

B7”like supermotif

B“0701

VLI(AMFWY) Y(VL1FWAM) VLI VLIMYFW

Position 2 P

Position 2 P B*3501 P 8*5101 P 8*5301 P

Position 9 VLI(A~FY~)

4 RepresentativeHLAmoleculeswhichare part of theHLA-B7supermotif:For each of the HLA molecules listed, positions 2 and 9 are primary anchor residues. Among these HLA-B7-related molecules, proline is the predominant anchor residue used at position 2 of the peptide. Peptides which bind to this group of HLA class I molecules tend to use a hydrophobic residue or aliphatic amino acid at the C terminus of the peptide. Given this significant overlap in binding motif, one peptide may be able to bind all HLA class I molecules withinthis group. Several such “supermotifs” have been described and may be useful in the design of peptide-based vaccines with application to a greater proportion of the general population. Adapted from Ref. 74.HLA, human leukocyte antigen.

e’s free energy of

on §ti~ulation wi

~ o l e c ~ l(2) e , di§pla~ed gnized by human CTL

affinity?

20 100 90

80 70 VI 3 ._. 60

M Immunogenic

CI

gCL

50

Non~mmunogenjc

g-

O 4 0

8

30 20

10 0

>3 hours

<3 hours Intermediate

~

~

i

~

Low

~

t

y

Contribution of Kd or Ke4 to immunogenicity in vivo: High-affinity HLA-A2restricted peptides almost invariably induce CTL responses in HLA-A2/H2-~bmice. It has been more difficult to predict the in vivo immunogeni~ity of intermediate- or low-affinity peptides. The right side of the graph shows the correlation betweenimmunoge~icityand the Ke4of peptide binding. Intermediate affinity indicates that between 50 and 500 n M of peptide is needed to compete off 50% of a radiolabeled high-affinity marker peptide from HLAA*0201. The low-affinity group requires between 5 0 0 nM and 50 pM in the competition experiment described. Onthe left portion ofthe graph, thesame previously defined “intermeKd on cell surface classI molecules. diate-” and “low”-affinity peptides were analyzed for the The K, is a more accurate predictor of in vivo immunogenicityfor these peptides. Immunogenic peptidesare shown in hatchedbars while peptides which did not induce a CTL response are indicated in black bars. The correlation of high-affinity peptides with CTL induction was the same for the two kinetic measurements. Adapted from Ref. 85. HLA, human leukocyte antigen; CTL, cytotoxic T lymphocyte.

nogenicity [88].In addition, stability of the MHC complex has also been shown to be a crucial determinant for immunogenicity forclass I1 complexes [89]. While the on rate of the peptide is important to effective competition within the ER, many peptide binding assaysusing cell lysates are conducted at4OC. The dissociation rate at this temperature is basically nonexistent; thus, the analysis of dissociation at the cell surface at 37OC is more physiologically relevant. Even inthe analysis of peptide loading within the ER, the measured solution on rates may be irrelevant to ER loading if the TAP/Tapasin complexcatalyzes the peptide loading process. Whendesigningpeptide-based vaccines, several parameters are important. While the primary anchor motifs have greatly aided in the definition of epitopes through reverse immunogenetics, applying such motifs does not ensure the generation of thepeptideduringcytoplasmicproteindegradation.It is imperative to determine whether the peptide is normally processed and presented. Also, the pep-

S

tide cannot be recognized if it appears too similar to a self-peptide. Even a weak detectable CTL response to the natural peptide suggests that the Tcell repertoire is able to detect the peptide as nonself. In addition, any detectable immune response suggests that the peptide is strong enough to be considered a potential target for immunotherapy. A goal of altering epitopes for inducing CTL activity is to improve peptide interaction with the MHC heavy chain without altering the TCR contactpoints. In this manner, a similar T cell repertoire may be induced by the improved peptide, which canthen recognize and respond to the natural epitope when a person becomes infected with the virus. Anobviousposition t o alterpeptides is at the anchor residues. Since anchor residues are completely buried in the structure of the cleft, alteration should only affect peptide affinity and should not adversely affect T cell recognition. The ability to alterpeptideorpocketresidues to compensatefor changes in the other has been intensively studied in our laboratory. Analysis of a -A*0201 from phenylalanine totyrosine at posin a decrease inCTL recognition [90]. A compensatory mutation atposition 2 of the peptide restored wild-type-peptide-specificCTL recognition [90]. This position 9 mutation is apparent in different subtypesof A2; therefore, such compensatory mutations should be consideredwhen designing peptide for different ethnic backgrounds [73]. Compensatory mutationsof position Kb showed the abilityto restore presentation of VSV peptides by restore alloreactivity of some Tcell clones [9l]. ecent analyses of melanoma peptides have defined a number of epitopes which are targets for tumor infiltrating lymphocytes (TILS). A close correlation exists between tumor regression and TIL-specific activity against tumor-associated peptides [92] The self-melanoc~e protein gpl00 contains three HLA-A2-restricted peptides. All three have acorrect amino acid at one, but not both, primary anchor positions [93]. Substitutional analysis of these peptides altered the anchor position residues. One peptide already displayed a high affinity for the HLA-A*0201 molecule; therefore, substitutions were unable to improve the binding orrecognition of this epitope greatly. The second and third peptides,G92, and 69280, both benefited from amino acid substitution. For 692809 simply changing the position 9 residue from an alanine to a valine allowed greater binding while maintaining CTLrecognition and IFN-.y production by CD8' cells. In addition, the altered peptide was able to stimulate in vitro growth of TIL which was able to recognize the natural epitope. ~ a methionine at position 2 in place Similar results were found with G 9 by~ placing of the natural threonine[93]. Another site for improving the interaction between heavy chain and peptide is at the position 1 (Pl) side chain. Initial evidence that P1 may be a potential site to improve epitopes came from studies investigating subtypes of HL * A single amino acidchangeat position 59 defines the differencebetween 705 *2703. This residue is located within the structure of the A pocket. In *2705 this amino acid is a tyrosin is involved in hydrog th theN terminus of the peptide. an leukocyte antigen a histidine at position 59 instead of the tyrosine, thereby losing a hydrogen bond interaction with the peptide. ~ o n s e ~ u e n t l ydifferent , position 1 residues may be preferredin order to compensate for the loss of the hydrogen bond. This was elegantly shown through the presentation of the HLA-~27-restricted epitope from .)

the nucleoprotein of

Analysis of binding kinetics showed that the reduced affinity for thepeptide. Alteration o to an arginine not only rest

restricted polepitope(aminoaci strains which have been analyze three single a ~ i n acid o variants displays an inter~ediate affinity reason for its inability to induce infected individuals. Alteration o (IlY) substitutions, the on rate

(Figure 7). This latter result

a favored secondary The ~ramatic reductio immunogenicity. ~tructuralanalysis of the tides showed that the P1 side c this side chain may be a potenti , CTL assays confir e wild-type pol[64]. tion by CTL lines, I1 in all individuals tested. Perip seropositive individuals were st The donors were selected for th 2 weeks of peptide stimulation lated a better wild-type pol-sp

phenylalanineandtyrosine whether the I1F improvemen ences or differences in T cell repertoire. The recent technology of sti~ulatingCTL

$z

100

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60 40

20

0.01

0.1

1

10

100

1000

100

1000

e ~ t i c~~centration ~e (p

1

10

Alterationof the terminal amino acid in the

HLA-~27-restrictedinfluenza

tion at whichhalf the molecules have assembled is an indication of the peptide’s relative types. (b) Alteration of the nucleoprotein peptide at the 5 or HLA-~*2703were analyzed for their ability to pres-

stimulated CTL. The peptides were expressed as minigenes in the p8901 vector. The percentfie lysis shownis at an CTL effectorto target ratio of5 . White bars indicate lysis of S while black bars show lysis of the ClR-l3*2703 transfectants. ,human leukocyte antigen; CTL, cytotoxic T lymphocyte.

2

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22

No peptide WT 11F IIY

;ie

10

0

5

10

15

20

25

30

Hours at 37OC

7 Complex stability is increased by amino terminal substitution of the HLA-142restricted HIV-1 pol epitope: Complex stability was determinedat the cell surface by adding exogenous peptide and ~2-microglobulin to T2.SinceT2cellsexpressHLA-A*0201,the exogenous peptides boundto cell surface molecules. The following peptides were used: wildtype pol (ILKEPVHGV), IlF (FLKEPVHGV), I1Y (YLKEPVHGV), and none. BrefeldinA was added to stop the egress of new molecules from the trans-Golgi to the cell surface. The stability of the cell surface MHC heavy chain/peptide complexes was determined over time by indirect immunofluorescence with the conformationally sensitive antibody BB7.2. The mean channel of fluorescence was determinedat each time point. The I1F and I1Y substitutions increased the half-life of the complex from approximately8 hr for the wild-type peptide to 24 hr. From Ref. 64. HLA, human leukocyte antigen; HIV, human immunodeficiency virus; MHC, major histocompatibility complex.

responses from antigen-naive blood may provide a method to analyze the improvement of epitopes for peptide-based vaccine work [95]. This approach, combined with advances in transgenic mouse models, should allow complementary analysis of immunomodulation of CTL activation.

The improved understanding of both peptide/ C class I interactions and TCR/ class I complex interactions provides the basis of specific and direct immunotherapy of disease [57]. Alteration of natural peptides may be applied to numerous disease models. While the design of antagonist peptides would prove harmful for viral vaccine design, antagonist peptides could be designed explicitly to prevent or halt an

autoimmune reaction. any autoimmune disorders,such as rheumatoid arthritis, display a T helper l (Thl) cytokine profile. Antagonists may preferentially induce a Th2 instead of a Thl cytokine profile or engage the TCR without inducing a complete signal [45-47,961. Direct targeting of disease-associated T cells may therefore result in anergy of the antiself Tcell response [97]. Initial studiesin the experimental autoimmune encephalomyelitis(EAE) model formultiple sclerosis have shown therapeuticbenefit of theadministration of an alteredpeptide.Single amino acid changes in the myelin basic protein peptide allowed reversal of disease symptoms, decrease in T cell influx into the brain, and reduction of y-interferon and tumor necrosis factor levels in the EAE mouse model [98-1001. Alteration of tumor antigens may also allow improvedtumor rejection. Single amino acid changes in oncogenes can sometimes lead to the recognition of tumor antigens. Such tumor-associated altered self-peptides have been described for6132A and p53 [loll. p53 ay be a profitable target for immunotherapy because of the proportion of tumors which carry mutationsin p53. HLA-A*0201/H2Kb transgenic mice injected with p53-derived peptides induced CTL responses which were ableto recognize a number of cancer cell lines of breast, colorectal, and Burkitt’s lymphoma origin [ 1021. Alteration of p53-derived peptides has also provided promising data for the induction CTLs which are protective against tumor challenge[1031

With the constant drive forward in the identification of new epitopes, viral, self, and tumor-associated, the outlook for the future of peptide-based immunotherapy is optimistic. The design of antagonistic peptides for the treatmentof EAE and the improvement of stimulating CTL precursors in HIV-infected individuals both provide evidence that such direct approaches to immunotherapy arepossible. Further analysis of delivery approaches, such as the string-of beads vaccinia constructs, DNA injection, liposomes, and peptide-pulsed dendriticcells, help to bridge the gap between in vitro T cell analysis and in vivo efficacy. Our increasing knowledge of peptide/MHC and HC/TCR interactions and peptide delivery allows us to design altered peptide-based vaccines for viral,intracellularbacterial, and autoimmune diseases which we hope will have significant therapeutic ramifications.

1. 2. 3. 4. 5. 6. 7. 8.

R Koup, J Exp Med 180:779, 1994. MG Brown, J Driscoll, JJ Monaco Nature 353:355, 1991. R Glynne, SH Powis, S Beck, A Kelly, L-A, K , J Trowsdale. Nature 353:357, 1991. CV Harding, J France, R Song, JM Farah, S Chatterjee, M Iqbal, R Siman. J Immuno1 155:1767, 1995. J Lowe, D Stock, B Jap, P Zwickl, W Baumeister, R Huber. Science268533, 1995. A Townsend, J Bastin, K Could, G Bownlee,M Andrew, B Coupar, D Boyle,S Chan, G Smith. J Exp Med 168:1211, 1988. J Driscoll, MG Brown, D Finley, JJ Monaco. Nature 365:262, 1993. M Gaczynska, KL Rock, AL Goldberg. Nature 365264, 1993.

9. N Shastri, T Serwold, F Gonzalez. J Immunol 155:4339, 1995. 10. L Van Kaer, PG Ashton-Rickardt 11.

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ck, AL Goldberg, PC Doherty, Fehling, W Swat, C Lap cience 265:1234, 1994. V Cerundolo,JAlexander, Gotch, A Townsend. Nature F Momburg, V Ortiz-Navarrete, J Powis, GW Butcher, JC Howard, P J Yewdell, C Lapham, I Bacik, T Spies, J Bennink. J Imm A Townsend, C OhlCn, J Bastin, H-G Ljunggren, L Foste 1989.

Cresswell. Nature 356:443, 1992. son, HMichel, K Sakaguchi, J Shabanowitz, E Appella, Englehard. Science255: 1264, 1992. 18. SJ Powis, ARM Townsend, EV Deverson, J Bastin, GW

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DE;

354528, 1991. 19. T Spies, R DeMars. Nature 351:323, 1991. 20. M Attaya, S Jameson, CK Martinez, EHermel, C Aldrich,JForman, K Fischer Lindahl, MJ Bevan, JJ M aco. Nature 355:647, 1992. 21 * J Trowsdale, I Hanson, I ockridge, S Beck, A Townsend, A elly. Nature 348:741 , 1990. 22. JJ Neefjes, F Momburg, GJ Hammerling. Science261:769, 1993. 23. MJ Androlewicz, P Cresswell. Immunity 1:7, 1994. 24. H-GRammensee, F Falk, 0 Rotzschke.AnnuRe 01 11:213,1993. 25. F Momburg,JRoelse, JC Howard,GWButcher,merling, JJ Neefjes.Nature 367:648, 1994. 26. J Androlewicz, P Cresswell. Immunity 5 : l , 1996. 27. PM van Endert, D Riganelli, G Greco, K Fleischhauer, J Sidney, A Sette, J-F 28 * 29. 30. 31. 32.

33. 34. 35. 36.

A Armandola, R Obst, J Brunner, G Hammerling. J Immuno1 156:2186, 1996. EA Hughes, B Ortmann, M Surman, P Cresswell. J Exp HJ Zweerink, MC Gammon, U Utz,SY Sauma, T Harrer, JC Hawkins, RP Johnson, A Sirotina, JD Hermes, BD Walker, WE Biddison. J Immunol150:1763, 1993. SA Hammond, RC Bollinger, TW Tobery,RF Siliciano. Nature 364:158, 1993. SA Hammond, RP Johnson, SA Kalams, Takiguichi, JT Safrit, RA Koup, RF Siliciano. J Immunol 154:6140, 1995. E Noessner, P Parham. J ExpMed 181:327, 1995. B Ortmann, M Androlewicz, P Cresswell. Nature 368:864, 1994. B Sadasivan, PJ Lehner, B Ortmann, T Spies, P Cresswell. Immunity 5:103, 1996. AL Peace-Brewer, LG Tussey, M Matsui,G Li, DG Quinn,JA Frelinger. Immunity 4:

505, 1996. 37 * AG Grandea 111, MJ Androlewicz, R§ Athwal, DE Geraghty, T Spies. Science 270: 105, 1995. 38. H-C Ljunggren, NJ Stam, C OhlCn, JJ Neefjes, P Hoglund, TNM Schumacher, A Townsend, K Karre, L Ploegh. Nature 346:476, 1990. 39. PM Day, F Esquivel, J Lukszo, JR Bennink, JW Yewdell. Immunity 2:137, 1995. 40. CC Norbury, LJ Hewlett, AR Prescott, N Shastri, C Watts. Immunity 3:783, 1995. 41. S Valitutti, S Muller, M Cella, E Padovan, A Lanzavecchia. Nature 375: 148, 1995. 42. A Viola, A Lanzavecchia. Science 273: 104, 1996. A Luscher, BH Barber, D Williams. Nature 352:67, 1991. 43.

44. 45. 46. 47 * 48. 49. 50. 51. 52. 53. 54. 55. 56. 57 * 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69.

0 deCampos-Lima, V Levitsky, J Brooks, SP Lee, LF Hu, ABRickenson, MG

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71. 72. 73. 74. 75.

Sproat, BEH Coupar, AA Scalzo, CA ripp, PC Doherty, DJ Moss, ASuhrbier. J ~mmunol157:822, 1996.

76. JL Whitton, N Sheng,MBA Olstone, TA McKee. JVirol67:348, 1993. 77. JA Berzofsky. Ann NY Acad Sci USA754: 16 l , 1995. CD Pendleton, S Sadegh-Nasseri,LRacioppi,RA 78. W-HBoehncke,TTakeshita, Houghten, JA Berzofsky, RN Germain. J Immunol 150:331, 1993. K Yokomuro, RN Ger79. H Takahashi, Y Nakagawa, CD Pendleton, RA Houghten, main, JA Berzofsky. Science255:333, 1992. 80. ME Ressing, A Sette, RMP Brandt, J Ruppert, PA Wentworth, M Hartman, C Oseroff, HM Grey, CJM Melief, WM Kast. J Immunol 1545934, 1995. 81. M Shirai, T Arichi, M Nishioka, T Nomura, K Ikeda, K Kawanishi, VH Engelhard, SM Feinstone, JA Berzofsky. J Immunol 1542733, 1995. 82. A Cerny, JG McHutchison, C Pasquinelli, ME Brown, MA rothers, B Grabscheid, P Fowler, M Houghton,FV Chisari. J Clin Invest95521, 1995. 83. P Wentworth, A Sette, E Celis, J Sidney,S Southwood, C Crimi, S Stitely, E Keogh,

NC Wong, B Livingston, D Alazard, A Vitiello, HM Grey, FV Chisari, RW Chesnut, J Fikes. Int Immunol8:651, 1996. 84. M Dupuis, SK Kundu, TC Merigan. J Immunol 155:2232, 1995. 85. SH van der Burg, MJ Visseren, RM Brandt, WM Kast, CJ Melief. J Immunol 156: 3308, 1996. 86. A Sette, A Vitiello, B Reherman, P Fowler, R Nayersina, WM Kast, CJ Melief, C Oseriff, L Yuan, J Ruppert. J Immunol 1535586, 1994. 87. V Levitsky, Q-J Zhang, J Levitskaya, MG Masucci. J Exp Med183:915, 1996. 88. GB Lipford, S Bauer, H Wagner, K Heeg. Vaccine 13:313, 1995. 89. CA Nelson, SJ Petzold, ER Unanue. Nature371:250, 1994. 90. M Matsui, JA Frelinger. Immunogenetics 40:66, 1994. 91. TJ Yun, MD Tallquist,EM Rohren, JM Sheil,LR Pease. Int Immunol6:1037, 1994. 92. Y Kawakami, S Eloyahu, C Jennings, K Sakaguchi, X Kang, S Southwood, PF Robbins, A Sette, E Appella, SA Rosenberg. J Immunol154:3961, 1995. 93. MR Parkhurst, ML Salgaller, S Southwood, PF Robbins, A Sette, SA Rosenberg, Y Ka~akami.J Immunol 157:2539, 1996. 94. RA Colbert, SL Rowland-Jones, AJ McMichael, JA Frelinger. Immunity 1:121, 1994. PA Wentworth, RW Chesnut, HM Grey, A 95. E Celis, V Tsai, C Crimi, R DeMars, Sette, HM Serra.Proc Natl Acad Sci USA91:2105, 1994. 1996. 96. P Chaturvedi, Q Yu,S Southwood, A Sette, B Singh. Int Immunol8:745, S Southwood, C Oseroff, T 97. J Alexander, K Snoke, J Ruppert, J Sidney, M Wall, Arrhenius, FCA Gaeta,SM Colon, HM Grey, A Sette. J Immunol150:1, 1993. 180:2227, 1994. 98. N Karin, DJ Mitchell,S Brocke, N Ling, L Steinman. J Exp Med 99. MF Samson, DE Smilek.J Immunol 155:2737, 1995. 100. S Brocke, K Gijbels, M Allegretta, I Ferber, C Piercy, T Blankenstein, R Martin, U Utz, N Karin, D Mitchell, T Veromaa, A Waisman, A Gaur, P Conlon, N Ling, PJ Fairchild, DC Wraith, A O'Garra, CG Fathman, L Steinman. Nature379:343, 1996. 101 PA Monarch, SC Meredith,CT Seigel, H Schreiber. Immunity2:45, 1995. 102. M Theobald, J Biggs, D Dittmer, AJ Levine, LA Sherman. Proc Natl Acad Sci USA 92:11993, 1995. 103. JI Mayordomo, DL Loftus, H Sakamoto, CM De Cesare, PM Appasamy, MT Lotze, WJ Storkus, E Appella, AB DeLeo.J Exp Med 183:1357, 1996.

9

Stan

ford ~niv~rsi~y, Stun

ford, ~aI~ornia

. I Individuals infected with the human immunodeficiency virus type l (HIV) suffer profound immunological changes. The clinical consequences of these changes include pathological infections by normally “mundane” pathogens, chronic and systemic inflammatory and oxidative stress, and increased incidence of lymphomas. Eventually, the burden on the immune system and the body is too great, and the individual dies. IV affects these widely disparate disorders, however, is still not underver, it is clear that HIV disease is accompanied by a wide spectrum of changes in virtually all cell types of the immune system. Even though HIV infection itself is restricted to cells bearing the CD4 molecule (i.e., almost exclusively CD4 T cells and monocytes), dysfunctions havebeen noted in every other compartment of theimmune system. Indeed,historically the firstobservation of immunological in patients with acquired immunodeficiency syndrome (AI cell activation [l]. S in cells of the immune system can be categorized into three partia~ly overlapping types: $e~$esentational(the absolute numberof a cell type), ~ ~ e n o t y ~ i cal (the collection of proteins expressed by a cell type), and ~ ~ n c t i o n (the a l set of responses of a cell type). A single defect may fallinto twocategories: for instance, a change in the regulation of expression of a cell-surface receptor is a phenotypic change but may also represent a change in the functional responsiveness of the cell. It is important to note that a changein the representation of a cell type may result in change in the functionality of the overall immune system-even though there is no change in thefunctionality of any given cell in the system. Therefore, we must determine whether any changes in functional measurements that are associated IV disease (or other disorders, for that matter) represent a change in the intrinsic functionality of cells in the immune system, or a changein the representation of functionally distinct cells within that system, or both. In this chapter, we demonstrate that accompanying HIV disease is a profound

change in the ~ e ~ ~ of functionally e ~ e distinct ~ ~ T~cell~subsets. ~ o ~ changes may account for the “immunodeficiency” that has been asc sponsiveness in in vitro assays or even in vivo. In addition, the changes that we discuss strongly support a model with of destruction the of the thymus asa pivotal event leadingto im

~uantitationof cell surface expression by flow cytometry is straightforward but requires rigorous attention to staining conditions. n principle, the amount of antigen expressed on a cell surface will be proportio a1 to the fluorescence obtained when cells stained are conjugated with a that to mo antigen. owever, care must be taken to ensure that the are reproducible and that the reagentused is at a saturating tite S for performing tive antigen density are discussed in detailelsewh any of the molecules expressed on cell the surface playroles in cell function: receptors,costimulato~y molecules, or other“environmental sensors.” Changes in the expression of these molecules withina single cell may herald a change in the functionality of the cell. Furthermore, the quantitation of the expression may reveal regulatory processeseffected at thetranscriptional level-i.e., changes in the activation state of transcription factors, both enhancers and repressors. is important toremember that a changein the expression of an antigenmay reflect a different frequency (representation) of unresolved underlying cell subsets that differentially express that antigen. For example, considerextheression 5, a protein expressed by v Itwell is known that S about two-fold more C amount of CD5 per T cell in a mixture of the proportion of each lineage in that mixture [3]. Figure 1 shows that theaverage expr reduced in human immunodeficiency vi T cells largely accounts etting of the cells to account for the differential representationof are a wide variety of changes in antigen densities of T cell molecules accompanying any of these occurred on ous cell populations: for instance, the expression of D4SR~’)CD8 T cells was reduced by almost 5 ~ (F~ 0 detectable change in the expressionof other molecules such as C hesion processes, this result may signify a change in the V-infected adults to perform certain homing functions. fficiently removed fro he cell surface on cell stimulation(through 2L expression may be caused by the systemic protein kinase C); the reduction in in~ammatorystress,manifestingreasedserum levels of molecules like tumor rentially affected on various expressed at lower levels on

I

0

00 300 200 100

20

40

60

80

100

0

Antigen density changes in HIV disease. (a, b) Antigen density changes in HIV disease may be potential surrogate markersof disease states. The expression of CD5 on all T cells (a) or CD62L on CD8 naive T cells (b) is correlated with disease progression. Both antigens show a progressive decline. the case of CD5, this decline is primarily due to the loss of CD4 T cells (discussed later). wever, for CD62L on the homogeneous naive CD8 T of surfaceexpression of this cells, this change may represent a change in the regulation adhesion molecule on a cell-by-cell basis. Different antigen density changes occur at different stages of disease;here,CD62Lexhibits thegreatestchangeduringtheearlystages.(c) Changes in antigen density may reflect changes in the representation of underlying subsets. CD5 expression is approximately two-fold higher on the surface of CD4 T cells than itis on CD8 T cells. Thus, measuring the average CD5 expression on T cells weights CD4 T cells greater than it does CD8 T cells. As is shown here, the average expression of CD5 is well correlated with the representation of CD4T cells among the totalT cell subpopulation. This conceptextends to other functional measurements as well: for instance,achangeinthe secretion of acytokinemayreflect no more than a change in the representation of the subset@) of cells normally producing that cytokine- even if there is no change inthe regulation of the production of that cytokine on a cell by cell basis. Thus, the extrinsic change in representationoffunctionallydistinctsubsetsovershadowsanyintrinsicvariationincell functionality that may or may not be present. (Adapted from Ref.3.) HIV, human immunodeficiency virus.

natural killer (NI()cells, at higher levels on monocytes, and unchanged on granulocytes [3]. This kindof differential regulation underscoresthe necessity for subsetting cells before quantitating antigendensity measurements.

We found more than a dozen different antigen density changes that were significant IV disease. Someof these changes occurred early in disease; some only occurred Thus, it is possible that these measurements have useful prognostic value for HIV disease. Indeed, Giorgi and colleagues have elegantly demonstrated the power of antigen density measurementsfor predicting progressionto AIDS. They ~uantitatedthe expression of an activation marker, CD38, on CD8 T cells and found that higher expression was correlated with faster progression to clinical AIDS [4]. In fact, the CD38 density measurementwas considerably more powerful than either CD4 counts or viral load measurements. Even incom~inationwith these two standard measurements, CD38 antigen density is a powerful predictor for rate of progression to AIDS. We have recently extended Giorgi and associates' findings by examining a ) . found cohort of patients that is relatively advanced in disease (C 4 < 2 ~ / ~ 1We that CD38 is a powerful predictor for progression to death in relatively advancedstage patients (Figure2).

100 80

60

Low CD38 20

40

~

0 0

200

400 800 600

ays sinee measur~ment

Antigen density measurements: prognostic value. The expression of CD38 on CD8 T cells has been shownto be the most powerful predictor of progression to AIDS by Ciorgi

and colleagues. Here, we showthat this parameteris also a powerful predictor of progression 200 CD4/pl. The survival rate (Kaplan-Meier to death in individuals who are already below analysis) of those who have high or low CD38 expression on day 0 is plotted; mortality rateis twiceasgreat for thosewithhighCD38expression.AIDS,acquiredimmunodeficiency syndrome.

One of the earliest markers for HIV disease progression is the absolute number of CD4 T cells in the periphery -i.e., accompanying HIV disease is a progressive decline in peripheral CD4 T cell counts. While the mechanisms accounting for this loss are still disputed, the loss itself serves as a relatively powerful staging tool. Contrary to theCD4 T cells, number of CD8 T cells is elevated early in disease and remain elevated untilrelatively late. T cells are not homogeneous subsets, however. They can be of to naive and memory T cells- functionally distinct stages differentiation that canbe identified phenotypically (Figure3). The memory subsets themselves can be further divided into several subsets. Indeed, the complexity of the T cell compartment is such that it cannot be resolved by measuring only three (or even four) surface antigens simultaneously (see Figure 3). For instance, u n a ~ b i g u o u sidentification of just the CD4 T cell compartment requires three measurements: CD3 (toidentifyT cells), CD4, and CD$. The latter is necessary so that the CD4+CD8+ population can be excluded from analysis, Since identification of differentiation stages (naive/memory) requires at least two more measurements (CD45RA and CD62L), it is apparent that a minimum of five antigens must be simultaneously measured to approach homogeneous populations. It is importanttorememberthatthere is asignificant population of CD4+CD8+(double positive [DP]) and CD4-CD8- (double negative [DN]) T cells in the periphery. The DP and DN cells are mature T cells that have functional m those of either of the CD4 or CD8 lineages - for instance, a P cells produce interleukin 4 (IL-4) in response to stimulation (Mitra and Roederer, in preparation). DP response can cause significant artefactin functional experiments thatuse only one or two-color phenotypingidentify to T cell lineages: DP cells willbe perforce includedin a typical CD8 gate (e.g., CD3+CD8+). This may lead to the erroneous conclusion thatCD8 T cells produce IL-4 (whereas S are contributing the cytokine). The complement of functions cells carry out remains to be determined- requiring at minimum three-color identification to identify those lineages uniquely. In order to resolve these populations, we recently developed eight-color fluorescence-activated cell sorter (FACS) analysis (for example, Figure 3), which gives us the capability to determine the phenotype of T cell subsets with a precision previously unattainable. The primary goal in developing this technology was to achieve the ability to resolve functionally and phenotypical~y homogeneoussubsets of T cells. In addition, we will be able to combine (for example)five-color phenotypic identification of T cell subsets together with three-color functional analysis (intracellular cytokine measurements, apoptosis measurements, etc.).

IN When we examined the fine Tcell subsets in our cohort of HIV-infected adults, we were sur~rised tofind a selective loss of CD8 naive T cells [S] despite an overall increase in CD8 T cell numbers (Figure 4). Indeed, the loss of the CD8 naive Tcells

seemed to occur at roughly the same rate as the loss of the total C d this hypothesis (Figure 5). Indeed, the rate of y identical to the rateof loss of naive C dicates that the memory CD8 compartment re elevated and remain relatively stable durund over a decade ago that there is a significant cells bearing an activation phenotype (CD38' es in the individual (a~ainst 8 T cells, those notinvolved athogens? These cells, after all, underlie the ects against future pathogenicinfections. At this time, thereis no way to identify these cells uniquely. Thus, we took the approach of enumerating unactivatedC 8 T cells as a measure of the re resentation of the re~ertoire that is notdirectedagainst shown in Figure6 , the absolute numberof resting during disease-much as the total C ' of these data, we conclude that all T cells which were present at the time of infection are progressively lost during disease and that these cells are not rs progressively with disease: a subset of 76 ast majority of peripheral $ T cells bear ividuals, however, this condition changes such that the V~l-expressing cells become the m ority 16-83. Indeed, in absolute terms, they disappear at a ratesimilar to that of 4 T cells, naive CD8 T cells, and unactivated memoryC findings These hav i~munopathogenesis for ions of CO causes cell loss through mechan~sms disease.primary The other than direct tion. cyto Therefore, there must be other ~ e c h a nostatic dysregulation, i s ~assuch s , or loss the of ability to generate cells. In addition, the o~servation that all T cell subsets are lost at the Same rate is

Phenotypic identification of T cell lineages and differentiation states: identificaore than a dozen phenotypically distinct subsets. T cell subsets in a single healthy a singlesamplestainedsimultaneouslywith individual are shownbrogressivegatingof antibodies against D4, CD$, CD45RA, CD62L, V61, and V62. Lymphocytes are T cells are selected on thebasisof CD3 identifiedbyforwsidescatter(topleft); ithin theCD3 T cells, V61 and V62 T cells are identified. TheCD4 om 6 - (a@")T cells are shown in the middle bottom. Here,CD4 and CD8 single-positiveTcells,aswellas 8" (DP) and CD4-CD8- (DN) T cells, each of these four bsets and the 6-expressing subsets, the and CD62L is shown (right).For the single-positive T cells, functional studieshaveidentified that the CD45RA"CD62L" cells are naive T cells; allothers are memory T cells(asshownin the outlined areas). The functional capacity of the subsets defined by CD45RA and CD62L for the DP, DN, and 6 subsets has not been determined.

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Correlated loss of naive CD4 and CD8 T cells. Cross-sectional analysis of almost 300 HIV-infe~tedadults, showing the correlation between naive CD8T cell counts and total (a) or naive(b) CD4 counts. The cross-sectional analysis suggests that CD8 and CD4 subsets are lost at similar rates during the progression of HIV disease. HIV, human immunodeficiency virus. highly suggestive that the same mechanism accounts for their loss. cause of the loss of CD4 cells is not due to a mechanism specifi ruling out hypotheses such asinfection and cytolysis, gpl20-med ate” signaling through the CD4 molecule, or superantigenlike stim protein.

d that the proportion of “activate disease. Why is it, though, that the whereas CD4 memory T cells do not? For the most part, th 8 -however, they do not express CD25, CD69, norCD cell activation. In addition, these cells are primed to apoptose, and once put into culture do not survive more than l or 2 days (data not shown). Perhaps the ex~andedCD8 subsetrepresents an unusualcell, even a differentlineage, thanthe“normal”thymicallyderivedCD8or cells. Another unusual population of CD8 T cells that increase in adults are CD57+CD28-cells [g]. These cells are thought tobe termin ated effector (CTL) cells, being large granular lymphocytes with detectable intracellular perforin expression. The expansion of these two cell types is product of the constant hyperactivationof the immune responseto it is noteworthy that these kinds of cells are also expanded in the immunereconstitution(withallogeneicbonemarrow), only to declineonce the 1” T cell population has recovered (Watanabe and Roederer, in preparation). ecently, we haveinvestigated several unusual T cell phenotypes that are significantly expanded in HIV disease. These phenotypes inclu and VG-bearing y6 T cells. Examples of these cell types are given in Figure 7.

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Longitudinal analysis of T cell loss. For individuals who were analyzed two to four times over a 2-year period, the rate ofloss of various 1:cell subsets was calculated and expressed relativeto the starting value. The percentage change in these subsets is shown in the bivariate plots; linear regression is shown (dark line). The linear fits are remarkably close to that expected by equivalent rates of loss (dashed lines). This confirms that hypothesis based on the cross-sectional data that all of these T cell subsets disappear at similar or identical rates in HIV-infected adults. HIV, human immunodeficiency virus. Unlike the disappearanceof the "normal" V62 cells (Figure 6),the V61 T cells expand in HIV disease, These two kinds of y6 T cells have completely different phenotypes: V62 are predominantly CD5'CD45RA-C~57-CD28', whereas V61 are CD5-/du"CD45RA+CD62L"CD57'CD28-. This latter phenotype is similar to that descri~ed for intestinal intraepithelial lymphocytes (iIELs). The iIEL T cells are educated and differentiate in the gut, in a thymus-indep ndent fashion. The V62 T cells, on the other hand, have a phenotype that is similar to that of "normal,'9 t hymic-derivedT cells. Another unusual population of T cells that increases in HIV disease are those that do not express CD5. These cells are quite rare in healthy HIV- individuals. While they were originally thought to be primarily a@ T cells [lo], we find that roughly half expressthe y6 receptor. Thus, thispopu~ationis only partially overlapping with the (increased)V61 subset noted.

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All “normal” T cell subsets disappear progressively in HIV disease. (a) The absober of unactivated (CD38-HLA-DR-) CD8 T cells is plotted against the absolute number of CD4 T cells. During middle to late stages of disease (CD4 counts dropping below 400), the resting CD8 compartment disappears at the same rate as CD4 T cells (see also the longitudinal analysis in Figure5). (b) They6 T cells that are most prevalent in healthy adults express the V62 gene, These cells are also progressively lost during HIV disease, at a rate that is correlated with the loss of naive CD8 T cells. HIV, human immunodeficiency virus; HLA, human leukocyte antigen.

ggests a hypothesis : that extrathymic differentiation is (partially) compensatingfor theloss of thymic-derived T cell lymothesis is more completely stated in three parts as follows: (1) disease at an early stage is an insult to the thymus that shuts y new naive T cells. There is histological evidence that the thymus is functionally destroyedin infected individuals; aswell, the T cells supports the lack of regeneration of this compartment. (2) memory T cells that are notinvolved in anti-HIV-mediated immunit normal kinetics: ably on the scale of many months for memory cells and years le there may be nondifferentiationaldivision occurrin~during be a stochastic loss of clones over time which underlies the turnover of the population. In healthy adults, the output of the thymus is sufficient to balance this slow loss of cells. The loss of memory T cells leads to progressive immunodeficiency (suscepti~ility to opportunistic infections); the inability to replenish the naive compartment renders thisimmunodeficiency permanent. The combinedloss of thymic activity and normal turnoverleads to progressive declines in all thymic-derived T cell populations. This may be an un of (3) an expansion of extrathymically derived T cell education that attempts to compensate for the loss of thymically derived cells. The full functional capacity of these cellsis unknown-are they capable of mediating the complexcascadeof responses that are associated with normal T cell immunity? It is likely that the answer to this is no-i.e., that these T cells are capable of only a limited set of

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responses. The limited immunity imparted by these cells may explain why there is only a limited set of opportunistic infections typically found in late-stage AIDS: other pathogens are sufficiently suppressed by the “unusual” Tcell subsets.

~nderstandingthe basis for the changesin the T cells in HIV disease is crucial for the evaluationof potential therapies. Alreadythere areseveral clinical trials planned or under way in which genetherapy of stem cells is carried out with the intentionof rendering the resulting T cells resistant to HIV infection. But if there is no thymic activity, such gene therapy is destined to fail, since no mature T cells will be generated. Further, because all lineages of “normal” T cells disappear during HIV progression-CD4, CD8, and a subset of +it is not at all evidentthat protecting cells from HIV infection (i.e., CD4 T cells) will correct the basic defect leading to this cell loss. Therefore, we must consider therapies that restore thymopoiesis if we are to restoreimmunefunction in IV-infectedindividuals.Suchtherapymayinclude thymus transplantation, an exciting prospect that is now being vigorously pursued by several groups. Alternatively, it may be possible to stimulate extrathymic differentiation to an even greater degree and attempt to regenerate more of the T cell responses. Such reconstitution is important even despite new, advanced antiretroviral therapies (such as protease inhibitors combinedwith reverse transcriptase inhibitors) which are extremely effective at reducing or even removing virus from the periphery. The preliminary evidence is that these therapies effect, at best, a mild increase in immune function-underscoring theneed to develop effective immunomodulatory therapy for this insidious disease.

S 1. HC Lane, H Masur, LC Edgar,AS Fauci. N Engl J Med 309:453, 1983. 2. A Kantor, M Roederer. Analysis of Lymphocytes. In: DM Weir, C Blackwell, L Herzenberg, L Herzenberg, ed. Handbook of Experimental Immunology, 5th ed. pp. 49.149.13. 3. M Roederer, LA Herzenberg, LA Herzenberg. Int Immunol8:1, 1996. 4. JV Giorgi. Phenotype and function of T cells in HIV disease. In: S. Gupta, ed. Immunology of HIV Infection. New York: Plenum Press, 1996, p. 181. 5 . M Roederer, JG Dubs, MT Anderson, PA Raju, LA Herzenberg, LA Herzenberg. J Clin Invest 95:2061, 1995. 6. A De Maria, A Ferrazin, S Ferrini, E Ciccone, A Terragna, L Moretta. J Infect Dis 165:917, 1992. 7. T Hinz, D Wesch, K Friese, A Reckziegel, B Arden, D Kabelitz. Eur J Irnmunol 24: 3044, 1994. 8. S Boullier, M Cochet, F Poccia,ML Gougeon. J Irnmunol 154:1418, 1995. 9. AL Landay, CE Mackewicz,JA Levy. Clin Immunol Immunopathol69: 106, 1993. 10. S Indraccolo, M Mion, R Zamarchi, V Coppola, F Calderazzo, A Amadori, L ChiecoBianchi. Clin Immunol Immunopathol77:253, 1995.

~ationalInstitute of Diabetes and Digestive andKidney Diseases, ~ationalInstitutes of ~ e a ~ t~ethesda, h, ~aryland

I. I The term “idiotype” denotes the array of antigenic determinants which can be serologically definedon a given antibody molecule [1,2]. When these antigenic determinants are shared among antibodies, the term cross-reactive idiotype is (CRI) applicable (3). Cross-reactive idiotypes can define a major proportionof a given antibody population. The designation CRIMis used to designate a major cross-reactive idiotype. These serological markers can be encodedby a germline gene and function in an immunoregulatory manner. When a small fraction of antibodies expresses a CRI, a minor cross-reactive idiotype (CRI,) is defined. The relative expression of idiotype assumes a level of’connectivity among members of the immune system cells, T cells) 141. It is also thebasis for the immunoregulatory aspects of the idiotypic immune network. The immunoregulatory aspect of idiotypy was originally proposed by Jerne [5] as a set of complementary interactions which form the basis for self-regulation of an autologous immune response (Table 1). Fundamental to thehypothesis was the dual nature of the antibody molecule. The primary antibody molecule recognizesand binds antigen through the antigen combining site. Also at this location is the expression of idiotypy.Thus, acting asantigens, idiotypic molecules (Abl) induce a second population of antibody molecules (Ab2). These Ab2 molecules are serologically complementaryto theAb1 antibody molecules. The Ab2 antibody populations are termedantiidiotypic. A unique subpopulation of antiidiotypic antibodies are those members that serologically mimic the initial antigen. This subpopulation is complementary to the antigen binding site of the Ab1 population, and binding to idiotypic antibodies is inhibited by antigen. As such, these molecules representan internal imageof the antigenic epitope [6,7]. It follows that these molecules, as internal images of antigen, may be candidates for antigenbased vaccine strate ies for the immunotherapyof infectious diseases 181. The immunological aspects of idiotypic-antiidiotypic interactions have been the subject of intense investigation. To date, onlylimited evidence existsthat idiotypic network interactions modify an autologous immune response [g]. In the daily

ic

Serological Aspects of Immunoglobulin,B, and T Cells" Idiotypic Ab1 Binds antigen Induced by antigen Expresses CRI Individual molecules may neutralize pathogen

'Ab,

Ab2 Binds idiotype Induced by idiotype Defines CRI

Ab3 inds antigen Induced by antiidiotype Expresses CRI and other idiotypes (expanded repertoire) Subpopulations may be in- Individual molecules may ternal image of antigen neutralize pathogen and (surrogate antigen) on population a basis may be more effective that Ab1

antibody; CRI, cross-reactive idiotype.

maintenance and regulation of the immune system, idiotypy has only been postulated to be functional. owever, substantial data implicate i~iotypic-antiidiotypic immunoregulation in bacterial, viral, and particularly, parasitic infections [1O-133. Thus, this chapter reviews the presence, immunoregulatiQn, and vaccine potential of idiotypic immune network componentsin schistosomiasis.

Approximately 200 millionpeopleworldwide are infected with the three major species of Schistosoma~S . ~ a n s o n i ,S . h a ~ m a t o b i uand ~ , S.japonic~m~ l ~ - l ~ ] ~ Epidemiologically, schistosomiasis japonica is a major health problem in both the epublic of the Philippines and the People's Republic of China, where between 10 S. ~ a ~ ~ ~ t o is b iendemic um and 50 millionpeople are infected. In ion by countries in Africaandtheeasternditerranean.In 52 countries in America, Africa, the Caribbean, and the eastern editerranean S. mansoni infection is endemic [ 161. Clinically and pathologically the two hepa forms of ~ c ~ i s t o soma infection, S. mansoni and S. japonicum aresimilar.wever,it is now clear that significant epidemiological [ 161, parasitQIogica1 [171, and immunological [18,19] differences exist between these twospecies.

Sc~jstosomajaponic~mis unique among the human schistosomes in that large nonhuman (wild and domestic animal)reservoirs exist. Thus, even with thedevelopment of praziquantel and subsequent mass chemotherapy programs, progress toward eradication of schistosomiasis japonica remains a formidable problem. In the ublic of thePhilippines,high levels oftransmission persi ening and treatment of the infected human population [20]. of the human populationclearly continues despite current controlstrategies. To this

point, experimental vaccines to prevent infection appear to be at best only partially effective in experimental animals [21]. Therefore, it is of critical importance for the purpose of reducing morbidity elucidate to the development and modulation of liver abnormality inducedin this human disease. S. j a p o ~ i infection, c~~ chronic parasitism causes development of severe plenic enlargement in a minority of the infected population ( 1 0 ~ 0 - 3 0 ~ 0 ) . als are children or young adults [22-231. Infected adults epatic enlargeme~t and granulomatous in~ammation as a long-term pathological consequencesof chronic parasitism. ions, in asymptomatic and hepatosplenic disease, hepatic hepatic egg granuloma [24]. The functionof the granul is to destroy the deposited egg at a minimal cost to adjacent host structures. j c ~ ~are hepatic enlargement from the clinical sequelae of S. j a ~ o ~infection inflammatoryrespoto periportallydepositedova andsubsequentdevelopment of hepaticfibrosis.patic abnormality is a result of boththeexuberant host immuneresponsetgantigensas well asthe directtoxiceffects of the eggs themselves on he~atictissues. Eggs contain living miracidia and when ova are impacted in host tissues, these living organisms release soluble egg antigens ~SEAs) through the pores of the egg shell [25]. The SEA molecules are immunogenic [25271 and evoke a delayed hypersensitivity reaction. In infected individuals both deiate hypersensitivity reactionsto SEA [28] and high immunoglobu, and IgE anti-SEA antibody titers develop [B]. In an analysis of the antibodyrepertoire induced by infection 1191, serum antibody binding charactercompared with those of antibodies derived from patients infectedin the epublic of China and the Republic of the Philippines. Similar enzymelinked im~unosorbentassay (ELIS end point titrations were noted, indicating a high level of antibody productivity. owever, limited cross-reactivity was noted on the basis of antigen binding profiles. Idiotypic, antiegg antibody reactivity to the specific subspecies antigen was conserved at a greater degree than idiotypic antiworm antibody reactivity. Thus, idiotype-based vaccine therapies for populations infected with S. i Q p o ~ j infection c ~ ~ have a greaterpotential for efficacy when based onegg antigen reactivity. The development of hepatosplenomegaly and the modulation of hepatic fibrosis vely in the murine model ofS. j a p o ~ i infection. c~~ These ne subspecies. They have shown that granulo~atousinamm mat ion is T cell-mediated [30,31] and is modulated (reduced) during chronic dulation of murine S . j a p o ~ i infection c ~ ~ appears mediclophosphamide-sensitive suppressor T lymphocytes [32] ntiidiotypic IgG 1 immunoglobulin molecules In S post infection)whenpeak inflammationand alitomycin- sensitive suppressor T cells are found in the spleens of infected mice [32]. These cells nonspecifically suppress SEA and conca)-induced blastogenic responses in vitro as well as granulomatous inflammation and portal pressure invivo [35]. These suppressor cells are not found in the spleens of animals in chronic infection (20 or 30 weeks post infection). In chronic infection, serum-mediated suppressive mechanisms develop. Serumof mice

taken at 20-30 weeks post infection has been shown to suppress granulomatous inflammation, portal pressures, and SEA-induced blastogenicresponsesinvitro [36]. This suppression is antigen-specific, as opposed to the immunosuppression noted in acute infection, which is nonspecific. The immunosuppressive molecules in serum obtained from chronically infected mice have been characterized [34,37] and include antiidiotypic IgCl antibodies. These antiidiotypic antibodies bindto specific regulatory idiotypes on anti-SEA immunoglobulin molecules of acutely infected mice, They also bind Lyl -t L3T4' lymphocytes from these animals. The antiidiotypic antibodies, derived from 30week infected mouse serum,profoundly suppressin vitro SEA-induced responsesof lymphocytes from acutely infected mice. These molecules are also antibody-antigen (SEA)-combining site-specific. Combining site-specific antiidiotypes are often referred to as antigen mimicsin that they can function as antigen (SEA) under certain S. j a ~ o n i acute c ~ ~infection are defined circumstances. The idiotypes found during by the immunosuppressive, internal image of antigen, 30-week antiidiotype. The idiotypes have been designated schistosomiasis japonica-cross-reactive idiotype major (SJ-CRIM). At concentrations of 0.31-5 pg/ml,anti-SJ M (30-week antiidiotype)suppressesSEA-inducedblastogenicresponses of SJ -positive T cells. In low concentrations (0.00$-0.0~5pg/ml) anti-SJ-CRIMacts li lates T cells obtained from acutely infected mice. Thus, SJ-C c ~ ~ is dose-de otypic regulation in murineS, j a ~ o n i infection During the course of infection SJ-CRIM also defines c idiotype expression. The expression of the SJ-CRIM both in serum antibody and to changes in hepatic by T cells of acutely infected animals has been compared immunopathological characteristics over time [37]. The disap arance of SJ-CRIM expression on B cells and T cells, and appearance of anti-SJ-C M antibodies parallel modulation of granulomatous inflammation and portal hypertension. Idiotype (SJ-CRIM) expression is high in acute infection, a time of elevated portal pressure and in~ammation.Antiidiotypic (SJ-CRIM) expression is elevated in chronic infection when portal pressure is low and hepatic inflammation has waned. These observations suggest that the expression of SJ-CRIM is modulated by antiidiotypic antibodiesdirected to the epitopes. Furthermore,themodulation of expression of SJ-CRIM on anti-SEA antibodies or Tcells may represent a maker and,potentially, a mediator fordisease modulation. To analyze the expression of murine SJ-CRI on a clonal level, a family of monoclonal antibodies were developed from the somatic cell fusion of infected or SEA immunizedmice with mouse myeloma P3NS1 [38]. The monoclonal antibodies were serologically characterizedand compared to the previously described naturally occurring polyclonal reagents. A family of anti-SEA (idiotypic) and antiidiotypic monoclonal antibodies were produced. They were characterized serologically for the expression of the SJ-CRIM immune network, for their ability to suppress SEAinduced blastogenesis in vitro, and for their ability to suppress granulomatous inflammation in vivo. From these data, it can be concluded that monoclonalSJ-GRIM antibodies appear to be the most immunosuppressive both in vitro and in vivo (using the lung granuloma model). It is also clear that other idiotypic molecules, serologically distinct from the SJ-CRIM network, can regulate SEA-induced blastogenesis in vitro and granulomatous inflammation in vivo. It is likely that these latter

moleculesinitiate other immune-mediatedsuppressormechanismswhich

do not

he in vivo effects of re ulatory murine monoclonal idiotypes and antiidioinfected mice. The monoclonal antiidiotypic anting site-specific antiidiotype, which reco was shown to reduce granulomatous inflammation e results demonstrate that a regulatory networkexists in infected by somatic cell ous componentsof this network can be “captured” fusion. expressingmonoclolantibodies(14 to date) and monoclonalantib S, we used these rea~entsto elucidate the antigens that initiate this regulatory immune network [38 a n t i - ~ E Amonocl a1 antibodiesboundtheidentical 9-12 prot estern blot analysis. hese same bands corresponded to periodic acid-Schiff on sodium dodecyl sulfate polyacryla pparent molecular weightsof bet antibodies to antigen was complete1 periodate t r e a t ~ e n tof antigen. These data suggest that the initiated by antigen(s) composed of carbohydrate-based epitopes. The mousemodel of S. j a p o ~ i infection c~~ continuesto offer investi~ational challenges including the precise characterization of the SJ-C I, antigenic epitopes, the determination of the cellular and subcellular mechanisms of idiotypic/antiidiotypic-mediated regulation, and the characterization of the antifibrogenic activity of chronic mouse serum(anti-~J-CRM). A summary of the immune network internoted to date in the murine model of schistosomiasis japonica is found in +

The immunopathological characteristics of schistosomiasis in humans have also been the subject of considerable investigation. Clinical disease (hepatome schistosomiasis japonica is not strongly linkedto intensity of infection [39]. gators have suggested that the development of clinical disease is under the control of certain human leukocyte antigen loci epitopes in humans 1401. Lymphocytes fr S. j a p o ~ i c ~ ~ - i n f e c patients ted proliferate vigorously in response to either pool gg or worm antigens. The cells responding to worm antigens are Leu 2a- 3a+ helper T lymphocytes [40]. umans with remote histories of S. j a p o ~ i infection c ~ ~ can be divided into hi and low responders with respect to their humoral responseto worm antigen [41]. fferences in T cell responsiveness are specific to schistosome anti ens and are associated with certain of infected hosts [42]. Using-compatible cells from high and low responders, it has been demonstrat o that antigen-specific responses are downressor T lymphoc blood mononuclear cells S) of low responders. CD$- T cell lines have be rproduced which p j a p o ~ i cSEA ~ ~ ,andsecrete interleukin 2 (IL-2 . One CD4” but was found to suppress luble suppressor factorwas

Summary of Idiotypic-Antiidiotypic Interactions ~inc ~ j s t o sj o ~~ a ~ o ~ j c ~ ~ Infection" Murine infection 1. SJ-CRIM -major cross-reactive idiotypeis expressed on anti-S -idiotype is expressed in acute infection. ype (anti-SJ-CRIM) antibodies are expressed in chronic infection that act as antigen mimics. 4. Immunomodulation of hepatic abnormality (granulomatousin~ammation) is caused by antiidiotypic antibodies. 5. SJ-CRIM is expressed on both B and T cells during infection. 6 . Specific members of SJ-CRIM idiotypic network can be captured as monoclonal antibodies derived from chronic infection. 7. SJ-CRIM is carbohydrate-based. Human infection 1. Idiotypic anti-SEA antigen binding reactivityis highly conserved between subspecies of parasite. 2. Idiotype expression varies among the antibodies expressed in patients with differing disease manifestations. 3. Idiotypic antibodies derived from patients with acute infection and no hepatomegaly are nonspecifically immunosuppressive. 4. Idiotypic antibodies derived from patients with hepatic abnor~alityare nonsu~~res~ive. 5. Antiidiotypic antibodies (defining HuSJ-CRIM) derived from chronically infected individuals specifically suppress antigen-driven T cell responses. 6 , Antibodies derived from EBV-transformed PBL express CRIM 7. Idiotypic antibodies derived from Chinese and Filipino patients are serologically and functionally distinct. 8. The antiidiotype (anti-SJ-CRI,) found in mice is serologically and functionally distinct from the human antiidiotype (anti-Hu-SJ-CRIM). "CRI, cross-reactive idiotype; SEA, soluble egg antigen; EBV, Epstein-Barr virus;PBL, peripheral blood lymphocyte.

were performed with the Japanese subspecies of S. j a o ~ i can~ ~ , these observations to immunoregulation of infectio cies and its idiotypic network is unknown. In init clones [43], we have isolated SEA-reacti of forming in vitro granulomas man T cells do not need to be an tions in vitro. hepresenceof an i ~ m u n enetwork (idioty~ic andan i o t y ~ i cmolecules) the course of the human disease has only recently been ablished in human S. j ~ ~ oinfection. ~ i cOur ~ recent ~ studies have focuse on the immunoregulatory function of these idiotypicandantiidio olecules, as well as the serological and functional nature of the murine and immunoregulatory networks. §erum antibodies bind in^ S. j a ~ o ~ antige~s i c ~ ~ have fied from clinically and parasitologically definedgroups.Thesepolyclonalmolecules c were specifically purifiedfromserumpoolsand were shown ess serologically distinct idiotypes in association with clinical manifestatio pecifically, theidio~ypes

ies of acutely infected children and adults, as well as those of children with and without hepatic enlargement, differed serologically. Serological also seen at the antigen binding level on the basis of Western en these idiotypic reagents wereaffinity-purified?antibodies from n with acute in~ection and no hepatomegaly were nonspecifically suppressive in the human blastogenesi say; i.e., both antigen- and mitogen-driven cellularresponses were suppress~d. S is ananalogous finding tothat in the murine system for i iotypic antibodies binding schistosome antigens. In findings notanalogous tothe mu em,purifiedanti-SEAidiotype,derived from children with primaryinfecthepatosplenicenlargement, was nonsuppressive in nterestin~ly? pur types derived from acutely infected adults with hepatic abnormality had a marked stimulatory effect on l? CS in the absence of antigen.Thislatterobservation was noted in S. r n f f ~ s Q ~ iction with reagents from chronically infected adults without abnormality. These results suggest that idiotypic differences exist between parasitologically and clinically defined groups in schistosomiasis. ies, immune network components were generated by Epsteinmation of circulating mononuclear cells of patient 51. In these experiments, cryopreserved cel transformed and the resultant polyclonal antibodies characterized by though these polyclonal idiotypic reagents represent a small spectrum of ies produced by that indi ual, a variety of polyclonal idiotypes have been produced by this method. The V transformation of cells derived from an acutely infected male patient resulted in two distinct polyclonal antibody populations.These antibodypreparations were idiotype-(antiantigen)-positive.They bound both egg and worm antigen. The antibodieswhich did not bind antigenwere tested for their ability to block antigen binding to idiotype. Polyclonal antibodies which block antigen bi ing to idiotype WO be potentialantigen-combiningsitespecificantiidiotypes.40-year-oldmalelipino with aremote S. jffpQ~~curn uced five distinct idiotypic populations as well as several antiidioiidiotypic polyclonal antib ies inhibited the binding of idiotype to ilipino derived antibodies. owever, the antiidiotypic antibodies did ing of idiotype, expressed on Chinese-derived antibodies,to antigen. cate that the Chinese and ~ i l i p i n oidiotypic antibodies are serologieveral other antiidiotypes were generated from chronically infected individuals. Cells from an individual produced an antiidiotype population which inhibited idiotype binding by 72%, thereby quantitatively defining a majo reactive idiotype expressed on Filipino anti-SEA antibodies and designated ived idiotype binding to antigen to the purified polyclonal idiots in the same clinical category patterns were observed for egg , as previously seen in the murine system, r Western blot egg antigen binding profiles. The polyclonal idiotypic and antiidiotypic antibody populations were tested for inhibition of antigen-induced blastogenesis using human cells from individuals with a history of past infection. The idiotypes produced by E V transformation

were shown to be nonspecifically suppressive. These findings were consistent with the data generated with the clinically defined, purified pooled serum samples. The transformed antiidiotypes, including the antiidiotype, which identifies CRIMPinduced antigen-specific suppression. These findings paralleled the antigenspecific suppression observed in the murine idiotype/antiidiotype network. Xenogeneic antiserum derived from immune rabbits has been tested in the human blastogenesis y as well [46]. Antiseracontaininantiidiotypicantibodies SJ-CRIMor murine SJ-CRIM wer compared. ~ntiidiotype serologicallydefining U SJ-CRIM was highly suppressive of only SEA-induced blastogenesis. On the other hand, antiidiotype muri -GRIM was not suppressive. The lack of function in thehumanassay(althoughmurine SJ-CRI, of themouseantiidiotype SJ is highly suppressiveinthe system) led tothe serologicalanalysis of the use-derived antiidiotypes. This analysis d that human antiidcannot inhibitacutemousesera (SJbinding to antigen. n together show that mouse idiotype is gically distinct from human idiotypic antibodies in S. j a ~ o ~infection. i c ~ ~The functional significance of this observationis that murine immunoregulatory reagentsdo not functionin the human system, Thus, it is unlikely in S. j a ~ o ~infection i c ~ ~that there is shared idiotypybetween humanandmurineimmunomodulatory molecules. Inother words,althoughregulatory idiotypicnetw ks exist in both infected mice and h u ~ a n s the , networks function in parallel a appear to be quite distinct functionally. ting the idiotypic dichotomy of the murine and human networks,an analy-SJ-CRIM is imperative. Initial studies have generated and characterized a lymphocyte of patients from endemic villages in the [46]. These IgG human monoclonal antibodies were individuals and exhibited an an *

m acutely infected

serologically related to nons

ica. If the latter observation stands the test of time, it would require all potentially therapeutic idiotype-based molecules for S. j a ~ o ~infection i c ~ ~to express human idiotypic serological characteristics.

The role of idiotypic/antiidiotypic interactions in the i m ~ u n response e to sc~istosos been o ~the ~subject of si~nificantinvestigation using the rodent ~ i ~ s~i sa ~ has

model and infected humans over the last few years. Several authors [48-511 have shownrecently that idiotypic/antiidiotypicinteractions are also involved in the protective immune response to S, ~ a ~ sinfection. o ~ i These studies show the use of internal image antiidiotypic vaccines for the protectionof rodents from achallenge by cercariae. A series of experiments analyzing the role of idiotypic/antiidiotypic interactions in the induction and regulation of immunopathological processes in murine and human S. ~ a ~ sinfection o ~ i have been performed [52-55]. In addition, recent evidence suggests that idiotypic/antiidiotypic molecules may cross the placenta during gestation and sensitize children in utero [59,60]. Thus, children born to infected mothers have been exposed to anti-SEA idiotypes and may have a modulated immune response[61]. The authors speculate that this modulation or in utero exposure might aid those children to adapt successfully to later infections. Such observations may have important implications to immunological adaptations by populations chronically exposed to schistosomiasis mansoni. In this regard, all children experiencing a primary infection are not at the same immunological starting point.Childrenborn to infected mothers may possess “inherited resistance’, to infection due to the previous exposure in utero. Thisis an important areaof investigation which has yet to be addressed inS. J a ~ o ~infection. i c ~ ~ Idiotypic and antiidiotypic immune network components have also been described using inbred mice infected with S. ~ a ~ s[62-631. o ~ i The antiidiotypic antibodies specifically appear to increase during chronic infection. Infected mice also have both antiidiotype positive T and cells. Recent studies have shown that Tcells specific for antiidiotypic antibodies can induce both cellular and humoral protective immunity [64]. In addition, at least two different T cell-derived suppressor factors have been isolated 165-66). Such molecules have been shown to be capable of modulating egg-induced granuloma formation in vitro. One factor appears tobe an antiidiotypic I-J positive, I-J restricted soluble T suppressor factor, while the other appears to be a dimer bearing a specific SEA antigen receptor and an I-J determinant. This lattermolecule was therefore idiotypic. In human S. ~ a ~ sinfection, o ~ i patients with different clinical forms of schistosomiasis appear to have different populations of anti-SEA (idiotypic) antibodies [67]. Although by ~ e s t e r nblot and ELISA analysis these anti-SEA molecules appear identical, they are significantly different by idiotypic analysis. Purified idiotypes are capableof stimulating lymphocytes directly without antigen[683. Patients with hepatosplenicdiseaseappear to have antibodieswithserologicallydistinct idiotypes compared to antibodies derived from patients in whom abnormality does not develop with chronic infection. Idiotypic antibody preparations from patients with hepatosplenic disease do not stimulate lymphocytes from patients in their or other clinical categories. Idiotypic anti-SEAantibodies derived from asymptomatic, chronicallyinfectedpatients appear to regulate autologous cellularproliferative responses to SEA, These patients regulate SEA responses as assayed by in vitro granuloma formation. Idiotypic antibodies at higher (20-40 pg/ml) concentrations stimulate the proliferation of presumed antiidiotype positive T cells found in the spleens of acutely infected mice or the PBMCs of chronically infected but asymptomatic humans. A concentration of 75 pg/ml of idiotype was required to suppress in vitro granuloma formation. Finally, idiotypes from asym~tomaticinfected patients (but not those with hepatosplenic abnormality) canbe used to induce cells to regulate anti-§EA-specific in vitro ~ranulomaformation [69-701. These idiotypic ~

molecules stimulate antiidiotype-positive T sup roliferation requires receptor cross-lin~ing (at purified T cells require a source for cytokine stimulation. The anti-SEA.idiotypic/antiidiotypicimmunenetwo s Q to~ i viously inhumans infected with S. ~ ~ ~ appears reactivity with the murine model of infection ~ 7 0 - 7 1 ~Spe . types from acutely infected mice appear to have similar anti-SEA idiotypes found in the serum of asymptomaticinfected humans. In addition, spleen cells from chronically infected mice which have modulat mality are also responsive to stimulation by idiotypes. These idiot have been observedin the serum of eitheracutely infected animals o infected humans. Patientswith hepato~plenicdisease do notexhibit these regulatory network components in their serum express or iton their cells. These observations taken together and summarized in Table 3 suggest that in humans who have significant abnormality (hepatospl ic disease) beneficial regulatory idiotypic/antiidiotypic responses fail to develop. a~dition9 both inbred mice and asymptomaticallyinfected h u ~ a n express s serologically related beneficial regulatory idiotypic/antiidiotypic networks. Thus, in S. ~ ~ infection9 ~ thes geneti-Q cally determined expression of idiotype may direct the clinical course of infection.

All infectious diseases can be thought of as parasitic in nature in terms of hostorganism interactions. Thereis an expanding literature regarding immune networks and infectious diseases. When they are considered in toto, it is apparent that idiotype and antiidiotypic molecules can play a dual role in the ecological relationships established between the host and invading parasite.~diotypic and antii~iotypic molecules can be components of the host adaption to infection, In cases of symbiosis or chronicinfection,immunenetworkcomponentscanmodifythe host’s immune response and promote a state of tolerance to harmful autologous autoimmuneresponses. Thus, the host may suffer only mild abnormality or illness. In this case, ~daptation to infection benefits both host andinfective agent. owever, idiotypic and antiidiotypic molecules can be more confrontational

i Salient Features of Idiotypic Antiidiotypic InteractionsS.in~ u n s o nInfections 1. Antiidiotypic antibodies functionas surrogate antigens and induce protection to reinfec-

tion. 2. Idiotypic and antiidiotypic antibodies appear during chronic infection. 3. Antiidiotypic T cell secrete suppressor factors which reduce granulomatous inflammation.

4. Patients with differing clinical formsof disease exhibit serologically distinct immune network components. 5 . Idiotypic sensitization occurs transplacentally. 6. Idiotypic antibodies stimulate human cell blastogenesis. 7. Serological cross-reactivity between murineand human idiotypi~/antiidiotyp~ networks.

~

athogen. Specific cross-reactiv iotypescanbeantiparasitic by binding igens. Immunolo 1 mechanisms promoting expressionof lify antigen-based immunotherapeutic approaches. In a simiof antigen, types acting as ant n mimics, or internal images vaccine strategy. attractiveadvantage in thiscase is the ability to generate, in quantity, clonal antiidiotypes with defined specificities. In addition, immunization m duced by antiidiotype vaccination in comparison to theuse of attenuat Thus in the classica nfectious diseases, two approaches have been ve shown the relevance of idiotype and antiidiotype to pathoegenerationof ac~uiredresistance to infectionthroughprotective to research on schistosomiasis, a large volume of work has anosomiasis. Sacks et al. [72] initially described the use of to generate protective i ~ m u n i t ythat was idiotype-specific. owever, the concept of idiotype escape, which has been eloquently described in themurinemodel of systemiclupusythematosus,wasnoted in these studies. Thus, the ability of the parasite to mo late surface antigenexpression by masking or variant-glycoprotein expressionis problematic in this form of therapy. In ~ $ y ~ a ( ~ m e r i c a ntrypanosomiasis) antiidiotypic immune rodents were noted roduction of antiparasitic antibody [73]. On infection, howimals were not protecte from infection despite high titers of serum hese studies show the need for oligoclonal antibodies for cessity to establish the mechanisms of protective immunity for each infective agent. nce established, i ~ m u n enetwork responses can be tailored to themost effective avenue of therapy. This latter point canbe most easily exemplified by two recent studies [74,75]. ice were shown not to be protected on exposure to imalsexhibited an antiparasite antibody response Zayi surface determinants; however, they exhibited nocellses to the larvae. In parallel studies,mice immunized with l1 immune response also showed an altered course infectio of ~ o ~chabau~i. i u ~These mice had a si nificant reduction in peak parasitemia levels in concert with no antibody titersto mune network components can be utilized to induce T cell suggest that antiidiotypic antibodies canbe used to induce cellular responses, which have been proposed to be a first-line defense e pathogenesis of parasitic diseases, autoantiidiotypic antiponses have been described in patients with Chagas disease [76]. The expression of the antiidiotypic components correlated with theclinical form of the disease. In these studies, it was shown that patientswith the clinical form of the disease exhibit high levels of stimulatory idiotypes. The authors suggest that these of severe Chagas disease is not due tojy: observations indicate that the pathogenesis cruzi antibody responses but to antiidiotypic antibody-directed immuneresponses. n other words, the clinical form of Chagas disease is potentially due to cellular hogenic idiotypes expressed on a population of h a hypothesis suggests a genetic basis for the of in parasitic infections. Individuals encoded with road spectrum

germline “pathogenic” idiotypes would experience the clinical sequelae of parasitic infection. ~lternatively, the lack of expression of these germlike genes, through either genetic deletionor induced immune tolerance, wouldresult in the asymptomatic form of disease.

In conclusion9 idiotypic and antiidiotypic molecules can modul te parasitic-induced immunity and disease. The studies outlined here, however, are only initial investigations. It is likely that i m m u ~ enetwork compone~tscascade their immunomodulatory effects into the world of cytokines and Tho, Thl, and Th2 cells. The latter field of study in itself is a complex array of intera~tions. Thus, theelucidation of immune-mediated disease and vaccine strategies for parasitic diseases remains a complex puzzle of interacting systems.

The author acknowledges the numerous colla~orativestudies performed with Dr. ichard Olds thanks andonilla secretarial her for assistance.

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30. AW Cheever, HE Bryam, S Heiwy, F von Lichtenberg, MW Lunde, A Sher. Immunopathology of Schis~osomaja~onjcumand S. mansonj in B cell depleted mice. Parasite Immunol7:399, 1985.

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yram, F von Lichtenberg. Immunopathologyof Sc~istosomajapon-

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AW Wisnewski, TF Kresina. S. japo~icumegg antigen specific human T cell line derived

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pathol, 65:325, 1992. Olds, JH Johnson, resina. Function expression and of ahumanidiotypicnetworkin schistosom~asis japonica. arasite Immunol 18:439,

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expressed as human cross-reactive id associated wtih immune function in S. 'a onicum infection. Hum Antib Hybridomas5 : 178, 1994. 48. Crzych, M Capron, H Bazin, A Capron. In vitro and in vivo effector function of IgC2a monoclonala n t i 4 ~ a n s oantibodies. ~i J Immunol 129:2739¶ 1982. 49. Crzych, M Capron, PM Lambert, C Dissous, S Torres, A Capron. An anti-idiotype vaccine a ainst experimental schistosomiasis. Nature (London)316:7 oussel,CVerwaerde, J M Crzych, A Capron.Protectiveeffects on anti50.

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71.

72.

*

73.

74.

75 * 76.

National rnstit~te Diabetes of and Di~estiveand Kidney Diseases, National rnstit~tes of ~ealthy 3ethesda, ~ ~ r y ~ a n d

Stanford Wniversity Schoolof ~ e d i ~ i nStan e y ford, ~ a l ~ o ~ n i a

*

Technologies that generate individual antibodies are important in biomedical research [l]. They provide useful toolsto determine antigen binding characteristics or biologicalactivities of specificantibodies. Currentmethodologies allow for the generation of human monoclonal antibodies from individuals with asymptomatic or symptomatic disease states. Thus, the clinical relevance of specific cloned human antibodies in the host’s immune response and disease pathogenesis can be readily investigated by using somatic cell hybridization techniques. Therapeutic interventions can alsobe devised by using human monoclonal antibodies.By using a combicell selection, in vitro polyclonal activation, and somaticcell hybridization techniques, one can generate virtually any antibody represented in the human cell repertoire. In addition, the random or specifically directed combination of immunoglobulin heavy and light chains can potentially generate a specificity of antibody that transcends that of the human repertoire. An alternate approach for this goal is the use of recombinant deoxyribonucleic acid (DNA) technologies and antibody engineering techniques [2,3] To identify biologically relevant antibodies of the human immune response toinfectious pathogens, somatic cell hybridization is the preferred approachto yield identical antibodies to those producedin vivo. For the generationof human monoclonal antibodiesusing cellular means, two basic methodological approachesexist [l]. First, immortalization of lymphocytes by Epstein-Barr virus(EBV) transformation and cloningby limiting dilution can result in the generation of long-term cultured lymphocytes secretingan individual immunoglobulin molecule. This technique hasbeen used successfully to generate antigen-

iquehasseriouslimitations, which cells and instability of antibody production fromthese cell lines. second,morepromisingmethodology to immortalize anti hrough the somatic cell fusion of lymphoid cells with long-t monoclonal cell lines. This technique thus oduces hybrid cell lines or “hybridomas.” The ideal fusion partner for lympho cells are stable cell lines that do not secrete immunoglobulin but possess the cell r machinery for antibody production and secretion. Cell lines that canbe readily selecte cells after fusion are the cellsof choice. relevant monoclonalantibodiesare actual1 as,theproduct of fused to an activatedhuman cell. Althoughnumerous cell sionpartners have been reported for the generation of h u ~ a monoclonal n antib es, the choice of a specific hybridoma fusion partner with the technique of som cell fusion results in dramaticallydifferent results [S]. Thesearametersincludefusion efficiency, level of antibody secretion of the hybridoma cells, and stability of the hybridoma cells in long-term culture.

uman monoclonal antibody p uction requires the immortalization of a single antibody-producing ell cannot be in inactivate~/resting an state but must be “activated” bulin synthesis at the time cell of fusion. Some studies [6] have reported the generationof immuno lobulin ting hybridomas without prior vitro inactivation. generation the of maximum numbers hybridomas of secreting indivi lobulin molecules, in vitro activation is required. In our laboratories, studies using peripheral blood ed from patients with infectious diseases exhibiting high ivo have generated only a limited number of hybridoma ina unpu~lishedobservations). After in vitro immunization, however, there is an increase in the number of activate^, antigen-s cells [7]. This limitation necessitates the utilization of in vitro stimulation of with polyclonal activators, therebyresulting in a greater number of hybrid0 cific disease-related antibodies. Corn polyclonal activators S are mitogens, pokeweed mitogen , and phytohemaggluS separated by Ficoll gradient centrifugation are cultured for 2 to 5 days in the presence of vitro.Somatic cell fusion is thenperformed.In general, a bell-shap generated when the number of fusion products versus days in culture i It is not clearly established whether higher number of antigen-reactive I culture of IgG secr hybridomas has been achieved on the basis of a 4 to 5 day mitogen activation [ has been extensively used as a primary step to generating

human monoclonal antibo ily infects human cells via binding of complement receptors and induces proliferation and antibod secretion. The percentage of cells activated is far g n u ~ b e of rthose likely to be infected transformed. or inued incubation of activated V results in cellular “i ” these cells tend to be unstable in maincommonly used approach is to activate llowed by cell fusion with an appropria

a frequency (l per lo4 cells of a given specificity are in general re~resented at low cell repertoire [lo, l l]. To increase the number of an enrichment procedureis frequently per taking advantage of the observati exhibit specific antibody on the cell surface. Using soluble antigens enriched by the technique of rosetting [12]. accomplished by “~anning”for cells on anti tion of this technique, a study[l51 has re orted the selection of act use of antigen-coated paramagnetic V activation has led to greater effici cell selection, activatio~, andclonal 1 has recently been described [16,1’7]. blood lymphocytes ferred by intraperitoneal injection to severe combined immunodeficiency human cells are able to survive for long periods in th of responding to in vivo immunization. The engrafted, “hu

specific fortheimmunogen9pep

190.

methodofferspromiseforobtaining

*

of human ly~phocytesagainst

I.

Further generation of antigen-specific cells, before fusion, can be performed by in vitro immunization of culturedcells. his is particularly relevant for the development of monoclonal antibodies to toxins and pathogens due to the obvious constraint of intentional humanexposure. The techniqueof primary in vitro immunization is becomingstandardized [21]. Specific re~uirements common to reported methodologies include(1) antigen, usually inthe formof antigen c molecule; (2)use of polyclonal stimulators (polkweed mitogen [P charide [LPS]);(3) use of B cell growth factors and T cell factors; and(4) use ofaccessory cells and T cells. By using these elements for in vitro activation, activated antigenspecific human B cells have been produced as a prerequisite for the production of human monoclonal antibodies. Somatic cell fusion with these activatedcells has resulted ina higher efficiencyof antibody-secreting hybridomas[22].

Afterantigen-specific cell activation and enrichment, the cell mustberendered immortalthroughhybridomaformation. As notedearlier,thisprocessoccurs through the somaticcell fusion of the lymphoidcell and theselected fusion partner. ~ u m e r o u shuman-mouse heteromyeloma cell lines have been generated for this purpose [5,9,23-281. These cells lines have the properties of nonimmunoglobulin and selectivity (usually hypoxanthane-guanosyl-phosphoribosyltransferPRT]-deficient,ouabain-sensitive). Humanhybridomas generated with thesefusionpartnershave been docum yield a level of1 to 100 pg/mlof immunoglobulin long-ter formation inma produced is by the physical activated fusion of edfusion cell partner. This is most often accomplished by mixing the cells together in the presence of polyethylene glycol (PEG) [23,24,28-311 asthe fusogen.Thisprocedurehasproducedmany antibody-secreting human hybridomas but with specific limitations. These include the necessity for relativ~lylarge numbers of activated cells (5 to 10 milli relatively low hybridoma formation efficiency (one suc ssful event per io5 and the generationof human hybridomas with unstable immunoglobulin secretion. These limitations have led to the development of electric field-induced cell fusion, or electrofusion techniques [3 l]. These techniques are capable of immortalizing a small number of cells and achieving a higher hybridoma formation efficiency than previously possibleby chemical means. The process is based on the observation that closemembranecontact between two cells can beestablishedin an alternating electrical field. Subse~uentapplication of a second direct electric current can then lead to a breakdown of the plasma and nuclear membranes, creating cytoplasmic and nuclear membrane bridges and leading to cell fusion. Using these two properties, electrofusion techniques have been shown to increase hy~ridomaformation 10- to 100-fold more than polyethylene glycol-induced cell fusion. With arr virus- (EB~)-activatedB cells, for example,hyb in therange of to lo-’(hybridomasperinput observed with PEG fusions. With standard electrofusion techniques, the efficiency rangesbetween and [32]. A comple~entaryadvanta~eof standard electrofusion techniques is the substantially lower number of input cells B needed to effect

the more efficient hybridoma formation. Polyethylene glycol-induced cell fusions generally require 5 X 106 cells, whereas electrofusion techniques are regularly performed with 1 X los cells. One of the major limitations in the production of antigen-specific human monoclonal antibodies is the rarity of relevant antigen-specific cells in peripheral d tissues. The general practice of identifying subpopulations of cells involves initial activationof cells in microtiter wells. Further expansion of the relevant cells may not be possible because of terminal differentiatimulation or theloss of secretion frequently associcells. Therefore, it is desirable to have techniques capable of immortalizing even a smaller number of cells. This further enhancement of hybridoma formation efficiency has been achieved with electrofusion (Figure l), especially when performed under hypoosmolar conditions [33]. The effect of hypoosmolar conditions in facilitating hybridoma formations has been postulated on the observation that the surface area of membrane contact between two adjacent

i

("-J t

ure 1 Electroalignmentandelectrofusion of cells:(A)Application of a low-voltage alternating current causes ionic separation in the cells. Inhomogeneity in the electrical field (B) Cells are aligned and tightly approxiresults in dielectrophoresis of cells toward one pole. mated on one electrode during application of the alternating field. B cells are opposed to of one or more short-duration, high-voltage pulses fusion partners randomly. (C) Application disrupts the cell membranes in the area of highest electrical field strength, i.e., at the cell "poles." Cell membrane intermingling ensues and fusion follows over a period of several minutes. Hybridomasare selected by conventional culture techniques.

cells aligned in analternating electric field is markedlyincreasedhypoosm conditions. This alignment provides a greater area for cell S contact subsequent ease in cell fusion. Another factor is the nuclear swelling associated with this condition, which facilitates nuclear fusionin the fused c contribute to a higher rate of hybri Electrical parameters defined in a hybridoma f o r ~ a t i o nefficien been accomplished with total inpu reduction in the required number is also associated with a hybridoma formation efficiency at hieved with a higher input cell number with isoosmolar condi factor in the development of this microfusion techniqueis the d ells and fusion partnersto the hypoosmolar c lectro~usiontechnique of hybridomaproductomesmore ed, it is clear that this technology has distin induced cellular fusion. Electrofusio extremely small numbers of activated large quantities of immunoglobulin listing of human monoclonal antibodies generated by using either the electrofusion technique.

As listed in Table 1, human monoclonal antibodies to many inf~ctious age~ts have been generated. These monoclonal antibodies have important potential uses in elucidation of the host’s immune response, identification of infection, diagnosis of specific pathogen, and therapy. Therapeutically, monocl man monoclonal antibodies can be used associated with S

to prevent

neoplasms within the body and for noninvasive internal organs [36).

The utility of human monoclonal antibodies for diagnostic purposes is illustrated by our investigation to identify and characterize immunogenic domains of human T-lymphotropic type I and type 11 viruses ( human retroviruses that T-lymphotropic man viru -cell leukemia/ ymphoma and a chronic neur

onoclonal Antibodies Derived from the Fusion of Human Lymphoid Cells and Heterohybridoma Fusion Partnersa HuMoAb

Fusion Immunoglobulin Reference technique subclass

Anti-T Cell Anti-VZV Anti-E~V lepra Ant~-~ycobacterium Anti-HIV Anti-HLA class I Anti-type 11 collagen Autoantibodies (multiple organ specifications) Antiplatelets Anti-Schistosoma mansoni Anti-squamous cell carcinoma Anti-T lymphocytic leukemia Anti-H~LV-I Anti-HLA classI Anti-Schistosoma ~ a n s o n i Anti-Schisto~omajapo~icum Anti-Cryptosporidum Anti-gp120 (HIV) Anti-hepatitis B Anti-hepatitis C Anti-stomach carcinoma Surface antigen Antitetanus toxoid Anti-Staphy~occusaureus Antirubella Antimeningococcal Anti-rheumatoid synovium

PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG PE6 PEG Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion Electrofusion

63 25,63-65 66 68 67,71 24 24 29,72 69 25 70 74 75 30,31

81 79 80 54-57 66 43,73 69 9 b

76 b

16 52 36

77 15 78 82 35

"HuMoAb, human monoclonal antibody; RBC, red blood cell; Ig, immunoglobulin; PEG, polyethyleneglycol;VZV,varicella-zostervirus;EBV,Epstein-Barrvirus; HIV, humanimmunodeficiency virus; HLA, humanleukocyteantigen; CMV, cytomegalovirus; HCMV, humancytomegalovirus; HTLV, human T-~ymphotropicvirus. %resina unpublished data.

thy or tropical spastic para uveitis and p o s s ~ ~ to l y poly TLV-I, diseases associated are just beginning to erne reportedonpatients wit

It has been also linked to I is structurally similar to ore difficult to identify and therinvestigators have recently E39-421 ike illnesses who are seropositive for

TLV-I1 and whose peripheral blood lymphocytes were demonstrated to contain TLV-I1 by polymerase chain reaction(PCR) analysis. Disease association studies of these viruses have been hindered by the need for multiple assays to confirm and differentiate infection between these vir approach this problem, our laboratory isolated and characterized a unique epitope with the human monoclonal antibody, 0.5 alpha [43-451. The lambda gt 11 expression vector system was used to generate an epitope library of HTLV-I envelope gene-encoded antigenic determinants. In this system, recombinant peptides are expressed as fusion proteins within the native bacterial be alactosidase molecule. By immunoscreeningwiththe 0.5 alphaantibody,multrecombinant epitopes were isolated. One of these recombinant proteins, designated MTA-1, has been identified with HTLV-1 infection but notwith HTLV-I1 infection or controls. To determine whether the analogous region of HTLV-I1 also contains recoga nized epitope, HTLV-I1 deoxyribonucleic acid (DNA) primers were designed and DNAfragmentscorresponding to different portions of tputativeepitope were synthesized by the PCR procedure [45]. The synthesized NAs were then cloned into the EcoR I site of gtl 1 and subsequently screened for e production of immunoreactive fusion protein by using a number of sera isolated from HTLVL-11- or HTLV-I-infected individuals. One of these recombinant proteins, designated K55 by Western blot analysis, reacted with virtually all HTLV-I1 sera and not HTLV-I or controlsera. In modified a Western blot assay usi V-I viral lysate and another recombinant protein, designated p21e [46], and K55 have been incorporated, By defining confirmation of either virus infection asthe presenceof antibodies to p24 gag protein and to p21e recombinant envelope protein, all HTLV-I and HTLV-1I sera were confirmed. Differentiation of the twoviruses was determined by reactivity to either MTA-I or K55.The inclusion of these recombinant proteinsin a modified Western blot hasallowed investigators to confirm and differentiate infection with these viruses in a single assay unambiguously. These results confirm the usefulness of human-derived monoclonal antibodies as an experimental approachto dissect the human humoral immune modulation viral to pathogens.

Viral hepatitis is a systemic infection in which hepatic cell necrosis and inflammaof hepatic sequelae. The multiple viral agents have tion result in a constellation distinct immunoserological characteristics, specific epidemiological attributes, and separate causes. No single histopathological lesion inthe liver is diagnostic for viral owever, there are common in~ammatorycharacteristics apparent in viral hepatitis. Acute portal hepatitis is characterized by edema and an inflammatory infiltrate comprising lymphocytes and histiocytes that enlarge portal tracts. In hepatitis C infection, focal bile duct lesions are common and in chronic infection canresult in degenerativechanges,includingnecrosisandperiductularfibrosis. Necrosis of periportal hepatocytes and merger of portal and lobular inflammation are characteristic of “piecemeal necrosis’’ and can occur in either acute or chronic active hepatitis. A further characteristic of viral hepatitis is lobular disarray where spotty necrosis occursand the in~ammatory cells accumulate in areas of h e p a t o c ~ e

necrosis [47], Thus, infection with hepatitis B virus or hepatitis C virus without subsequent resolution or cure results in hepatic in~ammation thatprogresses to chronic hepatitis, cirrhosis, and primaryliver cancer [48]. For acute hepatitis B virusinfection,approximately 90% of infections of adults resolve with the development of protective antibodies [49], Individuals vaccinated with viral surface antigen generate a substantial antiviral antibody response. On the basis of these observations, the concept of adoptive transfer of protective antibody-based immunity has been proposed [SO]. Adoptive transfer of protective immunity may represent a viable approach for application to immunologically nonresponsive individuals, such as liver transplantation recipients. Integral to this approach is the characterization of the protective antibody response and eplication as human monoclonal antibodies. Anumber of humanmonoclonalantibodiesspecificforhepatitisBvirus surface antigen have been generated. In a recent study [51], two human monoclonal antibodies were developed -lymphocytes from E derived from individuals with high titers of antis bodies. The transformed lymof human phocytes underwent large-scale fermentation to produce batch quantities monoclonal antibodies. Characterization of the antibody bindingepitopes indicated that one antibody bound a conformational epitope, whereas the other bound a linear epitope of hepatitis surface antigen. Only the latter antibody produces total inhibition of binding of hepatitis B surface antigen subtypes, suggesting potential clinical use. Previous studies had generated a number of human monoclonal antibodies specific for hepatitis B virus surface antigen by a novel method of B cell , activation, andclonal expansion in SCID-Hu mice[16]. Immunization of -peripheral blood lymphocyte (PBL) was followed by generation of antihepatitis B surface antigen antibody secretion hybridomas by the method of electro-

y using similar somatic fusion methodology, a human monoclonal antibody specific for hepatitis C virus (HCV) has been produced by hypoosmolar electrofucells from a patient with chronic sion of in vitro stimulated peripheral blood B HCV infection and chronic active hepatitis with end stage liver disease [52]. The d JRA1, secreted an IgM lambda monoclonal antibody 'v peptides. The antibody was positive on both first- and second-generation hepatitis C virus antibody analysis. Additional laboratories have generated monoclonal antibodies to the hepatitis C virus [53-57]. Siemoneit et al. [S31 have described the ene era ti on of two monoclonal antibodies to the 22-kDa nucleocapsid core protein. Epstein-Barr virus transformed peripheral blood lymphocytes were fused with the heteromyeloma cell line K6H6/BS5 resulting in heterohybridomas that secreted an IgGl/kappa monoclonal antibody and anI g ~ / k a p p a monoclonal antibody. Epitope mapping studies resulted in the identification of amino acids 24-39 of the core protein as the recognition sequence for the IgGl antibody. Additional studies by this group [S41 have provided human monoclonal antibodies to envelope protein E l of the hepatitis C virus. In these studies, the investigators screened the serumof anti-hepatitis C virus-positive blood donors for antibodies to the El glycoprotein. Thirty-two percent of the donors had positive findings for serum antibody to the E l glycoprotein, specifically the EP3 domain comprising amino acid residues 3 14-330. Four anti-E~3-producing heterohybrido-

massecretingeither kappaor IgG1 kappaandlambdahumanmonoclonal antibodieswere gene eIgG1kappaantibody was further able to detect an envelope protein of Western on blot. monoclonal antibodies have been also generated to the nonstructural (NS) of hepatitis C virus. The nonstructural protein NS3 possesses two enzymatic domains that are thought essential for the virus lifecycle. In addition, a human immune responseto the nonstructural region appears in acute infection. An IgC1 kappa antibody,which recognized a sequential epitope on the5-1-1 fragment of the NS4 region has been generated and specifically purifie [56], a human monoclonal antibody has with chronic hepatitis C virus infection n andspecifically recognizes a conform mino acids 1363 and 1454. uman monoclonal antibodies from both trans for me^ cell lines and bridomas which bind the nucleocapsid CO onent of the hepatitis C virus [57] have been identified.AnIgG1 kappaantibody,design 12.F8, recognized native nucleoprotein expressed in transfected eukaryotic Epitope mapping studies resolved a conformational epitope between residues 27 and 59, indicating cell epitope within the immunodominant nucleo-

gnized hepatitis C virus core protool for tissue localization of the virus. Furtherm displayed to phage particles, provided a basis for experiments of in vitro affinity maturation and selection of viral mutants.

The development of human monoclonal antibodies binding schistosome antigensis of multiple aspectsof schistoso~alinfection. essential to the understan~ing monoclonal antibodies are obviouslycrucial in the elucidation of parasite a uman immunoglobulin molecules, in particular IgE, are involved in human resisnce to infection. Also, human antibodies are believed to play a role in immunomodulation, reducing hepatic inflammation associated with S c ~ i s t o js ~~ ~ ~ o ~ i refore, human monoclonal antibo~iesrepresent molecules es contemplate antiinfection- or anti-disease-based vaccines. our laboratory has generated candidatemolecules for vaccine strategies, briefly listed in Table2. In S. j ~ ~ infection, o ~ immunoregulatory ~ c ~ ~ human monoclonal antibodies derived from patients infected with both the Chinese and Filipino strains of the parasite have been generated. In thestudies of infection with a familyof human monoclonal antibodieswere generated by tion/PEG fusion techniques.Splenocytes were the source antibodiesbound a 50antigenpresentin both schistoso ingly, however,thesemonoclonala antigenpreparations. S different immunoregulatory activity in vitro. Two antibodies, 1

oAb in Schistosomiasisa

Immunostage Schistosome Parasite Lymphoctye Fusion Clone China 14AB7 14AE10 14AB2 SJII-D SJII-E SJII-F SJIII-6 SJIII-14

S. japonicum S. japonicum S. japonicum

Spleen Spleen Spleen

Philippines S. japonieum S. japonicu~ S. japonicum S. japonicum S. japonicum Egypt Spleen S. mansoni Spleen S. mansoni

EG EG PEG EG EG EC

lectrofusion Electrofusion Electrofusion Electrofusion

SM23/11-l0

S. mansoni

Spleen

SM38El0

S. mansoni S. mansoni S. mans~ni

Spleen Spleen Spleen

lectrofusion Electrofusion Electrofusion

S. mansoni

Spleen

Electrofusion

+ arm, egg

-

Worm, egg

-

Worm, egg Worm, egg Worm, egg arm, egg Worm, egg

-

Worm Worm, egg, cercarie, schistosomula Worm, egg, cercarie, schistosomula Egg Egg Worm, egg, cercarie, schistosomula Worm, egg, cercarie, schistosomula

+ + ? ? ? ?

? ? ? ? ?

"HuMoAB, human monoclonal antibody;PEG, polyethylene glycol;PBL, peripheral blood lymphocyte.

isotype), showed no immunoregulatory activity. onoclonal antibody 14A (IgG1isotype)s~ecificallysuppressedparasite egg-antigeninduced 1 eproliferation by greater than 50%. These results clearly demonstrate I (iso-

ies were usedwhich e~pressedhigh levels of a cross-reactive idiotype associated with hepatosplenomegaly. These data suggest that some human mo~oclonalantibodies may be candidates foranti-disease vaccine strategies through the immunoregulation of schistosome-in uced hepatic disorders.

Further studies generated antiidiotypic human antibodies secreted by EBVtransformed lymphocytes of chronically infected patients. These antiidiotypic antibodies were shown to be serologicallyrelated to human monoclonal antibodies secreted by hybridomas. Such data reveal shared serological characteristics (idiotypes) of antibodies of different patients with S. japonicum infection. These human antiidiotypic antibodies were also immunoregulatory. They specifically suppressed antigen-inducedproliferativeresponses of P from Filipino S. juponicum-infected patients in vitro. The data taken togeth how at a clonal level the existence of an immunoregulatory human immune network (idiotypic/antiidiotypic antibodies) in the human infection [ S ] . The results of these studies support a therapeutic role for human monoclonal antibodies in regulating the hepatic immunopathological processes associated with human S. japonicum infection. These studies also demonstrate the importance of antibody serological characteristics as expressed by human monoclonal antibodies. Both the isotype and the idiotype appear to regulate S . juponic~megg-induced immune responses in humans. On the other hand, the serological differences in idiotypes as expressed by human monoclonal antibodies induced by different strains of S. juponicum may also partially accountfor thediffering levels of hepatosplenomegaly described in clinical field studies [60,61]. Human monoclonal antibodies are also beingused to studyprotection against infection with schistosomes. Murine monoclonal antibodies have identified schistosome antigens that can confer partial resistance to infection in mice. clonal antibody studiesmay confirm these molecules or identify novel antigens important in resistance in human populations. To this point, it has been assumed that the sameantigens play identical roles in human and murine infection. it should be realized that there may be differences between the antibody repertoires of humans and mice. Thus different antigens may be responsible for resistance to infection in these two hosts. Human antibody studies address this issue and should identify antigens that are involved in the human B cell response to schistosome antigens. A limitation tothis approach is the complicationof evaluating the human monoclonal antibodies in the murine modelof infection. However, recent advances instudieswith SCID mice mayovercomethisproblem [62]. The SCI provides a human immune system inside a murine habitat for infection, a nearperfect model system to screen for therapeutic reagents for human populations. To characterize S c ~ i s ~ o s omansoni ~a antigens, we gene ed human monoclonalantibodies by using bothPBLandsplenocytesasour cell source.Both V transformation/P~G and electrofusion, were used. With P derived before and after parasitic cure, ants E have been successfully obtained from S. mansoni-infected patien -transformed cell lines were noted to be extremely unstablewith regard to antibody secretion. As a result, from 12 PEG fusions we have generated only 2 stable human monoclonal antibodies secreting hybridomas. The fusion products initially secreted high levels of antibody but stopped secreting antibodies or were overgrown by non-schistosome-specific clones in the span of 2 months. This instability greatly reduces the efficiency of human monoclonal antibody production by PEG; as an alternative, our further studies used electrofusion techniques to generate human antibodies. fusion technique and PHA-stimulated splenocytes,we have been successful in generating a family of S. mansoni-reactive monoclonal antibodies from different pa-

tients [g]. These antibodies have been characterized by Western blot analysis and monoclonal showavariety of differentantigenbinding patterns(Table 2). antibodies thatshowbindingtoauniqueantigenexpressed instage of the parasite’s life cycle have been identified. Others show binding to several antigens expressed in various stagesof the parasitelife cycle. Such antibodies define epit whose antigenic expression is modulated during the life cycle of the parasite. cently, we have also identifiedone S. ~an~oni-infection derived antibody,23/11-6, which cross-reacts with separate and distinct antigens of both S. ~ a ~ ~ and o nS.i j a ~ o n i cworms ~ ~ (Figure 2). Identification of suchcross-reactiveepitopesmay form thebasis of a cross-species protective antibody-basedvaccine.

The characterization of human monoclonal antibodies recognizing schistosome antigens has elucidated the understanding of which parasite antigens can be specifically recognized by human monoclonal antibody reagents. Some overlap in reactivity with protective antigens previously identified by murine monoclonal antibodies is observed with the human antibodies developed to date. owever, uniquely reactive human monoclonal antibodieshave also been identified. As the human monoclonal antibody repertoire expands, unique insights into the human antibody repertoire induced by parasitism, as well as the principal parasite antigenic molecules, will be

Conserved antigenic epitopes between schistosome species: S. mansoni infectionS. derived antibody, 23/11-6, which cross-reacts with separate and distinct antigens of both mansoni and S. japoni~umworms.

I.

realized. This further understanding will complement the work already performed with murine monoclonal antibodies in identifying protective schistosome antigens.

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66. 67.

concept and evolution of the research project from targeted immunotherapy against hepatic cells which express viral antigen. Adv Drug Del Rev 17:3 17-320,1995. RA Heijtink, J Kruining, YA Weber, RA DeMan, SW Schalm. Anti-hepatitis B virus activity of a mixture of two monoclonal antibodies in an “Inhibition in Solution’’ assay. Hepatology 22:1078-1083, 1995. U ~immerman,L Love-Homan, P Gessner, D Clark, FC Johlin, CA Neil. Generation of a human monoclonal antibody to hepatitis C virus,JRAl, by activation of peripheral blood lymphocytes and hypo-osmolar electrofusion. Hum Antibod Hybridomas 6:7780, 1995. K Siemoneit, M da Silva Cardoso, A Wolpl, K Koerner, B Kubanek. Isolation and epitopecharacterizationofhumanmonoclonalantibodies to hepatitisCviruscore antigen. Hybridoma 13:9-13, 1994. K Siemoneit, M da Silva Cardoso,K Koerner, A Wopl, B Kubanek. Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoproteinEl. Clin Exp Immunol 101:278-283, 1995. C de Lalla, A Cerino, C Rosa, S Griva. Properties of a human monoclonal antibody specific for the NS4 region of hepatitis C virus.J Hepatol 18:163-167, 1993. MU Mondelli, A Cerino, P Boender, P Oudshoorn. Significance of the immune response to a major conformational B cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody. J Virol68:4829-4836, 1994. A Cerino, P Boender, N La Monica, C Rosa. A human monoclonal antibody specific for the N terminus of the hepatitis C virus nucleoprotein. J Immunol 15 1:7OO5-7015, 1993. G Esposito, E Scarselli, A Cerino, MU Mondelli. A human antibody specific for hepatitis C virus core protein: Synthesis in a bacterial system and characterization. Gene 164: 203-209, 1995. TF Kresina,LWCheever,MChireau,JJohnson,BRamirez,PPeters,GROlds. Human EBV-transformed lymphocytes of patients with Schistosoma japonic~minfection secrete idiotypically related immunoregulatory antibodies. Clin Immunol Immunopathol65:325-329, 1992. so, TR Olveda, GR PM Wiest, G Wu, S Zhang, J Yuan, PRS Peters, S McGarvey, Olds.Morbiditydue to schistosomiasis japonica inPeople’sRlic of China. Trans R SOC Trop Hyg 86:47-50, 1992. RM Olveda, ETiu, P Feridal, F Devegra, F Icatlo, E Domingo. Relationship of prevalence and intensity of infection to morbidity in Schistosomiasis japonica: A study of three communities in Leyte, Philippines. AmJ Trop Med Hyg 321312-1321, 1983. TF Kresina, A Wisnewski, L Love-Homan, B Ramirez, GA Neil. Induction of hepatic pathology is SCID-Hu mice engrafted with peripheral blood lymphocytes of patients J Inf Dis 170:733-736, 1994. with Schistosomi~sis japonica. SKH Foung, S Perkins, A Raubitschek, J Larrick, G Lizak, D Fishwild, EG Engleman, FCGrumet.Rescueofhumanmonoclonalantibodyproductionfrom transformed B cell line by fusion to a human-mouse hybridoma. J Immunol 7:83-90, 1984. A Fletcher, A Thomson. The introduction of human monoclonal anti-D for therapeutic use. Transfus Med Rev 9:314-326, 1995. SKHFoung, JA Blunt, PS Wu, P Ahearn, LC Winn,EGEngleman,FCGrumet. Human monoclonal antibodies to RhJD). Vox Sang 53:44-47, 1989. SKH Foung, J Blunt, S Perkins, L Winn, FC Grumet. A human monoclonal antibody to rh‘. Vox Sang 50:160-163, 1986. SKHFoung, PA Bradshaw,DEmanuel.Usesofhumanmonoclonalantibodies to J Larickeds. andvaricellazostervirus.In:CBorreback, Antibodies. New York: Stockton Press, 1990, pp 140-168.

68. SC Alpert, PJ Turek, SKH Found, EG Englemann. Human monoclonal anti-T cell antibody from a patient with juvenile rheumatoid arthritis. J Immunol 138:104-108, 1987. 69. EF Wallace, SKH Found, K Bradbury, SL Pask, FC Grumet. Generation of a human hybridoma producing a pure anti-HLA-A2 monoclonal antibody. Human Immunol28: 65-69,1990. 70. J Satoh, BS Prabhaker, MV Haspel, F Ginsverg-Fellner, AL Notkins. clonal autoantibodies that react with multiple endocrine organs. N Engl 220, 1983. 71. SKH Foung, S Perkins, C Koropchak, D Fishwild, A Wittek, EG Engleman, FC Grumet,AArvin.Humanmonoclonalantibodiesneutralizingvaricella-zostervirus.J fect Dis 152:280-285, 1985. 72. Banapour, K Rosenthal, L Rabin, V Sharma, L Young, J Fernandez, E Engleman, McGrath, G Reyes, J Lifson. Characterization and epitope mapping of a human monoclonal antibody reactive with the envelope glycoprotein of human immunodeficiency virus. J Immunol 1 73. KHadlock, S Perkins, S Production, characterizati ing human of monoclonal a antibody to HTLV-I and I1 specificrecombinantproteins to enhansensitivityandspeci HTLV immunoassays, 5th International Conference on uman Retrovirology: Kumamoto, Japan, May 11-13, 1992. 74. DJ Nugen. Human monoclonal autoantibodies to characterize platelet antigens in immune-mediated thrombocytopenia. Blood 5952-58, 1993. 75. AM Goes, RS Rocha, G Gazzinelli, BL Doughty. Production and characterization of ~ c ~ i s t o s~o a~~a s o nParasite i. Immunol11:695human monoclonal antibodies against 711, 1989. 76. TP Flanigan,AWisnewski, P Wiest, J Johnson, S Teipori, D Hamer, N Lam, TF Kresina. Human monoclonal antibodies againstCryptospori~iu~ p a r v u ~generated by hypo-osmolar electrofusion. Trans Assoc Am Physicians CVI:86-90, 1994. 77. RVV Glaser, S Jahn, R Grunow.Development of specifichumanmab’sbyasmall scale electrofusion technique: The influence of some physical and chemical factors on hybridoma yieldof human peripheral blood lymphocytes xCB-F7 fusions. Allerg Immuno1 35:123-132, 1989. 78 PGA Steenbakkers, FCM Van Meel, W Olijve. A new approach to the generation of human or murine antibody producing hybridomas. J ImmunolMethods,152:69-77, 1992. 79. T Kudo, S Kobayashi, K urakami, R Takano. A novel human monoclonal antibody ir to atumor-associatedantigen. Jpn JCancer Res84:760-769,1993. 80. aski, Y Fukuoka, T Kudo, H Saeki. A novel human monoclaonal antibody, - 1, reactive with T-lymphocytic leukemia cells.Int J Cancer62:42-47, 1995. 81. T Boldicke, B Haase, M Bocher, VV Lindenmaier. Human monoclonal antibodies to cytomegalovirus:Characterizationandrecombinantexpressionofag1ycoprotein-Bspecific antibody. Eur J Biochem 234: 297-405,1995. 82. AA Delvig, B Koumare, RW Glasser, JF Wang. Comparison of three human-murine heteromyeloma cell lines for formation of human hybridomas after electrofusion with humanperipheralbloodlymphocytesfrommeningococcalcases andcarriers.Hum ybridomas 6:42-46, 1995.

clences Centre,St. John’s, Newfound~and,Canada

Nutrition is a critical eterminant of immunocompetence and risk of illness. The of much research in the interactions of nutrition and immunity have been the focus last two decades. The critical role of nutrition in modulation of immune responses is based on physiological considerations. The severity and extent of dysfunction caused by malnutrition in various organ systems end on several factors, including the rate of cell proliferation, the amount and e of protein synthesis, and the ividual nutrients in metabolic pathways any cells of the immune system are known to depend for thei function on metabolic pathways that employ various nutrientsascrucialfactors.0thnutritional deficiencies and nutritional excesses influence various components of the immune system. The extent of immunological impairment depends not only on the severity of malnutrition but on the presence of infection and on the age of onset of nutritional deprivation, among other factors.

The immune system protects the host from the microbial invasion and is essential for survival. It is an im~ortant link in the chain of host defense mechanisms that are brought into action when the body faces external (e.g., bacteria, viruses, parasites) or internal (tumor cells, “forbi en clones” of autoantibody-producin~cells) forces of destruction. ~ r i m a r yimmu deficiency syndromes are well recognized as antecedents of rowth failure and high morbidity but are rarely encountered in clinical alnutrition is the most common cause of secondary A number of defensemechanismsprotect the host theentry of microordevelopment g of clinical infections (Figure ost resistance mechadivided e n into two main tiers: nonspecific antigen-specific. The nonspecificdefensesinclude the skin and mucous membranes, phagocytic cells, mucus, cilia, lysozyme, interferon, and other humoral factors. These innate processes are naturally present and are not influenced by prior contact with the infeche two main components of antigen-specific immune protection, hu-

A simple view of host defense as a protective umbrella. It consists of physical barriers (skin, mucous membranes), nonspecific mechanisms (complement, interferon, lysozyme, phagocytes), and antigen-specific processes (antibodies of five immunoglobulin iso1981.) types and cell mediated immunity). (Copyri~htARTS ~iomedical ~ublishers

moral immunity and cell-mediated immune responses, interact synergistically with several nonspecific factors of host resistance, such as complementor phagocytosis, to rest~ain theinvasion and multiplication of microorganisms and/or to eliminate them.

alnutrition is usually a complex syndromeof multiple nutrientdeficiencies. Imbalances of single nutrients are relatively ~ n c o m m o nin humans, and investi~ationsof the effect of various deficiencies such as deficiencies of protein and amino acids, vitamins, minerals, and trace elements generally are carried out in experimental animals. The role of trace elements in maintenance of immune function and their

casual role in secondary immunodeficiencyare increasingly being recognized. There is growing research concerning the role of zinc, iron, and other elements in immunity and the mechanisms that underlie such roles. The problem of interaction of trace elements and immunity is a complex one because of other frequently associated nutritional deficiencies; the presence of clinical or subclinical infections, which in themselves havea significant effect on immunity; and finally the altered metabolism resulting from theunderlying disease. Progressive reduction in body stores of many trace elements as a resplt of decreasing intake leads to immunological changes that in turn increase the risk of infection (Figure 2). There is extensive literature on trace elements and immune functions [l-31 and it is briefly discussed here. Observations in laboratory animals deprived of one dietaryelement and findings inthe rare patientwith a single nutrient deficiency have confirmed the crucial role of several vitamins and trace elements in immunocompetence.Severalgeneralconceptshave been advanced [4]: (l) alterations in immune responses occur early in the course of reduction in micronutrient intake; (2) the extent of immunological impairment depends upon the typeof nutrient involved, its interactions with other essential nutrients, severity of deficiency, presence of concomitant infection, and age of the subject; (3) immunological abnormalities predict outcome, particularly the risk of infection and mortality; (4) in the case of many micronutrients, excessive intake is associated with impaired immune responses (discussed separately); ( 5 ) tests of immunocompetence are useful in titration of physiological needs and in assessment of safe lower and upper limits of intake of micronutrients. Table l identifies some of the specific effects of singlenutrient deficiencieson cellular componentsof the immune system [5].

Of those trace elements essential to humans,zinc has been studied more extensively than most. The association of zinc with immunity was first ~ocumentedwith the discovery of human zinc deficiency by Prasad et al. [ 6 ] .The importance of zinc in human nutrition has been recognized only during the last two decades. Zinc is a

h

Decreasing nutrient intake

4

Impact of decreasing intakeof trace elements on immunity and infection.

Effects of Single Nutrient Deficiencieson Immune Functiona Primary immunologic impairment ~utrient

+ +

+ + + ita am in C

Iron agnesium Zinc Selenium

+ + +

+ + + +

+

+

+ +

+ + +

"These data include animal as well as human studies. Adapted from Ref.5. y metalloenzymes, thus, it is important for many aspects of cellular ficiency results in blunted cellular immunity, as measured by in vitro esponses to mitogensddelayed c~taneoushypersensitivity testing mic hormone activity gure 3) [7-9]. This effect is best illustrated in patients with acrodermatitis enteropathica, who have impaired lymphocyte response

I

Serum thymic factor activity in zinc deficiency and effect of zinc treatment.

to mitogens, decreased thymulin activity, and reduced delayed hypersensiti atients undergoing long-term parenteral alimentation have function (particularly cell-mediated immune responses), a with low serum zinc levels, which are associated in large part with adeq supplementation [1 11. In patientswith immunodeficiencydiseases serum zinc, zinc repletion is associated with restoration of man functions [12]. In laboratory animalmodels these findings can be addition one can demonstrate reducednumberof antibody-fo spleen and impaired T-killer cell activity [7]. There is much recent interest in the role of zinc in macroph deficiency results in phagocytosis. Zinc is p in stimulation of re oxidase through its e as a cofactor for p C . Zinc may stabilize 20 Zinc deficiency increases various organisms including ~ i s ~ ~eo ~~o ic y~ t o g e ~Important es. questions remain unanswered about zinc immunity:

Is leukopeniathe mainreasonforimmunodeficiencyobserved in zinc deficiency? . Are a n t i b o ~ yresponses to 1: cell-independentantigensnormal in zinc deficiency? 3. Are there significant shifts in the distribution ofcells bearing ~ifferent rface markers? hat is the molecular basis of impaired lymphocyte andp h a g o c ~ efunction inzinc deficiency? 1.

To date, the exact role of zinc in the immune response is not clearly ~ n o w n . Zinc plays an important le in growth and many physiological functionsincl immune c o ~ p e t e n c e[l 1. 0th a direct and an indirect roleof zinc in immuni on of zinc in regulatory activities related t o i ~ m u n i t y includeits functionin ning the biological activity ofthym kinase, ribonucleic acid polymerase, and deoxyribonucleic a merase aresome of theendent enzymes thatarecrucialcat thereplicationandtranscriptionof A during cell division. bviously, numerQus reactions contribute aneffective i ~ m u n response e where zinc metalloenzymes could participate.

Iron deficiency is the most prevalent nutritional problem worldwide[l]. deficiency is a well-recognized occurrencein developing nations and is k attributable to a combination of poor availability from diet high in tates aswell as chronic bloodlosses from intestinal parasites an dealing with susceptibility to infection, iron deficiencyis like a On the one hand, free ironis necessary for bacterial growth: removal of iron with the help of lactoferrin or other chelating agents reduces bacterial multiplication,

Most research concerning the role of iron in immunity hasbeen concentrated in the area of cell-mediated immune response, and several approaches have been used to study cell-mediated immunity in iron deficiency. Joynson et al. [l41 were the first to describe cell-mediatedimmune response (CMI) status of iron-deficient adult volunteers. They demonstrated impaired delayed cutaneous hypersensitivity reaction to C a ~ spp. ~ and ~ ~ purified a proteinderivative (PPD). Defectiveinvitro lymphocyte response to PPD was demonstrated in these subjects by assaying the [l51 production of microphage migration inhibition factor (MIF). Sawitsky et al. also found that the lymphocyte response to phytohemagglutinin (PHA) and pokeweed mitogens in adults with iron deficiency anemia was one-third that of control subjects. Does the amountof dietary iron influence the risk of infection? In the case of no other trace element is the discussion of deficiency and risk of infection so biased and controversial asis true of iron [16,17]. The concept of “iron nutritional immunity,” which emphasizes the effect of iron deprivation in limiting the multiplication of bacteria, is an attractivehypothesiswithconsiderableinvitroevidence, but clinical data do not support the suggestion that iron deficiency protects against infection or that correctionof iron deficiency particularly,if it is achieved gradually by oral iron therapy, increases the incidence or severity of infectious disease in humans.

In humans, isolated vitamin A deficiency seldom occurs,and it is usually associated with protein energy malnutrition (PEM). In animals vitamin A deficiency has been shown to depress antibody responses to immunizationsand decreasecellularmediated immunity [18,191. Vitamin A deficiency in animals resulted inatrophy of spleens and thymuses, with thymic cortices affected mostly and with complete depletion of lymphocytes[ 1 $1. The literature on the association between hypovitaminosis A and bacterial and viral infections has been reviewed [20,21]. Many pathological A mechanisms probably contribute to the increased risk of infection in vitamin deficiency, including tissue changes and altered specific and nonspecific immunity. Furthermore, infection itself can aggravate vitamin A deficiency. An incompetent immune system is one of the factors that add to the vulnerability of the deficient individual to infection. Usually, vitamin A deficiency occurs concomitantly with PEM, and in experimental animals, vitaminA deficiency causes inanition. There is good evidence of reduced immunocompetence in PEM [20,21]. If PEM is accompanied by vitaminA deficiency, the vitamin deficiency adds significantly to susceptibility to infection because of its additional detrimental effects on lymphoid tissues and organs. In vitamin A-deficient children, the associated presence of PEM and the reduction in the number of T cells [22] may be due to the concomitant PEM rather than vitamin A deficiency itself. In rodents fed a lowvitamin A diet, the level of serum thymic factor is unaltered (Figure 5 ) [23]. Decreased total leukocyte number has also been observed in vitamin A deficiency; the differential count revealed a relative increase in neutrophils and a decrease in lymphocyte number. Vitamin A also modifies the humoral response, especially to T-dependent antigens. Chandra and Au [24] reported that the number of plaque-

200

I50

100

50

Weeks

T

i

i

Control (A) Lymphocytestlationresponseinhealthyadultmengiven 300 mg zinc nt daily for 6 weeks ( compared with results in nonsupplemented controls (0); (B) Effects of zinc administration on lymphocyte stimulation index in mice. Columns marked with different number of asterisks(*) are significantly different from each other.

nutrition impairs immuneresponses and increases risk of several diseases, i on and cancer [30-321. Though zinc is a crucial nutritional c o m ~ o for the normal development and maintenance of immune functions, excessive intake is associated with significant impairment both humans (Figure 6A) [33] and laboratory mice (

2

of increasing zinc concentration in culture medium on oxygen consumption and bactericidal activity of neutrophils have been reported [33]. Excessive intakes of zinc interfere with copperabsorption andmay aggravate marginal copper deficiency [34]. Excessive intake of zinc also results in decreased platelet aggregation and poor histamine release by mast cells. Severe systemic iron overload occurs predominantly in individuals affected by geographically specific genetic mutations that permit the daily absorption from the diet of more iron thanis physiologically needed. The toxicity of iron at thecellular levels leads to dysfunction of various organ systems, and these clinical consequences of iron overload are similar, whatever the specific condition that gave rise to the excess accumulation. Hepatoxicityis the most common finding in patients with iron overload, since theliver is the majorrecipient of excess iron, even though the kidney may be a target of iron toxicity [35]. An increased risk of infection by several microorganisms, including Vibrio vu~n~icus, Listeria monocyto~enes,Yersinia ent~rocolitica,~ s c h e r i c ~coli, i a and ~ a n d i d aspecies, has been noted in various types of iron overload [36]. Iron overload may promote bacterial septicemia and increase the frequency of symptomatic malaria in endemic areas. Johnson et al. 1371 also reported that excess of iron impairs immune responsesto neurological dysfunction, cancer, and heart disease. Though an increased intake of vitamin E is beneficial, there is considerable evidence that excessive vitamin E supplementation may affect the cellular components of the immune system.Villa et al. 1381 reported that vitamin E supplementation in vitro caused a concentration-dependent inhibition of arachidonic-induced aggregation of PMNs and mononuclear leukocytes(MNLs) and suggested that the effect may bedue tointerference with lipoxygenase activity. Topinka et al. [39] also reported that the addition of alpha tocopherol to lymphocytes in vitro suppresses lipid peroxidation and oxidant damage to deoxyribonucleic acid (DNA) when induced by the catalytic system of Fe' '-sodium ascorbate. The mechanisms of the immunotoxic effects of these nutrients and vitamins are not clear, but in the case of zinc overdose, alterations in serum and cell-bound low-density lipoproteins, reduced levels of other nutrients, and changes in membrane structure and receptor expression possibilities. are

Both nutritional deficiencies and excesses influence various components of the immune system. The extent of immunological impairment depends not only on the severity of nutritional deficiency but also on the presence of infection. The immune system is an important link in the chain of host defense mechanisms and protects the host from the microbial invasion. Imbalances of single nutrients and vitamins are uncommon inhumans.However,whendeficiencies of zinc and iron occur either singly or in combination, significant depression of immunocompete~ce and increased risk of infectious disease result. Excessive intake of nutrients and vitamins impairs immune response and may be considered a of type malnutrition.

ify

1. RK Chandra, DH Dayton. Trace element regulation of immunity and infection. Nutr

Res 2:721-733, 1982. 2. A Bendich, RK Chandra. Micronutrients and immune functions.New York: New York Academy of Sciences, 1990. 3. RK Chandra. Trace elements and immune responses. In: Chandra RK, ed. Trace Elements in the Nutrition of Children11. New York: Raven Press, 1991. 4. RK Chandra. Micronutrients and immune functions: An overview. Ann NY Acad Sci 587~9-16,1990. 5. WR Biesel, R Edelman, K Nauss, RM Suskind. Single nutrient effects of immunologic functions. JAMA 24553-58, 1981. HH Sanstead, RM Schubert. Zinc metabolism in normals 6. AS Prasad, A Miale, Z Farid, and patients with the syndrome of iron deficiency anemia, hypogonadism and dwarfism. J Lab Clin Med 61537-549, 1963. 7. RK Chandra, B Au. Single nutrient deficiencyand cell-mediated immune responses.I. Zinc. Am J Clin Nutr 33:736-738, 1986. 8. PJ Fraker,CMZwicki, RW Luecke.Delayedtypehypersensitivityinzincdeficient adult mice:Impairment andregulation to dinitroflurobenzene.JNutr 112:309-313, 1982. 3: 118-125, 1987. 9. RK Chandra. Trace elements and immune response. Clin Nutr 10. RK Chandra. Acrodermatitis enteropathica: Zinc levels and cell-mediated immunity. Pediatrics 66:789-791, 1988. 11. RS Pekarek, HH Sandstead, RA Jacob, PE Barcone. Abnormal cellular immune responses during acquired zinc deficiency. Am J Clin Nutr 32: 1466-1471, 19’79. 12. S Cunningham-Rundles, C Cunningham-Rundles, B Duport, RA Good. Zinc-induced activation of human B-cells. Clin Immunol Immunopathol 16: 115, 1980. 13. NS Scrimshaw. Functional consequences of iron-deficiency in human populations. J Nutr Sci Vitamin01 36:47-63, 1984. 14. DHM Joynson, WD Murray, A Jacobs, AE Dolby. Defect of cell-mediated immunity in patients with iron deficiency anemia. Lancet 2:1058-1059, 1972. to phytomitogensiniron 15. BSawitsky,RKanter,ASawitsky.Lymphocyteresponse deficiency. Am J Med Sci 272: 153-160, 1976. 16. D Vyas, RK Chandra. Functional implications of iron deficiency. In: Stekel A, ed. Iron Nutrition in Infancy and Childhood.New York: Raven Press, 1984, pp 45-59. 17. CHershko,TEA Peto, DJWeatherall.Ironandinfection.BrMedJ296:660-664, 1988. 18. S Krishnan, UN Bhuyan, GP Talwar, V Ramalingaswami. Effect of vitamin A and protein calorie undernutrition on immune responses. Immunology 27:383-392, 1974. 19. KM Nauss, D Mark, RM Suskind. The effect of vitamin A deficiency on the in vitro cellular immune response of rats. J Nutr 109:1815-1823, 1979. 20. RK Chandra, PM Newberne. Nutrition, Immunity and Infection Mechanisms of Interactions. New York: Plenum, 1977. 21* RL Gross, PM Newberne. Role of nutrition on immunologic function. Physiol Rev 60: 188-202, 1980. 22. C Bhaskaram, V Reddy. Cell-mediated immunity in iron and vitamin-deficient children, Br Med J 3522, 1975. 23. RK Chandra, G Heresi, B Au. Serum thymicfactor activity in deficiencies of calories, zinc, vitamin A, pyridoxine. Clin Exp Immunol42:332-335, 1980. 24. RK Chandra, B Au. Single nutrient deficiency and cell-mediated immune responses.111. Vitamin A. NutrRes 1:181-185, 1981.

25. FA Oski. Vitamin E- a radical defense. N Engl J ed 303:454-455, 1980. 26. SN Meydani, M Hayek. Vitamin E and the immune response. In: Chandra

NutritionandImmunology. St. John's, Newfoundland, Canada: ARTS Bio 1992, pp 105-128. 27. Jensen, MFossum, C akkarainen. The effect vitamin ofthe onE cell-mediated immune responses in pigs. J Vet ed 35549-555,1988, 28. RE Harris, CABoxer,RLBaehner.ConsequencesofvitaminE-deficien phagocytic and oxidative functions of the rat polymorphonuclear leukocytes. 338-343, 1980. 29. JF Van Vleet. Current knowledge of selenium-vitamin E deficiency in domestic anied Assoc 176:321, 1980. 30. RK Chandra. Immunocompetence in overnutrition. Cancer Res41:3795-3796, 1981. 31. RK Chandra. Immunodeficiency in undernutrition and overnutrition. Nutr Rev 39:225231, 1981. 32. RK Chandra. Nutrition, immunity and infection: Present knowledge and future direc' S . Lancet 1:688-691,1983. Chandra. Excessive intake of zinc impairs immune responses. JA 33. 34. LM Klevan. The ratio of zinc to copper of diets in the United States. Nutr RepInt 11: 35.

Galleano, peroxikidney lipid and liver S Puntarol on

oad: Causes and consequences. Annu Rev Nutr7:485-508, 1987. 37 A Johnson, JG Fischer, BA Bowman, EVV Gunter. Iron nutriture in elderly individuals. FASEB J 8(9):609-621, 1994. 38. S Villa, A Lorico,G Morazzoni, D De Gaetano, N Semeraro. Vitamin E and vitamin C inhibit arachidonic-induced aggregation of human peripheral blood leukocyte in vitro. Agents Actions 19:127-131, 1986. AN Erin. The influence of alpha tocopherol and 39. J Topinka, B Binkova, RJ Sram, pyritinol on oxidative DNA damage and lipid peroxidation in human lymphocytes. Nutr Res 225:131-136, 1989.

36. f

The ~ i r i a m ~ o s p and i t a l Brown Universit~School of ~ e d i c i n eProvidence, , Rhode Island ~ h i l d r e n ~ ~ o s pBoston, i t a l , ~assachusetts ston, ~assachusetts

The range of immunomodulating agentswith therapeutic capability for gastrointestinal diseases is expanding. This chapter will discuss established agents in gastrointestinal disease and introduce newer agents that either are in early stages of clinical trials or are undergoing animal testing.

zulfasalzine, sulfasalazine) is a potent antiinflammatory agent effective in the treatment of ulcerative colitis and Crohn’s disease. The predominant activity of the azulfidine is due to the 5-aminosalicylic acid (5-ASA) component. Azulfasalzine was developed by Nanna Svartz in collaborationwith Dr. Willstedt in the late 1930s as otential treatment for rheumatoid arthritis and ulcerative colitis designed to attainhigh concentrations in connectivetissues and nflammatory and antibioticproperties. d salicylazosulfapyridine, which was employed colitis and rheumatoid arthritis (Figure 1). The clinical response was excellent in ulcerative colitis and of modest benefit in rheumatoid arthritis. Since the introduction of salicylazosulfapyridine, now called sulfasalazine, and marketed as Azulfidine, it has becomethe mainstay of outpatient medical management for patients with mild to moderately active ulcerative colitis or Crohn’s colitis. The drug is also effective in maintaining remissions in ulcerative esearch into the mechanism of action and pharmaco~inetics of azulfidine has led to the development of other ~-aminosalicylate (5-ASA) compounds now 7

.COOH

Sulfapyridine

5-Aminosalicylate

coon es~lamine

NaOOC

1 Thestructures of azulfidine,mesalamine,andolsalazinesodiumaredepicted. Azulfidine is a composite molecule composedof 5 aminosalicylic acid (5-ASA) linked by an azo bond to sulfapyridine. Mesalamine is the 5-ASA moiety alone; 5-ASA, olsalazine is two 5-ASA molecules joined by an azo bond.

available for the management of ulcerative colitis and Crohn’s disease (Figure 1, ecause of the rapid jejunal absorptionof orally ingested S-ASA, delayedrelease formulations were developed to deliver the drug to diseased distal small bowel and colonic sites for in~ammatorybowel disease treatment. This has been accomplished by coating 5-ASA with acrylic resinsor encapsulatingS-ASA in ethylcellulose microgranules. The acrylic-base resin (eudragit) dissolves at a pH greater than 6 , and the ethylcellulose is a semipermeable membrane allowingthe release of S-ASA, also in a pH-dependent fashion, as it traverses the smallbowel. Two other dimers joinedby an azo bond are olsalazine ( ~ i p ~ n t u m and ) balsalazide (Colazide). Olsalazine consists of two S-ASA molecules joined together;balsalazide is one S-ASA linked to an inert unabsorb~d carrier molecule. Simi~arly Azulfidine, to both drugs require colonic bacteriato cleave the azo bond and release the 5-ASA moiety. Therefore, similarly to Azulfidine, these agents are mainly active at colonic sites of disease. Although the precise mec~anismof action responsible for the clinical efficacy of S-ASA compounds is not known, in vitro investigations have identified any antiin~ammatory and immunosuppressive propertiesof S-ASA that suggest a multifactorial basis of therapeutic action.

. In 1972, Peppercorn and Coldmanidentified the mechanismby which Azulfidine is catabolized [3]. By measurin~ stool and serum concentrations of Azulfidine, 5ASA,andsulfapyridinein normal and bacteria-freemice,theyestablished that coliform bacteria kere necessary for reducing Azulfidine to 5-ASA and sulfapyridine through the enzymaticactivity of azoreductase. On oral ingestion, Azulfidine

1.

is partiallyabsorbedin the jejunum, the majority excreted intact into bile and delivered back to the intestinal lumen. Only a minority of absorbed Azulfidine is excreted in the urine. On reaching the large intestine, Azulfidine is reduced by the bacterial enzyme azoreductase to 5-ASA and sulfapyridine. The 5 poorly absorbed from the colon and is largely excreted in the sto sulfapyridine is rapidly absorbed from the colon, metabolized by the liver, and excreted in the urine with only small amounts remaining in the stool. If ingested separately, both 5-ASA and sulfapyridine are absorbed in the~roximalsmall intestine, metabolizedby the liver, and excreted into the urine, with onlya small fraction of either compound reaching the colon. Olsalazine also contains an azo bond isthat similarly reducedby colonic bacteria to release two 5-ASA molecules in the colon. Serum levels of Azulfidine and its components range between 0.025 and 0.0375 W after an average oral dose of 3-6 g/day [4]. After a d stoolconcentrations of Azulfidinebetween 1.25 and 2.0 concentration of 5-ASA is 0.006 stoolconcentration is a1 mucosal concentrations of 5-ASA are thought to rangebetween 0.5 , on the basis of direct measurements of 5-ASA in feline ileal and colonic mucosa [ 6 ] .These concentrations were consistent with the metabolism of Azulfidine. a

Azulfi~ineis a potent inhibitor of cyclooxygenase, whereas the metabolites 5 - A S ~ and sulfapyridine are less effective. Subsequent studies using mucosal specimens confirmedthisinhibitoryaction of Azulfidineand 5 on cyclooxygenase [7-91. Inaddition,Azulfidineandtosome extentsulfapyalsoinhibitedthe enzymatic activity of thromboxane synthetase, preventing the production of thromboxanes [IO-121. Since cyclooxygenase blockade did not alleviate colonic in~ammationin paof arachidonic acid metabolism tients, attention shifted to the lipoxygenase pathway 1131. Azulfidineand 5-ASA inhibit both5-lipoxygenase-and lipoxygenaseactivatingprotein ( F L A ~ )[14,15]. Thisactionblocks theproduction of leukotrienes, which function as chemotactic factors for neutrophils. In particular, mucosal levels of leukotriene I34 direct1 correlate with active in~ammation in inflammatory bowel diseasepatients.reliminary animalandhuman trials with lipoxygenaseinhibitors were promisibutsubsequentprospectiverandomized double-blinded clinical trials have not demonstrated a significant clinical response ion VII. D) [16-183. active metabolites of oxygen and nitrogen are ~ o t e n tmediators of tissue injury in manyinfectious and inflammatory conditions.Freeradicalscomprise partially reduced oxygen and nitrogen species with odd numbers of electrons. In general, free radicals are reactive because of their propensity to acquire electrons from surrounding organic compounds. Nonradical metabolites of oxygen and nitrogen are more stable but also react with organic substrates, albeit at a much slower rate. 5-Amino salicylic acid is one of the most potent free radical scavengers and antioxidants [19-251. These properties of 5-ASA are present at concentrations of 0.1-0.2 W, which are achieved within the intestinal mucosa 161. Interaction with

oxygen radicals results in the increased production of oxidized 5-ASA metabolites in the mucosa and stool of patients treated with Azulfidine [25]. These findings support the contention that free radical scavenging is an important antiinflammatory mechanismof

emonstrate profound antiprQliferative effects on T cells in in vitro cultures by preventing deoxyribonucleicacid (DNA) synthesis in the S phase of the cell cycle. 5-Amino salicylic acid appears to inhibit T-cell proliferation and subse~uent activation by preventing the accumulationof steady-state transcript levels of the early T cell activation gene interleukin 2 [26,27]. Sulfapyridine alter not does T-cell inhibiting DNA synthesis and, theregrowth the fore, cyc and 5-ASA prevent clonal expansion of potential pathogenic T cell populations. 5-Amino salicylic acid also inhibits im'n production by stimulated peripheral blood cells in dosea anner [27]. olymorphonuclear leukocyte ( N) functions, including chemotaxis, pha~ocytosis,andadhesion rties, are crucial to acuteinflammatory processes. Neutro~hilchemotaxis is m y ~nhibitedby Azulfidine and 5-ASAinvitro [28321. Similarly, chemotaxis and phagocytosis by monocytes are inhibited by AZ at concentrations achieved in the intestinal lumen 129, 331. The activation of myeloperneutrophils, a potent enzyme of the respiratory burst pathway, is

- cytokines- are locally proteins that, ate local immune responses. recently have the etabolites ontheproduction se potentinflam. Azulfidine and 5-ASA both block the production ulated peripheral blood mononuclear cells [34] and Thestimulation of tumor necrosis factor alpha r biolQgica1profile to that of IL-l,is also inhibited so appears to inhibit TNFa binding with its recepof s ~ b s e ~ u einflammatory nt responses[36]. ent chemoattractant for neutro ting monocyte chemoattractant protein l production in vivo [37]. In IL-l-stimulat S able to suppress IL-8 messe whereas sulfapyridine had n e influx of mucosal neutro

Soluble immune mediators

acute inflammation, ), predominantly an early product of T cell activation, cnne growth and differentiation factor. Azulfidine, but not rQduction from murinesplenocytes [38], whereas 5-ASA has been shown to reduce supernatant IL- levels in activated human peripheral blood likely to downmononuclear cell cultures 1261. Therefore, Azulfidine and 5-ASA are regulate inflammatiQn, in part by reducing the synthesis of proinflammatory cyto-

Azulfidine has proved to be efficacious in the treatment of active ulcerative colitis in a multitude of studies beginning in the1960s [39-421. The effective dose range is between 2 and 4 g/day, inducing a remission in from 50% to 80% of patients. Azulfidine has also provedto be effective in the maintenance of remission for over 1 year in ulcerative colitis over the placebo group in controlled double-blind trials [43-461. Azulfidine has also been effective in the treatment of active rheumatoid arthritis in several large multicenter studies 147-491, although it is not U.S. Food and Drug Administration (FDA) approved for this indication. Oral5-ASAcompounds,includingolsalazine(Dipentum)andmesalamine (Pentasa and Asacol), areeffective in inducing remissions in active ulcerative colitis (UC), with a response ranging from 50% to 71% at dosages of 2 to 3 g for olsalazine, 2.4 to 4.8 g for Asacol, and2 to 4 g for Pentasa [SO-551. Topical therapy with 5-ASA enemasis also beneficial inboth active distal disease,with remission rates as high as 93070 (4 g/day) for active disease and 75% for maintenance of remission [56,57]. Recent trials of oral 5-ASAagentssupport their use for both active and quiescent Crohn’s disease. In a 16-week trial, Pentasa (4 &/day) produced clinical improvement determined by the Crohn’s disease activity index in 64% of patients and full remission in 43%, when compared to a 36% improvement in the placebo group [58]. Although Azulfi~inehas not been proved to be effective in maintaining remission in Crohn’s disease patients, mesalamine has demonstrated efficacy in both maintaining remissions and treating active small and large bowel Crohn’s disease [59-621. Asacol and Pentasa have been approved by the FDA for patients with active ulcerative colitis; olsalazine hasbeen approved for ulcerative colitis in remission in those patients who are allergic to Azulfidine. Over the courseof 55 years since itsintroduction, Azulfidine has proved to be a safe and effective agent for the management ofulcerative colitis, Crohn’s colitis, and rheumatoid arthritis with minimal toxicity (Table 2). From the understanding of ~ u l f i d i n e ’ spharmacokinetics and the immunomodulatingproperties of its metabolite SASA, a new group of 5-ASA agents has been generated and proved as effective as Azulfidine in clinical trials. Of note, oral 5-ASA compounds are the first agentsto reduce the rateof clinical relapsein Crohn’sdisease [59,60,62]. At this time, it is not clear which of the large number of diverse pathways is central to driving the chronic inflammatory processin inflammatory bowel disease

Aminosalicylate (SASA)

Sulfasalazine

Pancreatitis Pericarditis Pneumonitis Colitis exacerbation Nephritis Fever Rash Watery diarrhea (olsalazine)

Pancreatitis Hepatitis Pneumonitis Nausea/headache Neutropenia/hemolssis Agranulocytosis Feverhash Male infertility

(IBD) that may be specifically targeted for the development of new therapeutic strategies. The fact that Azulfidine and5-ASA have multiple mechanismsof action suggests that their efficacy asantiinflammatory agentsrelies on a compositeof these properties. Futurepharmacologicalandtherapeuticstrategiesforinflammatory bowel disease may need to focus on the development of drugs capable of multiple mechanisms of action.

Corticosteroids are natural or pharmacologically modified 21 carbon atom derivatives of cortisol, which is synthesized from its precursors cholesterol and pregnenolone within the adrenal cortex. Soon after their introduction to clinical medicine in the mid-l940s, steroids became a cornerstone of therapy for IBD. Corticosteroids are also used in the treatment of a variety of gastrointestinal diseases, including eosinophilic gastroenteritis, liver transplantation rejection, autoimmune hepatitis, and graft versus host disease. In this section, we willfoccls on their uses in IBD.

Corticosteroids are potentinhibitors of the inflammatory response and immunomodulators via multiple nonspecific mechanisms. These mechanisms include inhibiting arachidonic acid release from cell membranes, cytokine release by immune cells, chemotaxis, and phagocytosis [63,64]. Corticosteroidsinhibitinterleukin 2 tranNA production by preventing association of the AP-1 transcription factor with its corresponding binding site on the lymphocyte’s interleukin 2 promoter [65]. ~lucocorticoidshave also recently been shown to stimulate the production of I K B ~which , binds to NF-KB, trapping it in the cytosol [66,67]. NF-K can activate many immunoregulatorygenes in response to proinfla~matory stimuli by translocating into the nucleus and acting as a transcription factor for these cytokine genes. Thus, inhibitionof NF-K activity by steroids may account for their multiple antiinfla~matoryeffects.

. Corticosteroids are well absorbed from the gastrointestinal tract, primarily in the proximal jejunum; up to 30% of corticosteroid may be absorbed from retention enemas [68]. Once absorbed, 90% of cortisol is bound to albumin and corticosteroid-binding globulin. The liver metabolizes cortisol and other corticosteroids by reduction and glucuronidation. Inducers of hepatic conjugation such as phenobarbital and rifampin can increase hepatic metabolism and excretion of steroids [69]. Although the plasma half-life of corticosteroids isless than 5 hr, the biological effects last much longer E691 Suppression of the hypothalamic-pituitary-adrenal axis can occurwith just 5 days of high-dose oral steroids andis inevitably seen with more than 2weeks of treatment [68,70]. Side effects seen with long-term high-dose administration include hyperten).

sion, fluid retention, diabetes, opportunistic infections,osteoporosis,cushingoid appearance, and striae (Table3) 1701.

7. ~ o ~Steroi~s i c ~ for ~ Topical steroids, like topical 5-ASA, have been successfully used in treating active distal colitis. Truelove first reported in 1956 that two-t~irdsof patients with active distal ulcerative colitistreated with120-m1enemas containing 100 mg of hydrocortisone had a favorable symptomatic response 1’711 Subsequent controlled trials have confirmed the efficacy topical of hydrocortisone and topical prednisolone compared to that of placebo 172,731. The most commonly used topical steroids are 100-mg hydrocortisoneenemasand 120-mg prednisoloneenemas, with responseratesof 0 about 7 0 ~ [74,75]. Poorly Absorbed/ etabolized Steroids Several studies have demonstrated appreciable absorption of topically administered steroi resulting in adrenalsuppression 176,771. This raises concernsaboutthepoten systemic sideeffects of topical steroids, especially with long-term use. Four steroids that have been the subject of recent trials are tixocortol pivalate, beclomethasone diproprionate, prednisolone metasulfobenzoate, and budesonide. These newer steroids are either poorly absorbed or rapidly metabolized on the first pass through the liver with either mechanismminimizing systemic side effects. Tixocortol pivalate (TP) is a 21-thiol derivative of hydrocortisone lacking glucocorticoid or mineralocorticoid activity [78]. Cont~olledtrials of 1250-mg 100-mg hydrocortisoneen absorbed and thus is enemas versus predni-

equal to that ofeither hydrocortisone newer steroids will provide patients the unwanted side effects. In addition they may be useful in the maintenance of n. They have not yet been approved foruse in the United States. udesonide is an analogue of hydrocortisone initially used in an inhaled formulationforthetreatmentma.Inbothoraland rectal forms, itsuppresses inflammation in the bowel. ver,because of extensive first-passmetabolism by thecytochrome P-450 enzy the liver,only10% to 15% of absorbedbudesonide has systemic bioavailabilty.The Canad ide effectivein inducing remissionin 5 1070 of a slow-release form for 8 weeks compared wit udesonide was foun se a dose-relatedreductioninbasal andadrenotropichormone- (AC stimulatedplasmacortisolconcentrationbut was notassociated with clinical1 rtant steroidsideeffects.Another studydemonted that budesonide was almost as effective as prednisolone in treating active with fewer corticosteroid side effects and less suppression of pituitary adrenal

Side Effects of Corticosteroid Therapy Cardiovascular Dermatological

Endocrinological

Gastrointestinal

Hematological Infectious

Neurological Muscular Ophthalmological

Orthopedic

Psychiatric Renal

Source: Refs. 69 and 70.

ypertension

. Atherosclerosis Cushingoid appearance oon facies Striae lopecia irsutism Acne Thinning/friability of skin Telangiectasia Impaired wound healing Adrenal suppression Impaired stress response Growth failure Diabetes mellitus/glucose intolerance Hyperlipidemia Nausea/vomiting Fatty liver Gastritis Peptic ulcer Pneumatosis intestinalis Pancreatitis Leukocytosis Lymphocytopenia Viral, especially varicella, herpes zoster Bacterial, especiallystaphylococcal/pseudomonal infections Fungal, especially candida, aspergillus Parasitic, pneumocystis Mycobacterial reactivation of tuberculosis Headache Pseudotumor cerebri Proximal myopathy Posterior subcapsular cataracts Increased intraocular pressure Papilledema Exophthalmos Eyelid swelling Osteoporosis Fractures Aseptic necrosis Spontaneous tendon rupture Depression Mania Sodium retention Nephrocalcinosis Hypercalciruria

function [93]. A recent Canadian studyof budesonide in patientswith C sion found that oral budesonide was well tolerated and prolonged remission compared to placebo [94]. However, there was no difference in remissionrate compared to thatof placebo at 1 year of follow-up. These newer topical and oral steroid preparations are expected to become available in the United States in the near future as ~ong-term studies demonstrate their safety.

2. ~ y s t ~~~t i~cr o i ~ s For moderate to severe UC and CD, systemic corticosteroids are the mainstays of therapy. In addition, flares of autoimmune hepatitisalsotreatedwith systemic steroids. Prednisone, prednisolone, and methylprednisolone are the options available. A starting dose ranging from 30mg to 60 mg orally each day is chosen, depending on the severity of the flare and previous steroid use/response. For patients with severe disease, intravenous steroids are favored since oral absorption may be impaired. Hydrocortisone, adrenocorticotropic hormone (ACTH), prednisolone, and methylprednisolone have all been demonstrated to be effective ['X]. Intravenous ACTH (120 U/24 hr) has been shown to be more effective than hydrocortisone inachieving remission in patients with severe UC whohave not received steroid therapy in the previous 30 days. However, in patients treated with steroids before hospitalization, hydrocortisone (300 mg/24 hr) was more effective in inducing remission[95]. ~ethylprednisolone(48 mg/24 hr) and prednisolone (60 mg/24 hr) are equivalent to300 mg of hydrocortisone; they can be given either as a continuous intravenous drip or in divided pulse doses, Although effective in treating acute flares of both CD and UC,corticosteroids are not effective in maintaining remission. Furthermore, systemic side effects encountered with long-term steroid therapy have limited its use. Thus, once a patient is re~overing from aflare of IBD, steroids should be changed to an oral form and slowly tapered while an alternate agent is used to maintain remission. An overview of the commonly used glucocorticoids and theirrelative potenciesis given in Table 4.

Commonly Utilized~lucocorticoidPreparations Equivalent dose (mg) Short-acting Cortisonea 30 25 ~ydrocortisonea 20 Intermediate acting 12-36 5 Prednisone 5 Prednisolone ~ethylprednisolone 12-36 4 Long acting Dexamethasone 0.5 etam met has one 0.6

Plasma half-life (min)

Tissue half-life (hr)

90

8-12

60 200

12-36

180 100-300

aStrongest mineralocorticoid (sodium-retaining) effects. Adapted from Ref. 70.

36-54

7

(6MP) are purine derivatives that are nthesis. Azathioprine is metabolized in ticnucleophilic attack by s u l ~ y d r a l containing compounds (e.g., glutathione) and its biological effects are identical to action and the toxicities of these those of 6MP. Thu both the mechanisms of drugs are identical. 0th drugs are useful in the treatment of liver transplantation rejection, as well as inthe therapyof inflammatory bowel disease and chronic active autoimmune hepatitis.

ptopurine is metabolized by three competing enzyme systems: xanthine oxithiopurine methyltransferase render inactive metabolites; hypoxanthineguanine phosphoribo GPRT) produces ribonucleotidesare that incorporated into the dividing cells, causing cell death. Their exact mechanism of action is u n k n o ~ nbut is thoughttoinvolve inhibitingT-helper lymphocyte activity. One study found that 6MP was a potent suppressantof immunoglobulin (IgG) and IgM production by pokeweed mitogen-stimulated peripheral blood mononuclear cells [96]. The absorption of 6MP from the gastrointestinal tract is variable, ranging from 10% to SO%, in contrast, azathioprine is well absorbed. 6-Mercaptopurine is primarily metabolized in the liver; however, up to 40% may be excreted in the urine. The principal route of catabolism of GMP is methylation by the enzyme thiopurine methyltransferase (TPMT). Alternatively, 6MP may undergo degradation by xanthine oxidase. The half-life of 6MP is triphasic, with the half-life of the final phase being 10 hours. Allopurinol increases body levels of 6MP by inhibiting xanthine oxidase metabolismof 6MP. In addition, probenecidmay increase the level of 6MP by inhibiting urinary excretion 1971.

3. ~ l i # se^ ~ ~ ~ l Inflammatory Bowel isease In 1980, alandmarkstudy by Presentand colleagues established the efficacy of 6MP with a 70% response rate in patientswith steroid-refractory Crohn’s disease [98]. The mean time of treatment required to produce a responsewas 3.1 months. In addition to decreasingdisease activity, 6 was found to have multiple beneficial effects in IBD, including stero healing and closure of fistulas, and maintenance of remission in both C Trials of A%A/6MP in children and adolescents with IBD reported response rates similar to thoseseen in adults [99,100]. Controlled studies of 6MP (1 .O-1 .S mg/kg/ 24 hr) or A%A (1 .O-3.0 mg/kg/24 hr) show efficacy in CD as long as the dose and duration of treatment are adequate [loll. A recent metaanalysis confirmed this ted that a therapeuticresponserequires at least 17weeks[102]. relitz found that patients with refractory Crohn’s disease treated with 6MP in whom leukopenia developed had a more rapid response time to remisweeks for patients with leukopenia9 compared with 14 weeks for patients

1.

without). The leukopeniain this group of patients was not associated with anyother signs of myelosuppression [ 103) Controlleddatain largetrials onthe use of AZA/6 in UC are lacking. everth he less, several studies have demonstrated the ef in chronic active LJC [104-1073. Adler and Korelitz have reported tients with ulcerativecolitis refractoryto steroidtapering demonstrat nical response to P (as defined by improvement in symptoms and decrease in steroiddosage) recent retrospective studyof long-term outcome in 105 UC patients treated P reported complete remission in 68 (65%), partial remission in 25 (24%), and no responsein 12 (l 1Yo) [l09]. The complete responders had35% a relapse rate and with remissionreestablished in 8 8 ~ 0withoutsteroids in most cases. In contrast , completeresponderswhodiscontinuedad an 87%subsequent relapse rate. Furthermore, there were few major toxicities associate umulative experience of 6 0 ~ to 0 80% efficacy and relative safety with AZA/ makes these agentsthe first-line therapies for refractory chronicactive UC and CD in addition to steroid-dependent complicated I by fistula(s). (.

Liver Azathioprine (1-2 mg/kg/24hr) is used in liver transplantation in conjunction with cyclosporine and prednisone. It is not effective in treating acute rejection. The use of azathioprine and duration of therapy vary with individual transplantation centers [ 1 lo]. A retrospective study by van oek et al. found that patients receiving azathioprine were significantly less likely to experience ductopenic rejection (DR) and vanishing bile duct syndrome; 14 of 66 patients (21 Yo) without azathioprine experienced D , compared to l of 98 patients (l Vo) receiving 2 mg/ kg/24 hr of azathioprine [l 1 l]. Complicating the use of azathioprine in liver transplantation recipients is the occasional developmentof azathioprine-induced hepatotoxicity, including cholesta, peliosis hepatis, nodular regenerative hyperplasia, and venoocclusive disease. istological findings suggesting this diagnosis include sinusoidal dilatation, centrizonal hemorrhagic necrosis, venulitis with en othelialcell damage, and cholestasis. ~ i t h d r a w a lof azathioprine in patients with azathioprine-induced hepatotoxicity results in improvementof liver function within aweek [l 12). Azathioprine has also been widely used in the treatment of steroid-refractory autoimmune chronic active hepatitis [l 131. Tr itionally, the therapy of this entity involves high-dose steroids with a slow taper. wever, many patients were found to experience a relapse once the steroid dose was tapered. I be reduced successfully, azathioprine at a doseof 1.5 to 2 the patient’s regimen, frequently enabling steroidsto be tapered CO et al. demonstrated that chronic active hepatitis could be kept in prolonged remission in patients continuing to take azathioprine[ l 14). Since 8 0 ~to 0 9 0 ~of 0 pediatric patients with autoimmune chronic active hepatitis experience relapse once all immunosuppression is tapered, azathioprine is also indi~atedin the treatment of pediatric type I and type I1 autoimmune chronic active hepatitis. The a d ~ i t i o nof azathioprinedecreasesthelong-termtoxicitiesandgrowth-impairingeffects of chronic corticosteroid therapy in childhood. Side Effects The most worrisome side effect of AZA/6 suppression in a dose-dependent fashion. Therefore, peripheral blood counts should be monitored during therapy. The initial doseused is 50 mg orally per day; this can

be increased gradually to a m ~ i m u mof 2.0 mg/kg/24 hr. If falling blood counts are observed, reducing the dose may allow counts to recover and patients to continue with therapy. Aretrospective review of 396 patients treated with6 a 15% incidence of side effects: infection ( 7 ~ 0 )pancreatitis , (3010)~feve Yo), bone marrow suppression ( 2 ~ 0 ) and , drug-induced titis ( 0 . 3 ~ 0[)1151. All ported toxicities were reversible, demonstratingthatcan beusedwith an acceptable level ofrisk.Additional side effectsreportedenauseaanddruginduced diarrhea. Although cerebral lymphoma hasbeen reported in two patients, e to suggest an sed frequency of malignancy (including patients onAZAcompared with that in untreatedcontrol patients.

an inhibitor of dihydrofolate reductase and results in deeducing agent tetrahydrofolate. Tetrahydrofolate is essen, a pyrimidine nucleoside essential in ynthesis and is cytotoxic to rapidly proliferating inhibition of cell proliferation, methotrexate also has a wide range of immunosuppressive effects on peripheral blood mononuclear cells in vitro [97]. bsorbed almost completely by the gut. owever, the higher the dose of oral methotrexate, the poorer the absorption, limiting thus its bioavailability and §uggesting saturation of intestinal transport mechanisms. Therefore, high-dose methotrexate is traditionally administered intravenously. It undergoes ?-hydroxylation in the liver and a small portion is excreted into the bile. The bulk of the excretion ( 8 0 ~ 0 - 9 0 ~occurs 0) in the urine, and therefore thehalf-life is increased in patients with renal failure. ethotrexate has a biphasic half-life, with the second phase being approximately 10 hours. Intracellular metabolism of methotrexate to polyglutamate derivatives also occurs and the metabolites also inhibit DNA synthesis [116]. at childhood leukemia in 1948. Subsere found tobe usefulin treating a variety udies have not shown efficacy for MTX or primarysclerosing cholangitis. X 25 mg/wk of for 12 weeks suggested efficacy in steroid sparing and induction of remission in patients CD [l 17,1181. Another uncontrolled trial with 15 mg/wkof reported similar results [l 191. A subsequent controlled trial compared5 mg of oral methotrexate given three times per week to placebo as a steroid-sparing agent in Crohn’s disease and found a 46% relapse rate for patients on methotrexate compared to an80% rate for those on placebo 11201. A larger controlled trial in 141 CD patients with steroid-dependent or §teroid-refractory disease reported that 39% of

the group treated with 25 m g h k intramuscularly (im) of TX were able to discontinuesteroidsandenterremissioncomparedtoonly19 of placebo-treatedpatients[121]. The results from an open trial in patientsinitiallyresponding to 12 weeks of X subsequentlytreated with oraat 7.5-15 mg/wkformainteon were disappointing. 0 UC patients and 5 l070 of 37 ce of patientsremainedinremission afteeeks of maintenancetreatment [122]. recent controlled trial in 67 patients with chronic steroid-dependent UC TX at 12.5 mg/wk reported no difference from the placebo group in ate, time to remission, or subsequent relapse rate [123]. Since the availX is less effective than A Z A / 6 ~ P be reserved as a modulator for patients with active om I treatment has failed with A Z A / 6 ~ Phas failed [loll. encountered effects Side with nts have included pneumonitis and abnormal liver findings. Several studies in patients with rheumatoid arthritis or psoriasisreceiving low-dose methotrexatesuggest that over a period of years fatty change in the liver, hepatic lobular necrosis, fibrosis, and cirrhosis may occur [ 1011. The progression to cirrhosis may in part relate methotrexate and polyglutamate metabolites within the !iver [116,l ost centers recommend serial liver biopsy examinations to monitor patients on chronic low-dose methotrexate since the progression to fibrosis and cirrhosis may be clinically silent. Biopsy specimens of the liver should be obtained with every 1 to 1.5 gofmethotrexate given cumulatively [125]. epatotoxins, particularly alcohol, should be avoidedby the patient receiving methotrexate. In addition, there have been multiple reported cases of patients on low-dose methotrexate who exhibited marrow suppression and an increased susceptibility to opportunisticinfections, includin~herpes zosterand ~neumocystiscarini~infections.

Cyclosporine A (CSA) is a cyclic 1 l-amino-acid peptide produced by the fungus ~ o l y ~ o c l a ~ iinflatum um ~ a m sThe . CSA molecule enters the cytosolof T cells and binds to thecytosolic protein cyclophilinA (Gyp-A). The CS -Cyp complexin turn binds to calcineurin, inhibiting its phosphatase activity. T cells treated i CSA exhibit a decreasein the level of nuclear factor of activated T cells protein important in promoting cytokine gene transcription. The CSA-treatedcells exhibit decreased interleukin 2 mRNA synthesis and release, and decreased expression of interleukin 2 receptors. Of note, only calcium-dependent pathways of T l y m p h o c ~ eactivation are affected by CSA; activation of T lymphocytesvia the cell surface molecule CD28 (a calcium-independent process) is not inhibited by CSA [126-1281. Suthanthiran et al. have proposed that CSA may also promote the production of transforming growth factor beta, which may actto inhibit T cell proliferation further [129]. Although the most pronounced e ts of CSA are on TLymphocytes,itmayalso modulateantibodyproductionblymphocytesand block the release of proinflammatory and chemotactic cytokines by mast cells [1301. Cyclasporine A is a lipophilic moleculethat requires bile flow and intestinal mucosal integrity to be readily absorbed. The oral bioavailability of GSA is quite low (ap-

proximately ~ O ~ Oand ) , it is further impaired in conditions that impair fat absorption, such as cholestasis or enteropathy [13l]. Coadministration of oral d-alphatocophery polyethyleneglycol 1000 succinate (a water-soluble vitaminE derivative) increases the oral bioavailability of CSA [ 1321. Neoral (Sandoz Pharmaceuticals, Basel, Switzerland) consists of a microemulsion of CSA mixed with surfactant, lipophilic, and hydrophilic solvents; it has been designed for oral absorption in conditions of reduced bileflow [1331. Cyclosporine A has a large volume of distribution (about 4 L/kg) and is bound primarily to plasma proteins, as well as erythrocytes. It is hydroxylated and demethylated in theliver and excretedin the bile, with almost no urinaryexcretion. The half-life in individuals without liver failure ranges from 6 to 12 hours [ Because of the hepatic metabolism, CSA can be used even if patients have renal failure. However, drugsthat modulate P450 microsomal enzymes affect CSA degradation and therefore affect serum CSA levels [ 13l , 1341. Even at therapeutic levels, CSA has a wide variety of adverse effects and toxicities (Table 5 ) . Nephrotoxicity manifestedby a decrease in creatinine clearance of patients. The and arise in serum creatinine level occurs in between 25% and 75% nephrotoxicity usually reverses on discontinuation of the drug or lowering of the dose, Other adverse effects include hypertension, neurotoxicity, diabetes, elevated transaminase levels, hirsutism, and gingival hyperplasia [135]. Up to 20% of liver transplantation recipients receiving CSA have infections with opportunistic pathogens such as cytomegalovirus,C a ~ ~species, i ~ a Epstein-Barr virus, and~ ~ ~ e ~ ~ i Z Z spp. Lymphoproliferative disease can occur in patients receiving CSA and is often associated withEpstein-~arrvirus infection [1361,

3. C l i ~ i ~Uses al Liver orPancreasTransplantationCyclosporinehas been utilized in liver transplantation recipients since 1979; gradually, dosage levels have been decreased in an attempt to minimize toxicity while preserving graft function. Cyclosporine A is typically used in conjun~tionwith prednisone with or without the addition of azathioprine, In liver transplantation recipients, CSA is typically begun on the first postoperative day at a dosage of 2-5 mg/kg/24 hr; this may be given either as a continuous infusion or individeddoses.SerumCSA levels are measured by high-performance liquid chromatography. Desired trough CSA levels vary between transplantation programs, although levels between 150-300 ng/ml are common. The dose of oral CSA is usually three times higher than the intravenous dose to provide the same blood level (usual oral dose 5-10 mg/kg/24 hr divided every 12 hours) [1371. ~ccording to data from three randomized trials comparing CSA to tacrolimus, 5 0 ~ 0 - $ 0of ~ 0liver transplantation recipients with a CSA-based immunosup regimen experience at least one acute rejection episode within their first year post transplantation [138-1401. It has not yet been determined whether the Neoral formulation of CSA will decrease the rateof acute rejection. Cyclosporine A hasbeen utilized in a similar manner as a maintenance immunosuppressive agent in pancreas transplantation recipients, though a significant subsetof patients require onv version to tacrolimus therapy[1411. Other Liver Disorders Liver diseases suspected to have an immune-mediated component contributing tohepatobiliary injury include autoimmune chronicactive hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis. Case reports

and smallopen-labeltrialssuggestpotential efficacy for CSA in these diseases [142-145]. In a randomized placebo-controlled trial of 29 patients, examined the effects of low-dose CSA in addition to supportive ther group demonstrated clinical and biochemical improvement in the dis a decreased incidence ofhistological progression [ 1461. * In~ammatoryBowel Disease randomized In a placebo-controlled rynskov et al. demonstrated that an oral dose of 5-7.5 mg/kg/24 hr pro remission of Crohn’s disease (CD) in 22 of 37 patients with steroid-refractory study, this . In CSA levels thein variable: patients responded clinically higherhad 3 treatment, and drug absorb the patients notdid at a lticenter placebocontrolled studies utilizing two diffe hr) of CSA as an adjunct to stan demonstrate any benefit of low-dose CSA in either inducing o sion [148,149]. These two trials can be criticized, however, in that they utilized a fixed drug dose rather than adjusting the dose to yield a therapeutic level. For ulcerative colitis (UC), Lichtiger et al. demonstrated in a ran study that CSA could successfully bring about remission and postpone surgery in steroid-refractory fulminant colitis. In this trial, 9 of 11 patients treated with CSA responded within 7 days, comparedto no patients of the 9 receiving placebo [150]. Other studies in adults and children have confirmed CSA’s efficacy in this situation wever, the relapse rate is > 50% once CSA is tapered. Therefore, it is that CSA be used as an induction agent in an attempt to convert a patient tomore definitive maintenancetherapywithagentssuchas6-mercaptopurine.

(AE) is an idiopathic ~ u t o i m m u n eEnteropathyAutoimmuneentropathy intestinal in~ammatorydisease occurring in children, characterized by secretory diarrhea, villous atrophy, and evidence of other systemic autoimmune disease (including glomerulonephritis, diabetes, and adrenal insufficiency). Immune abnormalities identified in AE include T lymphocyte activation and antienterocyte antihereascorticosteroidsandazathioprinehave limited efficacy in this disorder, CSA has improvedvillous histological characteristics, nutrient absorption, and growth [154,155].

Tacrolimus (FK-506) is a lipid-soluble macrolide lactone produced by the fungus ~ ~ ~ e ~~ ~ o ~ y~ Once c e internalized, ~ ~ the tacrolimus ~ molecule ~ complexes ~ with a group ofproteinstermed FK indingproteins (F . Inamanner similar lcineurin andinhibit to cyclosporine, the tacrolimus-FK P complex canb cytokinetranscriptionandlymphote activation/proliferation via the calciumdependent pathway of lymphocyte activation. As with CSA, the release of a wide variety of cytokines (including interleukin 2 [IL-23, IL-3, IL-4, IL-5; interferongamma. and tumor necrosis factor alpha) is inhibited by tacrolimus [ 156-15$]. In addition to its effects Tlymphocytes,tacrolimus caninhibit in vitro proliferationandantibproduction [ 129,1571.

~

imes more potent (by weight) than CSA and more reliably absorbed, even in cases of cholestasis or enteropathy. Once absorbed, tacrolimus is n tissues and in erythrocytes, with a volume of distribution of 0.85 L/ etabolism of tacrolimus is almost exclusively hepatic, with demethylation and hydroxylation occurring by the microsomal P450 system. The half-life of tacrolimus is variable, ranging on average from 8 to 16 hours. Hepatic dysfunction (but not renalinsufficiency) significantly prolongs thehalf-life. As with CSA, drugs that inhibit P450 metabolism raise tacrolimus levels, whereas those that accelerate ose seen with CSA, and are listed in Table 5 . Some side effects of CSA A, including hirsutism and coarsening of facial features and gingival hyperplasia, are not seen with tacrolimus. In addition, there may be a slightly lower incidence of hypertension and hypercholesterolemia with -506. The incidence of lymphoproliferative disease appears tobe comparable for individuals maintained on tacrolimus vs. CSA.

3. ~ l i ~ i~cs~els Liver Transplantation Initially tacrolimus was utilized as rescue therapy for CSA-refractory rejection afterliver transplantation. Studies utilizing tacrolimus for refractory rejection unresponsiveto CSAsuggested efficacy in controlling rejection and postponing retransplantation in at least 70% of patients [138,159]. The use of intravenous tacrolimusis often associated with high rates of renal or neuraltoxicity, so oral dosing is preferred. Thetypical starting dose for oral tacrolimus ranges from 0.1 to 0.15 mg/kg/dose given twice daily, with whole blood levels adjusted to give a level between 5 and 15 ng/ml [ 1601. Pediatric patients may require higher dosages to achieve therapeutic levels [ 1611. A major controversy in the literature is whether tacrolimus is a superior drug to CSA for liver transplantation,andwhether it shouldbe utilized asprimary therapy. Three large randomized trials comparing initial tacrolimus use to initial Side Effects of CyclosporineMTacrolimus (FK506) Therapy Nephrotoxicity Hypertension Gastrointestinal symptoms: nausea, vomiting Hyperglycemia/diabetes Opportunistic infections Lymphoproliferative disease/lymphoma Hepatotoxicity Increased bone resorption Hirsutisma Gingival hyperplasia" Hypercholesterole~iaa Hemolytic uremic syndrome Neurotoxicity: headaches, tremors, myalgias, seizuresb aMore common with cyclosporine therapy. bh40re common with tacrolirnus, particularly the intravenous preparation.

h el al.

CSA use (with tacrolimus rescue if necessary) have been published. The primary deficiency of all the randomized trials is the relatively short (1 year) period of follow-up. All three trials suggest patients receiving tacrolimus have a significantly lower incidence of rejection within the first year post transplantation, but survival rates are comparable in the CSA-treated group. Adverse effects may be slightly higher in patients treated with tacrolimus [ 138-1401. From the University of Pittsburgh, Todo et al. reported their experiencewith tacrolimus use in 1391 liver transplantation recipients, compared to 1212 historical controls given CSA, After 4 years of follow-up, patient survival rate was higher in the tacrolimus-treated group (children, 86Vo vs. 65%; adults, 71% vs. 65%). The numbers of rejection episodes and graft losses were also lower in the tacrolimustreated group [1621. In summary, although tacrolimus appears to be promising first-line therapy for liver transplantation, there is no definitive evidence suggesting it should completely replace CSA. Intestinal Transplantation Tacrolimus has been the mainstay of antirejection therapy for patients receiving isolated intestinal or combined liver and bowel transplantations [1631. The principal advantage tacrolimus has over CSA in this setting is its consistentabsorption in the presenceof gut rejection or inflammatory enteropecause of the increased risk of rejection, tacrolimus levels in these patients may be maintained as high as 25 ng/ml [ 1641. Pancreas Transplantation Tacrolimus has also been utilized as both primary immunosuppression and rescue therapy in patients on CSA. Rejection rates are generally higher than in liver transplantation recipients, approaching 5 0 ~ 0 - 6 0 ~6 0 months post transplantation. In these patients, tacrolimus levels are maintained at 15 ng/ml [141,165]. ther Liver Disorders Two open-label trials by Van Thiel suggest tacrolimus may provide efficacious therapy in autoimmune chronic hepatitis and primary sclerosing cholangitis [166,1671. The initial dose of tacrolimus utilized was 0.075 mg/ kg/dose given every 12 hours. In the chronic active hepatitis trial,levels of transaminases and bilirubin returned to the normal range within 90-1 80 days. In~ammatoryBowel Disease Little published experienceexists regarding the use of tacrolimus in Crohn’s disease or ulcerative colitis. Eighty percent of inflammatory bowel disease patients undergoing liver trans~lantation forsclerosing cholangitisstayinremission on their immunosuppressive regimens [168]. One case report suggests oral tacrolimus may be useful in the therapy of fulminant colitis [ 1691. Autoimmune Enteropathy of Childhood Tacrolimus may bring about remission in patients with CSA-resistant autoimmune enteropathy. The dose of oral tacrolimus utilized to achieve therapeutic levels may be significantly higher than in transplantation recipients [1701.

Rapamycin is anothermacrolideimmunosuppressantstructurallysimilar to but functionally different from tacrolimus. Rather than inhibiting cytokine synthesis, rapamycin blocks lymphocyte proliferation, Rapamycin binds to the family of FK

binding proteins ( F ~ B P s and ) ~ the rapamycin-F~BP complex inhibitsthe activation of the ~ 7 0 kinase ’ ~ ~ [171,172]. Subse~uently,there is decreased kinase activity of other cyclin proteins, and cell entry from the G1 to the S phase of the cell cycle is blocked [173,174]. The end resultis that the cellular proliferative responses induced by cytokines or growth factors (including & - l 9IL-2, IL-3, IL-6, and basic fibroblast growth factor [bFGF]) are dramatically reduced [174]. In addition to effects on Tlymphocytes, rapamycin can also inhibit B lymphocyte activation and differentiation [1751.

2. ~ ~ ~ r ~ ~ c o l ~ ~ y As rapamycin remains a drug in development, pharmacokinetic data are limited. The bioavailability of rapamycin in its current formulation is approximately 15%. Data in renal transplantation patients suggest that 40% of the drug is bound to plasma lipoproteins, and that the volumeof distribution averages approximately10 of the drug ranges from 35 to 95 hours, averaging L/kg. The terminal half-life around 60 hours [ 1761.

3. Clinic~lUse At this time, rapamycin is considered an investigational agent, and published data on in vivo immunosuppressive effects are limited to animal models. alone or in combination with other agents has been shown to prolong allograft survival in a number of animal models, including murine cardiac, islet cell, and porcine renal transplantations [1771. In addition, it has been shown to allow graft survival in hamster-to-rat cardiac xenografts[ 1781. Toxicities noted in animal models are principally hepatic (transaminase level elevation) and renal. Future clinical trials will determine the role of rapamycin in prevention of transplantation organ of action from rejection. It is hoped thatsince rapamycin has a different mechanism CSA or tacrolimus, combination drug therapy may achieve potent immunosuppression with less toxicity.

1. ~ e c ~ ~ofn~ ci t s ion ~

~ycophenolatemofetil (MMF) is an ester prodrug of mycophenolic acid, a potent inhibitor of the enzyme inosine monophosphate (I P) dehydrogenase, which is an o purine synthesis, converting essential enzyme in the metabolic pathway of de IMP to guanosine monophosphate. It was noted by Allison et al. that individuals lacking adenosine deaminase, another enzyme important in de novo purine synthesis, had severe impairment of lymphocyte function, leading to severe combined immunodeficiency. In a similar manner, MMF inhibits lymphocyte proliferation and differentiation by blocking purine and deoxyribonucleic acid (DNA) synthesis [ 179-1811. The effects of this drug arewide ranging; they include inhibited lymphocyte proliferation in response to antigen and mitogen, impaired T cell cytotoxicity, decreasedantibodyproduction by B cell decreasedglycosylation of adhesion molecules [ 179,1801. Inanimalmodels,hasproved effective in prolonging allograft survival in renal, cardiac,and pancreatic transplantation[18l].

~ ~ ~ r ~ ~ c o l o ~ y ycophenolate mofetil is almost completely absorbed from the intestine, and almost completely converted to its active-metabolite mycophenolic acid ( deesterification in liver and peripheraltissue. P um concentration occurapproximately 1 hourafter ingestion of , withthehalf-lif ranging from 8 to 12 hours. Mycophenolic acid is eliminated by glucuronidation in high-performance the liver, followed by renal excretion of the glucuronide [ 182 liquid chromatographic assay formeasurement of plasmaA levels has been developed, but the optimal plasma MPA level for transplantation patients is not esta~lished[1831. The principal adverse effects of MF are gastrointestinal. abdominal distension, gastritis, pancr itis, and upper gastrointe have all been reported. Other reported side effects include headaches, skin rashes, and cramps. As with other agents, opportunistic infections may occur [184-186]. 2.

3. ~ l i n i cUse ~l ycophenolatemofetilhas been principallyutilizedas therapy in individuals withrefractoryrenal allograft rejection andhas receive approvalfor this use. in 509'0Published studiesof phase I and I1 trials suggest allograft rescue can occur 70% of patients with refractory renal allograft rejection (as defined by a need for high-dose steroids and/or anti-CD3) [l 85,1861. In addition, primary maintenance immunosuppression with CyA was associated with a low rate of allograft rejection 118 F in human adults ranges from 2000 to 3500 mg/24 hr given one study suggested that higher doses resulted in a decreased incidenceof rejection [184]. At this time, MMF is being utilized to treat resistant liver allograft rejection, but there are no published data summarizing the drug's efficacy in this population. Two abstracts suggest that M F may have a role in maintaining hepatic allograft with lower tacrolimus or CSA dosages[187,188].

A synthesis and T and brequinar sodium QR) interferes with dehydrogenase, which ation. It inhibits t enzyme dihydrooro essential in the de novo synthesis of pyri in the lymphocyte. ~requinar sodiumdecreases. DNAand ribonucleic acid synthesis,lymphocyteproliferation> and cytokine and antibody production. In vivo transplantation experiments in rat and monkey animal models demonstrate that brequinar decreases proin~ammatory cytokine messenger RNA (mRNA) in allografts and prolongscardiac and liver allograft survival [189- 1911. The oral bioavailability of BQR approaches 1009'0. Once ingested, peak serum level approximately 2 hours after administration and has a half-life of approximately l5 hours. The long half-life suggests that once daily dosing may be feasible [189]. The drug is excreted in both the stool and the urine 11901. Plasma

levels can be measured by high-performance liquid chromatography¶ and levels are maintained between 1 and2 pg/ml [190]. are similar to those of other antiproliferative agents and include mucositis, gastrointestinal disturbances, and myelosuppression. m is an investigational agent that has not yetreceived FDA aphase I and phase 11 trials in renal transplantation recipients sugwas well tolerated and reduced the incidence of steroid-refractory rejection if substituted for azathioprine in a “triple therapy” immunosuppressive owever, further multicenter study suggested that it has a narrow re was no clear reduction in rejection episodes [ 1901. In theory, synerg equinar with CSA offers the potential advantage of cell inhibition; however, further clinical studies are neededto determine this agent’s role in immunosuppressive regimens.

l

Evidence suggests that activated cells are responsible for transplantation rejection episodes and likely participateinflammatorygastrointestinal diseases suchas inflammatory bowel disease [193-1961. Therefore monoclonal antibodies that target T cells and T cell subpopulations may be beneficial in treating active disease and preventing disease exacerbations. Three potentially useful antibody therapies target the pan cell T recept the helper T cell receptor CD4, and the alpha chain of the IL-2 receptor present only on activated cells. Anti-CD3 antibody (commercially known uromonab) is the first antibody treatment to receive approval for humans for thetreatment of transplantationrejection.It is addedwhenhigh-dosesteroids have failed to thwartrejection episodes. Antitherapycanabrogatehepatic ection in as many as75% of cases [l 97-1991. ithinhours of intravenousadministration,anti-CD3antibodies cause a uction in circulating 1: lymphocytes, suggesting that opsonized T cells are cleared by the reticuloendothelial system. Theresulting reduction in T cell PO Ulations and particular cytotoxic T cells account for its clinical efficacy. Antitreatment may be efficacious in gastrointestinal inflammatorydiseases where T play a role such assclerosing cholangitis and I Anti-CD4andanti-CD2milarlytargetThelper cells andactivatedT cell subpopulations¶ respectively. thsubpopulations havebeenimplicated in the chronic intestinal inflammation of IB [ 193,2001. Initial treatment with anti-C in small numbers of both IJC and Crohn’s disease patients has been somewhat effective [201, 2021, yet larger controlled trials are needed. Anti-CD25 therapyis as effective as antithymocyte globulin therapy in treating renal transplantation rejec. . 1 trial [203] but has notbeen tested in gastrointestinal diseases. tion i therapies that block leukocyte adhesion have been suggested for the treatment of intestinal diseases. CD18 is a molecule in the integrin family that forms heterodimers with CD1 l isotype molecules. CD18/CD11 is a leukocyte adhesion moleculeexpressed on vascularendotheliumandthebasolateral surfaces of

epithelial cells. Anti-CD18 antibody blocks the adhesion of leukocytes to these sites, preventing extravasation of leukocytes into tissues and across epithelial barriers [204,205]. Pretreatment with a n t i - ~ D l 8has been effective in preventingthe colonic inflammation in the TNBS rabbit model of colitis [206]; however, it remains untestedinhumans. Another integrin, very late a n t i ~ e n(VLA4), functions in the adhesion of leukocytes and hasbeen a target of monoclonal antibody therapyin the treatment of the cotton-topped tamarin animal model of ulcerative colitis [207].

. Cytokine-based therapies have rapidly evolved from the discovery of a group of low-~olecular-weightglycoproteins that have potent immune modulating properties roinflammatory cytokines enhance immune responses; other cytokines suppress immune-mediated events. Therefore, a number of antiinflammatory strategies take advantage of the biological properties of individual cytokines. These include providing naturally occurring antagonists, anticytokine antibodies, or soluble cytokine receptors that bind and renderinactive soluble cytokinesand providing immunosuppressive cytokines as recombinant proteins.

Interleukin 1 receptor antagonist (IL-lra) is currently the only reported naturally occurring cytokine antagonist. It bindsto the IL-1 receptor as a competitive inhibitor of the proinflammatory cytokine IL-l but has noagonist activity. Interleukin-l is produced predominantly by monocytes and macrophages but also by fibroblasts ) lymphocyte activaand endothelial cells. It promotes T cell and natural killer tion and B cell different~ation. Itis thought to be released in the inflammatory cascade and therefore to be a logical immunotherapeutic target. The antiinflammatory a~tivity of IL-lra hasbeen best studied in rabbit formalin-imm~necomplex colitis. It has proved to be effective in reducing the colitis if given prior to or within 43 hr of formalin administration[209]. In addition, antibodies directed to IL-lra in this model increased the mortality rate and prolonged intestinal inflammation [210]. In human ulcerative colitis there appear to be enhanced mucosal IL-l/IL-lra ratios with active IBD but not active self-limiting colitis [21l]. Therefore, it would be reasonable to attempt tocorrect this imbalance in iven the experimental rabbit data, IL-lra may be most effec-

T ~ m o rnecrosis factor alpha ~ T N F a )is another proinflammatory molecule with very similarbiologicalaction to IL-l. Since TNFa production is translationally regulated, TNFa mRNA levels do not increase with disease activity, but qualitative monocyte immunohistochemical rotei in measurements are enhanced in active UC and Crohn’s lesions when compared to those of normal controls 12121. Efforts to block TNF activity have included both anti-TNF antibody treatment and administration of soluble TNF receptors.

An open-labeled trial of a single infusion of anti-TNF human/mouse monoclonal antibody thera y in active Crohn's disease patients demonstrated a clinical response in 8 of the 10 patients treated, with an average duration of response of 4 months [213]. No adverse side effectswere reported with therapy. heth her a second dose in relapsing patients will afford a similar clinical response will need to be addressed in future trials [213]. A ~ t i - T N Fantibody therapy has no provenefficacy in ulcerative colitis.

Interleukin 10 (IL-10) is often categorized as an immunosuppressive cytokine, able to down-regulates inflammatory eventsby causing a reductionin the proinflam~atory T cell helper 1 (Tl) cytokines, y interferon (IFN-y), and IL-2. Since IFN-y up-regulates cell surface expression of the antigen presentingmolecules, major hisC) class I and class 11, on antigen presenting cells and tocompatibility complex ( e~ithelialcells, IL-10 has the effect of decreasing antigen presenting capabilities 12141. Interleukin 10 has been given topically to three patients with active ulcerative colitis in an uncontrolled pilot trial, with remission of disease in all three subjects [215]. This initial success coupled with thein vitro immunosuppressive ropert ties of this agent has prompted the initiation of a multicenter placebo-controlled trial in IBD.

Advances in molecular biology have spawned the generation of fusion toxin proteins that can be designed to target specific cellular subpopulations. One component of the molecule is the toxin (e.g., diphtheria, ricin, pseudomonas) that is joined to the receptor binding portion of a targeted ligand. The interleukin 2 diphtheria toxin (DAB38gIL-2) contains the cytotoxic portion of the diphtheria toxin catalytic domain, and the transmembrane domain is fused to the receptor binding portion of IL-2 [216]. Therefore,tagenttargetsIL-2 receptorbearing cells, which are mostly activated T cells. docytosis of the fusion protein and subsequent delivery of the c a t a l ~ i cdomain to thetarget cell cytosol arrests translation, resulting in cell death of activated T cells 12171. In vitro studies have demonstrated the specificity of DAB389~L-2 for activated T cells, leaving naive and memory T cells intact. This compound was created to treat allograft rejection and hasbeen effective in prolonging graft survival in animal models. In addition, this molecule has demonstratedefficacy in patients with hematological malignancies [218--2221,rheumatoid arthritis [223], and psoriasis [224]. In vitro studies have demonstrated its efficacy in killing activated lamina propria gesting therap~uticpotential in treating in~ammatorybowel disease 12251.

Thalidomide (a-~-phthalimidoglutarimide) is a glutamic acid derivative that was first described by the Swiss pharmaceutical firm Ciba in1953. It was found to have sedative hypnotic propertiesand prescribed as a tranquilizer in Europe. However, in

the early 1 9 6 0 thalidomide ~~ was removed from the market because of its profound teratogenic effects(especially dysmelia). Sincethen laboratorystudies have revealed several immunomodulating properties. Thalidomide inhibited lipopolysaccharide(LPS)-induced monocyte TNFa production, possibly by enhanc degradation [226,22~] Under similar experimental conditions th alter IL-6, IL-16, or granulocyte macrophage colony-stimulating factorpro~uction [226]. Thalidomide also alters integrin surface expression and functionon immune cells. Studies of thalidomide in marmosets and human volunteers demonstrated reduced surface expression andfunction ofleukocyte adhn moleculesi CD18 and CD54 (intercellular adhesion molecule 1 [ICA more, thalidomide suppresses T helper 1 (T1) activity by and increases IL-4 and IL-5 (T helper2) production in stimulated peri~heralblood mononuclear cells [230]. Although there are case reports of thalidomide benefit in intestinal graftversus-host disease [23 l], ehGet’s colitis [232], and ulcerative colit currently only FDA approved for the treatme~tof erythema nodos [234]. Besides teratogenicity at 45-55 days of pregnancy, side effects thalidomideinclude sleepiness, constipation, an reversible peripheral neuritis. Thus, it appears to have an acceptable side effect profile avoided. This fact coupled with the recent success of anti-T active Crohn’s disease [213, 2351 makes thalidomide an attractive agent for the treatment of Crohn’s disease.

ydroxychloroquine (Plaquenil) is an antimalari agent that has been found to be effective in thetreatment ofrheumatoidarthritisystemiclupuserythematosus(SLE). Its mechanism of action is to incre in intracellularvacuoles and thereby alter protein degradation, assembly of macrornolec lational modifications of pr eins in the Golgi apparatus [236]. tion of antigenic peptide-M C protein complexes is diminishe antigenpresenting capabilities to T cells, resulting in the do -regulation of the immuneresponse. Inaddition,hydroxychloroquineinhibits -la and IL-6 by monocytes and Tcells; IL-2, IL-4, TNFa, and I F N - ~ levels we Abnormal antigen processing and presentation have been implicated in the pathogenesis of IB Therefore, hydroxychloroquine (300 mg) was tested as a therapeutic in a pilot trial of active ulcerative colitis with 8 of 10 patients responding [237]. This result prompted a largerplacebo-controlled trial of hydroxychloroquine in 90 patients with ulcerative colitis. The clinical response in the treatment group, however, was not superior to thatof the placebo group [238]. Nevertheless, 12 of 15 patients who had a striking response were on hydroxychloro~uine,s u g g ~ s t i nthat ~ the drug maybe effective in a specific subgroup of patients.

15-~eoxyspergualin (DSC) is a synthetic analogue of the novel antibiot produced by the soil bacterium Bacillus laterosporus. I strated antitumor properties in animal tumor models

strated significant immunosuppressive activity, especially in animal models of xenograft survival [240]. The mechanismofaction of is not fully understood;however, preliminarywork indicates a variety ofce immunologicaleffectsdistinctfromthose and FK506. These effects include diminished release of superoxide and lysosomal enzymes by monocytes,modulation of IL-1 production,suppression of antibody formation, inhibition of alloreactive cytotoxic T lymphocyte induction, own-regulation of C class I and I1 antigens,decreasedexpression of d binding to heat shock proteins (Hsp70, Hsp9O) [239-2411. It is SG may exert its immunosuppressive effects via its interaction with heat shock proteins, resulting in altered antigen processing and interference with cytotoxic Tcell differentiation and antibody producing

Leukotrienes are important inflammatory mediators and chemotactic agents whose levels are elevated in I . Fish oils with 0-3 fatty acids areknowninhibitors of leukotriene synthesis. Thus, it has been postul that fish oils and other inhibitors of 5-lipoxygenase may be effective in treating . One small study in patients with tivity with 4.2 g of 0-3 fatty acids active UC found a 56% decline in mean disea compared with a 4% decline with placebo 12421. Another study in patients with active UC found that treatmentwith 3.24 g of eicosapentaenoic acid and 2.16 g of hexaenoic acid in addition to standard therapy with prednisone and sulfasalazine resulted in reductions in rectal dialysate leukotriene B4 levels, improved histological findings, and weight gain [243]. Therefore, dietary supplementation with fish oils may have arole in the treatmentof ulcerative colitis. Zileuton is a new drug that hasbeen studiedin the treatmentof asthma. It acts to inhibit 5~lipoxygenase, thuspreventing leukotriene synthesis. Findings of recent studies in patients with UC are conflicting. One study found a 25% remission rate uton 600 mg fouradaycompared withonly 7%for placebo 12441. leuton slightly more effective than placebo but not nce of remission in patients with ulcerative colitis rther evaluation of this agent is needed before any conclusions can be drawn.

The observation that smoking improves symptoms in ulcerative colitis led to the hypothesis that nicotine may have a role in treatment. A recent study found 17/35 patients treated with transdermal nicotine patches in addition to standard therapy had complete remissions compared with 9/37 in the placebo group [246]. The longterm efficacy and safetyof this treatment are unknown. The mechanism of actionis unknown and themost common sideeffects observed were nausea, lightheadedness, headache, and sleep disturbance.

The options for the treatment of various gastrointesinal diseases continue to expand. As more insight is gained into the roleof the immune systemin patho~enesis of these diseases, targeted immunomodulating agents will have an increasingly important rolein their treatment.

edical InstitutePhysicia ah was a HowardHughes S Bousvaros is supported in rt by a General Clinica ters Clinical Associate Physician Award from the National Institut Christopher Stevens is supported in part by an NI grant ( K ~ 8 ~ K 0 ~ 2 1 4 ) .

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67:1 - 17, 1993. 228. R Neubert,AC Nogueira, D Neu bert Archi Toxicolo 55:77-92, 1994. 229. AC Nogueira, R Neubert, H Helge, D Neubert. Life Sci 230. SM McHugh, IR Rifkin, J Deighton, AB Wilson,PJ Lachmann, CM Lockwood, PW Ewan. Clin Exp Immunol99:160- 167, 1995. 231. J Lopez, C Ulibarrena, J Garcia-Larana, J Odriozola, J Perez de Oteyza, JL Sastre, W Transplantation 11 :251-252, 1993. JL Navarro. Bone 232. H Larsson. Inter J 228:405-407, 1990. 233. MFR Waters, ABG Laing, A Ambikapathy, JE Lennard-Jones. Br Med J 1:792, 1979. 234. J Sheskin. Clin Pharrnacol Ther6:303-306, 1965. 235. HM van Dulleman, DW Hommes,J Meenan, F Celik,JA Bijl, J Woody, GNJ Tytgat, SJH van Deventer. Gastroenterology 106:A1054, 1994, abstract. na ArthritisRheum 23532-89, 1993. 236. Sachar. Gastroenterology 94:A293, 1988, abstract. 237. 238. L Mayer, DB Sachar, DH Present, Gastroenterology102:A661, 1992, abstract. 239. cad Sci696:123- 132, 1993. 240. r, K Soltys, SI Cho. Mount Sinai J Med61:51-56, 1994. 241. , C Mazzucco, C Singh, SL Kelley. Ann NY Acad Sci 685:136242. A Asian, G Triadafilopoulos. Am J Gastro 87:432-437, 1991. 243. WF Stenson, DCort, J Rodgers. Ann Intern Med 116:609-614, 1992. 244. Peppercorn, K Das, C Elson. Gastroenterology 106:A751, 1994, abstract. 245. awkey, M Gassull, K Lauritsen. Gastroenterology 106:A697, 1994, abstract. 246. hodes, S Ganesh. N Engl J Med 3305311-815, 1994.

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Johns ~ o p ~ i ~niversity, ns ~alti~or~,

~aryland

King ~ a ~ National a d Guard~ o s p i t a lRiyadh, , Saudi Arabia

Sepsis and its sequelae are major causes of significant ratesof morbidity and mortality in the critically ill, espite therapeutic advances with improved life-support techniques, the average mortality rate forsepsis ranges from 30% to 50%, and theincidence of sepsis is continuing to increase [l]. Sepsis also adds significantly to thecosts of health care by prolonging hospitalizationand increasing the expenses of therapy. The clinical syndromeof sepsis encompasses infection butis an ill-defined and heterogeneous concept that seems to extend beyond infection to include a variety of different causes with differing prognoses. Similar clinical manifestations occur regardless of whether the primary insult is invasive infection, massive trauma, or extensive tissuenecrosis.Until the mid-1980s gram-negative infection, mediated through endotoxin, was considered to cause the most lethal form of sepsis. The classical features of a warm, vasodilated, and hypotensive patient with a high cardiac output spiraling into irreversible multiple organ failure were thought to be entirely due to endotoxin. Endotoxin was recognized to be a component of the bacterial cell wall, and thelipid A component was a virulencefactor common to all gram-negative organisms' As lipid Ais common to all gram-negative o r g a n i s ~ s it , was thought to be an ideal target for modulating the response to severe sepsis and improving outcome, and the first attempts at nonantibiotic therapy were therefore aimed at counteracting the effect of endotoxin. This early and overly simplistic understanding of the septic syndrome has been superseded by a much more complex scenario, which has revolutionized our understanding of the mechanisms involved in the pathogenesis of septic shock and the host of targets that may be modulated by vaccines, antibodies, and other immunomodulating agents.

. In 1991 a consensus conference of the American College of Chest Physicians and the Society of Critical Care Medicine was held to develop a set of definitions that 7

could be applied to patients with sepsis and its sequelae (Table 1) 121. were urgently needed at this stage to provide both a practical framewo the variety of syndromes now falling under the general umbrella of ‘‘sepsis’’ could be categorized in order to facilitate the making of decisions regarding therapy and assistance with an understanding of the emerging pathophysiological characteristics of these syndromes. The elucidation of the inflammatory cascade has helped to identify targets for therapy, andnew definitions of sepsis have helped focus clinical trials of these new therapies. ~nfectionwas defined as a microbial phenomenon characterized by an inflammatory response to the presence of microorganisms or the invasion of normally sterile host tissue by these organisms [2]. Until this conference, the systemic response to infection had been termed “sepsis”; however, such noninfectious conditions as pancreatitis and massive tissue necrosis were now recognized to cause a conditionindistinguishable from that caused by severe infections, and the term “sepsis” was being applied to these conditions too, generating considerable confusion. It was therefore recommended that the term “sepsis,” which commonly implied a clinical response arising from infection, shouldbe replaced with systemic i ~ ~ Z a m m ~ ~ ~esponse o ~ y s y n ~ ~ o m[2]. e The systemic inflammatoryresponse syn (SIRS) is seen in association with a broad variety of clinical conditions, including infections, pancreatitis, multiple trauma, hemorrhagic shock, immune-mediated organ injury, and exogenous administration of mediators of the inflammatory response such as tumor necrosis factor. It was recommended that when the cause of SIRS was infection, the process be termed sepsis 121. actere~iawas defined as the presence of viable bacteria, in the blood; the terms viremia, fungemia, and parasitemia were to be applied to the relevant organisms. It was recommended that the term septice~ia,which had pr~viously been defined as the presence of microorganisms or their toxins in the blood, be abandoned, because of the nonspecific way in which it was used clinically and in the literature [2] The manifestations of sepsis were the same as those previously define SIRS, and in orderto identify them as manifestationsof sepsis, itwas recommended that it should be determined that theywere a part of the direct systemic response to the presence of infection and should constitute an acute alteration frombaseline in the absenceof other known causes of such abnormalities. Severe sepsiswas defined by the presence of organ dysfunction and hypoperfusion or the abnormalities associated with hypoperfusion such as lactic acidosis, oliguria, or an acute alterationin mental status 121. Sepsis * presence of a systolic blood pressure of < 9 g frombaseline in the absenceof other cau s ~ o isc a~subset of severe sepsis, defined as sepsis-induced hypotension, persisting despite adequate fluid resuscitation, along with organ dysfunction or evidence of hypoperfusion. The administrat~onof inotropes so that hypotension was no longer present would stillbe considered to be septic shockin the presenceof hypoperfusion abnormalitiesororgandysfunction.It was recommendedthattheterm “septic syndrome” should be abandoned, as it had been applied t o a variety of i n ~ a m m a tory states and was confusing and ambiguous121. These definitions are useful in helping to alert the clinician to the progression of the responseto injury/inflammation or therapeutic interventions. It is important

efinitions of the American College of Chest Physicians/Society of Critical Care nsensus Conference icrobial pheno~enoncharacterised by an inflammatory responseto the presence of microorganisms or the invasion of normally sterile host tissue by those organisms. The presence of viable bacteria the in blood. Systemic inflammatory responseto a variety of severe clinical insults. The response is manifested by twoor more of the following conditions: Temperature > 38OC or < 36OC, Pulse > 90/min, Respiratory rate > 2O/min, or hyperventilation as indicated by a PaCO, or < 32 torr (< 4.3 kPa) White blood cell count > 12,000 cells/mm3, <4,000 cells/mm3, or the presence of > 10% immature neutrophils. sis: The systemic response to infection. This systemic response is manifested by two or more of the following: Temperature > 38OC or < 36OC, Pulse > 90/min, Respiratory rate > 20/min, or hyperventilation as indicated by a PaCO, of < 32 torr ( C 4.3 kPa) White blood cell count of > 12,000cells/mm3, <4,000 cells/mm3, or the presence of> 10% immature neutrophils. Sepsis associated with organ dysfunction, hypoperfusion, or hypotension. and perfusion abnormalities may include, but are not limitedto, lactic acido an acute alteration in mental status. Sepsiswithhypotension,despiteadequatefluidresuscitation,alongwiththepresenceof perfusion abnormalities that may include, but are not limited to, lactic acidosis, oliguria, or an acute alteration in mental status. Patients who are on inotropicor vasopressor agents may not be hypotensiveat the timethat perfusion abnormalities are measured. od pressure of < 90 mm Hg or a reduction of > 40 mm Hg from baseline in the absence ofother causes for hypotension. Presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention. Source: Ref. 2.

to realize, however, that the outcomeof severe sepsis is extremely dependent upon the site of sepsis and underlying hostfactors. The definition of severe sepsis is also of less value in defining populations for the use of immunomodulators than was originally envisaged. From theclinical standpoint, the recognition of the onsetof organ dysfunction in any patient with sepsis is a very important indicator that the level of therapy needs to be escalated. Septic shock may also be influencedto a degreeby the site of sepsis and underlying host factors, but is more accurate in predicting death. It has therefore proved to be a much more useful term in defining populationsfor theuse of agents that modulate the inflammatory cascade. Mortality rate within this group varies depending on the site of infection, and its responsiveness to surgery or dkbridement significantly improvesthe prognosis.

With the increasing ability of life-sustaining technology to support thecritically ill, the occurrenceof progressive physiological failure of several organs/organ systems has been observed. However, these abnormalities have generally been described and defined in terms of organ failure rather than as a continuum of physiological derangement. In order to address this deficiency, and to allow recognition of the processes at an earlier stage, when therapy might still turn the process around, the consensus conference recommended that the detectionof altered organ function in the acutely ill be recognizedas a syndrome termedmu~tipleorgan ~ysfunction,with ~ysfunctionsignifying the inability to maintain homeostasis, Itwas hoped that this new definition would emphasizethe dynamic natureof the process and that changes in organ function over time coulduseful be in prognostication[2]. t was further recommended that organ dysfunction be described as primary or secondary: primary organdysfunction is the direct result of an insult where dysfunction occurs early and is directly attributable to the insult, whereas secon~ary as the consequenceof the host response within the contextof d ~ ~ f u n c tdevelops io~ ultiple organdysfunctionsyndrome(MODS),therefore,representsthe more severe end of the spectrumof SIRS andusually occurs after a latentperiod; it is most commonlyseen complicating severe infection. The mortality rate of ~ O ~ S / m u l t i p systems le organ failure (MSOF)exceeds 50%, and the syndrome accounts for alarge proportion of deaths among intensive care unit patients [3]. The number and durationof organ system failures areclosely related to hospital mortality, and the combination of organ system failures also has differences in the a n i m p o ~ a n teffect on survival [4], but there can be marked degree of physiological abnormalities, and these may partially explainthe variations in risk for patients with different types and combinations of organ failure. The patient’s admission diagnosis also influences the prognosis for those with multiple organ system failure, and hospital mortality rate is generally worse for those who [S]. have nonoperative diagnoses and those who have undergone emergency surgery

The inflammatory response is a protective host defense mechanism mediated by the activation of soluble plasma factors and immune competent cells, including neutrophils, monocytes, macrophages, and endothelial cells. It acts as part of the

systemic defense against both infectious and noninfective injuries. Infectious triggers of the inflammatory cascade include products of both gram-negative and grampositive bacteria, as well as viruses, fungi, and parasites such as malaria [G]. Noninfectious causes include pancreatitis, burns, massive tissue necrosis such as is seen with multiple trauma, and chemotherapy [7,8]. The extent of the inflammatory response depends upon the type and magnitude of the stimulus-the lipid A moiety of the gram-negative bacterialcell wall has been the most reproducible, predictable, and powerful stimulusidentified. The inflammatory cascadeis designed to upgrade the immunological response and assist ,with the eradication of microorganisms; however, an excessive response by inflammatory mediators leads t o the deleterious effects that are part of the syndrome of sepsis. eath due to sepsis is usually the result of an overwhelming inflammatory respon that causes shock or respiratory failure and mortality within hours, or a more delayed process resulting indeath from multiple organ dysfunction caused by either the initial inflammatory response or ongoing or repeatedstimulation of the inflammatory cascade. Deathis thought to be caused by excessive produring the inflammatory reduction of the potentially toxic mediators liberated sponse,includingcokines,kinins,eicosanoids,platelet-activatingfactor,and nitric oxide (NO) [6 ,101. This deleterious effect is largely focused on neutrophil and other inflammatory cell accumulation and endothelial cell dysfunction within involved organs, leading to organ dysfunction [l l]. The causeof the repeated inflammatory cascade stimulation and delayed mortality is not yet clearly delineated, and it is theorized that secondhit insults such as ventilator-associated pneumonia (VAP), phlebitis and other minor infections, as well as bacterial or endotoxin translocation from the gut may play cumulative roles. A sustainedhitphenomenonwithpersistence of initialinsultmayalso be a is associatedwith death in contributin~factor insome cases [121. Often V critically ill patients;however,adirectcausal re1 ship isuncommonandtechniques that reduce VAP, such as selective decontamination of the digestive tract t been shown to reduce the mortality rate[131. ut integrity with translocation of bacteria and endotoxin has also been implicated as b ing central to driving the host response toward the development of M O ~ S and , translocationof both bacteria and endotoxin hasbeen demonstrated in the portal and systemic blood during small animal experiments[14]. The evidence that this mechanism truly plays a role in humans is less clear [ 15-17]. The of SDI) would be expected to have a beneficial effecton thedevelopment S and mortality by reducing the intestinal bacterial load and translocation, as not been effective in achieving this result. It is now also well recognized that repeated stimulation of the inflammatory cytokines will lead to a down-regulation of their effects, either by increasing circuceptors or by promoting the production of antiinflammatory cytoW much protectionthis down-regulation providesis still unknown, as it has been shown that mortality in patients with both burns and the adult respiratorydistresssyndrome ( ~ R ~ isS related ) to persisting high levels of the proinflammatory cytokines[20-22]. The deleterious effects of the inflammatory cascade are mediated by excessive stimulation of the cellular proinflammatory cytokines, mainly tumor necrosis factor a, and interleukin 6, and the kinins,eicosanoids, and NO; the release of these

mediators leads to loss of capillary integrity9 distributiveS dysfunction [6,23,24]. The inflammatory response is best characterized by toxin in animal models and a clearly defined cytokine res initial rapid surge of production of tumor necrosis factor a l , followed by a slower,moresustained production of

*

i

with endo-

similar cascade is triggered by exotQxins, bacteria, or noninfectious substances such as kinins and bradykinins arising fr of cellular destruction. The initial stimulus pre macrophage system, but also neutrophils, an

that nitric oxide

The migration of neutrophils and the ongo

of the mechanisms increases; however, it i order to understand why interventions ma stimulus predominantly activates the mon trophils, and itstimulates pro~uctionof c ing factor, and complement both act as up- or down-regulatQrs itself may be modified by prio expression in different or

response that depends on receptornumbe addition, circulating free receptorsin theser

availa~ility. There maybe,in can bind to, andinactivate, the

A number of specific in~ammatorycytokines are now well recogni~ed to participate in the inflammatoryprocess, and their specific role in sepsis has largely been delineated.

7.

cytokine that plays a central role in the progression of se synthesis and release are the most powerful trigger of TNF production, also triggered by numerous other substances, exotoxins, viruses, parasites, fungi, and interleu [35,36]. Tumor necrosis factor is produced in nu tissue macrophages, lymphoc~es,neutrQphils, prohormone of 233 amino acids [37].

\ ... .

S (Endotoxin, exotoxins, proteases, peptidoglycans,etc.)

The inflammatory cascade.

3 ~timulus

Bradykinin

Endothellal cells, some neurons

Smooth muscle cell, platelets, some neurons

NO-

A

NO-

''2'.

lYnUn

NH

NH

NADPH, CaM

HiN

COO" L-arginine

NH

NO' nitric oxide

(a) Scheme showing constitutive release of nitric oxide. (b) Biosynthesis of nitric oxide from L-arginineand recycling of L-cirtulline.

4

amino-acid prohormone by cleavage of a residual signal peptide [38,39]. The prohormone is thetransmembraneform of TNF, which exposes thematureform extracellularly, allowing bioactivity to be expressed at a local tissue level [40]. The residual peptide may remain intracellular, and the free TNF molecule is a trimer composed of three 17-kDa polypeptides. The TNF gene that is located on the short arm of chromosome 6 is closely linked to the major histocompatibility complex [411. The synthesis and secretion of TNF aretightly controlled and the transcription of the TNF gene may be regulated by evanescent repressor molecules that usually control its release. Regulation at the posttranscriptional level involves the presence of a long sequence of adenosine and uridine residues in the three untranslated regions of TNF messenger ribonucleic acid (mRNA). This nucleotide se~uenceis also found in the m A of a number of other cytokines and is thought to control the half-life and translationof TNF The biological effects result fr inding to specific receptors that occur on almostall cells. Two distinctmembrane-associatedreceptors,termed very and TNF-R2 (75 kDa), have been recognized. They have lar domains and probably require different signal transduction paths. The different functions of the two receptors have been identified by using specific receptor antibodies. Stimulation of TNF-R1 results in cytotoxicity, expression of adhesion molecules on endothelial cells and keratinocytes, and activationof sphingomyelinase with formation of ceramide and activation of NF-KaB. Stimulation of TNF-R2 results in a proliferative response in mouse thymocytes and cytotoxic T cells, fibroblasts, and natural killer cells [44]. The extracellular domains of both TNFreceptors share 28% sequence identity,a similar homology shared with a or related protein, CD27, p of cell surface proteins, TNF re and FAS antigen,which B, nerve growth factor receptor, 51. These receptors inter part of a TNF receptor superfa relatedcytokines to ducedistinctivecellularresponses that usuallyoverlap one another, resultinginxtremelycomplexsystem. The two TNF receptors are both processed into soluble forms by the proteolyticcleavageof the extracellulardain of the transmembrane receptors.These soluble TNF receptors, both TNF , are found in the circulation of healthyhumans andare present on ry typeofcell,excludingredblood cells. They are, however, und in increased numbers in sepsis, as well as a number of noninfectious diseases 1. These soluble receptors retain their affinity for TNF and therefore compete wi ell-associated receptors for circulating TNF. The number of soluble TNF receptors depends on the amount of circulatin~ TNF and the production and clearanceof TNF receptors.Soluble TNF receptorsinhibit the activity of excessively high levels of circulating TNF with its short half-life, whereas their effect may augment the activity of TNF in body compartments where TN cleared slowly by stabilizing itsstructure andprolonging its effects[46-4.81. The administration of TNF to either humans or animals produces the pathophysiological changes seen in sepsis. They are identical to thoseseen after endotoxin administration;however,thetimeseqithTNF is earlier,suggesting a more directeffect. The effects arealsodose-ent:largerdoseslead to cardiovascular, cellular,hematological, and metabolicchanges if thetimecourse permits, whereas a flulike illness with fever only may occur with smaller doses. In sepsis, the

majority of patientsexhibitcharacteristiccardiovascularchanges,with peripheral vascular resistance and a normal to i ~ c r e ~ s ecardiac d output. ients this will progress to septic shock with a phases, depressionof left ventricula en demonstrated in animal experime of TNF or the induction cjf nitric oxide [ necrosis factor influences e intimately involved in leukocyte a leukocytecQngregation,adherence,migrationthroughend chemotaxis to sites of infection [52 TNF, causing increased expression neutrophil the on of t surface and inducing endothelial adhesio esion molecule 1 ( 1) [52].accu The e site of injury aids in the e l i ~ i n a t i oof~ pathogenic uction of reactive oxidants9proteinases, and antimic defense mechanism host agai the ve ability little to differentiate between foreign and host antigens9 and this activation may contribute to tissue and organ damage during sepsis. Tumor necrosis factor productiQn varies in experimental se tion of endotoxin or theinoculation of live bacteria mimics acute sepsis and results in the appearance of ~redictablyhigh levels of NF in the circulation within 6 minutes of injection; the levels peak at 90 minu S and return to baseline after

en in lethal peritonitis, TNF may not

umor necrosis factor plays a key role i there are so many stimuli that increase the pr would appear an ideal site for general pharma rate of mortality from severe sepsis; however, interactions between the various c ~ o k i n e sar circulating TNF may be ~eneficial or even essential to mount any form of host defense. Until the finer points of this casca e are more clearly may only be educated guessi~g. at-labileproteinswerefirstdescribe were then shown to produce monophasic fever when injected into either animals

r“leukocyticpyrogen.” §ubse~uent t endogenous pyrogen caused other decrease in serum iron and zinc levels, ts and colony stimulating fact entation of T cell responses. genous mediator.’, were induced by a single family of proteins, they ny molecules previously described their by biologin-l , mononuclear cell factor, lymphophils, osteoclast activating factor, melanomagrowthinhibitionfactor,andtumorinhibitoryfactor 2, havenowbeen ukin l by molecular analysis and cloning e~periments[62]. 1 roleinmediatingtheinflammatory ly, in concert with the other proinflamma-

erleukin 1 is produced

plasma of normal subjects, but it has been found in sepsis, organ rejection, and * is [M]. Someinvestigatorshavefoundacorrelationbetween outcome in sepsis [67].There is, however, a considerable discrepancy in thedetection of l b in atientswith sepsis, andthe reliability of the ‘S controversial. Interleukin l a is rarely , because of its primary cell-associated asaninhibitortobothIL-laand l b by co~petitive type I1 IL-lreceptors.Affinity of Ira for the type I receptor is similar to that of either IL-la or IL-lb, but it has no agonist activity

and re~uires 10- to SO-foldexcesses in concentration to inhi it type 2 receptors. Interleukin Ira is produced by the same cell types as IL-la and lb, butits synthesis and release are differently regulated [68]. The synthesis of IL-lra is regulated by complex transcriptional and posttranslational mechanisms, and the production of IL-lra may greatlyexceed the productionof IL-lb in humans after endotoxin stimulation. This difference may be due to other cytokines such as trans factor-beta ( T ~ F - ~IL-10, ), and IL-4, which increase IL-lra and production [69,70]. The two IL-l receptors differ considerably in distribution, with the type 1 receptor being found on the majority of cells, including T cells, fibroblasts, and endothelial cells, where it is important in signal transduction after binding to IL-l. The type I1 receptor is found mainly on neutrophils, monocytes, marrow progenitor cells. This receptor has no signal transduction function and is thought to act as a decoy receptor, to reduce the amount of activeIL-1.The extracellular domain of the type 11 receptor is also shed by neutrophils and monocytes as a soluble receptor, and these receptors bind with circ~lating IL-lb,thereby inhibiting its activity[7 l]. The antiin~ammatory cytokines and other agents such as dexamethasone may produce some of the antiin~ammatoryactivity by up-regulation of type I1 gene expression andincreasing the shedding of the receptor. The extracellular portion of the type I receptor is also shed by proteolytic cleavage to form a soluble receptor. ‘S receptor alsoantagonizes the of IL-1 by bindin to circulatingIL-1. ferentialbinding tothedifferen inesallowsthis sy m to bemoreaccuratelyregulated. Thetype recept I s withhighaffini o IL-lraandsheds type 11 receptors to inhibit the effects of IL-1tivelywithoutbinding toone another. The soluble type I1 receptor also prefer ly binds to IL-lb, thus inhibiting its effect. As with other cytokines, IL-l has both beneficial and detrimental effects. It stimulates nonspecific host defenses to infection by stimulating the recruitment and release of neutrophils and macrophages from the bone marrow also and stimulatin~ natural killer cells. t alsoproduces B cell ~roliferation increases antibody production.Like T F, itpromotesleukocytemigration t es of infection and increases adhesion by up~regulating thesynthesis of leukocyte adhesion molecules. It also causes increased synthesisof other biologically active substances, including the in~ammatory cytokines and m~diators, well as as hepatic acute phaseproteins. It leads to a decrease in free serum iron magnesi~m and levels, which inhibits growth of bacteria. It may also produce beneficial effects in cancer chemotherapy, by its stimulatory effect onmyelopoiesis. The main detrimental effect of IL-l appears tobe its role in the patho~enesis of septic shock and multisystem organ dysfunction. Here it plays a central role in of the effects of TNF alone andstrongly potentiating the effect r e p r o ~ ~ c i nmany g of TNF when given in combination [72]. It leads to increased vascular permeability, pulmonary congestion, and hemorrha~ic, ~istrib~tive? cardiogenic and shock. The infusion of either IL-la or IL-lb alone in experimental animals causes a profound shock-like state, with leukopeniaand thrombocytopenia [73]. Interleukin1 is alsoimplicatedinreperfusioninjury,where high levels, thought to be stimulated by oxygen free radicals, have been found. A number of

substances reduce thepro uction of IL-1. These include endogenous as glucocorticoids, corticotrophin releasing factor, TG exogenous drugs such as pentoxifylline. These substan of all the proinflammatory cytokines andthis, combined with the i of this system, canhave widespread effects on the modulationof the inflammatory cascade. leukin 6 levels in the circulation correlate better than either verity of disease in patients with injury, burns, sepsis, and n as surrogate markers of activation of both IL-l and TN inds to the IL-6receptor and triggers the cellular transduccomplexgpl3Oprotein.The gp13O protein serves as the signal transducer for IL-6, leukemia inhibitory factor, oncostatin neurotrophic factor; that role perhaps explains the many overlapping shared functions of thesecytokines. The signals provided through the membrane receptors affectthefunction of cells mainlythroughthemodulatione activity of sequence-specific transcriptionfactors; inIL-6thatfactor is IL-6, whichwas identified by IL-l induction of IL-6 expression. Interleukin 6 has a wide diversity of function but, unlike TNF or IL-I, does not induce shock or inflammation or have any effect on endothelial cell activation. Nor does its t i ~ u l a t eproduction of other proinflammatory cytokines. It acts synergistically with EL-1 to activate cells and T cells, stimulates he~opoieticstem cell growth, increaseshepaticacuphaseproteins, and stimulates IL-2 production. Production of IL-6 has been associated with atrial myxoma, rheumatoid arthritis, and other inflammatory conditions. This relationship suggests that IL-6 may be associated with the development of an autoimmune state. Tumor necrosis factor cx and IL-1 are also invariably associated with IL-6 production in inflammatory disI genes contain NF-IL-6 binding ease. The promoter regions of the sites; thus a s i ~ n athat l activates ~ F - I L - 6will simultaneously induce the expression of several inflammatory cytokines[77]. Interleukin 6 has also been implicated as a cause of aposi9s sarcoma, myeloma, and plasmac~omaas it acts as a potent growth factor in these cells. This may be triggered by stimulation of IL-6 by viral infections. Interleukin 6 is multifunctional and prod es both favorable and unfavorable effects on human health. ~ysregulationof IL production is linked to autoimmune disease and cancer, and itsblockademayhimportanttherapeuticimplications,In sepsis it appearsto play no significant role other than that its serum level acts as a measure of th severity of sepsis. nterleukin 8 (IL-8) is a proinflammatory cytokine that serves actant factor and activator of neutrophils; it is produ~edby many cell types after stimulation by IL-l, TNF, or microbial products such as endotoxin. Its releas awes neutrophil ~egranulation, andp r i ~ i n gby TNF seems to enhance IL-$-pro ted degranulation 1781. It has been detected in the urine of patients with urinary tract infections [7 1, and in the cerebrospinal fluid of patients ngococcal disease[SO] ty et al. demonstrated that IL-8 concentrations are also a marker of the exacerbation of c ~ o k i n eproduction and that concentrationsof IL-8 correlate with

severity and outcome of sepsis [SI]. indicator for the development of th

thoughthe anno Interleu~in also 8

as a n t i t ~ m o and r shock.

receptors for vaso

tension and myocar The increased pro harmful effects. Nitric Q de~ression[5 l].

ction, but its S stemic administration did not reduce

way,with

the numerous key biochemicalsub-

The systemic inflammatory response and multiple organ dysfunction syndromes are notdiseasestates or syndromesintheirownright;theyarethefinalcommon ng to death in the modern intensive care unit that provides organ are not treatable by specific therapy but can, in some instances, be pr~vented by appropriate care [l 19). The basic premise for treating anycritically ill patient is to deal with the primary inciting stimulus that has triggered the systemic inflammatory response syndrome. Such therapy may involve the drainage of abscesses, removal of necrotic tissue, or merely administration of antibiotics. ever, once the systemic inflammatory response has been triggered, the host response may be sufficiently overwhelming that organ dysfunction and death appear to be inevita~le.Innovative therapies insevere sepsis involve modulating the host inflammatory response or targeting the mediators of inflammation-such therapies have been in clinical trial the past 10-15 years. Careful discrimination of the relative roles of infection, andorgandysfunction is aprerequisite for designingtrials aimed at modifying the host’s inflammatory response.If the host responseis blunted in the face of overwhelming i~fection,this effect may be d e t r i ~ e n t a land allow further spread of the infection. Once the infection itself is under control, attenuating the inflammatory response may reduce the degree and extent of damage to other organs and have an overall beneficial effect. The magni e of the host response is wever, when evaluating acleardeterminant of the risk of mortality [120,121) suchtherapies it is important to realize thatthestudies were largely based on inhomogeneous patient populations with varying disease processes and unpredictableresponses to interventions. The diagnosticcriteriainthestudieshavealso so that divergent results for the same interventionhave been reported from

various centers, e.g.,the three studies evaluatingthe use of corticosteroids for sepsis he proinflammatory cytokines, particularly tumor necrosis factor a (TNFa) ukin l@(IL-P), have been shown clinically and exp most important cytokine mediators in the patho~enesis ofSI 261. I~terleukin6 appears to be a marker for the presence of proinflammaiators [76], and IL-8 appears toplay a major role in neutrophil activation [127]. These mediators stimulate release of a cascade of secondary mediators, includingarachidonic acid derivedprostaglandinGI2), thro~boxane latel let-activating factor(PAF),brady~inin, histe, serotonin,colon ing factors, and co~plement.The initiation of the inflammatory cascadeby infection also leads to the activation of regulatory mechanisms9 including acute-phase proteins, antiproteases, cyclooxygenase products, antiinflammatory cytokines, and specific anticytokine substances such asIL-1 receptor antagonist (IL-lra) andsolublec tokine receptors (e.g., soluble TNF receptors and soluble IL-l receptors) e administration of TNF and IL-1 alone or in combination has been shown to reproduce many of the physiological and laboratory changes observedin animal atients with sepsis syndrome. The central proinflammatory roles of in septic shockare related to their ability to activate endothelial.cells, induce the production of the other inflammatory cytokines, activate effector cells lymphocytes, neutrophils, monocyte-macrophages), and cause the release of platelet-activating factor, eicosanoids, and the other downstream mediators. Identification of the early eventsin the pathogenesisof septic shock suggests a number of interventional opportunities for blocking the cytokine cascade before extensive and irreversible changes have occurred in a variety of organ systems.

Historically endotoxin was the first mediator of the inflammatory cascade to be uring the 1960s, the structural featuresof ram-ne~ative bacterial lipopolysaccharide were identified: an inner core sugarregion, a common toxic lipid A. moiety, and an antigenicallydistinct O polysaccharide side chain, Experimental studies suggested that antibodies directed to epitopes in the inner-core sugar region of the lipopolysaccharide(which are widely shared by gram-negative bacilli) may be gents that neutralize or enhance the clearance of endotoxin and have been

1. Polyclonalhumanantibodies to the core region of endotoxin (J5 antiserum) humanized monoclonal antibodies to the lipid A. component 1A), respectively olysaccharide (recombinant ~actericidalperme4. Other treatments that aim to block the peripheral effects of endotoxin, whichincludepeptides andli~oproteinsthat bindotoxinandendotoxin-derived substances free of activating effects [l

~ubsequentstudies have shown that gram-negative bacteremia occurs in approximately 30~0 of patients with sepsis syndrome [124,13 1. There is, however, considerable evidence to support the hypothesis that the toxic manifestations induced by gram-negative bacteria are largely induced and mediated by endotoxin, particularly by its biologically active innermost component ,lipid A [1351. Initial interventions included the use of immunoglobulin and a polyclonal human antibody to the core region of endotoxin. Ziegler et al. reported a double-blind, placebo-controlled studyof 136 patients to assess whether treatment with antiserum c (15) ~ u t a ndecrease would t the to inner core of rate the of mortalit identified two 61. study This enefit fromsuchtherapy: (1) thosepatients with severe patient risk groups l sepsis and with gra~-negativebacteremia, in whom the mortality rate was 3 the placebo group compared with 22% in the J5 antiserum group ( p = 0.01 (2) those patients with septic shock who required vasopressors for more than 6 whomthemortalityrateforthose receiving pla was ~~~0 compared Vo inthose receiving J5antiserum ( p = 0.003). ever,thisstudy was demonstr~tethatthe effect of theantiserumetothe J5-specific antibody. ~riticismsof this study included the high mortality rate in the control group, but other studies to investigate the antiserum were initiated. The results in subsequent studieswere conflicting. No effect was observed whena single dose was used prophylacticallypatients with prolongedneutropenia [137]. In high-risk surgicalpatients treat prophylactically9 amarginallysignificantincrease in survival rate was found 81, butno significantimprovementinsurvival rate was reported for children with circulatory shock secondary to infectious purpura who received J5 [139]. A subsequent prospective, randomized double-blind study found no significant benefit in patients with gram-negativeseptic shock who received the antiserum [1401, whereas a small, unblinded randomized trial reported an improved atients withseptic shock but hasbeen criticized becauseof its h the increasing recognition of diseases transmitted by bloo blood products, theuse of J5 ceased.

The next step was the generation of monoclonal antibodies directe A region of the lipopolysaccharide; such antibodies are preferable to polyclonal preparations because they limit the risk of infection and are homogeneous, with well-defined antigen specificity. A human monoclonal antibody ( murine antibody(E5) were generated foruse in large multicenter placebo-controlled trials, which used similar entry criteria based largely on the definitions discussed [1429143]. ) monoclonal antibo to an epitope on lipi 00th lipopolysaccharide[1431 d the ~hysiological res~onses saccharidesinsheep [l441 and in another only slightly added to the protection provided by antibiotics in mice challenged with gram-negative bacteria[1

has been tested in two placebo-controlle ents with signs of gram-negative infectio were enrolled. The administration only in the patients with gram-n (137 patients). In this subgroup, of those who received E5 died w The second study enrolled 8 patients with gram-negative seps was noimprovementinthesurvivalrate of the 5 3 patients ~ gram-negative sepsis, although in a subgroup of patients with

out refractory shock had a re proved survival rate [ 1483. 11491.

was safe in. all ~ a t i e n t s~ r e s to~have ~ e ~

low prevalence of gram-negative bacteremia [lSl]. The expense of such therapy ethical dilemmas about the principles of social justice, t e fair utili~ationof resources according to medical need, an the patients who would benefit most from cal debates were premature, however, asin February l A"., announced that theF causingthestock of thecompany to fall by $675 millionin l day.The concluded that the data did not demonstrateefficacy of the product and requ further clinical data in order to consider the drug further. ospective randomized, A Efficacy in Septic tudy was to c o ~ p a r e all-cause mortality rate inpatientswith ebo in reducing the 14 gram-negativebacter , and to assess the safety of the drug in patients who did not have gram-negative~acteremia. Theresults of this study were fulfilledallenrollment

who patients

received

ratestality

in the patients

lity rate in patients with an increased mortality

*

nts who received placebo. It is also possible that the treatment

also suggested the study was underpowered because of the early stopping rule with a greater level of significance than is conventionally required in clinical trials to test the ~ifferences between the treatment groups. U~fortunately thesecond study had cient in many areas in which the previous collected oneach patient was kept to a underlying disease severity, nomeasure~entof ~ e r u m c ~ o k or i n een~otoxin levels, and no assessment of the appropriateness of anti~icrobialtherapy or of organ failure at entry [l54]. The choiceof all-cause mortality in an intent-to-t~eat analysis, to a reversal in a relevant endotoxin-induced physiological response, een too difficult an end~ointfor a potentially useful antisepsis product t may also have been unrealistic to expect any one therapeutic intervene of a complex multifactorial process to show statistically er fuel to the controversy,

a study using an animal model

to

investigate the therapeutic efficacy and microbiological andphysiological effects of HA-l A in sepsis found that only15 Vo of the dogs thatreceived HA- 1A survived 28 days compared with 57% of controls ( p = 0.05). At 24 hours the had lower mean arterial pressure ( p = 0.04) and cardiac index ( and higher lactate levels ( p = 0.05) than the controls. These parameters were also significantly more predictive of death, although all animals had similar levels of endotoxemia and bacteremia[155]. A further study raised still more questions asto -1A: this one, a post hocanalysis to determine the hemodynamic in the first multicenter randomized, double-blind study of the is study found that no changes over time attributable to the study drug were noted in the mean arterial pressure, heart rate, or need for vasopressor administration. No significant difference in cardiac index, oxygen delivery index, or left ventricular stroke work index between those receiving placebo and drug was demonstrable. When the patients were grouped according to whether or not they bacteremia, there still appeared to be no significant difference 1A in mean arterial pressure, heart rate, cardiac index, oxygen ft ventricular stroke workindex. has become more widely available for laboratory testing, some aised concerns regarding its binding affinity and specificity, as well as its unsatisfactory endotoxin neutralizing properties [157-1601 Initially the immunoglobulin ( I g ~ produced ) by this hybridoma wasdescribedasbinding specifically toaoadspectrumofsmoothlipopolysaccharidesand bacteriain an en~yme-linked immunosorbent assay [1501. However, subsequent reports have 1A binds poorly to smooth lipopolysaccharide in such assays, sometypes of smooth lipopolysaccharide in fluidphase as measured by rate nephelometry, but this technique may detect low-affinity as well gh-affinity interactions between antibody and antigen [161]. It was shown that purified from the hybridoma bound to gram-negative bacteria, but also to gram-positive bacteria, fungi, cardiolipin, and lipoproteins, raising doubts about its specificity [159]. It thus appears that the data on binding that were presented in the initial description of the antibody [l501 and endorsed in the study are substantially different from those more recently described by the pharmaceutical company and tigators. A further problem is that there is no experimental model in - l A has consistently protected animals from endotoxin challenge [161], g the practical problem that there is no established method to ensure quality control of the antibody since the characteristics of HA-1A that are related

A antiendotoxin antibody has beeninstructive for many reasons: it highlighted the importance of rigorous clinical trials in allowing critical evaluation of a new product, it drew attention to the ethical dilemmas that are becoming increasingly apparent in attempting to provide state of the art medical care to all patients, and it caused intensivists to reappraise what criteria could be used to identify patients most likely to benefit from a drug designed to benefit individuals with gram-negative sepsis. It also served to demonstrate that the cytokine cascade seen in sepsis is a highly complex, multifactorial process and that to expect any single intervention at any stage in this process to exhibit significant effects may be oversimplistic. Furthermore, an increased mortality rate was seen in patients who did not have gram-negative bacteremia in both studies with

tration is critical to its efficacy.

3 binding proteins, it remains to be seen whether this approach to the treatment of septic shock will be effective. There is reason for concern that the agentsmay only be effective if given before or at the timeof endotoxin administration,which is not a clinically reproducible scenario. It is also still not possible to predict accurately which patients with sepsis have a gram-negative cause. In addition, endotoxemia may persist over days, so that the antiendotoxin will require a long half-life or will need to be administered by continuous infusion [174]. Phase 1/11 clinical trials using human recombinant bactericidal/permeability-increasing protein have been initiated, but the results of such studies and whether such major obstacles can be successfully handled remainto be seen. Other antiendotoxin treatments aim to block the peripheral effects of endotoxin; they include peptides and lipoproteins that bind endotoxin, endotoxin-derived substances free of activating effects [133], binding substances such as polymyxin, and plasmapheresis [1331. Reconstituted lipoproteins had protective effects after endotoxin challenge in animals but also had neuro- and hepatotoxicity and increased the mortality rate [1331. Deacylated endotoxins and lipidX derived from thehydrolysis of lipid A and from a precursor of lipid A, respectively, inhibit some toxic effects of the native molecule but maintain their immunostimulatory properties [ 1751. Patients fulfilling criteria for sepsis and with measurable levels of endotoxin > 12.5 pg/ml only were enrolled in a controlled but unblinded study using a polyclonal immunoglobulin IgGMA preparation [ 1761. Nine of the 28 placebo group died (32%) compared with 1 of the 27 receiving IgCMA (4%). Although it was a small, unblinded study, it demonstrates that the use of an endotoxin assay may be an extremely powerful tool to select appropriate patients for the use of antiendotoxin therapy. The use of such an assay might well have made larger studies more convincing, if a treatment effect could have been documented in patients in whom endotoxin had been shown to be present.

Tumor necrosis factor alpha is critical in the host defense against infections, especially those causedby intracellular pathogens, but when produced in large ~uantities by the macrophage is an early and important componentof the endogenous mediator cascade that occurs as part of the systemic inflammatory response syndrome. Plasma levels of TNF, interleukin 1, and interleukin 6 are increased in experimental studies after challenge with endotoxin or bacterernic infection [ 177,1781, levels of TNF are increased after endotoxin challengein normal human volunteers [179], as well as in patients with both gram-positive and grarn-negative bacteremia [180-1821. High plasma levels of TNF or IL-6 are indicators of poor prognosis in patients with severe sepsis or septic shock [126,180,182]. Tumor necrosis factor is potentially an extremely attractivetherapeutic target as it is not specific to gram-negative sepsis, but released by the macrophage after any of the numerous stimuli that may trigger SIRS; this serves to provide protection from the injurious effects of TNF regardless of the natureof the infect in^ organism, or even in cases of noninfectious SIRS. Not only does TNF produce myriad proinflammatory cytokines that ultimately cause diffuse tissue injury, but IL-1 and y

interferon ( I F N - ~ )potentiate the physiological effects of TNF and may contribute independently to the cascade of inflammatory events that culminate in the sepsis syndrome. Anti-TNF monoclonal antibody therapy in primates and small animals has been demonstrated to reduce mortality caused by both gram-negative and grampositive bacterial infection [ 178,184,1851. The intrinsic capacity of each bacterial isolate to induce TNF production varies with the nature of its lipopolysaccharide phenotype, the quantity of o r g a n i s ~ s ,and the complement of endotoxins, as well as the cell wall constituents expressedby gram-positive organisms [ 186,1871. These results would suggestthat certain bacteriamay bemore potent inducers of cytokines thanothersand maymodifytheresponse toanti-TNFmonoclonalantibody therapy. Several studies of the use of anti-TNF monoclonal antibodiesin humans have now been reported. Fisher et al. reported an open-label phase I1 multicenter trial with escalating doses of a murine monoclonal antibody (C~0006)[ 1881. Eighty patients withsevere sepsis or septic shock were enrolled and treated with one of four dosing regimens in addition to the usual supportive care and antimicrobial therapy. The antibodywas well tolerated, despite the appearance of antimurine antibodies in 98% of patients, but no survival benefit was found for the total study population, and the overall 28-day mortality rate was 41%. Patients with increased levels of circulating TNF at study entry seemed to benefit from the high-dose treatment. It was also confirmed thatincreased levels of IL-6 predicted afatal outcome, although TNF levels in this study did not correlate with prognosis; failure of the IL-6 levels to decline within the first24 hours proved to be a more reliable predictor of outcome than the initialIL-6 levels. In patients who were shockedat study entry, TNFlevels were higherand the outcomewas poorer than in patients who wereseptic but not in shock. Plasma levels of TNF were greatest in patients with gram-negative bacteremia but were also substantial in those with bacteremic and nonbacteremic infections with gram-positive bacteria. Another murine monoclonal antibody (anti-TNF monoclonal antibody [Bay ~13511)has been evaluated in phase I and I1 studies. The phase I study included 20 patients at risk of sepsis; single doses of 1-15 mg/kg anti-TNF monoclonal antibody or two doses of 15 mg/kg were administered; there were also 16 sepsis syndrome patients who received a single dose of 15 mg/kg anti-TNF monoclonal antibody. The anti-TNF monoclonal antibodywas well tolerated and had a serumhalf-life of 50-54 hours [ 1891. Two large phase I1 multicenter randomized, controlled, double-blind studies of this murine monoclonal antibody havesince been performed. One of thestudies was conducted in North America and enrolled 994 patients from31 hospitals in the United States and Canada [ 1901. Of the enrolled patients 971 received placebo or two doses ofthe anti-TNF antibody(7.5 mg/kg or 15 mg/kg). Patients were comparable in degree of severity of illness, and the endpoint of the study was 28-day all-cause mortality, A planned interim analysis performed after 800 patients were enrolled demonstrated no benefit in the septic patients without shock. However, a dose-dependent trend toward decreased mortality rate was found in septic patients with shock, and both the North American (NORASEP~)and European/South African (INTERSEPT) trial protocols were amended, so that septic patients without shock were no longer enrolled and thosewith shock continuedto be studied.

it would therefore

es not have excessive kines (such as solub

ors: a toxic effect of

is a nat~rally ~ a t o r Ycells,

a Y

r

with IL-l@ and is a part of the body’s normal mechanism for controlling acute in~ammation.Increased concentrationsof this endogenous cytokine antagonist are detected in human sepsis or experimental inflammation, and the molecule has no intrinsicagonistproperties, even at increasedconcentrations. It inhibitsIL-1 by competing with it for cell receptor sites [ 1991. A recombinant 17-kDa form hasbeen produced and hasbeen found to prevent shock and reduce mortality rate in several rodentandbaboonmodels of sepsis [200]. Increasedsurvivalrates of animals challenged with endotoxinor gram-negative bacteria and improvement in hemodynamic parameters in animals challenged with gram-positive bacteria have been observed when IL-lra has been administered before or up to 3 hours after the septic challenge 12011. In humans with the sepsis syndrome, preliminary evidence suggested that a dose-related increase in survival rate occurred after treatment with IL-lra. In an unblinded phase I1 study of 99 patients with sepsis or septic shock, the mortality for those receiving 17 mg/hr of IL-lra; rate was 44% among placebo controls (32% 25%forthose receiving 67 mg/hr;and 16010 forthose receiving 133 mg/hr of IL-lra); thesurvival rate was a linear functionof the dose administered( p = 0.015) [202]. The r e s ~ l t sof a recently completed phaseI11 trial of IL-lra have been equivocal with respect to the value of this treatment [203]. In the trial 893 patients with sepsis or septic shock were treated with placebo or low-dose ( l mg/hr) or high-dose (2 mg/hr) IL-lra. Treatment had no significant effect on the two primary study endpoints: all-cause28-day mortality rate for the study group overall and mortality rate for the713 patients in shock. The28-day mortality ratewas 34% for the control patients, 319‘0 for the low-dose, and 29% for the high-dose group ( p = 0. the group with shock, the mortality rate for the controls was 36%; it was 3 both doses of IL-lra ( p = 0.23). A retrospective analysis of the phase I11 results was performed by using a model for estimating the baseline 28-day mortality risk for septic patients based on weighted risk factors derived from the patients’ Acute PhysiologyandChronicHealthEvaluation(APACHE 111) scare age,chronic health, days in the hospital/intensive care unit, underlying disease, blood p white cell count [204] This risk predi~tionmodel defined a subgroup of 593 patients with sepsis and high risk for mortality. Some, but not all, of these patients were in shock at study enrollment. With thisanalysis, IL-lra recipientswith a baseline mortality risk of >24% appeared to experience a beneficial effect, demonstrating an 18070 to 22% decrease in mortality rate. These data suggest that the benefits of IL-lra may accrue in the more severely ill patients. A follow-up phaseI11 study was started but, at the time of writing, had been discontinued because an interim analysis suggested that efficacy would be difficult to prove. There was no evidence to suggest a deleterious effect. These data neither prove nor disprove the IL-1 mediation hypothesisof sepsis.

The increased production of NO during septic shock may lead to several harmfuleffects.Nitricoxidemaybe largely responsible for sepsis-inducedhypotension [102,103], and invitrostudieshaveimplicated NO in sepsis-induced myocardial depression [5 l]. ~epsis-induced hypotension is an important predic-

tor of organ injury and death, and the useof NO synthase inhibitors might be expected to improve survival rate in septic shock through increasing the mean arterial pressure. Nitric oxide synthase inhibitors have been shown to be able to restore the responsiveness of the septic vasculature to catecholaminesinendotoxinchallenged animals [205] and to “normalize” mean arterialpressure in anestheti~ed animalschallengedwithendotoxin or TNT; withoutcausinghypertension [102, 1031. Nitric oxide also has well-documented cytotoxic effects [206,207], and itsoverproduction in septic shock may leadto direct tissue injury and organ failure. Inhibition of NO synthase has not been shownto prevent endotoxin-induced myocardial depression [208], and in vitro evidence has shown that NO may exert a p r o i n ~ a m matory effect during septic shock by enhancing cytokine release from phagocytic cells [209]. There may,however,also be beneficial effectsderived from NO in septic shock: it appears to play a role in maintaining visceral and microvascular blood l l] by counterregulating the mediators released during sepsis, such as e and endothelin-l, which cause vasoconstriction [212]. Its ability to block platelet aggregation [93] and leukocyte adhesion[94] helps to prevent microvascular stasis and thrombosis [213]. Another potential concern is the effect of N synthase inhibition on host defense caused by its antimicrobial activity [213] an im~unomodulatingeffects [209]. Nitric oxide synthase inhibitors have been used to treat hypotensionin patients with sepsis [214,215], but although alteration in mean arterial blood pressure was seen, no beneficial effects on clinical outcome, including survival rate, have been found in either animal or human investigations. Lorente et al. [215] described the hemodynamic effects of N-nitro-L-arginine, a NO synthase inhibitor, andL-arginine in 15 patients with sepsis syndrome. Compared with baseline values, N-nitro+ arginine increased meanarterial pressure, central venous pressure, pulmonary artery occlusion pressure, systemic vascular resistance index, and pulmonary vascular reowever, the drug also decreasedheart rate, cardiac index, and oxygen delivery index. Infusion of L-arginine appeared to reverse these effects. L-Arginine given alone to 7 patients produced decreases in mean arterial pressure, systemic vascular resistance index, and pulmonary vascular resistance index and increases incardiacindex,ox delivery index,and oxygenconsumption index. Theauthors concluded that plays a role in. theregulation ofsystemic and pulmonary vascular tone in patients with sepsis syndrome and suggested that NO synthase inhibitors and possibly L-arginine may be useful in treating patients with sepsis syndrome. These findings support the hypothesis that excess production of NO is responsible, at least in part, for the vasodilatation of septic shock and that N-nitro-L-arginine is able to block this excess production in a reversible fashion and may have potential benefit in treating the vasodilatation and preload abnormalities that are characteristicof septic shockin humans. Up to50% of patients who die of sepsis succumb to a h cardiac output form of hypotension that is refractory to vasopressors [216]. wever, Lorente et al.were appropriatelycautious in not drawing any conclusion as to whether NO production should be blocke mented in sepsis. The antivasodilatory effectof N-nitro-L-arginine infusion in their patients was accompanied by decreases in cardiac index and oxygen delivery; these effects of nitric synthase inhibitors have been observed in other studies [21l]. The

potential impact of this effect on the outcome ofseptic shock cannot be discerne n some animal models, inhibition of harmful. Administration of ~-methyl-L-argi inhibitor, to anesthetized rats and dogsincr decreased renal blood flow [218]. In model

rats caused cardiovascular c of NO synthase may be undesirable. The available inhibitors affect both the inducible an thases and may interfere with the homeostatic functions

o

argini~einhibitor appears to be somewha

induced isoform ofthe enzyme are likely to be developedand will possibly provide a powerful new means of treating septic shock andits many associated complications.

latel let-activating iator factor pathogenesis of sepsis [2 endot~elialcells, platelets, 1 release of inflammatory me sepsis [222,225] Concentr [226]. In a phase 11 study, however, infus cantly reduce mortality rate[227].

involved in the y of cells, including

m

nist did not

signifi-

Altho~gh the use of mediator antagonists for the treatmentof septic shock is based on a strong scientific ratio~ale, nonew therapy has shownclinical efficacy. mental evidence fro trials against antiendotoxin antibo clusive at best, and A-1A trials indicated that this ag may even be harmful to non-gram-negative bacteremic patien factor and IL-1 appear to be critical components in the systemic inflammatory

at leads toseptic shock, and in animalmodels, anti-TNF and pies have generally beeneffective; however, in studies in humans the ies have been inconclusive, and results of one study seemed to be associated with harmful effects in critically ill septic patients[ 1951. of the relative roles of infection, the systemic inflamorgan dysfunction is a prere~uisite for the design of clinical ate the host in~ammatoryresponse in a beneficial fashion. moreaccurate clinical andlaboratorydictorsare needed to identify whomaybenefit from any given treatm strategy, but the identification ly to benefit from new treatments of septic shock is an uch conditions as shock and organ dysfunction have been t patient pop~lations, but theresults have been i~consissuggest that the most severely ill patients derive the greatest benefit from these agents, e.g seemed to find septic shock an important criterion for defining gro a to benefit, and in the phase 111 , a predicted mortality rate based on weighted clinical factors found a group most likely to benefit [228]. owever, whether these determinants are based on differences in trial design or pro rties of the specific antagonists remains to be etermined. oratory markers may be more useful than clinical identifiers to select e more likely to benefit these therapies; e.g. ,endotoxin or TNF concentrations may need to be measur rior to the administration of therapeutic g and duration of administration also need to be more clearly ises of the therapeutic strategy itself may be flawed. Targeting of a single toxin, e.g., ay e not be a viable strategyfortreating a complex inflammatory respo gram-negative e bacteria. The strategy of inhibiting the host inflammat may not be beneficial because immune cells and play both pathogenic and protectiveroles. The use of combination therealing, as there arephysiologically many mediators acting in concert, but pressive processes of one or more antagonists may have a negative bacterial clearanc on effect of a combination of human a dimeric TNF 0 ~ 0 model of~ ~ s sepsis in IL-lra and gent alone conferred some

a1 with a microbial challenge. e septic response

ur knowledge of the complex timing of mediatorrelease and balance during

3 syndrome at this stage.At present the immunotherapyof sepsis remains an exciting, if elusive, goal.

1. RP Wenzel. The mortality of hospital acquired blood stream infections: Meed for a new vital statistic? J Epidemiol 17:225-231, 1988. 2. Members of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: Definitions for sepsis and organ failure and guidelines for theuse of innovative therapiesin sepsis. Crit Care Med 20: 864-874, 1992. 3. WA Knaus,EADraper,DPWagner, JE Zimmerman.Prognosisinacuteorgansystem failure. Ann Surg202:685-693, 1985. 4. PC Hebert, AJ Drummond, J Singer, GR Bernard, JA Russell.Asimplemultiple systemorgan failure scoring system predicts mortality of patients who have sepsis syndrome. Chest 104:230-235, 1993. 5. JE Zimmerman, WA Knaus, X Sun, DP Wagner. Severity strati~cationand outcome prediction for multisystem organ failure and dysfunction. World J Surg 20:401-405, 1996. 6. MR Pinsky, JL Vincent, J Deviere, M Alegre, RJ Kahn, E Dupont. Serum cytokine levels in human septic shock: Relationto multiple system organ failure and mortality. Chest 103:565-575, 1993. 7. RC Hock, R Rodriguez, T Manning. Effects of accidental trauma of cytokine and endotoxin production. Crit Care Med 839-845, 1993. 8. DD Tran, MA Cuesta, AJ Schneider. Prevalence and prediction of multiple organ system failure and mortalityin acute pancreatitis. J Crit Care 8:145-153, 1993. 9, X Guirao, SF Lowry. Biologic control of injury and in~ammation:Much more than too little ortoo late. World J Surg 20:437-447, 1996. 10. MP Glauser, G Zanetti, JD Baumgartner, J Cohen. Septic shock: Pathogenesis. Lancet 338:732-739, 1991. 11. G Schlag, H Redl. Mediatorsof injury and inflammation. World JSurg 20:406-410, 1996. 12. RC Bone. Toward a theory regarding the pathogenesisof the systemic inflammatory response syndrome: What we do and do not know about cytokine regulation. Crit Care Med 24:163-172, 1996. 13. Selective Decontamination of the Digestive Tract Trialists' Collaborative Group metaanalysis of randomised controlled trials of selective deco~ta~ination of the digestive tract. Br Med J 307525-532, 1993. 14. HWolochow, GJ Hildebrand, C Lammanna.Translocation of micro-organisms across the intestinal wall in rats: Effect of microbial size and concentration. J Infect Dis 116523-525, 1996. 15. GM Swank, EA Deitch. Roleof the gut in multiple organ failure: Bacterial translocation and permeability changes. World J Surg 20:411-417, 1996. 16. AM Munster, M Smith, M Meek, C Dickerson, RA Winchurch. Translocation: Incidental phenomenon or true pathology? Ann Surg 218:321-326, 1993. 17. S Bahrami, G Schlag, YM Yao, H Redl. Significance of translocation/endotoxin in the development of systemic sepsis following trauma and/or hemorrhage. Prog Clin S 392~197-208,1995. 18. W Ertel, JP Kremer, J Kenney, et al. Down-regulation of proinflammatory cytokine release in whole blood from septic patients. Blood85: 1341- 1347, 1995.

19. 20. 21 22. 23. 24. 25* 26. 27.

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218. CE Walder, C Thiemermann, JR Vane. The involvement of endothelium-derived relaxing factor in the regulation of renal cortical bloodflow in the rat. Br J Pharmacol 102:967-973,1991. NK Boughton-Smith. Role of nitric oxide in maintaining 219. xin-induced acute intestinal damage in the rat. Br J Pharmacol 101:815-820, 1990. oncada. The roleof nitric oxide in endotoxin shock: Effects 220. of N-monomethyl-L-arginine. J Cardiovasc Pharmacol20 (Suppl 12):S132-134, 1992. 221. SS Gross, DJ Stuehr, K Alsaka, et al. Macrophage and endothelial cell nitric oxide ective inhibition by N-aminoarginine, N-nitroarginine and Nm Biophys Res Commun 170:96-103, 1990. -Braquet, RH Bourgain, F Bussolini, D Hosford. PAR/cyto222. kinesautoregulatedfeedbacknetworksinmicrovascularinjury:Consequencesin shock, ischemia and graft rejection. J LipidMed 1:75-112, 1989. 223. M Sanchez-Crespo, S Fernandez-Gallardo. Pharmacological modulation of PAF: A therapeutic approachto endotoxin shock. J LipidMed 4:127-143, 1991. 224. 'naut, A Tenaillon, Y Le Tulzo, et al. Platelet activating factor receptor antago52021 inthetreatmentofsevere A randomiseddouble-blindplacebod, multicenterclinical trial. Crit ed22:1720-1728,1994. 225. SW Chang, CO Feddersen,PMHenson,elkel.Plateletactivatingfactormediates hemodynamic changes and lunginjury in endotoxin-treated rats. J Clin Invest 79: 226.

S Fernandez-Gallardo, MA Gijoin, M Sanchez-Crespo. Occupancy rs for platelet-activating factor in patients with septicemia. J Clin Invest 83: 1733-1738, 1989. 227. CJ Fisher, Y Zheng.Potentialstrategies for inflammatory mediator manipulation: Retrospect and prospect. World J Surg 20:447-453, 1996. 228. CJ Fisher, JFA Dhainaut, SM Opal, et al. Recombinant human interleukin-l receptor antagonist in the treatment of patients with sepsis syndrome. JAMA271:1836-1843, 1994. 229. Opal, AS Cross, JW Jhung, et al. Potential hazardsof combination immunotherapy in the treatmentof experimental septic shock. J Infect Dis 173: 1415-1421, 1996.

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~niversityof Cal~ornia-LosAngeles Schoolof ~ e d i c i n e , Los Angeles, ~ a l ~ o r n i a

The aberrant expression and/or mutation of various genes is known to play a central role in the development of human cancers. This aberrant gene expression can includeoncogeneoverexpression,amplification, and/or mutation; dysfunction antioncogene expression or the functional subversion of antioncogenes by viral encodedproteins;as well as the inappropriate expressionofgrowthfactors or cytokines and their receptors. The host environment also can greatly affect tumor development and growth. For instance, the host immune response to tumorcells, as well as other host responses, such as neoangiogenesis or nonspecific responses to tissue injury, can affect tumor growth and development and the eventual clinical outcome resulting from tumor growth. Various cytokines have been seen to play important roles in the development and growth of cancer [1-31. Interleukin 6 (IL-6)is a cytokine that has the potential to modulate the development! growth, and/or progression of several types of human cancer. It is a pleiotropic factor, with an unusually wide range of biological effects, which is produced by many types of cells, including many human tumor cells [4-71, and which may play a role in the pathogenesis of various forms of human cancer. In fact, IL-6 has been seen to act as an autocrine/paracrine growth and viability promoting factor for several types of human tumors, including multiple myeloma[8,9],ovariancancer[3],acquiredimmunodeficiency syn~r~meaposi’s sarcoma (AI S-ICs)[10-121, and renal cancer [ 131. The biological effects of IL-6 that may potentially promote tumor cell growth or development include the direct enhancement of tumor growth, either by acting as a growth factor or by enhancing tumor cell viability; the subversion of antitumor immune responses; and the modificationof the host environment (induction of the acute phase response, cachexia, and neoangiogenesis). Therefore, IL-6 may affect tumor growth directly, by acting as a paracrine or autocrine growth factor or by preventing tumor cell apoptosis, or indirectly, by modifying the host environment in a manner that would enhance or suppress tumor growth.

Although many of thebiological activities of IL-6 may result in the enhancement of the development and growth of human tumors, this cyto of potential use as a therapeutic agent for the treatment of hum because of its immune-enhancing effects or because of its ability to promote hematopoiesis [5-7,141. owever, the clinical usefulness of IL-6 as an antitumor agent has not been estab hed. The biological activities of IL-6, as well as the potential of several human role of thiscytokineinpromotingthegrowtdevelopment cancers,andinformationon in vivo levels o 6 in peoplewithcancer will be discussed later, focusing on recent studiescarut in our group examining IL-6 in ovarian cancer and in AI S-associated malignancies (lymphoma and

ytokine, which in ulatory factor 2 macytoma growth factor ( F), monocytegranulocyte fferentiation factor [4-71. his cytokine was described, prior to its being called I dinarily pleiotropic nature and its wide range of biologica biological activities that have been ascribedto IL-6 ation, support of plasmacytoma and myeloma gro receptorexpression,induction of modifferentiation,induction ofcytotoxic T cells, enhancementofnatural killer ( cell activity, induction reactants and stimulation of hepato induction of neuronal uction of mesangial cell growth, induction of keratinocyte growth, inhibition apoptosisorprogrammed cell dh,andsupport of hematopoieticstem cell differe~tiation[4-7,15,16], Clearly, -6 is a pleiotropic factor, with both growth and differentiation inducing properties. S been cloned and sequenced, and its S glycoprotein, consisting of 212 amino sequence deduced from cloned IL-6 copy deoxyribonucleic acid(c is differentiallymodifiedby glycosylation, esulting in thera weights seen for this cytokine. The gene forI -6 in humans is on chromosome 7; it consists of 5 exons and 4 introns, with the same exon/in on pattern as thegene for [4”7]. here is someamino acid granulocytecolony-stimulatingfactor(G-CSF) sequence similarity between IL-6 and G-CSF, suggesti . . . in the tertiary structure of these molecules.Also,

inding to asecondmolecule, a 130-k aprotein(gp130,or eved to be the primary signal-transduci molecule in the IL-6 30’gp130‘ is expressed constitutively on 6 is controlled by regulating the expression element of the rece tor complex. For example, the expreselement of the XL-6 receptor is regulated on cells, with activated increased numbe molecule and responsiveness to XL-6, 01. Interestingly, the soluble form of appears to enhance, than block or inhibit, IL-6-mediated an interact with IL-6 and cell-surface sduction [6,7,18,19]. ces in understanding XL4 receptor130 has been seen to be a common ine receptor complex, involved not the receptor complexes for several and IL-ll. The first stepin the ’IL-6R’, and C ~ l 3 0 ‘ g p l 3 0 ’ , and the homodimerizationof gp130[7]. Following this, theresulting gp130 homodimer interacts with and activates intracytoplasmic tyrosine kinases, the J kinases, which tyrosine-phosphorylate gp130, resulting in the attraction proteins. ~ T A T 3 ntyrosine-phosphorylatedbythe JA kinases, leading tothe formation of STdimers, which interactwithIL-6responseelements in the regulatoryregionarious IL-6 responsivegenes. Interleukin 6 can be produced by several types of cells, including T lymphocytes, monocyte/mophages,fibroblasts,epithelial cells, endometrial cells, and various tumor cells 71. Gene expression of IL-6 can be induced by a wide range of stimuli, includin ines (IL-l, tumor necrosis factor [TNF], and plateF]); microbial products, including lipopolysacchaS , including thehuman immunodeficiency virus 1 env protein, a virally encoded envelope protein ne fusion [4-7,211. The transcriptional regulation of IL-6geneexpressionhasbeenelucidated in recent work [4-7,221. The IL-6 promoter regioncontains several transcriptioncontrol elements with. potential activity: glucocorticoid-r a E), AP-1 binding an site, a serum-responsive element sine monophosphate- (CA ( responsive element, N an iresponse element sequence recognized by NF-IL6, a nuclear transcription factor protein has some homology with the fos and myc oncogene products, and interacts with the NF-IL6-binding motif (AGATTGCAC n the XL-6 gene regulatory region, inducing IL-6 gene expression. Normally, L6 is not expressed, but its expression is induced by exposure of IL-6-producing cells to various in IL-l , rapidly and transiently region, which is located wit -binding site; all may Various cytokines, in

production by monocytes[4-71. In recent work, we found that whereas exposure of S-activated human m o n o c ~ i ccell lines to both IL-4 and IL-10led to L-6 productionandgene expression,exposure IL-10 resultedin the adation of IL-6 messenger ribonucleic acid ( NA), and exposure to transcription of the IL-6 gene by affectin e, IL-4 and IL-10a p ~ e a r tinhibit o IL-6 production in monocytes by different mechanisms.

Interleukin 6 is produced by various types of human tumor cells. In an early study, abib~adeh andcoworkers used imm~nohistochemicalanalysis to detect IL-6 in sections of various types of tumor tissue and found that most human tumor tissues tested stained positive for IL-6, including primary squamous cell carcinomas; adenocarcinomas of mammary, colonic, ovarian, and endometrial origin; soft tissue includingleiomyosarcomaandneurofibrosarcoma;and Hodgkin’s and dgkin’s lymphomas [24]. However, this type of analysis could not distintween IL-6-producing cells and cells that displayed immunoreactive IL-6 taken up from their surroundings. In subsequent work, IL-6 was seen to be produced by cardiac myxoma tumorcells [25], prostate carcinoma cells 1261, epidermal cells andepidermoidcarcinoma cell lines [27], bladdercarcinoma cells [28,29], mesothelioma cell lines [30], gastric cancer cell lines [3l], malignant breast tissue [32], lung cancer cell lines 133,341, renal cancer cells [35], cervical ca melanoma cells [37], ovarian cancer cells [38], ~ I D S - ~ cells S [l l], ease 1391, andnonin’s lymphoma [40-421. any of these tumor cells also wereseen to express -6 receptor [l 1,29,34,43]. we foundthatmanyovariancancer cells ( S -3, C A O ~ - 3 as , well as various other ovarian cancerlines generated in our 0 cancers) produce ry) of epithelial origin (which constitute 9 ~ of~ovarian and secrete IL-6 [3910,38,439~]. Ovarian cancer cell lines of nonepithelial origin -1 and 222) did not secrete IL-6, although c ~ o p l a s m i cIL-6 was detected in -1,as well as in all epithelialovarian cancercell lines tested [38]. ~ r i m a r yovarian tumor cell cultures also produced substantial levels of IL-6, with primary isolates of ovarian tumorcells displaying intracellular IL-6by immunoperoxidase staining[38]. The IL-6 produced by ovarian cancercells was examined by radioimmunoprecipitation and found to correspond in molecular weight to monocyte/lymphoid-derived IL-6 [38], Interleukin 6 m R ~ A expression indicated that most epithelial ovarian tumor cell lines display detectable levels of IL-6 gene expression [38], and they also were seen to express CD1 26‘1L6-R , by Northern blot analysis 1431. Several cytokines (y interferon [IFN-y], IL-1, and TNF) were seen to up-regulate IL-6 production by these ovarian tumor cell lines 1383. However, work by others has shown that IL-6 ~roductionby ovarian cancercells is not dependent on the productionof IL-1 rimary cultures of normal human ovarian epithelium also were seen to produce IL-6[46]. These results indicatethat ovarian cancercells, including established ovarian cancercell lines, primary tumor isolates, and normal ovarian epithelial cells, produce ~iologically active IL-6 that is indistinguishable from theIL-6 produced by

peripheral blood mononuclear cells and express the 80 (CD126), the IL-6 receptor complexelement that is res tivity to this cytokine. If IL-6 is potentially to act as an aut cancer cells,cthese ~roducingdetectable quantities of creted IL-6 and IL-6 tor. Therefore, the observation that ovar compatible with the possibility that IL-6 acts as a growth/differentiation/viability factor forthese cells. In other studies, we found that variouscell lines derived from AID mens secreted substantialamounts of IL-6, contained intracellular IL-6 b peroxidase staining9 and displ els of IL-6 and ~Dl26’IL-6 [l 1,121. For example, althou 0.05 ng IL-6/ml) was det complete culture medium used passage supernatants the of this high levels of IL-6 (50 7.7immunosorbent ng measu assay IL-6/ml), (ELISA). Intrac~llularI monoclonal anti-IL-6 a cells wereposit strongly production, IL-6 for W isotype n immunocontrol globulin staining mon C (IgG) [l 1,121. Also, duced IL-6 the ically sinceactive, the endent ndiluted supernatant cell line: when the biological activity of the

*

13 unitdm1 IL-6 AIDS-KS cell lines tested expressed high levels of IL-6whereasperipheral blood m o n o ~ u c l stimulated A when IL-6, total RNA was obtained from freshly isolated KS tissue and from uninvolved andbrain specimens. S ng IL-6 mRNA expression wa S involved skin [11,12]. so, by using a monoclonal ant im~unoperoxidasestaining, IL-6 was detected in sections of AI to a lesser degree,int entnormal-appearingepithelial immunoprecipitation o -KS culturesupernatants9usingbothmon polyclonal anti-IL-6, resulted in the visualization of a 20- to 25-kDa s~onding to IL-6 [11 121. Subsequent work by others has confirmed th cells characteristically produce IL-6 [47]. When the IL-6 receptor (C126) issimultaneously expressed in IL-6 producing autocrine growth factor. Therefore, we D§-KS cell lines: these cells were seen to A [l 1,121. Similarly, substantial IL-6 isolated from a person with AI Aexpression was seeninuninvolv brain, or spleen taken from the sameindividual [l 1,121. o be expressed in Interleukin 6 and receptor also have malignancies, including dgkin’s disease dgkin’s [39] lymphom

els of this cytokine, as d mined by flow cytometr observations raise the p tocrine/paracrine factors for lymphoma) cells.

6 ~ o k i n e and s growth factors have been seen to play important roles in the development and growthof cancer [l-3,101.In fact,it hasbeen proposed that somecancers, suchasepithelial ovarian cancer,mayrepresent acytokine-propelled disease [50,51].Interleukin 6 has been shown to stimulate tumor cell growth and to act as an autocrine or paracrine growth factor in several typesof human tumors. Itacts as an autocrine/paracrine growth factor for human multiple myeloma cells [g],renal cancer cells [131, Epstein- rr virus transformed human T cell lymphoma cells [5] also acts as a costimulator genicity of acute myelogenous leukemia cell blasts [53]. I e were seen to develop plasmacytomas rapidly171. er observations suggest that IL-6 can play a role as a growth factor for ovariancancer cells. U and colleaguesobserved thatculturesupernatantsfrom activated human monocytes, which support ovarian tumor cell growth, contained significant amounts of IL-6 [54].Furthermore, anti-IL-6 serum inhibitedpart of this monocyte-produced growth-supporting activity. actas a paracrinegrowthfactorforovarianc examined the possibility that IL-6 acts an au as factor for ovarian cancer cells by inhibiting endogenous IL-6 gene oligonucleotides. Exposure of ovarian cancer ce 06-436) to various concentrations of a singles nucleotidespecific forasequence in thesec0 resultedindecreas IL-6 produ~tionand in a > $ 0 ~ 0 - $ 5 ~ inhibition 0 in cellular proliferation [43]. wever, theaddition of exogenousIL-6failed to restore the proliferation of the antisense-treated cells. Also, antibodies to IL-6 did not consistently inhibit cell growth, nor did recombinant ILconcentrations. These results suggest that exogenou the proliferation of ovarian cancer cells, although endog needed for optimalcell growth. , whichis amember of thefamily Variouscytokines,includingIL-6 and of IL-&like cytokines [6,7,18,19], have been seen to act a growth AIDS-KS r cells [ 11,12,55].In early studies sure of cells t o relatively high levels of human rec modest, but significant, increase in proliferation:30 units/m significantly increased the proliferation (>220~0)of the N [ 1 l]. The additionof rabbit anti-IL-6 serum partially suppressed exogenous hrIL-6-

llular proliferation:

150, inhibitionof endo~enous eotide, resulted in the inhibitio

herpesvirus 8), encodes a viral version of

~ n t e r l e u ~6i na

ests that activated, tumor-infiltrating immune cells, or other IL-6-producing cells, may enhance tumor growth by roducing IL-6, as well as other tumor * cytokines. Forexample,recentworkindicatesthatactivated varian tumor cell growth-enhancingfactors [M], and that rophagesisolated from h u ~ a novariancarcinomas release Part of this monocyte-p duced, ovarian tumor cell growthwas seen to be due to EL ition of a n t i - I ~ - 6serum e enhancementof tumor

ther biological activities of IL- su~gest that it may act to support tumor growth. ce a wide range of biologi responses in the host, including, but not the induction of the acute se response and cachexia [40] and angio. ~nterleukin 6 may act to mote (or inhibit) tumor growth in other ways. Interestingly,IL-6 been seen to increase cell motility anddecrease cellcell association in the T reast carcinoma cell lines and to lead to phenotypic changes reast cancer in cells are that IL-6 treated have fewer adherens-type cell junctions [59]. These IL-6-induced cellular changes le in tumor invasiveness and in the ability of tumor cells to metastasize. nterleukin 6 also has been seen to suppress programmed cell death, or apopcell h bridoma cells [15], as well as p53 tumor supprespoptosis in myelo leukemia cells [61]. In a recent report, Kawano showed that bon arrow stromal cells supported the differentiation of earlyplasma cells into mature plas cellsby se~retingIL- , whichprevented plasma cell apoptosis [62] Therefore, -6 production by tumor cells, i ~otentiallymay lead to increa immune cells, or surrounding stromal pressing the entry ofcells into pro rammed cell death. n recent studies, we IL-6 ha t may act as a viability-promoting factor for ovarian cancer cells. studies indicate that various tumor cells can be protectedfromnatural killer ( iated killing by IL-6 ( et al., unpublished observations). Cells ( C ~ O V - 3ovarian cancer cells) treated with dexamethasone, which results in greatly decreased IL-6 production [63], showed greatlyincreasedsensitivity to cell-mediated lysis, whereasaddition of exogembinant IL-6 to dexamethasone-treated cells restored resistance to N These p r e ~ i m i ~ a rresults y suggest that IL-6 (endogenously produced or exo~enous) can protect tumor cells from N -mediated killing. Inother studies, IL-6-producing ovarian cancer cells with IL-6 antisense oligonucl~oin the entry of these cells into a programmed cell death pathway: ex osure of the OC-194 or ~ C - ~ ovarian 36 cancercell lines to various ILe preparationsresulted in decreased endogenous fragmentation, with apoptosis partially prevent L-6 to the IL-6antisense oligonucleotide-treate nucleotide-induced a~optosisalso could be inhibited by acti~omye

Inother recentwork, we notedthatIcan

act as an ~utocrine/

er endogenous or exogenous Studies to determine the mechanism of viability are currently underway in our laboratory.

oma havebeenobserved to have 1 as hypergammaglo~ulinemia and

since IL-6 has been shownto be

ovariancancerpatieithmacroscopic disease, butinonly13% of thosewith of healthy control d o ~ o [69]. ~s mean serum micro§copic disease IL-6 concentration i f ovarian cancer patients with m (n = 21) was ~ . 2 6 nitdml, with§erumIL-6 levels reachingveryhigh levels (> l unit/ml) inpatients; some S nors were signifistudies have con771. Also, several s

1741; thisfinding is larly i ~ t e r ~ s t i n gsince , IL-6 is a potentstimulator megakaryocytopoiesis. In subse~uentstudies, we exami~edthe in vivo biological effects correlating levels of IL-6, acute phase rotei ins (C’-reactive protein [C

of

globin, and a2 macroglob ascitic fluid or serum (J. elevated levels ascites, when nonmalignant gynecological IL-6 levels (6.5 A 5.2 vs. increased levels of serum

CO

and in control subjects ( P of IL-6 present in patients wi of acute phase proteins and I ovarian cancer arebiological factor, inducing the differ potent inducer for the pro between elevated serum C reported by others [72]. In another recent stu obtained from women with

who did not have ovarian seen in ascites from the g lished observations). The

produce IL-6 (C. Casey ian cancer cells in vitro completely inhibited IL

of IL-l0 as a potential

significantly elevated from noncancer pati the most frequently IL-6, as well as TNF

increased levels of spontaneous IL-6 ~roduction,

relations have been

g that this may be a relatively early immune system change in

In recent work, we measured serum IL-6 levels in people in whom AIDSa developed and found that IL-6 levels ymphoma than in subjects matched on without l y m p h o ~ aeven , though both ofthese g had significantl~elevated levels of serum IL-6 when compared to those of

serone~ative control subjects The mean level of §erum IL l y m p h o ~ adevelopedwas 12 2 pg/ml, wh

a l t h o u ~ helevated serum TL-6 levels w

system molecules, in-

correlating to response to therapy.

ive control donors. ~pecimens were archival ; serum samples are collected from the study

defining opportunistic infections [ 1041. ~nfortunately’ the si’s sarcoma groupsin this latter study were not mat aposi9s sarcoma group displayeda significantly lower ection were correlated inversely with 11s [1041. Since serum IL den et al., unpublished observation), 4 number in other studies ave elevated serum IL-6 levels, when r whether people wit lar degree of immunesystem impaircompared to others with A ment. Various studies have idly implantation produced after of mice with various types of tumor cells [105-10~]. It vels in these tumor-implanted mice is n mor cells, but ratherto IL-6 productio detectable levels of serum IL-6 have been seento be induced in humans by in vivo ne study indicates that although both ovarian a1 cells produce IL-6 in ovarian cancer, thelevel the mesothelial cells is much greater, suggesting that these nent source of IL-6 in ovarian cancer-related ascites [ 1091. kept in mind that the high levels of IL-6 associated with many human cancers may be reactive d reflect the host response to these malignancies, rather than the production of 6 by the tumor cells.

The information presented here may be tak L-6 acts predominantly as a tumor growth enhancing factor. the overall role of IL-6 in the pathogenesis IL-6 was shown to act as an IL-l-induced, autocrine growthsuppressive factor for malignant melanoma cells [l lo]; in some instances, IL-l-induced growth suppression was found to be mediated via the induction of IL-6 production. Interleukin 6 has been seen to suppress the growth of human breast cancer cells and leu~emia/ nes [l 1l] and tolead to thedecreased appearance of tumor nodules with a transplantable9 chemically induced murine tumor [ 1123. Also, IL-6 has effects on some uman immune functions that may either enhance or inhibit antitumor immuneres nses. For instance, althoughIL-6 has been shown to enhance the growth of tumor filtrating lymphocytes isolated from human renal cell cancer [ 1131 modestly, it also has been seen to impair N cell function and to increase human lymphoblastoid cell t~morigenicityin nude mice [ 1141. Therefore, XL-6 can potentially have both direct tumor cell growth-enhancing and growthsuppressingeffects andalso mayhaveprofound effects onthe activation of the immune system in the tumor-bearing host, leading to either the augmentation or suppression of antitumor responses. In anycase, it is clear that the dysregulationof various biological and molecular factors, including oncogene overexpression, mutation or loss of antioncogenes’ growth factor and cytokine stimulation, and modification of the host environment and host antitumor immune response, canplay important roles in the development and progression of cancer. nderstanding the specific mechanisms involved in the development and growth of different human cancers will allow op~ortunities for

,

the rational design of effective antitumor t r e a t m e ~modalities, t inclu~ingtreatment aimed at inhibiting cytokine productionor responsiveness.

The author expresses his sincerest gratitude to ongoing collaboration in studies on IL-6 and hum

tions to the workpres

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Institute of ~eurology,London, ~ n g l a n d

asis is crucial for the health an survival of an organism. egulatory mechanisms that allow the bodyto adapt to exterances through interactions involving the nervous, endocrinep ve evolved. The fundamental tasks of the immune system are todistinguish “self” from 6‘nonself99 and to respond appropriately to any encounters with these antigens [l]. It is now accepted that the immune system does not function indepe~dentlyin coping with the potentially vast array of “foreign” and “self’ stimuli but communicates with cells of the nervous and endocrine systems; uality prevails in the use of neurotransmitters9 hormonesp m displays. In turn, the nervous and endocrinesystems can e immune response by both neural and humoral routes, including the release of immunos ressive glucocorticoids from the adrenal glands and the direct autonomic inne ation and modification of immune cells within their parent organ9 such as the thymus and spleen. sychological stressors, registered in higher centers of the brain, can also alter neuroendocrine output and modulate immune reactivity directly. Thus, numerous stressful events have been shown to cause immunosuppression in e~perimental odels and precede the onset of autoimmunedisease in the clinical setting [2,3]. The importanceof the hormonal milieu in determining immune statusis highlighted in the observat ibit an increased immune response and a greater prevalence of ases systemic lupus erythematosus (SLE) (female to male rati (19 : rheumatoid arthritis (3 : l), and multi le sclerosis (2 : 1). Further, the onset, clinical course, and severity of autoimm ses can be in~uencedbychanges ingonadalhormone levels suchas occur , duringpregnancy9postpartum,andduringmenopause.Experimental at animal models have also confirmed the importance of sex steroids to the developeases: e.gp in a mousemodelof SLE,fema opfatal ess they aretreated with theandrogendihy fiterone, whereascastration of prepubertalmalesanreatmentwithestrogen result in a similar mortalitypattern to that of females . Althoughsexsteroidsare clearly

important in determining susceptibility to autoimmunedisease, recent evidence suggests that a number of autoimmune susceptible mouse, rat, an chicken strains may also have a defect inthe reg f corticosteroneproduction,Thus,blunted - and cytokine-stimulated corticosterone seadrenocorticotropic hormone cretion, a decline in plasma corticosterone levels with age, and a decrease in free plasma corticosterone levels due to the elevation o orticosterone binding globulin are seen in comparison to those of CO he Lewis rat dysregulation of hypothalamic-pituitary-adrenal the ( anifest in the susceptibility to experime~tal autoimm~ne encephalomyelitis ( E ~ E )a9 model of the human linating disease multiple sclerosis. In this chapter the interactionof neuroen and immune systems is explored in relation to autoimmun~ disease, with particular reference to the roleof the HPA axis in modulating EAE.

The neuroendocrine and immune systems share a variety of humoral mediators and possess functional receptors for these agents. ~ i t h i nthe thymus a large panel of uitaryhormones, peptides, andtheir rec in addition to S, are expressed; there t epithelial cell function and t h y m o c ~ edifferentiation [$"l]. constitutively express uce, after stimulationwith lipopolysacch peptides historically associ otropin releasing factor en~orphin[IS]; receptors (or receptor messenger ribonucleic acids hormones,peptides,andneurotransmittersareexpresseddifferent lymphoc~esand macrophages [l6,17]. ~ l t h o u g hthe quantities ofpeptides synthesized and released are unli~ely tohave syste activation), they have localparacrin~effects. ety of immunosuppressive and immune enha and immune cell on which they act [ 18,191, Thus, macrophage phagoc~osisis [20],and agents thatblock the activity of endorphin exacerverse a plethora of immunosuppressive actions of the o [22]. Conversely, the localsynthesis of C F at sites of peripheral in e~ in rats and rhe as synovia^ joints inexperimentally i ~ d u c arthritis in humans suggests a deleterious paracrine effect[23-25]. ther endocrine organsin which n d glands, testes, and ovaries, centa produces increasing q u ~ t i t i e so ntri~ute to the increased corticosteroi~levels observed and modulate uteroplacental blood flow [ central nervous system (CNS) nuclei outside the paraventricular nucleus alamus CRF is found [28], and receptors for the peptide are located in high bellar cortex, neocorte~,b r ~ n s t e the putative role of

monokines such as tumornecrosis factor (TNF), interleukin 1 (IL-l), and ILendotoxin challenge [34], and receptors for immune me are constitutively expressed throughouttheneuroendocrine tissues [35,36]. ioninhibitory fachat prevents the random movement of macrophages, is secretorygranulesasAcorticotropesoftheadenoF alsoantagonizestheammatoryeffectsofglucocorticoids on cytokine production [37], Functionally, neural and endocrine modificationsattributable to cytokinesincactivation of the HPA axis, inhibition of the hypothalamic-pituitary-gonadal G ) axis via inhibition of gonadotropin and sex steroid secretion 138-401, fever induction, and promotion of slow wave sleep complexneuroendocrine-immuneinteractionsar relationship of glucocorticoids and IL-1 within the

. Interleu~in1 was originally described as a mediator of immune action, although it has clearly been shown to modulate the activity of the duced bya variety of cell types, partic~larlymacrophage stimulus [43], IL-1 stimulates the release of all hormonal components of the axis, although the most potent S are seen at the level of the hypothalamus on corticotropin releasing factor ) release [44,45]. A. direct effect of IL-l is onsidered that IL-1 does not cross thebloodnt amounts [46]. Entry to the CNS may take place nism E471 or through regions where the barrier is permeable, such as the circumventricular organs[48,49]. To this end IL-l@injected directly into median the emine F release into portal thebloodstream, increases circulating However, ACIL-l-containing fibers f hypothalamus the [53] and IL-1 innervate paraventricular the n receptor immunoreactivity has been located on ntaining neurons therein 1541, althoughreceptorbindingstudiesindicateeas t thehippocampusand anterior pituitary gland containlarge numbers of IL-1 bindingsites, these are scarce in the hypothalamus[35,55]. ecent reports which demonstrate that both endotoxin and stress induceIL-lsynt is inthehypothalamus [56,57] suggest that direct action on the hypothalamus is possible. The major source of IL-1 in the CNS appearstobethe microglia, alsoproduce IL-6 and TNT; [SS], whereasvascuothelial cells express for both IL-16 and IL-16 converting enzyme owever, anadditionaorcytokine to braincommunicationthat eliminates the need for IL-16 to access the CNS has also been suggested; it involves the peripheral activation of subdiaphra~maticvagal afferents in response to locally released IL-16 within the peritoneum [601. Although the role of IL-1 in mediating neural-immune communication has been emphasized, IL-6, TNT;, and interferon-~ can also alter pituitary hormonerelease either directly or via the hypothalamus and hence are potential mediatorsof immunomodulation [61-63]. Perhaps a more tenable argument is that cytokines stimulate C through indirect mechanisms and pathways involving neurotransmitters and second

messengers, such prostaglandins ( on the effect of L and cytokines o activation supporta stimulatory role for the eic in vivo measurement, using 66push pull,’ canula production is elevated in the hypothalamus [ response to either IL-1, IL-69 or endotoxin. from endothelial cells of the organu and acting on interneurons to activate d as an IL-1-mediated tr the hippocampus, a site rich in IL-1 receptors, was blockedby IL-1 receptor antagonist [72] suggests thatinterme es are involved in thecytokine regulation of higher braincentersthatcontroA axis activation, The hypothalamic PVN receives dense noradrenergic innervation, and direct synaptic contacts en hdemonstrated betwe noradrenergic neurons the basis depletion of immunoreactive to and vasopressin [73]. ~eafferentation adrenergic pathways oni [76], anta and participate to thoughtare in endotoxin, probably and locus ceruleus)

ydroxytryptamine the of

oleacetic acid (5-

Centrally acting IL-1 can also modify NA turnover and the immune response in the periphery. Alterations in sympathetic nervous system(SNS) activity, a ~ p a r e nafter t electrical stimulation of the ventromedial hypothalamus [82] or administration of IL-1 [83], increase splenic NA turnover E841 and profoundly suppress the cellular immuneresponse [ss]. Thus, spleenlymphocytenatural killer cell activity, the response to ph~tohemagglutinin and IL-2 production, is reduced after intracerebroventricular (i.c.v.) IL-l injection [86]. Furthermore these effects are abolished by axotomy, ganglion blockade, or depletion of peripheral NA butpersist after adrenalectomy [85,87], indicating a SNS rather than an adrenal steroid/catecholaminemediated im~unosuppressiveeffect.

tressors is associated iated at both central and gonadal levels by cytokines, notably IL-1 , and neurotransmitters. At the level of the gonadsa number of cytokines, includingIL-1, TNF-a, and interferons a and y (IFN-aand IFN-y), have the capacity to inhibit gonadotropin-

estrogen release both in vitro and in vivo [$$,90,91] and folliclestimulating hormone (FS )-stimulatedgranulosa 192,931 possibly by decreasing the binding capacityof the luteinizing hormone ) receptor [90,9~]and inhibiting the formation of gonadotropin-stimulated clic adenosine monophos~hate(c ) [90,91]. The effect of IL-1 appearsto ptors for this cytok ptor antagonist can ogesterone secretion decrease of gonadot e inhibitory effect of cytokines on b rogesterone-induced ats, and the proestrous su appear to be mediated through central rather than ~ e r i p ~ e rpathways al since these effects are seen after i.c.v. but not peripheral administration of IL-1,IL-6, and

. administration of hypophyseal-portal blood [1021.

~ d r e n a lsteroids,corticosteronein the rat and cortisol in humans, have potent effects on the neural, endocrine, and immune systems [lo?]. Endogenous glucocorticoidsexhibit autoregulation of the axis whereby excessive steroidsecretion is ive-feedback mechanisms effective at neural and pituitary le

manifest in the anterior pituitary gland, secretion and synthesis [1121 e

secretion by macrophages [ 113,l1IL-2secretion and IL-2receptor(IL-2 expression on T l y m p h o c ~ e s(lymphoproliferation) [l 151. ~ i r c a d i a nand stressinduced levels of corticosteroids regulate the migratory effects of lymphocytes in vivo, through aninverse relationship between steroid concentration and lymphoc~e numbers [116,1171. Adrenalectomy reveals the immunosuppressive and antiinflammatoryeffectsofendogenoussterone,asevidenced by the uncheckedproection [ 118-1201 andinduction of a lymphoduction of IL-1 and TNF after proliferativeresponse and clin E innormallyresistantBrownNorwayrats [ 1211. Furthermore, glucocorticoids have potent inhibitory effectson produ inflammatory mediators, including prostaglandins and leukotrienes [1221, tion of eicosanoid production in certain immune cells, such as macrop realized in part thro gh the induction and redistri~ution of the phospholipase A2 inhibitory protein li ocortin 1 11231; the inhibitory action of de~amethasone on nitric oxide production is also mediated by lipocortin l [1241. ~ i s t r i b u t e dthroughout nervous and endocrinetissue [125], lipocortin 1is also implicated in the negative feedback effects of glucocorticoids on the hypothalamus and pituitarygland [125els of lipocortin 1 in the spinal cord and cerebellum coincide with a simultaneous rise in plasma corticotin 1 suppressesT cell activation response in to myelin basic prot reover, lipocortin 1 level is also ' reasedinwhite matter and plaque tissue from patients withmultiplesclerosis S) [ 1301 and the protein confers protection in experimental neurodegenerative

Not only is the female immune system more active than that of males but so too is the activity of the igher corticosteroid response to stress is seen in . In males, castration enhances the corticosterone ent therapy with 5~-dihydrotestosterone ( presses the response [132-1351. The hyperresponsiveness of the female

further inhibit sex hormone output at the level of the adenohypophysis and gonads. Centrally acting IL-l can also activate the sympathetic nervous system (SNS), whichhasperipheral immunosuppressiveeffects on spleenandthymic cells vianoradrenaline (NA) release, whereas the parasympathetic nervous system can relay peripheral immune signals to the CNS via the vagus nerve. AI, A2, noradrenergic cell groups; Ab, antibody; AP, area postrema;

terminalis;PGE,prostaglandin Et;PMN, polymorphonuclear cell; Prog,progesterone; PVN, paraventricular nucleus; RN, Raphe nucleus; SFO, subfornical organ; Test, testosterone; TNF, tumor necrosis factor; 5HT, serotonin; -ve, negative feedback.

rogens te§to§terone (T) an have also been §hewn to

ocyte number, decrease bone

lation of either arm

7 l , 1721, and rheu-

a hyporesponsive ammatory media-

equel of a primary defect at the

transduction mechanism in the

GE2 and c A ~ P whereas 9 these were elevat to a lesser degree in the either strain showed significant increases i hypothalamic NA release (unpublished observations). These data suggest that disruption production within the hypothalamus may indeed underlierethe response, at least to IL-lp, in the LEW rat; the absence of c IL-16 in bothratstrains implies thatsomeotherine(perhapsTNF) may mediate theincreased hypothalamic NArelease after Although defective hypothalamicfunctioning is ht to haveamodulatory role in several T cell-mediated a u t o i ~ m u n econditions, in cal cell wall-induced arthritis, and thyroiditis, it is the e activation, i.e., glucocorticoids that are pivotal in determi rats exhibit reduced basal and stressise ease-resistant F/344 and Piebaldthe spontaneous recovery phase seen after relapses in chronic EAE in the mouse (Bolton et al., personal communication) is associated with a se in circulating corticosterone concentration. After adrenalectomy, are rendered susceptible to EAE, whereas in the L rat disease onset is more rapidand severe; in bothstrainsafat lysis ensues [ 1761. thenormal disease course in PVG andrats,respe~tively9 administration of a suitablecorticosteegimen.TherecoveryphaseofEAE is associated with a shift in cytokine production ofcells in in~ammatorylesions from T helper 1 (Thl) type ( T N F - ~ and IN^-^) to antiinflammatory cytokines characteristic of Th2cells (IL-10 and IL-4) 11841, a shift also stimulated in vitro glucocortiby . It is therefore apparent thatsusceptibility to and spontaneousremission are determinedby the cort ortlcosterone hyperresponsive vulnera~le are rats in experimental models of rheumatoid arthritis [ 186,1871, in contrast to EAE, although the clinical manifestations of the disease are much less severe than those exhibited by LEV4 rats.Henceitseemsthat,althoughimportant, susceptibility to immunemediated diseases is not solely attributable to glucocorticoid hyposecretion. Corticosterone replacement, however, is effective in attenu g spontaneous autoimmune thyroiditisinthe OS chicken,amodel of human s~imoto9s thyroiditis [1681. The OS chicken is glucocorticoid-compromised by virtue of the overproductionof corticosterone-binding globulin (transcortin) and the absence of a steroid response to an immune challenge. This latter characteristic is thought to be of hypothalamic or ~ituitaryorigin as adrenal function is normal in thechicken [ 1681. Sex steroid hormones regulate the development of murine LE-like disease with androgens exerting protective effects and estrogens enhancing thedisease process. In human SLE a higher incidence of disease is seen in women, and exacerbation of disease s y ~ p t o m has s been reported duringpregnancy oral contraceptives [138,139,141,188].~heumatoidarthritis, S are also more prevalent in females than in males, yet unlike in SLE, or estrogen therapy appears to have a beneficial effect [137,139,189,190]. The elevation of circulating estrogen, pro~esterone, and corticosteroid levels during pregnancy leads to a suppression of cell-mediated immune responses [4, 1921, a decrease in Thl-type cytokines, anincrease in Th2-type cytokines [ 1 and an enhancement of humoral immunity. Initiation of this immunosuppressive ~

strategy is thought to prevent rejection of the histoincompatible fetus by the mother, an effect readily reversed by the corticosteroid/progesterone receptor antagonist and abortive agent ~ 3 $ 4 8 Thus ~ . pregnancy might be expected to improve those autoimmune diseas t are primarily T cell-mediated (i.e., RA, MS, and thyroiditis), whereasthoseprecipitatedby humoralagents,includingpolyclonal activation and increased secretion of immunoglobulins/immune complexes (i.e. , SLE), are madeworse [ 1971.

protection against autoimmune conditions afforded by glucoutable to their immunosuppressive andantiin~ammatoryqualiprotective mechanism initiated by glucocorticoids is apoptosis of immune cells [198]. Apoptosis is an active physiological process whereby a reactive cell undergoes a series of characteristic changes, including DNA degradation, resulting in cell death [199]. This “programmed cell death’’ occurs in target cells in response to or after thewithdrawal of a stimulus that maybe endogenously generatedor a humoraltr 001. T cell receptor-mediated apoptosisoccursduring theelimination of a ive, immature T-cells in thethymus [201], and, therefore, a defective apoptotic mechanism may lead to the development of autoimmuring the fetal and neonatal stage of development corticosteroidssyntheare thymic epithelial cells, wheretheyantagonizeT cell receptor-mediated apoptotic death [ 10,2021; however, adrenal derived ~lucocorticoids are potent mediators of t h y m o c ~ e poptosis [203]. Thus, although the glucocorticoid milieu may prevent the development of “self” T cells in the mature animal, this effect dep on the maturation state of the t h y m o c ~ eand other costimulatory molecules. ture T cells are resistant to glucocorticoid-induced apoptosis [204] until they have reupon the sensitivity to apoptosis increases with repeated levels of corticosterone by either immune lants [208] induces apoptosis in vivo, and severe thymowas concomitant with the rise in endogenous corticosteed apoptosis of oligodendroc~es andT cells in the subsequently they reported that between 5% and 10% sions were apoptotic[Zl l]. In both active and transfer iously reported up to 49% T cell apoptosis in the is rats that was associated with recovery from the postulated the involvement of corticosterone in the process. ~e recently demonstrated that the profile of T cell apoptosis during EAEparalleled the expectedchanges teronesecretion9withtheabsolutenumber of apoptoticT cells rea ate st of peak clinical symptoms.The percentageofT cell (32.1%)concurrent with the begin apoptosis9however, aximum disease remission, a reported by Tabi al. et 12131. The imp0 of corticosterone in process c is highlighted in the observatio adrenalectomized uced raT cell apoptosis throughout the shortene course of the disease9 which is now fatal in these animals [214], It appears, there-

of the disease process.

SNS nerve endin act ~ o n o n ~ c l e a r

nd, by i n f ~ r ~ n ~ ~ ,

there is minimal myelin loss, are largely reversible [228]. T resolution of each pathological episode within the CNS bearing on the clinical course of the disease an the degree of neurological deficit. tention has been giv to putative abnormalities in immunoregulawever, it is becomin ncreasin~ly clear that i m m u ~ eresponses are ontrol by neuroendocrine athways, the i which must be studied in the whole o As endogenous corticosteroids to exert negative feed~ackon inflam-

normal circumsta~cesw o u l ~be suppres regulation is present in h u m ~ n s we , might expect th associated with elevated levels of cortisol, and that

be normal [229-2311; however, in found to be significantly increase in then u m ~ e or

trum of ~ n t i i n ~ a m m a t o reffects y of therapeutic doses of the steroid previously alluded to. A putative link between inflammatory processes and sy has beenproposed in t is generallyaccepte T sup~ressorcell fun S compromised, at ly a minimal decline in suppressor/cytotoxic cell population a threefold up-regulation of adrenergic receptor number is seen. The receptorsarefunctional, since exposure of sup~ressor/cytotoxiccells to t adrenergic agonist isoproterenol results in a parallel increase in the level of c within the cells [23 and IL-2 receptors that ~-adrenocepto the increase in IL ssion fre~uentlyprecedes it. Theinhi~ition of ILexpression after /3 stimulation tor in vitro S that 6-adrenocept up-regulationmay constitute a recoverymechanisms infunctional up-regulation of 6-adrenergic receptorsis observed when blood mononuclea mal control subjects arecul hy~rocortisone,whereast

confined exclusively to the CNS,

1. This chapter has highlightedsomeof the complexmechanismsinvolved in the reciprocal regulation of the neuroendocrine and immune systems. It is evident that inappropriate stimulation of either compartmentis deleterious and may contribute to the developmentof certain autoimmune conditions, for example,SLE, RA, and owever, the diverse nature of neuroendocrine-immune interactions suggests the possibility of more specific therapeutic intervention in the management of disease states.

The authors wish to thank Prof. manuscript.

. Louise Cuzner for her helpful discussionof this

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~esearch ~nstitute, PLJVA d.d., Zagreb, Croatia ~niversityof Otago, Dunedin, New Zealand The ~ n i ~ e r s iof t y ~ueensla~d, ~risbane, A ustraiia

~ynthetic,purified, and subunit antigens are poorly immunogenic and invariably require the help of an immunopotentiating agent to elicit a specific protective immune response. Unlike natural vaccines (killed bacteria), which contain inherent immunostimulatory molecules (e.g., muramyl dipeptide [MDP]) in addition to the antigenic epitopes, the synthetic or purified vaccines are frequently composed of relatively low molecular-weight (MW) substances that lack the support of natural adjuvants. During the last few decades, recombinant deo~yribonucleicacid nology has been in the forefront of developments in biology and medi generated unprece~ented numbers of bioactive materials, such as peptide drugs and vaccine antigens. Respite the availability of these new molecules, many have not found a practical application, in part because of lack of concurrent advancementin the design of appropriate formulations and dosage forms for their safeeffective and delivery. Any chemical entity, whethernatural or synthetic,with potential therapeutic or prophylactic properties remains merely a chemical entity until it is properly formulated and delivered to provide medicinal benefits. From the formulationview point, perhaps RNA and ribonucleic acid (RNA) encoded antigens have the potential to be the most advantageous and elegantadditions to the subunit class of vaccines because these immunogens have the potential to be directly injected or transfected into host tissues, thus obviating the need for an accessory delivery sysose, route, and frequencyof administration of an antigen all play important roles in ensuring an effective immune response is stimulated 1281, With killed, purified,andsynthetic vaccines, thetraditionalapproach is to

f o r ~ ~ l a t i oscienn

3

an el al.

Antigens solubilized in water and then emulsified in an oil phase as a water-in-oil (w/o) emulsion possess enhanced immunological effects. The best known emulsion adjuvant is Freund's complete adjuvant (FCA), which is a w/o emulsion containing c o ~ a c t e r i utu~erculosis ~ in the oil phase and the antigen in the water phase. Mycobacteria play a dominant role in enhancing the immune response because they contain immunostimulatory molecules. This is clearly evident from the poorer immunogenic effect of FIA, which is also a w/o emulsion but does not contain any mycobacteria. In a comparative study with a viomycin (VM)-protein conjugate, Wu et al. [4] reported stronger adjuvant activity of FCA than FIA in mice in boosting primary responses to the antigen. However, FIA was found more effective during booster injections. Later, the same authors [S] reported that the antigen emulsified with FCA elicited higher levels of antibodies to the antigen in mice than when the antigenwas delivered either mixed (but unemulsified) withFCA or dissolved in saline solution anddelivered through miniosmotic pumps capable of releasing the antigen continuously over a period of 1 month. Freund's complete adjuvant is highly potent in stimulating both humoral and cellular immunity and has been used as a standard for the comparisonof adjuvant activity for experimental purposes [18,19]; it is unacceptable for usein either humans or domestic animals.

The mode of adjuvant activity of emulsions is perhaps not different from that of the aluminum-based gels. The antigen entrapped in the oil phase is protected from rapid degradation because the oil is nonmetabolizable and highly viscous and thus performs a depot function in releasing the antigen. The emulsion can also carry antigen to multiple local sites in the lymphaticsystem and create granulomas at the injection site and atlocal sites that attract cells (macrophages, lymphocytes) of the immune system and induce the productionof antibodies and C I, The importance of the granulomas producedat injection sites and draining lymph nodes in maintaining serum antibodyresponses was demonstrated by Lascelles et al. [20] in anexperiment in which ovalbumin (OVA) and ferritin were emulsified separately in FIA and injected into twosides (left and right flank folds)of the prefemoralregion of sheep. Excision of the injection sites alone after 6-9 weeks did not significantly affect antibody titersspecific to the antigenin the seraof the majority of animals studied; however, excision of the injection sites and the draining lymph nodes resulted in a steep decline in the antibody levels in blood serum of the sheep, indicating the dominant role played by the lymph nodes in maintaining the circulating antibody response. About one-third of a dose of '251-labeledOVA/FIA injected into flank folds of the sheep was still present in the granulomas 20 weeks after injection, whereas only about 0.2% of the antigen was present in the regional prefemoral lymph node. An insufficient rate of release of the antigen from the granulomawas o u ~ h t tbe o responsible for the lack of effects of excision of the injection sites. owever, the number of injection sites used to deliver the total doseof the antigen also affects the antibody levels: a higher number of injection sites produces more granulomas with a larger number of sensitized i m m u n o c ~ e and s thus higher antibody titers [Zl].

Despite the strong humoral andcellular adjuvant activity of FCA, the usefulness of this adjuvant has been limited to research because of its localtoxicity and sensitization of the recipient to ~ y c o b a c ? e r i u ?ubercuZosis. ~ Moreover, some mineral oil emulsions were reported as suspected carcinogens [22] and are not approved for human use. The strong adjuvant actionof mineral oils stimulated concerted efforts to find ways to reduce the risk of local toxicity associated withthe nonmetabolizable mineral oils while still attempting to maintain their strong im~unomodulatory effect. The results have been mixed and empirical. Reports conflict on theefficacy of using metabolizable lipid emulsions composed of biodegradable materials such as glycerol and lecithin [23,24]. Part substitution (by50%) of the mineral oils used in FIA with fat-soluble vitamin E reducedlocal reactions but maintainedeffectiveness in enhancing immune responsesto theantigen [ZS]. Emulsions basedon natural oils (e.g., squalene and squalane) [26] and metabolizable vegetable oils (e.g., peanut or sesame oil) [27] induced significant antibodies in humans with insignificant local reactions. However, more recent studies in humans with some of these emulsions reported significant side effects, including local reactions, although these were primarily linked to other components added to the formulation for synergistic action 128,291. An o/w emulsion prepared from squalene as theoil phase and containing a commercially available influenza virus vaccine combined with muramyl tripeptide (MTP) produced significant local reactions when evaluated in humans in a pilot study [28]. Inclusion of adjuvant substances, which can be toxic, in emulsions seems unavoidable if strong adjuvant action of the formulation is required. Nonionic block copolymers have emergedlately as a safe and potent alternative to classical adjuvants for inclusion in emulsions for synergistic actions, with successful results in some preclinical models [30,31]. These polymers are nonionic amphiphiles consisting of hydrophilic blocks of polyoxyethylene and hydrophobic blocks of polyoxypropylene. They are currentlyused as excipients for various pharmaceuticals and cosmetic products and are thought to possess no pharmacological effect. The polymers are surface-active and have been speculated to be capable of binding antigens/peptides to the hydrophobic surface of oil drops if included in emulsions, thus facilitating presentation of antigen to thecells of the immune system [32,33], They can also activate host mediators, influence the production of cytokines, and affect the isotype andsubclass of the antibody response as a function of molecular weights (chain lengths) of their hydrophobes, possibly by influencing appropriate T cell subsets [30]. When added to emulsions to formulate antigen, certain nonionic block copolymers are proved to be better adjuvants than some classical immunostimulatory substances(e.g., lipopolysaccharide) [3l] The manufacturing procedure of emulsions is relatively simple and requires selection of appropriate emulsifier. The physicochemical properties of emulsions play an important role in vaccine delivery, and the properties of the antigen itself sometimes dictate the type of emulsion required. Some problems (e.g., viscosity of the oil phase) associated with single w/o type emulsions have been overcome by introducing multiple emulsions, such as w/o-in-water (w/o/w) emulsions. The controlled release effect of emulsions is dependent on such properties asviscosity of the external phase, phase volume ratio, and droplet size of the emulsion; the latter is substantially affectedby the choice of manufacturing method.

he humoral immune re-

or ~ r ~ s e n t a t i oton class 11 re-

~ ~ e m a tpresentation ic of mu~tilamellarlipo~omes indicatin~ the structure of ers and possiblelocations of encasulateddrugs or ~roteins/pe~tides.

Class II~Antigen

~ n d ~ Lysosom

m

Intracellular processing of exogenous and endogenous antigens. Exogenous antigen is processed in the endocytic pathway, which is sensitive to the treatment of weak base (1). Peptides are charged onto the class I1 molecules, which are exported from the ER (2) and then transported to the cell surface. Endogenous antigen or cytoplasmically delivered (endocytosed and fused with lysosomal membrane from within) antigen is processed in the cytosol by proteasomes (3) and selective peptidesare transported into the lumen of the ER (4). Peptides are loaded onto the newly synthesized class I molecules and exported to the Golgi a~paratus,which is sensitive to the treatment of brefeldin A (BFA) ( 5 ) . The class I peptide complex does not intersect with the endocytic pathway and is directly transported to the cell surface (6).ER, endoplasmic reticulum.(Source: Ref. 35.)

I

peptide

\

proteasoms A ~ T i ~ E N - P R E S ~CN ELn L~ ~

Schematic representationof proposed presentation of liposomal antigenin MHC C, major histocompatibilit~complex. (Source: Ref. 40.)

I-specific response; whereas acid-sensitive liposomes (unstable below p pared from a mixture of dioleoyl~hosphatidylethanolamineand palmitoylhomocysteine (also 4 : I m o l ~ r )delivered the encapsulated antigen into early endosomes; it was then processed by both class I and class I1 molecules of the system [42]. ified proteinantiguldbetargetedfor HC class I presentationby encapsulatingtheantigeninto -sensitive liposomespreparedfromequimolar amounts dioleoylphospha of ethanolamine and 1,2-dioleoyl-sn-3-succinylglycerol [41]. e as carriers of antigens and adjuvants, as depots for slow as targeting agents for delivery of novel antigens and adjuvants to antigenpresenting cells” (reviewed in ef, 47).There is ample evidencethat somes areaspotentas(or evenstrongerthan)FCAasimmunoadjuvants ,493,but they do not producea granuloma, are notviscous, and do not produce an adverse visible local reaction at the injection site. Liposomes have also been demonstrated as re potent adjuvants than aluminum gels and “FCA-like” adjuvants, e.g., Ribi TOX, a proprietary adjuvant consisting of 5 pg monophosph, 50 pg cell wall c~oskeletonof c ~ b a c t e rpi ~~l~e iand , 0.5 p1 squalane [45]. The immunoadjuvant properties of liposomes can be substantially strengthened by incorporating other adjuvants alongwith the antigen within the system for additive action. Some a n t i ~ ~ nwith s no immunogenic properties when combined with FCA or alum became highly immunogenic when the combination is delivered through liposome preparations (reviewed in elivery of toxic immunostimulat lecules, like choleratoxin(CT),tumor necrosis factor a ( T F ~ - ~ ) , and , encapsulated in liposomesfree oralong withantigens,reduce toxic activity and enhances the immune response to the antigen [SO-531.The MTP encapsulated in liposomes containing murine recombinant y-interferon (rIF~-y)

that the liposomalallergen can r

ori~inalchemical and physical (

. The leakage probl

L in sphingomyelin-egg

cleared from circulation and in fighting of infections in which the echni~uessuch as couplin~vector moleosome surfaceto target S to specific tissues or . Zhou et al. [53] were

uptake ofliposomes by omes with appropriate egatively charged lipo: 2, molar) were taken phosphatidylcholined orally to rats [66]. stability in the stomt body temperature (T is SPC are relatively stable

elivery of antigens, onses in some cases S to othermucosae, . ~ntranasal or oral ses to the antigen tigen preparation

hilic substances other

describe liposomeS. Like liposomes,

tides or ampiphilic antigens containing

3 [70,73,74]. Proteosomes had greater adjuvant properties than other commonly used adjuvants or vehicles with adjuvant properties, including liposomes, when used to deliver S.C. an ampiphilic carbohydrate antigen, G ganglioside [7Q].Like that of other adjuvants, their mechanism of action is not clearly understood. But in part they act like other particulate systems and have depot effects sustaining release of the antigen. orm mu la ti on factors such as antigen/proteosome ratio and proteosome size also play a role in their adjuvantactivity [?'Q]. ~ t r u c t u r ~ lsimilar ly to multilamellar vesicles (Figure 4) but prepared as multiple emulsions are vesicle-in-water-in-oil (v/w/o) emulsions. They are made from nonionic surfactants as bilayer-forming materials, by emulsifying an aqueous dispersion of the nonionic surfactant(niosomes) in an oil phase [71]. They differ from liposomes in that the external continuous phase of the v/w/o is oil, which is probably an important factorwhen controlledrelease of the entrapped materialis considered. Like liposomes, both hydrophilic and lipophilic drugs/antigens can be delivered through these vesicles, and the externaloil phase acts asa barrier for the solute to diffuse from a stable system. Their application to antigen delivery is relatively recent, and their potential as adjuvants has not been well investigated. The viscosity of the external oil phase should bea cause of concern in terms of local reactions at the injection sites. However, Yoshioka et al. E711 demonstrated that they had adjuvant activity in delivering TT to mice compared to TT delivered as either free antigen or vesicles; the authors confirmed the controlled-release prop~rties of the v/ w/oemulsions in vitro by usingmodelsolutes, carbo~yfluoresceinand 5flurouracil, compared to either a simple w/o emulsion or a solution of the solutes. The type of the nonionic surfactant and of the oil phase and droplet size play important roles in controlling the release process.

water

71.)

i

l

A diagrammatic representationof the vesicle-in-~~ter-in-oil system. (Source:Ref.

7

Next to liposomes, perhaps the most well-investigated delivery system for antigen are microparticles(microspheres or microcapsules) and nanoparticles, ~ i c r o p h e r e s are small sphericalparticles (monolithic-type matrices) of usually more than 1 pm in diameter (usually 1-200 pm), whereas nanoparticles are colloidal particles with a diameter less than 1 pm. The microcapsules are of similar sizes to microspheres but differ from the latterin that the internal matrixis usually encapsulated by an outer membrane (reservoir type) as the name implies. These systems have proved to be effective adjuvants when used to deliver antigens and appear promising for future development. The OVA entrapped in microparticles prepared from copolymers of lactic/ glycolic acids, poly (~,L-lactide-coglycolide)(PLGA), induced significantly higher levels of IgG antibodies in mice for up to 10 weeks during primary immunization after single intraperitoneal (LP.) or S.C. injections than did OVAin FCA; antibody titers in both groups were comparable after booster doses of OVA injected S.C. in icle as the primary injection for the microparticle group and using FIA group [191. Several workers reported on the adjuvant properties of serum albumin ~ o l y m e br ds used as biodegradable immunomodulating carriersto deliver antigens [75,76], bbit serumalbuminbeadswereshown to bepotent adjuvants when used to d er a bacterial protein antigen (~iostridiu~ botuiinu~, type D toxin) and a nonprotein antigen (~iebsieiia ~ n e u ~ o n i acapsular e, polysaccharide) in mice [76]. Comparable IgGantibodies were producedagainst virus particles when the antigen was injected into rabbits either emulsified in FGA or cross-linked into rabbit serum albuminbeads [75].

~icro~~~icl~s icles are prepared from mainly synthetic polymers that can be modulated accordingto the requirements of the formulator. From microspheres the release of the ~ntigenoccurs by either diffusion through pores in the matrix and/or erosion/degradationof the matrix. In the reservoir types, diffusion of the antigen through the polymeric membrane is the rate-limiting step in delivery. Among various polymers? lactic acid polymers (PLAs) and PLGA have been the most frequent choice to prepare microparticles for delivery of antigens [18,19,77-911 because of their safety record in humans. These polymers are biodegradable, have a long history of medical use, and have been approved by the U.S. d Drug Administration authority for use in therapeutic products [87]. ost studies suggest that microspheres have equal (or greater) effectiveness to FCA/FIA in inducingimmunetlack theadversereactionsoftheoiladju,encapsulated in PLGA microspheres and vants. Staphylococcal enteroto injected S.C. into mice, stimula murine g IgG antitoxin titers comparable to those obtained when the antigen was emulsified in FCA, but unlike in FCA the microspheres didnot induce any in~ammation and granuloma [78]. Like emulsions and liposomes? the microparticles induce both a humoral reI. ~ualitativelyantigens entrapped in microparticles were reported responses in mice superior to those induced by antigens emulsified in FIA 1901. In a comparative studyin mice with a 38 kDa antigen fromM , tuberculosis, antigen delivered from microparticles was reportedto produce antigen-specific S of

IgG levels comparable to those induced by microparticles induced higher levels of IgG 10-fold higher level of antigen-induced I N-y secretion invit icroparticles can be prepared in different ways, incl polymerizations. Some ofthese methods require th and long encapsulation processes that may enature the labile antigen molecules. To overcome such problem, absorption of the antigen from aqueous solutions into preformed gluteraldehyde cross-linked chitosan microspheres was attempted with limited success [92]. A new class of water-soluble polymers9 been suggestedas a solution to this organic solventaqueous solution of the polymer by ionic cross-li Ca2') producesahydrogelthathas been usedt rporateantigenand deliver it in theform ofmicrospheres [8].Their release eristics can bemodulated changing the polymer concentration within th , incorporatingappropri side groups to the polymer, or coating the microspheres with cationic substances like poly(L-lysine) [8]. The polymer also works as an adjuvant i its solution form when mixed and delivered with antigen; preliminary studies suggest that this polymer is a safe adjuvant [8].

2. As with other particulates, microspheres th isthe due to ease of their recognition erytheby of antigen; the however, thanstable more are they li ccordingly, microspheres can be designedto performa depot f u n ~ t i o nover a much longer periodthan liposomes. The uptake of microspheres by macrophages is strongly size-dependent [ 181, whereas the depot function depends on both the size of the micros and the material they are made from [87,91]. Therefore, the uptake process can be ~ a n i p u l a t e dby varying the microsphere sizes (discussed cess can be optimized by selecting both the matrix formi spheres. Therate of rele ~~A fromsome LGA micro sphere^^ determined in vary significantly as a functionof of the antigen occurred within 1-2 days, followed by a tric

rin [87] from microspheres prepared from blends of initial small burst release of the encapsulated materi near-linear release pro son of the release profiles obtained in different studiesis not va of the studies selected various manufacturing parameters to spheres and the select properties (e.g., parti release patterns oft signi~icantly influen compositionandloadingalsoaffectthe release profile studied the effect o microspheres prepa

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faster than microspheres witha higherPLGA. One advantageof ymer is that by varying the ratio of the monomers, lactic and glycolic acids, the rate of biodegradability and thus the release characteristic of the copolymer can be modified [79]; however, a contradicting report on in vitro release suggesting that the ratio of the monomers does notsignificantly alter therelease pattern exists; this was of the microspheresin the dissolution medium[sa]. explained as due to flocculation Other natural and semisynthetic substances, such as gelatin [95], polyacryl starch [96,97], and chitosan [92], have also been tested as matrix forming materials to prepare microparticles fordelivery of antigen. The adjuvant effect of microspheres made of gelatin (cross-linked with glutaraldehyde) containing human gamma globulin and injecteds . ~ into . mice was strongerthan that of FIA, and in contrast to FIA the gelatin microspheres induced minimal inflammatory response at the inoculation site [95]. ~

iority of microspheres over lipos erms of stability also makes them potentially better oral for delivery o rospheres may provide protection the to antigen against degradati ch, various enzymes of the GI tract, and intestinal bile salts [77, eres of 5-10 pm range are absorbed to a limited extent from the GI tract through Peyer’s patches, and microspheres of 1-5 pm range have been foundin the spleen and lymph nodes [77]. This perhaps explains why antigens encapsulated in microspheres are able to produce both serum and mucosal antibodies after oral delivery. microspheres containing purified9 formalin-inact influenza virus N2 and then boosted orally with the same micros antibodies comparable to those indu~edby systemic priming and boosting with the samemicrospheres. Theorallyboosted mice,however,had significantly higher [83]. Anorallyadministered levels of salivary antibodies than the other group microencapsulated human immunodeficiency virus l immunogen (branched peptide) induced high levels of both serum IgG and neutralizing antibodies against human immunodeficiency virus in guinea pigs [80]. Small microspheres (less than 1 pm) are able to cross the mucosal barrier and penetrate into the blood circulation when administered nasally, and in animal experiments such microspheres induced significantly higher levels of serum antibodies to the antigen thanwhen the antigen was administered freeto thenasal mucosae [loo].

S) constitute the so-called antigen delivery system with built-in adjuvant [loll introduced in 1984 by orein et al. [1021 as a delivery system for antigens with adjuvant properties. They use the basic principle of multimeric presentation of the antigen with an adjuvant in a particle [loll in an identical fashion to most traditional systems such as FCA. The system exploits the ad~uvantproperties of the immunostimulatory molecule, Qui1 A, which is complexed with cholesterol and embedded in the formof a matrix in thesystem, which in most cases also contains PLs. The matrix is a cagelike structure about 25-45 nm indiameterwhere antigen(s) areincorporated by hydrophobicinteractions [101,103-1051.

Research carried out on experimental models demonstrated that the ISCOM complex is a potent adjuvant when used to deliver various synthetic [ 1061, purified [1031, or subunit [104,1051 antigens and canindwe protective immunity in animals against lethalchallenge. The ISCOMs can induce botha humoral response andCM1 against the antigen and have the potential for vaccine formulation against viral infections [ 1041. They are also effective when used to deliver antigens via the oral route [105,10’7].The uptake of ISCOMs and their transportation to lymphoid tissues are relatively fast [ 1011, and they can cross cell membranes [ 1081, which may be the main factors contributing to the high immune responses obtained in some of action is not clearly understood and experiments, but their exact mechanism requires more investigation [loll. The toxicity issues of the saponin derivative Quil A, which is not approved for humanuse, are alsoa matter of concern, although the quantities of Quil A required in ISCOMs are often S%-lO% of the concentration of the incorporated antigen. This amount is sufficient to potentiate a 10-fold increase of the immune responseover that obtained with the antigen administeredvia other systems [102,103,109].

The vector- and carrier-mediatedsystems are based on linking the antigen to either a live vector or a carrier thatitself is an adjuvantby virtue of itsphysicochemical or biological properties. In vector-mediated systems the antigen or its immunogenic epitope is genetically attached with and expressed in attenuated live vectors such as bacteria fromSalmonella (e.g., S. typhl~[1101 or Lactobacillus (e.g., L. fermentum) [ 1 1 l] species. The vector facilitates the processing of the antigen by the respective cells of the RES because of its biochemical properties,and higher antibody titers are obtained than when the antigen is delivered in free form. S~lmonellais an enteric pathogen and is the most studied live vector. Oral delivery of a sperm antigen, SP10, expressed on attenuated Salmonella produced high titers of anti-SP10 IgG antibodies in serum and IgA antibodies invaginal secretions [l 121. Sal~onellahas also been used to deliver heterologous antigens for induction of immunity against several diseases. A vaccine prepared as a full-length fusion of the 28-kDa glutathione s-transferase (P28) of Schistosoma mansoni to a particular fragment of TT in live attenuated Sal~onellaty~himurium-inducedprotective immunityin mice from S~lmonel~a infection, tetanus, and schistosomiasis aftersingle-dose a administration of the vaccine [ 1 131. The mechanism of action of Salmonella is not clearly understood, but it hasbeen demonstrated in mice that M cells of the Peyer’s patches are a major route bywhich S. t y p h i ~ u r i ~invades m theintacthostandthebacteria use as carriers for preferentially interact with theM cells; that finding justifies their oral delivery of antigens [ 1 141 Based on a similar principle, the carrier-mediatedvaccine systems use various biological substances (mainly proteins) that are either genetically fusedwith or chemicallyattached to the antigen or its immunogenic epitopes. The biological substances, isolated from eitherlive vectors/pathogens or their synthetic analogues, are carefully selected for their functional roles in the biochemical processes of the immune system. Examples of these protein-based carrier systems that have been successfully used to deliver a number of antigens via various mucosal routes are the

subunit of heat-labile enterotoxin of oduced by the highly virulent bacte which is responsible for causing excessive secretionof elect infected humans. The CT molecule has a ringlike st subunits intowhich a toxic A nd the toxin to granulocyte macrophage 1 receptors o epithelium while the A subunit trans cells to cause excessive loss of elec licensed cholera vaccine, which uses t V. c~oZe~ae to immunize orally against the pathogen, has been reported to induce long-lasting protectiveimmunity in clinical field trialsand is safeforhumans [ 1 161. The high-affinity binding capacity of to the intestinal epithelial cells has been used to deliver foreign antigens using orseradish C peroxidase covalently coupled to CTB and delivered orally produced enhanced mucosal immune responses to the antigen [1171. Several studies demonstrated that CT- or -based foreign antigens can stimulate both mucosal and circulator antibodies in mice after mucosal immunization [1 181, (reviewed in [l 191 found the system suitable for both oral and S induce mucosal and systemic protective immunity. demonstrated that after oral administration n mice [1201 a minute ~ u a ~ t i of ty articulate or soluble antigen coupled to CT induced peripheral tolerance to the antigen, them e c h a n i s ~of w h i c ~was not clear. There are alsoconcerns about their suitability for repeated use because immune responses induced through vectorial antigens result in responses against both the vector and the antigen. An analogue to the carrier-mediated system is the receptor-~ediatedsystem, whichuses carrier proteins capable of facilitating endocytosis or uptake by the APCs of theRES when conjugatedto theantigen. In this case, the carrier is selected from a range of proteins that have specific receptors in the membranes of the host CS for effective internalization and processing of the antigen. Studies suggest that when ~2-macroglobulin,a protein secreted by macrophages, is linked to antigens, the resulting complexes are rapidly endocytosed by the macrophages via the specific receptors for this protein, and enhanced antibody responses to the antigen are induced [ 121-1251. Antigen complexed to human ~2-macroglobulin and injected into rabbitselicited IgG titers comparableto thoseinduced by the antigenemulsified in FCA and 10- to 500-foldhigherIgGtiterswhen compared to uncomplexed antigen controls[ 1251

. The desirability of providing complete protection against diseases like diphtheria, whoopingcough,tetanus, poliomyelitis, andhepatitisasa result ofa single health-care contact cannot be overemphasized and the development of vaccines suitable for single-step immunization against these killer diseases has become an integral part of the W O/UNDP immunization programme [126]. The substances with immunoadjuvant properties andmost of the antigendelivery systems described here either facilitate modification of antigen by the respective cells of the immune

system or rely on a simple depot effectto exert their immunomodulatory effect. The antibody titers induced by these methods are usually inadequate for providing a long-lasting protective immunity after a single administration. The possibility that “single-step” or “single-shot” immunization will become a reality was first given credence in the mid-1970s when Langer and his coworkers reported on the use of long-acting devices for the delivery of antigens [127,128]. The apparent efficacy of long-acting devices for delivering antigen changed the traditionalview regarding the requirement for multiphasic delivery of antigen to induce long-lasting immunity9 and different approaches to acquire long-lasting immunityhavebeen tested by various groups of researchers. Table 1 summarizes the recent trends and progress made in designing delivery systems for single-step immunization.

The concept of implantable therapeutic systems for long-term, continuous drug administration was pioneered by Lafarge in 1861 with the development of a S.C. implantable drug pellet (noted in Ref. 129). An implantable polymeric system made of silicone rubber capable of delivering a macromolecule (tri-iodothyronine) in a sustained-release manner was reported by Folkman and Long in 1964 [ 1301 and triggered the use of S.C. implants for controlled-release drug delivery. Almost a decade later, Langer and Folkman[ 1271 demonstrated that various macromolecular drugs could be delivered in a sustained-release manner for periods exceeding 100 days from implants prepared from a nonbiodegradable ethylene-vinyl acetate copolymer. In a follow-up study, Preis and Langer [l281 used this system to deliver antigens differ of ribonuclease (MW 14,000), BSA (MW 6$,000), and bovine y-globulin W lS$,OOO). The BSA delivered from these implants inducedIgG antibefor over 25weeks aftera single-dose S.C. administration; the response was comparable to the levels of antibodies produced by two injections of the antigenemulsified in FCA. The release of antigens from these implants occursmainly by diffusion, usually with an initial burst effect followedby a trickle continuousdelivery [ 1311. The rate of release of the antigen is affected by the particle size and loading doseof the antigen and its water solubility. The larger the particle size, and the higher the loading andwater solubility, the greateris the rateof release.

es of ~ o l y ~ l e~ pr l~~ ~n t s ng these implants forsingle-step immunization is their nonbiodegradability; a second visit to health personnel is required to remove the implant in an actual immunization program. This limitation prompted a search for biodegradable polymersto replace ethylene-vinyl acetate copolymer as construction material for implants carrying the antigen. The BSA delivered from a biodeimplantmadeof ~-benzyloxycarbonyl-L-tyrosyl-L-tyrosine -iminocarbonateinduced significant anti-BSAantibodiesfor in mice [ 132,1331. A biologically over 56weeks after a single-dose ad istration of the polymerCwas found to act as an adjuvant as potent in enhancing the immune response. Problems associated with implantabledelivery systems are the requirement for skilled personnel for surgical implantation of thedevice, the general discomfort for

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the host, and the possibility of infection at the site of implantation. A novel approach overcoming these problems is the in situ forming implantable systems that can be administered as injectable liquids using standard techniques but solidify on contact with body fluids [134,135]. A 1%-20010 (w/w) solution or suspension of PLGA copolymer preparedby stirring and heating the copolymer in an appropriate vehicle (triacetin or triethyl citrate)and then cooling to room temperature was found to form agellike matrix after S.C. or intramuscular(i.m.) injection into rats[134]. A model protein, myoglobin, incorporated into the PLGA injectable preparation resulted in a sustained release of the protein over 2 weeks in vitro. The efficacy of such a system in antigen delivery was later reported by Dunn et al. [ 1351. They used an in situ forming biodegradable implant (ARTIGEL) made from polymers such as PLA, PLGA, or copolymerof lactide with caprolactone preparedby dissolving the polymer in a water-miscible solvent such as ~-methyl-2-pyrrolidone or dimethyl sulfoxide. A model antigen (OVA) incorporated into ARTIGEL and injected S.C. or i,m. into mice stimulated antibody titers to the antigen much higher than those produced by the antigen delivered from saline or saline/alum system, and the antibody titers were comparable to those producedby the antigen delivered from FCA [135]. The efficacy of ARTIGEL in a single-step immunization procedure for providing long-lasting immunity remainsto be examined, The technique for the manufacture of polymeric implants that require the antigen to be treated with organic solvent may limit their practical application.The safety of using mostof these polymers in humans has not yet been well established.

In 1936, Shear first used lipid implants made of C containing a carcinogen, dibenzanthracene, to induce tumors in mice [ 1363. Since then, lipid implants have been used for delivery of various drugs including peptides and proteins [ 137-1431, but their utility has not been well investigated for the delivery of antigens, although in 1990 Beck [l441 noted in a patent the feasibility of using fatty acid esters such as glyceryl stearates to prepare implants fordelivering antigens to achieve prolonged immunity. Recently, we demonstrated the potential adjuvantity of C implants for delivery of antigen. The BSA delivered from C implants was moreeffectivein inducing anti-BSA antibodies in mice than either a single injection or three injections of the same total dose of BSA [1451. Blank Cpellets did not enhance antibody production when administered together with the injectable BSA solution. The release of the active component from lipid implants occurs mainly by diffusion through the intact matrix, although sudden burst release after a certain period due to disintegration of the system was reported insomestudies [137]. Naltrexone was released from a C-glyceryltristearate matrix by diffusion when implanted S.C. in rats and C acted as a diffusional barri for therelease process [ 1381 Our studies also demonstrated diffusional release o SA from lipid implants both in vitro [l461 and in vivo [145,147]. 7. Advantages and ~ j s a d v a ~ ~ a gofe~s j/ ~~ ~ j/ ~ ~ ~ t s Like nonbiodegradable polymeric implants, the C system has as its main drawback the persistence of the implants after delivery of the antigen. To make the system biodegradable, we further modified the implants by incorporating a PL (saturated

egg lecithin [PC]) into the matrix since C can be solubilized in PL bilayers through an interaction between the two components under certain conditions [ 148,1493. The BSA delivered from C-PC implants was able to induce and maintain an immune response in mice for at least 10 months after a single-dose administration (Figure 7) [147], and the rate of erosion of the implants could be manipulated both in vitro [l461 and in vivo [l471 by changing the C-PC ratio of the implants. The C-PC implants containing BSA induced levels of anti-BSA antibodies either equal to or higher than those inducedby the same total dose of the antigengiven in the formof three injections (14 days apart) in phosphate-buffered saline solution or as singledose C implants. A burst delivery (within 2-5 days) followed by trickle delivery of the antigen from the implants for up 7tomonths from someof these implants was observed in mice [147]. A significant correlation existed between the release of BSA and the erosion of the C-PC implants during in vitro studies under active hydrodynamic release rate. conditions, and increasing the PC contentof the implants enhanced the Among other factors, the hydrodynamic conditionsof the dissolution medium and the method used to mix C and PC in preparing the implantsgreatly influenced the 2 months 4 months 7 months 10 months

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ufe 7 Anti-BSA antibody levels in sera of mice after administration of BSAby singledose implants prepared from cholesterol alone (Chol.) and from cholesterol (C) and saturated egg lecithin (PC) and by three injections (Injection). The C-PC implantswere formulated at various C-PC ratios, which are indicated in parentheses. Control mice received blank implants (withoutBSA) with a C-PC ratioof 8: 1 (Blank). Vertical bars indicate standard errors of the means (n = 5 or 6 ) . BSA, bovine serum albumin. (Source: Ref. 147.)

owever,a lack of in vitrothese implants was noticeable dur ainly attributab~e to the fact that tions in vitro is a difficult task (reviewed in gested that variable release of peptides an l , 1421, results from ourin vivo studies wer he manufacturing technique of these implants required to prepare polymeric implants, and the antig heat or organic solvent treatment. These implants ca uvantssuchassaponin, , andperhapscytok these implants in delivering actual vaccine antigens responses induced, the release kinetics of the antigen, e implants require furtherinvestigation. In line with other implantabledevices, there are alsoconcerns about tissue responses to these lipid implants in the form of a fibrous tissue capsule surrounding thedevices [l 501.

In 1976, Chang [15 11 first suggested that a dosage form antigen, partly in solution form and partly in microencapsulate types of microcapsules, could be used for single-shot immunizati prepared from different polymerswouldbedegraded at release the antigen at predeterminedintervals. There is now either two or more delivery systems in the same dosage fo adjuvant substances or vector molecules with modified antigen delivery systems to sustain or control the release of the further modified to possess adjuvant properties in their own right. recombinant malarial antigen, be poorly immunogenic wh liposomes containing mon duced much hi her antibodies in hu )3 without liposomal encapsulation [152]. The presence of monoA in the liposomesplayeda dominantrole in enhancingthe antibody levels. Similar results were obtained with another recombinant malarial al. E1541 wereable to use a mixtureloaded T to induceantibodiesinmiceafter le-dose injection thatwere comparable to those induce by a conventional multidose immunization regime. The depot effectof liposomes e n c a p s u l a t i ~ ~ antigen an was extended substantially in rats by encapsulating the liposomes into alginate- oly(L-lysine) microcapsules [l SS]. The antibodyresponses inducedby the al~inate-poly( sules were two- to three-fold higher than those induced by th from either liposomes (without encapsulation), saline solution h i ~ hantibody levels were maintained for over 150 days after aS tration (Figure 8). bout 30010 of the total i2sI-labeled antige these microencapsulated liposomes could be recove d from the injection sites 80 days after injection, whereas no radioacti be ~etectedin rats with the antigen in saline solution, liposomes, or FC

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Time (days)

umoral response to BSA in rats immunized with antigen in saline solution U, liposomes 0, or micro~ncapsulatedliposomesThe data pointsaretheaverage (Lts.d.) of 5 animals, at a serum dilution of 1: 100. Antibody values are corrected for preimund.BSA,bovineserumalbumin;FCA,Freund’scomplete adjuvant.

icrospheres of various sizes and/or made of various materials anddelivered in a single-dosage form have been used frequentlyto obtain multiphasic delivery of the antigen. Eldri ge et al. [l81 reported that a combination of two microspheric preparationscoul release vaccineantigen atdifferenttimes,providing discrete primary and booster doses after a single-dose injection. A mixture of 1- to 10-pm pheres, made of PLGA (50 a primary and an anamnesti single-dose injection in mice. An antifertility antigen (a heteroS a-ovine of lutei * * and &human chorionic hCG] linked to encapsulated into two types microspheres of made of two different polymers and delivered together induced equal or higher levels of antigen-specific antibodies in monkeys after a single-dose administration than that produced by the same total dose of the antigendelivered three times (l month apart) adsor~ed onalum ( 9) [89]. The microspheres were preparedfrom either ( 1 ~ ~ polymer 0 ) o 35) copolymer and mixed at a 1 :ratio 1 i -30% of the mic vehicleinjectio for the rest were within the range of 10-90 pm. Afte the antibody titerswere high for up to225 days and com the three injections. A booster injection of the further in both groups of animals. In an earlier S specific-anti~odylevels in rats were reported a

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Days after immunization Antihuman chorionic gonadotropic hormone (hCG) response in bonnet monkeys (~acaca ra~~ asameasured ta) by hCG binding capacity. The monkeys were immunized with three injections at 1 month intervals of 300 pg of a hetero species dimer (HSD) of a-ovine luteinizing hormone and P-hCG linked to DT (HSD-DT) adsorbed on alum, followed by a booster of 300 pg at day 225. Another groupwas immunized witha single injectionof 900 pg of HSD-DT loaded ina combination (1:l) of microspheres prepared from PLGA and PLA, followed by a booster of300pgof the microsphre-loaded HSD-DT at day 225. The data points are average values (n = 4) andverticalbarsindicate sad. PLGA, polyD,L-lactide (Source: Ref. 89.) coglycolide; PLA, lactic acid polymer.

antigen-loaded PLGA microspheres; the response was comparable to that of the antibodies induced by two injections (30 days apart) of the alum-adsorbed antigen [ 1561. The micros~heresfell within the range of 5-70 pm with most 20-40 pm. eterogeneous microparticulardelivery systems with multiphasiccontrolled-release properties for antigens have been patented by various authors[144,1571. As described earlier, the uptake of microspheres by macrophages is strongly size-dependent, whereas the release of the active component from these systems is dependent on the size of the microspheres, the material they are made from, and some manufacturing process parameters. It has been suggested that microspheres < 10 pm in diameter are phagocytosed and release the antigen at a substantially accelerated rate when comparedto thelarger microspheres contributingto theinitial immune response [183. The release of the antigen from larger (> 10 pm) microspheres is mainlygoverned by therate of theirdegradationandoccurs slowly because theyare notphagocytosed by the APCs[l 81 The delayed and slower release of the antigen from thelarger microspheres seem to contribute to thesecondary or prolonged immune response. A prolonged high-level immune response (during 192 days) was also obtained in mice after a single-dose administration of BSA-loaded PLGA microspheres of 10-100 pm diameter (Figurelo), which showeda near-linear

release profile in vitro (see Figure 6) [91]. Assuming such a release profile in vivo, the results suggest that aninitial burst release of the antigen may not be a prerequisite for initiating the immune response, although this can only be confirmed by studying the in vivo release profile of the microspheres since the in vitro-in vivo correlation is always a matter for concern(reviewed in Despite the fact that some of the heterogeneous delivery systems described here require organic solvents in the manufacturing process that may denature the antigen, the experimental results obtained in some of these studies are prom is in^. These results have also drawn the attention of the WHO, which has acknowledged the potentialof these systems for thesingle-step immunization program [ 1261.

The traditionalview on antigendelivery is that a priming dose is required to initiate the immune process, to be followed by several further exposures to lower levels of the antigen to evoke lasting immunity.Limited objective data, however, are available to allow conclusions to be drawn on what constitutes an optimal delivery profile for antigen. Indeed, the optimal delivery profile may be different for each antigen or antigen-adjuvant combination. A few studies with soluble antigen demonstrated that several small doses of antigen are likely to be more effective than

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IgG immune responses of mice induced by a single S.C.injection of 5 0 0 pg BSA loaded in microspheres prepared from a blend (0.5/0. l , w/w) of PLGA (75:25) and PLA (MW 2000). The absorbance readings were obtained from ELISAon diluted (l :1600) sera in Tris-buffered saline solution. Vertical bars indicate standard errors (n = 6). IgG, immunoglobulin G ; s.c.; subcutaneous; BSA, bovine serum albumin; PLCA, poly D,L-iactide cogly( ~ ~ ~ ~ c e : colide; PLA, lacticacidpolymer;ELISA,enzyme-linkedimmunosorbentassay. Ref. 91.)

either a single inoculum or a few large dosesin stimulating an that the primary dosage of the antigen should be greater th injecte subsequent doses [158, 1591. Low and high doses of antigen several weeks have also been reported not to amplify the i cause tolerance to the antigen [160,1611. Large doses of daily for several weeks induced antigen~specific immunolo Its obtained from most of the controlled- or sustaine ditional belief that multiple-dose administration quired to evoke lasting immunity. Theevidence emergingfrom most of these studies demonstrates that an initialpulse release followed by a continu as is the case with both polymeric [128,131-1331 and lipid [14 someofthemicroparticulate delivery systems [S equally well in inducing prolonged immunity. Conti delivery of antigen througha miniosmotic pump for up to as effective as either two or three inject rimingdosesofantigen not significantly im ults andthe high antibo iters obtained by S with near-linear in vitro release kinetics of antigen suggest that a p * ' an essential requirement for induction of an imm the immune system, such as follicular dendritic c tissues, are known to trap and retain antigens over 165), and persistence of easily degradable antigen year in lymphoid follicles of mice [ 1641. It is believed that both the persistence of antigen and the level of specific antibody are responsible for maintaining andregulating antibodies; persisting antigen is unable to stimulate further synthesis of antibodies if the specific antibody levels are already high, and if the level falls below a critical point, the presence of antigen new cycle of antibody synthesis (reviewed i antigen to the cells of the immune sy from sites of administration are important for inducing an immune becausememory cells that generate secondar titer, and affinit require repeatedexposuret livery systems capable of presenting antigento theimmune system overan period have been recommended for vaccine formulation [91]. The in vivo release pattern of the antigen from thedelivery systems described has not been well investigated. The lack of concrete information on what constitutes an ideal release profile for antigens to induce high levels of pro1 indicates that more work is required on the effectsof zero-order a profiles on the immune response before optimal systems can be a step immunization.

The adjuvant properties of individual immunostimulatory substances of biological origin (e.g. , mycobacteria, mycobacterial fragments) or their synthetic analogues of sufficient (e.g., TP) may be adequate to induce immunostimulation

to providean effective immunity;however,theycan human use, and, moreover, may not provide vants, such as nonionic block polymers, may native to the classical adjuvants, but they often require the support o system such as an emulsion to confer their adjuvant properti and their potential een well investigat a omi in ant role to p *

profiles to induce long-lasting ifficult for formulation scienieving lasting immunity. The livery systems for anti ens to is not only to m o to improve their lus trickle delivery [ 128,13 l] demonstrates that a seconthout a secondary burst delivery effect. to achievemultiphasic delivery have 181. These datain though achievingthisgoal djuvants participate in the i m m ~ n eprocess and a clearer perception of “optimal” delivery profiles. Formulation scientists will then be in a better positionto design appropriate delivery systems for antigens to achieve long-lasting immunity by using asingle-dose admi~istration of the anti~en.

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58. N Arora, SV Gangal. Int Arch Allergy Appl Immunol91:22, 1990. 59. N Arora, SV Gangal. Allergy 46:386, 1991. FRDenys, AK Berry, JH Eldridge, JH McGhee, 60. SM Michalek, NK Childers,J

RCurtiss.CurrTopMicrobiol uno1 1465l, 1989. 61. E Harokopakis, NK Childers, SM Michalek, SS Zhang, M Tomasi. J Immunol Methods 185:31, 1995. 62. TDMadden, MBBally,MJ Hope,PRCullis, HP Schieren, AS Janoff. Biochim Biophys Acta 81757, 1985. 5: 143, 1987. 63. G Gregoriadis, D Davis, A Davies. Vaccine 64. MC Finkelstein, G Weissmann. Biochim Biophys Acta 587:202, 1979. 65. C Kirby, J Clarke, G Gregoriadis. Biochem J 186591, 1980. 66. Y Aramaki, H Tomizawa, T Hara, K Yachi, H Kikuchi, S Tsuchiya. Pharm Res 10: 1228, 1993. 67. MC Woodle, G Storm, MS Newman, JJ Jekot, LR Collins, FJ Martin, FC Szoka Jr. Pharm Res 9:260, 1992. ng. Advanced Drug Delivery Rev 3:343, 1989. 68 N ~rahmbhatt,DC White,MW Fountain, M Rohde, J Wehland,KN 69. Timmis. Microbial Pathogen 14:149, 1993. 70. PO Livingston, MJ Calves, F Helling, WD Zollinger, MS Blake, GH Lowell. Vaccine 11: 1199, 1993. 71. T Yoshika, N Skalko, M Gursel, G Gregoriadis, AT Florence. J Drug Targeting, 2: 533, 1995. 72. C-0 Rentel, JA Bouwstra, B Naisbett, HE Junginger. Proc IntSympControl Re1 Bioact Mater 23:77, 1996. 73. GH Lowell,WR Ballou, LF Smith,RA Wirtz, WD Zollinger, WT Hockmeyer. Science 240:800, 1988. 74. GH Lowell, LF Smith, RC Seid, WD Zollinger. J Exp Med 167:658, 1988. 75* JB Dewar,DA Hendry, JFE Newman. SA Med J 65564, 1984. 76. C Langhein,JFE Newman. J Appl Bacteriol63:443, 1987. 77. JH Eldridge, CJ Hammond, JA Meulbroek, JK Staas, RM Gilley, TR Tice. J Controlled Release l 1205, 1990. 78. JH Eldridge, JK Staas, JA Meulbroek, TR Tice, RM Gilley. Infect Immun 592978, 1991. 79. DT O’Hagan, H Jeffery, SS Davis. Int J Pharm 103:37, 1994. 80. DT O’Hagan, JP McGee, R Boyle, D Gumaer, X-M Li, B Potts,CY Wang, WC Koff. J Controlled Release 36:75, 1995. 81. R Edelman, RG Russel, G Losonsky, BD Tall, CO Tacket, MM Levine, DH Lewis. Vaccine 11:155, 1993. 82. 2: Moldoveanu, JK Staas, RM Cilley, R Ray, RW Compans, JH Eldridge, TR Tice, J Mestecky. Curr Top Microbiol Immunol 146:91, 1989. 83. 2 Moldoveanu, M Novak, W-Q Huang, RM Gilley, JK Staas, D Schafer, RW ComJ infect Dis 167:84, 1993. Eldridge, G Ludwig, JK Staas, JF Smith, RM Gilley, SM Michalek. 84. Vaccine 13:1411, 1995. 85. T Uchida, S Goto, TP Foster. J Pharm Pharmacol47:556, 1994. 86. T Uchida, K Yoshida, A Ninomiya,S Goto. Chem PharmBull 43: 1569, 1995. 87. H Sah, R Toddywala,YW Chien. J Controlled Release 35: 137, 1995. 88. M Singh, A Singh,GP Talwar. Pharm Res 8:958, 1991. 89. M Singh,0 Singh, GP Talwar. Pharm Res 12: 1796, 1995. 90. HM Vordermeier, AGA Coombes, P Jenkins, JP McGee, DT O’Hagan, SS Davis, M Singh. Vaccine 13:1576, 1995. 91 H Sah, YW Chien. J Pharm Pharmacol48:32, 1996. a

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in. Infect Immun 59:2909, 19 rmendia. Vaccine. 12:1349, 1

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MD Drew, A Estrada-Correa, BJ Underdown, ermott.

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CT Chu, SV Pizzo. J Immunol 150:48, 1993. Chu, TD Oury, JJ Enghild, SV Pizzo. J Immunol 152:1538, 199 Aguado. Vaccine 11596, 1993. anger, J Folkman. Nature 263:797, 1976. I Preis, RS Langer. J Immunol ethods 28:193,1979. YW Chien.NovelDrugDeliverySystems. New York: ekker, 1992, p 441. J Folkman, DM Long Jr. Ann NY Acad Sci 11 1:857, 1964.

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J Shear. Am J Cancer ~~V1:322, 1936.

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icroencapsulation 13589,

1996.

1997.

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University ~ o s p i t a l ~ p p e n dUniversity or~ of amb burg, amb burg, Germany Baylor Collegeof ~ e d i c i n e ,ousto on, Texas , York, New York ~ o u nSinai t School of ~ e d i c i n eNew

Tumor transplantation studies, the developmentof inbred mouse strains, and new diagnostic procedures give evidence that a broad variety of cancers in humans as well as in animalmodels induce host immune response[ 1-41. This immune response does not needto be directed against antigens exclusively expressed in malignant cells but also involves antigens shared by nontransformed cells [5-81. Enhancing this process of host immune response to attack preexisting metastatic tumors successfully and togain prolongedsurvival are the objectivesof cancer immunotherapy. New techniques in gene transfer and eukaryotic gene expression over the last decade made it feasible to test and evaluate the therapeutic benefit of a variety of cytokines involvedin the antitumoral hostresponse. In mousetumor models significant tumorregression in vivo has been shownfor interleukin l (IL-l)[9], interleu~in 2 [ 10-131, tumor necrosis factor cy [12,141, granulocyte-colony stimulating factor (G-CSF) [ 15- 171, and interleukin 12 [18,191. Cytokine treatment dramatically enhanced the host immuneresponse and caused tumor regression. Recombinant interleukin 2 has also been usedsuccessfully in the treatment of humans with metastatic renal cancer or melanoma [20-223. Considering the complexity of host immune response mechanismsit is not surprising that only patients with certain malignancies benefit from treatment with recombinant IL-2 (rIL-2) and even among those with renal cancer and melanoma many show incomplete or no response [22]. New therapeutic approaches based on introducing cytokine genes rather than the recombinant proteins wereestablished in order to obtainhigh and localized cytokine gene expression, increasing the therapeutic index. Cytokine gene therapy is the transductionof either tumorcells or immunecells involved in the antitumoralhost response. Thesegenetically engineered cells express either cytokinesor antigens stimulating the antitumoral response.Ceii~lar a~optive therapy requires isolation ofcells involved in the host immune response and in vitro stimulation with cytokines or transduction with cytokine or antigen encodinggenes. 7

11s are then transferred back into the cancer patient, usually in combinaapplication of recombinant cytokines,

Tumor antigens are encoded by normal cellular genes, mutant cellular viral genes. It has been shown that normal gene absolutely silent in non cells, can be activated in malignant cells 1231. ome tumor antigens are also expressed on normal cells during a certain stageof differentiation and are considered differentiation markers. These glycoproteins and glycolipids are tu and not tumor-specific 124,251. Expression of these antigens can levels 10- to 100-fold. Embryonic and fetal antigen expression resu tion or depression of fetal or embryonicgenes, silent in normal a the best studied embryonic tumor antigensis the carcinoembryon expressed in human colon carcinoma and as an embryonic antigen restricte~ to fetal gut ,liver, and pancreas[24] Tumor antigens encodedby mutant cellular genes are the result of mutations caused by physical or chemical muta malignant normal andbehavior of ce rotooncogene an p53 suppresser gene mutations127,281,

induction of malignant tran on. These shared regardless of tumor origin an proviruses into the host genomic tion withchemicalcarcinogens,

virus-specific tumor an

adult Tcell leukemia. Tumor antigens arepresented to the host immune system by two majormechanisms: histocompatibility major ( antigen presents3. Almost all cells present endogenous and viral ~eptides on the cell surface C class I molecules after processing the proteins in the endoplasmic reticulum [31] and thereby signaling immune cells. A subset of cells utilizes 1 soso sed endogenous proteins. Those class I1 molecules 132,331. The anddendritic cells, and activated T cells uced in a variety of norma been demonstrated that some p HC class I molecules [34]. cytes recognize antigens presente molecules. These lymphocytes T d into two major subgroups according to the surface markersCD8 and

si~ultaneous~y the to es ~olymorphical/a

accumulating natural killer cells, monocytes, and neutrophiles.

S,

such as IL-4, IL-5, IL-6, h2propertiesand

a distinct subgroup of lymphocytes capable of killing

cells has been demonstrated in vitro by leukemia line YAC and the human eryth-

cells cancer cells

of renal cell carcinoma and

tumor cells, macrophages can be activated in vitro to cause ling is mediated by secretion of cytotoxic optive transfer of macrophages activated phage activation in vivo can reduce metastasis in some

he MHC and non-MHC restricted host immune cells cooperate to achieve tumor rejection. CD8 and CD4+ T lymphocytes may recognize specific tumor antigens presented by MHC class I and I1 molecules either on the tumorcell surface or after uptake of tumor-derived proteins and display on antigen presenting cells such as macrophages and dendriticcells. Tumor cells are thendirectly lysed by CD8 cells. Alternatively, activated CD4 and CD8 C cells secrete cytokines such as TNF-a, interferon y (IFN-y), granulocyte macrophage colony stimulating factor (GM-CSF), and IL-2. These cytokines induce Tcell proliferation, activation, and accumulation of LAIC cells, macrophages, and neutrophils. Activated accessory immunecells can kill tumor cells independently of specific tumor antigen recognition. Even if tumor cells, after deletion or hypoexpression of MHC class I or 11, are not recognized by T cells, they remain susceptible to activated accessory cells like NK cells, LAK cells, or macrophages. Considering the tumor antigen presentation by normal host cells, release of cytokines rather than directcytolysis by T cells helps prevent initiation of autoimmune destruction of the host cells. Released cytokines can initiate thespecific killing of neighboring tumor cells by LAK and macrophages.

+

+

+

T Tumor growth-affecting cytokines are released by either the tumor or surrounding host cells. Cytokines provide signaling between cells and are usually expressed transiently. Tumor cells can constitutivelysecrete significant amounts of different cytokines. High local concentrations of cytokines can lead host immunecells to infiltrate the tumor vicinity and/or release other cytokines. Macrophage chemotactic proteins, released by the tumor, can attract macrophages1781. These macrophages can release other growth factors and cytokines, such as platelet-derived growth factor DGF), resulting in enhanced tumor growth [79]. Other cytokines, such as transforminggrowthfactor @ (TGF-@), canenhancetumorgrowth by inductionof angiogenesis [80,8 l]. Tumor cells can be transformed to produce significant amounts of certain cytokines. Cytokines like C-CSF [82] and TNF-cu [83,84] expressed withinthe tumor can inhibit tumor growth even in the absence of T cells. Other cytokines, such as IL-1 [85], IL-2 [86], and interferon-y [10,871, show growth inhibition in transduced tumor cells in the presence of T lymphocytes. Tumor rejection usually follows T cell-mediatedimmunity.Althoughcytokine release is assumed to induce T cell immunity, other mechanisms, such as impaired growth of transduced tumor cells and therefore prolongedexposition of tumor antigens, remainpossible.

Expression of cytokines within the tumor cells to induce or enhance host immune response is appealing and simple in concept. Recently a variety of molecular techniques emerged to introduce andexpress foreign DNAin tumortissue. i here as the delivery of DNA to human tissues is rather difficult, viral vectors are much more promising for efficient gene transfer since in viruses efficient mechanismshave

evolved to introduce and express their nucleic acid into recipient cells 28 challenge of using viral vectors is inducinghigh and long-lasting expressionof the exogenous gene while minimizing the host defense. Retroviruses offer stable introduction of foreign genes into the host genome[89,90]. owever, low transduction efficiency and the requirementof cell division make this gene tra feasible only in vitro with the opportunityto select transduced cells. viral gene transfer is one of the most attractive techniques for c therapy, in vivo genetherapy requires high titers and vectors with high transduction efficiency. Implantation of retrovirus packaging cells resulting in local release of retroviruses has been demonstrated to be effective in animal tumor models 2911. Ade~oviruses cansucces lly transduce tumor cells in vivo, resulting in high exogenous geneexpression [ Sinceadenovirus-transduced cells donotintegratethe S gene and expressed adenoviral antigens induce host immune res ne expression is transient. Since the expression of suicide and cytokine genes in cancer gene therapy is intended to kill tumor cells directly or by activated host immune cells, permanent gene expression is, in contrast to gene therapy of metabolic diseases, not required. L

The efficacy of an adenoviral combination gene therapy approach with a suicide gene and the interleukin 2 genewas investigated in our lab by using a murine metastatic colon cancer model [93]. colon carcinoma was generated in the liver by intrahepatic implantatio 26 tumor cells. These colon cancer cells were chemically induced in e, poorly differentiated and highly tuof 3 X lo5 cells into the left morigenic in the syngeneic host ection lateral tip resulted in tumors measurin~5 to 7 mm in diameter after 7 days. These tumors were then treated by intratumoral injection of various combinations of recombinant, replication-deficient adenoviruses, expressing the herpes simplex virus thymidine kinase suicide gene Ad.RSV-TIC, mouse interleukin 2 Ad.RSV-IL-2, and galactosidase Ad. irusexpressingminterleukin2was synthesi p andpAd.~SV-mIL-2 in by calciumcoprecipitationoftheplasmids transformed h u ~ a nkidney cells. pJ 7 is a plasmid ~ontaining thecomplete humanpathogenAd5adenovirusgene,andpAd.RSV-mIL-2wasgenerated by insertion of the rous sarcomavirus long-terminal repeat promotor and the gene for murineinterleukin2intopXCJL.1, k' ovided by FrankGraham, University. Coprecipitation results in adenovirus with deletion o generegion 1 (El)and replacementberleukin2gene. Thus these recombinant adenoviruses can onlyreplicate in transformed 293 cells providing the E l gene and are replication-deficient in normal and tumor tissue. To reduce the risk of recombination with wild-type adenovirus, resulting in replication-competent recombinant adenovirus, the E3 region was point mutated first-generation and deleted in second-generationrecombinantadenovirusesAd.V-IL-2 was amplifiedfrom a single plaque and purifiedby using discontinuous cesium gradient ultracentrifugation. The titerof infectious particles was determined as plaque forming units (p,f.u,) utilizin~a plaque assay in 293 cells. The synthesis of the suicide gene adenovirus

d

Adwas similar to synthesis of thecytokine expressinga [96,97]. bee Thefunctionality of theadenovirus SV-IL-2 wasdemonstrated by transcells in vitro and measuring the mIL-2activity by using a T cell reformed tumors were injected at day

-1L-2, 6 X lo8 p.f.u. Animals were then treated for the 6 days with ganciclovir at 35 mg X kg" intraperitoneally (LP.) perday.aftercompletionof ganciclovir treatment animals were killed. esidual tumor areas were measure ric point count analysis of the maxim

cancer cells and syngeneic -@Galcontrol gro ormed. Ad.RSV250

n

W

150

100

50

0

S

Residualtumorareasaftervarioustreatmentmodalities.Theresidualtumor area reflects the computerized morphometric analysis of the maximal cross-sectional tumor area.Animal per group: Ad.RSV-~GAl:n = 13, A~.RSV-mIL-2:M = 8, A d . ~ S V - ~ G A L AD.RSV-mIL-2: n = 7, Ad.RSV-TI(: n = 12, andAd.RSV-TI( ~D.RSV-mIL-2:n = 10. (~eproducedfrom PNAS with permission.)

+

+

-1L-2 were 100% (616) protected against that protectionagainst

mice treated with Ad.

cific because none ofthe cha ne cell types involvedin this rejection further in vitro, cytotoxic ) assays were performed. Splenocytes isolated from untreated animals as well as with the control virus A d . ~ S V - ~ did ~ a not l show CTL activity CA-26target cells. Combinationt merit witfa Ad.RSV-T resulte~in a significant increase of dated from mice after treatment

couldcompletelyabolish not show any effect.

in vitro

e reduction and neumor is associ-26 tumor cells. h o u ~ han effective antitumoral T cell response developed in the animals after + mIL-2 vector, the cytolytic activity could not persist. Therefore, long-term

1

n U

@-gal

IL-2

B-gal

+

IL-2

TK

TK

+ IL-2

Treatment groups

Systemic immunity against parental tumor cell challenges at distant sites. Subcutaection of tumorigenic doses of CA-26 and MOD. Evaluation 14 days after challenge. (Reproduced fromPNAS with permission.)

80

tk+mlL-2

60

40

tk

20

mlL-2 normal &gal + mlL-2 8-gal

0

Cellular immunity against parental cancer cells after various treatment modalities using an in vitro CTL assay.E / T ratio represents the proportion of#effectorcells (activated splenocytes)totargetcells(tumorcells).Targetcelllysis is quantified bychromium-51 release. CTL, cytotoxic T lymphocyte. (Reproduced from PNAS with permission.) survival could not be achievedby this combination treatment.To achieve long-term protectionagainstrecurrenceandmetastases,cytokinesthatcanregulateother immune effector cells, e.g., N cells, macrophages, and a n t i g e ~presenting cells, are imperative for thisprotective immunity. The adenovirusesexpressing K - l 2 and -CSF were tested in this metastatic model. 40 ro,

8 30

anti-CD8 anti-CD4 NO Ab

._. v)

20

10

0

Blocking of the CTL response against parental cancer cells in vitro using antibodies directed against CD4+ cells and CD8+ cells. Splenocytes were isolated from animals binantinterleutreatedwith Ad.RS~-TKand AD.RSV-mIL-2, in vitrostimulated wit kin 2, andincubatedwithtargetcells i hepresence of either antiantibodies (RM4-5 andRN63-6.7,Phaingen)atincreasingprote cell ratio of 50: 1. CTL, cytotoxic T lymphocyte.( eproduced from PNAS with permission.)

vi Interleukin 12 has been shown to stimulate the growth and activation of Tcells but also natural killer cells [99-1021. Systemic toxicity resulting from the applicationof recombinant interleukin 12 in clinical studies is a severe limitationin this experimentaltreatmentmodality [103-1061. Cytokineexpression andsecretion within the tumor or its vicinity can potentially minimize the toxicity observed when recombinant interleukin 12is administered systematically. Interleukin 12 is mainly produced by antigen presenting cells (APCs) [ 107-1091 and facilitates the proliferation of Thl cells [ 110-1 121. The antitumoral activity is basically mediated by T cells and NK cells producing y interferon [ 113-1151. Furthermore,aninhibitionoftumormediatedangiogenesis by aninterferon-inducibleprotein 10 hasb leukin 12 is a heterodimeric proteinconsisting of a 40 th subunits are required for functional activity. A recombinant adenovirus expressing murine interleukin 12 wasgenerated and tested in an established animal model [l 191. The cDNAof the subunitsp40 and p35 was cloned by RT-PCR from total RNA after pokeweed mitogen stimulation of mouse splenocytes. TheDNA wassequenced andbothfragmentslinked to an encephalomyocarditis virus internal ribosome entry site (IRES), obtained from NOVAGEN (pCITE-l), The resulting construct p40-IRES-p35 was then ligated into the multiple cloning site of the E l deleted adenovirus backbone plasmid pAd.1/ RSV. The recombinant adenovirus Ad.RSV-IL-l2was generated by copreci~itation of pAd.RSV-IL-l2 with pBHG10 in 293 cells. The functionalassay was performed by incubation of -26cells withAd.RSV-IL-l2andtransferofthesupernatant after 48 hours to splenocytes. After 48 hours y interferon wasdetermined by enzyme-linked immunosorbent assay (ELISA)( E ~ D O ~ E N ) . Preformed hepatic tumors measuring 4 x 4 to 5 x 5mm after injection of MCA-26 cells were treated by local injection of either5 X lo8p.f.u. Ad.RSV-IL-12 or a control virus, Ad.DL312. Tumors in the treatment group showed significant mor volume reduction (Figure 5 ) with a mean tumor volume reduction of 8 0 ~ 0 . istological analysis revealed replacement oftumors by fibrosis with strong inflammatory response basically caused by infiltrating lymphocytes, macrophages, and neutrophils.Treatmentwith interleu~in12-expressing adenovirus resulted in a highly significant increase of long-term survival (p < 0 . ~ 1 over ) treatment with the controlvector (Figure6 ) with 25% (3/12) surviving animalsafter "70days.

Granulocyte macrophage colony stimulating factor (GM-CSF) has a broad range of functions as a growth and survival factor as well as an enhancerof the function of mature blood cells [120-1221. Most of its properties affect progenitors of granulocytes, monocytes, and eosinophils as a growth factor, extending their lifespan and augmentingtheirfunctionalcapability, ~eutrophilsandmacrophagesshow enhanced anti~ody-dependentcytotoxicity. In addition, GM-CSFis an important immunopotentiating factor, expanding potent antigen presenting cells [123-1251 as well as augmenting antigen presenting abilities of mature macrophages [126,1271, and therefore dramatically enhancing the host response to antigen.

1. 1250

1000 A

750

500

250

0

Ad.2DL31

Ad.RSV-mlL12

Tumor volume analysis after intratumoral injection of Ad'RSV-mIL-12. Seven days after tumor cell inoculation, tumors ~easuring4 x 4-5 x 5 mm were injected with either 5 X IO8 p.f.u.Ad.DL312 (n = 4) or 5 X IO8 p.f.u.Ad.RSV-mIL-12 (n = 7). Animals were killed14 days after adenovirus injectionand tumor volumes were measured (analysis of variance [ANOVA],p < 0.0001). (Reproduced from PNAS with permission.)

Long-term survival of animals treated with Ad.RSV-mIL-12. Seven days after tumor cell inoculation tumors were injected with either Buffer(G), n = 4; Ad.DL312 (F), n = 7; Ad.RSV.mIL-12 (E), n = 12. Animals were observed for survival, log rank test, p 0.0001. (Reproduced from PNAS with permission.)

o obtain in situexpressionof G -CSF within thetumor a recombinant adenovirus was constructed by delivering the gene for murine G cells. The therapeutic effect of intratumoral administration of was investigated in a CC36 murine metastatic colon cancer model [ 1281. ease the viable tumor volgical examination revealed that massive infiltranohistochemical analysis revealed that infiltrating cells. Tumors injected with infiltration. In addition, CTL in mice treated with the

+

V-IL-2, consistent with enhanced ous challenge remained up to 150 days, significantly longer than in animals treated with Ad. -2. Systemicimmunity is also reflected in the outcome of long-term survival dies. ~ n i m a l streated with the triple combination survived longer than those e Cp C 0.01) and 25% of the treated animalsachieved n demonstrated that the additional administrationof es the initial inflammatory response, mostlikely activates antigen presenting cells, and synergistically to IL-2 activates and enhances the proliferation of cytolytic T cells, resulting in long-term survival.

. that kills virtually any target is independent of antigenicity their tumor target within hours, in se the same tumor target in several is is induced by exocytotic granules d LAK cells as adoptive therapyin combination with recombinant interleukin 2 resulted in clinical responses for a variety of different 91 but improved survival attributed to LAK was only observed in kin 2 therapy alone, and compared n to recombinan * further improve survival. administration of cells (malignant ascites) is currently under investigation[ 1401.

ize tumorantigens

expanded fro in vitro using recombinant in

in either a

[TILS]) hocytes

by

They are usually cultured for 30 to 60 days, resulting in a lO,O~-foldexpansion

with more than 10" cells available for therapy. To study in vivo distribution, TILS were indium-l l l-labeled and administered to patients. After pooling in liver and [ 151,1521. Crosslung,progressiveaccumulation in tumor deposits was found reaction of human TIL against melanoma suggested shared human melanoma antigens [153-1551. Analysis of shared melanoma antigens recognized in a class I fashion is under investigation; several of these antigens have been discovered, inc~uding -peptide, presented in human leukocyte antigen A1 ( LA-A1) [156-1601. The direct therapeutic value of TIL remains to be evaluated, whereas shared tumor-specific antigens may be discoveredby using TIL. The localization of micrometastases using radiolabeled TIL is also of clinical value. Strategies to improve adoptive therapyusing cultured autologousT lymphocytes includegenetic modificationandcombination withchemotherapy or coadministrationwithcytokines [148,161,162].

. Activated monocytes or macrophages increase their phagocytic activity and tumoricidal properties by a variety of agents (IFN-7, bacterial proteins) [163vated macrophages kill tumor cells independently of specific antigens or killing requires days rather than hours. Peritoneal macrophages have been shownin a mouse model to be protective against €316 melanoma micrometastasis after in vitro activation with lymphoc~e-derived supernatants [ 1661. With advanced cell separation techniques and leukapheresis it became possible to isolate 1-10 X lo8 tumoricidal macrophages per week in humans. Several phase I clinical trials have been initiated but with no therapeutic benefit so far [167]. This experimental approach is limited to the quantityof tumoricidal macrophages that can currently be prepared;furthermore,macrophages,onceadministered,cannotbefurther expanded like LAK with recombinant interleukin 2[138].

In the development of new approaches to cancer treatment gene transfer technologies have become the most promising and exciting tools available. The first gene transfer approach was the introduction of the NeoR marker gene into TIL using retroviral gene delivery systems. Ongoing clinical trials are using this technique to transfer TIL after in vitro transduction with cytokine encoding genes and to improvethetherapeutic efficacy of suchanapproach.Otherapproaches in gene therapy of cancer involvethe direct modification of tumorcells in vivo, either to kill them by the expression of suicide genes, to increase their immunogenicity, or to activate and expand host immune cells. Recombinant adenoviruses with the possibility of high titer concentrations and high transduction efficiencies are invaluable tools for suicide and cytokine gene transduction in vivo. Preclinical studies using interleukin 2 and interleukin12 expressing adenoviruses showed increased cytotoxic activity in T cells and NK cells, resulting in significant survival benefit. The discovery of tumor antigens opensnew perspectives in cancer treatment, and the incorpo-

ration of genes encoding for these tumor antigens into viruses may offer new options in cancer treatment.

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P

Infectious pathogens have developed mechanisms that not only establish a close interactionwithc tissues of the infected hostbutalsoallowfortheevasion . Evasionfromtheadaptiveimmunesysteminvolves variofhostimmunee ous mechanisms,ginductionofimmunesuppression and escape from immune recognition. Thus, theinfecting agent prevents its elimination and the destruction of the host before the pathogen has reproduced and propagated into another host [l]. Ac~uiredimmunodeficiency syndrome (AIDS) provides a spectacular illustration of the capacity of a pathogen to induce a progressive and complete loss of etence. In the infected host, the death of a lymphocytic population, helper cell, results in a loss of the control of the adaptive immune response [2,3]. There are two major ways in which cells are knownto die. The firstrecognized form of cell death, necrosis, is always associated withdisease and is a conse~uence of cell destruction by several agents,suchascomplement,chemicalagents,or ring necrosis, cell swelling and rupture of the cell membrane lead to the proteases and other intracellular toxic enzymes. An inflammatory reaction and a scarring process occur that disorganize the architecture of the tissue or In contrast, programmed cell death involves an orderly process of isintegration that has been termed apoptosis [6]. This process includes shrinkageof the cell and thenucleus, condensation andfrag~entationof the nuclear chromatin, maintenance of plasma membrane integrity despite membrane blebbing, disruption of the mitochondrial transmembrane potential, and segmentation of the cell into apoptoticbodies that are rapidlyingested by neighboring epithee most important difference between programmedcell death and necrosis is en a cell undergoes necrosis, the only way to prevent death is to remove the causative agent. In contrast, programmed cell death is regulated by signals provided by the local environment. Programmed cell death can be induced or suppressed in most cell populations by the withdrawal or addition of defined

activation signals. Its inductionor prevention depends on theexpression of defined genes [4,7-91. Thus, cell death can beprevented in most casesby the modulation of cell signaling. In this review, we will discuss the role of cytokines in the regulation of programmed cell death in human immunodeficeiency virus (HIV) infection. Specifically, AIDS may be related to a form of tolerance caused by the dysregulation of physiological T celldeath. Thisis a consequence of interference by the HIVwith the intercellular signaling requiredfor cell survival. The HIV-mediated dysregulation of programmed cell death thereby represents a particular and dramatic example of a strategy that has evolved in infectious pathogensto manipulate the immune system.

uman immunodeficiency virus infection usually leads to AIDS, which is characterized by cell loss and tissue atrophy in several organs. The major pathological features of AIDS are the progressive collapse of two complex regulatory networks of the human body, the immune system and the central nervous system [2,3,10]. Acquired immunodeficiency syndrome induces the loss of CD4 T cells in the immune system as well as neurons in the brain. Dysregulationof programmed cell death has been hypothesized to explain the two major defects of CD4 T cells from HIVinfected individuals that lead to immune i ompetence: (1) early in vitro dysfunction and (2) late in vivo depletion [l 1-131. sregulation of programmed cell death also provides a possible explanation for two paradoxical features in HIV-infected individuals. Thefirst is thepersistenceof a lo ercentageofperipheralblood CD4+ T cells thatare productivelyinfectedb V andthat expressviralribonucleic acid (RNA) [14,15]. The second is the death of neurons in the brain. In the brain, neurons, in contrast to CD4T cells, are not targets forWIV infection. In the central nervous system HIV infection is expressed primarily in cells of the macrophage lineage, suggesting that it can cause the death of cells that are uninfected [16,171. In AIDS, immune system cell dysfunction occurs priorto the detection of cell depletion. CD4+ T helper cell defects are characterized by a selective loss of memory function. These qualitative defects include a failure of CD4 T cells to mediate delayed-type hypersensitivity reactionsto major histocompatabilitycomplex classI1 restricted antigens and a selective loss of T cells to proliferate in response to T cell receptor stimulation either by recall antigens, by antibodies directed to the CD3/T cell receptor complex, or by defined polyclonal activators suchas polkweed mitogen [18-231, A paradoxical feature ofcell dysfunction in HIV-infected individuals is the chronic activation state of the immune system. This activated state occurs in the presence of a dysfunctional CD4 T helper cell population. Immune activation involves cell populations that are either permissive for HIV (monocytes) or nonpermissive (B cells and CD8 T cells). Persistent B and CD8 T cell hyperactivation may be a truncated and inappropriate form of helper immune function provided by the dying CD4 T cell. Since programmed cell death is an activepr associated with transient lymphokine secretion [24] and release of nucleosomes [25] The initialfinding by Grouxet al. E261 was that abnormal levels of pro-

programmed cell death induction alsoinv CD8+ T cells can

Whole body irradiation

T cells [27-311, The death of of HIV-infected individuals

of primates infected with the simian

'

sive induction of programmed cell death that involves CD4 thymocytes more than thymocytes occurs 1351. These observations are consistent with the hypothesis in the induction of abnormal apoptosis of mature T cells a mechanism may be as effective in inducing programmed cell death in immature precursors, thus interfering with T cell renewal. This possibility is further supported by the observation that similar thymic depletionis noted in rhesus macaques infected with a pathogenic strain of SW, but not in the macaques infected with a nonpathogenic molecular clone of SW. In bothcases, the thymus is an early target ofviral infection [36,37]. Thus theessential question, Does programme^ T cell death play a central role S pathogenesis? Or is it merely a consequence of an ongoing and ineffective stimulation of the immune systemin a chronic lentiviral infection? To address this question, we have compared the induction of in vitro programmed T cell death of IV-1 infected individuals as well as programmed T cell death in various primate models of HIV infection. This comparison allows one to discriminatethe biological featuresassociatedwithpathogenicversusnonpathogenic chronic lentiviral infections [31]. In infected and uninfected primates, the fol experimental systems were used: chimpanzees experimentally infected with in which disease does not develop; African green monkeys naturally infe SIVagm in which disease does not develop; and rhesus macaques experimentally infected with either a viral strain of SIVmac that induces AIDS or a recombinant molecular clone of SIVmacthat does notlead to disease (Table l). Abnormallevels of activation-induced programmedcell death of the CD4 T cells were only observed in theinfections leadi to AIDS:rhesusmacaques infected wit41 a pathQgenic strain of SIVmacandIV-l-infected individuals. Incontrast,enhanced in vitro activation-induced programmed cell death of the CD8 T cells could be detected in both pathogenic and nonpathogenic chronic lentiviral infections. Similar findings have also been reported by other 1aboratQries. Abnormal levels of in vitro programmed cell death of peripheral blood T cells have been shown in primate and of pathogenic lentiviral infections that induce~IDS-relateddiseases, IV-l-infectedchimpanzeesinwhich disease doesnotdevelop [27, 38-401

AIDS and 1:Cell Apoptosisa AIDS IV-

Human

1

Host IV- 1 SIV mac251 (isolat) SIV mac251

Viruses

African agm SIV green monkey

+ -

+ -

Apoptosis CD4 CD8

+ _.

+ I

-

+ + + + +

"AIDS, acquired immunodeficiency syndrome; HIV, human immunodeficiency virus; SW, Simian immunodeficiency virus Source: Ref. 3 1 .

Takentogether, these datasuhatabnormalpriming cells for programmed death occur ~-l-infectedhumans ~ifferentprocesses. Ononehand, ell deathappears clos pathogenesis, whereas C 8 T cell death may be an indirectconsequence of immune cell priming may occur during both patho~enic and nonpathotions. A pattern of similar abnormal in vitro programmed Tcell death restricted the to C individuals arewho ~haracterized as long-term nonprogressors or long-term survivors ha quier et al., unpublished observation^. Inappropriate cell signaling through the b i n ~ i nof~the glycoprotein recep r may induce abnormal programmed ytopathic effect of IV-1 mediated by the viral envelope pr by the addition of an a nvelope antibody that interfered with the binding of the IV envelope interaction to the C e induction of programmed deat ~ubsequentfindings [45-471 haveindi ressed by infected or transfected cells, and t T cells induces programmed cell death. This selective inhibitors of T cell activ do not inhibit bindingo may act by modifying indicated that thecross gp 120 plus anti-gp 120 antibodies primed purified programmed death. The programmed cell death owever, programme^ cell death of ed in the absence of T cell receptor

considered in the broader context of the primate models of patho~enic and nonic lentiviral infection. In these mo Is, the binding of the viral envelope to olecule is a feature that is requir cell infection and is shared

by all lentiviruses. Therefore, one is postulate that subtle differences in are sufficient for the radical differmolecules or in thelentiviral n in the capacityof envelopeeraction to induce programmed cell tal in vitro system using envelopes death. Such a pos§ibility awaits an 11s from different primatespecies. from nonopathogenic lentiviruses an Inaddition tothe infection of , a generalproperty of lentiviruses is their tropism for accessory cells such as monocytes/macrophages. Lentiviral infection of monocytes/macrophages may inducea defect in their accessory cell function. This may lead to programmed T cell death through the inappropriatedelivery of activation cosignals required for normal Tcell activation. he two-signal hypothesis of T cell activation implies that T cell differentiation and proliferation require both T cell receptor stimulationby antigen and appropriate cosignaling provided by anti en presenting cells. Antige resenting cells, such as monocytes, dendritic cells, ry molecules, such as cells, provide membrane athogenic lentiviruses le factors such as cytokine grammed T cell death, simply by altering accessory cosignals required to prevent T cell deathin response to a given activation signal. Oralternatively,pathogenic lentiviruses may lead to anincrease in the intensity and durationof T cell activation that renders inoperative preventive cosignals present in the T cell environment. Some observations argue that V-l infection of accessory cells may indeed play a role in programmed T cell dea nduction. It has been proposed 1381 but is as of yet unconfirmed [51] that all primate lentiviruses fail to infect chimpanzee monocytes. Thus, the lack of in vivo pathogen~sisof V- l-infected chimpanzees as well as the lackofpriming of peripheralbloodT S for in vitroprogrammed cell death induction in this model would not berelated to benign cytopathic propertiesof these viruses but to a lack of infectivity of monocytes. Other studies [52] using SCI mice reconstituted with t human T cells and monocytes and infected with ous molecular clones of -1 show similar data. These studies suggest thatmolecul that infect monocytes and ar opathic for CD4 T cells nd profound in vivo depletion T cells as compared to at are highly cytopathic forCD but poorly infect monocytes. mode this In cell depletion by programmed death 4 T cells results when cells are in the

+

neurons do not seem to be nfected have been shownto eurons normally depend on signals provided by other cell populations in order to prevent programmed cell death induction. Therefore, the inappropriate delivery of survival fected accessory cells may represent a unifying mechanism by which infected macrop promote immunological and nonimmunological defects (pathogenesis) in T cells, hematopoieticprogenitors,andneurons.

Cytokines play a critical role in the immune system’s response to pathogens [l]. 4 T helper cells have been shown to segregate into two functional subsets, based on their cytokine secretion pattern. The Thl cells secrete IL-2 and

y interferon, promote macrophage activation, and induce cell-mediated immunity and delayed-type hypersensitivity reactions. The Th2 cells secrete IL-4, IL-5, and IL-10. These cytokines favor the optimal activation of B cells to secrete antibody ‘ such as immunoglobulinE and promotemast-cell and eosinophilactivation 155-581. Therefore, differentiation of CD4 T cells into Thl or Th2 cells in response to invasion by an infectious pathogen represents a crucial event in determining the nature of the immune response and outcome of the infection, These can vary to include persistenceof the pathogen, protection of the host, or immunopathogenesis. The Thl- and Th2-defining cytokines can be produced by cells other than CD4 T cells, including differentiated CD8 T cells and activated accessory cells such as B cells, macrophages, natural killer cells, basophils, and mastcells. Cytokines secreted by accessory cells activated by the invading pathogen play an essential role in the decision of naive CD4 Th precursor cells to differentiate into Thl or Th2 cells. In particular, IL-l2 secreted by activated macrophages favors Thl-cell induction. Interleukin secre 4, S , basophils, mast and cells, together with IL-10, secreted by e, favors Th2 induction. Several studies have suggested tha e pathogenesis and persistence of various chronic bacterial, viral, or parasitic infections involve a Thl to Th2cytokine switch [59-621. Assimilating these postulates with the hypothesis that costimulatory si~nalsof accessory cells play a key role in the control of T cell survival and T cell death, we have explored the possibility that pathogen-mediated down-regulation of the Thl cell lve programm~d T cell death induction. tion, the early functional defects of cell-mediated immunity that T cell depletion involve the CD4 Thl cell population. They lead to a on of delayed-typehypersensitivityreactions. Incontrastto G. Shearer and S. Clerici’s hypothesis that the loss of CD4 Thl cell function may be due to aprogressive shift of Thl to Th2[63,64], we have observed a p Thl response. Stimuli that induce programmed death in T cells from individualsprovide levels of IL-2 and y interferon secretion that V-inf~ctedindividuals and healthy controls.No significant IL-4 or IL-5secretion S detected in HIV-infected individuals up to several days after in vitro stimulation. I~terleukin 10 secretion was similar in activated T cells from HIV-infected individuals and controls [65,66]. This observation of a lack of Th2 cell expansion is er results describing cytokine messenger RNA analysis of the -infected individuals [67,68]. These data suggest that the progressive loss of in vitro and in vivo Thl cell response, which characterizes AIDS progression, is not related to an absence of Thl CD4 T cells. Nor is it related to a down-regulation of Thl cells by an expanding Th2 cell population. The loss of a Thl cellularresponse is due to the stimulation of Thl cells fromHIV-infected that induces rapid death by apoptosis. V infection, the addition of antibodies blocking Th2 cytokine IL-l0 or IL-4 binding or the additionof the Thl cytokine IL-12 hasbeen reported to restore the early defectivein vitro proliferative response of T cells to stimuli [63]. Moreover, the addition of antibodies to IL-l0 or the additionof IL-12 has a preventive effect on abnormal programmed cell death induction in response to in vitro stimulation. These observations further support the postulate thatin HIV infection the early T cell functional defects are related to abnormal programmed T cell death induction [65,66,69]. Together9these observations imply that T cells from

related to animbalanc of environmental survival signals, or toa terminal differentiation stage of the T cell. owever, defensive mechanisms have evolved in so lymphocyte su~§et§. In pa cular, the perforin-granule exocytosis pathway of T cell killing mayensurethe classical defense resp against infected cells, T cells may be directed as-mediated apoptosis that canbe inducedby tivated syngeneic lymphocytesand have a ro mmunoregulation P31. t a major pathway in the encesuggests thatas/FasLmayrepre such, this pathway may regulation of cellular function in the body [94 cytes, and participate in control homeostasis, eliminate normal activat autoimmune dise ne privilege [l05- 1071 g

leads to programmed eel either antibodies to Fas, cells exture T cells constitutively express ssion and makes theT cell sensi-

are able to kill Fas-expressing activated lymphoc The Fas and FasL may interact on the same cell 1 as cyclosporin A inhibit the induction of FasL expre§sion on T helper§ubpopulations, Thl mongthe classical nd lyse, in Fasa manner, more readily than [88,91]. The Th2 cell re less sensitive to Fas-mediatedprogrammed cell death. They express Fap-l , ed phosphatase im~licatedin the prevention of death of Thl effectors is dependent on Fas and FasL expression, whereas the slower Th2 death is observed even from Fas- and cells, which are professional cytotoxic T cells,

S is enhanced by a protein synthesis ins are required in the control of the Fas sensitivity. ~nterestingly, our observation indicates thatcytokines may control eviously, IL-l2 upl apoptosis. It also ed individuals [66].

Activated cell

\Fas ligand

CD4 T cefls

CD8 7: cells

I Apoptosis Apoptosis Cytokines participate in the controlof Fas-mediated T cell apoptosis: Cytokines in a specific manner prevent CD4 + and CD8 + T cell apoptosis after Fas ligation mediated by soluble Fas ligand or an agonistic anti-Fas antibody.

induced immunodeficiency syndrome in mice. In human peripheral blood lymphocytes, the cross-linking of CD4 mole~ules, either by an antibody or by the HIV envelopeproteingp160,up-regulates the expressionof Fas. Thisup-regulation closely correlates with the occurrenceof apoptotic cell death. The injection ofmice with antibody to C 4 leads to rapid Fas-based apoptosis of T cells. In addition, human T cell lines transformed with WIV are more sensitive to Fas-mediated apoptosis than the parental cells [l 12-1 141. 8 T cells from HIV-infected individuals undergo programmed cell ~ e a t hin response to not only Fas li~ationbut TCR stimulation. The use to FasL or antagonisticFas a n t i b o ~ yprevents C either antisense oligonucleotides T cell death but not CD8 cell death 1081. These data, in addition to theanalysis of A expression from ndividuals [110,1151, suggest that the TC programmed cell T cells involves a Fas-mediated signaling ~athway,whereas TCR stimulation of C 8 T cells leads to a different death process. It has been reported that TCR-induced death of 8 T cells occurs through a p75 T~F-receptor-mediated mecha~ism ition of TNFa did not induce apoptosis in CD4 and CD8 T uals. The a ~ d i t i o nof a n t i - T ~ Fantibodies showed mediated programmed cell death. Therefore, additional experiments are required to determine the pathway ofTC cell apoptosis. Ligation of Fasor binding of TNT; to its receptor hasrecently been implicated as si nalingintermediaryproteins.Proteinssuchas T beardeathdomains resemblingthose in CD95 an 7: expression of these proteins induces apoptosis (S 119). The formation of complexed proteins through Fas or TNFactivation induces

the cell death machinery including the protease cascade. At the apex is FLICE/ MACH1, recruited by FADD/MORTl, which primes for a second transcleavage. Activation of interleukin 1 converting enzyme (ICE) family-related proteases, the ICE subfamilly members formedby ICE, ICE re1 111, and ICE rel I1 (also known as TX and ICH2), and the NEDD-2 family members ICH1, leads to a second protease cascade. One critical target of this cascade is CPP32p (also known as Yama, and Apopain).OthertargetsincludesubfamilymembersCPP32,Mch2,Mch3(also knownasICE-LAP-3andCMH-l),and Mch4. The targets of theseproteases include the nuclear enzymes poly(adenosinediphosphate[ADP]-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK), both involved in aspects of DNA damage sensing and repair. Additional targets are U1 small nuclear ribonucleoprotein and the nuclear lamins (lamins A, €31/B2, and C), and, in the cytoplasm, Gas:!, a component of the microfilament system, protein kinase C6 (PKC6) and various componentsof the cytoskeleton, such as actin and thesterol regulatory element-binding proteins SREBP-1 and SREBP-2 [120,121]. ICE and CPP32 are highly selective in their proteolytic activity [122-1271. A series of peptide analogue substrates has elucidated the role of these proteases in the programmed cell death process. It has been shown that ICE cleaves a sequence composed by the tetrapeptide YVAD, whereas CPP32 cleaves a DEVD sequence (Figure3). Fas antibody-mediated and soluble Fas ligand-mediated apoptosis of CD4 and CD8 T cells from HIV-infected individuals maybe prevented by pretreatment with the Ac-YVAD-CMK peptide, an irreversible competitiveinhibitor of the ICE cysteine protease family[1081. Additional experimentsusing reversible aldehyde competitive inhibitors of cysteine protease specific to ICE (Ac-YVAD-CHO) and CPP32

Ac-YVAD-CH0

Ac-DEVD-CH0

Apoptosis

Figure 3 Cyteine proteases and apoptosis: T cell activation inducing Fas ligand expression

results in Fas signaling that involves cysteine protease cascades whose ICE and CPP-32 play a major role. Catalysis involves a typical motif composed by the sequence QACRG. These in aspecificmanner by antagonists YVAD and DEVD twoproteasesmaybeinhibited tetrapeptide and offer new approaches to modulate Thl apoptosis. ICE, interleukin 1 converting enzyme.

cise n a t u r ~of the rot eases involved, our findin~sstrongly r o ~ r a ~ cell ~ e death d and protease activations in -infected individuals.

YtO

Thl apoptosis and therapeutic approaches, Three different approaches may be to prevent Thl apoptosis: (l) blocking the interaction between Fas and Fas Ligand; (2) mo~ulatingFas sensitivity through the use of cytokines;(3) inhibiting the protease cascade P32 etc.), mediators of cell death. ICE, interleukin 1 converting enzyme.

tant to explore

whether the recentlydescribedbeneficialin

vivo effect o

and potent regulatory role of cytokines on the activation and defined T cell populations. cell sensitivity to Fas-medi

dif

Viruses that induce programmed cell death include pathogenic lentiviruse a multitude of pathogens with different degree and tropism such as intra pathogens, bacteria, or extracellular parasites can in uce programmed cel Therefore, although cell survival dysre~ulationmay not in itself induce disease, it may play an important role in allowing pathogen survival. If that is true, strategies aimed at countering pathogen-mediated dysregulation of programme may have important therapeutic implications for the control of infectious diseases.

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Case Western Reserve University, University ~ o s p i t a lof s Cleveland, Cleveland, Ohio The ~ i r i ~ao s~p i t a lBrown , ~niversity, ~roviden~e, Rhode Island

1.

I

The rationale for trials of immune-based therapies for treatmentof human immunodeficiency virus l (HIV-l) infection and its complicationsis based upon the inextricable relationship of HIV-l to immune responses and immunologically competent cells. In this regard there are four fundamental observations that provide the underpinnings for these treatment trials: First, HIV-l infects immunocompetent cells and as an obligate intracellular pathogen must collaborate with host elements for successful completion of its life cycle. These host elements may providelegitimate targets for antiviralstrategies. Second, HIV-l infectionis associated with laboratory and clinical evidence of immune activation, and immune activation potentiatesHW-l infection in vitro [l]. Thus strategies designed to modulate immune activation in the context of HIV-l infection may directly (or indirectly) result in down-modulation of HIV-1 propagation. Third,much of themorbidityof l infection is attributableto virusinducedprogressiveimmunedysfunctio theattendant risk ofopportunistic infections. Treatment of HIV-l infection with highly active antiretroviral therapy ( H A A ~ T is ) associated with evidence of partial immune reconstitution [Z], but to date there is little evidence that the profound perturbations of the immunologica~ repertoire (at least as measured by distribution of V beta families) is corrected with antiviral therapies [2,3]. For this reason, trials of immunological therapiesdirected at restoring immune function are warranted. Finally, immune-based therapeutic trials provide an important mechanism to l pathogenesis in the most relevant model for HIV-l infecd person. Carefully designed immune-based therapeutic trials can simultaneously test treatmentstrategies and clarify andextend pathogenesis hypotheses generated in the laboratory andin epidemiolo~icalstudies.

. Early treatment of attributable to several factors. First, the first g as defined by progress

an obligate intracellular pathogen,

ri~onucleicacid

ated by host polyme substantially longer generation time. in escape of a cell fr survival of this cell

gp120, is an essential ele-

facilitating recognition of cell antigen receptor complex. Trials of soluble this cell-membrane-b te achieving potentially inhibus infusions of soluble

combination have

st 10~0-20~0 of ~aucasiansare

rsons with this genotype are overrepresented -1 who are uninfected [ 1l ,121, and peripheral

obtained from such individuals demonstrate in vitro resistance to

S

may provide a useful

NAlysllL, to initiate zed dinucleotide triphosdeoxyribonuc~eic phates for aci y, hydroxyurea, inhiban itor of ribonucleotide red~ctase, hasbeen shown to inhibit IV-l propagation in vitro,particularlywhen ap * epresence of other reverse transcriptase inhibitors, especially didanosine . ~ l i n i c astudies l of hydroxyurea ad~inistration in combination with ddl have resulted in substantial, nearly 2 log inhibition of levels that exceed the inhibition predicted to be a result of ddl *

therapy alone [l 51. Ongoing comparative trials are comparing the antiviral effects of ddl and hydroxyurea alone and in combination and alsowill attempt to ascertain the durability of hydroxyureaeffects.

et Human immunodeficiency virus 1 ut transcriptional enhancer sequences such as nuclear regulator domains, ingSp-l sites, and NFAT-1 sites that are homologous to regions found in the promoter enhancer regions of a number of immunologically relevant genes, such as the genes controlling tumor necrosis factor, interleukin 2, and the interleukin 2 receptor alpha chain. Targeting these elements may provide antiviralactivity but may also impair important host immunecapabilities. ~lutathione-repleting agents,such as N-acetyl cysteine, L-5-oxothiazolidine-2 carboxylic acid (procysteine), and glutathione esters, can inhibit HIV-1 propagation in vitro through mechanisms thought to be dependent upon inhibition of N F - k ~ activation [16]. Clinical trials of these agents have not demonstrated evidence of antiviral activity. Severaltopoisomeraseinhibitors,topotecan,andcamptothecinhave been shown to inhibit HIV-1 transcriptional activation [17,18]. These agents also have antitumor activity, and trials of these agents orderivatives in HIV-l-related malignancy may testthe antiviralactivities of these agents. The HIV-l Tat protein is an important transcription enhancing factor that binds to early HIV-l RNAs and forms a multimolecular CO lex with a number of hostproteinsthat is thoughttoenhance processivity of -1 transcription. A familyofbenzodiazepine derivatives blockstheTat-medtranscriptional enhancement in vitro but does so without binding Tat or its RNA target, the HIV-1 TAR region. These agents inhibit HIV-1 propagation in vitro, and virus can be passed in the presence of these compounds for many months without the development of resistance, suggesting that these agents target a host element that participatesinTat-medi transcriptio~alenhancement 1191. A clinical trial of one of these substances, 4-7429, failed to demonstrate evidenceofantiviral activity, although plasma levels of Ro-24-7429 exceeded in vitro inhibitory concentrations 01. These findings have been taken to suggest that Tat may IV-1 activation particularlyin the contextof c~okine-mediate This conclusion is premature. In fact, ~o-24-7429is highly protein-bound, and this cteristic of the compound may underlie the failure of this reagent to block -1 expression in vivo [21]. Therapeutic strategies targeting Tat or host proteins to which it binds should not be abandoned on the basis of the dataavailable to date. e

Cyclosporine A is an immunosuppressive agent commonly used to prevent transplantation rejection andto treat autoimmune diseases. In vitro, cyclosporineA can IV-l propagation, and this inhibition of IV-l replication may be mediated throughoneormore mechanisms.First, theimmunosuppressiveeffectsof cyclosporine A may be mediated through inhibition of interleukin 2 (IL-2) receptor expression via interference with the activity of the nuclear factor NFAT-1, which

7

alsobindstothe HIV-1LTR [22]. Thisregionmaybedispensable for HIV-1 transcriptionalactivation,however.Perhapsmoreimportantly,cyclosporineA blocks the activity of cyclophilins A and B, which facilitate protein transport and IV propagation to facilitate polymerizationof the HIV-1 gag polyprotein [23]. Finally, through inhibition of T lymphocyte activation, cyclosporine A may decrease the susceptibility of CD4' T lymphocytes to completion of the HIV-1 propagation cycle. Resultsof clinical trials of cyclosporineAadministration to persons with HIV-l infection have not been promising. Early studies among French volunteers with advanced HIV-1 infection reported mixed results [24]. In contrast, a controlled trial of cyclosporine A administration to Canadians with advanced of accelerated acquired immunodeficiency syndrome (AIDS) was halted because disease progression in the treated arm [25]. The AIDS Clinical Trials Group will be initiating a trial of low-dosecyclosporineA(ACTG 334) to persons with early HIV-l infection and no evidence of clinical immune deficiency. The intent of this trial is to ascertain whether lower dosesof cyclosporine A can diminish the heightened immune activation state that characterizes all stages of HIV-1 infection and can thereby indirectly (or directly) decrease plasma levels of HIV-l.

Human immunodeficiency virus 1 proteins must undergo a number of posttranslational modifications. Proteolytic cleavage of the gag-pol preprotein is effected by viral protease, but cleavage of the gp 160 envelope preprotein is effected by a host protease. Envelope proteins are gycosylated, and several HIV-l proteins are fatty acylated by host enzymes. A recent trial of the glycosylation inhibitor SC-49483 (ACTG259) was unsuccessful largely because of intolerable gastrointestinaltoxicity without clear evidence of antiviral activity. Host-directedtherapiesforHIV-1are in the early stages ofdevelopment. Although these agents are likely to have predictable toxicities, targeting host elementsneeded for viral propagation mayrenderescapemutation unlikely and thereby provide a durable antiviral effect. Of the approaches discussed, targeting the HIV-1 chemokine coreceptors has generated the greatest enthusiasm, and compounds with activity against these receptors arein development.

Progressive and inexorable deterioration of immune defensescharacterizes the natural history of HIV-l infection, and it is the failure of host defenses that underlies the enhancedrisk of opportunistic infections (andpossibly malignancies)that characterizes most of the clinical manifestations of AIDS. Highly active antiretroviral therapy (HAART) can decrease therisk of opportunistic infectionsin persons with HIV-l infection [26], and recent data demonstrate that short-term administration of HAART can result in some evidence ofpartial immune restoration as characterized by increases in circulating naive and memory CD4' lymphocytes, increases in naive CD8' lymphocytes, increases in B lymphocytes, and enhancement of delayedtype hypersensitivity responses and lymphocyte proliferative responses [2]. None-

theless, perturbations in the the increase in circulating ly cytes orthefailure oflymphocytedeathor, if enhanonandmaturation underlie these Qbservations~ that repertQire perturb -1 infection occurs at a site of cell maturation and selection (e.g., thy pertur~ationsof the T cell a~timicrobialprophylaxis earlier mandate^ prophylaxi replication will pe it more complete immune r tion will be requir or even effective) in restori lymphocyte respo receive active antiviral therapies before immun This implies that persons at risk be encouraged since immune deterioration canprogress in the a illness until profound im threatening opportunistic infection. For the timebeing, therefore, efforts arebeing directed at the development of strategies designed to restore immune responses in persons with a infection.

there has been interest in trials of replacement of these cells in an effort to restore T lymphocytes in the immuneresponses. Ex vivoexpansion of autologous presenceof combinations of antiviralagentsthathasd in apparenteradica-

donors is ongoing. This strategyis also notlikely to expand the repertoire of antigen sponses, and it remains to be seen how long these infusedcells r e ~ a i nresistant to IV-1 infection. ~dministrationof allogeneic cellsIVl -~eronegativedonors may procomplete e repertoi sponsiveness, but by donor-recipient incompatibilities. e infusions followedby bone marrQw transplantation from an IV- l -seronegative donor to his IV-l-infected human leukocyte antigenentical twin [33]. Some return delayed-type hypersensitivity could be d after the infusions, but the benefit was short-lived. In the prepatients were treated with monthly infusions of era, 16 matched haplotype-mismatched lymphocytes obtained fro this unco~trolledtherapeutic trial, substantial increases in the numbers of circulat-

een, and surprising~y therewas no evipersonal communican no con fir me^ reports atients transfuse^ with ecently infusions cytes together with intravenous immunotantial increases in circulating lymp~ocyte e expansion of autologous cells or lly matched ~ymphocytes, susce~timiting step for cellular

enefit of the treatment; infusions revealed that approximutations in the Nef ~ e p t i d e of these cells exerted a selection ecific CTL expansions and reinther oli~oclonal e~pansions will

C compati~ility, and ntiviral therapies has

V-l can infect thymic tissue ncern that appropriate this i ~ p a i r ~ ewould nt r cells or bone marrow trans-

reliminary studies have been examining the feasibilityof allogeneic thymic transplantation, capitalizing on the reported success of thymic tissue transplantation in children with DiGeorge syndrome.~re~iminary data in DiGeorge syndrome indicate that recipient T lymphocytes matured in a donor thymus recognize recipient MHC antigens as self and can respond to peptides presented by recipient antigen presenting cells [35]. Whether thymic transplantation will permit T lymphocyte reconstitution in persons with advanced HIV-l infection remains to be determined.

. 1. Cyto~ine lnhi~ition in the Treat~ent of ~ I V - lise ease The proin~ammatorycytokines tumor necrosis factora (TNF-a) and interleukin 6 (IL-6) are not structurally related but have considerable overlap in their activities. In addition to costimulationof immune responses, both can increase HIV-l replica‘ in vitro and have been proposed to play a role in different manifestations of disease. Reviewing the in vivo trials of inhibitors of these cytokines can help us rstand cytokine network regulation, the role of cytokines in the pathogenesis of HIV-1 disease, and the potential usefulness of their inhibition for the treatment of HIV-l disease or its complications. Tumor Necrosis Factor a Tumor necrosis factor a is a homotrimer produced by many cells, but its primary source are macrophages. Both TNF-a receptors are expressed on all nucleatedcells, explaining thiscytokine’s broad range of actions. In immune cells, TNF-a increases synthesis of IL-6, IL-l, and other cytokines; enhances monocyte microbicidal activities, and is important in defense against intracellular pathogens [36-381. BothTNF-a levels and soluble TNF receptor levels infected patients. Levels of TNF-a aredirectly correlated wit and inversely correlated with CD4 and CD8 cell counts [40]. levels are predictors of HIV disease progression [41]. The relationship between HIV-l replication and TNF-a production seems to be bidirectional. Invitro,HIVinfectioninducesexpressionofTNF-a [42]. As mentioned, TNF-a levels are directlycorrelated with IV RNA levels, and treatment with antiretroviral agents decreases TNF-a levels [43,44]. The decrease in TNF-a levels correlates with the decrease inWIV RNA levels. Exogenous TNF-a activates expression of HIV-1in chronically infected T cell lines through activation of NF-KB[45]. In vivo, intravenous TNF-a administration to five HIV-infected patients with Kaposi’s sarcoma produced increasesin p24 antigen levels of more than 50% in three patients 1461. Decreases of HIV RNA levels er TNF-a inhibition would further S stantiate the role of TNF-a in i ~ ~ r e a s i n g V-l replication, and consequently, i IV disease pathogenesis. Activity of TNF-a can be antagonized by either inhibiting its production or binding the cytokineto prevent interaction with its receptor. Thalidomide is a modest but selective inhibitor of TNF-a production. In asymptomatic HIV-infected patients with CD4counts greater than 400 cells/pl, thalidomide at a doseof 100 mg given daily produced undetectable blood levels and no change in ex vivo cytokine production [47]. Most recently, thalidomide has been proved an effective therapy for idiopathic aphthousulcers complicating HIV disease 1481. Nevertheless, it is not

S

ide’s effects are related toTNF-ainhibition becauseplasma TNF-a levels and plasma evels increased after its administration [NI. TNF-a production, has been shown to -amessenger levels modestlypatients in with advanced ad, as assessed by different methods, was these agents to decrease viral load may be agents tested. In another study, patients with no op~ortunisticinfections but advanced IV disease, as as pl, were given a chimeric huma though serum immunoreactive levels were seen. Another IV-l replication is that replicationmaybeupis inhibited, vitro, In ) infection induces . Specific inhibition high levels of proin~ammatorycytokines like of any one of these cytokines through the use e receptors does not

tion.

num~roustrials of

cell e~evations remains

in modest but sustained increases in

be conducted? These are criti uestions since the clinical utility of IL-2 must be confirmedin a clinica oint trial. If trials are conducted among personswith early disease and high 11 counts,largecohorts a ill be required because the frequency clinical of events in this solution may be to conduct a hase 111 trial in areasof the tuberculosis, which occurs with increased frequency in intermediate-st ease, is highly prevalent. Interleukin12TheheterodimerIL-12 is produce by activatedmonocytes macrophages after stimulation by bacteria, bacterial roducts, and some intrarasites 1961. Interleukin 12 promotes the development of type l cytokine l effect is the induction cells andT cells[97]. produces modest effects on peripheral mononuclear cell tion, and induces the expression of other cytoki granulocyte macrophage colony stimulating factor Cells obtained from IV-infectedpatientsproduce less IL-l2 than cells obtainedfromcontrolswhenstimulatedwith ~ta~~yloco~c o ~ l a s ~~ a~ n[99]. d ~However, i in vivo IL-12 responsesto e are not impaired in IV-infecte~ subjects as compared to responses o otoxin administrationto controls [loo]. In vitro incu~ationwith I cell cytotoxicity [loll and lymphocyte proliferative responses [ cells obtainedfromI~-infectedsubjectsand death lo^]. It is also conceivable that deficient hanced programmed cell death and contribute to t patients to infectionwithintracellular prove may beneficial for these patients of approach this is that in incubation vitro from activated tion IL-12 with ca

drop in the absolute lymphocyte count and body-~ependentcell cytotoxicity were seen

load, and apoptosis. Interl~ukin15 Interleukin 15 is produced by macrophages, fibroblasts, an2 epitehelial cells, but not T cells [107]. It has a secondary structure similar to that of IL-2 and shares with that cytokine the ability t o enhance T cell proliferation, incytotoxicity9actasa T hemoattractant,and up-regulate theproduction of TNF-a, ~-interferon9 and IL-2 in assays of lym~hocytepr induce l y m p h o c ~ eproliferation in the context of T cell receptor activation, IL-2 * duces it in the absence of T cell receptor e IV-1 replication from latently infected cells. tion [I lo]. ~ l t h o u ~noh clinical trials with I

rect defective lymphocyte pro~ifera~ymphocyte count. ~otential toxicities

fected patients with CD4 cell counts lls/pl received a single infusion of IL-10, a transient but asma viral load was seen [l 151. Levels of proin~ammatory

im~ortantly,clinical outcomes when used in conjunction with potent antiretroviral cytokines could prove helpful in reconstituting immune lication.Interleukin 2, the one most extensively clinical trial,thedesignof whichis earlier stages of clinical trial developA. as a surrogate marker when l cytokine therapy or cytokine inhibition provi any additiona~ ~enefithighly to active antiretroviral therapy?

l immunogens and has the

cant biomedical obstacles to the developme~tof most successful viral vaccines, exhi~iti oped from live attenuated virus. For taken this approach to develop a prot uals .

potentially more expensive thera

t of attenuat~d

IV/SIV viruses, have

1301, protection from

ells, are largely limited to antiviral on is the direct killing of virally

over time, although some suggested.

progression. The use of killer infection is derived from three clinical ation in peripheral blood monocytotoxic activity is detected in fection; and (3) C ction and expande doptive transfer of autologous n these individuals both total er treatment. Since IL-2 was cellular effect is difficult to a did not changesignificantly clinical improvements in opportunistic infections were

e been shown in rima ate studies [l course of disease. Thus, it rema

re~iication,reduc and transmission.

rotei in enco~ing ~ene(s) coul gle or several viral rotei ins CO

have shown thefeasibility of this latter concept. In rodents, original studies showed IV epitope-specific cellular humoralimmune responsescouldbegenerated in vivo by intramuscular injec of a gp 160 DNA construct. The neutralizing antibody responses and cytotoxic cellular responses were shown to protect the organism from a lethal tumor (HIV antigen-specific) challenge. A similar antibody ne response has been observedin primates 11401. A recent clinical DNA construct has ben abstracted; the report indicates that 15 IV-infected individuals have been safely vaccinated and boosted. In addition, an ne response may have been generated in er clinical trials have immunized with candidate envelope and core proteins, with the hope of generating neutralizIV and/or cytotoxic T cells. In the initial study, immunization of iduals with a gp160 subcomponent vaccineresulted in a specific increase in antibody to HIV-1gp160 and antigen-specific in vitro blastogenesis responses [141]. Sincethistrialneither was placebo-controlled nor comprised a comparisonvaccination arm, only limited conclusionscanbedrawn.Asecond 11421. Imcontrolled trial utilized a gpl20-depleted inactivated IV- 1 preparation munization resulted in a significant increase in serum antibody level as well as increased lymphocyte proliferation to p24 antigen. The rate of CD4 cell decline lower in the vaccinated than the control group. In the most recent infected individuals with CD4 counts > 6 ~ / m mwere immunized in uble-blind, placebo-controlled trial using an envelope subcomponent vaccine [1431. An immunization and boosting protocol was chosento obt results showed no significant difference in . The lack of a beneficial impact on the imm or virological course of infection of vaccination may be due to the selection of a poor vaccinecandidate.Alternativelypostinfectionimmtion strategies may notprove to be useful in themanagement of established infection.Further studies are needed foroptimal selection of candidate,adjuvant,immunization route, and protocol.

In conclusion, vaccine studies for IV are in their infancy, and the data generated to date indicate that immunization of naive or infected persons can result in the generation of new virus-specific immune responses. The tremendousresearch effort has generated a wealth of information on the relevant immune responses for virus neutralization. Increasing evidence has indicated that broadly based cellular immune responses and possi lso high-titer neutralizing antibody responses are keys in long-termsurvivors W IV infection. heth her vaccine strategies will beable to induce or enhance these relevant responses to host-specific strains will await the results of ongoing studies.

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the two-signal concept of self/nonself discrimination, es of immunological hypotheses that accommodate all relevant therefore enunciate principles, as opposed to concise “small theoonly a few facts [l]. Cohn’s points are exemplified by transplantation, which developed thr ghout much of its modern history in the absence of a unifying general principle he inability to evolve an encompassing explanation for observations made in hu n and animal allograft recipients, or in surrogate in rove m u c ~of transplantation research down the same reductionist pt can be traced to two premises into the still fragile base of transplantation immunology be. The first was that the events following transplan ns: host versus graft efensible framework from which ever, when the first premise was t whole organ allograft “accepchimerism-dependent acquired 2,3], the result was derivative dogma that we have called “the one-way paradigm”141.

~ronically, the first examples of successful human renal transplantation were directly responsi~le forestablishment of the intellectually disorienting one-way paradigm.Until 1959, theproductionofchimerism by donor leukocyteinfusion in preparationfororganallotransplantationhad been a muchanticipatednatural ] of the neonatal tolerance models [2,3] and the adult rodent ana3

ent conditioning by cytoa~lationwas re threat of raft-vers this strategy to closely histo when long survival of func su~lethallyirradiated reci~ients and then regularly (from l ti^^ under continuous ph rism and theneed for r ~ c i ~ i ecytoablation nt s e e ~ i n ~had l y been eliminatedas condichism of organ transplantation, casualty of the consensus was a f, and Zeiss [20], who ~ostulated cells in neonatally tolerant mice oming mutually nonreactive while retainheses of tolerance experimenta~ supportc ~othesishad been adv clonal deletion was bei ~lantationtolerance. clonal se~ection as th closure with the ulti

response E303 (Figure1A).hen the one-way in vitrotestsofimmune reactivity were developed [31,323, they were automatica~lyaccepted as “minitransplant mo els,” the results from which were assumed to bedirectly applicable to invivo circumstances.

, it alsoseemed logical to interpret the cyte [2,3] or subsequently bone marrow amples of the one-way paradigm (Figure by the immunologically corn ss there was a high degree o the major histocompatibility comp~exab~ationand“repla~ernent’~[7,8] that rrow transplantation in hu uency after 1968 [34-37). achievements encouraged the belief that cytoablation (or cytoreductio space” was a necessary condition for donor leuko ef. 38), in spite of early [39,40] and recent eviden earlier with the engraftment of human kidneys, the ne marrow transplantation strengthened the grip of the oneway paradigm, notwithstanding its inability to explain why organ transplantation rned by different rules than those for bone marrow transplantation( sine qua non root of the dilemmalay the assumption that chimerism, the of bone marrow transplantation, was irrelevant to an explanation of successful organ transplantation.

. A connection between the chimerism of hematolymphopoietic transplantation and the successful engraftment of whole o r ~ a n was s made with the discovery in 1992 of persistent donor leukocytes (microc~imerism~ up 30 to years postoperativel~in the perip~eraltissues or blood of human kidney, liver, and other organ recipien 461 (Figure 1C). The donorcells were few in number, requiring sensitive imm tochemical and polymerasechain reaction ( ) techniquesfordetect~on. theless, we were able to postulate that the donor-derived leukocytes const one limb of anta istic but ultimately reciprocally attenuated or abrogated (rejection)and G reactions [42-47] In this context, disruptionof the leukocyte interactionby the host cytoablation used to prepare bone marrow recipients, but not therecipients of whole organs, was res~onsible for the disparitiesin the two different kinds of transplantation (Table l). The cancelling effect of the two immunocyte populations under the conditions of postoperative immunosuppression used for organ trans poor prognostic discrimination of human leukocyte antigen as the rarity of GV after the engraftment of immunologically active organs such .)

C

3

etween Conventional Transplantation one Yes

c-

Common Tolerance

c-

cytoablationa Recipient

-+

NO

Not critical Rejection Rare

Term for success

-+

"Acceptance"b

"All differences derive from this therapeuticstep, which in effect establishes an unopposed in the bone marrowrecipient,whosecountervailingimmunereaction is C, major histocompatibility complex; GVHD,graft-versus-host disease. bOr"operational tolerance."

as the intestine an resolution, first o

obvious that the characteristic cycle of crisis and ug-immunosuppressed kidney recipients and most hanges in allograft function [16], was the product re 2). At the same time as the peripheral migration influx of recipient cells that did not cause graft te im~unosuppressionwas given (Figure 1C) [44,45,48-521. uccessful, both the allograft and the recipient became genetic S

composites. sed that bone m a r r o ~and organ transplantation were, in sulting fromthedrasticallydifferenttreatment strategies has been supporte~by reports describing a trace residual ~ u ~ o c y t in e sessentially all human bone marrow recipients,

Immune reaction

""""""""

+ Time after transplantation

Contemporaneoushost-versus-graft(HVG)andgraft-versus-host(GVH)reactions in the two-way paradigm of transplantation immunology. After the initial interaction, the evolution of tolerance of each leukocyte population to the other is seen as a low-grade stimulatory state that may wax and wane rather than a deletional one. (By permission of ~ m m u n o l o ~~yo d a y . ~

who previously were thought to have complete donor cell chimeris

153,543.

The microchimerism fo~lowing organ transplantation hasbeen studied extensively in a variet of rat and mouse allograft mode lthough limited tissue or always notorgan bearing patients blood reveal the microc ,70-73], experimental failure the thethat own to find donor ~eukocytes in a long survivingorgan allograft recipient simply reflects an inadequate search [59,6~,671.The cumulativeevidence from the experimental studies has all but eliminated the ~lternative possibility that the microchimerism may be an effect rather than the cause of organ allograft acceptance [7 ,753. The answer to this “chicken or egg first?’, question 1761 was the same asin historical investigations of florid chimerism[77-$01. lso in accordwith classical studies,thequantityan lineage composition of the donor leukocytescontainwithin the transplanted rodent organs strongly influencedtheir survival in the cipient anddeterminedwhetherthechimerism ced wasassociatedwithlethal a1 cells of the liver (the most tolerogeni bone marrow cell suspensions n that both contained hi leukocytes and cells of myeloi origin than the lympho intestinal allograft orallogenic l y m ~ hn Thus, the difference between the row transplantation and that produce has appeared to be largely sem profile of the ‘‘passenger leu line with this conc~usion, thechange§ following transplantation to noncytoab~ated ients of the hind limb with its rich bone marrow content are much the same as those after~ngraftmentof visceral organs [$l].

A protective umbrella of immunosuppression is usually needed for the survival of theminoritypopulation E661 (~igures1C and owever, this maybeonly a temporary requirement after outbred canine liver [SZ]and kidney tran§plantat~on models too numerous tocite of liver, heart, lung, and kidney ermanent acceptance of liver allografts is po§sible with no treatment at all in a significant percentage of outbred pigs [87-9 t strain [91,92] and virtually all mouse strain combinations [B]. 931 and kidneyallografts [9 ] are also accepted ~ i t h o u tthe need for immunosuppression in a much more ~imited number of stances. In all of these ~ n i m a lmodels, the allograft pass resolving rejection on the way to toleran i ~ b l yextends to other t antidonor reactivity [6 tained ( s ~ l i tolerance) t

0

10

lmmunosuppr~ssion Time on and off of immunn(gray)

20

30

No i~~unosuppression

of 12 longsurvivingliverrecipients 1995. Patients 150 and 169 stopped medicaof noncompliance. The others were munosuppression. These 12 patients represent continuously borne hepatic allografts for 15 to 25-5/6

years.

*

unretiable foiiowup = 4 physician panic = 3 biliary tract disorder = 2 PBC iecurrence = 1 renal failure = 1 Steatohepatitis = 1

Summary of the first 80 liverrecipients in a pros~ectiveweaning trial by the of thepatients edicalCenter (n = 80). Note thatanhalf redu~tion.Therate of are completelyoff drugs or are on an uninterruptedschedule weaning has been slowed from that used originally (see text). There have been no patientor graft losses ance was detected or in the presence of intrinsic liver disease, including the reemergence of autoimmune disorders. (Table 2). and 4, whose mixe depressed reactio ashowed drug wit~drawal[45], had prompt re§toration of re§ponse state but withno evidence of rejection.

The ~er§istenceof multilineage dono to 30 years after tran§plantation has presence of hematopoietic stem andp Discontinuance of Immunosu~pressionin Lon Recipientsa aplotype Indication for Patient mismatch weaningb Tx post Years

3 (JN) 4 (Jvv)" 5 (DS)"

drugs off ears 33 32 32 32 33

0

NC

1

Comp

30 15

0 2 1

NC

29

Comp Comp

'These are 5 of the 16 longest functioning allografts in the world. bComp, complications:skincancer,warts,infection,hypertension, Nc, noncompliant. 'These were children at the timeof transplantation.

3-1/2

3

obesity, orthopedicproblems;

his belied the widely held belief that malian life require a bone marrow , it has been shownthat all hematomice (9.5 Gy) can be reconstituted sionofstem cells isolated from It liver and in other organs experimentation. pic liver transpla eterotopic heart transplantation O6 infused donor bone marrow nt ~ematopoieticreconstitution f survival of almost all other

row engraftment by suppressis factor cy [TNF-a], T etc.) released in respo typical in the posttransplant id progenitor cell promoting F) and granulocyte macro-

t of donor/reci~ientleukocyte

f tolerance can be broughtto bear on problemsof transof such ex~erimentshave been one-way paradigmatic, immune cell su

ic reconstititution

cell tolerance” has been the term U for successful ex~erim~ntal mani~ulations ectrum of highly controlled CO ions. here has been m u c ~ ~vidence,howlym~hocytes aredirected by s ~ e c i a l i z e ~ i m mregulatory u~e leukoor unknown lineage (i.e., veto E1141 and s u ~ ~ r e s s o[l151 r cells). 1. [l 161 have isolate^ a circulati~g donorleukocyte of u n k n o ~ n li~eagein a tolerant human kid~ey recipi~nt with such p rful veto function th a single cell caneutralizetheinvitro activity of 10 recipient cytotoxic

*

uch clues are intriguing, but it is u~likely thatallograft a c c e ~ t a ed from the results of stu ies of i n ~ i v i ~ u1eukoCyte al Freemartin cattle (1945)

~ra~iosis

Organ Tx

(1960)

.. .. .. ,

1

Biliingha~/~rent ~lavin/Strober(1977) Medawar Ildstad/Sachs (1 984) Thomas (1 987) (1 953)

~ontinuumof chimerismfrom o~servation~ of the discovery in 1992 of mi~rochimerismin organ recipients.

wenin f r e ~ ~ a r tcattle i n to

ing to this state suggest learning (cognitive) an functions of the immune system that are roperties of the networks 11, Coutinho C221 lerance to nonlivi mably would be multimechanistica and other factors. hypotheses have been advance in the past in the conat first with little onsideration of the far eory articulated by et al. [ 1301 and ~ c h w a r t z[131]. nonlethal clonal silencin was elaborated and experimentally supported by Nossal edassurviving in a ~uiescent, ng to a~optosis[ 1 3 ~ - 1 3 ~ 1Con. se involved in the merger of two systems of the two-way paradigm, rimentally verifiable events may be

The role of the thymic versusperipheralmechanisms [ l under clinical circumstanceshasbeencontroversial. The the recipient thymus after orga articular interest of the strikingly tolero c effect in rodents of intrathymic inoculation or leukocytes [ 137,1381. ever, thymectomy in adult rates does not influence either the chimerism or aneous tolerance induced akhsh- ones et al. [l391 have shown that after n adult mice reconstituted with~urifiedhematodeveloped no differently thanin control animals

patients had no clinical advantage or disadvanta~e.

nts havebeen put to good usein transplantation research (nonreactive) leukocytes that repopulate an organ du iary allogeneic host dis~ualify the graft’s r e t r ~ ~ ~ ~ l ~ ~ study of c o m ~ l e xtolerance mechanisms. In addition, the leukocyte replacement during the p a r ~ i n gperiod is not complete. Even at 1 year of residence in a tolerant

nt, 10% of the nonparenc essentially fixed from day 1 the second-stage procedure have beenunlnterpreta n s i m ~ l e rexperiments inv donor irradiation, both the toler allografts [68] are abrogated or infusion of donor strain spleno transplantation [ 1441. The necess also has been unequivocally sh and in several 0th

a state resembling ave recently shown in these chimeric animals that the abilityof dono in response to a skin graft challenge was a mor

successfully long after either organ or bone ma

realized that all examples of allograft 6‘acceptance,9’whet not, were variations on the theme originally ith this finding, it was possible to defineboth success andfailureafter transplantation in a different way than before. Success “ tolerance or graft acceptance [meant] that a characteristic 1 cell chimerism had been introduced which may be stable either without further treatment, or only when continued immunosuppression is connoted a state in which “an unstable graft and its migr rejected or cause GV [42]. Itrequirednoition to see theuninterrupted thread of chimerism fromtheobservations by en [l471 of naturaltolerancein freemartincattle tothedonor-derived leukocyundnearlya half centurylater inhumanandanimal recipients of organ allog etween these brackets lay the rodent neonatal [2,3], cytoablation-~ependent more complicated “mixedchimerism’’ toleranc One of the lessons emphasized in our first [42] and all subsequent reports of organ transplantation-associated chimerism was that the rapidly evolving drug-free

u~ocytesin a few days in rodent clinical expectations [76,l031. In e of the twocell populations necesarms from destroying the other has er transplantation weaning trials.

ely that excessive therapy may diverse sites of action [l 521, do success by allowing alternaan xpressed. Chimerism and the ous in numerous rodent mode cause (chimerism) and the hen liver transplantation is o matter what the means of

nottolerance, cause b

I

us t I

,

I

No immunosuppression

Time between cause (chi~erism)and effect (donor-specific tolerance) after liver lantation in different species. Note that immunosuppression is not universally required in three of the five species shown.( y permission of no no logy ~ o ~ u y . )

It would be equallyabsurd tobelieve that theevents after transplantation?including the develo~mentof tolerance to alloantigens, are not under genetic control. This genetic framework S been too well worked out uring the last 40 years to warrant extensive cit~tion. wever, most of the evidenc of a recision effect o the outcome of organ transplantation in large mammal conditions of a perfect or near-~erfectdonor-recipient ingly little fall-off in survival expectations of human le r levels of mismatching, no matter how extreme the bone marrow transplantation, an unam~iguous d has de~ended onsimulation of the immunolo~ic n e ~ ~ a ttolerance al conditions of t cytoablation [33-379157,15~~ nder all other circumstances, the results after trans h ~ m a n st o mouse [ 5 ~ and ] rat models [ e not beencongruentwiththe predic~tions from genetic analyses. The two-way paradigm ex~lainswh ractical purposes, the one constantfactorforinduction o ing trans~lantationtoleranceha in strength and CO quently, successful conditions that llow assimilation of the fragment of or i ~ ~ ~ n o l o g i c a l lograftintothe preexisting network, r and vice cal modulationof the large ithin feasibility boundaries that are ~ e n ~ t i c a l lproy n his recent review of two books on the history of immunology ~ ~ 6 0 9 1 6 1 ] ,

self.” Tauber hassuggested

igm allows ~ r e d i ~ t i o about ns what can (and cannot) be accom1 of which are att~mpts to alter the with tolerance inducing reci~ientleukocyte interac n establish~d that when both immunocyte ~ o ~ u l a t i ocom~etent, are ns pression is d~livered theto

two arms equally, the microchimerism produced by organ transplantation can be greatlyaugmented by thecoadministrationof 3-6 X 108/kgunmodifieddonor significant risk of C . The presenceof donor deoxyribonucleic acid ) in the myeloid and olonies generated from mononuclear cells ( using y standard clonal 1 assays provided unequivocal evidence een confirmed and extended by Garci hematopoietic progenitor cells assays. Despite the increase of persistent chimerism to levels many times higher than that in control patients neither the timing,severity, nor frequency of acuterejection has been different than in non-marrow-augmented control patients [163-1651 (Figthese endpointswouldbealtered. ure 7). Ithadnotbeenanticipatedthat hypotheses being tested are, first, that the threat of chronic organ rejection will be reduced, and, second, that the frequency of ultimate drug indep~ndence can be increased by achiev g a higher persistent level of chimerism. An efficacy evaluation is expected to take o l0 years, roughlythe S eframe (Figure 6 ) delineated by three decades clinical experience with -incompatible liver and bone marrow transplantation [103]. stration of colony stimulating hematolymphopoietic growth facSF) or drugs like lisofylline (discussed earlier) is predicted to be without significant risk of GV if suchchimerismaugmenting treatment is imposed on. both cell populations ally. In contrast, procedures that alter only one of the interacting arms must be approached with caution, as exemplifie~ by the after cytoablation and bone marrow transplantaleukocyte- or T cell-specific depletion of intestinal

40

20

p-0.533 (Log-rank test) 0

I

0

6

12

18

24

Cumulativeincidenceofrejectioninbone rnented organ allograft recipients.

30

36

42

marro~-augmented and -nonaug-

1.

2. 3. 412, 1956.

4. 5.

6.

hornotransplantation of kidneys and of fetal liver and spleen after total body irradia152:354-373, 1960.

Prehn.Successful skin homogra tion and hornologous bone rnarro

7. 1955. 8. 9. 10.

11.

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homotransplantation in man after radiation of the recipie

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plantation of kidney in patientstreated by preoperative local irradiation and postoper19. 20. 21.

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Ouse liver transplantation tolerance: The role of hepatocytes and nonparenells. Transplant Proc27509-510, 1995. Y Shimizu, S Goto, Lord, G Vari,CESmith, S Chiba, D Schlect, M ano, N Kamada.Restoration of tolerance of rathepaticallografts by spleenved passenger leukocytes. Transplant Int9:593-595, 1996. JW Streilein. Neonatal tolerance of H-2 alloantigens: Procuring graft acceptance the “old fashioned,’ way. Transplantation52:l-10, 1991. P Alard,JAMatriano, S Socarras, A Ortega, JWStreilein.Detectionofdonorderived cells by polymerase chain reaction in neonatally tolerantmice: ~icrochirnerism 60: 1 125-1 130, 1995. fails to predict tolerance. Transplantation RD Owen,Immunogeneticconsequencesofvascularanastomosesbetweenbovine twins. Science 102:400-401, 1945. C Martinez, F Shapiro, RA Good. Essential duration of parabiosis and development of tolerance to skin homograftsi mice. Proc SOCExp Biol Med 104:256-259, 1960. S Slavin, S Strober, Z Fuks, HS aplan. Induction of a specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: Long term survival of allogeneic bone marrowand skin grafts.J Exp Med 146:34-48, 1977.

150.

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151.

cyte globulin and donor 152.

153.

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155. 156.

136:1080-1097, 1972. 157.

158.

159.

160. 161.

162, 163.

164.

165.

or xenogeneic

ers smith, ˆ on don, ~ n ~ l a n d

sease, affecting 0.5 070 to l Yo of the popula-

tribute to additional drug-related of the disease spectrum is nce, and premature death s self-evident and has ug development based on deeper knowledge of the pathogenesis ofdisease.

ay be viewed as a systemic disease that localizes to joints cesses appear tobe occurring simultaneously and originate int capsule and periosteum [3, 41. The lining layer of the synovial membrane, norma~lyone to two cells thick, becomes hypercellular as a result of an increase in both type of precursor cells. It is considered that the type A from the marrowvia the blood, predominate and f accumulation. The locally resident type typic characteristics of fibroblasts, also hypertrophic lining layer acts as a secreeate the tissue interstitium and joint fluid. blood vessels, lying immediatelyunderneaththe , from which leukocytes (mainly neutrophils) miS a result of anincrease in vascularpermeabil-

Etiopathogenesis of Rheumatoid Arthritisa I. Initiation -Genetic factors Suggested population, by family, sibling, and twin studies, e.g., shared epitopeof HLAof DR4 DRP chain expressed by subtypes and DR1 -Environmental factors Not known; bacterial and viral infections suspected 11. Perpetuation and chronicity -Immune response joint, the In involves antigen presenting cells and CD4'T cells. Antigen not yet identified; tissue-specific (e.g., chondrocyte) antigens favored: could explain disease localization cell e.g., rheu-Autoimmunity Well demonstrated at €3level, matoid factor, antifillaggrin antibodies, but role in pathogenesis andrelat~onshipto T cell response unresolved 111. Tissue damage and inflammation -Cytokines Important regulating in leukocyte traffic into and from joints, promotingcel~u~ar interactions, and inducing angiogenesis and production of mediators of tissue damage -OtherinflammatorymoleculesProstaglandins,matrix met~lloproteinases, free radicals, nitric oxide "HLA, human leukocyte antigen.

ity, serum proteins also leak into the joint space, contributingto an increase in the volume of an exudativegranulocyte-rich joint fluid. In active stages of disease the membrane is covered with fibrinous material and assumes a villous appearance as aresult of a second process. This results in an increase in newly vascularized tissue and, as a result of transmigration from blood, the formation of a locally organized lymphoid structure. The components of the structure include perivascular aggregatesof lymphocytes, mainly C interaggregate areas are found CD8'T cells, activated B and plasma cells (with occasional appearance of germinal centers), and an increasein macrophages, fibroblasts, and dendritic cells. The third process is characterized by the formation of pannus, whichis a destructive invasive tissue, continuous with the synovial membrane, overlying articof tissue damage, ular cartilage and over time forming a slowly advancing zone which also extends beneath the articular cartilage, eroding subchondral bone.

~ t t e n t i o nhas focused on thehypothesis that the disease process, havinglocalized to joints, is anti~en-driven, and thisimmunological response is linked to the chronic in~ammatoryreaction. An immunological concept of pathogenesis is not new anddatesfromthe discovery of i~munoglobulin ) r h e u ~ a t o i dfactor

F) in the blood of patients with A over 50 years ago [ F of all isotypes [6], and analysis of 'v genes 7). Anotherautoconsistent with ananti n-dependentresponse (reviewed in antibodyproduced by ells in RAjoints of IgG class is diagainst cartilagespecific collagen 11 [$]. Claims have been made that the diagnostically specific Ig of antiperinuclear factor in bucca/mucosa antibodies responsible for e detection cells and antikeratin antib staining of stratified rat epithelium by indirect i m m ~ nofluoresent tests are both directed against the antigen profillaggrin [9,10]. ever, the role of all A-associated autoantibodies in disease pat~ogenesisis unclear, andtheidentity o antigens reco~nizedby T cells in jointsdoesnotappear to correspond to that recognized by antibodies. The list of candidate T cell antigens it includes collagen I1 [l l] heat shock protein 65 [l21 deoxyribonuNAj) [13], and human chondrocyte gp39 [ 141. For the present, the lack of consensus on the nature of the " A antigen'' imposes a constraint on the ~evelopmentof antigen-specific therapies, The importance of T cells in the pathogenesis of RA is emphasized by the ( association of the disease with certain human leukocyte antigen DR T cells in the synovial membrane of terized by the presence of a pentapeptide sequence (the shared epitope)on the0chain of the molecule that forms partof the antigen-binding groove [l63 and is involved in antigen presentation to T cells. CD4'T cells are of the memory phenotype and bear activation and adhesion markO', CD7 dim, C very late antigen (VLA)-l , -4, and -5 [17]. On the basis of the paucity of local production of T cell cytokines, such as y interferon (y-IFN), it has been argued that T cells are unimportant in the chronic i n f l a m ~ a t o r yphase of established disease [l$]*

anycytokines areoduced locally in thejoint.These intercellular messenger molecules are involv in localization andperpetuationoftheimmuno tory response and in mediation of destruction of cartilage and bone. locally in the synovial membrane appear to regulate a numberof biological effects [l91 and may beclassified into the following groups. 7. l ~ f ~ r l 7~ u ~? ui ~ o r Interleukin 1 (IL-1) and tumor necrosis factor a (TNFa) are key cytokines implicated in degradation of cartilage [20]. They appear to mediate their effects singly and in combination and are implicatedin tissue destruction by various me~hanisms. These activities are discussed fully in a later section. ~~~

include IL-8,

'

*

families have been found in hi1 activating peptide 78 , regulated upon activati , m o n o c ~ echemoattractantproteinl

motactic influence on neutrophils, monocytes, and lymphocytes and, t the up-regulated adhesion molecules, create a microenvironment that p ficking of blood-~ornecells into the extravascular compartment (synovial membrane and jointcavity).

oduction of reactive teins characteristically produced to the joi and maybeinvolved in inducing these proteins, via the gp 1 0 receptor chain, shared with theIL-6 receptor. acrophage colony-stimulat and not only are i S and monocyte fu extensively in thedisease [23].

a mixed ~icture.Some, IL-13 [ 2 ~ ]

is a subject of debate and some c

disease 1321.

are those that

bind to their specific m

rapeutic aims of interv~ntionsmay be summa-

by deletion of the f a predomi~antlyT ances in biotec~nology, the number of r, discussion will be the stageof c l i n ~ ~ a l

interest as therapeutic targets and

velo~ment,which occurred against

~ u e n t i ain~ strengthening the effector mediators already

f synthesis of cartilage maadhesionmolecules, presenting cells and lymtween mono-

The Rationale and Stagesof Concept Validation of AntiTNFa Therapy for Rheumatoid Arthritis” __

Stage I: Defining a molecular target In vitro activityof TNFa -Induces PGE,, collagenase, nitric oxide, free radicals 1983 (33) - Inhibits matrix synthesis by chondrocytes 1986 (34) - Induces osteoclastic bone resorption 1988 (35) -adhesion molecules Induces 1991 (36) In rheumatoid arthritis,TNFa 1988 (37) -Overproduced in joints -Colocalizes with TNF-R in synovium and (38) 1992 pannus - Regulates otherproin~ammatorycytokines, e.g., IL-l, GM-CSF, IL-6, IL-8 pro1989-95 (39-41) duction Stage 11: In vivo experimental systems

erosions arthritis

Animal models invivo -In collagen-induced arthritis, anti-TNF Mab suppressesinflamm~tion,reduces joint - In transgenic miceTNFa causes a rheumatoidlike Stage 111: Clinical trials

1992 (42) 1991 (43)

Initial clinical trials with anti-TNFa antibody (cA2) (Centocor, Inc., Malvern, PA) -Open label phase 1/11: large effect 1993 (44) - Randomized placebo-control~ed trial80% response to high dose (versus 8070 placebo) 1994 (45) possible therapy epeated (46) 1994 Confirmatory trials -CDP571 an ti-TNa dose ranging versus 1995 (47) placebo -TNF-R p75-IgG efficacy (Im~unex) 1996 (48) -TNF-R p55-IgG efficacy (Roche) 1996 (49) “TNF, tumor necrosis factor; PGE,prostag~andinE; IL, interleukin; GMCSF, granulocyte macrophage colony stimulating factor; IgG,immunoglobulin G.

other cytokines, results in the production of a competitive inhibitor of TNF and serves as a “feedback” homeostaticmechanism [ S O ] . A high-affinity dimeric TNF-R linked to immunoglobulin (IgG) has indeed been used as aTNT; bloc~ingtherapeutic agent. ur studies, based on anin vitrotissue culture system and employing enzymatically disaggregated mixed monoclonal cell populations obtained from surgically excised synovial membranes from RA joints, provided further strengthto the growing conviction of the importance of TNFa. Not only was T N F a o v e r p r o ~ u c e ~ in the culture supernatants, together with other cytokines (without an additional

stimulus), but in a key experiment, neutralization of TNFa with a specific antibody virtually abolished IL-1 production [39]. Results of this and later experimentsled to the hypothesis that TNFa was of central importance in regulating production of other potentially important proinflammatory cytokines in A9 such as IL-1, G CSF 1401, IL-8, and IL-6 [41]. Clinical studies that delineated the importanceof TNF included the documentation of its presence in joints by a variety of techni~uessuch as assays on joint fluids 1371, in situ h y b r i ~ i ~ a t i o[51], n and immunohistological analysis [52]. That Fa could exert its presumed pathological effects in the local milieuwas supported by the coexpressionof both p55 (type I) and p75 (type 11) TNF receptors on cells in macrophages in the lining layer and at the pannus-cartilage junction, but also a proportion of macrophages and lymphocytes in perivascular aggregates and vascular endotheliumitself [38]. In the next stage of development, the role of TNFa was studied in animal hese studies, in the setting of more complex biological systems, demonbenefit of blockade of TNFa with monoclonal antibody receptor, in the form of a soluble TNF- IgG fusion protein 142,531. mouse expressing t human TNFa genewith a globin substitution of the 3’ end was constructed by ollias and colleagues and experienced rheumatoidlike arthritis [43]. The arthritis of this mouse was attenuated by administration of a n t i - T N ~ antibody. Taken together with in vitro data, these results provided a compelling case for clinical trialsof anti-TNFa therapy in RA. In 1992, the first trial of a monoclonal anti-TNFa antibody(cA2) was begun; it was followed by arandomizedplacebo-controlled trial in 1993-94(described later). In 1995-96 a further phase 11 trial, which sought to establish whether repeated therapy with the antibody were possible and whether its immunogenicity would prevent its use for long-term control of RA, was completed. seeking clinical efficacy, a major objective of these trials was to examine the safety of the therapy and define indications for the prescription of the antibody in routine clinical practice.

The biological functions of IL-l relevant to the pathological characteristics of have beenextensivelyreviewed [19,54] and are sim in many respects to thos . Their action is buffered by 0th IL-16 and IL-la are overproduced in two types of inhibitors, namely, interleukin l receptor antagonist (IL-lra), which soluble IL-l receptors, blocks the binding of both forms to IL-l receptors, and shed which act as competitive inhibitors by bindingto IL-1 [20]. Both types of inhibitors have been used as biological therapy inclinical trials in

3. Illre is a debate about the role 6

in RA disease with both pro- and a n t i i n ~ a m wever, amonoclonalanti-IL-6antibodyand dy have beenused in clinical trials in RA.

was one of the first biologicals to enter trials in RA, although the rationale for its use was not clear. This cytokine appears tohave paradoxical activities in possessing both proinflammatory and antiin~ammatory properties, cliniand

to anti-inflam-

tran§migration into §ynovium, although enhanced cellular interactionsbetwee tigen present in^ cells and ly phocytes could also be important.

.

ited re§ources available for health care, pharmacoeconomic issues are increa§in

, is derived from a muri ed to constant d o ~ a i n of s

n

cacy. Nevertheless, manyimportant conclusions could bedrawn. First, the infusions were extremelywell tolerated with no changein hemodynamics or symptomsassociated with cytokine release observed with some therapeutic monoc~onal antibodies. ~ e c o n dpatients , showed an impressive improvement, which was statistically significant and began within 1-2 days and peaked at 3-4 weeks in both subjective and objective measurements of disease activity; these parameters include duration of stiffness of joints onwaking, patient’s and physician’s as disease activity, and counts of swollen and tender joints. Third, a dramatic reduction in the concentration of circulating C-reactive protein (C ), a laboratory index of in~ammatoryactivity in RA, was observed within days and was a modest reduction in the erythrocyte sedimentation rate (ESR). reduction in IL-6 levels wasobserved,supportingthe on that TNFa reg~lated this cytokine andindirectly reduced the production of The next clinical trial was amulticenter randoplacebo”contro1le~trial [45]. atients’ treatment on 73 patients in London, Erlangen, Vienna, and Leiden was stabilize^ on a fixed dose of nonsteroidal antiin~ammatorydrugs and corticosteroids for4 weeks before therapy. After this, asingle intravenous infusion, which consisted of either a high dose (10 mg/ g) or a low dose (1 mg/k 2) or a placebo ( 0 . 1 ~ 0human serum albumin), was of patients. Equal numbers of patients in each group received an infusion on day0 and were observed at frequent intervals over the next the end of thefourth week,patientscategorizedasnonresponders by a preset definition received a second infusionat variousdose levels. After termination of the trial and decoding, 14 patients were found to have received 3 mg/kg c providing an additional dose level in addition to 1 and 10 surements of disease activity were determined and d as ‘~responders” if they satisfied the index requires improvement in four t in tender and swollen joint scores, ~ u r a t i o nof morning stiffness, and ESR, and a 2 grade (out of 5) improvement in the patient’s and observer’s assessment of disease severity 1701. The results were impressive. It could be observed that at 1 week after cA2, ~ 0 ~ 0 - 7 of 0 ~patients 0 had respon ed to 1, 3, and 10 mg/kg of with placebo treatment. At 4 week the proportion respondingin had declined somewhat, whereas the proportion of patients responding in the 3 10 mg/kg groups had increased slightly, to between 70% and 8 0 ~ 0 onding to placebodidnotchange(Figure 1). These were highly ~ifferences. The clinical significance of the responsewas indicated by the of the clinical response and was assessed by examining individua~ featuresof disease 2). These data demonstrated that in the group receiving high-dose dy, the meani~provementwas of the order of6 0 ~ 0 - 7 0(~307 ~ for 0 g with all clinical response measurements, nts receiving cA2 and showed maximal cha sponse exceeding 4 weeks at the high dose also observed in hemoglobin and platelet counts. In summary, 0-controlled trial was the first demonstration that blockadeof a cytokine could be effective in a chronic in~ammatorydisease in humans.

ek 1

eek

0 70 60

50

0 30 0

10

0 0

1

3

1

0

0

1

3

1

0

Percentage of patients responding accordingto 20% Paulus criteria 1 and 4 weeks after placebo (0.1% human serum albumin) or anti-TNFa chimeric monocIona1 antibody (cA2) in a randomized, double-blind trial. Placebo (0): n = 24; 1 mg/kg: n = 25; 3 mg/kg: n = 14, 10 mg/kg: n = 24. (Source: Ref. 45.) TNF, tumor necrosis factor. The reduction in swelling of joints was associated with a reductionin cellularity on sequential biopsies ofsynovium taken before and after therapy. Further studies on the synovial biopsy specimens have suggestedthat the reductionin cellularity (including a reduction in the number of CI34’ T lymphocytes in the synovial membrane) is due to altered patternsof trafficking of cells - most likely as a result of the blockingof ingress of leukocytes.This appears to beaconsequenceof deactivation of vascular endothelium anda reduction of production of chemokines in the joint. This hypothesis is supported by a reduction in the level of expression on molecules E-selectin, ICA 1, and vascular cell adhesionmolecule 1 l) in sequential synovial biops pecimens (Figure 4) and by a corresponding increase in blood of the number of circulating lymphocytes (Figure 5 ) . A reductheserumconcentrationsofsoluble-E selectin andserumIC -1) (Figure 5 ) , which are TNFcu- and IL-l-inducible, provides further support for this concept. More direct evidence has been sought by tracking radiolabeled granulocytes9andapreliminarystudyhasshown a reductionin U over “hot” jointsafteranti-~NFcutherapy(TaylorandPeters,unpu~lished). ever, circulating granulocyte counts, unlike lymphocytes, are reduced after anti-TNF therapy, suggesting that more complex cell kinetics are involved, for example, as a result of reduced maturation and release from the marrow subsequent to reduction in G-CSF Given these interesting data,thequestionthat arises is whether antitherapy is a form of ~ M A ~that I 3 could be effective in long-term control of Since the duration of response to a single infusion of antibody is dose-dependent, lasting a median duration of 3, 6 , and 8 weeks after a single infusion of l , 3, and 10

n= 1

3

3 2 1

Post

Pre

0 Post

Evaluation of (a) E-selectin, ( istologicalanalysis of synovial TNFa therapy. n = number of patient sections by two independent observ intercellular a ~ ~ e s i molecule on (Adapte

iii

70

Pre cells by er antiof each biopsy specimen vascular cell adhesion molecule;IC

mg/kg cA2 [69], long-term treatment would require repeated infusions of cA2. In an open study in which seven patients from the first “open label trial” received two or three further repeat cycles of cA2 after relapse of disease, with an observation period, in some cases, extending over a period of more than 1 year. se~uentialinfusion, a reproducibl linical response of the same magni initial infusion was observed [46]. wever, there appeared to be a trend toward a shortening of the response period, and in half the patients anti-human chimeric antibody directed against the murine idiotype developed. These results were encouraging in respect to the continuing dominance of TNFa over the cytokine networkin but were inconclusivein respect to the possibility of con ti nu in^ therapy. Further ical trials were necessary to determine the efficacy of repeated therapy with a monoclonal antibody, not yet reported for any clinical indication9 and to ascertain whether the immunogenicity of cA2 would be alimiting factor. Studies of longer-term control have begun with a multicenter study on 101 patients who received multiple infusions of 1, 3, or 10 mg/kg cA2, with or without methotrexate9 which was completed in 1996. A preliminary analysi has shown a positive res [73], encouraging the belief that ant viable long-term option.rthertrialsare in progress in the U Lipsky. If the results in the murine model of an be extrapolated to the n disease, thenone might predict thatthethe wouldnotonly exert an antiin~ammatoryeffect, but possibly protect joints from erosive and destructive changes ( F i ~ u r e6). It is axiomatic that trials of proof of principle are verified by independent studies. In relation to TNFa, these studies have been performed with three additin 1 TNFa blocking agents: ahumanized mono 71 (Celltech), a p S 5 - T ~ F - R - I gfusion ~ protein sion protein (Immunex). A randomized controlled trial with C 57 1 led to significant clinical improvement and a reduction in CRP 1471. E~uivalentdoses (1 mg/kg and 10 mg/kg) to

The antiin~ammatoryeffect (paw thickness) and joint protective effect (proximal angealjoint [PIP] erosion) of nti-TNT;therapyin CO en-inducedarthritisin controlmAb; 300 pganti-TNT;mAb; mice, D, 50 pganti-TNT;mAb;isotype 500 pg anti-TNT; mAb.TNT;, tumor necrosis factor; mAb, monoclonal antibody. (Source; Refs. 42 and 61.)

ver, the clinical responses were not i ad little effect on theswollen joint cou reduction of about 3 0 ~ 0 from baseline. In mized placebo-controlled trial, at l f a mean swollen joint count scoreof abou

subclass, It is possible that clear~nce two antibodies aredissimilar and that this characteristic has an in~uence on their pharmaco~ineticproperties. Alternatively, the

n of such differences may be valuable in analyzrently similar biolo ical a ents.

reduction in painful and swollen joint counts in a ~ose-dependent manner. At the uction from baseline of 6 4 ~ 0and Ss%,compared witha r with placebo, was observed (p < 0.001 for both). The A response was achieved by 149'0, 3 3 ~ 0 46070, , and 75% of patients in the 0, and 16 mg/m2 group. Theseresults are compara e to cA2 results, but the duration of benefit, after cessation of therapy with TNF fusion protein, was shorter than -1gG fusion protein (Lenercept, a variety of trials also prese ns do not allow strict comparisons ut differences from placebo were recorded in randomized ~49,74,75~. The magnitudeof clinical response and proporing wereless than those reported with p75 TNFfusion proteins. The reason for thedifferences is not obvious.

0 observed in the groupreceiving the maximum doseof IL-lra. about 3 0 ~was Ina placebo-controlledtrialpublished in abstractform, 472 received 150 mg IL-lra daily by self-administered subcutaneous injections. At the

1.

st extensively teste

r e s ~ ~ n scriteria e versus 2 0 ~ 0receiving

lus), was used in exte~sivetrials with variable schedules and assess~e~~s.

ells. After intravenous infusion of a ukocytes are depleted. A humani~ed rials with promising and significant ntinued because of re renceofinfections,someofthem allegedly serious. Since an increased rate of serious infections is a characteristic of the disease [97] it is not possible to evaluate whether termination of trials forthis reason was justified. IgC2a monoc~onal antibody .5) directed agai . Thirty-tw~patie ntibody at initiation followed 40, or 80 mg for 4 days [98). Eight days later, 13/25 patients satisfied predefined response criteria. At90 days after the infusion, 3 patients were still responding. In a study 10 patients with relatively recent onset A were treated with the -1 antibody [99]. Clinical response by the previously used criteria was 5 cQntinued to demonstrate a observed in 7 of the 10 patientsatlmQnthand si~nificantresponse on day 60. The data are of considerable interest and results of placebo-controlled trials should beilluminating.

, has been in clinical trials in

From this review it will be apparent that a large number of patients with taken part in clinical trials with biological agents in the past 5 to 10 year modalities in current use there is increasing evidence for the efficacy of TNFa and results in placebo-cQntrolled trials with anti-T cell therapies Intheanti-TNFatrialsthereare similarities anddifferencesbetweenthe agents in use thatcouldnothave beenpredicted.reoveranti-TNFatherapy seems more effective than IL-lra therapy, a experimental systems IL-1 This difference may be dueto

beamoreattractivetherapeutic objective than indiscri~inate elimination of T cells that include populatthat mediate both proinflam~atory and (beneficial) antiinflammatory effects. h more detailed follo -up study is re~uired thanmay prove possible witha sing nt in trial when facewith the rapid develop~entof

refinements (e.g. , humanized monoc~onal antibodies) and emergence of exciting new therapeutic targets, such as agents that block interactions. Since even the best therapeutic approaches available thus far appear to offer only temporary contro of symptoms, it is necessary to devise new strategies of treatment for a cure of f this aim requires repeated therapy, then the potential immunogenicity of the icals becomes an issue thatcould limit their use. Two alternative approaches are immediately apparent: The first is to devise methods for reducing the immunogenicity of the biological and/or suppressing the i m m u ~ e response of the recipient. The second is to develop a chemical drug that simulates the effect of the biological by the advanced methods now available in the pharmaceutical industry. Our prediction is that both approaches will practice, but current biologicals have a lead in this process. administration of chemical drugs (by mouth rather than injection) offers advantages, as does thelikely lower cost to the consumer. Safety issues have become increasingly important in licensing of drugs. A have increased rates of serious infections, lymphomas, and other complications as effects of the disease (or as a result of previously used or concurrent medications), theevaluation of adverseeventswith new therapies requires better information on relative risks of such complications thanis available. The lack of adequate information can place increasing demands, ethical and financial, on sponsors and investigators alike and hinder progress. Shared databases could provide a way forward in this regard. Finally, it seems likely that a multifactorial and multistep disease like not be amenableto monotherapy. Combinations of new biologicals (or a bi and anexisting drug) targeting different points in pathogenesis of disease may prove more beneficial than a single agent. In animal models, for example, thereis already evidence of such synergies (e.g., by a combination of a n t i - T ~ Fand ~ anti-T cell therapy [61]. There can be little doubt that the field of immunomodulation has set in motion an accelerated phase of deve~opmentof new interventions, the outcome of which could benefit patients in the foreseeable future.

matism Council forResearch of Great Lindsay Roffe and Annette Winter fo the manuscript.

1. T Pincus, LF Callahan. Rheum Dis Clin North Am 19:123, 1993. CC Erhardt, PA Mumford, PJ Venables, RN Maini. Ann Rheum Dis 48:7, 1989. JC Edwards,ERPettipher,eds.Mechanismsand 3. AJ Freemont.In:BHenderson, Models in Rheumatoid Arthritis. London: Academic Press, 1995, p 83. 4. SA Allard, MT Bayliss, RN Maini. Arthritis Rheum 33: 1170, 1990. 5. E Waaler Acta Pathol Microbiol Scand 17: 172, 1940. 2.

, G Goldstein. Lancet 2839, 1981. m 30:1205, 1987. 18. G § Firestein, 19. Feldmann,F

olini, CE Newdin, T h, JC Burrows, GK

Immunol21:2575, 1991.

Naturedy.

319516, 1988.

, §J Invest Leibovich. Lab

528

Main/ and Feldmenn

71. P P Tak, PC Taylor, FC Breedveld, TJM Smeets, MR Daha, PM Kluin, AE Meinders, RN Maini. Arthritis Rheum 39:1082, 1996. 72. EM Paleolog, M Hunt, MJ Elliott, JN Woody, M Feldmann, RN Maini. Arthritis Rheum 39:1077, 1996. 73* RN Maini, FC Breedveld, JR Kalden, JS Smolen, D Davis, J D Macfarlane, C Antoni, B Leeb, MJ Elliott, J Woody, T Schaible, M Feldmann. submitted. 74. F Hasler, L van de Putte, M Baudin, E Ludin, L Durrwell, T McAuliffe, P Van der Auwera. Arthritis Rheum 39(suppl):S243, 1996. 75. D Furst, M Weisman, H Paulus, K Bulpitt, M Weinblatt, R Polisson, P St Clair, P Milnark, M Baudin, E Ludin, T McAuliffe, M-R Kahny, W Lesslauer, P Van der Auwera. Arthritis Rheum 39(suppl):S243, 1996. 76. GV Campion, ME Lebsack, J Lookabaugh, G Gordon, M Catalano, and the 11-1Ra Arthritis Study Group. Arthritis Rheum 39: 1092, 1996. 77. B Bresnihan, J Lookabaugh, K Witt, P Musikic. Arthritis Rheum 39(suppl):S73, 1996. 78. I Watt, M Cobby, and the Amgen rhIL-lra Clinical Research Product Team. Arthritis Rheum 39(suppl):S123, 1996. 79. BE Drevlow, R Lovis, MA Haig, JM Sinacore, C Jacobs, C Blosche, A Landay, LA Moreland, RM Pope. Arthritis Rheum 39:257, 1996. 80. D Wendling, E Racadot, J Wijdenes. J Rheumatol20:259, 1993. 81. J Wijdenes, E Racadot, D Wendling. J Interferon Res 14:297, 1994. 82. FC Breedveld, PA Van der Lubbe. Br Med Bull 51:493, 1995. 83. MJ Elliott, RN Maini. Int Arch Allergy Immunol 104:112, 1994. 84. LA Moreland, WJ Koopman. In: V Strand, DL Scott, LS Simon, eds. Novel Therapeutic Agents for the Treatment of Autoimmune Diseases. New York: Marcel Dekker, 1996. p 41. 85. C Herzog, C Walker, W Muller, et al. J Autoimmun 2:627, 1989. 86. G Horneff, GR Burmeister, F Emmrich, J F Kalden. Arthritis Rheum 34:129, 1991. 87. D Goldberg, P Morel, L Chatenoud, C Boitard, CJ Menkes, P H Bertoye, J P Revillard, J F Bach. J Autoimmun 4:617, 1991. 88. C Reiter, B Kakavand, E P Rieber, M Schattenkirchner, G Riethmuller, K Kruger. Arthritis Rheum 34525, 1991. 89. EH Choy, IC Chikanza, GH Kingsley, V Corrigall, GS Panayi. Scand J Immunol 36: 291, 1992. 90. D Wendling, E Racadot, B Morel-Fourrier, B Wijdenes. J Clin Rheumatol 11542, 1992. 91. LW Moreland, RP Bucy, A Tilden, PW Pratt, AF LoBuglio, M Khazaeli, MP Everson, P Daddona, J Ghrayeb, C Kilgarriff, ME Sanders, WJ Koopman. Arthritis Rheum 36: 307, 1993. 92. LW Moreland, PW Pratt, MD Mayes, A Postlethwaite, MH Weisman, T Schnitzer, R Lightfoot, LH Calabrese, DJ Zelinger, JN Woody, WJ Koopman. Arthritis Rheum 38: 1581, 1995. 93. D Wendling, E Radacot, J Wideness, and the French Investigators Group. Arthritis Rheum 39(suppl):S245, 1996. 94. PA Van der Lubbe, BAC Djikmans, HM Markusse, U Nassander, FC Breedveld. Arthritis Rheum 38:1097, 1995. 95. R Levy, M Weisman, C Weisenhutter, D Yocum, T Schnitzer, A Goldman, M Schiff, F Breedveld, A Solinger, B Macdonald, J Lipani. Arthritis Rheum 39(suppl):S122, 1996. 96. NJ Olsen, J J Cush, PE Lipsky, EW Sinclair, G Cannon, WJ McCune, V Strand, T Lorenz. Arthritis Rheum 37:S295, 1994. 97. F Wolfe, DM Mitchell, JT Sibley, J F Fries, DA Bloch, CA Williams, PW Spitz, M Haga, SM Kleinheksel, MA Cathey. Arthritis Rheum 37:481, 1994. 98. AF Kavanaugh, LS Davis, LA Nichols, SH Norris, R Rothlein, LA Scharschmidt, P Lipsky. Arthritis Rheum 37:992, 1994. 99. A Kavanaugh, R Jain, J McFarlin, L Nichols, P Lipsjky. Arthritis Rheum 37(suppl): s220, 1994.

25 lmmunomodulation at Mucosal Surfaces: Prospects for the Development of Antiinfectious and An tiinflammatory Vaccines Cecil Czerkinsky Herriot Hospital, Lyon, France

Jan Holmgren University of Goteborg, Goteborg, Sweden

1.

INTRODUCTION

Mucosal surfaces covering the aerodigestive tract, the urogenital tract, the eye conjunctiva and the inner ear, and the ducts of exocrine glands represent the largest (nearly 400 m2) organ system in upper vertebrates. Being the most frequent portals of entry of common microbes and environmental substances, these surfaces provide a most sophisticated barrier against entry of exogenous matter. Endowed with powerful mechanical and physicochemical cleansing mechanisms, they are further protected by a specialized immune system that guards them against potential insults from the environment. Although the mammalian immune system is remarkably diverse, there is strong evidence that certain specialized types of immune responses preferentially take place at mucosal sites as opposed to systemic sites such as the skin, bone marrow, and peripheral lymphoid organs. The mucosa-associated lymphoid tissue (MALT), the largest mammalian lymphoid organ system, represents a well-known example of a compartmentalized immunological system as evidenced by (1) the existence of defined lymphoid microcompartments within mucosal tissues and glands [1,2]; (2) phenotypically and functionally distinct B cell, T cell, and accessory cell subpopulations [2]; and (3) restrictions imposed on lymphoid cell recirculation potential to (and from) a given mucosal site or even within various regions of the same mucosal organ [3,4]. Through the compartmentalization of its afferent and efferent limbs, MALT functions essentially independently from the systemic immune apparatus. This notion may explain why systemic injection of immunogens is relatively ineffective at eliciting ant immune response in mucosal tissues. Apart from the need for maintaining self-tolerance, the MALT has three main functions: to protect against colonization and invasion by the large number of potentially dangerous microbes encountered each day, to prevent uptake of unde529

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.

~raded anti~en§ i n c l ~ ~forei in~ sal mi~roor~anism§, and, mos tially harmful immune respo irnrn~nea ~ ~ a r a twhich ~ § , is

and exha~§tion.

ucosal sites have a propensity

to

diated byT lymphocytes (cytoytQtoxicity, and natural killer he e p i t h e l i u ~of the gastroineic cells and viruses has been es and other mucosal tissues fected cells, their presence in ole as a first line of defense the host, a possibility that owever, knowledge regard-

development of such responses will have a profound impact on the selection and cQnstruction of ap ropriate antigen delivery systems to protect the host against pathogens colonizing or entering throughmucosal epitheliae.

once taken up in the intestine by cialized epithelial cells covering the appendix, and analogous in the rectum 114.1, can be (parenchy~alintestinal macrosed and presented directly by epithe~nhaled upt antigens are S,

lial cells to underlyi mechanisms in the

elements also harbors substantial numbers of dendritic c of the antigen with accessory cells and cognatehelper T icroenviro~ment, an immune re several factors, including the nature of d lymphocytes involved, and the genetic

background of thehost.The sensitized immunocytes, in particularantigencells but presumably also Tcells, leave the site of initial encounter with antigen, transit through the thoracic duct, enter the circulation, and then seed that er distant yet privileged mucosal sites [16]. In their new locations, cells may further differentiate in plasmocytes producingsecretory This latter condition appearsto be effected t h r o u g ~siteg’, receptors) on mucosal lymphoidcells plus the presence of complementary structures (“addressins”) associated with tissue-specific vascular cell endothelial cell surfaces [4]. ifferentialutilization of organ-ficendothelial recognition mechanisms culating precurso byimmunoblasts has recently been demonstrated in humans [171. studies ith in the murine system [4],this finding may explain both the unification of immune responses in diverse mucosal sites an the physiological segregation of mucosal from nonmucosal immune mechanisms. similar migratory behavior has been suggested for mucosal T cells [181 and also for antigen-bearing intes dendritic cells [191, although the conse~uences of suchbehavior(immunity antolerance) have not yet been examined. n the basis of the concept of a common mucosal immune system through which afraction oflymphocytes activatedin a givenorgan, e.g., th of antigen, can disseminate immunity not only in that same organ a1 and glandular tissues, there is currently much interest in the possiblity of ping oral vaccines against, e.g., infections in the buccal, occular, respiratory, nital mucosae. Indeed, enteric delivery of immunogens is the most practical safe immunization route and may bea most efficient means t against patho~ensgaining entry by various mucosal sites. studies involving immunizationsof human and nonhuman primat type ~ u c o s a immunogen, l cholera toxin (CT), and its various routes (peroral, rectal, genital, intranasal, intrat a possibility and indicate in fact that a degree of subcompartmentalization relating to homing of plasma cell precursors exists within the mucosalimmunesystem. hus,whereas oral immunization may induce strong antibody responses in the proximal segment of the small intestine, in the ascending colon, and even in distant exocrine glands such as the salivary glands, it is relatively inefficient at evoking an antibody response in the distal segments of the small and large intestines, in the tonsils, or in the female genital tract mucosa 1161. Conversely, rectal immunization evokes local antibody responses in the rectum but little if any response in the small in test in^ and in the colon [161. Taken together ~ i t seminal h studies by Ogra and cowor~ers [20], these studies demonstrate thatwithin the contextof the commonmucosal immune organs may privilege the development of Ig~-committedprecursor progeny is preferentially destinedto particular mucosal locations.

It is now almost axiomatic thatin order to be efficacious, vaccines against mucosal infections must be ableto stimulate themucosal immune system, and that this goal etter achieved by delivering immunogens via a mucosal route rather than

parenterally [20921]. owever, stimulation of mucosal immune responses by, e.g., the oral consumption, inhalation, ortopical deposition of most nonviable antigens is often inefficient, requiring multiple administrations of large (milligram to gram) quantities of immunogens and yielding, if any, modest and short-lasting antibody responses~ It has thus been widely assumed that onlylive vaccines would efficiently stimulate amucosal i ~ m u n eresponse. The use of live attenuated reco~binant viruses that canbe genetically engineered to express unrelated antigens cated. Such an approach has obvious advantages since it is theoretically possible to package the same recombinant organism with oligonucleotides encoding several unrelated antigens, and since n ~ t u r a infection l with live microorganis~sis known to induce persis dstrongimmuneresponses in bothmucosaland systemic compartments.er, in bacterialvectors,suchas Sal~onella,~ s c ~ e ~ inia en~e~ocolitica, S ~ i ~ e and l l ~~9a c t o ~ a c i l l speus cies, antigens may be expressed in insufficient quantities9in limited size, and/or in an inappro~riate form (nonglycosylated) or must await bacterial death to be released from the periplasm. In addition, the response to thevector is often dominant and may preclude the effectiveness of subsequent booster immunizations with the same recombinant organism. Further, as is the case with bacille Calmette-Gu~rin CG), potentially harmful delayed-typ ) reactions demay velop reexposure on to this bacterium. f vaccines based on recombinat attenuated strains of viruses ropism, such adenoas viruses,polioviruses9 and poxviruses, is especially attractive since these allow insertion of relatively larger antigenic fra~ments thatcan be expressed as integral surface or envelope com~onents thatcan be glycosylated on replication in target cells and stimulate broader types of responses, especially cytotoxic responses. Their main disadvantag~s include the fact thatimm~nity to the viral vector may develop and thus as for bacterial vectors may interfere with and/or booster subsequent eir persistence in the imm~nizedhost is yet poorly controllable, and ce potential side effects associated with tissue-damaging in as has been reported with vacci~iaviruses and adenoviruses. Live strategies of antigen delivery include liposomes, biodegradable microspheres such as copolymers of poly m-lac oglycolide, polyphosphacenes, polyalginates, cellulose starchwithincorpo or surface-adsorbedantigens have been utilized, but their preparation generally requir and/or harsh conditions resulting in potenti lectin-like molecules endowed with i ~ m u n o s toxin (CT), the most potent mucosal immuno~e its analogue, ~ s c ~ e r i coli c ~heat-labile i~ enter with either unconjugated or conjugated antigen cosal and systemic antibody responses. This is due to alarge extent to the ability of ganglioside on cell sur faces including epithelial subunit, and to anotherextent to the adjuvant properties of the toxin, which appear torequire the adanosine diphosphate-(^ ribosylatingaction of the enterotoxic unit [dl’]. Several formulationsbasedon chemicalcouplingorgeneticfusion o with selected antigensornucleotidesare nowbeingevaluatedaspotentialvaagainstsexuallytransmittedchla human immunodeficiency virus

n ad~ition to orinstead of inducing local S inhalation of antigens may also result in the immunolo~icaltolerance, a situation characterized by the fa sponses in n o n ~ ~ c o stissues al will not develop even if the anti tration of antigens is in fact a long-reco~ni~ed method of i

I

J Mestecky, JR McGhee. Adv Immunol40:153, 1987. Kaetzel, ME Lamm, D Fletcher, JG Nedrud. Proc Natl Acad Sci USA 86:6901, 1992. ertzbaugh, JH Eldridge, M Hirasawa, H 7.

5.

6.

riere, I Durand, F Rousset, J Bancherea

8.

175:671,1992. 9. 10. 11. 12. 13. 14.

v Immunol7: 145, 1989. i. J Exp Med 148:1661, 1978.

Mucosal Immunol Update 4:1, 1993.

15.

16. C Czerkinsky,J Holmgren. Immunologist3:97, 1995. 17. 18. 19. 20. 21. 22. 23. 24.

L Weiner, A Friedman, A Miller, SJ Khoury, A Al-Sabbagh, L Santos, M Sayegh,

25. 26. 27. 28. 29. 30.

31. C Whitacre, C Gienapp, IE Orosz, D Bitar. J Immunol 147:2155, 1991. J Pediatry 138:341, 1982. 32. 33. Weiner, 34. HL DA Hafler. Science 259: 1321, 35* DE Trentham, RA ~ynesius-Trentham,EJ Orav, D Combitchi, C Lorenzo, 36. LA Rawlings, JM Lynch. J Immunol 122:2261, 1979. 37. ad Sci USA 91:10795, 1994. 38. 39. 0 Elson. J Immunol 152:4663, 1994. 40. HJ de Aizpurua, GJ Russell-Jones. J Exp Med 167:440, 1988. 41. Lycke, C Czerkinsky. Vaccine 11:1179, 1993. 42. alding. J Irnmunol 133:2892, 1984. 43 J Holmgren, C Czerkinsky, N Lycke, A- Svennerholm. Am J Trop 1994. 44. ask, T Olsson, J Holmgren, C Czerkinsky. Proc Natl Acad Sci USA. 93: 45. I Bergeron, C Ploix, J Petersen, V oulin, C Rask, N Fabien, M Lindblad, A Mayer, olmgren, C Czerkinsky. Proc Natl Acad Sci USA 94:4610, 1997.

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~ a t i o n aCancer l rnstitute, Rockville, ~ a r y l a n d

rogress in basic research is expediting the identification of promising therapeutic agents. Clinical evaluation ofnew agents is inherently complex and time-consuming, however. The specification of a coherent and targeted clinical development plan, the identification ofclear trial objectives, and theutilization of efficient clinical trial designs are crucial for keeping clinical development time in control. In this chapter we will review some recently developed clinical trial designs that can be considered for use in theclinical development of innovative therapeutics.

PhaseItrialsare usuallyconducted to determinethemaximumtolerateddose TD) of an agent. ts, however, may be most effective when administered at a dose less t . Hencefortrials of biologicals, determiningthe of biological response is also an important relationship between dose and a measure objective. The traditional phase designs I used in oncology trials are basedon cohortsof three to six patients per dose level. Usually three patients are treated ata dose level and observed for 3 weeks. If none experiences dose-limiting toxicity (DLT), then the next cohort of three patients receives the next higher doselevel. If, however, two or three of the cohort experience DLT, then the MTD is considered to have been exceeded. If one of the three patients experiences DLT, then three additional patients are assigned the same dose level.If none of them experiences DLT, then escalation of dose continues for the next cohort. Otherwise the MTDis considered to have been exceeded. Usuallysix patients are placedat the dosebelow the one that resulted in two or more cases of DLT. Several newer approaches to the design of phase I trials have also been described [l-31 for determining the MTD in oncology trials. herea as cohorts of three to six patients may be appropriate for roughly deter-

ts

mining the MTD, there is no reason to believe that they aresufficient for determining the relationship between dose and biological response. If one has treated n patients in each of k dose levels, one question of interest is whether biological responses for all dose groups are equivalent A widely used nonparametric test for this purpose is the ruskal- alli is test. Nonparametric tests are useful for continuous endpoints that are not normally distributed. iological endpoints are often of this type. The co~putationsinvolved in the Kr kal- alli is test are described in statistical texts [4]. The sample size requirements for this type of analysis are shown in Table 1, which indicates the required numberof patients per dose groupas a function of the number of dose groups and the difference in mean response b e t ~ e e nthe lowest and highest dose groups. The difference in means is expressed in standard deviation table i s based on statistical power calculations €or normal distributions, uskal- alli is test is valid more generally. The table is also based on an alternative hypothesis that the intermediate-dose groups have equally spaced means intermediate between those of the lowest and highest groups is]. An alternative nonparametric statistical test for such trendalternatives is the Johnckheere test [4]. Table 1 shows that only very large effects can be detected with small sample sizes. The size of effect is measured in standard deviation units, and there are two components to this variability. One is assay variability, which should be minimized by identifying and controlling the factors that contribute to it. The other is interpatient variability. If baseline patient factors predictive of the endpoint can be identified, then stratified analyses that will increase statistical power and reduce the required number of patients can be performed. If the endpoint for analysis is binary, response or nonresponse, then the appropriate analogueof the Kruskal- alli is test is Fisher’s exact test for 2 X k contingency tables. A chi-square test should not be used when. the sample size is small asit often is in phase I trials of biologicals. Fisher’s exact test is not easy to compute; however, it is included in many statistical packages. The analogue of the Johnckheere test for trend with binary response data is the Cochran-~rmitage test [6] for linear trend in response probabilities. Table 2 shows the sample size requirements for this test as a function of the number of dose groups, thedifference in response probabilities betweenthe lowest and highest dose groups, and the response probability for the lowest dose group. As for Table 1, the response probabilities for the

8Ooi0 Statistical Power Number of Patients per Dose Group for with 5% Significance for Detecting Difference in Means Using ~ruskal- all is Test with Continuous Endpointa

std

in

Difference in mean between lowest and group highest 0.75 1

2

37

36 21

Number of treatment groups 2

3

4

30 17 4

5

21 5

aPower calculated for normal distributions with linear trend in means.

Number of Patients per Dose Group for 80% Statistical Power with 5% Significance for Detecting Difference in Response ochran-Armitage Test for Trend with Binary

4

Differenceinresponseproba-Number bility between lowestand high3 est 2 0 54 69

.30 28 30 32 .40

34 37 16

of treatmentgroups 2 58 74 69 83 32 37 40 20 23 23

18 20 21

15 12 15

14

61 79

18 18

.50 12

14

aPower calculated with linear trend in response probabilities. For each cell in table, top entry represents number of patients per arm if the response probability for the control arm (or lowest dose arm) is 0.10. Middleandlower entries correspond to response probabilities in control arm of 0.20 and 0.30, respectively.

intermediate doses are assumed to be equally spaced and intermediate between the two extremes [7). The tests described are used for testing the hypothesis that the biological response is the same forall k dose levels. If this hypothesis is rejected withp < .05, then pairwise comparison of dose levels is an appropriate next step. The tests used for pairwise comparisons are the Wilcoxon2 sample test and Fisher’s exact test for 2 X 2 tables, respectively. It is clear that the sample size required to detect a biological relationship among groups may be much greater than that required to determine the ~onsequently,ien useful to splitthetwo objectives intotwoseparate clinical trials.Afterthe orthetolerablerange is determined,onecanthenrandomize patients to dose levels in order to evaluate biological response. If one expects a monotonic relationship between dose andbiological response, then it is only necessary to test patients in the lowest and highest dose groups d u r i n ~this randomized stage in order to determine whether there is any dose response. Assigning patients to intermediate dose levels is less efficient for simply determining whether a dose response exists but may be important for estimating the smallest dose at which maximum response exists. etermination of this smallest fully effective dose, however, may require many more patients that the numbers indicated in Tables l and 2 because it may require distinguishing a dose group at which full response occurs from one atwhich half of that response level occurs. An alternative approach to estimati~g MT or biological dose response is to

use a titration design. If the patient stays on study for multiple treatmentcourses, then the dose is titrated upward until a maximum acceptable level of toxicity or biological response is achieved. As long as there are no cumulative dose effects, this is a safe and efficient approach. Titration designs are sometimes criticized because the resulting data areanalyzed incorrectly. It is essential to analyze the data using a statistical model that distinguishes data for different courses of the same patient fromthosefordifferentpatients.ithcontinuous biological responsemeasures, one appropriate modelmight be Y, =

+

( d , CXDij) + L yi + ( d , aD,)

+

y , is the observed biological response in the jth course of treatment of the ith patient. d, is thedoseadministered to patient i in the jth course,and D, is the cumulative dose administered to patient i in all courses previous to j . a Is a cumulative-toxicity parameter that will be estimated from the data. In many cases it will have the value 0. denotes the maximum level of biological response achievable, L denotes the level existing without administering any dose, andyi denotes the dose level at which patient I achieves half ofthe maximum response. The parameter yi is permitted to vary among patients and may be assumed to have a normal distribution with meanp and variance2 to be estimated from the data. If biological response is defined asincrease over baseline level, then L equals zero. This modelis a generalization of the model describedby Sheiner et al. 181. There are two main advantages of a titration design. The first is that it is efficient to utilize multiple courses of treatmentto explore dose response for individual patients. The efficiency of the design will depend, however, on the amount of intrapatient variability of biological response, as well as the number of courses of treatment that patients receive. The second is that it provides information on interpatient variability of thedose-response relationship. Sample-size requirements have not been well studied for titrationdesigns. One approach is to use the titration design during the course of the phase I trial in order to determine the TT), using traditional cohorts of three to six patients starting at each dose level. At the end of this phase, the toxicity and biologic are analyzed by using a model suchas the onedescribed above, an additional patientsto be studied determined. For oncology trials isit generally not possible to include a placebo or untreated group. In other areas of drug develo~ment, however, use of such control groups is standard. Whenever possible, a control group should be used becaus value in distinguis~ing among transient variations in biological en tions due to disease, and true treatment effects.

The traditional objective of a phase I1 design is to determine whether the agent has sufficient antidisease efficacy to warrant further development.hase I1 trials are also used to select dose levels or schedules based on short-term endpoints.

Simon’s optimal two-stage designis widely used for phaseI1 oncology trials [91. One selects a response ratep , representing a low level of biological activity which would not be of interest for further development of the agent. One also selects a higher level pt representing a response rate of substantial interest. One alsospecifies error rates a and p, a represents the chance of concluding that the agent is promising if its response probability is only p,, p represents the chance of failing to consider the agent promising if its true response pro~abilityis p * . Often a = 0 = .l0 is used. These parameters determine a design. The design is specified by values n19 n2 r l , and r. In the first stage of the trial n , evaluable patients are treated.If there arert or fewer responses, then the agent is rejected as not promising and the trial is termitherwise anadditional n, evaluablepatients areaccrued.Atthat time eases and the agent i ejected as not promising if the total number of responses is not greater than r. herwise the agent is considered sufficiently promvelopment. The optimal design minimizes the expected number to anagent when the true response probability is only p,. Simon ax” designs that minimize the maximum number of patients subject to the constraints on the error rates. Some optimal ble 3 for a = p = .lo. A more extensive table is shown in [91 and a convenient interactive program for personal computers is available from the author to generate designs for nontabulatedvalues of the parameters.

Two-stagedesigns are particular~y convenient for multicentertrialsbecause the logistics of data collection and review are complex. Often, however, there is a desire to monitor the interim response data more intensively. In such cases the statistical Simon’s Two-Stage Optimal PhaseI1 Designsa Reject drug if response rate

.05

.IO f20 .30 .40 .50 .60 .70

.25 .30 .40 .50 .60 .70

.80 .90

0/9 1/12 3/17 7/22 7/18 11/21 611 1 6/9

2/24 5/35 10/37 17/46 22/46 26/45 26/38 22/28

14.5 19.8 26.0 29.9 30.2 29.0 25.4 17.8

.63 .65 .55 .67 .56 .67 .47 .54 ~~

~

”Probability of recommending as promising regimen with true response probability less thanPois .lo. Probability of recommending as promising regimen with true response probability at leastp , is .90: n, denotes the numberof patients treated in the first stage;n denotes the maximum numberof patients treated; EN@,) denotes expected sample size if true response probability is po; PET@o) denotes probability of early termination if true response probabilityis po.

design should account forsuch monitoring. Thall and Simon have developed ian designs for continuous monitoring and analysis of phase I1 trials with binary endpoints [lo]. These designs, like the optimal two-stage designs, were developed for oncology trials and hence do not allow for use of a control group. With the ayesian designs the response rateexpected for the control groupC is summarized by specifying two paramet p is the average of the distribution of response rate ~ 0 that the for the control group and is the range in which one is 9 ~ confident response rate for the control groupwould lie. At the start of the phase I1 trial one alsomust specify thepriordistribution of responserate to expected forthe experimental treatment E. Thalland Simonhaverecommen that a relatively “uninformative” prior distribution be used. uring the course of the phase I1 trial, patients are treated with the experimenmen. The data can be reanalyzed after each new patient is evaluable for ne combines the number of responses and number of nonresponses observed in the study with the prior distribution to obtain t 66posterior distribution” of response pro~ability for the ex~erimental treatme~t Thedistribution of response probability for the control group C remains unch ed since no control patients are included in the trial. e can compute mathematically the probability r than it is with C. If that probability is high, that the response rate with E is gr e.g., greater than .95 or .99, one may decide t o terminate the phase I1 trial and conclude that the regimen is worthy of phase 111 evaluation. It is alsoeasy to compute the probability that the response rate with E is not greater than the response rate with C by at least an amount A. f this probability is, say, larger than .95, one may decide to terminate the phase 11 tria nd conclu~e that theregimen is not sufficiently promising. Inthiscalculation,wouldrepresentthe smallest improvement over the control response rate considered medically or scientifically important. The analyses described can be made at many points during the trial. A maximum sample size should be specified in advance, but the trial may terminate much earlier. Thall and Simon suggest setting the maximum sample size to correspond to a 9 5 ~ probability 0 interval of widthA for the posterior distributionof the response probability for the experimental treatment Thall et al. also provide continuous monitoring ayesian designs for multiple binary endpoints that may correspond to efficacy an toxicity measurements [1l]. They have also s t u ~ i e dthe probabilities that their designs provide an ambiguous r accepting nor rejecting the experimental regimen. of the prior distribution for response rate of the hase II design without a controlarm will not be efficient and ambiguous results are likely. If, rate expected for the control group can be accurately predict sian continuous monitoring designs with A in the rangeof 15 rcentage pomts can be quite efficient. of the Thal sian designs for r generalization A I1 trials with multip~e dose group straightforward. domized among the k dose groups. At any point in the trial the tion of response rate for each dose group is computed. e can calculate for each dose group the probability that its response rate is not greater than that for the control groupby at least A. ~e might drop any dose group for which that probability is greater than .95. At any point we can also compute the probability that the

response rate for one dose group is greater than that for another, or the probability that there is a dose-response effect. The ayesian model is very flexible for addressing such questions. The Thall-Simon Bayesian designs have not, however, been extended to randomized phase 11 trials witha control group. i.

ndomized phase I1 trial design for selecting the most promising regimen to be further developed. All k arms ( k 3 2) would consist of experimental regimens (perhaps different doses or schedules of the atients are randomlyassigned t o each of thek arms, the arm with the greatest number of responses at the end of the trial isselectFd for further develo~ment.Only one arm is selected even if it differs from the next best arm by only one response. he sam le size is determined so that if one arm has a true responseprobability at leas greaterthanthat of the next best arm,thenthe roba ability of selecting the b arm will be at least P*, for a suitably high value of P*. Table 4 shows thenumber of patientsperarmrequiredfor , 3, or 4. This approach requires manyfewerpatients for ising regimen, but it assumes that oneis indifferent to which regimen is selected if the response probabilities are equal. Thusit ignores potential oxicity or cost. Selection designs have been described for time-toThe selection idea has been furtherdeveloped in combined phase II/III selection-testing designs by Thall, Simon, and Ellenberg[ 14, 151.

. Strauss and Simon [l61 described an approach to therapeutics development in settings where innovative regimens are being created more rapidly than they can be

Number of Patients per Treatment Group for Randomized PhaseI1 Selection Designs" Number of treatments Smallest response probabilit~ 0.10

0.20 0.30 0.40

0.50 0.60 0.70

0.80

2

3

4

14 20 23 24 24 21

23 32 38 41 40 36 29 18

28 40 48

17 11

51

50 45 36 22

robab ability thatthebesttreatmentwillhavethelargestobserved response rate is 0.85 when the true response probability is 15 percentage points larger than that for the second best treatment.

to a new treatment or compares two new

ay bedesi~nedas a variantof the regimen

patients are randomized to each treat-

sample size n per arm ineach trial ment selected after a totalof some

many new treatme~tswill be explored. The precision of estimation of the response rate for treatment is reduced,however,as n decreases. Consequently there is a value of n depends somewhat on the horizon size N a n d also types of treatments available. If there are some very good r test, then smalln is favored in order to increase the chance

y; another is that this a p ~ r o a c hdoes not provide cantimprovementforany selected treatment.

y be worthyof consideration in rapidly evolving roach foropti~izing the sample size for individ-

Suppose that oneis developing a hematopoietic growth factor to reduce th of thromboc~openia aftercis-platinum-based cancer chemotherapy and t

target population is patients undergoing chemotherapy for canc of the compound will be most clearly seen in subjects n

conducted sequentially with dose increased for subse~uent cohorts of S size of the cohorts depends on the incidence of toxicity that one wish For example9if one wants the incidence of “significant” toxicity at the observing at least one case of 44significant99 toxicity amo when the true roba ability of toxicity is 0.20. If one on cumulative

starting dose for the phase I trial would d

though itmay not reflect the efficacy of the compound after chemotherapy. The phase I trial in human subjects would be followed by a cancer patients undergoing chemotherapy. In designing this trial it defineitsprimary objective(s) carefully. One objective mightb whether the dose selected from the previous t cancer patients undergo in^ chemotherapy. Although the initial tri clear picture of toxicities in normal subjects, cancer patients wi organ statusmay experienceadditional complications c o m p o u n ~in cancer patients undergoing chemother ble, however, without a control group of cancer pat but not receiving the study c o m p o u n ~ . ~ o n s e ~ u e n t l y , randomized trial. If the previous study identified an serum concentration at that dose is approximate1 efficacy in animals~ then the second study could be restricte If no dose-limiting toxicity was two dose levels in the second tri ized two- or three-arm trial. In additionto assessing toxicity of the com administered to cancer patients undergoing chemotherapy, the trial WO be designed to evaluate the efficacy of the compound in a covery.

To plan the number of patients required for the second trial, one needs data on the duration of thromboc~openiaexperienced by cancer patients receiving the chemotherapy to be studied. Ideally one should haveindivid~alpatient data on time to recovery of platelet count to aspecified level (e.g., 150,000 cells/ml) for patients receiving the target chemotherapy. If those data appear symmetricallydistributed, then one might use a normal distribution approximation and estimate the number of patients per treatment groupas

n =2j where z, is the percentile of the normal distribution determined by the significance level a, zp is determined by the desiredstatisticalpower 1 - p, A denotesthe difference in mean times to platelet recovery for the control group and the group receiving the compound of interest to detect, and a denotes the standard deviation in time to platelet recovery among the patients in the control group. Suppose that past data are symmetrically distributed with a mean of 12 days to recovery and a standard deviation of 6 days.We might wish to design the trial to have 8 0 ~ 0 statistical power for detecting a A = 4 day reduction in average time to platelet recovery, which represents a 33% reduction; thus A/a = 4/6 = 0.67. The specifi0 power correspondsto zp = 0.84 .For a one-sidedstatistical cation of 8 0 ~ statistical significance level of 5070, we have z, = 1.65 . Consequently the preceding formula gives a sample size of approximately 28 patients per arm for the trial. This is very similar to the value obtained from Table 1 under the assumption that A/a = .75 and that a non-parametricsignificance test will be used. If only one doselevel is to be studied, then the total samplesize will be approximately 56 patients. If two dose levels are tobe studied, then it is advisable to use zff = 1.96 in order to account for the two comparisons to the control group that will be made. In that case, the formula gives 35 patients per arm. If the data available for time to platelet recovery for previous patientsreceiving the target chemotherapy indicate that the distribution is skewed, rather than symmetric, then it is advisable to take logarithms of the recovery times and to calculate the standard deviation of the logarithms. The preceding formula can be still be used but with A representing the difference in logarithmof the meanrecovery time for the control group and the logarithmof the largest mean recovery time for the treated group. In some cases historical data will not be readily available for estimating the mean and standard deviationof recovery time for a control group or fore~amining its distribution. It is important, however, to attempt to obtain such information.If this in~ormationis impossible to obtain, there are several alternative approaches. One is to plan the trial as a two-stage procedure. The first stage might consist of 1015 patients per arm. The interpatient variability seen in the first stage will be used for determining the total sample size needed. A second approachis to use Table 2 to plan the trial for detecting a difference in the proportion of patients with platelet recovery at a specified point in time. For example, one might say that at the time when the control group has30% recovery, we want to be able todetect a difference corresponding to 6 0 ~recovery 0 for the treated group. Table 2 gives a samplesize of 40 patients per arm for a two-arm trialwith this specification. This approach is not

very attractive, however, because it is not efficient to analyze the data in terms of the proportion with recovery at a specified time. In cases where platelet counts will be measured only infrequently, however, it may be appropriate. A third alternative to sample size calculation in cases where historical data are notavailable is to assumethatthedistributions of timeto platelet recovery are exponentially distributed. The variance equals the mean for the exponential distri~ution, and the resulting sample size estimate may be viewed as conservative. With exponential distributions, the number of patients per arm is approximately

by the where 6 represents the median timeto recovery for the control group divided median time to recovery for the group receiving the compound and ““l” denotes natural logarithm. For example, if we want to be able to detect a 33% reduction in median time to recovery, then 6 = 1.5 and n = 75 patients per arm. The conservatism of this approximation is apparent. On the basis of these considerations, the second trial might be a randomized trial with a control group not receiving the compound and one or two active dose groups.Thesample size would be about 30-35 patients per armbasedonthe assumed variability and size of effect to be determined. Since the sample size is relatively modest for a comparative trial, it may be advantageous to include two dose groups if there is any question about the most appropriate dose. The modest sample size results from the relatively small variability in time to platelet recovery assumed. In any actual trial, this variability mustbe estimated from previous data, and, asindicated, obtaining such datais important. The methods used for computing sample size are a somewhat oversimplified representation of the analyses that will be performed at the end of the trial, but they help ensure that theresults will be reliable, In the actualanalysis, time to recovery data for more than one course of chemotherapy may be available for some patients and the protocol should indicate how data for the later courses will be analyzed. The investigator may be tempted to use a cross-over design in which all patients receive the compound in one course and chemotherapy alone in another course, with the order selected by randomization. Cross-over designs can be very efficient in some situations, but they tend not to be useful in circumstances in which many patients go off study after one course and where there may be carry-over effects between courses. Hence for the trial described, a cross-over design would not be recommended. We might call this a randomized phase I1 trial, because the endpoint used is not a direct measureof clinical benefit. After this trial, onewill ideally have a good assessment of biological efficacy. If the compound is not effective for speeding platelet recovery, then its development might be redirected to a different clinical situation or abandoned. If the compound does demonstrate biological efficacy in this trial, then one should be in a good position to design a phase 111 trial at a single dose versus a control group for determining whether the compound improves clinical measures suchas transfusion requirements, outcome of chemotherapy treatment, and costs.

variety ofnew designs for clinical trials. Clinical trials are jectives, patient populations, therapeutic interventions, u n o m o ~ u l a t i nagents ~ are so varied that the example ot be viewed in any way as a typical drug developmentplan. It however, some considerations in the development of one type of the clinical development of immunomodulating agents is exre c o ~ m o key n design issues to focus on. Common problems nition of objectives, meaningful endpoints, appropriate consize, and prespecified hypotheses. Clinical evaluation can be of success enhance by thoughtful planning, clever design,

1.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

F Thall, R Simon, E Estey. Stat Med14:357-379, 1995. Simon, RE Wittes, SS Ellenberg. Cancer Treat Rep69: 1375-1381, 1985.

6-mercaptopurine clinical uses, 277 mechanism of action, 277 pharmacology, 277 adhesion molecules therapy of rheumatol~arthritis, 5 15 adjuvants, 161,386 adrenal steroids, 367 adrenocortical responses, 3'74 a~renoco~icotropic homone, 84 adrenal axis, 75 adult respiratory distress syndrome^ 305 AIDS T cell apoptosis, 442 T cells, 209,2 13 aposi's sarcoma, 35l allergen, 2 1 allergic asthma, 24 allergic reactions, 2 1 alpha melanocytestimulating hormone, 85 aluminum-based delivery systems, 385 androgen, 9 1 receptors, 9 1 androgens, 92,370 antibody response, 79 anti-en dot ox^ t r e a ~ e n t s3, 16 antigen delivery sytems immunomodulation in, 385

antigen density

in EAE, 373 arginine vasopressin 9 atherosclerosis, 137 autoi~munediseas systemic, 129 azathiopri~e clinical uses, 277 mechanism of action, 277 pha~acology,277 Azulfidine clinical efficacy, 272 immunological actions, mechanism of action, 270 pharmacolo~y,26 bacteremia, 302 bereavement, 149

bone m a ~ o wtransplantation, 485 ~ r e ~ u i nsodium ar clinical use, 287 mechanism ofaction, 286 pha~acology,286 calcitonin gene-related peptide, 101 cancer cells cytokines in, 354 growth by cytokines, 350 ovarian, 348 cancer immunity effector mech~isms,353 effector mechanisms, CD4cells, 423 effector mechanisms, CD8cells, 422 effector mechanisms, LAK cells, 423 effector mechanisms, macrophages, 423 effector mechanisms, NK cells, 352 ca~ier-mediatedantigen delivery systems, 40 1 CD4 T cells, 4,5 cell-mediated im~unity,79 cel~ularadoptive therapy, 42 1 LAIC cells, 43 1 T lymphocytes, 43 l tu~oricidalmonocytes, 432 chemokines, 509 chimerism, 485

mun no modulating agents for, 539 r ~ d o m i ~ phase e d I1 design, 543 statistical designs, 544 therapy of rheumatoid arthritis, 5 15, 522 clonal deletion, 35 colony-fo~ingunit spleen, 64 corticosteroids ~linicaluses, 274 mechanisms ofaction, 273 pha~acology,273 therapy, 275 co~icotropinreleasing factor, 83 Crobn's disease, 267 cross-reactive idiotype, 22 1

~ ~ p t o s p o r i d~i u ~ ~175 ~ u cyclosporine clinical use, 28 l, 460 mechanism ofaction, 280 pha~acology,280 cytokine-based therapy, 288 IL-1 RA, 288 I t - 10,289 IL-2DPT fusion protein, 289 TNF, 288,464 cytokine blockage in sepsis, 3 15 cytokine gene therapy, 421 metastatic colon cancer, 425 vectors, 424 cytokine immunomodulation infectious diseases, 466 cytokine receptors, 10 cytokine-based immune system, 175 cytokines, 8,507 therapy of rheumatoid arthritis, 507, 51 1 cytolytic immune responses infectious diseases, 187 cytotoxic T cells antigen presentation and MHC, 188 infectious diseases, 187 cytotoxic T lyrnpbocytes activation, 192 class I presentatio~,195 epitope binding, 193 MHC binding, 196 TCR binding, 196 deoxyspergualin,290 dose-limiting toxicity, 539 electrofusion, 24 1 emulsions, 388 advantages and disadvantages, 389 mode ofa d j u v ~action, t 388 endocrine organs, 364 endotoxin, 301 monoc~onalantibodies to, 3 17 polyclonal antibodies to, 3 17 endotoxin antagonists in sepsis, 3 16 endotoxin neutralizing protein, 323 e~thropoietin,76

~

3 estrogen, 9 1 receptors, 9 l Fas-mediated programmmedcell death, 450 FK506. See tacrolimus flow cytometry, 209 follicle s t ~ u l a t i n ghormones, 90 gas~ointestinaldisease, 267 gastrointestinal nematodes specific and nonspeci~cimmunity, 176 gastrointestinalworms, 170 gene therapy adenovirus-mediated interleukin 12 in, 429 combination therapy in, 429 cytokines in, 430 immunomodulation, 15,42 1 genetic vaccination, 14 germline transcription, 29 glucoco~icoids,1 1, 85 in the neuroendocrine system, 367 graft-versus-host disease, 487 granulocyte colony-stimulatingfactor, 61,76 granulocyte-macrophagecolonys t ~ u l a t i n gfactor, 76 granulomatous in~ammation,223 growth hormone, 76, 102

H e ~ i ~ ~ o s o ~p ooi ~ e~s r17~1,s172 , h~mopoieticstem cells, 55 hepatic cell necrosis, 244 hepatic fibrosis, 223 hepatitis, 237 hepatitis C virus, 245 he~atosplenomegly,223 heterogeneous antigen delivery systems, 410 HIV disease, 2 12 bone marrow andprogenitor cell replacement, 463 cellular therapies, 462,463 cytokine therapies, 464 ~ m u reconsti~tion, n ~ 46 1 progression, 2 13 thymic ~ansp~antation, 464

HIV infection apoptosis in, 440 chemokines in, 459 cytokines in, 439 glycosolation inhibitors, 46 1 HIV binding and cell entry, 458 immune-based therapies, 457 immune therapy in, 457,472 reverse transcription, 459 targeted transcriptional activation, 460 HIV therapy attenuated vaccines, 470 chimeric vaccines, 47 1 passive immunotherapy, 47 1 host versus graft, 487 HTLV infection, 237 human immunodeficiencyvirus, 460 human monoclonal antibodies E3 cell selection, 239 infectious diseases, 242 production, 238 hybridoma formation, 240 hydroxychloroquine, 290 hypothalamus-pituitary-gonadal axis, 366 ~ypothalamus-pi~ita~-adrenal axis, 103, 365 hypothalmic, 1 1,364 idiotopes, 5 idiotype, 22 1 i d i o ~ e - ~ t i i d i o t y pinteractions? e 22 1 idiotypes, 35,36 idiotypic-specificT cells, 36 idiotypic heavy chains, 40 idiotypic light chains, 40 idiotypic peptides, 35 idiotypic-speci~cCD4 cells, 49 IgE response, 21 immune cell interations cognate and noncognate, 6 immune function secretory, 95 immune modulation, 196,222 immune regulation principles, 2 immune response, 6

[immune response] anergy, 4 immune system changes representation, phenotypic, and ~nctional,209 immunization for HIV therapy, 472 immunoadjuvants,386 immunocompetence, 147 immunoglobulin class switching, 28 heavy chain, 28 immunology ofT cells, 5 10 immunomodulation ofmucosal T lymphoc~es,529 immunopathogenesis AIDS, 2 15 immunostimulatingcomplexes, 400 immunosuppression in transplantation, 488,495 ~fectiousdisease, 131, 164 infectious pathogens, 439 inflammatory bowel disease, 273 insulin, 82 insulin-like growth factor, 77 interleukin, 76 interleukin l, 3 1 1 interleukin 1 receptor antagonist, 327 in rheumatoid arthritis, 52 1 interleukin 6, 1l, 3 13, 345,465 in cancer pathogenesis, 345 receptor, 348,353 interleuk~6 therapy in rheumatoid arthritis, 5 1 1 interleukin 8 in sepsis, 3 13 interleukin 11,60 interleukin 10 in sepsis, 3 14 interleukin 1l , 60 interleukin 12, adjuvant, 161 enhanced antibody levels, 165 in infectious disease models, 164 systemic delivery, 163 treatment, 162,468 intracellular parasites, 132, 175 interleukin 12, 161 intracellular signaling, 25 iron, 259

iron overload, 264 JAK-STAT pathway,25 Janus kinase, 25 kidney fibrosis, 135

~ ~ ~ s ~ mmajor, a n i a170 lipid implants, 408 advantages and disadvantages, 408 liposome-like vesicles, 395 liposomes advantages and disadvantages, 39 1 antigen delivery to mucosae, 395 immunotherapy9394 mode ofaction, 390 lipoxygenase inhibitors, 29 1 liver disease, 281 liver transplantation, 283,483 l u t e ~ i z hormones, ~g 90 lymphohemopoieticstem cells, 55 lymphoid tissue autonomic innervation, 374 macrophage inflammatory protein la, 68 malnutrition, 255 trace elements and vitamins, 256 MAP kinase, 77 marital discord i m m u n i ~150 9 mate~al-fetalsuppression, 45 melatonin, 98 methotrexate clinical use, 279 mechanism of action, 279 pha~acology,27 MHC class I, 41 HC class II,41 MH~/peptideinteractions, 199 ~icrochimerism,483 microparticles advantages and disadvantages, 397 mode ofaction and release, 398 microspheres delivery of antigen to mucosae, 400 monoclonal antibodies human, 237 monoclonal antibody therapy, 287 MOPC 315,36

mucosa-associated lymphoid tissue, 529 mucosal i m m u n i ~ c o m p ~ e n t s53 , l mucosal surfaces immunomodulation, 530 multiple organ dys~nctionsyndrome, 304 multiple sclerosis, 125, 375 mycophenolate mofetil clinical use, 286 p h ~ a c o l o g y286 , natural i m m u n i ~ intracellular parasites, 175 natural killer cells, 423 neonatal tolerance, 49 nerve growth factor, 99 neuroendocrine immune system, 363 neuroendocrine pathways, 146 neuroendocrine system, 106 therapy, 12, 107 neuroendocrine systems, 10 neuroendocrine-inducedimmune modulation, 75 autoimmuni~,363 neuroendrocr~e dys~nction, 37 l autoimmuni~,37 1 neuroi~munoregulato~ network, 10, 375 neuropeptides, 80, 100, 105 neurotransmi~ers,11 nicotine, 29 1 niosornes, 395 ~ ~ ~ o s t r o n~~r al su~ ~ s i e n170, s i s , 171 nitric oxide, 328 nonobese diabetic mice, 127 nutritient defficiences, 256 nu~ition,255 immunocompetence,255 opioid peptides, 84 oral tolerance, 543 organ transplantation, 485 therapeutic implications,496 parasites i m ~ u nand i ~ mixed infection, 181

parasitic diseases, 23 1 phagocytic cells, 8 1 phase I clinical trials, 539 phase I1 trial design, 542 ~ i ~ i t agland, r y 102 pituitary hormones, 102,364 pi~itary-thyroidaxis, 97 placental lactogen, 76 plasmacytoma, 48 latel let-activating factor, 330 polymeric implants, 403 progenitor cells Thl, 22 Th2,22 progesterone, 94 programmmed cell death, 439 FasRasL in, 446 in AIDS, 440 Th1 and Th2 cytokines, 443 p~oin~ammatory cytokines, 306 prolactin, 76,102 proteosomes, 395 psychological stress, 145 immune competence, 145 psychoneuroimmunology, 145 randomized clinical trials phase I1 design, 545 rapamycin clinical use, 285 mechanism ofaction, 284 pharmacology, 285 receptor-mediated gene transfer, 15 rheumatiod arthritis, chernokines in, 509 cytokines and T cells in, 5 14 cytokines in, 509 immune response in, 508 immuno~odulation,5 13 pathogenesis, 507 rheumatoid arthritis, 128,267 schistosomiasis human monoclona~antibodies, 246 immunomodulation,222 immunopathology,225 schistosomiasisjaponica, 222 schistosomiasismansoni, 228

sepsis, 30 l pathogenesis, 304 sepsis induced hypotension, 302 septic shock, 302 sex hormones,75 sex steroid hormones, 369 signal transduction pathway, 26 single-step ~munizations,402 somatic cell fiusion, 240 somatostatin, 10 1 stem cell cycling, 63 frequency, 57 specific growth factors, 59 stem cell factor, 60 c-kit, 60 stem cells, 56 in transplantation, 49 l stress immune-related disease, 145 stress-immune reactions, 153 substance P, 100 sympathetic nervous system, 366 systemic in~ammatoryresponse s ~ d r o m e302 , systemic steroids prednisone, 276 T cell deletion, 5 1 T cell dynamics AIDS, 213,218 1:cell receptor, 5 T cell receptor transgenic mice, 41 T cell selection, 35 T cell subset HIV disease, 2 16 T cell surface receptor, 2 10 T cell therapy in rheumatoid arthritis, 522 T cell tolerance in ~ansplantation,493 T cells AIDS, 213 differentiation, 23 therapy of rheumatoid arthritis, 5 14 T helper cell subsets, 16 1, 169 tacrolimus mechanism ofaction, 282 pharmacology, 283

Tapasin molecule, 191 targeted inte~ention,12 testosterone, 370 Tho cells, 22 Th1 cell development, 170 Thl cells, 6, 8,21, 161 Thl cytokines, 3, 169 Th2 cells, 6, 8,21, l61 Th2 cytokines, 3, 169 thalidomide, 289 thyrotropin releasing hormone, 97 thymocyte deletion, 49 thymocytes idiotypic peptides, 43 negative selection, 46 positive selection, 43 thymus, 42 growth, 78 thyroid stimulating hormone, 97 thyroxin, 97 t r a n s f o ~ i n ggrowth factor beta, 68, 12 1 activation, 122 kidney fibrosis, 135 lymphocyte development and differentiation, 123 ove~roduction,134 receptors, 122 transgenic mice gene targeting, 29 transgenic T cell receptor, 41 transplantation tolerance, 483,494 ~ r i ~muris, ~ ~ 17rl, ~172 s triiodothyronine, 97 tumor antigen presentation in gene therapy, 422 type 1 immediatehypersensitivity, 21 tyrosine kinase, 25 ulcerative colitis, 267 vaccine, 163, 196 vaccines mucosal, 529,532 vasoactive intestinal peptide, l 0 1 vector-mediated antigen delivery systems, 40 1 vitamin A, 86,261 vitamin D, 86 vitamin D3,96, 104

57

vitamin E, 262 supplementation, 264

zinc, 257 zinc overdose, 264