Advances in Immunology Volume 68

ADVANCES IN

Immunology VOLUME 68

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ADVANCES IN

Immunology EDITED BY

FRANK J . DIXON The Scripps Research Institute La Jolla, California ASSOCIATE EDITORS

Frederick Alt K. Frank Austen Tadamitsu Kishimoto Fritz Melchers Jonathan W. Uhr

VOLUME 68

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CONTENTS

ix

CONTRIBUTORS

Posttranscriptional Regulation of mRNAs Important in T Cell Function JAMES

S. MALTER

I. Introduction 11. 111. IV. V. VI. VII.

Measurement of mRNA Decay Rates Measurement of Translation mRNAs Regulated by Posttranscriptional Control cis Elements tran.s Factors Concluding Remarks References

1 1 3 4 18 29 36 37

Molecular and Cellular Mechanisms of T Lymphocyte Apoptosis JOSEF

M. PENNINCER A N D GUIDO KROEMER

I. Introduction 11. Degradation Phase of Apoptosis 111. Effector Phase of Apoptosis IV. Initiation Phase of Apoptosis V. Conclusions References

51 54 65 89 122 124

Prenylation of Ras GTPase Superfamily Proteins and Their Function in lmmunobiology

ROBERTB. LOBELL I. Introduction 11. The Ras Superfamily Members 111. The GTPase Cycle IV. Downstream Signaling Effectors: Ras and the Rho/Rac Connection V. Rho/Rac Effectors VI. Prenylation of the Ras Superfamily Members VII. Preriylation and Processing of CaaX Substrates V

145 145 147 148 150 150 152

vi

CONTENTS

VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII.

CaaX Prenyltransferases CaaX Protease and Carboxymethyltransferase Rab GGTase-I1 Role of Prenylation in Membrane Binding and in Protein-Protein Interactions Role of Ras GTPase Family Members in Immunobiology: The Ras Pathway The Rho/Rac Pathway and Leukocyte Function Regulation of the Neutrophil NADPH Oxidase by Rac and Rap Re lation of Phos holi ase D by RhoA Ro e of C-Termina Met ylation of Prenylated Proteins in NADPH Oxidase Regulation and Other Leukocyte Functions Role of Rab Proteins in Membrane Transport in Leukocytes Regulation of Vesicular Transport by Rho Proteins Other Prenylated Proteins Prenyltransferase Inhibitors Effects of Prenylation Inhibitors on Leukocyte Function Conclusion References

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152 157 158 162 166 168 169 171 171 172 174 174 175 178 179 180

Generation and TAP-Mediated Transport of Peptides for Major Histocompatibility Complex Class I Molecules

FRANK MOMBURG A N D GUNTER J. HAMMERLING I. Introduction 11. TAP as the Principal Peptide Supplier for MHC Class I Molecules enic Peptides from Endogenous Antigens 111. Generation of Class I Molecules in the ER VII. VIII. IX. X. XI. XII. XIII. XIV. XV.

TAP as a Member of the ABC Transporter Superfamily Structure of TAP Molecules In Vitro Assays for Pe tide Binding and Transport by TAP Substrate S ecificity o Peptide Transport Biochemic Characteristics of Peptide Transport Linking TAP Structure and Function Export of Peptides from the ER Involvement of TAP in Diseases Concluding Remarks References

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191 192 199 205 209 211 213 214 215 218 228 231 233 234 239 240

Adoptive Tumor Immunity Mediated by Lymphocytes Bearing Modified Antigen-Specific Receptors

THOMAS BROCKER AND KLAUSKARJALAINEN I. Ado tive Tumor Therapy 11. Sing e-Chain Fv Receptors 111. New Approaches References

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CONTENTS

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Membrane Molecules as Differentiation Antigens of Murine Macrophages

ANDREWJ. MCKNIGHTAND SIAMON GORDON I. Introduction 11. Differentiation Antigens Expressed by Murine Monocytes and Macro hages 111. Use of Di erentiation Antigens to Characterize Macrophages in Situ and in Vitro IV. Conclusion References

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271 271 298 303 305

Major Histocompatibility Complex-DirectedSusceptibility to Rheumatoid Arthritis

GERALD T. NEPOM

I. Introduction 11. Mechanisms to Account for the Association of the Shared Epitope with RA 111. Clinicd Applications References

3 15 318 326 327

Immunological Treatment of Autoimmune Diseases

J. R. KALDEN,F. C. BREEDVELD, H. BURKHARDT, A N D G. R. BURMESTER

I. Introduction 11. Cytokines and Anticytokine-Related Treatment Principles in Autoimmune Diseases 111. Anti-CD4 mAb in the Treatment of Autoimmune Diseases IV. Monoclonal Antibody Treatment against Cell Surface Antigen of T Cells (with the Exception of Anti-CD4) V. Immunological Treatment Principles in Animal Models of Autoimmune Disease References INDEX OF RECENT VOLUMES CONTENTS

333 337 347 353 365 397 419 43 1

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CONTRIBUTORS

Nuinhers in parentheses indicate the pages on which the authors’ contributions begin

F. C. Breedveld (333),Department of Rheumatology, Leiden University Hospital, Leiden, The Netherlands Thomas Brocker (257), Basel Institute for Immunology, CH-4005 Basel, Switzerland H. Burkhardt (333),Department of Internal Medicine 111 and Institute for Clinical Immunology, University Hospital Erlangen-Nurnberg, Germany G. R. Burmester (333),Department of Internal Medicine 111, Medical Faculty of the Humboldt University, Berlin, Germany Siamon Gordon (271), Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom Gunter J. Hammerling (191), Department of Molecular Immunology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany J. R. Kalden (333),Department of Internal Medicine 111 and Institute for Clinical Immunology, University Hospital Erlangen-Nurnberg, Germany Klaus Karjalainen (257), Basel Institute for Immunology, CH-4005 Basel, Switzerland Guido Kroemer (51),CNRS-UPR 420, F-94801 Villejuif, France Robert B. Lobell (145), Merck Research Laboratories, Department of Cancer Research, Merck and Company, Inc., West Point, Pennsylvania 19486 James S. Malter (I),Department of Pathology and Laboratory Medicine, University of Wisconsin Hospital and Clinic, Madison, Wisconsin 53792 Andrew J. McKnight (271), Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom Frank Momburg (191), Department of Molecular Immunology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany Gerald T. Nepom (315),Virginia Mason Research Center and Department of Immunology, University of Washington School of Medicine, Seattle, Washington 98101 Josef M. Penninger (51),The Amgen Institute, Ontario Cancer Institute, and Departments of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario M5G 2C1, Canada ix

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ADVANCES IN IMMUNOLOGY, VOI.. hH

Posttranscriptional Regulation of mRNAs Important in T Cell Function JAMES S. MALTER Deparhnent of Parhobgy and laboratory Medicine, University of Wsconsin Hospifal and Clinic, Madison, Wisconsin 53792

I. Introduction

The development and activation of T lymphocytes involves substantial and complex alterations in gene expression. In this way, T cells can prepare for or react to changes in their environment. A great deal of attention has focused on understanding the transcriptional events that underlie T cell gene expression during differentiation or activation. Relatively little is known as to the extent of or mechanisms of posttranscriptional gene regulation under these conditions. Despite the infancy of this field, it is clear that a variety of critical genes are dominantly regulated at the level of posttranscriptional control. It is the intent of this review to discuss how posttranscriptional gene regulation, especially alterations in mRNA stability, contributes to the ultimate phenotype of a T lymphocyte. In some cases, I will extrapolate from other cell systems to T cells where insufficient data are available but likely applicable. Posttranscriptional regulation is often employed by cells or tissues that must respond quickly to changes in their environment with corresponding changes in gene expression. For most genes, transcription takes several hours to initiate and in the case of extremely long mRNAs (such as dystrophin) may take many hours to complete. Posttranscriptional control mechanisms, however, can be evoked in seconds to minutes, permitting cells much more control over mRNA and protein abundance and function. Therefore, posttranscriptional regulation affords greater speed in responding to stimuli. Second, such regulation is extremely versatile. As will be described later, cells can selectively regulate the stability, quantity, and translatability of individual mRNAs. Thus, modulation of a subclass of target mRNAs can be accomplished without affecting other cell functions. These features mandate the existence of regulatory systems capable of discriminating among different mRNAs. II. Measurement of mRNA Decay Rates

A variety of methods have been developed for the measurement of how rapidly mRNAs decay within cells. The rate of mRNA decay is equal to 1

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the production rdte (transcription) minus net accumulation. Therefore, in the absence of ongoing transcription, the rate of mRNA decay equals the change in mRNA amount over time. Thus, all methods for the measurement of inRNA decay must quench ongoing transcription or mask its presence. Current methods include pulse chase, transcriptional blockade, and a variety of inducible promoter systems. The measurement of decay of abundant mRNAs can be accomplished by puke chase (Alterman et at., 1984; Levis and Penman, 1977). Radiolabeled ribonucleotides are added to the culture supernatant and allowed to be incorporated into elongating transcripts. After a time adequate for radiolabeling of nascent mRNAs, a vast excess of unlabeled nucleotides are added to chase the radiolabeled homolog. This effectively produces a wave or pulse of radiolabeled mRNAs whose abundance can be followed over time. Although this method is effective for the measurement of high-abundance mRNAs such as globin, its application is quite limited. In addition to being insufficiently sensitive for low-abundance mRNAs, it remains unclear whether the instantaneous dilution of radiolabeled nucleotide occurs upon addition of unlabeled homolog. However, this methodology does not perturb the cells under investigation and, thus, where usable, remains the gold standard. Alternative methodologies have been developed to measure the decay rates of less abundant mRNAs. The most commonly employed use drugs that block transcription. Under such conditions, the amount of target mRNA remaining over time (as determined by Northern blot or RNAse protection) equals the decay rate. Transcriptional blockade can be accomplished with RNA polymerase I1 inhibitors, such as actinomycin D (Act-D) (Singer and Penman, 1972), cr-arnanitin (Chen et al., 1993), or the adenosine analog 5,6-dichloro-l-/3-~-ribofuranosyldencimidazole (DRB) (Sehgal et aZ., 1975). Act-D blocks transcription by intercalating between DNA stands, whereas DRB appears to function by interfering with kinases necessary for RNA polymerase I1 activity. The effective concentrations of these agents vary for different cell types, necessitating control experiments demonstrating that transcription has been inhibited. It is worth emphasizing that transcriptional blockage with these agents is global. Therefore, the measurement of decay of a particular mRNA occurs in the context of complete shutdown of the transcriptional apparatus. In addition, the effects of these drugs on other metabolic pathways are poorly characterized. Recent data have demonstrated that c-fos (Shyu et al., 1989; Chen, C. Y. et al., 1995), erythropoietin (Goldberget al., 1991), transferrin receptor (Seiser et al., 1995), and granulocyte macrophage-colony stimulating factor (GM-CSF) (Chen et al., 1993) mRNAs were stabilized in cells treated with Act-D. We have recently evaluated the effect of Act-D on

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3

the decay of capped, polyadenylated GM-CSF mRNA transfected into resting lymphocytes (Rajagopalan and Malter, 1996). GM-CSF mRNA was precipitated onto gold beads and accelerated into resting lymphocytes by particle-mediated gene transfer. After cells were washed to remove external, exogenous mRNAs, decay was assessed in the presence or absence of Act-D. When present, Act-D rapidly stabilized exogenous GM-CSF m R N A by greater than 10-fold. We also observed that the synthesis and secretion of GM-CSF protein was inhibited for approximately 2 hr when compared to untreated cells, although treated cells caught up by 10 hr. Therefore, Act-D has a profound effect on the mRNA decay machinery, as well as the translational apparatus. It remains unknown at this time if DRB induces similar effects. In order to avoid problems associated with the use of metabolic poisons, like Act-D or DRB, several inducible promoter systems have been developed. One of the most popular utilizes the c-fos promoter (Kabnick and Housman, 1988; Shyu et al., 1989). The presence of a serum response element within the promoter permits transient activation in response to serum. Transcription is self-limited, lasting approximately 15-30 min (Greenberg and Ziff, 1984; Kruijer et al., 1983), which produces a pulse of mRNA whose decay can be monitored by standard techniques. Functionally, this is equivalent to pulse chase but with a major advantage that the cDNA for any low-abundance mRNA can be used. However, in order to induce a brief pulse of transcription, cells harboring the transgene must undergo serum starvation for at least 12 and often 24 hr ( S h p et ul., 1989). Under such conditions, protein synthesis, gene transcription, and mRNA decay are reduced. For the study of c-fos mRNA, which is normally induced by serum, such issues are probably irrelevant. However, insufficient data exist to determine the appropriateness of these manipulations to study the decay of other mRNAs. Recently, other regulatable promoters have been employed for producing a controlled pulse of mRNA. These include the metallothionine metal response element (Hurta et al., 1993) as well as those activated by antibiotics such as tetracycline (Eldredge et al., 1995; Grossen and Bujard, 1992). The former is rather leaky, difficult to control, and requires cell culture in high concentrations of &valent metals. However, the tetracycline response promoters are gaining widespread use because these antibiotics have no apparent effects on eukaryotic cell metabolism, can be rapidly shut down, and are sufficiently active to produce a large bolus of niRNA on demand. 111. Measurement of Translation

The measurement of mRNA translation in intact cells is somewhat less problematic than mRNA decay. Clearly, the production of a previously

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JAMES S. MALTER

absent protein either in cell pellet or supernatant is a direct and irrefutable sign that translation has occurred. Protein can be detected by a variety of means including Western blotting, ELISA, or bioassay against a responsive cell line. It is important to point out that the rapid appearance of protein in a cell supernatant may reflect mobilization of preformed stores rather than de novo synthesis. Incubating the cells under study with 3sS-labeled amino acids with subsequent incorporation provides incontrovertible evidence of de novo protein synthesis. Another commonly used technique to evaluate translation is polysome profiling. Intracellularly, mRNAs can be partitioned to the soluble, ribonuclear protein fraction (mRNP) or attached to single or multiple ribosomes (so-calledpolysomes). Movement of an mRNA from soluble mRNPs to polysomes often precedes enhanced translation. Such changes often occur in response to changes in environmental conditions or cell activation. Translation can also be enhanced by cytoplasmic elongation of the 3’ polyadenylate tail. Well-described examples include vasopression mRNA (Carranza et al., 1988; Waller et al., 1993) in response to dehydration or water restriction. Finally, enhanced translation is often associated with increased activity of proteins important in translation initiation (Boa1 et al., 1993), which is the rate-limiting step. For example, on cell activation, eukaryotic initiation factor 4e (eIF4e or cap-binding protein) is phosphorylated, which increases its activity (Ma0 et al., 1992). Translation can be globally arrested by a variety of agents of which cycloheximide (CHX) is the most widely used. This drug freezes polypeptide elongation. Cycloheximide has profound effects on mRNA abundance by inhibiting mRNA decay. Many cytokine and protooncogene mRNAs, including GM-CSF (Rajagopalan and Malter, 1996), IL-2 (Shaw et al., 1988), IL-1 (Gorespe et al., 1993),cfos (Wilson and Treisman, 1988), and c-myc (Reed et al., 1987),are stabilized by CHX suggesting decay requires ongoing protein synthesis or the replenishment of a labile protein that destabilizes these m RNAs. IV. mRNAs Regulated by PosiiranscripiionalControl

The ability to rapidly modulate cytoplasmic mRNA quantity and translatability is critical for T lymphocytes to effectively respond to environmental change. Such change might be the binding of cell surface cytokine, interactions between the T cell receptor and specific antigen, differentiation, or in vitro manipulation with a variety of activating agents, including phorbol ester, ionophore, or lipopolysaccharide. During the past 10 years, our understanding and appreciation of posttranscriptional regulation has grown immensely. A large number of T lymphocyte and other cell mRNAs are

POS'ITRANSCRIPTIONAL REGULATION OF mRNAs

5

dominantly controlled by alterations in their stability and/or translatability rather than by transcriptional control. In the next sections, I will identify and discuss mRNAs under such regulation. A. PROTOONCOGENES The ability to increase or decrease protooncogene levels is integral to appropriate progression through the cell cycle, proliferative response, and apoptosis. Generally, mRNAs coding for protooncogenes are exquisitely unstable in resting T lymphocytes, due in large measure to the presence of cis-acting destabilizing domains. The best characterized of these is the AUUUA motif (see Section V). As cells move from Gointo GI,protooncogene mRNAs accumulate dramatically (Reed et al., 1987). The accumulation of c-myc mRNA occurred rapidly in normal B and T lymphocytes stimulated with phorbol ester, ionophore, or phytohemagglutinin (Reed et al., 1987). After stimulation with PHA, steady-state levels of c-myc mRNA increased 20- to 40-fold within 1 hr with only modest increases (3- to 5-fold) in the rate of transcription. It was also noted that c-myc mRNA stabilization was cycloheximide sensitive. C-fos mRNA is expressed rapidly and transiently following stimulation with growth factors, membrane depolarizing agents, neurotransmitters, and phorbol esters (Greenberg and Ziff, 1984). Transient expression of cfos mRNA was due to rapid decay mediated by a 3' untranslated region (UTR) AU-rich element (Wilson and Treisman, 1988) and a coding region instability determinant (Shyu et al., 1989). In fibroblasts the half-life of serum-induced c-fos mRNA was approximately 9 min, but can be indefinitely prolonged by the addtion of protein synthesis inhibitors such as cycloheximide (Rahmsdorf et al., 1987). In addition to cycloheximide, phorbol ester superinduced c-fos mRNA by approximately 10-fold,whereas calcium ionophore (A23187) had little, if any, effect (Shigeoka and Yang, 1990). In macrophages, c-fos mRNA can be induced by phorbol ester, lipopolysaccharide(LPS),or calcium ionophore. These effects were antagonized by interferon-y ( IFN-.)I),which enhanced c-fos mRNA degradation without affecting its transcription rate (Radzioch and Varesio, 1991).Others have shown that c-myc mRNA can also be downregulated by interferon-y (Harel-Bellan et al., 1988). Because interferon-y typically induced the expression of a variety of genes, c-fos and c-myc mRNA downregulation appears somewhat atypical. Pokeweed mitogen (PWM) or anti-CD3 antibodies significantly increased c-jun mRNA in T lymphocytes (Chauhan et al., 1993). The elevation of c-jun mRNA was maximal after 15-30 rnin of exposure of T cells to PWM. Although nuclear run on assays demonstrated enhanced transcription, c-jun mRNA was also stabilized. Cycloheximide treatment had no

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JAMES S. MALTER

effect on c-jun mRNA induction by PWM. Others have demonstrated that cycloheximide superinduced cjun in 3T3 fibroblasts (Lamph et al., 1988). They also observed upregulation of c j u n mRNA after phorbol ester treatment, although specific effects on mRNA stability were not assessed. The expression of the serine-threonine kinase pim-1 mRNA has been investigated in mitogen-treated ovine lymphocytes (Wingett et al., 1991). After a 4-hr stimulation with Con A and phorbol ester, a fourfold induction of pim-1 mRNA was observed. By 17 hr post stimulation, pim-1 mRNA had decreased by 50%. pim-1 mRNA half-life was 80 min at 4 hr poststimulation and fell to 35 min after 17 hr. In addition, cycloheximide superinduced the stability of pim-1 mRNA consistent with its function on other AUUUA-containing, protooncogene mRNAs. pim-1 mRNA is expressed as an alternatively spliced, 2.4-kb transcript in germ cells that lack AUUUA motifs. The decay of this mRNA was substantially slower than the AUUUAcontaining full-length message, consistent with a destabilizing role of the adenosine-uridine (AU)-rich element (ARE). IL-2 caused a transient increase in pim-1 mRNA in the IL-2-dependent murine CTLL-2 line (Dautry et al., 1988). The protooncogene c-kit encodes a transmembrane tyrosine kinase receptor whose ligand is the recently described stem cell factor. IL-3, IL4,and GM-CSF downregulated the expression of c-kit in murine myeloid mast cell progenitor cells (Sillaber et al., 1991). In normal CD34 bone marrow progenitor cells, phorbol ester downregulated steady-state levels of c-kit transcripts and stem cell factor receptor surface expression (Asano et al., 1993). The primary effect of phorbol ester (TPA) was to accelerate the decay of c-kit mRNA by fourfold, which was concentration dependent with a maximum at 10 mM. These effects were antagonized by cycloheximide. Transforming growth factor /3 (TGF-/3)treatment of murine hematopoetic progenitor cell lines also destabilized c-kit mRNA. Within 2 hr of TGF-P treatment, c-kit transcripts decayed with an accelerated half-life of approximately 1 hr. The decrease in c-kit mRNA was associated with a decrease in cell surface receptor expression (Dubois et al., 1994).As can be seen from the previous examples, cell activation with a variety of agonists often modulated the stability of rotooncogene and cell cycle progression factors. The tyrosine kinase p56k! ' was transiently downregulated upon T cell receptor/CD3 complex engagement (Paillard and Vaquero, 1991). Northern blotting and nuclear run-off assays revealed low basal transcription and accelerated decay, which combined to reduce steady-state p56Ick mRNA levels. This effect was antagonized by cycloheximide, which superinduced ~ 5 6 mRNA "~ levels by enhancing its stability. Thus, p56IckmRNA was downregulated by signal transduction pathways that usually increased lymphokine mRNA levels.

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Glucocorticoids induced Go/G1arrest of lymphoid cells by decreasing the abundance of cyclin D3. When dexamethasone was added to P1798 murine T lymphoma cells, cyclin D3 mRNA was reduced by 50% within 2 hr and 80% within 4 hr (Reisman and Thompson, 1995). The effects of glucocorticoids were reversible, however, with a return to control levels within 2 hr after their removal. Interestingly, there was no change in the transcription rate of cyclin D3 within 6 hr after the addition of glucocorticoids suggesting posttranscriptional regulation. Measurement of the decay rate of cyclin D3 showed a half-life of 8 hr in mid-log phase cells that was reduced to less than 1 hr after glucocorticoid treatment. These effects were antagonized by actinomycin D. Other cyclin mRNAs show considerable variation of expression throughout the cell cycle. Cyclin B1 peaks in G2/M with minimal levels in GI. BI mRNA can be induced transiently in HeLa cells after y-irradiation coincident with the development of a G2 block. Measurement of cyclin B, mRNA stability during different phases of the cell cycle revealed that the half-life varies from 1.1 hr in GI to 8 hr in S and 13 hr at the GJM interphase. Irradiation decreased the stability of cyclin B1 mRNA through an unknown mechanism (Maity et al., 1995). Similar regulation of the halflife of p53 mRNA was observed after the activation of peripheral blood mononuclear cells with a combination of phytohemagghtinin (PHA) and TPA (Voelkerding et al., 1995). At the GO/GIinterface, p53 mRNA was rapidly degraded with a half-life of 1 hr. Cycloheximide treatment superinduced p53 levels by stabilizing the mRNA. Cells driven into the cell cycle showed progressive stabilization of p53 mRNA to a half-life of 6 hr after 24 hr of stimulation. The combination of PHA and TPA was a more potent stabilizer than were TPA or PHA alone. This common theme of mRNA accumulation and stabilization on transit through the cell cycle also applies to mRNAs encoding c-rel (Gruniont and Gerondakis, 1990), B-myb (Reiss et ul., 1991), rufl (Colotta et al., 1991), the transcription factor spi-1 (PU.l) (Hensold et al., 1996), and the protein tyrosine phosphatase PTP-S (Rajendrakamar et al., 1993). In all cases, dramatic increases in mRNA levels were driven by enhanced mRNA stability.These effects were manifest at different points of the cell cycle corresponding to physiological requirements for these particular proteins. In most cases, cycloheximide antagonized regulated stability and typically increased steady-state mRNA levels. The mechanism of cycloheximide action remains obscure but has been ascribed to a requirement for a labile protein integral to mRNA destabilization, Conversely, cycloheximide and other protein synthesis inhibitors may directly interfere with polysome based mRNA decay. An additional level of regulation has been demonstrated for protooncogenes coded for by alternatively spliced mRNAs. Both c-re1 (Grumont and

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Gerondakis, 1990) and the human splicing factor PR264SC35 are coded for by multiple mRNAs which differ in their 3' untranslated regions. When measured simultaneously, these mRNAs exhibited dramatically different half-lives (Sureau and Perbal, 1994).

B. CYTOKINES Posttranscriptional gene regulation plays a critical role in the regulated expression of cytokines upon activation or differentiation. In many cases, increased cytokine mRNA stability preceded cytokine elaboration and constituted the dominant means by which T cells enhanced the production of these critical molecules. Although this field remains relatively new and certainly far less intensively studied than transcriptional regulation, the number of cytokines controlled through alterations in mRNA stability is striking. GM-CSF is a hematopoietic growth factor produced by a variety of cells, including T and B cells, monocytes, endothelial cells, and fibroblasts. A variety of activating agents, such as antigen, plant lectins (PHA, PWM, and Con A), mitogenic anti-cell surface antibodies, and phorbol esters (TPA), induced GM-CSF mRNA in T cells (Granelli-Piperno et al., 1984; Shaw and Kamen, 1986; Lindsten et al., 1989; Thorens et al., 1987). Thorens et al. (1987) demonstrated that mouse peritoneal macrophages can be induced to accumulate GM-CSF mRNA and release GM-CSF by inflammatory agents, phagocytosis, or adherence to substrates coated with fibronectin. GM-CSF mRNA accuinulation was blocked by dexamethasone and IFN-y. After activation, GM-CSF mRNA transcription rates were unchanged with accumulation of message entirely dependent on enhanced cytoplasmic stability. Shaw and Kamen (1986) and Caput et al. (1986) identified the presence of multiple AUUUA motifs in the 3' untranslated regon of GM-CSF. They demonstrated that GM-CSF mRNAs intrinsic instability in resting cells was due to these repeats, which could confer instability on a chimeric mRNA such as globin. However, Thorens et al. (1987) showed that c-sis, which codes for the chain of platelet-derived growth factor (PDGF), was differentially regulated from GM-CSF after macrophage adherence or LPS treatment. c-sis also contains AU multimers in the 3' untranslated region suggesting this class of unstable mRNAs may not be coordinately regulated at all times. After these initial observations, a number of groups have confirmed and extended them to better characterize the kinetics, types of activating agents, and responsible cis-acting elements. Lindsten et al. (1989) demonstrated that GM-CSF, IL-2, IFN-y, and tumor necrosis factor-a (TNF-a) mRNAs displayed differential regulation depending on how T cells were activated. Antibodies directed against the T cell receptor/CD3 complex induced

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upregulation of cytokine transcription but had no affect on cytoplasmic mRNA stability. However, when anti-CD28 and anti-T cell receptor antibodies were jointly employed, cytokine mRNAs were stabilized and markedly accumulated. Under such conditions, cytokine transcription rates were not enhanced. Interestingly, c-fos and c-myc mRNAs remained unstable despite activation through CD28. These data suggested that mRNA stability could be induced through specific signaling pathways. Just as mRNA stabilization appears necessary for cytoplasmic accumulation and gene expression, reinstitution of rapid decay must occur to quench cytohne production. Treatment of mitogen-activated peripheral blood lymphocytes as well as immortalized T lymphocyte lines with vitamin D3 downregulated GM-CSF mRNA by50 and 90% at 6 and 48 hr of exposure, respectively (Toebler et nl., 1988). The effects of vitamin D3 required protein synthesis as they were antagonized by cycloheximide. Nuclear runoff assays demonstrated that GM-CSF gene transcription was unchanged, but that mRNA decay was accelerated by approximately 10-fold. The immunosuppressive drugs FK506 and rapamycin selectively enhanced the degradation of GM-CSF and IL-2 mRNAs (Hanke et al., 1992). These drugs downregulated the IL-2 and GM-CSF promoters as well as enhanced the degradation of these mRNAs. Interestingly, neither the stability of IL2 receptor nor GAPDH mRNAs were affected by FK506 or rapamycin. These data suggested that commonly employed immunosuppressants may partially exert their effects by destabilizing cytokine mRNAs. Similar effects have been observed in activated lymphocytes treated with glucocorticoids or dexamethasone (Fessler et al., 1996). Finally, GM-CSF mRNA can be downregulated by cytokine treatment. In long-term bone marrow cultures stimulated with IL-1, TNF-a, or endotoxin, IFN-a inhibited the expression of GM-CSF mRNA (Gollner et al., 1995).The effects of IFN-a were dose and time dependent with maximal inhibition at 500 U/ml, which started approximately 90 min posttreatment. Transfection of GM-CSF promoter fragments revealed IFN-a had no affect on GM-CSF transcription but accelerated GM-CSF mRNA decay. Irradiation of cells induces the stress response associated with cytokine production. Hachiya et al. (1994) showed that irradiated fibroblasts increased their production of GM-CSF mRNA and protein. Irradiation was capable of potentiating the effects of phorbol ester stimulation. Removal of IL-1 bioactivity partially effaced the GM-CSF response suggesting IL1 contributed to the irradiation effect. Although runoff analysis revealed that the rate of transcription was increased, stability studies showed GMCSF mRNA half-life increased greater than fivefold in irradiated cells. These data suggested that GM-CSF can be upregulated through nonprotein kinase pathways.

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In addition to the previously described effects of phorbol ester, antigen, mitogens, and irradiation, calcium flux can also trigger GM-CSF expression. Treatment of EL-4 thymoma cells (Iwai et al., 1993) with the calcium ionophore A23187 induced a 12-fold increase in GM-CSF mRNA stability. These effects were mediated by the AUUUA boxes in the 3’ end of GM-CSF, although an upstream region approximately 160 bases away also contributed. IFN-y, like GM-CSF mRNA, can be superinduced by the treatment of cells with inhibitors of translation as well as low-dose y-irradiation (Lebendiker et al., 1987). Increased IFN-y mRNA levels could not be accounted for by increased transcription or decreased mRNA turnover. These data are in contradistinction to those of Lindsten et al. (1989), who demonstrated IFN-.)I was stabilized when T cells were activated with a combination of anti-CD28 and anti-CD3 antibodies. Peritoneal macrophages treated with LPS and IFN-y showed increased levels of IFN-P mRNA (Gessani et al., 1991). Measurement of IFN-/3 transcription failed to show an enhancement after LPS and IFN-.)I treatment despite a 20fold increase in steady-state mRNA levels. These authors attributed IFN-/3 accumulation under these conditions to enhanced stability. The recently described IL-12/NK stimulating factor has potent effects on human T cells. Addition of neutralizing anti-IL-12 antibody to PHAstimulated peripheral blood mononuclear cells markedly reduced both IFN-y protein and mRNA levels. Conversely, treatment of purified T cells with PHA and recombinant IL-12 increased IFN-y mRNA stability and protein production, whereas IL-2 mRNA levels were unaffected (Nagy et al., 1994).Interestingly, accessory signaling through CD40 synergized with IL-12 to upregulate the production of IFN-y as well as a variety of other TH1 and TH2 cytokines (Ping et al., 1996). These agents increased both the protein and the mRNA levels; however, determination of IFN-y mRNA half-life was not done. Purified human T lymphocytes activated by CD38 ligation also secreted a variety of cytokines, including IL-6, GM-CSF, IFN-y, and IL-10 (Ausiello et al., 1996). IL-7 stimulation of T lymphocytes synergized with anti-CD3 or anti-CD3/anti-CD28 to induce IFN-y and IL-4 mRNA expression (Borger et al., 1996a). At optimal concentrations, IL-7 (5 ng/ml) increased IFN-y mRNA levels by &fold and IL-4 mRNA by 3-fold. These effects could not be blocked by anti-IL-12 antibody. IL-7 induced the stabilization of both IFN-y and IL-4 mRNAs as well as enhancing their transcription. The regulated production of IL-la and -P has been intensively studied. In human fibroblasts and fibrosarcoma cells, IL-la and -p are constituitively transcribed but fail to accumulate. These data are consistent with a rapid decay rate, which has been estimated at approximately 20 min.

POS’ITRANSCRIPTIONAL REGULATION OF mRNA\

11

Treatment of fibroblasts with TNF-a, cycloheximide, or phorbol ester stabilized IL-1 mRNAs (Gorospe et al., 1993). Similar protein kinase C (PKOdependent IL-1 mRNA stabilization has been observed in human peripheral blood monocytes (Smith et al., 1991).However, downregulation of PKC by prolonged phorbol ester treatment followed by LPS stimulation revealed IL-la and -p mRNA levels can be regulated by an alternative signal transduction pathway. Stimulation of neutrophils with a combination of IL-1 and TNF-a upregulated IL-IP mRNA and protein (Marucha et al., 1991). These treatments induced large increases (30- to 90-fold) in the transcription rates of the IL-@ gene along with a modest (3- to 5-fold) increase in IL-1 mRNA stability. Retinoic acid enhanced IL-1 mRNA levels by altering the processing of precursor transcripts (Jarrous and Kaempfer, 1994). Under these conditions, IL-10 mRNA stability was not changed. Based on chimeric globin mRNAs containing the 3’ UTR of IL1, the 3’ AUUUA motifs were responsible for rapid IL-1 decay in resting cells (Kern et al., 1997). However, an LPS response element could not be mapped to the AU-rich determinant, suggesting another region of the 3’ UTR participated in this effect. Along with TNF-a, IL-1, and GM-CSF, IL-2 mRNA regulation has been intensively studied. Shaw et al. (1988) showed that IL-2 mRNA has a half-life of 1 hr in Jurkat cells or resting PBLs. IL-2 mRNA degradation was sensitive to cycloheximide as well as to actinomycin D. Lindsten et al. (1989) demonstrated that IL-2, GM-CSF, IFN-7, and TNF-a mRNAs were stabilized in normal T cells by mitogenic combinations of anti-CD28 and anti-CD3 antibodies. IL-2 mRNA accumulation can also be upregulated by treatment with PHA and TPA (Nordmann et al., 1989).Cyclosporin completely blocked IL-2 transcription but had no apparent effect on IL-2 mRNA decay. Garlesi and Mastro (1992) showed that pretreatment with phorbol ester for 10 hr blocked a response to subsequent mitogenic challenge with Con A or Con A plus TPA. Under such conditions, IL-2 mRNA accumulation was inhibited, which could be partially counteracted by cycloheximide. Conversely, mitogenic treatment with Con A plus TPA in the absence of pretreatment with TPA caused augmented IL-2 mRNA accumulation. These data suggested that protein kinase C activation induced IL-2 mRNA stabilization and accumulation (Dill et al., 1994). These authors noted that different mitogens differentially stabilized IL-2 mRNA. A combination of phorbol ester plus Con A appeared maximal and increased IL-2 inRNA levels by >20-fold and the half-life by >5-fold. Phorbol ester plus ionophore increased IL-2 mRNA by >100-fold and the half-life by 10-fold. In addtion to mitogens, IL-2 mRNA was stabilized by the treatment of Jurkat cells with substance P (Calvo, 1994).

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JAMES S . MALTER

Interestingly, substance P-mediated stabilization was abolished by concominant treatment with cyclosporin A, actinomycin D, or cycloheximide. Laff et al. (1995) evaluated the kinetics of IL-2 mRNA production and stabilization in mouse T cells stimulated through the CD28 and T cell receptors. CD28 signaling increased IL-2 mRNA levels by 20-fold compared to T cell receptor signaling alone. In the absence of CD28 costimulation, IL-2 mRNA rapidly decreased secondary to accelerated IL-2 mRNA decay, However, CD28 costimulation also involved nuclear effects as the levels of unspliced IL-2 mRNA were increased, Gerez et al. (1995) have shown that mitogenic induction of IL-2 gene expression also involved nuclear accumulation of precursor IL-2 mRNAs. The net production of mature message from precursor was greatly facilitated, causing a superinduction of cytoplasmic mRNA. The CD28 costimulatory pathway can be blocked by treatment of T cells with glucocorticoids (Fessler et al., 1996). No changes in transcription rate were observed in the presence of dexamethasone, demonstrating that the IL-2 mRNA levels must be controlled by posttranscriptional mechanisms. In addition to glucocorticoids, FK506 and rapamycin selectively enhanced the degradation of IL-2 mRNA (Hanke et al., 1992). Although it was not demonstrated, these data suggested that other AUUUA-containing cytokine mRNAs may be similarly affected. Destabilization was antagonized by okadaic acid, demonstrating the importance of phosphorylation in FK506-mediated degradation of IL-2 message. TNF-a is a critical cytokine produced by many cells of the hematopoietic and lymphoid systems. It has wide-ranging systemic effects and plays a central role in the pathophysiology of cachexia, inflammation, and septic shock. At the cellular level, TNF stimulated the production of cytokine by lymphocytes and macrophages. Sung et al. (1988) showed that TNF-a mRNA was stabilized in T cell lines and normal peripheral blood T lymphocytes by costimulation with phorbol ester and anti-CD3 antibody. Phorbol ester can substitute for CD28 stimulation in providing necessary signals for TNF-a mRNA stabilization. Protein kinase C inhibitors destabilized TNF-a mRNA in virus-infected astrocytes (Lieberman et al., 1990).These data supported a role for kinase-mediated regulatory systems in the control of TNF-a mRNA stability. Using a reversibly bound, protein kinase C activator, Sung et al. (1991) confirmed that TNF-a mRNA was stabilized through PKC-mediated events that could be antagonized by removing the agonist or adding the protein kinase inhibitor H-7. TNF-a mRNA can also be induced by ionizing y-irradiation (Weill et al., 1996). Because these effects were observed within 30 min, it is likely they involved some degree of posttranscriptional control. A variety of cytokines and drugs also modulated TNF-a mRNA stability. Treatment of murine macrophages with IL-4 plus LPS enhanced the degradation of TNF-a message, which was not observed

POSTTRANSCRIPTIONAL REGULATION OF m R N A s

13

after IFN-.)I plus LPS (Suk and Erickson, 1996). Thalidomide accelerated the decay of TNF-a message (Moreira et ul., 1993). The effect of this drug appeared specific because other LPS-induced monocyte cytokines were unaffected. In addition to the previously discussed mRNAs, a number of other cytokines are also controlled at the posttranscriptional level. IL-10 is produced spontaneously by monocytes and B cells but not by T cells of healthy donors. Cycloheximide treatment of normal T cells superinduced IL-10 inRNA levels through mRNA stabilization (Stordeur et al., 1995). IL-4 mRNA can be induced in resting T lymphocytes by Con A (Dokter et al., 1994). Treatment of Con A-stimulated T cells with IL-7 enhanced IL-4 inRNA levels by increasing its stability. Antibodies against IL-1 and TNF-a had no effect on the IL-7-induced enhancement of IL-4 mRNA suggesting a direct effect of IL-7. IL-4 mRNA was induced through antiCD3 or anti-CD3 plus anti-CD28 treatment of human T lymphocytes (Borger et al., 1996b). Treatment of stimulated cells with CAMP as well as prostaglandin E2 blocked mitogen-induced IL-4 mRNA accumulation. These effects were dominantly transcriptional when Con A was used to activate T cells, whereas the modulation of IL-4 inRNA stability was the dominant feature in CD3KD28-activated T lymphocytes. Interestingly, TPA plus calcium ionophore-induced IL-4 mRNA expression was insensitive to the effects of CAMPand prostaglandin E2.Treatment of lymphocytes with cross-linked TCR with vasoactive intestinal peptide inhibited IL-4 production at the posttranscriptional level (Wanget al., 1996).These effects were antagonized by recombinant IL-2. IL-3 is transiently produced by T lymphocytes stimulated with mitogen or antigen. Stimulation of mast cells with calcium ionophore or phorbol ester stabilized IL-3 rnRNA without affecting its transcription rate ( Wodnar-Filipowicz and Moroni, 1990). These effects disappeared when stimulating agonists were removed, demonstrating a necessity for ongoing signal transduction. In activated human T cells, IL-7 stabilized IL-3 and GM-CSF rnRNAs (Dokter et al., 1993). The IL-7-mediated effect was independent of protein synthesis and had no effect on the transcription rate. Enhanced mRNA levels led to 4-fold increases in IL-3 and GM-CSF protein secretion. In addition to blocking IL-2 transcription, cyclosporin A destabilized IL-3 mRNA (Nair et al., 1994). Interestingly, IL-4 and IL6 transcripts that were coexpressed with IL-3 mRNA were not affected by cyclosporin. All three cytokine rnRNAs contain 3’ AUUUUA motifs, suggesting selective regulation of IL-3 must occur through an additional mechanism. IL-6 has recently been shown to be regulated at a posttranscriptional level. Kuo et nl. (1996) showed that IL-lP added to c-kit ligana IL-10-stimulated mast cells prolonged the half-life of IL-6 m R N A by

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JAMES S. MALTEH

approximately 4 hr and induced a 50-fold increase in the level of IL-6 protein. IL-11 can be stabilized in phorbol ester-stimulated primate bone marrow stromal cells, which were susceptible to protein synthesis inhibition (Yang et al., 1996). Chimeric transcripts demonstrated that the 5’ UTR coding region and 3’ UTR contributed to regulated IL-11 mRNA decay. IL-8 mRNA was stabilized by prolonged (24-hr) treatment of human T cells with LPS (Villarete and Remick, 1996). Vascular endothelial growth factor (VEGF) is expressed by CD3-positive peripheral blood T cells. This potent endothelial cell mitogen and angiogenic factor can be induced in CD3-positive T cells by hypoxia (Freeman et al., 1995). Levy et al. (1996) demonstrated that hypoxia increased VEGF mRNA stability from 43 to 106 min. Several hypoxia-induced proteins were identified that bound specifically to 3’ UTR VEGF mRNA sequences and whose activity increased with message stabilization. Other members of the TNF-a family, including lyinphotoxin and lymphotoxin /3, have shown variable levels of posttranscriptional regulation in activated murine T cell clones (Millit and Ruddle, 1994).In anti-CD3-activated T cells, lymphotoxin and lymphotoxin fi mRNAs were stabilized. However, these two mRNAs showed differential sensitivity to cycloheximide, with lymphotoxin superinduced but lymphotoxin /3 unaffected. Other monocyte/macrophage-derived chemokine mRNAs, including HILDNLIF, GRO-a, and GRO-/3, can be synergistically induced with a combination of phorbol ester, LPS, and vitamin D3 (Anegon et al., 1991; Iida and Grotendorst, 1990). These mRNAs can be superinduced with cycloheximide and appear to be stabilized by mitogens. Finally, colony stimulating factor-1 (CSF-1) can be induced by phorbol ester, TNF-a, or cycloheximide (Koeffler et al., 1988). The half-life of GCSF mRNA was increased 16-fold in cells cultured with TNF-a, TPA, or cycloheximide. C. CELLSURFACERECEPTORS Just as cytokines are regulated by alterations in inRNA stability, cell surface cytokine receptors often show similar regulation. When T cell clones were anergized with high concentrations of peptide in the presence of antigen presenting cells (APCs) cell surface CD28 was decreased. This was accompanied by accelerated CD28 mRNA decay (Lake et al., 1993). The early lymphocyte-activation antigen, CD69, was rapidly induced during lymphoid activation. This molecule can transmit stimulatory signals in T and B lymphocytes, NK cells, and platelets. In phorbol ester-activated T lymphocytes, CD69 mRNA declined rapidly with a half-life of less than 60 min. The 3’ UTR of this mRNA contains AUUUA motifs that, when fused to a previously stable globin transcript, conferred instability (Santis et al., 1995). In the S1A T lymphoma cell line, polyunsaturated lipids

POSTTRANSCRIPTIONAL REGULATION OF inKNA\

15

enhanced Thy-1 inRNA and protein levels (Deglon et nl., 199Fj).Increased Thy-1 mRNA was entirely due to enhanced stability and appeared to be mediating by the coding region alone. IL-2 receptor-a (IL-2Ra) chain was upregulated in T lymphocytes activated with combinations of antiCD3 or anti-CD2 plus anti-CD28 antibodies. The costimulatory effect of dual-receptor ligature resulted in enhanced stability of IL-2Ra mRNA (Cerdan et a l , 1995). Combined treatment of human inonmytes with IL-2 and IFN-y has also been show to stabilize IL-2Ry mRNA (Bosco et al., 1994). CD7 is a 40-kDa member of the immunoglobulin superfamily expressed earIy in T cell development. Ligand binding to CD7 can deliver a costimulatory signal with CD3-mediated activation. Treatment of peripheral blood T cells with a nonmitogenic ionophore increased CD7 transcription without altering CD7 mRNA stability.After stimulation with mitogenic doses of ionomycin, PHA, and anti-CD3 antibody, CD7 mRNA stabilitywas enhanced (Ware and Elaynes, 1993). IL-4 receptor mRNA accumulated in human T cells after activation with Con A, TPA, calcium ionophone, or a combination of these agents (Dokter et nZ., 1992). Mitogens increased IL-4 receptor mRNA stability by two or threefold, which could be further enhanced by treatment with IL-4. The CSF-1 mRNAs can be upregulated in phorbol ester-stimulated monocytes. These effects were antagonized by cotreatment with dexamethasone and cyclosporin, which destabilized CSF-1 mRNA (Chambers et al., 1993). IL-6 treatment of the human monocytic line THP-1 stabilized IFN-y receptor inRNAs (Sancau et al., 1992).These data were in contradistinction to a dominantly transcriptional effect mediated by TNF-a treatment alone. mRNAs coding for the CD45 isoforms were controlled through alterations in stability (Deans et nl., 1992). After T cell activation high-molecular-mass isofornis of CD45 were preferentially expressed for approximately 2 days followed by rapid downregulation with increased expression of a low-molecular-weight isoform, CD45 KO. The switch from high- to low-molecular-mass isoforms appeared to be controlled by rapid mRNA degradation sensitive to cycloheximide. Specific ligation of a cytokine to its cell surface receptor has also been shown to alter receptor mRNA stability. IL-1 downregulated cell surface expression and mRNA levels of the IL-1 receptor type 1 (Ye et nE., 1992). When 2-15% of the IL-1 surface receptor was occupied, IL-1 receptor mRNA stability was reduced from 6 to 1 hr. These effects were blocked by cycloheximide suggesting de izovo protein synthesis may be necessary for decreased RNA stability. The IL-6 receptor is encoded by two distinct mRNAs of different lengths that vary only in the 3' untranslated region (Bowman et al., 1990). The longer mRNA contains multiple AUUUA motifs, suggesting that IL-6 receptor expression can be controlled by mitogens as well as alternative splicing to vary steady-stable levels. Expression

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JAMES S . MALTER

of the transferrin receptor is controlled by 3’ untranslated region iron response elements (Teixeira and Kuhn, 1991). In addition to iron levels, IL-2 treatment of the murine T cell line B6.1 induced transferrin receptor mRNA by 50-fold when added to arrested, IL-2-deprived cells (Seiser et al., 1993). In these cells, IL-2 increased the binding activity of the iron response element-binding protein, which prevented transferrin receptor mRNA degradation. CD2 is an important T cell surface receptor capable of transmitting proliferative signals. The stimulation of human peripheral blood mononuclear cells with PHA, anti-CD3 monoclonal antibody, and TPA rapidly increased CD2 mRNA levels by stabilizing CD2 mRNA by 4-fold (Malter et al., 1988). The upregulation of CD23 by IL-4 can be antagonized by IFN-.)I (Lee et al., 1993). The inhibitory action of IFN-.)I required new protein synthesis and occurred by decreasing the stability of CD23 mRNAs. D. OTHERmRNAs REGULATED BY ACTIVATION A well-known phenomenon associated with T cell activation is enhanced mRNA translation, which can increase by approximately 10-fold (Boa1 et al., 1993). Resting T lymphocytes express low levels of critical initiation factors, including eIF-2a, 4E, and 4A mRNAs, compared to proliferating T cells. Activation resulted in a rapid, 20- to 50-fold increase in the level of these three mRNAs (Ma0 et al., 1992). New protein synthesis was not required for increased initiation factor mRNA level, nor could transcription upregulation account for these changes. Therefore, eIF-4 mRNA stability was likely enhanced during T cell activation. For eIF-2a, the expression of alternatively spliced mRNAs with different 3’ UTRs may account for some of this effect. The 1.6- and 4.2-kb transcripts differed in their stability with the larger message more stable in activated cells (Miyamoto et al., 1996). The regulated expression of molecules critical for normal immune response, antigen recognition, and humoral immunity are partially or fully controlled by posttranscriptional regulatory pathways. CD3 is a multisubunit assembly associated with the T cell receptor. The CD36 gene produces three distinct, mature mRNAs of 0.7, 1.5, and 2.5 kb (Wilkinson et al., 1989).Cycloheximide treatment increased the expression of all three CD36 transcripts, which along with variably spliced mRNAs that differ only in the 3’ untranslated region, suggested a component of posttranscriptional control. Accumulation of the TCRa gene can be induced by phorbol ester, calcium ionophore, or protein synthesis inhibitors ( Wilkinson and Macleod, 1988). Treatment with multiple agonists increased the level of TCRa message that could not be suppressed by cyclosporin. HLA class I1 expression was also regulated at a posttranscriptional level in human T cells.

POSTTRANSCRIPTIONAL REGULATlON OF rnRNAs

17

Although resting T cells do not express detectable, cell surface class 11, activation with PHA and TPA caused a rapid appearance (Caplen et al., 1992).Northern blotting revealed constitutive expression of class I1 mRNAs that could be superinduced with cycloheximide. Del Pozzo and Guardiola (1996) showed that in vitro, HLA class I1 mRNAs associated with polysomes derived from cells treated with puromycin or cycloheximide were more rapidly degraded than those in the absence of protein synthesis inhibitors. Therefore, it appeared that ongoing translation was required for the stabilization of HLA class I1 mRNAs. Class I expression may also be partially controlled by regulated stability. Compared to HLA-A and -B, HLA-C showed low levels of cell surface expression. Northern blotting revealed that HLA-C mRNA was expressed at lower levels than HLA-B mRNA and that this difference resulted from faster degradation of the HLA-C mRNA (McCutcheon et al., 1995).A 3’ UTR domain approximately 600 bp downstream of the stop codon appeared responsible for this effect. In mature B cells, steady-state immunoglobulin mRNA levels were increased by approximately 50-fold over earlier B cell progenitors. Early stage lymphomas degraded p inRNA in approximately 2 hr, which was a 9-fold increase in stability in hybridomas (Genoviese and Milcarek, 1990). Cox and Emtage (1989) demonstrated a 6-fold stabilization of p mRNA as B cells differentiated into plasma cells. Enhanced stability was almost sufficient to account for the differences in steady-state mRNA levels between the two cell lines. Similar data have been shown by Reed et al. (1994). E. DIFFERENTIATION Very little information is available concerning alterations in mRNA stability in the developing thymus. In the microenvironment of the thymus, cytokines are probably carefully and precisely regulated to ensure the appropriate differentiation of T cells. Le et al. (1991) have shown that thymic epithelial cells produce IL-1 and IL-6. Primary cultures of normal human thymic epithelial cells treated with EGF or TGF-a increased IL-1 and IL-6 mRNA levels. In both cases, transcription rates were unchanged, but IL-1 and IL-6 mRNAs were stabilized. Because both EGF and TGFa can induce tyrosine phosphorylation, enhanced cytokine mRNA stability was likely dependent on tyrosine kinase cascades. In the most complete study to date, Takahama and Singer (1992) evaluated the regulation of CD4 and CD8 mRNAs as thymocytes transited from dual- to single-positive cells. T cell receptor cross-linlang induced such differentiation, which was entirely dependent on enhance degradation of CD4 or CD8 mRNAs. Interestingly, the VDJ recombinase gene, RAG-1, is also tightly regulated at the posttranscriptional level (Neale et al., 1992). RAG-1 and RAG-2 are

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JAMES S. MALTER

both expressed only in immature lymphocytes. Treatment of immature lymphocytes with phorbol ester caused a rapid elimination of RAG-1 and RAG-2 mRNA. In addition to blocking the transcription of RAG-1, phorbol ester accelerated the decay of RAG-1 mRNAs by more than twofold. V. cis Elements

Given the vast number of mRNAs that coexist in any cell at any point in its life cycle, mechanisms must exist by which the cellular machinery can discriminate one mRNA from another. The ability to localize mRNAs to distinct subcellular fractions, selectively degrade or stabilize, as well as load them onto polyribosomes at different times under different conditions requires elaborate regulatory capacity. This regulatory machinery appears to require both cis-acting elements embedded within mRNAs and trans factors capable of interacting with these cis elements. Cis elements can be produced by unique, contiguous nucleotide sequences, secondary structure such as stem loops, or higher folding between distant regions separated by as many as several thousand bases. The diversity of structural elements reflects the intrinsic ability of mRNAs to fold, bringing nearby or distant regions close together for the creation of regulatory sites. The location of cis elements can be predicted by the alignment of mRNA sequences coding for a single or a family of genes from divergent species. The alignment of mouse, rat, and human mRNAs often reveals unexpected homologies, especially outside of the coding regions. The conservation of 5’ or 3’ UTRs strongly suggests the presence of a regulatory element. For example, cytokine mRNAs typically show substantial homology throughout their entire length. Although the coding regions would be expected to share substantial homology at the nucleotide level, the 5’ and 3’ UTRs can be >90% identical (Shaw and Kamen, 1986). In general, conservation in the 5‘ UTR often implies the presence of translational control elements, whereas that in the 3’ UTR typically suggests domains important in mRNA stability or localization. In some cases, these domains may interact as well. The previous discussion is not meant to imply that all mRNA cis elements reside in untranslated regions. Recently, cfos (Kabnick and Housman, 1988; Shyu et al., 1989; Schiavi et al., 1994; Shyu et al., 1991; Wellington et al., 1993) and c-myc (Bernstein et al., 1992; Prokipeak et al., 1994) mRNAs have been shown to contain a destabilizing coding region element. Such domains are difficult to identify by homology search but tend to become apparent after other potential elements have been experimentally manipulated without loss of the expected phenotype.

POSTI'RANSCRIPTIONAL REGULATION OF mRNAs

19

A. ADENOSINE-URIDINE-RICH ELEMENTS The putative identification of regulatory domains by homology search necessitates experimental demonstration of their functionality. In 1986, Shaw and Kamen, coincident with Caput et al. (1986), identified a conserved nucleotide sequence consisting of repeated, tandem AUUU boxes within the 3' UTR of mRNAs encoding inflammatory mediators, cytokines, and protooncogenes. They were usually organized as AUUUAUUUA repeats varying in number from several to eight (Shaw and Kamen, 1986). These domains showed remarkable conservation across species lines and appeared restricted to cytokine and protooncogene mRNAs. The repeated AUUUA motifs (also known as Shaw-Kamen boxes, AU-rich elements, or AREs) failed to demonstrate obvious relationships to either the stop codon or the poly A tail. In addition, some mRNAs, such as IFN-.)I, contained two or three AUUUA tandem pentamers separated by 20-50 dissimilar bases followed by another cluster of AUUUA repeats. GM-CSF mRNA, on the other hand, showed much tighter packing of these domains. Measurement of GM-CSF mRNA decay in a T cell line after transcriptional blockade with Act-D revealed very rapid degradation with a half-life (&) of approximately 45 min (Shaw and Kamen, 1986). TNF-a mRNA decay showed similar kinetics (Caput et al., 1986). P-Globin, however, was supremely stable, with a calculated ti > 15 hr (Shaw and Kamen, 1986). Therefore, AREs destabilize mRNAs that contain them. Mutagenesis of the AUUUA repeats present in GM-CSF, TNF-a, or cfos mRNAs dramatically reduced the decay rate of the mutant mRNAs (Shaw and Kamen, 1986; Rajagopalan and Maker, 1996; Iwai et al., 1993). Conversely, chimeric globin mRNAs fused to the AUUUA motifs derived from GM-CSF or c-jios greatly accelerated the decay of this previously stable transcript from 17 hr to approximately 45 min (Shaw and Kamen, 1986). In addition, protein synthesis was required for the rapid decay of wild-type GM-CSF or chimeric globin GM-CSF mRNAs because it was prevented by cycloheximide (Shaw and Kamen, 1986). Finally, labile mRNA decay was coupled to PKC-regulated events because TPA treatment of T lymphocytes or fibroblasts stabilized most, but not all, AU-containing mRNAs (Shaw and Kamen, 1986; Koeffler et nl., 1988). Thus, these pioneering studies demonstrated a link between mRNA turnover mediated through AU motifs, translation, and PKC-mediated signal transduction pathways. These data showed that regulatory pathways that modulated transcriptional events coordinately controlled posttranscriptional events. Database searches have revealed many mRNAs with single reiterations of the AUUUA motif. P-Globin mRNA, for example, contains a single motif. Given the known stability of globin mRNA, these data suggested that

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JAMES S . MALTER

single reiterations of the AUUUA motif were unlikely to be a destabilizing element. Because multiple, often tandem reiterations of AUUUA were found in rapidly degraded mRNAs, this suggested that a higher order or reiterative structure containing multimers of this sequence might be the true element. Recent work (Zubiaga et al., 1995; Lagnado et al., 1994) has conclusively shown that the nonamer UUAUUUA (UIA) (U/A) is the true destabilizer formed by reiterations of the AUUUA motif or a single motif in a U-rich context. Interestingly, somewhat greater instability was conferred by reiterations of this nonamer as found in GM-CSF, IL-2, and TNFa mRNAs. Reiterations of AUUUA (AUUUAAUUUA)are not destabilizing (Lagnado et al., 1994) suggesting that multiple interior purines interfere with the dominantly U-rich element. Whether this destabilizing element assumes higher order structure remains unknown. Computer-assisted folding has failed to demonstrate a stable stem-loop structure and ribonuclease mapping has yet to be reported. Thus, current data suggest that the AUUUA motifs function as a primary sequence. Additional work has demonstrated that the 3’ untranslated location of the AU motif is not coincidental. P-Globin GM-CSF chimeras showed identical stability to wild-type P-globin if (i) the mRNA could not be translated, (ii) the AUUUA motifs were disrupted by guanosine or cytosine substitutions, and (iii) the stop codon was mutated, allowing polyribosomes to be translated into or through the 3’ untranslated region (Savant-Bhonsale and Cleveland, 1992). Untranslatable, chimeric mRNAs remained associated with cytoplasmic mRNPs rather than polyribosomes. This was in marked contrast to wildtype GM-CSF mRNAs containing functional stop codons that were associated with very large (>20 S) translation-dependent destabilizing complexes. Finally, ARE-containing mRNAs remained unstable only when fully translated. Inhibition of ribosome translocation as the result of the insertion of a stable stem-loop structure in the 5’ UTR prevented AREmediated destabilization (Aharon and Schneider, 1993). Somewhat unexpectedly, a stable stem loop within the 3’ UTR upstream of the ARE also blocked rapid decay (Curatola et aZ., 1995). These data suggested that ARE-mediated decay likely involves ribosomeassociated, translation-dependent decay factors that require particular topography only achieved under conditions of normal translation. These could include alterations in secondary or tertiary structure assumed by the 3’ untranslated region or possibly the juxtaposition of particular protein factors such as a ribonuclease to the mRNA target. However, Chen, C. Y. et aZ. (1995) showed that P-globin c-fos chimeras were rapidly degraded in the absence of translation. When chimeric mRNAs containing a c-fos ARE and a 5’ iron response element to permit regulated translational

POSTTRANSCRIPTIONAL REGULATION OF mRNAs

21

initiation were evaluated, the chimeric message also decayed rapidly in the absence of translation (Koeller et al., 1991).Finally, there have been recent reports that GM-CSF inRNA decay can occur in the absence of ongoing translation (Chen, C. Y. et al., 1995). Whether these discrepancies reflect true differences between the cfos ARE and GM-CSF remains unresolved. Other possible explanations include the types of cells used and the presence of metabolic poisons, such as actinomycin-D, used for the measurement of mRNA decay. Although the use of chimeric mRNAs has been invaluable to demonstrate the function of cis elements, such studies must be interpreted cautiously. For example, the Yj’ UTR or coding region may contain additional ancillary information necessary for appropriate regulation. The demonstration of coding region destabilizing elements in cfos (Shyu et al., 1989) and c-myc (Bernstein et al., 1992) mRNAs demonstrates that such regions should not be ignored. Finally, long-range interactions within the 3’ UTR of the insulin-like growth factor I1 mRNA (Meinsma et al., 1992; Scheper et al., 1995) show how distant sequences can assemble to form a functional destabilizing domain. Finally, chimeric mRNAs containing AREs are usually nonresponsive to the effects of phorbol ester or other agents known to stabilize their wildtype counterparts (Akashi et al., 1994). Therefore, it is highly likely that the AREs, although active as dominant destabilizers, may lose some functionality without additional ancillary information provided by nearby or distant sequences within the body of the mRNA. Given the paradigms of GM-CSF, IL-2, or TNF-a mRNAs, how do the AREs direct rapid decay? The biochemistry of this process remains largely unknown. Clearly, once initiated, decay is extremely rapid. Only on rare occasions have intermediates in this process been identified (Stoeckle, 1992), suggesting that once the process begins it occurs with extreme speed. Based on the requirement for translation, mRNA decay likely occurs on a polysome or in close association with it. Wilson and Treisman (1988) were first to demonstrate that c-fos mRNA decay was initiated by poly A tail shortening. They observed progressive loss of the polyadenylate tail before disappearance (with presumed cleavage) of the coding region. Shortly after the deadenylation to approximately 30 A residues, the mRNA body disappeared as assessed by Northern blotting. These data have been confirmed by many other investigators and suggested that deadenylation was the first step in labile mRNA decay. When the A tail reached a critical length of approximately 30 residues, destruction of the mRNA body was triggered. Such destruction could be the result of progressive and continued 3’ to 5’ exonuclease action or of internal endonuclease digestion. Using an in witro system to study c-myc mRNA decay, Brewer and Ross (1988) identified an mRNA decay intermediate whose termini was near to or at

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the AUUUA motifs. These data were interpreted to suggest that c-nzyc mRNA was cleaved by an endoribonuclease that may recognize the AU motifs. Several putative ribonucleases potentially specific for AUUUAcontaining mRNAs have been partially or completely purified (Astrom et al., 1992; Huaet al., 1993;Wennborget al., 1995; Caruccio and Ross, 1994).

B. APPROACHES TO IDENTIFYING NEWcis ELEMENTS Although the AU-rich element remains among the most intensively studied mRNA instability determinants, it is by no means the only one. As discussed herein, there appear to be dozens, and perhaps hundreds of mRNAs that are regulated partially or dominantly at the posttranscriptional level. Typically, nuclear runoffs fail to account for changes in mRNA abundance, suggesting alterations in mRNA stability may be operative. Because many mRNAs regulated at this level lack AU-rich elements, there must be additional domains with distinctive sequences, structures, and shapes capable of mediating regulated and selective decay. Clearly, the presence of shared elements among coregulated mRNAs provides a powerful means for coordinated control. Thus, both experimental data and common sense suggest that a plethora of distinctive cis elements exist. Identification of novel cis elements can be performed in a variety of ways. As mentioned previously, homology searches between divergent species can reveal unexpected conservation. This approach was used to identify the AUUUA motifs in cytokine and protooncogene mRNAs (Shaw and Kamen, 1986; Caput et al., 1986). Second, the availability of “natural experiments” can point to the presence of a previously undefined element. For example, IL-2 (Henics et al., 1994) and IL-3 (Mayo et al., 1995; Algate and McCubrey, 1993) mRNAs accumulate in MLA-144 and FL5.12 cell lines, respectively. Sequencing these mRNAs revealed the insertion of retroviral long terminal repeats (LTRs) into the 3’ UTRs. The LTRs disrupted the endogenous AUUUA motifs causing message stabilization. In some cases, the coopting of cellular protooncogenes into retroviruses has been associated with the loss of similar regulatory domains. v-fos lacks the terminal 3’ untranslated region of its cellular homolog c-fos. When measured, the decay rate of v-fos mRNA is far slower than that of c-fos, contributing to its accumulation and transforming activity (Rahmsdorf et al., 1987). Finally, the identification of protein binding sites may pinpoint the location of regulatory domains. We have used this approach to identify an instability determinant present in the amyloid protein precursor (APP) mRNA (Zaidi and Malter, 1994; Zaidi et al., 1994). In this case, a large 3’ untranslated region (1.2 kb) made classical mutagenesis approaches impractical. Therefore, we produced radiolabeled APP mRNA and used

POSTTRANSCRIPTIONAL REGULATION OF inRNAs

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it for mobility shift assays with cytoplasmic lysates from neuronal cell lines. APP mRNA was previously shown to be abnormally stable in these lines. We were able to identify multiple mRNA-protein interactions that ultimately were mapped to a 29-base domain approximately 200 bases from the stop codon. After mutagenizing this domain, APP mRNA was stabilized, demonstrating the functional significance of the protein binding site (Zaidi et al., 1994).

C. OTHERcis ELEMENTS Because many posttranscriptionally regulated mRNAs lack AU repeats, additional instability determinants clearly exist. There is no a priori reason why such domains need be AU rich. In this section, I will discuss some of the better defined mRNA instability elements found in mRNAs expressed by T lymphocytes. In many cases, such regulation has not been demonstrated in T cells but is likely. APP mRNAs are expressed by T cells as well as by most other nucleated human cells. At least five alternatively spliced APP mRNAs exist that code for amyloid precursor protein from which P-amyloid is proteolytically derived (Beyreuther et al., 1991). Overproduction of 0-amyloid is likely pivotal for the development of Alzheimer’s disease. In addition to overproduction of APP and 0-amyloid, a subset of patients also overexpress APP mRNA (Jacobsen et al., 1991). Elevated levels could be the result of enhanced transcription or decreased m RNA degradation or both. Therefore, we examined if APP mRNA was regulated at the posttranscriptional level by measuring its half-life in resting and activated peripheral blood mononuclear cells. In resting cells, APP mRNA decayed with a half-life of approximately 4 hr, which could be increased to >12 hr after cell activation with a mitogenic combination of phorbol ester and phytohemagglutinin (Zaidi et al., 1994). These data suggested that APP mRNA contained a domain through which its stability was controlled. By the use of protein binding and mutagenesis, a 29-base region approximately 200 bases from the stop codon was implicated in APP mRNA regulation (Zaidi and Malter, 1994).This region is highly conserved between murine and human APP mRNAs with 26 of 29 bases in common. Homology search has failed to reveal any additional mRNAs containing like sequences. At the nucleotide level, the regon is 61% AU but not organized into AUUUA motifs. Computer modeling revealed the presence of a potential stem-loop structure, although ribonuclease mapping has yet to be performed. Insertion of guanosine in place of adenosine and cytosine in place of uracil completely ablated the functionality of this domain, rendering APP mRNA constitutively stable (Zaidi and Malter, 1994).

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The coordinated regulation of intracellular iron metabolism occurs at both transcriptional and posttranscriptional levels. The iron response element (IRE) is a stem-loop structure common to several mRNAs whose levels vary with the iron status of the cell. The IRE is highly conserved from oocytes to mammals (Harford et al., 1994; Klausner et al., 1993). In particular, a single IRE is found in the 5‘ untranslated region of ferritin mRNA, whereas five copies are present in the 3’ untranslated region of transferrin mRNA. IREs have also been described in several other mRNAs that code for proteins involved in iron metabolism, such as ALA synthase. Based on computer folding, this sequence forms a stable stem loop (Harford et d.,1994; Klausner et d.,1993). The length of the stem appears variable, but there remains an absolute requirement for a conserved cytosine residue on the 5’ end of the stem, 5 nucleotides from the loop (Harford et al., 1994; Klausner et al., 1993). The loop itself is highly conserved and composed of 6 nucleotides with a conserved cytosine residue at the 5’ end. When this element is present in the 5’ UTR of ferritin, it confers translational regulation. When present in the 3’ UTR the IREs modulate transferrin receptor mRNA stability. The position of the element in respect to the 5’ cap in ferritin mRNA is critical for its function (Gray and Hentze, 1994). As it is moved toward the start codon, it becomes progressively less active, suggesting that it can inhibit the assembly of ribosomal components but not block the movement of a completely assembled ribosome. [For a more complete discussion of the IRE, please see one of the many recent reviews (Hentze and Kuhn, 1996)l. Ribonucleotide reductase is a highly regulated, rate-limiting enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides (Wright et al., 1987). The enzyme is composed of dissimilar heterodimers, referred to as R1 and Rz, that are encoded by different genes. R1 is itself a homodimer with an aggregate molecular weight of 170 kDa that contains substrate and allosteric binding sites, whereas Rz is also a homodimer that binds iron (Kabnick and Housman, 1988; Verma and Sassone-Corsi, 1987). Appropriate enzymatic activity requires both R, and R2 subunits, whose levels change during proliferation (Weber, 1983) as well as after treatment with TGF-P1 (Hurta et al., 1991) or TPA (Choy et al., 1989). After stimulation with phorbol ester or TGF-P, both R, and Rz mRNA levels increase due to elevated stability of the coding mRNA. Through careful mutagenesis as well as the construction of chloramphenicol acetyltransferase R1 and Rz mRNA chimeras, Wright and coworkers (Chen et al., 1994) have conclusively demonstrated that regulated R1 mRNA stability was determined by a 49-nucleotide region located at the distal portion of the 3‘ UTR. In unstimulated cells, removal of this domain minimally destabilized R, mRNA, but its loss prevented

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25

phorbol ester-mediated stabilization.Therefore, the 49-nucleotide element mediates TPA-induced R1 stability but appears dispensable for rapid R1 mRNA decay in resting cells. Like R,, R2 mRNA levels are controlled by regulated stability. R2 mRNA contains an 83-nucleotide element located in the mid-3’ untranslated region that appears to function as a TGF-P1 response element (Amara et al., 1996a). In the presence of TGF-P, R2 mRNA was stabilized. Interestingly, the R1 and R2 3’ UTR cis elements are dissimilar and without sequence homology to other known stability elements. Functional mapping of these large domains has recently been reported (Amara et al., 1996b). The inevitable cellular production of superoxide and hydrogen peroxide as a result of oxygen-based metabolism requires abundant antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase; Fridovich and Freeman, 1986). In newborns, exposure of the lungs to hyperoxia results in increased antioxidant enzyme activity, which appears to be mediated by changes in catalase mRNA stability (Clerch et al., 1991). The response element has been putatively mapped to a conserved 240-base domain sequence within the 3’ UTR of catalase. The 3’ UTR contains two elements, one of which is a 36-base, stem-loop structure and the other a CA dinucleotide repeat. These domains together appear both necessary and sufficient for the specific binding of a cytoplasmic, catalase-specific, RNA-binding protein (Clerch, 1995). Despite the identification of an RNAbinding protein, it is yet to be shown (by deletion studies) that this domain confers regulated stability to catalase mRNA. Insulin-like growth factor-I1 ( IGF-I1) is a 67-amino-acid polypeptide structurally related to IGF-I and insulin (Rinderknecht and Humbel, 1978). IGF-I1 mRNA undergoes developmental and tissue-specific regulation by differential activation of four promoters and alternative splicing (de PagterHolthuizen et al., 1988; Van Dijk et al., 1991). In addition to full-length IGF-I1 mRNA, an uncapped 1.8-kb, 3’ cleavage product with an intact poly A tail has been identified (Meinsma et al., 1991). The cleavage of IGF-I1 mRNA is directed by 3’ UTR sequence elements separated by approximately 2 kb (Scheper et al., 1995). Each element is approximately 300 nucleotides long with the distal element encompassing the cleavage site. The downstream element was necessary for cleavage, whereas the upstream element controlled the rate at which cleavage occurred. When introduced into a chimeric mRNA (P-globin), these two elements directed cleavage of the chimera, suggesting they are necessary and sufficient. These interacting domains are to date the most widely separated and clearly demonstrate how distant regions can cooperate to form a functional element.

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The existence of extremely stable mRNAs could be accounted for by the absence of destabilizing elements or the inclusion of domains that retard mRNA decay. It appears that P- and y-globin mRNAs contain stabilizing domains. Globin mRNAs are produced in a tissue-restricted pattern by erythroid cells and eiythroid precursors. Liebhaber’s group has demonstrated the presence of distinct stability determinants within the 3‘ untranslated region of both a- and P-globin (Weiss and Liebhaber, 1995; Russell and Liebhaber, 1996). The a-globin domain appears to be composed of three cytosine-rich regions in which base substitutions cause message destabilization through a translationally independent mechanism (Weiss and Liebhaber, 1995).When ribosomes were permitted to translate into the 3’ UTR by mutagenesis of the stop codon, the stability element was nonfunctional. These data have been interpreted to imply that the aglobin determinant must be spatially organized and can be disrupted by ribosomal read-through or targeted mutation. The P-globin element showed similar sensitivity to ribosomal read-through, demonstrating that an unperturbed 3’ untranslated region was also critical for its normal function. However, unlike a-globin, antiterminated P-globin mRNA was unstable irrespective of whether it was translated (Russell and Liebhaber, 1996). The stability of P-globin may be further modulated by 5’ untranslated region sequences that, when mutated, also influence mRNA stability (Ho et al., 1996). Effector T cells release cytolytic enzymes such as perforin, a 70-kDa protein with cytolytic activity. Interestingly, cytolytic cells downregulate perforin and esterase mRNAs upon exposure to targets (Bajpaiet al., 1991). This decrease in perforin inRNA content was caused by accelerated mRNA decay that was unaffected by protein synthesis blockade by cycloheximide (Goebel et al., 1996),suggesting cotranslational degradation was not operative. Mutagenesis as well as the production of reporter chimeric mRNAs revealed perforin mRNA instability determinants were present in the coding region rather than the 3’ UTR. Based on the inability of small fragments of the coding region to confer regulation to the chimera, the authors concluded that the element consists of multiple domains that likely interact to form a functional unit. Interestingly, NK cells treated with IL-2 and IL-12 show enhanced stability of perforin mRNAs (Salcedo et al., 1993). Whether regulated perforin mRNA stability is solely dependent on the coding region determinant remains to be shown. Intracellular adhesion molecule-1 (ICAM-1; CD54) is one of several cell surface molecules belonging to the immunoglobulin superfamily. ICAM-1 serves as a ligand for Pzintegrins, lymphocyte function-associated antigen1 (LFA-1; CD-11A/CD-18) (Diamond et al., 1991), and MAC-1 (CDllB/ CD-18), which is critical for a variety of immune functions including T

POSTTRANSCRIPTIONAL REGULATION OF mRNAs

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cell-mediated killing, T-helper responses, as well as leukocyte trafficking and adherence to vascular endothelium and epidermal cells (Springer, 1990). ICAM-1 is inducible on a variety of cells but is dramatically upregulated at sites of inflammation (Springer, 1990). Many inflammatory mediators, such as IL-l, IFN-7, and synthetic mimetics such as phorbol esters, increase ICAM-1 expression (Carlos and Harlan, 1994). Sequence analysis revealed ICAM-1 mRNA contains multiple reiterations of AUUUA motifs that appear responsible for rapid decay (tt < 1 hr) in unstimulated monocytic cell lines (Ohh et al., 1994). Consistent with the role of AUUUA motifs in the posttranscriptional regulation of ICAM- 1, cycloheximide, phorbol ester, and IFN-y all stabilized ICAM-1 mRNA (Ohh et al., 1994). Mutagenesis revealed that the AUUUA motifs function as destabilizers in resting cells and appear to act as the TPA response element. However, IFN-7 stabilized ICAM mRNA from which the AUUUA motifs had been deleted (Ohh and Takei, 1994). The IFN-.)I response element was subsequently mapped to an 87-nucleotide region upstream of the AUdestabilizing motifs. The mRNA coding for protooncogenes cfus and c-myc is regulated at both transcriptional and posttranscriptional levels. The immediate-early response gene, c-fos, is dramatically upregulated (50- to 100-fold) during the transition of fibroblasts from Goto S phase (Muller et al., 1984). Similar upregulation has been observed when cells are exposed to ultraviolet light (Angelet al., 1986). Finally, stimulation of semm-deprived fibroblasts with serum or PDGF rapidly upregulated cfus mRNA transcription, which returned to prestimulation levels within 1 hr (Greenberg and Ziff, 1984). Because cfos mRNA accumulation largely mirrored transcriptional activity, it was clear that cfos mRNA must be exquisitely unstable, with an estimated half-life of between 5 and 20 min (Greenberg and Ziff, 1984). Sequence analysis of the 3' UTR of c-fos mRNA revealed the presence of three AUUUA motifs within a U-rich 67-base region. This regon was highly conserved between murine and human cfus genes, which differed by only two nucleotides (Van Straaten et al., 1983). The removal of this domain greatly enhanced the transforming potential of c-fos by stabilizing cfos mRNA and increasing the amounts of Fos protein. Of note, the viral homolog offos (vfus) lacks the 67-base destabilizing domain (Meijlink et al., 1985). Extensive mutagenesis of the 3' UTR has revealed the presence of two destabilizing domains (Chen, C. Y. et al., 1994). Domain 1is located within the proximal 49 nucleotides of the AU-rich element and contains 3 AUUUA motifs. This domain can function independently as an mRNA destabilizer. Domain 2 is a 20-nucleotide U-rich sequence 3' to the AU motifs that in and of itself cannot function as a destabilizer. However, this region enhanced the destabilizing activity of domain 1 and appeared to

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buffer potentially stabilizing mutations within domain 1. These data are consistent with a two-step mechanism for c-fos mRNA decay, with deadenylation followed by body cleavage, and are consistent with data showing that the initial steps in c-fos mRNA decay involved shortening of the polyadenylate tail prior to body cleavage (Wilson and Treisman, 1988). Despite the presence of the potent AU-rich element as a 3’ untranslated region destabilizer, c-fos mRNA contains a second domain within the protein coding region that appears to function independently as an instability determinant (Shyu et al., 1989). When inserted into P-globin mRNA, this region destabilized the chimeric message. As c-fos mRNA accumulated when translation was inhibited, deadenylation and decay appeared coupled to translation. The coding region determinant within c-fos mRNA encodes the nucleic acid binding and heterodimerization domains. This region is purine rich and approximately 60 bases in length. When inserted into a heterologous mRNA in frame, the resultant chimeric message was partially destabilized suggested the participation of additional coding region or 3’ UTR determinants. Finally, the coding region determinant was insensitive to actinomycin-D, which has been shown to impede the functionality of the AU-rich element. The c-myc protooncogene encodes proteins involved in transcriptional regulation and possibly DNA replication (Luscher and Eisenman, 1990). c-myc mRNA is exquisitely unstable and normally decays with a t 4 of approximately 15 min. Like c-fos mRNA, it contains two AU-rich regions preceding dual polyadenylation signals. Each of these domains is approximately 50 bases long and contains at least one or more AUUUA pentamers. Both lymphomas and myelomas have been identified with translocations causing the loss of the 3’ UTR of c-myc mRNA. In these cells c-myc RNA levels were elevated through message stabilization (Hollis et al., 1988). However, the translocations added additional heterologous sequences to both the 5‘ and the 3‘ ends of c-myc mRNA. Thus, the additional sequences may have altered c-myc mRNA decay. Indeed, 5’ UTR-truncated myc transcripts derived from fusions with immunoglobulin sequences were more stable than the wild type (Eick et al., 1985), demonstrating that additional heterologous sequences stabilized c-myc mRNA rather than the deleted 5’ UTR contained an instability determinant. Some investigators have been unable to show that the deletion of the AU-rich domains enhanced c-myc mRNA stability (Laird-Offringa et d.,1991). These authors also showed that complete translation was not necessary for rapid decay of c-myc mRNA. These observations suggested that an additional mRNA instability determinant may be present within c-myc mRNA. Recently, Wisdom and Lee (1991) demonstrated that the protein coding region confers instability on c-myc mRNA. Herrick and Ross (1994) demonstrated

POSTTRANSCRIPTIONAL REGULATION OF mRNAs

29

that this region can destabilize the normally stable p-globin mRNA when fused in frame. Interestingly, the coding region determinant coded for the nucleic acid binding and heterodimer-forming regions of c-myc protein -but was nonfunctional in the presence of actinomycin-D (Wisdom and Lee, 1991). In addition, depending on the cell type used for analysis, the coding region determinant may be silent (Yeilding et al., 1996). Thus, depending on the transcriptional blockers employed as well as the cell types used, the c-myc instability determinants may or may not be active. VI. hcrns Factors

Given the large number of both cis elements and mRNAs that contain them, cellular mechanisms must exist for the identification and regulation of specific RNAs. It seems inconceivable that ribonucleases, in and of themselves, can adequately discriminate between stable and unstable mRNAs. Therefore, many investigators have proposed the existence of cytoplasmic proteins capable of interacting with cis elements. Such proteins could function as destabilizers either by recruiting ribonucleases to a particular mRNA or by containing ribonuclease activity themselves. Conversely, proteins could act as stabilizers by directly interacting with mRNA targets and blocking ribonuclease recognition. A more complex model can also be envisioned whereby trans factors interact with mRNA outside of a putative cis element. Under such conditions, folding or conformation of the mRNA may be changed, obscuring or potentially increasing the presentation of a ribonuclease recognition site. Such binding need not be close to the cis-acting element but rather could be hundreds or even thousands of nucleotides away. The presence of RNA-protein interactions can be detected by the use of mobility shift assays or filter hybridization (Northwestern blotting). The most commonly employed assay has been the electrophoretic mobility shift assay. This technique is very similar to the DNA mobility shift assays commonly employed to detect DNA-binding proteins. Radiolabeled RNA is prepared in vitro and incubated with cytoplasmic extracts derived from the tissue or cell of interest. Assay conditions are critical, with most investigators favoring near-physiologic pH and relatively low ionic strength buffers. Nonspecific competitors, such as tRNA, poly-I, poly-C, poly-G, or heparin, are also included to decrease nonspecific interactions. After brief incubation at room temperature (5-30 min), ribonuclease (T-1 or A) is typically added to destroy any free RNA probe as well as portions of bound RNA that are not protected by the interacting protein. Samples can then immediately be electrophoresed on nondenaturing acvlamide gels, usually under low ionic strength conditions (0.25-0.5 X TBE) or ultraviolet light

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cross-linked and electrophoresed on denaturing SDS acrylamide gels (SDS-PAGE). Controls typically include the untreated RNA ligand and the RNA ligand treated with ribonuclease in the absence of cytoplasmic protein. Using a combination of native and SDS-PAGE, we were able to identify several distinct mRNA-binding proteins that interacted with the AUUUA motifs (Malter, 1989; Gillis and Malter, 1991), the APP 29-base element (Zaidi et al., 1994), and an erythropoietin mRNA destabilizer (Rondon et al., 1991). It is important to remember that the observed mass of the complex represents a combination of both protected RNA and protein, making definitive calculation of the protein’s molecular weight approximate. In addition, the differences in buffer and irrelevant competitors (tRNA vs. heparin vs. poly-I : poly-C) can lead to vastly different results using identical cell lysates. Northwestern blotting has been infrequently employed due to difficulties in achieving native folding of immobilized RNA-binding proteins. We observed a loss of binding specificity when nucleolin and hnRNP C proteins were immobilized on membranes (Zaidi and Malter, 1994). Others, however, have been successful in employing Northwestern blotting for the direct cloning of mRNA-binding proteins from cDNA libraries expressed in Escherichia coli (Qian and Wilusz, 1994). Thus, most investigators begin the search for trans factors interacting with their mRNA of interest by mobility shift assays. Using this approach, many RNA-binding proteins have been identified and their target sequences determined. In only a few cases have these proteins been cloned, but it is likely that within the next few years many more cDNAs will be obtained.

A. AU-SPECIFIC RNA-BINDING PROTEINS Due to the large number and critical nature of mRNAs containing AUUUA motifs, many investigators began to search for the existence of specific binding proteins. As previously discussed, such proteins could stabilize or destabilize AUUUA-containing mRNAs. Functionality might be determined by their affinity for RNA and their subcellular location, intracellular concentration, and activity. The first definitive report of an AU-specific mRNA-binding protein was made by Malter (1989). RNA mobility shift assays were performed with cytoplasmic lysates derived from log phase Jurkat cells (J32,T cell leukemia) and an 80-base radiolabeled, in uitro transcript containing four tandem repeats of the AUUUA motif. Cytoplasmic lysates were incubated in the presence of radiolabeled ARE containing RNA for 10 min in low ionic strength buffer at physiologic pH with a vast excess of nonspecific tRNA competitor. After 10 min, reaction mixtures were treated with RNase A or T-1 to cleave unprotected RNA, followed by nondenaturing polyacrylamide gel electrophoresis or ultraviolet cross-linking and SDS-PAGE.

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31

Using these two approaches, a dominant RNA-protein complex with a molecular mass of approximately 36 kDa was identified. This complex could be specifically competed by unlabeled AUUUA-containing RNA but not by irrelevant competitors. Based on binding specificity, this protein was dubbed the AU-binding factor or AUBF. Finally, it was demonstrated that AUBF must have very high affinity for AUUUA RNAs because complex formation was nearly instantaneous in solution phase. Interestingly, very short RNAs of less than 30 nucleotides, but containing multiple AU repeats, failed to interact with AUBF (Gillis and Malter, 1991). This suggested that secondary or higher order structures were involved in presenting the primary sequence to AUBF. Computer folding of AU-containing mRNAs has not revealed a thermodynamically preferred structure. This is not to say that such a structure is not adopted by the AU-rich RNA in solution, however. In addition, RNA ligands with less than three AUUUA repeats failed to interact with AUBF. Therefore, AUBF would not interact with mRNAs such as globin that contain a single AUUUA motif. Mutagenesis of the AUUUA repeats dramatically reduced the ability of AUBF to bind. Especially deleterious were conversions of the middle U to G (binding decreased by more than 95%) or U to C (binding decreased by approximately 80%) (Gillis and Malter, 1991). These data suggested that AUBF recognition was highly sequence specific. If the ribonuclease machinery that normally recognized and rapidly degraded AU-containing mRNAs has similar specificity, mRNAs with mutations in the AU-rich regions would escape ribonuclease surveillance and likely be long-lived. Indeed, transformed cell lines containing viral insertions that disrupt the AU-rich 3' UTRs of IL-2 (Chen et al., 1985)or IL-3 (Algateand McCubrey, 1993; Hirsch et al., 1993) create cytokine mRNAs with 5- to 10-fold greater half-lives than normal. Truncation of the c-fos mRNA has also been reported to stabilize this message and facilitate cellular transformation (Meijlink et al., 1985). Because cytokine mRNAs are stabilized in lymphoid cells by activation with phorbol ester (Shaw and Kamen, 1986), PHA (Shaw and Kamen, 1986), LPS (Thorens et al., 1987), ionophore ( Wodnar-Filipowicz and Moroni, 1990), cytokines ( Wodnar-Filipowicz and Moroni, 1990), or antigen (Takahama and Singer, 1992), changes in binding protein quantity or activity would provide important insight into their function. AUBF was inactive in resting lymphocytes (Malter and Hong, 1991). After treatment with PHA, TPA, or ionophone, activity was rapidly upregulated and maintained for at least 8 hr. Even in the presence of actinomycin-D and cycloheximide, similar upregulation of AUBF activity was observed. This suggested that preformed AUBF existed in an inactive state that, through posttranslational modification, acquired binding activity (Malter and Hong, 1991).

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Other nucleic acid-binding proteins including NF-KB can be similarly regulated (Kopp and Ghosh, 1995). Because phorbol ester and ionophore stimulated AUBF activity, PKC was likely involved in modulating the mRNA decay machinery. PKC or a kinase downstream of PKC might phosphorylate AUBF, causing alterations in binding activity. Such data would be consistent with the observed upregulation of AUBF activity in the context of both transcriptional and protein synthesis inhibition (Malter and Hong, 1991). This was tested directly by treating active cytoplasmic lysates with potato acid phosphatase (PAP), with subsequent measurement of AUBF activity. Under such conditions, PAP completely ablated AUBF activity, suggesting a requirement for phosphorylation. In addition to the PKC pathway, activation with 8-bromoCAMPalso increased AUBF activity (Stephens et al., 1992).Thus, it appears that multiple phosphorylation cascades can modulate AU-specific binding proteins. An interesting observation first made with the bacteriophage R17 RNAbinding protein was sensitivity to oxidants (Starzyk et aZ., 1982). A similar requirement has been noted for the iron response element-binding protein (Hentze et d.,1989) suggesting this might be a generalized phenomenon. Treatment of cytosolic lysates with oxidants such as diamide or N ethylmaleimide (NEM) completely ablated AUBF activity (Malter and Hong, 1991). After diamide oxidation, incubation with reducing agents such as 2-mercaptoethanol fully restored binding activity. These data suggested that redox changes within the cytosol, along with phosphorylation, regulated AUBF activity. Redox sensitivity suggested the presence of critical sulfhydryl groups that participated directly or indirectly in RNA binding. Reduced sulfhydrylscould be present in the active site or stabilize AUBF’s secondary or tertiary structure by chelating metals. The so-called zinc finger proteins chelate zinc through a coordination complex formed by cysteine and histidines. In order to differentiate between these two options, AUBF-AUUUA RNA complexes were treated with NEM. If cysteines were directly bound to the RNA ligand, they would be unavailable for modification by NEM and complexes would be maintained. Conversely, if sulfhydrylswere chelating metal ions at a site distant from that interacting with AUUUA RNA, NEM would block complex formation. Because NEM treatment inhibited preformed AUBF-AUUUA RNA complexes, it was likely that critical cysteine residues were distant from the RNA binding site and probably interacted with divalent metals. After extensive dialysis of AUBF-containing cytoplasmic lysates against EDTA and EGTA, a variety of divalent or trivalent metals were added back prior to binding activity assays (Malter et al., 1990). Only magnesium and calcium ions were able to reconstitute binding activity suggesting that

POSTTRANSCRIPTIONAL RECUJATION OF mRNAs

33

these metals likely interacted with AUBF and are important for its function. Based on these data, the authors proposed that AUBF was regulated by reversible phosphorylation as well as by reductiodoxidation. Given the absence of AUBF activity in resting lymphocytes and its rapid upregulation after phorbol ester, ionophore, or CAMPactivation, conditions that enhanced the stability of AUUUA mRNAs, it was proposed that AUBF might function as an AUUUA rnRNA stabilizer (Malter and Hong, 1991). In order to test this hypothesis, polysomes were isolated from mitogenactivated peripheral blood mononuclear cells and used to determine GMCSF mRNA decay in vitro (Rajagopalan and Malter, 1994). Under such conditions, GM-CSF mRNA decayed with a half-life of approximately 90 min. When polysomes were depleted of AUBF by RNA affinity chromatography, GM-CSF inRNA decay was accelerated fivefold. Although it is possible that proteins in addition to AUBF were removed by affinity chromatography, these observations support the proposal that polysomebased proteins specific for the AUUUA determinants of cytokine and potentially protooncogene messages can stabilize them. Direct confirmation of this conclusion must await cloning and expression of recombinant AUBF.

B. OTHERAUUUA-BINDING PROTEINS Polysomes from log phase tumor cells have long been used as an in vitro mRNA decay system (Brewer and Ross, 1988). Because in vitro decay on polysomes can discriminate between AU- and non-AU-containing mRNAs, they are a logical location for potential trans factors involved in regulated decay. As described previously, AUBF has been localized to polysomes (Rajagopalan and Maker, 1994). Brewer showed the presence of a destabilizing activity in the S130 soluble fraction obtained after the centrifugation of polysomes through sucrose gradients (Brewer and Ross, 1988). Using density gradient centrifugation, this activity was isolated, cloned, and denoted Auf-1 (Brewer, 1991). Auf-1 consisted of 37- and 40-kDa isoforms. Cloning of the 37-kDa isoform revealed two nonidentical RNA recognition motifs (Burd and Dreyfuss, 1994). The larger isoform appeared to contain an additional 19 amino acids located N terminal to the first RNA recognition motif (Ehrenman et al., 1994). In vitro binding assays with both cellular and recombinant Auf-1 showed high-affinity association constants in the low nanomolar range (DeMaria and Brewer, 1996). Indeed, DeMaria has shown that the affinity of Auf-1 to different ARES reflects the potency of ARE as an mRNA destabilizer. These data have been interpreted to support a role for Auf-1 in ARE-mediated decay. In addition to c-fos and c-myc mRNAs, Auf-1 interacts with GM-CSF, glucose transporter-1, and p-adrenergic receptor mRNAs (Pende et al., 1996).

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Because Auf-1 activity increased as AU-containing mRNA decay accelerated, it has been proposed to function as a destabilizer. Although the mechanism is unknown, Auf-1 may alter the secondary structure of target mRNAs, directly recruiting ribonucleases or in some other way making target mRNAs more susceptible to decay. Auf-1 has not been shown to possess intrinsic ribonuclease activity. Despite these associations, there is yet to be an unambiguous demonstration, either in uitro or in uiuo, of the functionality of Auf-1. Shortly after the description of AUBF, Bohjanen and coworkers (1991, 1992) identified a series of proteins termed AU-A, AU-B, and AU-C that bind to mRNA with AUUUA multimers. AU-A resembled AUBF as well as a similar protein described by Vakalopoulou et al. (1991) and migrated as a 34-kDa protein when associated with RNA. AU-A was primarily localized to the nucleus although it was also detected in the cytoplasm. This protein was capable of interacting with the 3’ untranslated region of GMCSF, IL-2, TNF-a, and c-myc mRNAs. A second RNA-binding protein, called AU-B, was not present in unstimulated T lymphocytes but was rapidly induced following engagement of the TCR-CD3 complex (Bohjanen et al., 1991).This protein has a predicted mass of 30-kDa and interacts with the AU-containing mRNAs listed previously with the exception of cmyc. AU-B appeared localized exclusively to the cytoplasm. Based on protease mapping, AU-A and AU-B are likely distinct proteins. A third activity, denoted AU-C, appeared structural related to AU-B. Both AU-B and AU-C required three or more AUUUA repeats for efficient and highaffinity binding and, like AUBF, were intolerant of mutations within the AUUUA recognition motif (Bohjanen et aZ., 1992). Because AU-A was constituitively present and tolerated wider variation of sequence than AU-B or AU-C, it has been proposed that AU-A may play a more general role in the metabolism of U-rich or AU-rich mRNAs. The upregulation of AU-B/AU-C upon CD3-TCR activation, along with their narrow sequence specificity, suggested that they may participate in the regulation of cytoplasmic inRNA decay. However, because TCR-CD3 activation is insufficient to stabilize GM-CSF, IL-2, and INF-.)I(Lindsten et aZ., 1989), conditions that upregulated AU-B and AU-C, it remains unclear what additional regulatory steps might be involved. Several other groups have described AU binding activities with molecular inasses of 30-50 kDa. These include a 32-kDa AU binding factor described by Vakalopoulou et ul. (1991) and Myer et nl. (1992), a group of four Urich sequence-binding proteins (You et al., 1992), and a group of three proteins designated A, B, and C that are capable of interacting with the TNF-a AU-rich element (He1et aE., 1996).The 32-kDa protein described

I'OS'ITRAN SCHIPTION A L HEGU LATION OF

111 R NA\

35

by the Steitz lab has recently been cloned and been designated hnKNP 0 (Meyer and Steitz, 1995). In addition to the novel activities described previously, it has recently become apparent that abundant nuclear proteins, such as heterogenous nuclear ribonuclear proteins A and C (hnKNP A and C), can be found in the cytoplasm, associated with polysomes and based on mobility shift assays, and able to interact with AU-containing mRNAs (Hamilton et nl., 1993). These data suggest that RNA-binding proteins may have distinctive functions depending on subcellular location. Changes in phosphorylation or other posttranslational modification rnay enhance their transit to the cytosol, where they can participate in labile rnRNA decay.

C OTHERCYTOKINE A N D PROTOONU
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JAMES S. MALTER

tained the C-terminal RNA recognition motifs were able to bind, however, suggesting that nucleolin autocleavage might represent an integral regulatory step in the activation of this binding protein. Recently, APP mRNA could be stabilized in rabbit reticulocyte lysate by the addition of hnRNP C and nucleolin (Rajagopalan et al., 1997). Two distinct binding proteins have been described that interacted with the RI and Re mRNAs coding for ribonucleotide reductase. Amara et al. (1996b) have shown that a 75-kDa, sequence-specific Cytoplasmic protein binds selectively to the 83-nucleotide cis-acting element of Rz mRNA. p75 RNA binding activity required new protein synthesis and cannot be detected until cells are stimulated with TGF-01. Because the appearance of p75 binding activity paralleled the accumulation of R2 mRNA, the authors proposed that binding leads to stabilization of Re message (Amara et al., 199613). After phorbol ester treatment of 3T3/fibroblasts, the Re mRNA was stabilized threefold. A 20-nucleotide TPA response element (distinct from the aforementioned 83-nucleotide domain) specifically bound a 45-kDa protein (Amara et al., 1996a). This binding activity was detectable in unstimulated cells and markedly downregulated after phorbol ester treatment. Therefore, the authors proposed that p45 may be linked to rapid decay of Rz mRNA in unstimulated cells. Finally, R, mRNA stability appears to be modulated by a 57-kDa protein that interacted specifically with a 49-nucleotide region of the R1 3’ untranslated region (Chen, F. Y. et al., 1994). The p57 was downregulated by phorbol ester and okadaic acid but upregulated by PKC inhibitors (Chen, F. Y. et al., 1994).Therefore, both R, and Rz mRNA stability appears to be modulated by several cytoplasmic proteins that can destabilize or stabilize the mRNA under appropriate conditions. VII. Concluding Remarks

As discussed previously, a large and growing number of human genes are regulated at the level of mRNA stability. This pathway can rapidly and effectively increase or decrease levels of coding mRNA, modulating gene expression as needed. The underlying mechanism for these effects relies on cis-trans interactions that identify and target particular mRNAs. A large number of important mRNAs are regulated in this way, including cytokines, protooncogenes, and cell surface receptors. It is likely that as this field advances, many more candidate mRNAs will be identified and their regulation characterized. Dysregulation of these pathways has been associated with cellular transformation and immune dysregulation. Thus, events outside of the nucleus may have profound effects on cell metabolism and regulation.

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ACKNOWLEDGEMENTS The author thanks Ms.Gloria Scalissi for expert secretarial help, the National Institutes of Health (Grants AC10675 and HL.56396) for ongoing support, and the inquisitive and thoughtful members of my research laboratory.

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A J N A N L E 5 IN IMMLINOLOL\

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Molecular and Cellular Mechanisms of T lymphocyte Apoptosis JOSEF M. PENNINGER’ AND GUIDO KROEMERt ‘The Amgen lnrtiiufe, Ontario Comer Institute, and Deporhnenk of Medical Biophysics and Immunology, Universily of Toronto, Toronto, Onterio M5G 2C1, Canado; ond tCNRS-UPR 420, F-9480 1 Villejuif, France

I. Introduciion

According to the French philosopher Albert Camus, the primordial question of philosophy is whether life is worth living (1).It appears that this existential problem also affects various cell types in the multicellular organism. Apoptosis is now generally conceived as a strictly regulated (“programmed”) device for the removal of superfluous, aged, or darnaged cells within the immune system as well as in all other organ systems. It is fundamental for development, throughout embryogenesis, organ metamorphosis and organogenesis, including synaptic interactions of neurons, and repertoire selection of T lymphocytes. The process of apoptosis has attracted much attention in immunology, given that lymphocytes are continuously at risk of comitting suicide during positive and negative selection processes. In addition, acquired or genetically determined dysregulations of apoptosis have a major pathogenic impact, underlining the physiological importance of apoptosis control for the normal immune function. Enhanced resistance to apoptosis induction can lead to the persistance of self-reactive or mutated cells, which in nonnal circumstances would be eliminated to avoid the development of autoimmune diseases, lymphocyte hyperplasias (lymphadenopathy + splenornegaly due to lymphocyte acculeukemia). In mulation), or lymphocyte-derived tumors (lymphomas contrast, an enhanced susceptibility to apoptosis can cause a numeric or functional immunodeficiency. An ever-increasing number of spontaneous or experimentallygenerated genetic models of irninunopathologyare linked to dysregulations of apoptosis (Table I). Froin an immunologist’s point of view, another interesting facet of apoptosis concerns the handling of autoantigens. It is now generally agreed on that apoptosis is a physiological means of cell removal in which dying cells undergo subtle changes in membrane physicochemistry that trigger their recognition and phagocytic removal by normal adjacent cells. Classical apoptosis de facto precludes the release of cellular contents into the interstitiurn, thereby avoiding secondary inflammatory or autoimmune responses. Perturbation of apoptotic cell removal thus might play an important role in the initiation and/ or perpetuation of autoaggressive diseases. Nonetheless, this review will

+

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JOSEF M. PENNINGER AND GUIDO KROEMER

TABLE I EXAMPLES OF H O M O L O G O U S R E C O M B I N A T I O N OF GENES AFFECTINGLYMPHOCV~E ApopTosIs IN THE MOUSE Knockout

Phenotype

Fds/CD%-’-

Lympoproliferative syndrome, liver hyperplasia, autoimmunity Defect in deletion of peripheral CD8+ T cells Defect in negative selection, large thymus Immunodeficiency, polycystic kidneys, hair hypopigmentation, and other defects Impaired lympocyte maturation, massive neuronal apoptosis Lymphoid hyperplasia, hypospermia Enhanced apoptosis in response to CD3 and Fas embryonic lethal

TNF-R1-lCD30-/BcI-2-’Bc1-X-IBtWSEKF

Reference 578 266, 579 256 418,419 420 580 380

focus on the genetic and molecular mechanisms of T lymphocyte apoptosis rather than discussing the death of antigen presenting or target cells. The modem era of apoptosis research has been initiated by the discovery that glucocorticoid-treated thymocytes undergo characteristic morphological changes involving shrinkage and chromatin condensation, accompanied by a characteristic DNA fragmentation into mono- and oligomers of -200 bp. This finding gave rise to the obvious speculation that endonuclease-mediated DNA fragmentation was the decisive event of the cell death process (2), and one of the first models of apoptosis regulation suggested that an augmentation of cytosolic and nuclear Ca2+concentrations would function as second messenger linking the initial trigger (glucocorticoid ligation) to the crucial biochemical event of (Ca2+-dependent) endonuclease activation (3). This simplistic model constitutes a good example how scientists tend to order isolated pieces of existing knowledge to construe theories explaining complex systems. In the meantime, it has been discovered that apoptosis-triggering stimuli can use an ever-increasing number of distinct signal transduction modules, that Ca2+ elevation is not decisive for apoptosis to occur in most models, and that endonuclease activation is a late event that actually is dispensable for apoptosis. Moreover, multiple additional elements of apoptosis regulation have been discovered during the past decade and have led to the refinement of the actual cell death researcher’s worldview. This review constitutes another attempt of ordering the known pieces of the apoptotic puzzle. According to current understanding, the process of apoptosis can be subdivided into at least three different phases (Fig. 1) (4,5). During

53

MECHANISMS OF APOPTOSIS

Inducer

Inducer

Inducer

central executioner

manifestations

Fie. 1. Schematic view of the three phases of apoptosis. During the initiation phase, different inducers trigger disparate pathways that finally trigger the central executioner. These pathways are “private” in the sense that they depend on the initial apoptosis trigger. The common phase of apoptosis has two distinct phases; the effector phase, during which the central executioner is activated and which is subject to regulatory mechanisms, and the degradation phase (beyond the point of no return), during which apoptosis becomes manifest at the levels of morphology and biochemical catabolism.

the initiation phase, cells receive apoptosis-triggering stimuli. Such death inducers include ligation of certain receptors [Fas/APO-l/CD95, tumor necrosis factor receptor (TNF-R), transforming growth factor receptor (TGF-R), etc.] or, in the case of obligate growth factor receptors, the absence of receptor occupancy. In addition, interventions on second messenger systems (Ca”, ceramide, kinases, etc.), suboptimal growth conditions (shortage of essential nutrients and hypoxia), mild physical damage (radiotherapy), and numerous toxins (reactive oxygen species, chemotherapy, and toxins strict0 sensu) can induce apoptosis. Virus infection and attack by cytotoxic T cells or NK cells is another way of apoptosis induction. Nonspecific or receptor-mediated death induction involves a stimulusdependent (“private”) biochemical pathway, and it is only after this initiation phase that common pathways come into action. It is generally assumed

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[OSEF M. PENNINGER AND GUIDO KROEMER

that the execution phase of apoptosis defines the “decision to die” at the “point of no return” of the apoptotic cascade. During the execution phase the “central executioner of apoptosis” is activated. It is at this level that the different private pathways converge into one (or few) common pathway(s) and that cellular processes (redox potentials and expression levels of oncogene products including Bcl-2-related proteins) still have a decisive regulatory function. Once the cell has been irreversibly committed to death, the different manifestations classically associated with apoptosis such as DNA fragmentation become detectable. This degradation phase is similar in all cell types. It is characterized by the action of catabolic enzymes, mostly specific proteases (caspases) and endonucleases, within the limits of a near-to-intact plasma membrane. Thus, the cell actively contributes to its removal in a “suicidal”fashion and undergoes stereotyped biochemical and ultrastructural alterations. This review will focus on the various phases of apoptosis initiation, execution, and degradation of lymphocytes. First, we will elaborate on the common aspects of the late phase of the death process, which actually produces the phenotypic appearance of apoptosis. Second, we will discuss the actual conception of the effector stage, during which the cell activates the central executioner. Third, we will detail upstream (private) signal transduction pathways that regulate resistance or susceptibilty to death, as well as those proapoptotic signals that activate the central executioner. Thus, this review aims at integrating common principles of apoptosis executiom‘degradation with particular pathways of death control prevailing in lymphocyte (path0)physiology. II. Degradation Phase of Apoptosis

The degradation phase of apoptosis is probably very similar in all cell types. Electron microscopic features of apoptosis include a variety of changes in cellular ultrastructure. These changes are most prominent at the level of chromatin and nuclear structure. At the level of the nucleus, easily recognizable changes include condensation of chromatin that appears divided into compact and diffuse areas, progressive chromatin condensation that eventually involves all the nucleus with homogeneously electron-dense areas, reduction in nuclear volume (pyknosis), destruction of the nuclear envelope with disappearance of nuclear pores from the nuclear envelope that surrounds the compact areas of chromatin, and fragmentation of the nucleus (karyorrhexis). In contrast, morphological changes in the cytoplasma are less impressive: rounding up of the cell, blebbing of the plasma membrane, increased vacuolization, reduction in cell volume, and later cytolysis with signs of secondary necrosis (i.e., necrosis after apoptosis).

MECHANISMS OF APOPTOSIS

55

Organelle ultrastructure appears to be roughly preserved in cells that have undergone full-blown nuclear apoptosis. One important feature of apoptosis concerns the plasma membrane, which remains near-to-intact until late stages of the process. Thus, in contrast to primary necrosis (i.e., necrosis without apoptosis), apoptotic cells do not release their content and may form membrane-surrounded apoptotic bodies that contain organelles, nuclear fragments, and parts of the cytosol. Today’s overwhelming consensus is that these obvious changes in cellular structure occur well after that cells have “decided” to undergo cell death, during the so-called degradation phase of apoptosis. Obviously, the apoptotic process is accompanied by major changes in cellular biochemistry involving the activation of catabolic enzymes, mostly proteases and nucleases. In addition, major changes in energy metabolism, redox potentials, and ion homeostasis occur during the apoptotic degradation phase. Some particularly striking features of apoptotic degradation phase will be discussed in this section. However, this review will not cover all aspects of apoptotic degradation.

A. ENDONUCLEASES ACTIVATEDDURINC: APOPTOSIS During apoptosis, DNA fragmentation occurs via the activation of nucleases that generate either large fragments 2 5 0 kbp of DNA or monoand oligomers of 180-200 bp corresponding to the length of the nucleosome. This latter fragmentation, which is due to inteniucleosomal DNA cleavage, is generally referred to as oligonucleosomal DNA fragmentation. Apparently, different enzyme systems are involved in both types of DNA fragmentation, as suggested by the existence of cell lines that undergo high-molecular-weight DNA fragmentation without oligonucleosomal DNA fragmentation (6). Moreover, ZnZt typically inhibits only the oligonucleosomal type of DNA fragmentation (7).Typically, DNA froin apoptotic cells demonstrates double-strand breaks with single-base 3’ overhangs as well as blunt ends. It appears that single-base 3’ overhangs, as they are generated by some but not all nucleases, are seen in apoptotic but not in necrotic cell death (8).DNases that generate this type of cleavage include DNase I and cyclophilins A, B, and C (9,lO).Both DNase I and cyclophilin C induce 250 kbp DNA fragmentation but not oligonucleosomal DNA fragmentation ( 11). Nucleosomal-size DNA fragmentation may involve nuclear, -30- and -97-kDa endonucleases, as well as an -65-kDa cytoplasmic endonuclease, which are all inhibited by Zn2+(12, 13). Endonuclease G, which is also found in the mitochondria1 matrix, has recently been isolated from thymocyte nuclei (14). However, it appears that mitochondrial DNA is not degraded during apoptosis (15).

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Endonucleases contained in the nucleus may be activated in uitro by a number of different stimuli includng increases in Ca” or Mg2+ (16) concentrations, low pH (17),proteases such as trypsin (18),mitochondrial apoptosis-inducing factor (AIF) (19),and a heterodimeric (40/45 kDa) socalled DNA fragmentation factor (DFF) that is activated by caspase-3 (20). In addition caspase-mediated cleavage of actin may abolish actin’s inhibitory action on DNAse I (21). The relationship between DNA fragmentation and chromatin condensation is not clear. According to one report (22), chromatin condensation of rat thymocytes treated with lysates from Fas/APO-l-stimulated Jurkat cells requires ATP, whereas chromatin cleavage into 250-kbp and oligonucleosomal fragments does not, suggesting that both changes can be dissociated. That this is the case is also suggested by experiments involving Schizosaccharomyces pombe in which overexpression of proapoptotic genes from mammals (bax)or Caenorhabditis elegans (Ced-4)induces chromatin condensation without major DNA fragmentation (23, 24). Based on the fact that certain endoiiuclease inhibitors, such as Zn2+and aurintricarboxylic acid, can inhibit apoptosis in some models, it has been inferred that endonucleases are responsible for cell death. This is certainly not correct because some cell types and cell lines undergo apoptosis without endonuclease activation (6, 25) and because anucleate cells can be stimulated to undergo cell death (26, 27). Instead, it appears that Zn2+, which has pleiotropic effects on multiple enzyme systems, acts on yet undefined upstream events of the apoptotic cascade (28). This is also true for aurintricarboxylic acid, which inhibits, among other enzymes, calpain (29) and topoisomerase I1 (30). Thus, endonucleases probably are not central to the apoptotic process and rather fulfill the function of “cleaning up after death” (31).To our knowledge, the only case in which endonucleases may constitute a prime mechanism of cell death is provided by certain Mycoplasma species. Mycoplasma penetrans may cause cell death via the hrect action of pathogen-encoded endonucleases (32, 33). This type of cell death lacks certain features of apoptosis such as chromatin condensation and early disruption of mitochondrial functions (32). Advanced endonuclease activation occurs only at a late stage of the apoptotic process. Thus, after injection of glucocorticoidsin viuo, no major DNA fragmentation can be seen among lymphoid cells, even in conditions in which the cellularity of thymocytes and splenocytes is strongly declining (34). In situ, almost all thymocytes showing DNA fragmentation are localized within other cells (35),indicating that heterophagic recognition of apoptotic cells occurs before DNA fragmentation begins.

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57

B. CASPASES Overwhelming evidence suggests the involvement of specific cysteine proteases cleaving after aspartic acid (“caspases”),which catalyze a highly selective pattern of protein degradation, in apoptosis (36-40). At least 14 different caspases exist in humans. All caspases are synthesized as proenzymes (zymogens),which are proteolytically processed to form active heterodimeric enzymes. The cleavage sites for proteolytic maturation of procaspases are themselves cleaved by caspases, suggesting that caspases may engage in a cascade of proteolytic activation and amplification steps, much as this is known for the complement system. Despite a notable similarity in structure, different members of the caspase family possess distinctive activation requirements, substrate specificities, and inhibitory profiles (Table 11). Some, but not all, caspases are endowed with the capacity of autoactivation.Thus, for instance, caspase-1 cleaves procaspase1. Moreover, caspases can activate others following an ordered sequence. Thus, caspase-10 cleaves caspase-6, -7, and -8, which in turn do not cleave caspase-10, suggesting that different caspases may act at distinct levels of the apoptotic cascade. Gene knockout experiments have demonstrated an essential role for capase-1 in Fas/Apo-l/CD95-induced apoptosis (41, 42) as well as a role of caspase-3 in the regulation of neuronal apoptosis (43). However, these knockouts have no major impact on apoptosis in general. Thymocytes from either caspase-1 or caspase-3 knockout mice undergo normal glucocorticoid-induced apoptosis (41, 43), a finding that is widely interpreted to mean that caspases can function in a redundant fashion. The following findings underline the role of caspases in the apoptotic degradation phase: Addition of caspases to cell-free extracts is sufficient to trigger nuclear apoptosis. To obtain this effect, caspases have to trigger endonuclease activators contained in the cytosol(44-46). One such endonuclease activator is DFF, a heterodimeric protein that is activated by the proteolyic action of caspase-3 (20). Induction of apoptosis is accompanied by activation of caspases, which can be measured using fluorogenic substrates such as a fluorogenic substrate containing the cleavage site WAD [4-(4’-dimethylaminophenylazo) benzoic-YVADAPV-5-[-2-aminoethyl)-amino] naphtalene-l-sulfonic acid] or DEVD (Ac-DEVD-amino-4-methylcoumarin) (45). The activation of caspases cleaving DEVD appears to be a general phenomenon, whereas activation of YVAD-cleaving caspases is less frequent (47, 48). In apoptotic cells, some but not all caspases are found in the activated, proteolytically cleaved form. This applies in particular to caspase-3. Moreover,

TABLE I1 PKINCXPAL FEATURES OF CASPASES 1-10 No.

cn a3

Synonyms (special signs)

Optimal substrate (inhibitors)

Activators

1

ICE

WEHD [WAD.cink] [crmA]

2

ICH-1 Nedd-2 CPP32 Yama Apopain

DEVD

Granzyme B

DEVD [ DEVD.cmk]

Granzyine B Cytochroine c Caspase-6, -7, -10

3

Substrates Prointerleulan-lp Interferon-y-inducing factor a-Spectrin PITSLRE kinases Procaspase-1, -2, -3 PARP Procaspase-2 Sterol regulatory element binding protein Huntingtin DNA-dependent protein kinase a-Spectrin Protein kinase Cy (PKC-y) Actin B Fodrin Gas-2 Heteronuclear ribonucleoproteins C PITSLRE hnases PARP Retinoblastoina protein (Rb) U l 70 kDa PAK2 Procaspase-3, -6

5

Tx ICH-2 ICErel-I1 ICErel-111

6

TY Mch-2

4

I

8

in

9

Ls

10

Mch-3 ICE-LAP3 CMH-1 Mch-5 FLICE Mach- 1 [ FADD domain] ICE-LAP6 Mch-6 Mch-4 [FADD doinain]

WEHD

WEHD (IN/L)EXD

Caspase-10 Caspase-3

DEVD

Caspase-10

(IN/L)EXD [crniA]

Caspase-10 DISC

(IN/L)EXD

Granzyme B

(IN/L)EXD [crmA]

DISC

PARP Laniin A Heteronuclear riboniicleoproteins C PARP Sterol replator). element binding protein All known caspases

All known caspases

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JOSEF M. PENNINCER AND GUIDO KROEMER

a number of caspase substrates found in the nucleus [lamin, poly(ADPribose) polymerase (PARP), U1-70 kDa, etc.] and in the cytoplasma (e.g., fodrin) are constantly cleaved in apoptosis, at the same site of the primary sequence as that recognized by caspases. The subcellular localization of caspases has not been investigated in detail. It appears that some caspases are localized in the cytosol (procaspase-3 and -9) (49), whereas active interleukin-fl converting enzyme (ICE) distributes to the plasma membrane (50). It is unclear how caspases gain access to the nucleus to cleave nuclear substrates including PARP, U1-70 kDa, and the nuclear lamins. Inhibitors acting on a wide range of caspases, such as the baculovirus protein p35 (51-53), N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk) (54-62), its truncated analog Boc-Asp(0Me)fluoromethyl ketone (B-D.fmk) (59), or acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-DEVD.cmk) (63), inhibit the acquisition of apoptotic morphology in a wide range of experimental models. According to one report, some cell lines (e.g., CTLL-2 cells) respond poorly to Z-VAD.fmkmediated inhibition of apoptosis but do respond to B-D.fmk (59).However, the overall consensus is that inhibition of caspases by z-VAD.fmk can fully prevent several aspects of classical apoptosis including endonuclease activation. The previous findings have suggested that caspase activation and apoptosis are near to synonymous. This idea has received apparent support by the fact that at least two procaspases (caspase-8 and -10) possess long N-terminal prodomains with homology to the so-called FADD/MORTl molecule, allowing its interaction with and activation by the Fas/APO-l/ CD95 or TNF-R complexes (64-66). Moreover, numerous natural inhibitors of apoptosis are inhibitors of caspases. Such caspase inhibitors are encoded by baculovirus (p35) (51),cowpox virus (crmA) (67), Kaposi’s sarcoma-associated human herpesvirus-8, or human molluscipoxvirus (68). Caspases are also inhibited by nitric oxide (NO), which acts via specific S-nitrosylation of cysteine residues important for proteolytic function (69). Multiple different proteins can be digested by caspases (Table 11).Some of these proteins have been considered to have a particularly important role in causing cell death. Thus, the “death substrate” PARP has been thought to be particularly important because cleavage of PARP causes enzymatic activation with consequent NAD depletion. However, cells from PARP knockout mice undergo apoptosis with normal kinetics (70). Actin cleavage has been thought to be important for the changes in overall cell morphology. However, actin is not cleaved in all cell types undergoing

MECHANISMS OF APOPTOSIS

61

apoptosis (71). The role of lamins, which are cleaved by a number of caspases in the apoptotic process, has also been overestimated. Thus, transfection of cells with caspase-resistant lainin B only retards the process of nuclear apoptosis (72). Functionally, more important caspase substrates may be DFF (20),which can stimulate endonucleases, and PAK2. Transfection of cells with caspase-3-resistant PAK2 abolishes formation of apoptotic bodies although it leaves unaffected endonuclease activation and phosphatidylserine exposure (73). Full inhibiton of caspase activation with consequent absence of protein degradation and endonuclease activation does not always rescue cells from death. In a series of different models, z-VAD.fmk does not prevent membrane blebbing, does not maintain the clonogenic potential of cells, and ultimately does not prevent cytolysis (60-62, 74) (Fig. 2), suggesting that, at least in some models of apoptosis, the activation of caspases, although necessary for acquisition of apoptotic morphology, occurs after the decision to die has been made. It thus appears that the activation of both endonucleases and “downstream caspases” occurs after the point of no return of the apoptotic process. Only in certain circumstances do caspases act in signal transduction pathways that may be linked to apoptosis induction. This applies, for instance, to caspase-8, which links the Fas/APO-l/CD95 receptor to induction of a number of kinases (35,38,46, and 54 kDa) including the stress-responsive mitogen-activated protein kinase p38/HOG (75, 76) and the dephosphorylation of retinoblastoma protein (77). Similarly, a crmA-inhibitable caspase links TNF-R-mediated signaling to ceramide generation (78). In synthesis, it appears that caspase activation is an obligatory corrolary of the apoptotic mode of cell death. In several cases, caspase activation links signal transduction pathways to apoptosis induction. However, caspases are not (or not always) involved in the initiation and effector stages of apoptosis. Moreover, cell death can occur in the absence of caspase activation.

C. OTHERPROTEASES In addition to caspases, a number of different proteases have been implicated in the apoptotic process. Putative apoptosis-triggering proteases include serine proteases, calpains, and proteasomes. Evidence for the involvement of these enzymes is based on studies using inhibitors that can prevent apoptosis induction in some models of cell death. These data are difficult to interpret because supposedly specific inhibitors of a protease can act on other enzyme systems. For instance, “calpain inhibitor I” is a relatively efficient inhibitor of proteasomes. Probably, most of these noncaspase proteases are only involved in some particular types of apoptosis induction, Thus, proteasome inhibitors such as lactacystin and MG132

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FIG. 2. Caspase inliibition can inhibit apoptosis without preventing cell death. Mouse thyinocytes were stiimilated for 12 hr with etoposide (2 p M ) ,either in the absence (A) or in the presence (B) of z-VAD.fink (100 p M ) . Note the typical apoptotic nuclear morphology (condensed, homogeneously electron-dense nuclei) that is visible with etoposide alone (A). This morphology is found in a cell that has undergone secondary necrosis ( i t . , necrosis after apoptosis). In the presence of etoposide plus z-VAD.fink (B) cells undergo cytolysis without acquiring an apoptotic morphology (i.e., primary necrosis). Similar results are obtained when, instead of etoposide, glucocorticoids are used as apoptosis inducers. For details see Ref. (62).

protect thymocytes against a number of different apoptosis inducers (glucocorticoids, etoposide, etc.) (79), have no effect on camptothecin-induced apoptosis of HL60 cells (SO), and induce apoptosis in proliferating cell lines (81, 82). How proteasome-mediated degradation of ubiquinated proteins can regulate apoptosis is not understood. Similarly to proteasome inhibitors, inhibitors of calpain and serine proteases prevent cell death in

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63

some but not all systems of apoptosis induction (83, 84). The inhibitors of calpain (benzyloxycarbonyl-Leu-Leu-Tyrdiazomethylketone and acetylLeu-Leu-Nle aldehyde) and serine proteases (3,4-dicliloroisocoumariiiand tosyl-Phe chloromethylketone) may actually induce apoptosis (85). Altogether, these data suggest that proteasomes, calpains, and serine proteases are not involved in the common phase of the apoptotic process; these enzymes rather participate in private pathways upstream of the common effector phase. In addition to these upstream effectors, some iioncaspase proteases may be involved in the degradation phase. Thus, inhibition of the nuclear scaffold-associated serine protease by AAPFcmk prevents lamin breakdown (86).A number of different serine protease inhibitors have been shown to inhibit oligonucleosoinal DNA fragmentation without affecting

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JOSEF M. PENNINGEH AND CUlDO K R O E M E R

formation of 250-kDa fragments and chromatin condensation (83). Inhibition of this serine protease does not prevent cell death, as might be expected. Currently, the molecular nature of these downstream proteases remains elusive.

D.

OTHER FEATUHES OF TIIE

DEGRADATION PHASE

1 . Cell Membrane Changes In normal intact cells, lipids contained in the plasma membrane are distributed in a rigorously asymmetric fashion. Phosphatidylserine and phosphatidylethanolamine are only found on the inner side of the plasma membrane. Apoptosis is accompanied by a loss of membrane asymmetry with consequent “flipping out” of phosphatidylserine and phosphatidylethanolamine, which become detectable on the surface of apoptotic cells (87, 88). This is a general feature of apoptosis regardless of the initiating stimulus (89-91). Phosphatidylserine exposure is prevented by caspase inhibition and occurs in anucleate cells (61, 92, 93), indicating that is independent of the nucleus but requires caspase activation. Chelation of extracellular (but not intracellular) Ca2+has a partial inhibitory effect (94). The exact mechanisms of phosphatidylserine exposure are elusive. It might involve the inhibition of an ATP-dependent aminophospholipid translocase (“flippase,” which would counteract the entropy of the outer membrane) (95) or activation of a Ca2+-activated“scramblase” (which would actively perturb the plasma membrane) (96-98). Alternatively, it might be due to the caspase-mediated derangement of the cytoskeleton with fodrin cleavage, thereby disrupting the anchoring of phosphatidyl serine residues on the internal side of the plasma membrane (99). The exposure of phosphatidylserine on the cell surface typically occurs before the membrane becomes permeable to vital dyes such as ethidium bromide or trypan blue. It appears, however, that some changes in plasma membrane permeability occur relatively early (100) and possibly facilitate the outflow of glutathione (101) and potassium (102). Phosphatidylserine exposure is a functionally important event because phagocytes possess receptors for phosphatidylserine ( 103).Thus, surface exposure of phosphatidylserine facilitates the recognition and removal of apoptotic cells. Phosphatidylserine also causes activation of procoagulant enzymes (104).

2. Redor Status Apoptosis is generally associated with major changes in the redox status (105). Such changes include a loss of nonoxidized glutathione (106, 107), which is extruded from the cell (101, 108) and/or may be oxidized during the process of apoptosis. Hyperproduction and/or reduced detoxification of reactive oxygen species is generally found in cells undergoing apoptosis

MECHANISMS OF APOPTOSIS

65

(109). Accordingly, membrane lipids tend to be oxidized in apoptotic cells (110). 3. Ion Fluxes During apoptosis, changes in subcellular Ca’+ distribution have been investigated in detail. It appears that during the late degradation phase, cytosolic Ca2+levels tend to increase to supraphysiologicallevels (>1 p~ ) (107), whereas K+ levels decrease (102). These changes may have some functional impact because they can participate in the activation of endonucleases. Whether they correspond to a general dysfunction of plasma membranes or a specific dysregulation of specific ion transporters is not clear. In conclusion, it appears that most if not all structures of the cell are severely perturbed during the apoptosis degradation phase. Thus, catabolic enzymes, mostly proteases and nucleases, become activated and the overall entropy increases, causing a loss of membrane asymmetry and later a disruption of membrane barrier function. Because direct inhibition of proteases and nucleases or interventions on other manifestations of the degradation phase (e.g., oxidative processes or increases in cytosolic Ca2+ levels) fail to preserve the cell’s integrity, with the exception of some special cases, these changes are not (or not always) decisive for the cell’s fate and rather become manifest after the cell has decided to die. The nature of this decision process-the effector phase of apoptosis-will be discussed in the next section. 111. Effector Phase of Apoptosis

As pointed out in the Introduction, the common phases of apoptosis, which includes the effector phase (regulated) and the degradation phase (beyond regulation), are likely to be the same in all cell types. Therefore, this section will discuss data obtained in different experimental systems, without any specific consideration of lymphocyte physiology.

A.

THE

“CENTRAL EXECUTIONER”: A THEORETICAL CONCEPT

Because apoptosis is induced by a myriad of different inducers (5, 111) but demonstrates a stereotyped pattern of morphological and biochemical changes, irrespective of the cell type and the initial trigger, several investigators have postulated the existence of a so-called central executioner (112) or “death machine” (38), colloqually also referred to as “great integrator” or “apostat.” Activation of the hypothetical central executioner during the effector stage would allow the decision to die to be made and to streamline

fifi

JOSEF M PENNINGEK AND GUIDO KIiOEMER

the many private pathways of apoptosis into one common pathway. The nature of this hypothetical entity has long remained elusive. What would we expect from the central executioner, based on functional considerations? In other terms, what criteria should a change in cellular biochemistry fulfill so that it may be considered a part of the central executioner?

Chronological criterion: For obvious reasons, the central executioner should become activated before alterations classically associated with the degradation phase occur (full-blown activation of the caspase cascade with lamin degradation, endonuclease activation, phosphatidylserine e.xposure on the plasma membrane, etc.) (Fig. 3A). Functional criterion: When apoptosis is induced, activation of the central executioner and of the degradation phase should be undissociable (Fig.3B). Criterion of conuei-gence: The central executioner should function as the great integrator. In other words, it should sense multiple proapoptotic signal transduction cascades as well as damage pathways, all of which should converge onto the central executioner (Fig. 3C). Criterion of coordination: Triggering of the central executioner should suffice to induce the entire spectrum of apopotic changes at the levels of the nucleus, the cytoplasrna, and the plasma membrane. In other words, the central executioner, once activated, should have pleiotropic effects on several organelle systems (Fig. 3D). Criterion of universulity: Because apoptosis follows the same end stage pathway in all cell types, the central executioner should be the same in lymphocytes and in all nonlymphoid cells. Similarly, the central execution should constitute a critical event of apoptosis that is independent of the cell death-initation stimulus (Fig. 3E). Criterion of vitality: All cells can be driven into apoptosis. This applies to primary cells from various tissues, to tumor cells, as well as to transformed

FIG.3. A heptalog of criteria that should be fulfilled by the hypothetical central executioner. (A). Criterion of chronology: The central executioner should be activated before the different facets of apoptotic degradation become apparent. (B). Functional criterion: During naturally occurring apoptosis, activation of the executioner and the manifestation of apoptotic degradation should be undissociable. (C). Criterion of convergence: Very different damage pathways (symbolized by lightnings) or receptor-mediated signal transduction pathways should converge on the central executioner. (D). Criterion of coordination: Activation of the central executioner should have pleiotropic effects on different organelle systems, thereby causing the full spectrum of apoptosis-linked changes. (E).Criterion of universality: The central execution should be the same in different cell types and in different models of apoptosis induction. (F).Criterion of vitality: The central executioner (or its components) should exert functions vital for normal cell survival. ( G ) .Criterion of the switch: The central executioner should be a self-amplifying device that is either in the on or in the off position.

A Criterion of chronology

B

Functional criterion

C

Criterion of convergence D

I

E

Criterion of universality

F

Criterion of vitality

G

Crit. of coordination

I

central executioner

I

Criterion of the switch

Growth factor withdr. Ceramide etc. Pro-oxidants (ROS, NO) Ischemidreperfusion Excitotoxiq calcium Metabolic toxins, etc. mnifertations olapoptosis

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JOSEF M. PENNINGER AND GUIDO KROEMER

cell lines that have been cultured during decades in vitro (113).Based on this premise, it may be speculated that the structures necessary for apoptotic cell death are also indispensable for normal cell survival; otherwise, mutated cells that are completely resistant to apoptosis induction would have arisen during in vivo or in vitro selection for tumor growth (Fig. 3F). Criterion ofthe switch: Cell death is an all-or-nothing process. There is no such thing as a “half-dead’ or “half-alive” cell. Accordingly, the central executioner should be activated in an all-or-nothing fashion. In other biological systems such irreversible ordoff decision processes are obtained by positive feedback loops that lock the cell in a committed state. [This applies, for instance, to differentiation processes involving transcription factors that stimulate their own transcription ( 114)].Accordingly, the central executioner should have self-amplifying properties so that it can behave like a switch that is either in the on or in the off position (Fig. 3G).

B. A FEWERRONEOUS PROPOSALS ON THE NATUREOF THE CENTRAL EXECUTIONER Since the rediscovery of apoptosis by Andrew Wyllie and colleagues (115),numerous studies have claimed the discovery of the universal mechanism of apoptosis. Most if not all of these theories have been invalidated. Unfortunately, it takes a long time for the scientific community to dismiss wrong hypotheses. Thus, the recent literature on apoptosis is still plagued with a number of erroneous a priori ideas on apoptosis that have been en vogue during short periods. Here we will discuss some of these incorrect assumptions on the general mechanism of apoptosis (Table 111).

1. “Killer Genes” and “Death Programs”? Based on the observation that inhibition of transcription and translation can prevent apoptosis in several models, it has been widely assumed that apoptosis would require the expression of killer genes or the realization of genetic death programs. This idea was apparently supported by the observation that numerous genes regulate apoptosis susceptibility. Stimulated by the hypothetical existence of so-called killer genes, hundreds of groups have attempted to isolate genes specifically expressed during apoptosis using differential display and subtracted libraries. The final result of these efforts has been extremely deceiving. No universal killer gene has been discovered in mammalian cells. The few genes that have been implicated in death induction are only involved in particular induction pathways but are dispensable for apoptosis induction in most systems. Thus, for instance, nur77, whose expression is involved in T cell receptor (TCR)-mediated thymocyte deletion, is not required for glucocorticoid-

69

MECHAR‘ISMS OF APOPTOSIS

EmovEous ASSEHTIONSO N

THE

TABLE I11 NAIUIIE01;T H E CENTRAI. EXECUTIONER OF APOITOSIS

Assertion

Invalidation

Apoptosis requires the expression of “killer genes” or “death programs” Apoptosis is an abortive cell cycle or mitotic catastrophe

Apoptosis c m Iw induc.ed in all cells in tlw presence of cyclohexiinide (113) Apoptosis can he induced dnring any pliase of the cell cycle [reviewed by Ref.

Apoptosis is a nuclear process

Cytoplasts (anucleate cells) can uudergo receptor-tnedi~itrdcell death (26, 27) Apoptosis can be induced in/by the al)sence o f oxygen, in a Bcl-2-regiilatetl fhshion (135, 1%) Ca” depletion can induce apoptosis ( 143); nuclear apoptosis can be induced in Ca”-free inedia (147, 179, 581) I n some inodels. acidification inhibits cell death (150) Bax and Bak overexpression intluces death in the presence of a caspase inhibitor (60, 61); Bcl-XI,overexpression can resciie cells i n which caspases have cleaved PARP (162) 111 some cases, necrosis is inhibited by the apoptosis regulator Bcl-2 (167, 169); classical apoptosis inducers provoke necrosis i n the presence of caspase inhibitors (Tuhlr IV) or in conditions of ATP depletion (171, 582) Cell-free systenis of apoptosis require mitochondria or mitochondria1 products (19. 147, 179)

(511 Apoptosis requires the action of reactive oxygen species Apoptosis requires the elevation of Ca’+ i n sonie subcellular conipartnient Apoptosis requires cytosolic acidification Apoptosis always requires the action of caspses

Apoptosis is fundainentally different froin necrosis

Apoptosis does not involve initochondria

induced thymocyte apoptosis (116, 117). In the meantime, it has become clear that protein neosynthesis is not required for apoptosis induction in most systems (118, 119). On the contraiy, it appears that all primary cells can be driven into apoptosis by a combination of the kinase inhibitor staurosporine A and the protein synthesis inhibitor cyclohexiinide (58, 113).This iinplies that all proteins and probably all nonprotein structures necessary for the acquisition of the apoptotic phenotype are constitutively expressed. 2. Apoptosis as an “Aliortizje Cell Ciple” of “Mitotic Catastrophe”? A few gene products that regulate apoptosis susceptibility (e.g., p53, Bcl-2, Bax, c-Myc, E2F-1) have marked effects on cell cycle control (120-

70

JOSEF M. PENNINGER A N D GUlDO KHOEMER

127).In addition, in some special cases, apoptosis induction has been linked to a determined phase of the cell cycle (128).These findings suggested that apoptosis might constitute a sort of abortive cell cycle. Apoptosis and mitosis share some characteristics, such as cytoskeletal changes, rounding up of the cell, nuclear envelope breakdown, and chromatin condensation, thus favoring the speculation that programmed cell death (PCD) might represent an aberrant cell cycle, involving out-of-phase expression in postmitotic cells of mitotic activities with lethal consequence; PCD would be a mitotic catastrophe (129). Nonetheless, it appears that the link between apoptosis and cell cycle, if such a link truly exists, must be loose. Although in some special cases apoptosis induction is linked to a perturbation of cell cycle advancement, in principle apoptosis can be induced both in resting and in proliferating cells, during any phase of the cell cycle ( 5 ) . Recent studies have also shown that disassembly of nuclei is radically different in mitosis and apoptosis. In mitosis, p34'd"-mediated phosphorylation of lamins results in their reversible depolymerization, whereas in apoptosis lamin is irreversibly degraded by proteases f 130).Thus, apoptosis is not an aborted mitosis.

3. The Nucleus CIS a Prime Target of the Apoptotic Process? Because changes in nuclear morphology are the most spectacular ultrastructural features of apoptosis, it has been assumed for a long time that the nucleus would be a prime target of the apoptotic effector phase. This idea was strengthened by the fact that endonuclease-mediated DNA cleavage was the first biochemical alteration specifically associated with apoptosis. In the meantime, several independent groups have reported that cytoplasts (anucleate cells) can undergo receptor-induced regulated cell death that shares several features with normal apoptosis: regulation by Bcl-2 (26,27),caspase activation, phosphatidylserine exposure (92,131), and disruption of the mitochondria1 transmembrane potential (131, 132). Apoptosis-like death of cytoplasts has been induced by a variety of stimuli including growth factor withdrawal, cross-linking of Fas, staurosporine, ceramide, and granzyme B (26, 27, 92, 131-133). This indicates that the nucleus cannot be part of the central executioner and rather constitutes a downstream target of the degradation phase.

4. Reactive Oxygen Species as Universal Apoptosis Effector,s? Most events of apoptosis are accompanied by dramatic changes in the cellular redox balance: depletiodoxidation of glutathione ( 101, 106, 107), hyperproduction of reactive oxygen species (109, 110), and oxidation of cellular constituents including lipids (110). Moreover, in many (but not all) cases, antioxidants prevent apoptosis, whereas prooxidants induce or facilitate apoptosis ( 105).Although these findings suggested that apoptosis

MECHANISMS OF APOPTOSIS

71

might be an oxidation process, it has been shown that apoptosis induced by a variety of different stimuli (staurosporine, growth factor withdrawal, and anti-Fas) can be induced in cells kept under anaerobic conditions, in the manifest absence of reactive oxygen species (ROS) (134, 135), and that hypoxia can be an apoptosis-triggering condition (136). Only in some pathways (e.g., in glucocorticoid-induced apoptosis) does the formation of ROS appear to be essential for apoptosis induction (137). Thus, redox processes constitute one way of regulating apoptosis but cannot be central to the apopoptotic process.

5. Ca2+:An All-Explaining Cation? Increases in cytosolic Ca2+can induce apoptosis, and chelation of intracellular calcium prevents apoptosis induction in a number of models (138). This also applies to TCFUCD3- or glucocorticoid receptor-triggered thymocyte apoptosis, and Ca" has also been proposed to be the factor responsible for nuclear endonuclease activation (139, 140). Although Ca2+may serve as a proapoptotic second messenger, several examples have been reported in which apoptosis is induced in or by the absence of extracellular Ca2t (141-143) or in the presence of intracellular Ca2' chelators such as BAPTAAM (144). Although cytosolic Ca2+tends to increase to nonphysiological levels ($10 W M ) during the late stage of apoptosis (107) and Ca2+can activate endonucleases (140, 145), many cell-free systems of apoptosis do work in the presence of CaZt chelators (20, 45, 146, 147). Thus, Ca2+ elevations are unlikely to be essential parts of the effector or degradation phases. 6. Cytosolic Acidijication: The Panacea?

The degradation phase of apoptosis is accompanied by major changes in ion homeostasis including a cytosolic acidification (148). Attempts have been undertaken to reduce the death/life decision to a question of pH regulation (149). Nonetheless, in certain cell types, e.g., thymocytes, cytosolic acidification actually inhibits apoptosis (150).Altogether, the present data suggest that changes in ion fluxes and compartmentalization are a byproduct of apoptotic degeneration downstream of protease activation rather than essential elements of the process (151, 152).

7. Caspases: Decisive for Cell Death? During the past few years it has been widely assumed that caspases might constitute the central executioner (36-38, 112). This is suggested by numerous observations: Caspases are constantly activated during apoptosis (see Section 11).

72

[OSEF M. PENNINGER A N D CUIDO KROEMER

Transfection-enforced overexpression of caspases causes apoptosis, at least in several models (153-156). Addition of caspases to cell extracts can induce nuclear apoptosis in uitro (45, 46). Inhibition of caspase activation by broad-spectrum inhibitors precludes acquisition of the apoptotic phenotype (60, 61, 135). Several apoptosis-inducing pathways, including those activated by ligation of TNF-R p55, Fas/APO-l/CD95 (64,65),or granzyme B (157, 158), are directly coupled to caspase activation. Caspases can engage in a cascade of sequential (and sometimes mutual) activation, suggesting that they can participate in (aut0)amplification processes (36, 37, 40, 158). Nonetheless, a few facts argue against the obligatory participation of caspases in the central executioner: Caspase can exert other functions than those involved in apoptosis. Thus, caspase-1 is necessary for the processing of interleukin-lp (159) and interferon-y-inducing factor (160). Caspase-3 activation can be observed in the absence of apoptosis (161),and the caspase-mediated digestion of the nuclear substrate PARP has been observed in cells that maintain their clonogenic potential (162). In several systems, inhibition of caspase activation using the broadspectrum inhibitor z-VAD.fmk prevents acquisition of the apoptotic phenotype but does not inhibit cell death. This applies to a number of different models (Table IV), suggesting that caspase activation is decisive for the degradation phase but dispensable for the effector phase of apoptosis. In synthesis, caspases are not (or not always) involved in the effector phase of cell death. 8. Apoptosis in Opposition to Necrosis? Conventional textbook knowledge insists on the opposition between apoptosis and necrosis. However, several facts weaken the idea that apoptosis and necrosis involve fundamentally different mechanisms:

After apoptosis, cells undergo necrosis. The same toxin can induce apoptosis (at low doses) and primary necrosis (at high concentrations) (111, 163). Many pathologies labeled as “necrotic” are now known to involve apoptosis. This applies to myocardial infarction, cerebral apopleloj, and excitotoxin-induced neuronal cell death (164-166).

73

MECHANISMS OF APOPTOSIS

TABLE IV MODELSOF APOPTOSISI N WHICH THE CASPASE INHIBITOR z-VAD.fmk FAILSTO INHIBITCYTOLYSIS Inducers of Apoptosis Overexpression of bax

'

c-myc overexpression and serum withdrawal or overexpression of bak Protonophore, protoporphyrine IX, dexamethasone, etoposide, or nitric oxide CTL granule exocytosis

Effect of z-VAD.fmk Disruption of the mitochondrial transmembrane potential, followed by death without DNA fragmentation Cytoplasmic blebbing for hours, followed by cytolysis without DNA fragmentation Disruption of mitochondrial transmembrane potential; retarded nonapoptotic cytolysis in the absence of caspase or endonuclease activation (see Fig. 2) Inhibition of nuclear apoptosis without prevention of target cell Iysis

Reference 60

61

62, 216

74

Bcl-2 overexpression can inhibit apoptosis and, at least in some cases, necrosis (167-169). Inhibition of caspases can induce a switch from the apoptotic to the necrotic mode of cell death, as discussed previously (60-62, 74). Manipulations of the ATP level can influence the choice between the two modes of cell death. Thus, in cells in which the ATP level is lowered, for instance, by withdrawal of glycolytic substrates and inhibitors of mitochondrial ATP generation, stimuli that conventionally induce apoptosis (e.g., Fas- cross-linking) cause necrosis (170, 171). Altogether, these recent findings imply that apoptosis and at least some examples of necrosis share a common effector pathway. 9. Lack of Mitochondria1 Involvement in Apoptosis? Several studies have reported that cells lacking mitochondria1 DNA could undergo full-blown apoptosis (172, 173). Although these data were correctly interpreted by the authors of these studies, many investigators have short-circuited them to assume that cells without mitochondria would retain the capacity of undergoing apoptosis and that therefore mitochondria would not be important for the apoptosis effector stage. Nonetheless, cells without mitochondrial DNA do possess morphologically normal mitochondria that just lack some components of the respiratory chain complexes I, 111, IV,and V encoded by the mitochondrial genome and thus are respira-

74

JOSEF M . P E N N I N C E R A N D GUIDO KROEMER

tion deficient. Therefore, the assumption that mitochondria are not relevant to apoptosis, as based on the aforementioned experiments, is certainly incorrect [for review see Ref. (174)]. Indeed, numerous recent finding suggest that mitochondria do have a central role for the apoptotic process. C. THECENTRAL EXECUTIONER OF APOPTOSIS:A MITOCHONDRIAL HYPOTHESIS Mitochondria undergo major changes in their structure/function early during apoptosis [for review see Refs. (163)and (175)-( 177)].Two different major changes in membrane permeability have been observed. On the one hand, the electrochemical gradient built up on the mitochondrial inner membrane dissipates during apoptosis (109, 178). On the other hand, proteins that normally are sequestered in mitochondria are released through the outer mitochondrial membrane. Such proteins include cytochrome c (179-181) and a so-called apoptosis inducing factor (19, 147). These two proteins both activate caspases and trigger nuclear apoptosis in cell-free systems. The exact molecular mechanisms and the cause/effect relationship between the increase in inner and outer mitochondrial membrane permeability are a matter of debate. We have advanced the hypothesis that opening of the so-called “mitochondrial permeability transition pore” (also called “mitochondrial megachannel”), which is formed by apposition of proteins within the contact site of the inner and outer membranes, might be closely linked to both the dissipation of the inner transmembrane potential (A?,,) and the cytochrome c release (163, 175-177). Importantly enough, it appears that proteins of the Bcl-2 family, many of which are selectively enriched in the mitochondrial innedouter membrane contact site, regulate apoptosis via affecting the mitochondrial permeability transition (177).In most experimental systems, Bcl-2 must be present in the mitochondrial membrane to inhibit apoptosis (182,183).Overexpression of the apoptosis-inhibitory genes bcl-2 and bcZ-X, prevents the A!Pm collapse and/or the release of apoptogenic activities (cytochrome c, and AIF) (Table V). This result has been observed in intact cells as well as in isolated mitochondria (19, 132, 147, 162, 177, 178, 180, 181, 184) (Fig, 4). Conversely, proapoptotic members of the Bcl-2 family such as Bax favor the loss of mitochondrial function (60). Interestingly, several members of the Bcl-2 family have been shown to have a channel-like function, when incorporated into artificial membranes (185, 186). Whether this function is related to their regulatory effects on mitochondria remains unknown, It thus appears that mitochondrial membrane permeability is a prime target of apoptosis regulation by Bcl-2-like proteins. Bcl-2 has been speculated for a long time to act on the apoptotic effector stage (4,187-189). Does this mean that the central executioner involves the mitochondrion?

75

MECHANISMS OF APOPTOSIS

TABLE V A ~ ~ I ~ T O S I S - ~ N REGIMES D U ~ : I NI N~ :WHICH BCI-2 PRESERVES MITOCHONUHIAL, FUNC:TION

Cell Type

Inducers of Apoptosis

Effect of Bcl-2 on Mitochondria

Reference ~

Thymocytes or T cells

T cytoplasts B cells

PC12 cells

Fibroblasts HL60 cells

Glucocorticoids, ceratnide ter-Butylhydroperoxide Protoporphyrine IX Etoposide (VP-1G) y-Irradiation, doxorubicin Cytosine arabirioside inClCCP (protoiiophore 1 Cerainide Surface IgM cross-linking Cyclosporin A, etoposide ( VP- 16) y-Irradiation, doxorubicin Cytosine arabinoside, adriamycin ter-Butylhydroperoxide Ceramide, senim withdrawal Cyanide, rotenone Antiinycin A. etoposide (VP-16) Calcium ionophore p53 Etoposide, staurosporine

Stabilizes AT,,,

Partial A*,,, stabilization Stabilizes AT,,, Stabilizes AT,,,

109, 203 19 199 132 132 132 19 131 132 132 132 132 Unpublished Unpublished

Stabilizes AT,,,

Stabilizes A q , , , Prevents cytoehrome c release and ATn,

184 184 184 S83 180

In the following paragraphs we will examine the question of whether changes in mitochondrial membrane permeability fulfill the seven criteria of the central executioner that we have previously discussed. 1, Chronological Criterion If the mitochondrial membrane change constitutes part of the central executioner, it should occur before the degradation phase of apoptosis becomes manifest. The mitochondrial transmembrane potential (A?,") results from the asymmetric distribution of protons on both sides of the inner mitochondrial membrane, giving rise to a chemical (pH) and electric gradient that is essential for mitochondrial function (190). The inner side of the inner mitochondrial membrane is negatively charged. As a consequence, cationic lipophilic fluorochromes, such as rhodamine 123, 3,3'dihexyloxacarbocyanine iodide [DiOC6(3)], chloromethyl-X-rosamine (CMXRos),or 5,5',6,6'-tetrachloro-l, 1',3,3'-tetraethylbenzimidazolcarbo-

76

JOSEF M. P E N N I N G E R AND GUIDO KROEMER

FIG.4. Bcl-2 effects on cells, cytoplasts,and mitochondria. Transfection-enforced overexpression of Bcl-2 prevents rnitochondrial changes associated with apoptosis in intact cells, cytoplasts, and isolated mitochondria. This indicates that the antiapoptotic effect of Bcl-2 does not require the nucleus and that Bcl-2 affects mitochondrial function due to its local presence within the mitochondrial membrane.

cyanine iodide (JC-l), distribute to the mitochondrial matrix as a function of the Nernst equation, correlating with the A",,, (Fig. 5 ) . Using a cytofluorometer or a confocal microscope, these dyes can be employed to measure variations in the AqInona per mitochondrion or per cell basis. Cells induced to undergo apoptosis manifest an early reduction in the incorporation of A*",-sensitive dyes, indicating a disruption of the A*,, . This A q I ncollapse can be detected in many different cell types including lymphocytes, irrespective of the apoptosis-inducing stimulus (19, 60, 109, 131, 132, 147, 162, 174, 175, 178, 191-201). Upon induction of apoptosis, the A",, disruption is also found in cells lacking mitochondrial DNA (which have a normal steady-state A",)) (174, 196). It addition, it appears that early during apoptosis, mitochondrial intermembrane proteins such as cytochrome c leak out into the cytosol (179-181). Whether this loss of the outer mitochondrial membrane function is a cause or a consequence of, or without any relationship to, the disruption of the inner membrane is still unknown. In a number of different models, the A*,, disruption becomes manifest before cells aberrantly expose phosphatidylserine (PS) on the outer cell membrane leaflet, fragment nuclear DNA, hyperproduce ROS, or manifest a massive dysre ulation in ion homeostasis (107, 109, 131, 178, 193, 202, 203). Only (but not AWmhlgh)cells contain proteolyticilly activated caspase-3, indicating that the activation of caspase-3 is probably secondary to the A",,, disruption (203).Cells that have reduced their A",,, are irreversibly committed to undergo death, even when the apoptosis-inducing trigger is withdrawn (109). Thus, the A",, collapse marks the point of no return

77

MECHAEU'ISMS OF APOPTOSIS

DiOC6(3) (3,3'dihexyloxacarbocyanlne iodide)

HE

ethidium

hydroethidine (= dihydroethidium)

distribution into mitochondrial matrix

cationic lipophilic

hydrophilic fluorescent

lipophilic non-fluorescent

CMXRos (chloromethyl-X-rosamine) R

R

thiol-

conjugstion

cnsi + w - ~ p t i e

cnm-psptie

lipophilic cationic

JC-1

fiiable

NAO (nonyl acridine orange)

(5,5',6,6'-letrrchloro-1,1',3'3,3'-

tetraahylbsndmidazolca~ocyanine iodide)

cn3

Cn3

I Cn,

I CHZ

R-cc-mi, I

w-cwcn I

n

HCDCOR'

I

n,c-~po,~n,C.fflH,o-Po~~cn~ I

OH

Cardiolipin (diphosphatidylglycerol) cationic, lipophilic green monomer red aggregate

distribution into mitochondrial matrix

F I ~5.. Structures of five fluorochromes allowing for the assessment of apoptosisassociated mitochondrial alterations. DiOC6(3),CMXRos, and JC-1 are lipophilic cationic fluorochromes that distribute through intracellular membranes as a function of the Each of these dyes has different properties: DiOC6(3) is nonfixable, CMXRos reacts with thiol to produce aldehyde-fixable thiolesters, and JC-1 can be used as a ratiometric probe because it emits two fluorescence wavelengths, depending on its concentration. HE and NAO incorporate into cells independently from the A",,,. Both are nonfluorescent and react with superoxide anion or nonoxidized cardiolipin, respectively, to form fluorescent products. The fluorescence produced by HE (i.e.,the conversion HE + Eth) is proportional to the time of incubation and the generation of reactive oxygen species. The fluorescence produced by NAO is directly proportional to the cell's content in nonoxidized carcholipin. Changes in HE and NAO fluorescence are found at a later stage of apoptosis than changes in the incorporation of AY,,,-sensitivedyes. For details see Ref. (589).

78

JOSEF M . PENNINCER AND GUIDO KROEMER

of apoptosis but precedes all common signs of the apoptotic degradation phase: nuclear apoptosis, PS exposure on the membrane, activation of CPP32-related caspases, Ca2+influx, K+ loss, and cell shrinkage. To understand the mechanism by which cells undergoing apoptosis lose their ATl,,, experiments have been performed in which cells were first labeled with Aq,,,-sensitive fluorochromes and then purified in a fluorocytometer, based on their AT,,,. In appropriate conditions, this procedure allows for the purification of cells with low ATl,, values and a still normal DNA content and morphology (= preapoptotic cells) or, alternatively, of cells with a high ATIl,that will lose their AT,,, upon a short-term (30-120 min) culture period (109, 178,204).A!Pl,1" (but not ATmh'gh) cells undergo oligonucleosomal DNA fragmentation upon short-term (3060 min) culture at 37°C. The AT],, loss of Aq)Rh cells is prevented by three inhibitors of the mitochondrial PT pore: cyclosporin A (CsA), the nonimmunosuppressive CsA derivative N-methyl-Val-4-CsA, and bongkrekic acid (178, 198, 204). These data indicate that the permeability transition (PT) accounts for the AT,,, collapse observed during early apoptosis. According to one report, cytochrome c release can occur before the AT,,, collapse (which is indicative of irreversible opening of the inner membrane PT pore) (180). In contrast, opening of the PT pore in isolated mitochondria causes the release of cytochrome c (205), during both the initial (reversible) and the advanced (irreversible) stages of megachannel opening (G. Kroemer, unpublished results). As a possibility, an inital "flickering" of the PT pore (that would not cause a manifest Aq,disruption) may lead to cytochrome c release in cells before the AWm dissipates. The relationship between cytochrome c release and PT requires further clarification.

2. Functional Criterion The PT-mediated A*,,, reductions should be undissociable from the subsequent degradation phase, if this mitochondrial change formed part of the central executioner. Apparently, the Aql,l reduction and later nuclear apoptosis are closely linked with each other. Very different inducers of apoptosis provoke the same sequence of events (mitochondrial followed by nuclear alterations). Moreover, whenever an inhibitor interrupts the cascade of signals leading to apoptosis, both the mitochondrial and the nuclear changes are abolished. This is illustrated in Table VI for thymocytes but also applies to other cell types, including primary peripheral lymphocytes, T cell hybridoma cells, lymphoma cells, as well as nonlymphoid cell types. In one particular case, in thymocyte cell death induced by CD99 cross-linking, cell death occurs in the absence of full-blown nuclear apop-

79

MECHANISMS OF APOPTOSIS

TABLE VI APOPTOW I N D U C INC, REC.IMLS IN THYMOCYTES THATCAU5.E A AT,,,DI~RKJFTIO~ PHEC E I ~ NCn c M A R DNA FRAGMENTATIO~ A N D THEIRI N H I B I T I O ~ Inducer of Apoptosis Gliicocorticoids

DNA diunage (etoposide or y-irradiation )

Anti-Fas antibody

TNF-a, cerainide Thapsigargine Negative selection or the absence of positive selection Menadione (ROS generator) ter-Butyhydroperoxide

NO Diamide

Protopoi-phyrine IX

Inhibitor of Apoptosis

Reference

RU38486 (receptor blockade) 192, 194, 198 Actinomycin D (transcription 131 inhibitor) Cycloheximide (translation inhibitor) 131 TLCK (trypsin inhibitor) 198 Lactacystin, MG132 (proteasome 163 inhibitors) N-acetylcysteine (GSH precursor) 178 Catalase ( H 2 0 2detoxifying enzyme) 107 Bongkrekic acid (ANT ligand) 198 Monochlorobiinan (thiol reactive) 200 Chloromethyl-X-rosamine (thiol 200 reactive) p 5 3 null mutation 131, 198 Actinomycin D (transcription 131 inhibitor) Cyclohexiniide (translation inhibitor) 131 TLCK (trypsin inhibitor) 198 Lactacystin. MC,132 (proteasoirie 163 inhibitors) Bongkrekic acid (ANT ligand) 198 Monochlorobiinane (thiol reactive) 200 Cliloroiiiethyl-X-rosaniine(thiol 200 reactive) Ar-WAD.cmk (caspase-1inhibitor) 131 Unpublished Cyclosporin A 584 194

Monochlorobinian (thiol reactive) Ch loromethyl -X- rosain ine ( t h iol reactive) Cyclospoiin A Bongkrehc acid Monochlorobiman (thiol reactive) Chloroniethyl-X-rosaiiiine(thiol reactive) Bongkrekic acid (ANT ligand)

194 200 200 216 216 200 200 199

80

JOSEF M. PENNINGER AND GUIDO KROEMER

tosis, but the AT,,, disruption precedes phosphatidylserine exposure (206). In most experimental systems, it appears that PT is both sufficient and necessary for nuclear apoptosis to occur. Induction of PT by agents acting on the PT pore complex causes apoptosis, and inhibition of PT by pharmacological agents of overexpression of Bcl-2 prevents apoptosis (62, 132, 147, 198-200). PT inducers that also induce apoptosis include inhibitors of the respiratory chain, protonophores, and ligands of the peripheral benzodiazepin receptor. Pharmacological PT inhibitors with antiapoptotic effects include cyclophilin ligands (cyclosporin A, N-methyl-4-Valcyclosporin A) and bongkrekic acid, a ligand of the adenine nucleotide translocator (62, 147, 198-200).

3. Criterion of Convergence The mitochondrion should be capable of integrating very different proapoptotic signal transduction and damage pathways. In this context it appears important that the PT pore is a dynamic multiprotein complex located at the contact site between the inner and the outer mitochondrial membranes, one of the neuralgic sites of metabolic coordination between the cytosol, the intermembrane space, and the mitochondrial matrix. Although the exact composition of the PT pore complex is not known, it is currently thought to involve cytosolic proteins (hexokinase), outer membrane proteins (peripheral benzodiazepin receptor; porin, also called voltage-dependent anion channel), intermembrane proteins (creatine kinase), at least one inner membrane protein (the adenine nucleotide translocator; ANT), and at least one matrix protein (cyclophilin D) (207-211). Speculatively, it may also interact with proteins of additional multiprotein complexes, namely, the Tim (transporter of the inner membrane) complex, the Tom (transporter of the outer membrane) complex (212), and the Bcl2 complex (177) (Table VII). Irrespective of its exact composition, the PT pore complex contains multiple targets for pharmacologd interventions and is regulated by numerous endogenous physiological effectors (Table VIII). Such effectors include ions (divalent cations, mainly Ca2+and Mg2+),protons, the AT,,,, the concentration of adenine nucleotides (ADP and ATP) (208, 209), the pyrimidine redox state (NAD vs. NADH,; NADP vs. NADPH2),the thiol redox state (controlled by glutathione) (213), reactive oxygen species and nitric oxide (214-216), the concentrations of lipoids (lipid acids, acyl-CoA, and perhaps ceramide and derivatives) (208,209,217),the concentrations of some peptides (amphipathic peptides and perhaps signal sequences of peptides targeting proteins to the mitochondrial import machinery) (218, 219), and changes in the composition or function of the Bcl-2 complex (109,147,177).As a general rule, it appears that any major change in energy

MOLECULESIN

THE

TABLE VII PT PORECOMPLEX AS TARGETS FOR ENDOGENOUS EFFECTORS AND PHARMACOLOGICAL AGENTS Nomd Funchon

Molecule (Topology) ~

Adenine nucleotide translocator (ANT) (inner membrane)

Matrix thiols (within the inahix side of the ANT?)

Peripheral henzodiazepin receptor (PBR) (outer membrane) Porin (outer membrane) Cyclophilin D (matrix)

Ca%ensitive sites Tim23 (and other proteins of the Tim and Tom wmplexes?) Hexoldnase (cytosol) Creatine kinas? (intermembrane)

Modulators and Role in PT

Helerence

~

ADP and ATP inhibit PT Bongkreldc acid (binds to matrix site): favors m-state and inhibits PT Atractyloside (hinds to intermembrane site): favors c-state and induces PT Thiol oxidation (e.g., phenylarsenine oxide) and disnlfide Thiol sensor for redox potentials (in quilibnum with bridge formation (e.g., diamide) favor FT glutathione) Thiol derivatimtiun hy monochlorohiman or chloromethyl-Xrnsamine prevents FT Protoporphyin N:induces PT Reeptor for endozepin (= CoA-binding protein) PK11195, FGIN 1-27. chlorndiazepam: facilitate PT Voltage Voltage-dependent anion channel, ATP transport Cyclosprine A: inhibits interaction with inner membrane Peptidyl prolyl isomerase (chaperone function) N-methyl-Val-4-cyclosporine A (nonimmunosuppressive) acts like cyclosporine A Low pB: prevents interaction with inner membrane Calcium favors PT Sensor for divalent cations Transport of proteins through the outer (Tom) or iniier (Tim) Signal peptides of proteins targeted to the mitochondrial matrix inhibit a FT pore-like channel nutochondrial membranes Facilitates or regulates IT? Phosphorylates hexasacchandes (mainly glucose) while hydrolysing ATP Inhibits FT? Transfers phosphate from creatine phosphate to ADP or from ATP to creatine

ATPIADP antiport

210.230

200. 213

223, 233 21 1 586

208, 209 219 211 211

TABLE VIII FUNCTIONS OF THE PERMEABILITY THANSITION PORE Function Voltage sensor Thiol sensor Sensor of pyridine oxidation Matrix pH sensor

Principles of Modulation

Example

Low ATl,, induces PT High AT,,, inhibits PT Oxidation of a critical matrix dithiol (in the ANT?; regulated by GSH) induces PT Oxidation of NAD(P)H2 favors PT (in equilibrium with GSH oxidation)

Anoxia, respiratory inhibitors induce PT Hyperpolarization (nigericin) inhibits PT Prooxidants and thiol cross-linking induce PT Prevention of thiol cross-hnlang prevents PT NAD(P)H2prevents PT Antioxidants prevent PT Prooxidants favor PT Akalinization (pH = 7.3)of matrix favors PT Neutral or acidic matrix pH inhibits PT Increase in matrix CaZ+induces PT M$+ and Zn2i prevent PT Extra ATP (glycolytic substrates) prevents PT Oligomycin (F, ATPase inhibitor) favors PT Caspase-1 induces PT Calpain-like enzyme may favor PT Palmitate and stearate induce PT Carnitine prevents PT Mastoparan induces PT Peptide ligands of Tim 23 inhibit PT?

ADPIATP sensor

Reversible histidine protonation (of cycophilin D?) prevents PT Ca2+induces PT Other divalent cations inhibit PT ADP (and ATP) inhibits FT '

Protease sensor?

Direct action of proteases on outer membrane proteins

Lipid acid sensor?

Long chain lipid acids induce PT

Peptide sensor?

Amphipathic peptides induce PT

Cation sensor

Note. For references and details consult main text.

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balance (the absence of oxygen, depletion of ATP and ADP, depletion of NAD(P)H?, or disruption of tlie A*,,,) or in redox balance (oxidation/ depletion of nonoxidized glutathione or NAD(P)H2and hyperproduction of reactive oxygen species) can provoke PT. This implies that PT functions as an integrator of stress responses and that major damage of cells will invariably cause PT. In addition, a number of signal transduction pathways triggered via intracellular or cell surface receptors can result in PT (Fig. 6). Thus, PT is facilitated by a nuniber of second messengers: increases in cytosolic Ca2+ concentration (220-222), ceramide (109,203,217,223),and some caspases such as caspase-1 (but not caspase-3) (203). Because PT is regulated by the Bcl-2 complex, changes in tlie stoicliioinetry of this complex (e.g., enhanced synthesis of tlie Bcl-2 antagonist Bax) or signal transduction pathways culminating in posttraiislational modifications of the Bcl-2 complex can also facilitate PT. The phosphorylation of Bcl-2 or Bcl-2 honiologs

I

Changesinthe

1

Redox catastrophe

Slgnai transductlon Bioenergetic catastrophe

Fic:. 6. Inducers of perineability transition. Different signal transduction pathways can promote the activation of caspnses, increases in cytosolic Ca” levels, and nitric oxide, ainphipatliic peptides, or lipid mediators (e.g..ceraniide) provoke F’T. In addition, hyperexpression of the Bcl-2 antagonist Bax or posttranslational effects affecting the function of the Rcl-2 complex can induce F’T. Major changes in the cellular redox and energy balance also trigger PT. Note that PT is a s e l f - q M ) i n g process (hvo-headed arrows). Certain PT inducers (increases in cytosolic (=a‘+. reactive oxygen species, etc.) may be involved in “private” patliways of apoptosis indiictioii and tliiis function as facultative PT inducers. yet constitute constant by-products of the apoptotic process (striped boxes). This explains why several conseqnences of PT are constant by-products of :ipoptosis, hut itre not necessary for apoptosis to occur in all instances.

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(e.g., Bad), as well as the subcellular distribution of Bcl-2-related proteins (e.g., Bad) or Bcl-2-associated proteins (e.g., Bag-1 and Raf-1), is regulated by growth factor receptors (177, 224, 225), thus suggesting how growth factor withdrawal can trigger PT via changes in the composition of the PT-regulatory Bcl-2 complex (177). Speculatively, an additional pathway that may converge at the level of mitochondria may involve the Src-like kinase substrate HS-1, a proapoptotic molecule (226) that interacts with the mitochondrial outer membrane protein Hax-1 (227). The functional significance of pathways leading to the formation of a mitochondrial p38 neoantigen (228) in apoptotic cells still remains elusive. Altogether these findings suggest that, in addition to integrating various damage responses, PT can be triggered via receptor-connected pathways. It thus may constitute the crossroad of both nonspecific damage responses and responses mediated via specific receptors. 4. Criterion of Coordination The central executioner should be capable of coordinating the different manifestations of apoptosis at the levels of the nucleus, the cytoplasm, and the plasma membrane. Hence, the question is whether and how the disruption of mitochondrial membrane integrity may provoke the entire spectrum of apoptotic degradation. Does PT coordinate the apoptotic degradation phase? Pharmacological inhibition of PT prevents all manifestations of apoptosis, at the levels of the nucleus, the plasma membrane, and the cytoplasma (198, 200), emphasizing that PT can indeed function as a central coordinating event. How does PT cause cell death? In normal circumstances, the inner mitochondrial membrane is near-to-impermeant. This feature is required for building up and mantaining the inner transmembrane potential ( A q m ) .Opening of the PT pore allows for the diffusion of solutes with a MW of I 1 5 0 kDa, according to gross estimations based on the use of polyethylen glycol polymers (208, 209). Prolonged opening of the PT pore causes the dissipation of the A q mwith consequent loss of mitochondrial RNA and protein synthesis, cessation of the import of most proteins synthesized in the cytosol, release of Ca2+and glutathione from the mitochondrial matrix, uncoupling of oxidative phosphorylation with cessation of ATP synthesis, oxidation of NAD(P)H2and glutathione, and hyperproduction of superoxide anion on the uncoupled repiratory chain (208) (Fig. 6). Accordingly, cells undergoing apoptosis manifest an arrest of mitochondrial biogenesis, at both the transcriptional and translational levels (191, 229), and perturbations in mitochondrial electron transport (230). Multiparameter fluorescence analyses revealed that the preapoptotic A*,,, collapse is closely linked to major changes in cellular redox potentials, namely, NAD(P)H2 depletion (192), GSH depletiodoxidation (107), and

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later increases in superoxide anion generation (109) and massive cytosolic Ca2+elevations (107). Superoxide generation is reduced in several models by rotenone, an inhibitor of the respiratory chain complex I, suggesting that it is formed on the uncoupled respiratory chain (109, 193). The bioenergetic and redox changes of PTs themselves are sufficient to cause cell death by necrosis (220, 223, 231-234). How does PT trigger apoptosis? PT allows for the release of proteins that are usually confined to the mitochondria1 compartment. Thus, PT causes the release of cytochrome c from the intermembrane space into the cytosol(205).The protein precursor of cytochrome c (apocytochrome c) is synthesized in the cytosol and transported into the intermembrane space, where the heme lyase attaches a heme group to generate holocytochrome c (235). Holocytochrome c (but not its precursor apocytochrome c, still lacking a heme group) can interact with other yet unknown cytosolic factors to activate caspase-3 (179-181). Caspase-3 then can activate the DFF, which in turn acts to activate nucleases (20). In addition to cytochrome c, mitochondria undergoing PT release AIF, a protein of approximately 50 kDa that suffices to cause nuclear apoptosis and activation of caspase-3 in cell-free and cytosol-free systems (19, 203). As also true for cytochrome c, this activity appears to be ubiquitous and preformed. Exhaustive studies have identified an inhibitor of AIF: z-VAD.fmk (19). This protease inhibitor abolishes all activities of AIF on the nucleus. z-VAD.fink is also a universal inhibitor of nuclear apoptosis occurring in intact cells, irrespective of the cell type (54-62), thus emphasizing the possible in vivo relevance of AIF. In summary, mitochondria contain several proteins endowed with the capacity of stimulating at least some facets of the apoptotic program in cell-free systems. Currently, it appears that at least two biochemical pathways may link mitochondria to nuclear apoptosis (AIF; cyctochrome c + factor X + caspase 3 + DFF). It is not known which among these pathways prevails in vivo or whether both pathways are complementary. If PT can stimulate both primary necrosis and apoptosis, what does make the difference? As a possibility, the bioenergetic and redox catastrophe ensuing PT (which would induce necrosis) and the activation of catabolic enzymes (caspases and nucleases) might compete among each other in a sort of race. Cells would only die from primary necrosis when apoptogenic proteases fail to come into action, either because they are inhibited (e.g., by addition of z-VAD.fmk or by natural compounds such as NO) (60-62, 69) or because the time frame of the process is too rapid to allow for protease activation. This view of cell death would be compatible with the observation that many substances induce apoptosis at low doses (when PT is induced smoothly, perhaps first in a fraction of mitochondria, and cells can activate proteases) but necrosis at higher doses (when PT is caused

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abruptly and cells lyse before proteases come into action). Finally, it would explain how maintenance of cytosolic ATP levels can influence the decision between the two modes of apoptosis induction, with high ATP levels favoring apoptosis and low ATP levels favoring necrosis (170,171),whereas inhibition of proteases or manipulations that reduce cellular ATP levels favor necrosis over apoptosis (Fig. 7). It may be noteworthy that, in contrast to other cell types such as neurons and myocardiocytes, lymphocytes undergo apoptosis rather than necrosis in response to most death-inducing regimens (111).This may be due to the fact that ATP generation in lymphocytes mainly relies on glycolysis.

5. Criterion of Uniuersality The central executiorer should be operative in all cells and in all events of apoptosis. Molecules such as the ANT [which exchanges matrix ATP for cellular ADP on the inner membrane (236)] or porin [which allows for the exchange of ATP on the outer membrane (237)] are essential for oxidative phosphorylation and thus must be expressed in all living cells. For

I

mitochondria1 permeability transition

i

it of redox potentials and bioenergetic catastroph

I

primaKy necrosis (without apoptosis)

[necrosis) secondary necrosis

FIG7 . Hypothetical model of the apoptosis/necrosisdichotomy. Disruption of initochondrial function caused by mitochondria1 permeability transition can cause necrosis via its consequences on redox and enerQ metabolism. Alternatively, the liberation of apoptogenic mitochondria1 proteins, such as cytoclirorne c and AIF, causes the proteolytic activation of different cytosolic caspase precursors, as well as the activation of DNA fragmentation factor (DFF) via caspase-3. Caspases, DFF, and AIF can act on the nucleus (and on cytoplasmic stmctures) to cause apoptosis. Depending on the pathway that prevails, necrosis oc'ciirs before apoptosis (primary necrosis) or after apoptosis (secondary necrosis).

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obvious reasons, the apoptogenic factor cytochrome c, which participates in the respiratory chain, also must be present in all respiring cells. AIF-like activity has also been detected in many different tissues (19), suggesting that it may be ubiquitous. Although mitochondrial PT pores have been classically studied in hepatocytes (208, 209), it appears that mitochondria of very different cell types possess cyclosporin A-sensitive PT pores: heart muscle cells (238),neurons (211,221,222,239),kidney cells (240),lymphocytes (175), and fibrosarcoid cells (223).This suggests that PT pores are ubiquitous. Moreover, it appears that mitochondrial membrane changes affecting the outer and/or inner membrane occur in all cases of apoptosis investigated thus far (175, 176, 241).

6. Criterion of Vitality The central executioner or the compounds that compose it must have some function(s) that is essential for normal cell survival. Some if not all PT pore components are essential for cellular metabolisms, and this also applies to cytochrorne c. However, it is not clear whether the PT functions in physiological situations not related to apoptosis. According to Brdiczka and co-workers (211), the PT pore might be important for the handling of ATP and metabolic control. This idea is based on the fact that many of the proteins involved in formatiodregulation of the PT pore specifically interact with ATP and ADP: porin, hexokinase, and the ANT. Bernardi and Petronilli (209) propose an alternative physiological role for PT. Short spikes of PT may be involved in the periodic outflow of calcium from the mitochondria1 matrix, and this would be necessary to avoid excessive calcium accumulation in mitochondria. Kinnally and colleagues (212, 242) suggest that the PT pore might be identical with a mitochondrial multiple conductance channel modulated by peptides responsible for targeting mitochondrial precursor proteins. This would imply that the PT pore participates in the import of nuclear gene products into mitochondria. If any one among these hypotheses was confirmed, PT would be essential for normal mitochondrial function.

7. Criterion ($the Switch The central executioner should function as a switch that is either off or on and thus should be endowed with the capacity of self-amplification. Many of the metabolic consequences of PTs themselves trigger PT (Table IX). Two types of self-amplification can be conceived, one at the level of the organelle and the other at the level of the cell. At the level of single disruption, which mitochondrion, the immediate consequence of PT, in turn Favors PT, is likely to lock the organelle, once the is below a critical threshold, in an irreversible stage of damage. It appears that PT

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TABLE IX SELF-AMPLIFYING FEATURES OF MITOCHONDRIAL PERMEABILITY TRANSITION Inducer of PT

AT,,,reduction facilitates PT (587) Calcium causes PT (208, 588) Thiol oxidation of matrix proteins facilitates PT (213) Oxidation of NAD(P)Hz favors PT (213) Reactive oxygen species induce FT (208) Proteases can cause PT (198) AIF induces PT (203)

Consequence of PT PT causes AT,,, disruption (208) PT causes outflow of matrix calcium and ATP depletion, thereby disrupting calcium homeostasis (208) PT causes depletion of nonoxidized glutathione, thereby favoring protein thiol oxidation (208) PT results in NAD(P)H2oxidation (192) PT causes hypergeneration of superoxide anion on the uncoupled respiratory chain (109, 191) PT results in protease activation (19, 179) PT causes AIF release (19)

is accompanied, at the local level, by a loss of mitochondrial matrix glutathione and ADP/ATP (which extrude through the PT pore), two conditions that facilitate PT (208, 243). In addition, local oxidation of mitochondrial compounds, such as cardiolipin (109, 244) and the PT-induced extrusion of cytochrome c (205,245), are likely to interfere with respiratory function, thereby preventing recovery of the mitochondrion that has undergone PT. Several among the consequences of PT affect the whole cell and thus could act on other mitochondria than those undergoing PT via a sort of domino effect. These changes include oxidative changes in the redox potential (107, log), increases in cytosolic free CaZt concentrations (probably due to Ca" influx through the plasma membrane) (107), and caspase activation (203), which all induce PT (208, 243; J. Penninger and G. Kioemer, unpublished data). AIF released from mitochondria undergoing PT can also induce PT (203).Thus, PT initially occurring in some mitochondria of a cell is likely to affect the metabolism in a way that causes PT in all remaining mitochondria. The capacity of self-amplification renders PT an attractive candidate to constitute the (or a) death switch. In conclusion, it appears that mitochondrial PT (or a closely associated mitochondrial process) fulfills most if not all criteria of the central executioner. As a word of caution, it should be mentioned that additional processes including caspase activation (37, 38, 112) have been suggested to form part of the executioner. It is possible that the central executioner involves an intricate interplay between mitochondrial membrane alterations, caspase activation, changes in redox metabolism, and ion fluxes rather than the mitochondrion alone. Be that as it is, it appears plausible that mitochondria exert a decisive role in the effector stage of apoptosis.

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IV. initiation Phase of Apoptosis

The preceding sections of this review dealt with the common pathway of apoptosis. Here, we will describe private pathways; that is, pathways that operate in response to determined signal and that thus are activated in a cell type- and trigger-specific fashion. Whenever possible, we will concentrate on pathways operating in T cells and in their precursors. Cell fate decisions in developing and mature T cells depend on signal transduction via the antigen-specific TCR and additional receptors in a complex interplay. Thus, the same TCR can signal for survival or cell death depending on the affinity for the ligand and second modulatory signals. Here, we will concentrate on signal transduction pathways that link surface receptors to the death effector machinery. In particular, the role of signal transduction via TNF-R superfamily proteins, stress kinases, tyrosine kinase-based receptors, NFKB, PI3'K, and protein kinase B in the decision between apoptosis and survival will be discussed. A. THYMIC T CELLDIFFERENTIATION AND CLONAL SELECTION: GENERAL PRINCIPLES T ceU development within the thymus is a well-defined process during which precursor thymocytes divide, rearrange, and express TCR. Thymocytes undergo two selective processes: positive selection and negative selection. Mechanisms governing positive and negative T cell selection are critically dependent on physical interactions between the antigen-specific TCRs on developing thymocytes and major histocompatibility complex molecules (MHC) expressed on thymic stromal cells (246-248). Recognition of MHC class I molecules by the TCR commits thymocytes to the CD8' lineage, whereas interactions with MHC class I1 molecules determines the generation of CD4+ T lymphocytes (Fig. 8) (249). Recognition of self-MHC molecules expressed on thymic epithelial cells by immature thymocytes and subsequent differentiation into CD4+or CD8' T cells are the basis for positive selection (246-248, 250). Thus, positive selection generates a T cell repertoire that is restricted to self-MHC. Positive selection mechanisms must trigger a survival signal in developing thymocytes, which subsequently migrate from the thymus to commence their life as mature peripheral T cells. Thymocytes expressing TCRs that recognize self-antigens on bone marrow-derived cells with high affinity/avidity are clonally deleted, leading to the removal of T cells that express T cell receptors with potentially harmful self-reactivity (248-251). The outcome of both positive and negative selection events is directed by the specificity of the T cell receptor expressed on the developing thymocyte (Fig. 8) (250). Developing thymo-

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JOSEF M . PENNINGER AND GUIDO KROEMER

Bone Marrow precursers (adult) Fetal liver (embryo)

Thymic cortex

CD4-CDB-

RAG-1IRAG-2 Rearrange TCRa locus

NeProllferatlon TCRap.int

RAG-l/RAG-2 Rearrange TCRa locus

I T cell selection

~

TCRaphbh

TCRaPl9h

Switch off RAG-11RAG-2

Thymic medulla FIG.8. Outline of thymocyte differentiation. Positive, negative, and default selection occur at the CD4+CD8’TCRcr/3’”ghstage of differentiation and depend on the affinity of the TCR for the peptide/MHC ligand and on specific second signals provided by thymic strornal cells. The second signals for positive and negative selection appear to be biochemically distinct, and these signals are mediated by distinct cell populations of the thymic microenvironment. The signals involved in selection and development of the second T lymphocyte lineage expressing y8 TCRs are still elusive and involve the signal transduction via the protein tyrosine kinases ~ 5 6 ’and ‘ ~ SYK and the protein tyrosine phosphatase CD45.

cytes that express a TCR with low affinity for self-peptides in association with MHC molecules die via neglect (nonselection default). It appears that more than 80% of developing thymocytes undergo death by default

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due to expression of a low-affinity TCR (35). Apoptotic cell death thus ensures the survival of only those T cells that express a self-MHC-restricted and self-tolerant TCR. Although default selection and negative selection pathways are fundamentally different, both pathways trigger morphological and biochemical changes typical for programmed cell death, indicative of a common death effector pathway. Thus, thymocytes undergoing apoptosis in response to a variety of different stimuli manifest the key features of the effector stage, including a disruption of the mitochondria1 transmeinbrane potential (A?,,,) (131, 194, 200), and the typical characteristics of the degradation phase, including caspase and endonuclease activation and phosphatidylserine exposure (5, 111). In contrast, a number of regulators of apoptosis have a restricted effect on thymocyte apoptosis, depending on the apoptosis trigger. Thus, transgene-enforced overexpression of bcl-2 prevents glucocorticoid or irradiation-induced thymocyte apoptosis but has little if any effect on negative selection of thyinocytes (252,253). Similarly, knockout of the tumor suppressor gene p53 only affects apoptosis induced by genotoxic stress but fails to interfere with selection-induced apoptosis or glucocorticoid-mediated thymocyte apoptosis (254, 255). In contrast, a member of the TNF-R family, CD30, appears to be necessary for TCW CD3-triggered thymocyte apoptosis but does not influence the induction of cell death by damage pathways (256). These examples illustrate the existence of multiple pathways of apoptosis induction that may or may not be influenced by ~ ~ 5bcl-2, 3 , or members of the TNF-R family. B HOMEOSTASIS OF MATURE T CELLSGENERAL PRINCIPLES Activation of mature T cells induces the switch from high- to lowmolecular-weight CD45 isoforms. The activation-induced transition from CD45RB"g'l'' to CD45RO""Rh' and the transition from CD45RA+ to CD45RO' T cells correlates with a decrease in Bcl-2 expression and IL-2 synthesis, whereas the expression of Fas, the Hodgkin's lymphoma antigen CD30, and the low- and high-molecular-weight TNF-Rp55 and TNF-Rp75 is increased in primed T cells (Fig. 9) (257, 258). In addition, IL-2 and fibroblasts can prevent apoptotic cell death, presumably by enhancing Bcl2 expression or by affecting cycle progression, respectively. These data imply that activation of T cells generates lymphocytes that are prone to apoptosis, unless these cells are rescued by IL-2 or reside in a specialized environment (257-261). Thus, activated lymphocytes undergo programmed cell death by default in the absence of rescue signals mediated by cytokines or cell-cell contacts. This mechanism maintains leukocyte homeostasis in vivo and may explain why memory T cells, which often display a CD45 phenotype similar to primed T cells (CD45ROt), may

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JOSEF M. PENNINGER AND GUIDO K R O E M E R

FIG.9. Proposed correlation between activation and programmed cell death in mature peripheral T cells. Regulated expression of Bcl-2, Fas, CD30, and both TNF receptors confers susceptibility or resistence to apoptosis and survival signals, depending on the state of activation. Besides cell death due to the absence of continuous stimulation by peptides or cytokines, it appears that apoptosis of activated T cells is mainly triggered through ligation of Fas, TNF-Rp55, and TNF-Rp75.

depend on continuous stimulation or the presence of antigen in vivo (257, 258). Antigen-dependent peripheral deletion of peripheral T cells is mainly mediated via the death receptors Fas, TNF-Rp55, and TNF-Rp75, and perhaps additional molecules of the TNF-Rp55 family (262-266). Although in determined circumstances T cell death can be induced by starvation from IL-2, this lymphokine can also facilitate T cell apoptosis. Thus, pretreatment with IL-2 renders T cells susceptible to TCR-induced apoptosis, perhaps by dysregulated activation of the cell cycle (267) or by influencing Fas/Fas-L signaling (268). Based on these data, a scenario emerges in which antigen recognition by naive peripheral T cells triggers IL-2 production, proliferation, and functional differentiation (Fig. 9). Dysregulated activation of the antigen receptor during cell cycle progression may lead to programmed cell death, a mechanism that would maintain clonal tolerance through inactivation of bystander T cells that have been unspecifically activated by IL-2. Only cells that receive signals in the correct

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temporal order and within the proper spatial confinements are allowed to expand and differentiate into effector lymphocytes. After completion of the effector functions, for instance, after clearance of a virus infection, primed T cells undergo programmed cell death to maintain leukocyte homeostasis, with the exception of a few T cells that are rescued by unspecific stimuli such as IL-2 signaling or by the persistance of antigen. This model also implies that activation of self-reactive T cells that are frequently found in mice and humans can only occur in rather exceptional circumstances: in the context of a temporally and/or spatially dysregulated activation of both the TCR and additional signals (259, 260, 269).

SIGNALS TRIGGERED VIA THE TCWCD3 C APOPTOSISREGULATORY RECEPTORCOMPLEX Specific recognition of antigens by T cells is mediated by the TCWCD3 receptor complex and nonspecific accessory molecules such as CD4 and CD8 glycoproteins. Antigen receptor-induced activation of T cells initiates a cascade of signal transduction molecules that results in transcriptional activation or repression of a variety of genes involved in T cell activation and development (Fig. 10) (270-272). These newly transcribed genes can encode for surface receptors, transcnption factors, or cytokines that in concert regulate T cell proliferation and coordinate the immunological response (273). Crucial players for TCWCD3-mediated signal transduction include the Src-family protein tyrosine kinases (PTK) p59'p and p56Ick, which physically interact with the TCWCD3 complex and CD4CD8 accessory molecules (271,274-276); the cytoplasmatic PTK ZAP70, which associates with the phosphorylated CD35 chain via SH2 domains (277); the guanine nucleotide exchange factor p9Sav(278, 279); the ring finger containing protooncogene ~ 1 2 0 '(280); ~ ' p50'jk,which negatively regulates the catalytic function of Src-family kinases (281);protein tyrosine phosphatases (PTPase) such as CD45 (271); and probably other molecules (270, 272, 273). After an initial wave of tyrosine phosphorylation and tyrosine dephosphorylation events, phospholipase C-yl (PLC-yl)is activated and mediates hydrolysis of phosphatidyl inositol into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (InsP3) (Fig. 11). DAG leads to activation of protein kinase C (PKC) and InsP3 mediates an increase in the concentration of cytoplasmically free Ca2+through binding and opening of InsP3-regulated receptors in the endoplasmatic reticulum (282). Besides PKC and PLCy l , avariety of other signal transducing molecules, such the protooncogenes p21rd7and p74' rd, mitogen-activated serinekhreonine kinases (MAPK), or the stress-activated protein serinelthreonine kinases (SAPK, also termed Jun-N-terminal kinases or JNKs) are activated (Fig. 10) (270-273, 283,

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FIG.10. Schematic model of antigen receptor-mediated signal transduction. Other signaling molecules that are phosphorylated on tyrosine residues after TCR activation are CD3l, ZAP70, SPL76, SLAPl30, the molecular adapter p120"c"', Ras-CAP, the regulatory p85 subunit of the P13' kinase, or the cyoskeletal proteins ezrin, talin, and vinculin (not shown).

284). Antigen receptor-induced signal transduction is organized in cascades and ultimately results in the activation of transcription factors including c-fos, c-jun, or NF-AT (273). TCFUCD3 signaling is intrinsically complex because its final outcome is influenced by multiple parameters: concentration, affinity, and avidity of the ligand; chronology of receptor ligation; activation stage; differentiation stage; cell type; and simultanous ligation of other receptors. In addttion, many of the consequences of TCWCDS signaling will affect the expression level of other receptors (e.g., Fas/Apo-l/CD95 and IL-2Ra) or ligands (e.g., Fas-L and IL-2), thereby having indirect effects on the cell's fate via cross talk with additional receptors and signal transduction cascades.

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FIG.11. Model for antigen receptor-indiiced activation of PLC-yl and PKC. Stimulation of TCWCD3 molecules and accessory receptors triggers the rapid activation of tyrosine kinases, in particular p56''k and p59'". which then leads to tyrosine phosphorylation and subsequent activation of PLC-yl. Interactions between PLC-yl and PTKs are mediated via SH2 domains present in PLC-yl. Active PLC-yl translocates to the inetnhwne and hydrolysis inembrane-hound phosphatidyl inositol (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (InsP3). InsP3 triggers the release of Ca" stored in the endoplasmatic reticnlum through activation of' InsP.3-sensitive receptors { InsP3-H). Increased oytoplasmaticillyfree Ci2+probably mediates phosphorylation of InsP3 to generate inositol 1,3,4,S-tetraphosphate (InsP4), which triggers the opening of Ca2+channels located at the p h n a metnhrane. Elevated Ca2+levels in concert with DAG activate the serine/threonine kinase PKC, wliich translocates to the membrane. To coinpensate for the increase of cytosolic Ca", K' channels are opened that pump K t from the cytoplasm into the extracellular spaces. Speculatively, this might explain the fact that K' channel blockers such as the hormone somatostatin can inhibit T cell activation and proliferation.

1. TCR Signtiling and Thyinocyte Selection: The Impact of lntmcellular Ca2+and InsP3 Receptors TCR-dependent interactions between immature thymocytes and thymic stromal cells presenting peptide antigens or superantigens trigger an activation signal that results in functional changes of the developing T cell and expression of activation markers (271,285-287). Antigen-specific activation of thymocytes can mediate signals that lead to either positive T cell selection

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or activation-induced programmed cell death (261, 288, 289). In vitro and in vivo cross-linking of the TCWCD3 complex expressed on immature CD4'CDS' double-positive thymocytes can induce TCR-mediated signal transduction and increase the concentration of cytoplasmatically free Ca2' (Fig. 11) (285, 286). In addition, the catalytic activity of two Srcrelated PTKs, ~ 5 6 ' and ' ~ p59'y", is increased in transgenic TCRaP T cells undergoing positive selection (290), and activation of thymocytes can be blocked with PTK inhibitors (291). Thus, similar to mature T cells, antigen receptor-mediated signaling in developing thymocytes is associated with Ca2+mobilization and the activation of tyrosine kinases (270, 271, 272). Whereas antigen receptor stimulation preferentially leads to proliferation of peripheral, mature T lymphocytes, activation of the TCWCD3 signaling cascade in immature thymocytes leads to programmed cell death by "poptosis (292). Thymocytes undergoing negative selection and programmed cell death have been shown to contain elevated levels of Ca2+ in the cytoplasm (139,291). Moreover, increased Ca" levels correlate with negative selection, and antigen receptor-triggered clonal deletion of thymocytes can be inhibited using CaZt-chelating agents (293). Interestingly, cyclosporin A, which blocks activation-induced cell death (294) by inliibition of the Ca2+-dependentphosphatase calcineurin (295), does not block negative selection of developing thymocytes (296)but rather blocks positive T cell selection (297). Increased expression of the type 3 inositol 1,3,4-triphosphate receptor ( InsP3R3), an InsP3-gated Ca2+-release channel in the endoplasmatic reticulum (Fig. 11) (282), has been linked to antigen receptor and glucocorticoid-induced apoptosis in T and B lymphocytes (298). Antigen receptor-mediated activation induces physical interaction between the TCR-associated protein tyrosine kinase ~ 5 9 and ~ ' InsP3 receptors in T cells, and InsP3R can be tyrosine phosphorylated and activated by p5gfP (299). Moreover, it has recently been shown that type 1 inositol 1,3,4triphosphate receptor-deficient Jurkat cell lines are protected from antiTCWCD3, anti-Fas, and dexamethasone-induced apoptosis and that induction of apoptosis in Jurkat cells is dependent on opening of intracellular InsP3R-gated Ca2+channels (300). This suggests the possibility that increases in cytosolic Ca2+levels secondary to the action of InsP3R-gated Ca" channels may be a major signal transduction pathway contributing to the induction of apoptosis. Obviously, Ca2+is a pleiotropic second messenger, and the link between increases of cytosolic CaZt concentrations and apoptosis may be complex. In a number of experiinental systems of cell death, Ca2+elevation is necessary (and sometimes sufficient) to induce opening of the mitochondrial megachannel

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(220, 221, 239, 301). Indeed, Ca2+has been known for a long time to induce or to fkilitate mitochondrial PT in isolated mitochondria (208, 209). Thus, Ca" may provide a direct link between receptor triggering and activation of the mitochondrial executioner. Whether this also applies to T cells remains to be elucidated.

2. Protein Tyrosine Kinases: p56lCkand p59fy" Elevation of Cap+levels after TCR ligation in mature T cells depends on upstream events including the activation of PTKs (282, 299). The Srcfamily protein tyrosine kinases ~ 5 6 'and ' ~ p59fp are associated with the cell membrane through a myristoylated glycine residue and mediate very early events in antigen receptor-induced signal transduction because the transmembrane TCWCD3 complex does not possess any intrinsic tyrosine kinase activity (270-272, 274-276). The PTK ~ 5 6 ' interacts '~ noncovalently with cysteine residues in the cytoplasmic region of both CD4 and CD8 molecules (274-276), whereas p59fv"can directly associate with the TCW CD3 complex (302). Negative selection of thymocytes expressing an MHC class II-restricted TCR is not prevented by PTK inhibitors (291),indicating that PTK-mediated signal transduction may not be required for the induction of thymocyte apoptosis. However, PTK inhibitors can block programmed cell death in T cell hybridomas (303), and activation-induced cell death of proliferating peripheral T cells correlates with alterations of tyrosine phosphorylation (304). In addition, cross-linkingof CD4 molecules using anti-CD4 antibodies (305) or HIV gpl20 protein (306) renders mature T cells susceptible to apoptosis. Moreover, transgenic overexpression of CD4 molecules impairs positive and negative T cell selection of CD8' thymocytes expressing the transgenic H-Y TCR presumably by sequestrating ~ 5 6 ' (307). '~ Because CD4 molecules are noncovalently associated with the Src-family kinase ~ 5 6 ' "which ~ is critically involved in TCR-mediated signal transduction (274-276, 308), it appears possible that ~ 5 6 ' 'and ~ similar kinases may be involved in activation-induced cell death. Recently, it has been demonstated in ~ 5 6 ' " (308, ~ - 309) or p59fy" (310, 311)-deficient mice that these PTKs relay TCWCD3-mediated signals to the apoptotic machinery. In a model of superantigen-driven cell death, ~ 5 6 ' is ' ~not required for the deletion of CD4' cells but influences the death of CD8' T cells (312). Because superantigens are presented by MHC class I1 molecules and thus are more efficient stimulators of CD4' than CD8' thymocytes due to differences in affinity, ~ 5 6 appears "~ to be an important signal transduction molecule involved in the deletion of superantigen-reactive CD8' T cells and perhaps in CD4' thymocytes expressing a low-affinity TCR on the cell surface. CD4' thymocytes expressing TCRs with higher affinity for ligand can, however, undergo nega-

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tive selection in the absence of ~ 5 6 ’(312). ‘ ~ This interpretation is corroborated by results in CD4-’- mice (313)and CD4-’-CD8-’- double-knockout mice (314) in which cloiial deletion occurs in thymocytes expressing TCRVP chains with high affinity for superantigens but not in thyinocytes expressing low-affinity TCRVP chains. Altogether, these data are consistent with a model in which quantitative differences in the TCWligand interaction and/or coreceptor signaling determine the requirement for ~ 5 6 during “ ~ positive or negative selection events. Besides the Src kinases ~ 5 6 ’ ‘and ~ p59fy”, it has recently been demonstrated that the CD3c-associated protein tyrosine kiiiase ZAP70 (315),and the protooncogene product p95”“ ( J. Penninger, unpublished data), an SH2 and SH3 domain-containing guanine nucleotide exchange factor for the small Ras-family proteins Racl, CDC42, and RhoA (316), may have a direct role in TCR-mediated apoptosis in thymocytes. Thus, a number of different tyrosine kinases are essential signal transduction modules linking the TCR to downstream effectors of the apoptotic cascade. Currently, it appears unlikely, however, that p56ILkand p59fy” are direct activators of the central executioner. Thus, addition of these molecules to a cell-free system of apoptosis fails to trigger the apoptosis machinery (317),whereas the addition of another molecule possessing a Src homology 2 domain, Crk, can accelerate apoptosis in such a system (318).

3. The CD45 Protein Tymsirie Phosphatase in the Regulation of T Cell Apoptosis Signal transduction by the antigen receptor in T cells depends on the balance between protein tyrosine kinases and protein tyrosine phosphatases (PTPases) (271). Antigen receptor-induced signal transduction in both T and B lymphocytes requires expression of the transmembrane receptor PTPase CD45 (319-322). The intracellular CD45 PTPase domain dephosphorylates negative regulatory tyrosine residues of ~ 5 6 and “ ~ p59‘”’ (but not of ~6,”“)(271, 322). CD45 is expressed on the cell surface of all nucleated hematopoietic cells and their precursors. CD45 is expressed in various isoforms with a molecular weight ranging from 180 to 235 kDa. These isoforms arise from alternative splicing of variable exons (exons 4-7) that encode sequences at the amino-terminal domain. Almost all sites for N- and 0-linked glycosylation are located in a serinehhreonine-rich region corresponding to exons 3-8 of the CD45 molecule. Changes in the expression of the variable exons modify the molecular architecture of CD45, as well as the amount of negatively charged sugar residues of the extracellular domain. Expression of distinct CD45 isoforms also correlates with different hematopoietic cell lineages and depends on the state of differentiation (271, 322). Thus, all B lymphocytes express the high-molecular-weight

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isoform of 220 kDa (also termed B220), which includes all CD45 exons. By contrast, immature CD4+CD8' thymocytes mainly express low-inolecularweight CD45 isoforms, whereas mature CD4' or CD8+ thymocytes and peripheral CD4' or CD8+T cells can express multiple isoforms (323-326). Expression of different CD45 isoforms changes during T cell activation. For instance, naive T cells switch from high-niolecular-weight to lowmolecular-weight CD45 isoforins upon stimulation (327,328). Beside exon switching, CD45 glycosylation depends on the cell type. T and B cells have different patterns of CD45 glycosylation due to differences in enzymatic glycosyltransferase activities within the Golgi apparatus (271, 322). Although the biological function of CD45 isoforms might be different, TCRmediated signal transduction can be restored in CD45-defective cell lines with chimeric molecules containing only the intracellular CD45 PTPase domain (329, 330). Thus, it appears that expression of cytoplasmic CD45 PTPase domain alone is necessary and sufficient for TCWCD3-mediated signal transduction to occur in uitro. Similar to the activation event in peripheral T cells, alternative splicing of CD45 exons changes upon receptor ligation in thymocytes undergoing positive and negative selection (325).Immature CD4'CD8+ thymocytes express lower-molecular-weight CD45 isoforms, whereas mature CD4t or CD8+ T cells express higher-inolecular-weightCD45 isoforms due to exon switching (325,331).Alterations in CD45 isoform expression could change the enzymatic PTPase activityvia interactions with distinct ligands or association with different coreceptors on the cell surface (271,322).Alternatively, different CD45 exon switching may be required for T cell migration from the thymus and homing to distinct peripheral organs (309) or could simply correlate with the generation of mature unprimed T cells (271,322). Mice lacking the alternatively spliced CD45 exon 6 (321) or the CD45 exon 9 (332) exhibit a block in T cell development at a late stage of development, namely, at the transition of immature CD4+CD8' doublepositive to mature CD4' or CD8' single-positive thymocytes (321, 332). Accordingly, it appears that CD45 can participate in the control of positive selection of thymocytes expressing transgenic TCRaP heterodimers (309, 333).Moreover, the PTPase CD45 is crucial for the induction of immature thymocyte apoptosis by superantigen (309, 312, 321), anti-CD3 crosslinking (332), and antigenic peptides ( J. Penninger, unpublished data). Transgenic overexpression of the low-molecular-weight CD45 isoform, CD45R0, in mice can augment antigen receptor-mediated cell death and negative selection of thymocytes expressing the autoreactive H-Y antigenspecific ap TCR (334). Interestingly, the endogenous lectin, galectin-1, which is produced by thymic epithelial cells (335), can induce apoptosis of developing thymocytes and activated human T cells (336). Because

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galectin-l-induced apoptosis depends on the expression of the lowmolecular-weight CD45RO isoform on T cells (336),differential expression of CD45 isoforms may determine the interaction with receptors expressed on cells of the thymic microenvironment, thereby influencing the fate of thymocytes. Because CD45 isoforms can regulate the function of adhesion receptors on thymocytes (337) and peripheral lymphocytes (338),it is possible that different CD45 isoforms mediate physical interactions of thymocytes with different thymic microenvironments. Deregulated positioning of thymocytes may trigger cell death due to temporally or spatially “wrong” interactions with thymic stromal cells. Apoptosis as a control mechanism for cell positioning has been recently termed “anoikis” (homelessness) and appears to be controlled by integrin receptors (339). Thus, activation of integrin receptors (VLA-5) with fibronectin can induce apoptosis in hematopoietic cells (340) and VLA-4 and VLA-5 expression is differentially regulated on developing thymocytes (341). CD45 can control the activity of integrin receptors (337) and different CD45 isoforms preferentially associate with the integrin receptor LFA-1(342). These findings suggest another possibility of how CD45 may influence life and death in the thymus. A wrong CD45 isoform is expressed on abnormal T cells accumulating in mice homozygous for the MRL-lpr or gld mutations, namely, mutation of the Fas surface receptor (343) or the Fas ligand (344), respectively. Such mice develop severe lymphadenopathy and systemic autoimmune disease (345, 346). The lymphadenopathy is caused by the accumulation of TCRaPtCD4-CD8-HSA+ T cells expressing B220, i.e., a glycosylated isoform of CD45 normally expressed on the surface of B cells. Similar to systemic autoimmunity in Iprlgld mice, “disproportional” expression of certain CD45 isoforms on T cell subsets has been implicated in the pathogenesis of various autoimmune diseases such as diabetes (347) or experimental allergic encephalomyelitis (348). It is conceivable that signal transduction by different CD45 isoforms may be directly or indirectly involved in the regulation of apoptotic cell death or induction of autoimmunity. To directly test whether CD45 exon switching and expression of different CD45 isoforms are required for T cell development and thymocyte selection, CD45-’- mice have been reconstituted with transgenes encoding the low- or the high-molecular-weight isoforms of CD45, CD45R0, and CD45ABC, respectively (334,349). Both CD45 isoforms can restore maturation of TCRaPt thymocytes, indicating that the CD45 PTPase activity alone is sufficient to generate a positive selecting signal, in accord with the fact that the extracellular CD45 domain is dispensable for antigen receptor-mediated signal transduction in cell lines (329,330). Interestingly, the main signal transduction defect in CD45-’- thymocytes is inappropriate

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activation of PLC-71 and opening of Ca2+channels, implying that Ca2+fluxes might relay TCWCD3-triggered PTK and PTPase signaling to the apoptotic rnachinery during thymocyte selection ( J. Penninger, unpublished data). D STRESSKINASES During the development of all inulticellular organisms, cell Fate decisions determine whether cells undergo proliferation and differentiation or apoptosis. Distinct and evolutionarily conserved signal transduction cascades mediate survival or death in response to developmental programs and environmental triggers. Multiple stimuli for differentiation and cell growth activate the MAPK, aIso known as the extracellular signal-regulated kinases ERKl and ERK2 (350-353), which translocate to the nucleus and regulate the activity of transcription factors (354). MAPKs are activated by the phosphorylation of a threonine and a tyrosine residue by the dual-specificity MAPK kinases MEKl and MEK2, which relay Ras and Raf signal transduction to MAPKs (Fig. 12) (355-358). A parallel signaling cascade leads to the activation of SAPWJNK (Fig. 12) (357,359).SAPKs/JNKs are activated in response to a variety of cellular stresses such as changes in osmolarity and metabolism, DNA damage, heat shock, ischemia, shear stress, inflammatory cytokines such as TNF and IL1,or ceraniide (360-368). When activated, SAPKs/JNKs phosphorylate cJun, thereby activating the transcriptional complex AP-1 (369). SAPKs/ JNKs are activated by the phosphorylation of tyrosine and threonine residues in a reaction that is catalyzed by the dual-specificity kinase SEKl (also known as MKK4 and JNKK) (359,370-372). SEKl/MKK4 transmits signals from upstream activators such as Rac-1, CDC42, PAK65, MEKK1, ASK1, HPK1, or MLK3, to SAPK activation (Fig. 12) (360, 361, 371, 373-377). Although SEKl is structurally related to MEKl and MEKB, MEKs do not activate SAPKsIJNKs and, conversely, SEKl does not activate ERKs. This implies that parallel and independent signaling cascades exist for MAPK and SAPWJNK activation (378,379),thus introducing a dichotomy into hnase cascades. 1. Distinct Stress Kinase Pathways .for D i f f rent Types of Cellular Stress Genetic data in SEK1-deficient embryonic stein (ES) cell clones (380) and results using chromatographically fractionated extracts (381,382) indicate that SEK1-dependent and SEK1-independent intracellular signaling pathways for SAPWJNK activation exist, and that different types of stress trigger distinct signaling pathways for SAPWJNK activation. Thus, SEKl is the critical activator of SAPKdJNKs in response to the protein synthesis inhibitor anisomycin and heat shock, whereas SAPK activation in response

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MAPK

SAPK

hwosina klnasa Receptor

p38/HOG

1 Iachernlaraperfuslon,Heat shock, Irrrdlatlon, UV, hyparosrnolarlty

GrbUSOS Pyla

Vav

\r Racl, CDC42 HPKl

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Tpl-2

MEKKl

MLK3

SEKllMKK4 SEWKK7

ASK1

?

MKK3 MKK6

+

ERK2

p9ORsk GSK-3

Fos

Elk-1

c-Jun

ATF2

Mef

kinase2

FIG. 12. Signal transduction pathways for MAPK, SAPK, and p38/HOG induction. Various cross talks exists between these pathways, and distinct pathways have multiple upstream regulators. For example, Ras can activate Racl/CDC42via PI3’ kinase (not shown). Theoretically, distinct upstream activators might relay signals from different stresses or receptors. This is, however, complicated by the fact that, for example, MLK3 can activate SAPK and p38/HOG, whereas HPKl is a direct activator of MLK3, but HPKl can only activate SAPK. Thus, by analogy with yeast signaling in reponse to osmolarity changes, a hypothetical mammalian scaffolding protein has been proposed to spatially confine and channel similar signal transduction pathways to distinct downstream effectors such as SAPK or p3WHOG. The downstream molecules that relay Pyk2 signaling to MAPK and SAPK activation are not known. Only few downstream molecules are known to be substrates of MAPK, SAPK, and/or p38/HOG. A forth parallel signaling pathway that involves MKK5 + ERK5 has been omitted from the diagram. HPK1, hematopoietic protein kinase1; GCK, germinal center kinase; ASK1, apoptosis-inducing kinase-1; MLK3, mixed-lineage kinase-3: SOS, son of sevenless; PAK, p21-activated kinase.

to osmolarity changes, UV irradiation, y-irradiation, and cerarnide is mediated via a novel dual-specificity kinase termed SEKYMKK’I (Fig. 13) (380). The second activator of SAPKdJNKs operates independently from SEKl and defines a distinct signal transduction pathway. The substrates of SEKUMMK7 are likely to overlap with those of SEKl because overex-

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Oarnotic shock

+

v

?

MEKKl

3

i

SEKllMKK4 I-kB

NF-kB SAPK/JNKcr,p,y

t

Survival

I Survival

FIG.13. Two distinct signaling pathways relay different types of stress to SAPK activation. The liypothetieal point ofbifiircation, i.e., MEKK1, between the SAPK and NF-KBsignaling pathways, is indicated. For details see text. Although SAPK activation has been extensively implicated in the induction of apoptosis, recent genetic data indicate that SAPK activation could protect from premature T cell death during development and after activation.

pression of dominant inhibitory SEKl in COS cells can inhibit the SEKW MKK7-dependent activation of SAPWJNK in response to anisomycin, sorbitol, and UV irradiation (365). Interestingly, in transfection studies using COS cells, SAPWJNK responses to anisomycin are not affected by dominant-negative inhibitors of Racl or CDC42 (373-375). Moreover, UV irradiation and sorbitol-mediated osmolarity changes, but not anisomycin, trigger tyrosine phosphorylation of Pyk2 (also known as related adhesion focal tyrosine kinase or CAP-P), a tyrosine hnase that has been linked to SAPWJNK signaling in PC-12 cells (383). Thus, it appears that Racl, CDC42, and Pyk2 are involved in SEK1-independent SAPWJNK activation in response to sorbitol and UV irradiation but not anisomycin, adding to the multiplicity of stress kinase activating pathways. Besides activation of SAPWJNK signaling cascades in response to many types of cellular stress, SAPWJNK activity is induced in response to growth factors, heterotriineric G-proteins, phorbol esters, or costimulatory activation of T lymphocytes (284, 359-361, 384-386). Moreover, activation of SAPWJNKs leads to phosphorylation of c-Jun and activation of JudFos heterodimeric AP-1 transcriptional complexes, generally believed to be positive regulators of transcription (3Fj7, 369, 370, 372, 387). In T Iymphocytes, ligation of the TCR results in rapid activation of the Ras 4 Kaf + MEK -+ MAPK -+ Fos signaling cascade (Fig. 10) (358, 378,388). How-

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ever, activation of MAPK cascade is not sufficient for effective IL-2 production and proliferation, and T cells require a second costimulatory signal (389). Recently, it has been shown that coordinate stimulation of the TCR/ CD3 complex and the costimulatory receptor CD28 correlates with the activation of SAPKs/JNKs,phosphorylation of c-Jun, and AP1 activity (284). Activation via the TClUCD3 complex and CD28 costimulation can be mimicked using phorbol esters (PMA) and Ca2’- ionophores, respectively (390), and the simultaneous treatment with PMA and Ca2+-ionophore leads to SAPWJNK activation in T lymphocytes (378). These biochemical data imply that T cells utilize two distinct signaling cascades for antigen specific activation, a TCR-triggered Ras -+ Raf + MAPK + Fos cascade and aTClUCD28-induced SEKl -+ SAPWJNK+ c-Jun cascade. Recently, it has been demonstrated that the guanosine nucleotide exchange factor ~ 9 5 ” (316) ~ ’ and/or the TClUCD3-activated kinase Pyk2 (383, 391) might link TClUCD3 signaling to SAPK activation. Failure to activate SAPW JNKs in T cells might result in clonal anergy and induction of immunological tolerance (392-394). To determine the role of SEKl/MKK4 in SAPKs/JNKs activation in response to CD28 costimulation (284) and CD40 signaling (395), SEKl-’-RAG-’- chimeric mice have been generated (380). S E K P RAG2-’- chimeric mice exhibit a partial block in B cell maturation, whereas their peripheral B cells display normal responses to IL-4, IgM, and CD40 cross-linking. However, IL-2 production and proliferation are impaired in SEKl-’- T cells in response to suboptimal concentrations of CD28 costimulation and PMA/Ca2+ionophore activation, indicating that the stress signaling kinase SEKl is a downstream effector involved in TCW CD3 and/or CD28 auxiliary receptor signaling. The impairment of T cell growth and IL-2 production is not complete in response to CD28 costimulation and PMA/Ca2+ ionophore treatment, and strong activation via the TClUCD3 complex done leads to normal proliferation of SEKl-’- T cells. Whereas CD28 is absolutely crucial to generate vesicular stomatitis virus (VSV)-specific germinal centers, SEKl-’-RAG2-’- chimeras can mount a protective antiviral B cell response, exhibit normal IgG class switching, and make germinal centers in response to VSV. Interestingly, PMA/Ca2+ ionophore stimulation, which mimics TClUCD3 and CD28-mediated signal transduction, triggers SAPWJNK activation in peripheral T cells, but not in thymocytes, from S E K P mice (380). These data provided the first genetic evidence that SEKl is an important effector molecule that links CD28 signaling to IL-2 production and T cell proliferation. Most important, these results also show that signaling pathways for SAPK activation are developmentally regulated in T cells. Thymocytes use the SEKl/MKK4

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signaling pathway and peripheral T cells utilize SEKUMKK7 for SAPK activation in response to the same stimuli (380). 2. Stress Kinases: Universal or Restricted Inducers of Apoptosis? It has been proposed that SAPWJNK activation triggers apoptosis in response to many types of stress, including UV and y-irradiation, protein synthesis inhibitors (anisomycin),high osmolarity, toxins, ischemidreperfusion injury in heart attacks, heat shock, anticancer drugs (cisplatinum, adriamycin, or etoposide), ceramide, peroxide, or inflammatory cytokines such as TNF-a (363, 365, 367, 368, 396, 397). Moreover, nerve growth factor (NGF) deprivation in PC12 pheochromocytoma cells leads to sustained SAPUJNK activation and the induction of apoptosis (398). The overexpression of dominant-negative SEKlIMKK4 can block the induction of cell death by heat shock, irradiation, anticancer drugs (cisplatinum, adriamycin, and etoposide), peroxide, ceramide, or cytokine deprivation (364, 365, 397, 398). In addition, overexpression of inactive c-Jun or dominant-negative MEKKl was found to inhibit the induction of cell death by irradiation, ceramide, or heat shock in U937 and BAE cells (364) and to protect PC12 cells from apoptosis after NGF withdrawal (398). Overexpression of the novel SAPK-activating MAPKKK homolog apoptosis signal-regulating kinase-1 (ASK-1) can mediate apoptosis in mink lung epithelial cells (MvlLu cells), human 293 embryonal kidney cells, A673 rhabdomyosarcoma cells, KB epidermal carcinoma cells, and Jurkat T cells (399).ASK-1 is a familymemberofthe MAPKKKs Raf-1, Ksr-1, Tpl-2, Tak1, and MEKK1, and ASK-1 mediates SEKUMKK4 and SAPK activation in response to TNF-(r (Fig. 12). SAPK activation has also been linked to induction of ICE/CED-3-like protease and apoptosis in response to the DNA-damaging anticancer drugs etoposide (VP-16)or camptothecin (400). In addition to its importance in stress-induced cell death, SAPWJNK activation correlates with Fas-mediated apoptosis in human T lymphocytes (401, 402). The previous results might suggest that the ASK-UMEKKl +. SEKl +. SAPWJNK + c-Jun signaling cascade is a common pathway required for the induction of apoptosis. This possibility, however, is invalidated by experiments involving cell Iines or animals in which the SEKl gene has been inactivated. Apoptosis does occur in S E K P ES cells, SEK1-’thymocytes, and S E K P splenic T cells in response to anisomycin, serum depletion, UV and y-irradiation, sorbitol-mediated changes in osmolarity, heat shock, anticancer drugs (etoposide, adriamycin, and cisplatinum), CD3/CD28 ligation, and PMA/Ca2+ ionophore with similar kinetics and at similar doses as in SEKl+’+and SEKl+’- cells (380). In the absence of SEK1, anisomycin, heat shock, and PMA/Ca2+ ionophore treatment of

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thymocytes do not induce any detectable SAPWJNK activity, as would be expected. Similarly,in other models of apoptosis, cell death can be observed in the absence of SAPK activation; this applies to death induced via TNFRp55 (403, 404) or Fas (405). These results collectively invalidate the hypothesis that SEK1-mediated activation of SAPWJNK is required for the induction of cell death in response to all apoptosis inducers. Rather, this pathway must operate in a signal-specific (and perhaps cell typedependent) fashion. The question that remains to be examined is whether stress kinases may be involved in negative selection of thymocytes. It has been shown that overexpression of dominant-negative MEKl can inhibit the differentiation of CD4-CD8- double-negative (DN) thymocyte precursor cells to immature CD4+CD8+double-positive (DP) thymocytes (406), whereas the expression of activated Ras suffices to promote the differentiation of CD4-CD8- DN precursor cells to CD4+CD8+DP thymocytes in RAGBcomplementation assays (407). In contrast, it has been shown that positive thymocyte selection and maturation of immature DP thymocytes to mature CD4+ or CD8+ single-positive (SP) thymocytes is impaired in mice transgenic for dominant-negative Ras, Raf-1, and/or MEKl (408-411). However, negative selection via TCWCD3-mediated apoptosis is independent of Ras and MEKl(408-410), implying that signals for positive and negative selection are biochemically different (256). Because SEKl and SAPKs/ JNKs had been implicated as a common pathway required for the induction of apoptosis (363,365,367,368,396,397), the SAPWJNK signaling cascade was considered a prime candidate for the induction of cell death in thymocytes. What is the effect of a SEKl deficiency on thymocyte differentiation? SEK1-deficient RAG-’- chimeric mice have normal numbers and ratios of CD4’ and CD8+ T cells in lymph nodes and spleen (380). However, the thymi of S E K P chimeric mice were four to five times smaller than those of age-matched 129/J mice or SEK1’” chimeras. This reduction in thymus size was due to a significant decrease in the population of DP thymocytes and a relative (but not absolute) expansion of mature SP thymocytes. Moreover, the total and relative numbers of DN thymocytes were not increased in SEK1-I- mice, indicating that SEKl was not required for the progression of DN precursor cells to DP thymocytes. Surprisingly, SEK1-I- thymocytes and peripheral T cells were more susceptible to apoptosis in response to the physiological stimuli CD3/TCR and Fas (380). These data show that SEKl actually protects T cells from Fas- and C D 3 d TCRap-mediated cell death. Thus, the SEK-1 + SAPWJNK pathway appears dispensable for negative T cell selection.

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How is it possible that the S E K - 1 4 SAPUJNK cascade actually reduces thymocyte susceptibility to apoptosis induction? In the context of this question, it may be useful to recall that TNF-a, ionizing radation, or UV irradation do not only induce SAPK but also trigger the activation and nuclear translocation of NF-KB, a pleiotropic transcription factor that is important for lymphocyte responses to antigens and cytokines (412). Interestingly, the stress kinase activating MAPKKK MEKKl has been identified as a key regulator of NF-KBactivation through site-specificphosphorylation of the NF-KB inhibitor, I - K B ~which , binds and retains NF-KB in the cytosol (Fig. 13) (413). Thus, MEKKl is a critical component of both the SAPK and NF-KB stress response pathways. Moreover, NF-KB has been identified as a crucial survival factor and can protect human and mouse fibroblasts and T lymphocytes from apoptosis in response to ionizing radiation, the anticancer drug daunorubicin, and TNF-a, but not Fas, activation (414-416). Because TNF-a-mediated apoptosis is enhanced in many cell lines by drugs that inhibit protein synthesis, these results indicate that TNF-a mediates two signaling pathways, one that induces cell death and another that leads to the NF-KB-dependent transactivation of cytoprotective genes. It is conceivable that, in an analogous way, SEKl deficiency might entail the induction of antiapoptotic genes, which would account for the partial protection from anti-CD3- and anti-Fas-induced apoptosis observed in SEKl-’- thymocytes (380).

E CROSSTALKBETWEEN SIGNAL TRANSDUCTION MODULESA N D MOLECULES OF THE Bcl-2 COMPLEX

Bcl-2 is known to belong to a growing fiainily of apoptosis-regulatory gene products that may either be death antagonists (Bcl-2, Bcl-XL,Bcl-w, Bfl-1, Brag-1, Mcl-1, and A l ) or death agonists (Bax, Bak, Bcl-Xs, Bad, Bid, Bik, and Hrk) and that regulate the effector stage of apoptosis (4, 188, 189, 417). Knockout studies have revealed that Bcl-2-like apoptosisinhibitory proteins exert essential cytoprotective functions; their deficiency entails the ablation of determined cell types, for instance, that of lymphoid cells in Bcl-2-/- (418, 419) and that of postmitotic neurons in Bc1-X-l- mice (420). Transgene-mediated hyperexpression of Bcl-2 can protect lymphocytes against physiological and pathological insults in vivo (189), whereas overexpression of Bax favors lymphocyte apoptosis (126). The ratio of death antagonists (Bcl-2, Bcl-XIJ,Bcl-w, Bfl-1, Brag-1, Mcl1,arid A l ) to agonists (Bax, Bak, Bcl-Xs,Bad, Bid, Bik, and Hrk) determines whether a cell will respond to an apoptotic signal. This deatulife rheostat is mediated, at least in part, by cornpetetive dimerization between selective pairs of antagonists and agonists (417, 421).

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Activation of various growth factor receptors, antigen receptor stimulation in T and B lymphocytes, CD28-mediated costimulation in T cells (422, 423), or sensing of DNA damage via p53 (424-427) can modulate the expression levels of Bcl-2 family protooncogenes (189). However, the relative expression level of Bcl-2-related death agonists and antagonists is not the sole parameter to determine the resistance or susceptibility to apoptosis. Thus, in addition to changing the expression level of Bcl-2related proteins, signal transduction can influence the composition of the Bcl-2 complex and/or induce posttranslational modifications of death agonists and death antagonists of the Bcl-2 family. The homo- and heterodimers of Bcl-2 homologs mainly localize to the outer mitochondrial membrane, in particular to regions of inner-outer membrane contact sites, and face the cytosol, where they constitute targets of cytosolic effectors including signal transduction molecules. One of the main signal transduction molecules involved in growth factor receptor- and antigen receptor-mediated cell proliferation is the small GTP-binding molecule p21R"(428,429). P2lRaS is active in its GTP-bound form and GTP-Ras can directly bind to the serinekhreonine kinase Raf1. The growth-promoting activity of Ras is primarily due to Raf-1 + MEK1,2 + MAPK activation (354,430).Although expression of activated v-Ha-Ras and v-Ki-Ras can trigger apoptosis in fibroblasts and Jurkat T cells and although Ras-induced cell death can be prevented by overexpression of the death-protective protooncogene product Bcl-2 (431), it is generally assumed that the Ras protooncogene product and its downstream target Raf-1 mediate signals that protect cells from apoptosis (432, 433). p21R" (431), Raf-1 (434), and the Ras-related molecule R-ras p23 (435) coimmunoprecipitate with Bcl-2. Recent data imply that this interaction reflects a cross talk between the Ras/Raf-1 pathway and Bcl-2-like molecules (Fig. 14). Bcl-2 can target the Raf-1 kinase to the mitochondrial membrane (224) and mitochondria-associated, but not plasma membrane-bound, Raf1 can stimulate the phosphorylation of the death-promoting Bcl-2 family protein Bad (436), thereby favoring its distribution from the mitochondrial membrane to the cytosol. Growth factor (IL-3) receptor occupancy stimulates the phosphorylation of Bad on a serine residue, thereby favoring the sequestration of Bad in the cytoplasm through binding to the 14-3-3 molecule (which binds phosphorylated Bad but not nonphosphorylated Bad). (225). In contrast, growth factor withdrawal leads to dephosphorylation of Bad, thereby causing its release from 14-3-3 and subsequent translocation of Bad to the mitochondrial membrane where Bad interacts with Bcl-2 family members and ultimately triggers cell death (225). Another signal transduction pathway that alters the composition of the Bcl-2 complex influences the subcellular distribution of the Bcl-2 binding

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MECHAKISMS OF APOPTOSlS

A

* *

Growth factor receptors occupied

+ +

Ras+

P13’K

Raf-1

PKB

PT-pore closed No apoptosis

B Growth factor withdrawal

p 1 i

1

Ras+

1

P13’K

Bcl-2 BCl-2

i

PT-pore open Apoptosis

FIG.14. Posttranslational niodificatioriof Bcl-2 and BAD through serine phosphorylation influences resistance or susceptibility to apoptosis. (A) Signaling via an essential growth factor receptor, e.g.,IL-3R, leads to phosphorylation of Bad and sequestration of phosphorylated Bad in the cytosol through binding to 14-3-3. Serine phosphorylation of Bad is probably regulated via the kinase Raf-1. The role of PKB in this phosphorylation events is purely hypothetical. In addition, growth Factor receptor occupancy (HGF-R and PDGF-R) may allow the death inhibitory molecule Bag-1 to activate the Raf-1 kinase. (B) Withdrawal of essential growth factors (IL-3) leads to inactivation of PKB and Raf-1 and subsequent dephosphorylation of Bad. Dephosphorylated Bad is released from 14-3-3binding and forms heterodimers with Bcl-UBcl-X,,,thereby triggering apoptosis. Moreover, growth factor (CFD and PDGF) withdrawal leads to recruitment of the death inhibitory molecule Bag1 to the plasma membrane and inactivation of Bcl-2 by phosphorylation. The direct effect of Bcl-2 molecules on the PT pores is speculative.

protein Bag-1, which cooperates with Bcl-2 to proIong cell survival (437). Bag-1 can interact with the cytoplasmic domains of the receptors for hepatocyte growth factor and platelet-derived growth factor. Upon growth factor withdrawal, Bag-1 binds to these receptors and hence Bag-1 is not

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available for binding to mitochondria1 Bcl-2 (438). In T cells, Bag-1 is upregulated in response to IL-2 stimulation (439). Bag-1 can also bind to and activate Raf-1 (431), suggesting the existence of complex interactions between members of the Bcl-2 family and kinases. Bcl-2 itself can also be the target of posttranslational modifications via serine phosphorylation (Fig. 14). Phosphorylation of Bcl-2 occurs within a 60-amino acid loop without defined structure (440). Although the functional significance of Bcl-2 phosphorylation is still disputed, recent data imply that Bcl-2 phosphorylation reverts its antiapoptotic effect, that is, phosphorylated Bcl-2 does not protect from apoptosis (441-445). The ability of chemotherapeutic agents that act on microtubules (taxol,vinblastine, and vincristine) to induce phosphorylation of Bcl-2 has been also implicated as a mechanism by which these anticancer drugs can neutralize the antiapoptosis function of Bcl-2 and promote death. The kinase responsible for Bcl-2 phosphorylation has not been identified yet and is probably not Raf-1 (445).Thus, the exact nature of the kinases acting on Bcl-2 (and other members of the Bcl-2 family?) remains elusive. Most studies suggest that Bcl-2-related proteins have to localize to mitochondria in order to regulate apoptosis (183,446,447). In addition to their probable pore-regulatory function (19, 147, 185, 186,448), Bcl-2 and BclXL can determine the subcellular localization of apoptosis regulators with which they interact. This applies to Raf-1, calcineurin (449), and CED-4. Homodimerized Bcl-2 and Bcl-XLcan bind to the mammalian homolog(s) of CED-4, a molecule that was originally identified to be involved in the cell death pathway in the nematode C. elegans (450-452). CED-4 may function as an adapter between the mitochondrial-bound Bcl-2/Bd-XL and some procaspases with large N-terminal prodomains (e.g., procaspase-1 and -8).As a possibility, CED-4 keeps caspases in an inactive state, provided that it is bound to BcI-YBcI-XL (Fig. 15). Disruption of the trimolecular complex between CED-4, Bc1-2/Bcl-XL, and the caspase would lead to the activation of the inactive zymogen. Heterodimerization of Bcl-2/Bcl-XL with the death promoters Bax, Bad, Bak, Bcl-Xs,or Bik has been speculated to disrupt binding of CED-4 to Bcl-YBcl-XL, thereby releasing CED-4, which might activate caspases and/or act on the nucleus to provoke chromatin condensation and other apoptosis-like changes (24, 450-452). Based on the findings detailed previously, the antiapoptotic effects of Bcl-2 and Bcl-XI,overexpression could be explained in terms of a ternary molecular complex involving, in addition to Bcl-WBcl-XL, the mammalian CED-4 analog arid procaspases (453). As an alternative possibility, the binding partners of Bcl-2 might influence its capacity to regulate mitochondrial membrane permeability. In this hypothetical scenario, Bcl-2 and its homologs would function as mere targets of signal transduction pathways

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Death

Mitochondrion Fic:. 15. Hypothetical model of Bcl-2. CED-4, and caspase interactions at the mitochontfrial nienibrane. Binding of CED-4 to Bcl-WBd-Xl,results in caspase inactivation.Activation of effector caspases, e . g , caspses 3 and 6, requires release of cytochroine c and/or AIF from the mitochondria. Caspases involved in the induction phase of apoptosis such as caspase-8 (FLICE/Mach-1)relay death receptor signaling (DISCS)to Bcl-2 family inembers to open the PT pore and release cytochroine c and AIF. Moreover. such activating caspases iniiy caiise the direct proteolytic activation of downstream easpases.

involving caspases, CED-4, and other molecules (kinases, and phosphatases, including calcineurin), which modulate the composition of the Bcl2 complex and hence determine its capacity to open and close pores in the mitochondria1 membrane (448).

F PI3’ KINASEA N D Akt/PKB ACTIVATION THE KEY TO SURVIVAL? Recently, a novel signaling pathway, activation of the serinekhreonine

PKB (also known as Akt), has been shown to rescue cells from apoptosis (Fig. 16) (454).PKB is activated by the PI3’ kinase (455),which is involved in a variety of transmembrane receptor signaling cascades including signaling via antigen receptors in T and B cells, CD5, CD28 and CTLA4, receptors for IL-2, IL-4, insulin and insulin-growth factor, epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor, or signaling via Gapy-coupled seven-membrane spanning receptors such as the major mitogenic factor present in fetal calf serum, LPA (454, 456, 457). Among the four distinct PI3’ Ks, three PI3’ Ks link tyrosine lanase receptor signaling to downstream effects and one is activated by induction of GPy subunits from G-protein-coupled receptors (458-460). PI3’ kinases are regulated via direct binding to transmembrane receptors or via Ras or

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FIG.16. The role of PI3’ kinase and protein kinase B (PKB or A h ) in growth factormediated survival. PKB might prevent apoptosis through inactivation of caspases. However, other downstream substrates of PKB might also have a critical role in the survival function of this kinase. GF-R, growth factor receptor; PtdIns-3,4-P2, phosphatidylinositol-3,4diphosphate. The scheme is adapated from Ref. (454).

R-Ras (461) and PI3’ kinases can induce a variety of downstream signaling events including p47phoXactivation required for cellular transformation, actin polymerization (462), and PBK/Akt activation (455). PI3’ kinase can phosphorylate the D3 position of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4-P), and phosphatidylinositol4,5-diphosphate (PtdIns-4,5-P2) to generate phosphatidylinositol-3phosphate (PtdIns-3-P), phosphatidylinositol-3,4-diphosphate(PtdIns-3,4P2), and phosphatidylinositol-3,4,5-triphosphate (PtdIns-3,4,5-P3), respectively (457). PtdIns-3,4-P2 can also be generated from PtdIns-3,4,5-P3 through the action of a 5’ phospholipid phosphatase such as the inositol polyphosphate 5-phosphatase SHIP, a negative regulator of B cell receptor and IgE recepter signal transduction (463-468). PtdIns-3,4,5-P3 and PtdIns-3,4-P2 interact with the plekstrin homology domain of PKB/Akt with high affinity (457, 469). High-affinity binding of PtdIns-3,4-P2 to PKB/Akt leads to the recruitment of PBWAkt to the plasma membrane, where PKB/Akt undergoes a conformational change and becomes phosphorylated on serine 473 and threonine 308 (454,457). Interestingly, the MAPK kinase p3WHOG-activated MAPKAP kinase 2 can phosphorylate serine 473 in PKB/Akt under conditions of cellular stress that do not activate P13’ kinase (470, 471), implying that the stress signaling kinase p38/HOG (258,378,472) can regulate PKB/Akt activity. The MAPK family kinase p38/HOG is activated by a plethora of different stimuli, such as irradiation, toxins, ischemia, heat shock, ceramide, or ligation of the TNF-

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Rp55 (Fig. 12) (258, 378, 379, 472). P38/HOG-regulated PKB/Akt activation provides another example of the multiple cross talks that exist between different signal transduction cascades. Overexpression of activated PI3’ kinase or membrane-targeted PKB/ Akt can protect Rat-1 fibroblasts and COS cells from UV-B light-mediated apoptosis (473). Insulin-like growth factor- 1 ( IGF-1)-mediated survival of neurons appears to depend on a functional PI3’K -+ PKB/Akt signaling pathway, and IGF-l-mediated neuronal survival can be blocked by overexpression of dominant-negative PKB/Akt (474). PKB/Akt signaling can also prevent NGF-induced apoptosis of pheochromocytoma PC12 cells (454). Moreover, Ras activation of PI3’ kinase suppresses c-Myc-induced apoptosis in fibroblasts.This protective effect depends on the activity of PKB/Akt (475). However, in the same experimental system Ras activation triggers apoptosis through induction of the Raf-1 kinase (475), implying that Raf1 activation does not necessarily lead to the suppression of cell death. Rather, Ras/Raf-1 can mediate antithetical intracellular signals for cell fate decisions. These data indicate that certain signals including the growth factor IGF1 can promote cellular survival via activation of the Ras + PI3’ kinase + PKB signaling cascade. The Ras-induced survival pathway is independent of MAPK activation and independent of ~ 7 0 ~and ~ GSK3, ~ ~ ” which ~ ’ ~are the only known downstream effectors of PKB/Akt activity (458). Thus, the identification of the downstream apoptosis regulatory targets of PBWAkt will be of the utmost importance. G. THETNF RECEPTORSUPERFAMILY The ever-growing TNF-R family includes the low-molecular-weight TNF-Rp55, the low-molecular-weight TNF-Rp75, LTPR (476), FadAPO1 (CD95) (343, 477), designated death receptor-3 (DR3 or wsl-1) (478, 479), designated death receptor-4 (DR4) (480), the cellular receptor for the cytopathic avian leukosis sarcoma virus termed CAR1 (481), ATAR (482), CD27 (483), 41.BB (484, 485), CD40, 0x40, nerve growth factor receptor (486), the Hodgkin’s lymphoma antigen CD30 (487),or the newly identified inhibitor of osteoclast differentiation osteoprotegerin (488).The TNF family of receptors is characterized by sequence homology at an extracellular cysteine-rich domain. In contrast, the cytoplasmic domains of these molecules lack homology, suggesting that the members of the family share similar ligand recognition properties but differ in signal transduction (485,489-491). The members of the TNF-R superfamily mediate a plethora of biological responses including induction of cellular proliferation, induction or suppression of programmed cell death, costimulation in T and B lymphocytes, osteoclast differentiation from bone marrow

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precursors, development of Peyer’s patches and lymph nodes, and inflammatory immune responses. Activation of mature T cells upregulates the expression of receptors that can receive death signals (Fas, CD30, and the low- and high-molecularweight TNF-Rp55 and TNF-Rp75) and simultaneouslycauses downregulation of apoptosis inhibitory genes such as bcl-2. Thus, antigen-driven activation and expansion generates T lymphocytes that are prone to apoptosis (257, 258, 492). This mechanism maintains leukocyte homeostasis in uiuo (Fig. 9). Activation-induced cell death of peripheral T cells is mainly mediated via the death receptor Fas, TNF-Rp55, and TNF-Rp75 (262266, 346, 477). Although these receptors have little if any impact on physiological thymocyte selection, another member of the TNF-R family, CD30, appears to determine negative selection in the thymus (256). The following sections will focus on the role of Fas, CD30, TNF-Rp55, and TNF-Rp75 in lymphocyte development and function and the general principles of signal transduction pathways that link these receptors to the central executioner or the survival factor NF-KB.

1 . TNF Receptors, Fas, and CD30: Impact on Central and Peripheral Tolerance The biological functions of TNF-a and TNF-P are mediated by three distinct surface receptors (TNF-RpSS, TNF-Rp75, and LTPR). TNF-a and TNF-P bind to either TNF-Rp55 or TNF-Rp75. These effects are pleiotropic, ranging from cell proliferation via costimulation to induction of programmed cell death and inflammatory immune responses (489-491, 493). The third distinct receptor that interacts with TNF, LTPR, preferentially interacts with TNF-/3 and controIs the development of Peyer’s patches and lymph nodes (494). Both TNF-Rp55 and TNF-Rp75 are expressed at low levels on the cell surface of resting peripheral T cells but upregulated upon antigen-specific activation, implying that these molecules may share a common mechanism for transcriptional induction and may function in a similar manner (491). Similarly, expression and function of other TNF receptor family members such as Fas, CD40, CD27, CD30, 41.BB, or OX-40, or their receptors are highly regulated and depend on the T cell lineage and stage of differentiation of thymocytes (489-491, 493). Within the thymus, both mature and immature T cells produce TNF-a constitutively (495, 496). TNF-a can augment IL-6-induced thymocyte proliferation or induce apoptosis of CD4CD8- precursor and mature single-positive thymocytes. Because murine, TNF-a but not human TNF-a, can induce thymocyte apoptosis (human TNF-a only binds to the inurine TNF-Rp55 but not the murine TNFRp75) (496), the apoptotic effect of TNF in thymocytes is most likely

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mediated by TNF-cdTNF-Rp75 interactions. However, t h p o c y t e development and negative selection are not altered in TNF-Rp55 (497), TNFRp75 (498), or LTPR (494) gene-deficient mice. In contrast to thymocyte selection, peripheral T cells from TNF-Rp55- and TNF-Rp75-deficient mice do exhibit a partial resistance to activation-induced cell death (264266). This phenomenon becomes particularly manifest when animals lacking both the TNF-R and the Fas receptor are analyzed (264,265), suggesting that various members of the TNF-R superfamily may be involved in the same pathway in a redundant fashion. Within the thymus, Fas is highly expressed in CD4+CD8+TCR""""'".''' cells, whereas Fas expression is downregulated in mature CD4' or CD8' thyniocytes (499, 500). Although Fas is probably not directly involved in negative selection of thyinoctes responding to peptide antigens (265, 501, 502) or superantigens (503),the peak of Fas expression correlates with the stage of thyniic maturation at which T cell selection occurs. Interestingly, CD4+CD8- or CD4-CD8- thyrnocytes expressing N K 1 . l and the TCRaP heterodimer are cytotoxicagainst CD4+CD8+thymocytes and this cytotoxic activity is mediated by Fas/Fas ligand interactions (504). Moreover, antiFas cross-linking can induce cell death of immature thymocytes in vitro (41,505) and in thymic organ cultures (506), implying that Fas might-at least to some extent-modulate death of thymocytes. However, most data suggest Fas is predominantly involved in peripheral T cell tolerance. A wealth of data point to a role of Fas in the maintenance of peripheral cell numbers and apoptosis of activated T ly~nphocytes(345, 346, 477, 507). Fas is rapidly upregulated upon stimulation of peripheral T cells. However, activated T lymphocytes are only susceptible to Fas-triggered apoptosis several days after the initial activation event (477, 508, 509). In addition, anti-CD4 induced apoptosis of peripheral T cells in vivo and Ca2+-independentT-cell cytotoxicity appears to depend on Fas expression (510, 511). In this context it is interesting to note that Fas does not only induce cell death but also can provide an IL-2 independent signal for proliferation of peripheral T cells and thymocytes (512). The 120-kDa glycoprotein receptor CD30 molecule is a member of the TNF-R superfamily and was originally identified as a diagnostic marker for Hodgkin's disease, the most frequent lymphoma in humans. CD30 is found on multinucleated Reed-Sternberg cells and Hodgkin's cells in Hodgkin's disease and on a variety of other tumor cells including embryonal carcinoma, melanoma, and some mesenchymal tumor cells (513-515). CD30 is also expressed on non-Hodgkin lymphomas and some virally transformed T and B cells. In addition, it has been suggested that CD30' T cells play a role in HIV pathogenesis, Epstein-Barr virus infections, measle virus infections, atopic disorders, or autoimmune diseases such as

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systemic lupus erythematosus and rheumatoid arthritis (516). In normal lymphoid tissues, CD30 is expressed on activated T and B cells and medullary thymocytes (517-519). Moreover, the CD30 ligand is expressed on activated T cells (520). Because some tumor cell lines undergo apoptosis after CD30 cross-linking, it has been proposed that the CD30 surface receptor triggers signal for cell death (521). By contrast, CD30 may also function as a receptor in activation and differentiation of T and B cells (518,522,523). Moreover, it has been proposed that CD30 is differentially expressed on Th2 T cells (516) and that CD30 induction through IL-4 might have a role in the differentiation and/or maintenance of Th2 cytokineproducing T lymphocytes (524, 525). Recent studies involving CD30 gene-deficient mice suggest a role for CD30 in central immune tolerance (256). In CD30-’- mice, thymocyte numbers are increased. Whereas positive selection and dexamethasone and y-irraditation-induced cell death of immature thymocytes are normal, CD30-’- thymocytes are resistent to anti-CD3-induced apoptosis in vitro and antigen-driven negative T cell selection is impaired in two different TCR transgenic mouse models in viva These results show that TCWCD3induced cell death can be mediated via the CD30 receptor in developing thymocytes. Despite impaired negative selection, peripheral T cells from CD30-’- mice are still tolerant to self-antigens, implying that multiple mechanisms control the induction and maintenance of immunological tolerance (256).However, these data provided the first evidence for a receptor that specifically interferes with negative thymocyte selection. The data for CD30-’- mice also confirmed that distinct signal transduction pathways exist for positive and negative selection of T cells. By contrast, CD30 can activate T and B cells (518,522,523) and may have a role in Th2 cytokine production (524, 525). It thus appears that CD30 signaling can induce contradictory signalingevents in terms of fate decisions for survival or apoptosis. 2. Death Domains and Death-Inducing Signaling Complexes Similar to most other transmembrane receptors, signaling of TNF-R family molecules requires clustering of the receptors after ligand binding (Fig. 17). Receptor clustering induces conformational changes, the recruitment of signal transduction molecules to the cytoplasmatic domain, and subsequent induction of cellular responses such as proliferation andlor apoptosis. The ensemble of signal transduction molecules that bind to the oligomerized death receptors and mediate cell death have been termed the “death-inducing signaling complex” (DISC) (65, 526, 527). One of the principles of DISC formation is the interaction between socalled death domains (DDs).The D D is a region ofhomology first identified

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FIG.17. Model of TNF-RpSS and Fas-mediated signal transduction for apoptosis (Fas and TNF-Rp5S) and NF-KB activation (TNF-Rp55). DD, death domain; DED, death effector domain: ICE, ICEKED homology domain. RAIDD also contains a DD domain and interacts with RIP via homotypic DD domain binding (not indicated in the figure). For more details see text.

in TNF-Rp55 and Fas in an approximately 80-amino acid-long cytoplasmic domain that is crucial for the induction of cell death (Fig. 17) (489, 528). Additional transmembrane receptors possess similar cytoplasmicDDs. This applies to DR3 (wsl-l),DR4, the chicken CAR1 protein (481), and possibly the p75 NGF-R (375,529,530). DDs are also found in cytoplasmic signaling molecules, namely, in the receptor interaction protein (RIP) (531), the Fas-associated protein with a death domain (FADD) (532, 533), the TNF-R associated protein with a death domain (TRADD) (534, 535), and the RIP-associated ICH1-homologous protein with a death domain (RAIDD) (536). Death domains are homotypic protein-interaction domains that permit binding of DD-containing intracellular molecules to the DD domains present in the transmembrane receptor. Thus, oligomerization of the TNF-

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Rp55 triggers the binding of the molecular adapters TRADD and FADD to the cytoplasmic tails of the TNF-Rp55 through homotypic D D interactions (532-535, 537). FADD contains a death effector domain (DED) that interacts with the DED domain of caspase-8 (46,65,526,538). Procaspase8, formerly called FLICE ( FADD-homologous ICEKED-3-like protease)/ Mach, contains a DED domain for binding to FADD and an additional region with high homology to ICE/CED-3 caspases (65, 538). During activation and assembly of the DISC, procaspase-8 gets proteolytically cleaved between the DED and the ICE homology domain and the activated caspase-8 can stimulate apoptosis induction. Similarly, the DR3 receptor triggers apoptosis via DD domain recruitment of TRADD and FADD and activation of caspase-8. The death-inducing signaling machinery associated with the DR4 receptor after binding to its ligand TRAIL has not been eluciated, but it does not appear to involve TRADD (65, 538). Similar to the TNF-Rp55, oligomerization of Fas through binding of its trimeric Fas-L induces the formation of a DISC. However, FADD binds directly to the D D of Fas and recruits caspase-8 (FLICE/Mach-1) via homotypic DED domain interactions (Fig. 17). Cleavage of FLICE at the assembled DISC then leads to the release of the active caspase component of caspase-8 and subsequent induction of Fas-mediated cell death (65, 538) in a pathway that may involve activation of other upstream caspases including caspase-1 (formely called ICE) (42, 45, 539). Caspase-1 may induce mitochondria1permeability transition in a fashion that is not antagonized by Bcl-2, at least in CEM-7 cells, (203), in accord with the fact that Bcl-2 is an inefficient inhibitor of Fas-induced apoptosis (540-542). Besides the recruitment of FADD and caspase-8, an additional molecule, RIP, interacts with the DD of Fas. RIP contains a DD at the NH2 terminal and a serinekhreonine kinase homology region at the COOH-terminal end (543). Moreover, a Fas-associated protein tyrosine phosphatase, FAP, has been identified that can directly bind to the COOH-terminal 15 amino acids of Fas and might protect cells from Fas-induced apoptosis (544). Although RIP can function as serinekhreonine kinase in vitro, the functional significance of this enzymatic activity is unclear. RIP interacts with the adaptor molecule RAIDD (536). RAIDD has an unusual bipartite architecture comprising a carboxy-terminal death domain that binds to the homologous domain in RIP and an amino-terminal domain homologous with the sequence of the prodomain of two procaspases, human caspase2 (ICH-1) and C. eleguns CED-3. Thus, homotypic interactions mediated by caspase prodomains can determine the specificity of binding of caspase zymogens to regulatory adaptor molecules (536). Selective activation of RAIDD by Fas might explain earlier reports that Fas and TNF-Rp55 can

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employ distinctive signal tranduction pathways to trigger programmed cell death (173, 545). Although clear evidence exists that CD30 plays a role in antigen-driven negative selection of thymocytes, the cytoplasmic domain of CD30 has no obvious homology to death domains present in Fas, TNF-Rp55, DR3, and DR4 receptors (see the following section). Nonetheless, CD30 activation leads to the recruitment of the TNF receptor-associated proteins TRAF1, TRAFB, and TRAFS and induction of NF-KB (546,547). In a Hodgkin's lymphoma cell line, ligation of CD30 is mitogenic, depending on activation of a protein tyrosine kinase and the MAPK signaling pathway (548). Thus, CD30, like many other receptors, can trigger both activating and Iethal signals.

3. NF-KB Activation by TNF: Activation of u Survival Signal Activation via TNF does not always induce apoptosis, and many cell types are fairly resistant to TNF-mediated cell death. Most TNF-resistant cells, however, are hlled by TNF in the presence of protein synthesis inhibitors. This indicates that TNF induces two pathways, one that mediates apoptosis and a second that induces transcription of cytoprotective genes. One crucial molecule that is activated by almost all TNF-R superfamily receptors (with the exceptions of Fas and DR4) is the transcription factor NF-KB. NF-KB is important for lymphocyte responses to antigens and cytokine-inducible gene expression (412). NF-KB activation can protect fibroblasts and T lymphocytes from apoptosis in response to ionizing radiation, the anticancer drug daunorubicin, and TNF-(I!treatment (414-416). Activation of NF-KB appears to be mediated by TNF-R-associated factors (TRAFs) (Fig. 17) (549). Six distinct TRAF molecules have been identified to date. TRAFs bind via their COOH-terminal TRAF domains to the TRAF domain of TRADD or directly to the cytoplasmatic region of the receptor (for example, in the case of TNF-Rp75) (550-554). Thus, activation of the TNF-Rp55 leads to recruitment of TRADD via the DD domains and TRADD recruits TRAFB via homotypic TRAF domain interactions. By contrast, TNF-Rp75 oligomerization leads to the recruitment of TRAFl and TRAFB to the cytoplasmatic region of the receptor and TRAFl and TRAFB forin heterooligomers through interactions of their TRAF domains. Similarly, CD40 activation leads to the recruitment of TRAF2, which then forms oligomers with TRAF3 (555). In addition to the TRAF domain, some TRAF molecules, e.g., TRAFB and TRAFS, contain a NH2terminal ring-finger domain that is required for NF-KB induction. Importantly, a doininant-inhibitory mutation of TRAF2 leads to TNF-mediated apoptosis instead of activation, i.e., in the absence of a TRAF2-mediated signal, TNF-treated cells trigger a default signaling

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pathway that culminates in cell death (552). Moreover, it has been shown that TRAF2 mediates TNF-induced activation of SAPK (403, 404). The bifurcation between TRAF2-induced SAPK and NF-KB activation is probably the serinekhreonine kinase MEKK1, a MAPKKK that directly activates SEKl and NF-KB through site-specific phosphorylation of the NF-KB inhibitor, I - K B (Fig. ~ 13) (413). Recently, it has been shown that a variety of other molecules bind to the signaling complex that relays TNF-R stimulation to cell death and NFKB. For example, a novel TRAF-interacting protein, I-TRAF, has been identified that binds to the conserved TRAF-C domain of TRAF1, TRAFB, and TRAF3. Overexpression of I-TRAF inhibits TRAF2-mediated NF-KB activation signaled by CD40 and both TNF receptors. Thus, I-TRAF appears as a natural regulator of TRAF function that may act by maintaining TRAFs in a latent state (549). Moreover, four mammalian molecules, termed cellular inhibitors of apoptosis (c-IAPs), have been identified that interact with TRAFl and TRAF2 (552, 556), suggesting the existence of multiple endogenous inhibitors acting on specific apoptosis induction pathways. Although c-1APs have homology to viral inhibitors of apoptosis such as the baculovirus p35 protein, the physiological role of c-IAPs in mammalian cells is still elusive. 4. TNF-Receptors, Sphingomyelinases, and Ceramide

TNF-Rp55 signal transduction for NF-KB activation does not only involve TRAFZ but also sphingomyelin breakdown into ceramide by the acidic sphingomyelinase (557, 558). The second messenger ceramide is produced through either the induction of sphingomyelin (SM) hydrolysis via cytosolic acidic or membrane-bound neutral sphingomyelinases (SMase) or de novo biosynthesis (559, 560). Ceramide transduces signals mediating differentiation, growth, growth arrest, apoptosis, cytokine biosynthesis and secretion, and a variety of other cellular functions (559). Rapid sphingomyelin hydrolysis to ceramide correlates with irradiation, Fas, and TNF-R-induced apoptosis in various cellular systems (561-563). In particular, it appears crucial for irradiation-induced apoptosis because lung epithelial cells, but not thymocytes, from mice deficient for acidic sphingomyelinase and some human cell lines from patients lacking this enzyme fail to undergo apoptosis in response to y-irradiation (564). Ceramide is a second messenger that induces a membrane-bound ceramide activated serinekhreonine protein kinase (CAPK) (565) and a cytoplasmic ceramide-activated protein phosphatase (566). Ceramid can also lead to the induction SAPK (357, 494) and p38/HOG (472). Ceramidemediated SAPK activation has been functionally linked to the induction of apoptosis (363, 364, 397, 567). Other downstream targets for ceramide

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action include Cox, IL-6 and IL-2 gene expression, PKCS, Vav, the retinoblastoma protein, c-Myc, c-Fos, and a variety of other transcriptional regulators (568).Although a number of ceramide-activated effector molecules have been described, it will be important to identify additional cellular targets that link the activation of SMases to the regulation of nuclear events. Cross talk between ceramide-induced signal transduction cascades and other signaling pathways adds to the inherent difficulty in distinguishing the specific effects of complex and multifaceted signal transduction pathways (559, 569). The human TNF-Rp55 initiates at least two independent ceramide signaling cascades via activation of acidic or neutral sphingomyelinases (570). The acidic sphingomyelinase (A-SMase) pathway involves a phosphatidylcholine-specific phospholipase C , an endosomal A-SMase, and controls expression of multiple TNF-responsive genes through induction of transcription factors including NF-KB (557,558,571).Acidic SMase-triggered NF-KB activation is probably mediated by rapid degradation of I - K B ~ through a serine-like protease. Activation of A-SMase by TNF-Rp55 stimulation was mapped to the region of the death domain (572) and may require a crmA-inhibitable caspase (78). The neutral sphingomyelinase (N-SMase) pathway comprises a membrane-bound N-SMase, CAPKs, and phospholipase A2 and appears critical for the inflammatory responses induced by TNF. An ll-amino acid region (aa 309-319) of the human TNF-Rp55 is necessary and sufficient for activation of N-SMase and a molecule termed FAN has been cloned that couples the TNF-Rp55 to the N-SMase signaling pathway (571). The N-SMase activation domain is distinct from the death domain and incapable of induction of A-SMase, NF-KB, and cytotoxicity suggesting that N-SMase and A-SMase control nonoverlapping pathways of TNF receptor signal transduction (573). Interestingly, CAPKs can phosphorylate and activate Raf-1 (574) and mediate MAPK activation (575), implying that N-SMases initiate the proinflammatory actions of TNF-a via a CAPK + Raf-1 -+ MAPK signaling cascade. In C. elegans and Drosophila melanogaster, a potential CAPK has been genetically identified as the kinase suppressor of Ras (KSR) (576). In synthesis, it appears that ceramide derived from neutral SMase activation mediates proinflammatory responses through the activation of CAPK (KSR), Raf-1, and MAPK, whereas ceramide generated through acidic SMase activation appears to be primarily involved in NF-KB and SAPK activation and correlates with the induction of apoptosis (567).It is tempting to speculate that neutral, membrane-associated sphingomyelinases may be involved in cell proliferation and proinflammatory responses, whereas the induction of acidic sphingomyelinases may mediate cell death. In contrast

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to this hypothesis, however, irradiation of bovine aortic endothelial cells induces rapid sphingomyelin hydrolysis and apoptosis through a neutral sphingomylinase pathway (561). Moreover, CD28 costimulation, which results in T cell proliferation and prolonged survival, leads to the activation of an acidic sphingomylinase (568). The mechanism by which activation of shingomyelinasesand the generation of ceramide activate the central executioner is unknown. According to one report (217),ceramide can induce mitochondrial permeability transition, but this finding has not been confirmed by another group (577), who reports that ceramide enhances the mitochondrial generation of reactive oxygen species. In a cell-free system that includes mitochondria and nuclei, ceramide itself is an inefficient inducer of nuclear apoptosis. However, cytosols from cells treated with ceramide contain an activity that provokes mitochondrial permeability transition in vitro (203). Thus, ceramide may either have a direct effect on mitochondria or provoke the generation of second messengers that activate the mitochondrial executioner. V. Conclusions

DeatMlife decisions are crucial for the homeostasis of both the thymic and the peripheral compartments of T cells. During the past few years, major progress has been achieved in understanding how signal transduction pathways may influence this decision. Whereas certain signal transduction pathways are mainly involved in conferring apoptosis resistance (e.g., the P13’ kinase +. PKB signaling cascade and activation of NFKB),others may have a major role in mediating death signals. One such pathway involves activation of stress-activated protein kinases (SAPWJNK)and/or p38HOG kinase via a plethora of different stimuli, such as irradiation, toxins, ischemidreperfusion, heat shock, ceramide, or surface death receptors such as the TNF-Rp55 and Fas may trigger apoptosis. However, recent genetic evidence indicates that SAPK signaling prevents premature T cell death during activation and development. Another predominantly deathinducing pathway involves raises in cytosolic Ca2+levels, which together with additional but ill-defined factors can trigger apoptosis. Recent data indicate that the stoichiometry of Bcl-2-related death agonists and antagonists is not the sole parameter to determine apoptosis regulation of these proteins. Posttranslational modifications of Bcl-2-related proteins, such as BAD and Bcl-2, have a major impact on apoptosis regulation and might be a critical crossroad that links surface receptor signals to the regulation of apoptosis. Multiple signaling pathways may feed into the posttranslational modification of these molecules, in particular signaling via the serine hnase Raf-1. A series of receptors belonging to the

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TNF-R superfamily, namely, TNF-Rp55, Fas, DR3, and DR4, assemble a death-inducing signaling complex upon ligation. Oligomerization of these receptors leads to the recruitment of death-inducing adaptor molecules through so-called death domains and eventually triggers certain caspases. It would be an oversimplification to asssuine that these receptors mediate only apoptotic responses. Thus, the TNF-Rp55 triggers conflicting signals that either induce apoptosis (caspase activation and ceramide) or trigger activation of the apoptosis-inhibitory transcription factor NF-KB. Altogether, it becomes increasingly clear that the initiation of apoptosis mostly does not involve just a simple linear sequence of biochemical events. Rather, multiple parallel and sometimes antagonistic pathways are activated. Cross talk between different signal transduction pathways is frequent and contributes to augment the complexity of the system. Recent progress suggests that after the heterogeneous, signal transduction-dependent initiation phase, apoptosis employs one (or a few) common pathways, in line with the fact that the biochemical and ultrastructural features of apoptosis are similar in all cell types, independent from the initial apoptosis trigger. The common pathway can be subdivided into two phases: the effector phase, during which the central executioner is activated, and the degradation phase, beyond regulation, during which cellular catabolism gives rise to the apoptotic phenotype. The central executioner involves major changes in mitochondrial membrane permeability, including the opening of so-called PT pores. Recent evidence suggests that a number of signaling molecules facilitate such changes in mitochondrial membrane permeability: Ca2+,reactive oxygen species, nitric oxide, perhaps ceramide, and some caspases that are activated in particular pathways of apoptosis (e.g., caspase-1). In addition, mitochondrial membrane permeability is regulated by the members of the Bcl-2 family and/or Bcl-2 associated proteins, and thus constitutes a target of multiple pathways that affect the level of expression of such Bcl-2-related proteins, influence their subcellular distribution, or introduce posttranslation modification affecting their apoptosis regulatory potential. At this latter level, lunases and phosphatases may act on Bcl-2 and Bcl-2-binding proteins. The involvement of mitochondria in the central executioner is attractive for several reasons. First, it opens the possibility of detecting apoptosis at a relatively early stage by monitoring mitochondrial membrane integrity. Second, the mitochondrion can function as a sensor for changing (stressful) metabolic conditions as well as for certain second messengers. Third, the molecules involved in the mitochondrial executioner cannot be mutated because they are necessary for normal cell function, thus avoiding the selection of tumor cells that would be completely resistant to apoptosis induction. Fourth, the mitochondrial membrane changes may have some

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self-amplifying properties, which may allow them to act in a switch-like fashion. Finally, activation of the mitochondrial executioner has multiple lethal consequences. Once the mitochondrial membrane permeability has been perturbed and vital mitochondrial functions are disrupted, the cell is irrversibly committed to death. At this stage, mitochondria release apoptogenic proteins that are normally well secluded. Such proteins trigger the activation of downstream caspases and endonucleases and thus induce the typical pattern of morphological and biochemical changes that accompanies the late stage of apoptosis. Inhibition of caspases and endonucleases does not prevent cytolysis and rather determines a necrotic type of cell death. Thus, the degradation phase is probably not a useful target for pharmacological interventions. Any attempt to modulate apoptosis must aim at interfering with apoptosis-triggering signal transduction pathways and/or the activation of the central executioner.

ACKNOWLEDGMENTS We thank Dr. Maurice Geuskens (Free University of Bruxelles, Belgium) for electron microscopic data and Drs. Catherine Brenner, Didier Decaudin, Tamara Hirsch, Philippe Marchetti, Isabel Marzo, Patrice X. Petit, Santos Susin, Naoufal Zamzami, Young-Yun Kung, Klaus Fischer, Ivona Kozieradzki, and Takehiko Sasaki for sharing unpublished data and helpful discussion. This work has been partially supported by Agence Nationale pour la Recherche contre le Sida, Association pour la Recherche contre le Cancer, Centre National de la Recherche Scientifique, Fondation de France, Fondation pour la Recherche MBdicale, Institut National de la SantB et de la Recherche MBdicale, Ligue Franqaise contre le Cancer, North Atlantic Treaty Organization, and the French Ministry of Science (to GK), and grants by the Medical Research Council and Amgen (to JMP).

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561. Haimovitz-Friedman, A,, Kan, C.-C., Ehleitner, D., Persaud, R. S., McLoughlin, M., Fuks, Z., and Kolesnick, R. N. (1994).J. Exp. Med. 180, 525-535. 562. Kolesnick, R. N., Haimovitz, F.-A,, and Fuks, 2. (1994). Biochem. Cell B i d . 72, 471-474. 563. Kolesnick, R., and Fuks, Z. (1995).J. Exp. Med. 181, 1949-1952. 564. Santana, P., Peih, L. A,, Haimovitz-Friedman, A,, Martin, S., Green, D., McLoughlin, M., Cordon-Cardo, C., Schuchman, E. H., Fuks, Z., and Kolesnick, R. (1996). Cell 86, 189-199. 565. Mathias, S., Dressler, K. A., and Kolesnick, R. N. (1991). Proc. Natl. Acad. Sci. USA 88, 10009-10013. 566. Dressler, K. A., and Kolesnick, R. N. (1990).J. B i d . Chem. 265, 14917-14921. 567. Pena, L. A,, Fuks, Z., and Kolesnick, R. (1997). Biochem. Pharmacol. 53,615-621. 568. Boucher, L. M., Wiegmann, K., Fuetterer, A., Pfeffer, K., Machleidt, T., Schuetze, S., Mak, T. W., and Kroenke, M. (1995).J. Exp. Med. 181, 2059-2068. 569. Testi, R. (1996). Trends Biochem. Sci. 21,468-471. 5 70. Dressler, K. A,, Mathias, S., and Kolesnick, R. N. (1992). Science 255, 1715-1717. 571. Adam, D., Wiegmann, K., Adam, K.-S., Ruff, A., and Kroenke, M. (1996).J. B i d . Chem. 271, 14617-14622. 572. Machleidt, T., Wiegmann, K., Henkel, T., Schuetze, S., Baeuerle, P., and Kroenke, M. (1994).J. B i d . Chem. 269, 13760-13765. 573. Wiegmann, K., Schutze, S., Machleidt, T., Witte, D., and Kronke, M. (1994). Cell 78, 1005-1015. 574. Yao, K. S., Clayton, M., and O’Dwyer, P. J. (1995).J. Natl. Cancer Inst. 87, 117-122. 575. Raines, M. A., Kolesnick, R. N., and Golde, D. W. (1993).J. Bid. Chem. 268,1457214575. 576. Zhang, Y. H., Yao, B., Delikat, S., Bayoumy, S., Lin, X. H., Basu, S., McGinley, M., Chanhui, P. Y., Lichenstein, H., and Kolesnick, R. (1997). Cell 89, 63-72. 577. Garcia-Ruiz, C., Colell, A,, Man‘, M., Morales, A., and Fernindez-Checa, J. C. (1997). J. Biol. Chem. 272, 11369-11377. 578. Adachi, M., Suematsu, S., Kondo, T., Ogasawara, J., Tanaka, T., Yoshida, N., and Nagata, S. (1995). Nature Genet. 11, 294-300. 579. Pfeffer, K., and Mak, T. (1994).Annu. Reu. Immunol. 12, 367-411. 580. Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A., and Korsmeyer, S. J. (1995). Science 270, 96-99. 581. Newmeyer, D. D., Farschon, D. M., and Reed, J. C. (1994). Cell 79, 353-364. 582. Nicotera, P., and Leist, M. (1997). Cell Death Differ. 4,435-442. 583. Guend, I., Sidoti-Defraisse, C., Gaumer, S., and Mignotte, B. (1997). Oncogene, 15, 347-360. 584. Beaver, J. P., and Waring, P. (1996). Cell Death Differ. 3,415-424. 585. Halestrup, A. P., Woodfield, K.-Y., and Connern, C. P. (1997).J. Biol. Chem. 272, 3346-3354. 586. Nicolli, A,, Basso, E., Petronilli, V., Wenger, R. M., and Bernardi, P. (1996).J. Biol. Chem. 271, 2185-2192. 587. Bemardi, P. (1992).J. B i d . Chem. 267, 883448839, 588. Szab6, I., Bemardi, P., and Zoratti, M. (1992).J. B i d . Chem. 267, 2940-2946. 589. Kroemer, G., Lisardo, B., Zamzami, P., Hortelano, S., and Martinez-A,, C. (1997). In “The Immunology Methods Manual” (R. Lefkovitz, Ed.), Chapter 14.2.,pp. 11111125. Academic Press, San Diego.

This chapter was accepted for publication on July 2, 1997.

ADVANCES I N I M M U N O L O G Y . V O L 68

Prenylation of Ras GTPase Superfamily Proteins and Their Function in lmmunobiology ROBERT B. LOBEU Merck Research labomtories, hporhnent of Cancer Research, Merck and Company, Inc., West Point, Pennsybanio 19486

I. Introduction

The Ras superfamily of GTPases comprises a diverse group of proteins that play critical regulatory roles in a variety of cellular processes involved in immune system function (see Fig. 1).Although members of this family play diverse roles in cells, they carry out their functions via similar biochemical mechanisms. First, these proteins cycle between GDP and GTP-bound states and rely on accessory proteins to regulate this GDP/GTP cycle. Second, many members of the Ras superfamily can regulate multiple signaling pathways through interactions with different downstream effector molecules. Third, these proteins all function at membrane surfaces and are localized to membranes via C-terminal lipid moieties that are added to the protein posttranslationally in a process commonly referred to as prenylation. Lipidation of the Ras superfamily of proteins involves a family of prenyltransferases, which attach isoprenoid-derived lipids consisting of 15 carbon units (farnesyl) or 20 carbons (geranylgeranyl) to C-terminal cysteine residues. In contrast to membrane insertion via transmembrane domains, membrane association via prenylation can be a transient and regulatable phenomenon. This transient association is essential to the function of some of the prenylated GTPases, in particular the Rab GTPases, which catalyze the intracellular flow of membrane compartments and that must cycle on and off membrane sites to function. This review will give an overview into the biochemistry and function of the Ras superfamily members, discuss the enzymology and functional consequences of protein prenylation, detail specific roles of the three major subfamilies of the Ras superfamily in immune system function, and discuss inhibitors of protein prenylation and their effects on the function of the Ras superfamily of proteins. II. The Ras Superfamily Members

The Ras superfamily can be subdivided into three major subfamilies, the Ras proteins, the Rho/Rac proteins, and the Rab proteins (Boguski and 145

Copynght 0 1998 by Acdrmir Press All nghts of reprmiuction in Any form reservc=d W65-277fiN8 $25 00

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FIG.1. Some ofthe functions of Ras superfamily proteins in immune cells. Ras superfamily members are indicated by the solid circles.

McCormick, 1993).The Ras proteins play a key role in signal transduction processes that regulate cell proliferation, activation, and differentiation. The Ras subfamily includes the mammalian ras alleles Harvey (H), N-ras, and Kirsten (K). The K-ras gene codes for two alternatively spliced variants, K4A and K4B, which are distinguished by the presence of a highly charged C-terminal region in K-Ras4B known as the polybasic domain (Barbacid, 1987). The expression of the different rus alleles vanes in different tissues (Leon et al., 1987), and there is some evidence to suggest that these different forms of Ras have somewhat different biochemical activity because mutant forms of these proteins differ in their ability to transform cells (Maher et al., 1995). The Ras proteins are closely related to the Rap proteins (RaplA, -1B, -2A, and -2B) and the R-Ras, RalA, RalB, and TC21 proteins. The Rho/Rac subfamily of proteins play many regulatory roles, including regulation of the actin cytoskeleton (Ridley, 1995),and regulation of the c-Jun kinasehtress activated protein kinase ( JNWSAPK) pathway.

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147

The Rho/Rac family includes RhoA, RhoB, RhoC, RhoD, RhoE, RhoG, Racl, Rac2, CDC42Hs, and TC10, which are -50% identical with each other and share -30% identity with other Ras-like GTPases (Nobes and Hall, 1994). The Rab subfamily of proteins consists of -30 members, which regulate the trafficking of intracellular membrane compartments (Pfeffer, 1994);this includes a role in regulating endocytosis and exocytosis, two types of membrane transport that are particularly important in immune cell function. 111. The GTPase Cycle

The members of the Ras superfamily of GTPases function as on-off switches that cycle between GTP-bound and GDP-bound states. They are in the “resting state” or “off” position when bound to GDP and activate their respective cellular processes when in the GTP-bound state. Turning these molecular switches on and off requires accessory proteins that are specific for the different members of the family (reviewed in Boguski and McCormick, 1993). The activation step involves guanine nucleotide exchange factors (GEFs),which facilitate dissociation of the bound GDP. Dissociation of the bound GDP enables the GTP to bind due to the high concentration of GTP in the cell relative to GDP (Bourne et al., 1991). The signaling pathway leading from transmembrane receptors such as the EGF or platelet-derived growth factor (PDGF) growth factor receptors to the activation of Ras is fairly well understood and proceeds through the activation of the guanine nucleotide exchange factor SOS. The initiating event is the binding of ligand to the receptor, which induces tyrosine autophosphorylation of residues in the receptor’s intracellular domain. The phosphorylated tyrosines of the receptor serve as docking sites for adapter proteins, such as Grb2 or Shc, which bind to the phosphotyrosines via their SH2 domains (Burgering and Bos, 1995).These adapter proteins also contain SH3 domains that mediate a binding interaction with polyproline stretches found on SOS (Quilliam et ul., 1995).Thus, receptor activation recruits the Grb2-SOS complex to the membrane, leading to guanine nucleotide exchange and activation of membrane-bound Ras. GTP binding is thought to induce a change in conformation that exposes the so-called effector domain, allowing the Ras protein to interact with downstream signaling effectors. A fairly detailed molecular description of the activation of Ras and its interaction with downstream effectors has been provided from crystallographic studies of Ras and Ras-related proteins (for a review, see Wittinghofer and Nassar, 1996). The members of the Ras family of proteins remain activated until their bound GTP is hydrolyzed. The Ras family members have weak intrinsic

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GTPase activity and require an interaction with another auxiliary protein, called a GTPase activating protein (GAP), to hydrolyze the bound GTP. In the case of Ras, Ras-GAP can accelerate the Ras GTPase reaction by almost five orders of magnitude (Gideon et al., 1992). Rho family members are activated by Rho-GAP domains, which are found in a variety of large, multifunctional proteins (Lamarche and Hall, 1994). The crystal structure of the Ras-GAP domain of p12OGAP, and the Rho-GAP domain from p50rhoGAP, have been solved recently (Barrett et al., 1997; Scheffzek et al., 1996). The deactivated GDP-bound form of the protein remains dormant until the proper activation stimulus is received and the protein can repeat the GTPase cycle. In the case of the Rho and Rab proteins, a third type of auxiliary protein, called a guanine nucleo'ide dissociation inhibitor (GDI),is involved in the GTPase cycle. GDI binds the GDP-bound form of the protein and, as the name implies, inhibits the dissociation of GDP. More important, the interaction of GDI with the Rho or Rab proteins prevents their binding to cellular membranes and, additionally, GDI can extract the Rho or Rab proteins from membranes (Wu et al., 1996). The ability of GDI to extract Rab proteins from membranes is critical to Rab protein cycling. GDI functions in retrieving the Rab protein from an acceptor membrane after a membrane vesicle fusion event has occurred and in delivering the Rab protein back to the donor membrane for another round of transport (Pfeffer, 1994; Soldati et al., 1994; Ullrich et al., 1994). IV. Downstream Signaling Effectors: Ras and the Rho/Rac Connection

A common feature of the Ras superfamily of GTPases, exemplified by both the Ras and Rho/Rac proteins, is the ability to activate multiple downstream effector pathways. Proteins that interact with the Ras effector domain include the Raf serinekhreonine kinase, phosphoinositide 3'kinase, MEK kinase (a kinase in the JNUSAPK kinase cascade), Ras GAP, Ral-GEF, and two proteins of unknown function (Rin, for Ras interacting; and Rsb; for Ras binding, Marshall, 1995; Wittinghofer and Nassar, 1996). Although the physiological importance of many of these effector interactions to the function of Ras remain unclear, the activation of the mitogenactivated protein kinase (MAPK) pathway via the Ras-Raf interaction is clearly important in transducing growth proliferation signals. The Ras-Raf interaction serves to localize Raf to the plasma membrane (Moodie et al., 1993; Van Aelst et al., 1993; Vojtek et al., 1993), where Raf itself becomes activated (Stokoe et al., 1994).One mechanism of Raf activation is through phosphorylation by a ceramide-activated protein (CAP) kinase (Yao et al., 1995). Recently, CAP kinase was shown to be the KSR (kinase suppressor

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of Ras) protein (Y. Zhang et al., 1997), which had been identified through genetic studies in Caenorhabditis elegans and Drosophila mlanogaster as being a modulator of the Ras-Raf pathway (Kornfeld et al., 1995; Sundaram and Han, 1995; Therrien et al., 1995).Once activated, Raf phosphorylates MEK (Dent et al., 1992; Howe et al., 1992; Kyriakis et al., 1992),a tyrosine/ threonine kinase that in turn phosphorylates MAP kinases such as Erk2. Phosphorylated Erk2 can then translocate to the nucleus, where it can phosphorylate transcription factors such as Elk-1. Elk-1 in turn can activate genes involved in cell growth, such as cfos. The ras genes, particularly K-ras and N-ras, are frequently mutated in a variety of human cancers; for example, N-ras in the case of acute myelogenous leukemia, and K-ras in carcinomas of the pancreas, lung, and colon (Bos, 1990).These mutations inactivate the GTPase activity of Ras, leaving the Ras switch stuck in the “on” position. The inability to turn off Ras leads to the transformed phenotype of the cancerous cells because they no longer require growth factor-induced transmembrane signals to initiate the signaling pathways leading to cell proliferation. In addition to stimulating uncontrolled proliferation via activation of the MAPK pathway, oncogenic versions of Ras also have a profound effect on cellular morphology. Ras-transformed cells growing in monolayer cell cultures are not contact inhibited as are normal cells, and they have a refractile appearance in the light microscope. The effect of Ras on cell morphology is most likely mediated through the Rho/Rac family. The involvement of Rho and Rac proteins in Ras-mediated cell transformation is illustrated by the ability of dominant-negative inhibitors of these proteins to block Ras transformation (Khosravi-Far et al., 1995; Prendergast et al., 1995; Qiu et al., 1995a,b). Although the exact molecular link between Ras and the Rho/Rac pathway is unclear, mutations in the Ras effector domain reveal that the Ras-Rho/ Rac connection is independent of the Ras-Raf interaction (White et al., 1995).One such Ras mutant is defective in its interaction with Raf and is unable to stimulate the MAP kinase pathway but still causes the change in cellular morphology typical of activated Ras (Joneson et al., 1996b; Khosravi-Far et al., 1996). Several mechanisms have been proposed to account for the effect of Ras on cell morphology. One possible mechanism is through stimulation of a Rho-GAP activity; the Ras effector domain can interact with Ras-GAP, which in turn has been shown to bind the p190 Rho-GAP protein (Foster and Hu, 1994). Alternatively, the Ras effector, phosphoinositide 3’ kinase (PIS-K), might be involved in linking Ras to the Rho/Rac pathway (Rodriguez-Viciana et al., 1997). This is indicated by the correlation between the ability of Ras effector domain mutants to affect the actin cytoskeleton and to bind to PI3-K, and by the finding that inhibition of PI3-K blocks Ras induction of membrane ruffling.

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V. Rho/Rac Effectors

Like Ras, the Rho family of proteins have multiple cellular effectors. These proteins were first noted for their effects on the cell cytoskeleton. Microinjection studies in fibroblasts have shown that Rho induces actin stress fiber formation, whereas Rac and Cdc42 induce membrane ruffling and the formation of filopodia and lamellipodia (Nobes and Hall, 1995). In addition to their regulation of the cytoskeleton, Rho family members regulate several different protein kinases. For example, CDC42, as well as Racl, regulates the JNWSAPK kinase cascade via an interaction with the P6EiPAK kinase (Coso et al., 1995; Minden et al., 1995). Racl and Cdc42 bind and activate the 70-kDa S6 kinase (Chou and Blenis, 1996),which has been shown to play an important role in cell cycle progression in many cell types including lymphocytes. Other downstream signaling targets of the Rho/Rac family include the pl2OAcKtyrosine kinase (Manser et al., 1993) and the p160HoCK serinekhreonine kinase (Ishizaki et al., 1996). Rho family members also function through signaling systems that involve lipid metabolism. For example, stress fiber formation induced by growth factors involves leukotriene generation via the metabolism of arachidonic acid. An activated (GTPase-defective) Rac mutant induces stress fiber formation and leukotriene generation in a growth factor-independent manner, and leukotriene synthesis inhibitors abrogate Rac-induced stress fiber formation (Peppelenbosch et al., 1995). Additionally, Cdc42Hs binds to the p85 subunit of PI3-K and regulates its activity (Zheng et al., 1994), whereas RhoA has been implicated in phospholipase D activation (see below). The list of Rho/Rac effectors is likely to grow because Burbelo et al. (1995) have identified a motif found in the GTPase binding sites of p120ACK and P65PAKthat is present in more than 25 proteins from a variety of eukaryotic species. Similar to what has been found for Ras, mutations in the effector domain of Rac and Cdc42 have been defined that prevent their ability to interact with P6SPAK and activate the JNK kinase pathway but do not affect their ability to regulate the cytoskeleton (Lamarche et al., 1996; Joneson et al., 1996a). VI. Prenylation of the Rar Superfamily Members

Since 1980 it had been known that Ras localized to the plasma membrane of cells and that this localization required posttranslational modification of the protein (Lowy and Willumsen, 1993). In 1984, it was shown that a cysteine residue in a CaaX motif found at the C terminus of Ras played a role in its membrane localization because a Cys to Ser substitution

151

PHEKYLATION OF R a GTP'sr PROTEINS

abolished membrane binding (Willumsen et al., 1984). Furthermore, this Cys to Ser mutation abolished the transforming ability of a GTPasedefective H-Ras mutant, suggesting that membrane localization was critical to the function of Ras. A number of observations made in the mid-1980s led to the definition of the nature of the Ras C-terminal posttranslational modification. It had been shown that an inhibitor of HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway (Fig. Z), blocked entry of cells into the S phase of the cell cycle (Schmidt et al., 1982). Furthermore, metabolites of exogenously added [3H]mevalonate, an intermediate in the cholesterol synthetic pathway, were incorporated into proteins (Schmidt et al., 1984).In 1986, genetic studies in yeast showed that posttranslational modification of yeast Ras and the yeast a-mating Factor, which also contained a CaaX at its C terminus, was controlled by the same genes (Powers et al., 1986).In 1988, the precise chemical structure of yeast a-mating factor was elucidated and shown to contain a C-terminal cysteine that was farnesylated via a thioether linkage and methylesterified

1 HMG-COA

1 I--

Lovastatin

Mevalonate

Cholesterol

1 1

Other

FTI

GGTl

- FTase

e GGPP

I

11

RPI

Methyl

___*

GGTase-l -CxC

GGTase-lI -cc

Methyl Transferase

~

,

-c-

s-9s

S - geranylgeranyl -C-OMe

s-99 s-99 , -C-X-C-OMe

s-99 C -OH

Frc;. 2. Biosynthetic pathway of prenylated proteins. The pathway is shown initiating from an early intermediate in the cholesterol biosynthetic pathway (HMG-CoA), leading to the prenylation enzyme snbstrdtes farnesyl diphosphate (FPP) and geranylgeranyldiphosphate (GGPP).The pathway branches through the three different prenyltransferase enzyrues and the subsequent processing enzymes. Sites of inhibition by existing pharinacologic agents, including HMG-CoA reductase inhibitors, FTIs, GCTIs, the CaaX protease inhibitor BPI, and the methyltrailsferase inhibitor AFC, are indicated. Abbreviations used: S, serine: M, rnethionine; Q, glutamine; L, leucine: X, any amino acid; gg, geranylgeranyl: -OMe, methylated carboy terminus.

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on its carboxylate group (Anderegg et al., 1988). From this precedent, similar modifications on the Ras C-terminal CaaX were demonstrated (Casey et al., 1989; Hancock et al., 1989; Schafer et al., 1989). VII. Prenylation and Processing of CaaX Substrates

Protein prenylation involves the covalent addition of two types of isoprenoids, farnesyl pyrophosphate or geranylgeranyl pyrophosphate, to cysteine residues at or near the C terminus. The farnesyl isoprenoid, a 15-carbon lipid, is an intermediate of the cholesterol biosynthetic pathway and is derived from the basic 5-carbon isoprenoid unit, isopentyl pyrophosphate (Fig. 2). Geranylgeranyl pyrophosphate contains an additional isoprenoid unit and is derived directly from farnesyl pyrophosphate. Three different enzymes, or prenyltransferases, have been identified that carry out these modifications (Zhang and Casey, 1996). Farnesyltransferase ( FTase) and geranylgeranyltransferase type-I (GGTase-I) are sometimes referred to as the CaaX prenyltransferases, because they catalyze the addition of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), respectively, to the cysteine residue in the sequence CaaX found at the C terminus of prenyltransferase substrates. A variety of proteins are substrates for the CaaX prenyltransferase enzymes (Table I), many of which are members of the Ras superfamily of small GTP-binding proteins. The third prenyltransferase enzyme, known as Rab geranylgeranyltransferase or geranylgeranyltransferase type I1 (GGTase-I1), adds geranylgeranyl groups to each cysteine in the XXCC, CCXX, and XCXC motifs at the C terminus of Rab proteins. VItI. CaaX Prenyltransferases

FTase and GGTase-I are both heterodimeric proteins that share a common 48-kDa a! subunit (Reiss et al., 1990; Seabra et al., 1991). The p subunits of FTase and GGTase-I are 46 and 43 kDa, respectively, and are approximately 30% identical at the amino acid level (Zhang et al., 1994). The genes for the yeast and mammalian prenyltransferases have been cloned and expressed in heterologous systems (Chen et al., 1991a,b; Fujiyama et al., 1987; Kohl et al., 1991; Omer et al., 1993; Powers et al., 1986). The a and /3 subunits of mammalian FTase are 30 and 37% identical to the corresponding yeast enzyme, encoded by the RAM2 and RAMl genes, respectively (Chen et a!., 1991a,b; Kohl et al., 1991; Omer et al., 1993). Mutations in either RAMl or RAM2 abolish the activity of FTase in yeast. FTase and GGTase-I both recognize the cysteine in CaaX motifs as the site for prenylation. In general, whether a protein is prenylated by FTase

PRENYLATION OF Ras CTPase PROTEINS

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or GGTase-I is defined by the X residue in the C a d motif; proteins with = serine, methionine, or glutamine are FTase substrates, whereas X = leucine for GGTase-I substrates (Casey et d.,1991; Moores et al., 1991; Yokoyama et al., 1991). The specificity of prenylation of CaaX substrates by FTase or GGTaseI is not always absolute because some proteins, such as K-Ras4B, can be both farnesylated by FTase and geranylgeranylated by GGTase-I in vitro (James et al., 1995; Moores et al., 1991). However, the catalytic efficiency (ke&,,,) for farnesylation of K-Ras4B by FTase is -140-fold greater than that for the geranylgeranylation of the protein by GGTase-I (F. L. Zhang et al., 1997). The preference for farnesylation of K-Ras4B is also reflected in vim, in which the protein is normally found in the farnesylated state (Casey et al., 1989). Other Ras isoforms, including K-Ras4A and N-Ras but not H-Ras, are also prenylated by both enzymes in uitro, with farnesylation being the preferred reaction (F. L. Zhang et al., 1997). The RhoB protein is another exception to the prenyltransferase specificity “rules” because this protein is both farnesylated and geranylgeranylated in vivo (Adamsonet al., 1992). RhoB is not an FTase substrate in vitro but rather is both farnesylated and geranylgeranylated by GGTase-I, with farnesylation being the preferred reaction (Armstrong et aZ., 1995). Some proteins, such as the heterotrimeric Gia subunit, contain an apparent CaaX motif but are not prenylated (Mumby et al., 1990). In the case of Gia, sequences upstream of its CGLF CaaX box apparently inhibit prenylation because the CGLF sequence confers prenylation when transferred to rus sequences (Cox et al., 1993). These data illustrate potential inaccuracies in predicting the nature of the prenyl group attached to a putative CaaX substrates based solely on prediction from the sequence of the CaaX. Characterization of the prenyl group on a CaaX substrate can be suggested by analysis of FTase and GGTase-I prenylation reactions on the substrate in vitro but should ultimately rely on characterization of the protein from cells or tissues. One commonly used method for characterization of the prenyl group involves labeling cells with [3H]mevalonate,which will incorporate into both farnesylated and geranylgeranylated proteins. After isolation of the labeled protein of interest, its labeled isoprenoid can be released via chemical means and identified by chromatographic separation and coelution with known standards (Casey et al., 1989). The potential for cross-prenylation of CaaX substrates in vivo, i.e., the farnesylation of GGTase-I substrates by FTase and vice versa, is suggested by the ability of proteins such as K-Ras4B to be prenylated by both FTase and GGTase-I. Studies in yeast illustrate the potential for cross-prenylation. It was shown that overexpression of the GGTase-I p subunit partially suppressed the growth defect of cells lacking FTase-P (Trueblood et al.,

X

TABLE I A CATALOG OF PRENYLATED PROTEINS Protein Ras proteins H-Ras N-Ras K-Ras4A K-Ras4B Ras-related proteins RaplA RaplB Rap2A Rap2B R-ras RalA RalB TC21 Rheb Rho proteins RhoA RhoB RhoC RhoD RhoE RhoG Cdc42Hs Racl Rac2 TClO

C terminus

Prenylation

CVLS CWM CIIM CVIM

F F F F

CLLL CQLL CNIQ CVIL CVLL CCIL CCLL CVIF CSVM

GG GG F GG GG GG GG GG F

CLVL CCKVL CPIL CCLAT CTVM CILL CVLL CLLL CSLL CLIT

GG FIGG GG F F GG GG GG GG GG

Protein Heterotrimeric G proteins yl (bovine, transducin) Y2 (ui2 a i3 Nuclear lamins Lamin A Lamin B cGMP phosphodiesterase (asub.)

C terminus

Prenylation

CVIS CAIL CGLF CGLF

F GG Not prenylated Not prenylated

CSIM CAIM

F F

CCIQ

F

Phosphorylase kinase (rabbit) CAMQ PXF (CHO cell) CLIM Interferon-inducible GTP binding proteins GBPl CTIS GBP2 CNIL Yeast YDJl CASQ CQTS Human HDJ2 Hepatitis delta virus large antigen CRPQ Protein tyrosine phosphatase, PRL-1 CCIQ Inositol triphosphate 5' phosphatase CWQ 2',5'-oligo(A) synthetase CTIL

F F F GG F F F F F GG

Rab proteins (selected members) Rabla cc cc Rab2 Rab3a CAC Rab3b csc Rab4a CGC Rab5 CCSN Rab5b CCSN Rab6 csc Rab’i dog csc Rab8 CVLL Rab9 dog CC RablO dog cc Rabll CCQNI

cli-GG di-GG di-GG di-GG di-GG di-GG di-GG di-GG di-GG GG di-GG di-GG di-GG

Note. The C-terminal amino acids (in standard amino acid code) and the prenylation state of each protein [farnesyl ( F ) or geranylgermyl (GG)]are indicated

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ROBERT B. LOBELL

1993). Similarly, overexpressionof two essential GGTase-I substrates, Rho1 and Cdc42, allows for growth of GGTase-I P-deficient cells: presumably, overexpression allows the cells to survive due to at least some level of farnesylation of these proteins (Ohya et al., 1993; Trueblood et al., 1993). Cross-prenylation has important implications in the context of pharmacological inhibition of prenyltransferases; alternative protein prenylation might rescue protein function in cells treated with a specific prenylation inhibitor (see Section XX). Structural information on prenyltransferases will further our understanding of the determinants of substrate specificity of FTase and GGTase-I and, in this regard, the structure of Rat FTase was recently solved by X-ray crystallography (Park et al., 1997). Although the crystal structure was determined in the absence of either substrate, the location of the active site of the enzyme was surmised based on the location of a bound zinc atom. It has been shown that the cysteine thiol of a CaaX peptide substrate coordinates to the zinc metal in a ternary complex consisting of enzyme, peptide, and FPP, indicating a direct role of the zinc in catalysis (Huang et al., 1997). The structure showed that the zinc atom of FTase is in close proximity to a hydrophobic pocket found in the P subunit of FTase. This hydrophobic pocket is likely the binding site for FPP because it is of sufficient length to accommodate FPP but not the larger GGPP molecule, consistent with the observation that FPP binds 15-fold tighter than GGPP to FTase (Yokoyama et al., 1997). In addition, Park et al. (1997) proposed a model for the interaction of the CaaX motif with active site residues of FTase. The model was based on the location of the nine C-terminal amino acids of the P subunit, which for some reason inserted into the active-site region of the adjacent aJP dimer in the crystal structure. Although this model provides a useful starting point for further studies, it is not supported by recent site-directed mutational data, which showed that mutation of three residues, Ser159, Tyr362, and Tyr366, changed the substrate specificity of yeast FTase to that of GGTase-I (DelVillar et al., 1997). The crystal structure model did not implicate these residues as being directly involved in CaaX substrate binding, although it cannot be ruled out that mutation of these residues changes substrate specificity through indirect effects on neighboring residues. Evidence from circular dichroism analysis indicates that FTase undergoes conformational changes upon binding CVIM peptide, FPP analogs, or tetrapeptide inhibitors of the enzyme (Wallace et al., 1996). Thus, the structure of the apoenzyme might not accurately reflect the structure of the active site with ligands bound. Further structural information, particularly data derived from enzyme-substrate or enzyme-inhibitor complexes, will enable a more precise

PRENYLATION OF Ras GTPase PROTEINS

157

definition of the molecular interactions involved in substrate recognition by FTase and GGTase-I. IX. CaaX Protease and Carboxymethyltransferase

Proteins modified by FTase and GGTase-I undergo additional Cterminal processing steps (Fig. 2). The C-terminal aaX is cleaved from the protein by a microsomal protease and the resulting C-terminal prenylated cysteine is carbolo/methylated. A protein activity that binds both farnesylated and geranylgeranylated proteins that contain an intact aaX C terminus has been postulated to play a role in these additional processing steps by localizing prenylated proteins to the membrane surfaces where the CaaX protease and methyltransferase activities reside (Thissen and Casey, 1993). Two Saccharomyces cerevisiae genes, Rcel and Afcl, are required for the C-terminal proteolysis of prenylated proteins (Boyartchuk et al., 1997). The AFCl protein is a zinc-dependent metalloprotease that is required for proteolysis of the yeast mating pheromone, a-factor, but is not essential for processing of yeast Ras. RCEl is essential for processing of both afactor and Ras. The mammalian protease activity responsible for processing of prenylated proteins has only been partially characterized. Proteolytic activity capable of releasing the Val-Ile-Met tripeptide from the tetrapeptide substrate, N-acetyl-S-farnesyl-L-Cys-Val-Ile-Met, is localized to membranes, is not affected by standard protease inhibitors, and displays properties consistent with a serine or cysteine protease (Ma et al., 1993). This proteolytic activity requires detergent for its solubilizationfrom membranes and chromatographs as a single peak of activity over gel filtration and anion exchange chromatography (Chen et al., 1996). Tetrapeptide inhibitors of the CaaX protease, such as RPI (Fig. 3), have been developed and have been reported to block Ras processing and function in cells (Chen et al., 1996). The mammalian enzyme(s) responsible for C-terminal methylation of prenylated proteins is also incompletely characterized. In yeast, the methyltransferase for CaaX proteins has been identified as Stel4 (Hrycyna and Clarke, 1990; Marr et al., 1990).Prenylcysteine-directed mammalian methyltransferase enzyme(s) is associated with microsomal membranes and utilizes S-adenosylmethionine as the methyl donor (Stephenson and Clarke, 1992). Methylation reactions are potentially reversible and, in this regard, a methylesterase has been described that is selective for prenylated cysteines containing methylesters (Tan and Rando, 1992). The ability to add or remove methyl groups from the C terminus of prenylated proteins suggests that these events could regulate the function of prenylated proteins, and there is some evidence to suggest that this does occur (see Section XIV).

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FPP

P

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L-704,272

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BMS-186511 Fic;. 3. Representative inhibitors of prenyltransferases and other enzymes in the biosynthesis of prenylated proteins. Shown at the top are the substrates for the farnesylation of k-Ras (FPP) and the k-Ras CaaX (CVIM). See text for details and references.

X. Rab GGTase-ll

Rab proteins are digeranylgeranylated by GGTase-11, a heterodimeric enzyme containing a 50-kDa a subunit and a 38-kDa 6 subunit that share approximately 30% identity in amino acid sequence with the cdfi subunits

CVlM

CaaX Competitive FTl‘s

I

L-731,734

I L-739,749 (kCH3) L-744,832 (R=CHz(CH&

FTI-276 (R=H) sFTI-277 (R=CH3)

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8956 (R=H) 81086 (R=CHa)

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SCH44342

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ROBERT B. LOBELL

GGTase-I lnhlbitor

GGTI-286: R=OCHB

I

GGTI-287: R=O-

HMG-CoA Reduction Inhibitor

Lovastatin

L

CaaX Proteare Inhibitor

Carboxymbthyitransferaselnhibltor

t?+n L-AFC(1)

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FIG.3-Continued

of FTase and GGTase-I (Armstrong et al., 1993).GGTase-I1 adds geranylgeranyl groups to each cysteine residue at the C terminus of Rab proteins, which end in CCXX, XXCC, or CXC sequences (Farnsworth et al., 1994).

PRENYLATION OF

Ray

GTPase PROTEINS

161

Rab proteins are not proteolyzed at their C termini, and only Rab proteins

with the CXC motif are carboxymethylated (Smeland et al., 1994). Prenylation of Rab proteins by GGTase-I1 requires a third protein called Rab escort protein or Rep. Rep functions by presenting the unprenylated Rab protein to the catalytic GGTase-I1 d p heterodimer (Andres et al., 1993; Seabra et al., 1992). Mutational analysis of Rab proteins has shown that in addition to the C-terminal cysteines, internal Rab protein sequences are involved in the prenylation reaction (Wilson and Maltese, 1993). The effect of these mutations may be due to effects on the Rab-Rep interaction. Rep binds both unprenylated and prenylated Rab proteins; due to this property, the GGTase-I1 reaction in uitro is limited by the concentration of Rep. MonogeranylgeranylatedRab protein remains tightly bound to Rep, even in the presence of detergents, ensuring that the second geranylgeranyl group can be added by GGTase-11. Although digeranylgeranylated Rab exhibits a somewhat greater propensity to dissociate from Rep in the presence of detergents or phospholipids (Shen and Seabra, 1996) in vim the prenylated Rab likely remains bound to Rep until it is delivered to the correct intracellular membrane compartment (Alexandrovet al., 1994). In uiuo, delivery of the Rab protein to the correct membrane compartment presumably facilitates the dissociation of the prenylated Rab protein from Rep, although it is not known what directs the Rab protein to its correct membrane compartment. Another aspect of the Rab-Rep interaction that is not well understood concerns the low affinity of Rep for Rab-GTP. Because GTP is found at much higher concentrations than GDP in cells, newly synthesized, unprenylated Rab might bind GTP and then would be less able to bind Rep. One possibility is that a chaperone protein might bind newly synthesized Rab in a conformation that prevents GTP binding and allows the Rab to bind Rep (Desnoyers et al., 1996). Two Rep proteins, Repl and Rep2, have been identified. A defect in Repl function is responsible for choroideremia, a human retinal degenerative disease (Andres et al., 1993). Repl and Rep2 are 75% identical and are generally redundant in activity except for two known examples. Rab27, a protein found in high levels in the retina, has a somewhat higher affinity for Repl compared to Rep2, which might explain why the effects of choroideremia are limited to the retina (Seabra et al., 1995). Additionally, the prenylation rate of Rab3a is lower when Rep2 is the escort in the reaction (Cremers et al., 1994). Rep proteins are 30%identical in amino acid sequence to the Rab-GDI protein (Wu et al., 1996). X-ray crystallography of Rab-GDI has revealed that two of the most highly conserved regions between Rep and GDI are found on one face of GDI (Schalk et al., 1996). Mutational analysis of GDI suggests that this protein surface is involved in the interaction of

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GDI, and presumably Rep, with Rab proteins (Wu et al., 1996). Although there are similarities to the interaction of Rep and GDI with Rab proteins, there must be significant differences because only Rep can bind unprenylated Rab and thus only Rep can facilitate the geranylgeranylation reaction (Pfeffer et nl., 1995). In addition, GDI but not Rep apparently interacts with the effector domain on the Rab protein. A single mutation in the RablB effector domain abolishes the GDI-Rab binding interaction but does not affect the geranylgeranylation reaction, indicating that the mutant protein retains the ability to interact with Rep (Wilson et al., 1996). In cells, this RablB effector domain mutant was targeted to the correct intracellular compartment but was unable to cycle between membrane and cytosolic compartments (Wilson et al., 1996). This result is consistent with a model in which Rep functions in the delivery of prenylated Rab proteins to donor membranes, whereas GDI functions specifically in the recycling of Rab back to donor membranes after the vesicle fusion process has occurred. XI. Role of Prenylation in Membrane Binding and in Prokin-Prokin Interactions

Ras proteins containing Cys to Ser mutations in the CaaX are not prenylated and are not membrane bound (Willumsen et nl., 1984). Although farnesylation of Ras is clearly a critical component to its membrane localization, the proteolytic cleavage of the a& and the methylation of the farnesyl cysteine at the mature C terminus are also important. An in vitro system utilizing rabbit reticulolysates, reconstituted with or without microsomal membranes containing CaaX protease and methyltransferase activity, can produce Ras in various states of posttranslational processing. Utilizing this system, it was found that farnesylation of K-Ras4B in the absence of proteolysis and methylation results in only 20% of the K-Ras4B protein being associated with membrane fractions (Hancock et al., 1991a). Forty percent of farnesylated and proteolyzed K-Ras4B associated with membranes, whereas addition of the carboxymethylation activity led to 80% of the fully processed protein associated with the membrane. Similarly, a KRas4B CaaX mutant that can be farnesylated but not further processed is approximately 50% membrane associated in cells compared to a >90% association of the wild-type protein (Kato et ul., 1992). The importance of the carboxymethylation event to membrane binding is further illustrated by in vitro analysis of the binding of prenylated peptides to liposomes. Farnesylated peptides bind poorly to liposomes unless the farnesyl cysteine is methylated, whereas geranylgeranylated peptides bind reasonably well in the absence of methylation (Silviusand L’Heureux, 1994).The difference between farnesylated and geranylgeranylatedpeptides in their requirement

PRENYLATION OF Ras GTPasv PROTEINS

1ti3

for carboxymethylation for binding to membranes reflects the greater lipophilicity of the geranylgeranyl group. In addition to C-terminal farnesylation, proteolysis, and methylation, other mechanisms, including the addition of other lipid moieties in the case of H-Ras or the presence of multiple-charged amino acid residues in the case of K-Ras4B, contribute to the binding of Ras and other prenylated proteins to cellular membranes. A specific palmitoyltransferase covalently modifies H-Ras and N-Ras on cysteine residues in their C-terminal region with the 16-carbon lipid, palmitate (Liu et al., 1996). The palmitoylation reaction apparently requires that the proteins are first farnesylated and further processed because nonfarnesylated, bacterial-expressed H-Ras is not a substrate for the palmitoyltransferase (Liu et al., 1996). C h a n p g the two cysteines in H-Ras that are normally palmitoylated to serine residues prevents palmitoylation, results in a 10-foldreduction in membrane binding compared to the palmitoylated protein (Hancock et al., 1990), and significantly impairs its signaling ability (Dudler and Gelb, 1996).The C-terminal polybasic domain of K-Ras4B, which contains a stretch of six lysine residues adjacent to the CaaX, was shown to contribute significantlyto its membrane binding in cells (Hancock et al., 1990).Changing these lysines to glutamine impairs the membrane binding of K-Ras4.B; as the number of lysines in the polybasic region is progressively decreased, the affinity of the protein for membranes is also progressively decreased. Other prenylated proteins in the Ras superfamily also contain polylysine stretches adjacent to their Cadi. These studies indicate that all the C-terminal processing events, including prenylation, proteolysis, carboxymethylation, palmitoylation, as well as the polybasic domain of K-Ras4B, all contribute to the membrane binding of Ras proteins. However, mutations that abolish palmitoylation, proteolysis, or the polybasic domain only slightly impair the transforming ability of Ras proteins, whereas Cys to Ser mutations in the CaaX completely abolish the transforming ability (Hancock et al., 1990; Kato et al., 1992). These mutational studies show that the only processing event that is absolutely critical to the transforming ability of Ras is the farnesylation step. However, these transformation studies should be interpreted with caution because they involve overexpression of the Ras protein. A mutant version of Ras that is partially impaired in its membrane binding might reach the threshold level of signaling at the plasma membrane that is required for transformation of the cell only when the protein is overexpressed. Although the C-terminal lipidation of Ras is critical for its function, this requirement can be abrogated by artificially targeting the protein to membranes via introduction of a lipid functionality at the N terminus of the protein. This has been accomplished by introducing the v-Src N-

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myristoylation sequence at the N terminus of Ras (Buss et at., 1989). Furthermore, it has been suggested that the sole function of Ras is to tether the signaling molecules downstream of Ras to the plasma membrane. This is supported by the finding that a Raf kinase construct containing a CaaX motif is transforming to cells even in the presence of a dominantnegative Ras protein that can normally inhibit transmembrane signaling to the MAPK pathway (Leevers et al., 1994; Stokoe et al., 1994). Although the processed, prenylated C terminus of Ras mediates its membrane association, it is unclear what directs Ras to the plasma membrane rather than to other intracellular membrane compartments. It would seem that other signals are required for targeting Ras to the proper membrane compartment because farnesylated proteins other than Ras can be localized to other membrane structures such as the nucleus in the case of the lamins (Brown et al., 1992; Chen et al., 1991b; Reiss et aE., 1991) and the cytoplasmic surface of peroxisomes in the case of a protein of unknown function known as PXF (James et al., 199413). Although prenylation of Ras is important in localization of the protein to membrane surfaces, prenylation also plays a role in protein-protein interactions. For example, yeast Ras2 regulates the enzyme adenylyl cyclase, and farnesylation is required for this interaction. The interaction of unprocessed Rase with solubilized adenylyl cyclase is approximately 100fold less than when Ras2 is farnesylated (Kuroda et al., 1993). The interaction of H-Ras and K-Ras with the guanine nucleotide exchange protein, SOS, is influenced by prenylation (Porfiri et al., 1994; McGeady et al., 1997). SOS fails to catalyze nucleotide exchange of unprocessed H-Ras and K-Ras, and addition of the 10-carbon geranyl group fails to reconstitute the interaction. The exchange reaction occurs with Ras modified with farnesyl, analogs of farnesyl such as tetrahydrofarnesyl, and geranylgeranyl groups, and proceeds to a greater extent when it is fully processed (McGeady et al., 1997). Although these results indicate that SOS interacts, at least in part, with the prenyl group of Ras, they do not exclude the possibility that the prenyl group induces a structural change in Ras that enables it to interact with SOS. The contribution of prenylation and carboxymethylation to membrane binding is further illustrated from studies of other prenylated proteins, notably the heterotrimeric G-proteins. These GTPases are localized to the plasma membrane via myristoylation and palmitoylation of the G, subunit and prenylation, either farnesylation or geranylgeranylation, of the Gy subunit (see Table I and Higgins and Casey, 1996). Transducin, a Gprotein found in the retina, contains a farnesylated G, that is found in both methylated and unmethylated forms. Both farnesylation and methylation

PRENYLATION OF Ras GTPase PROTEINS

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of G, contribute to the membrane binding of this protein (Parish and Rando, 1994). Prenylation also plays an important role in many aspects of proteinprotein interactions involving heterotrimeric G-proteins, including intrasubunit interactions, interactions between the G-protein and the transmembrane receptor, and perhaps interaction of the G-protein subunits and downstream signaling effectors. Heterotrimeric G-proteins dissociate into G, and Gpysubunits when ligand binds to the seven-transmembrane receptor to which the G-protein was originally bound. The G, dimer is extremely stable, and its initial assembly appears to be influenced by prenylation. The assembly of the Gpycomplex is thought to occur prior to prenylation and proteolytic processing of G,; this is suggested by the finding that proteolysis of the a& of prenylated G, impedes complex formation with G, (Higgins and Casey, 1994). The high-affinity interaction of Gp, with G, requires the prenylation of G, (Higgins and Casey, 1994) and the myristoylation of G, (Linder et al., 1991). Farnesylated peptides correspondmg to the C terminus of G, can inhibit the interaction of Gpywith G, and the degree of inhibition increases as the hydrophobicity of the prenyl group is increased by either methylation of the farnesylated peptide or geranylgeranylation of the peptide (Matsuda et al., 1994). The prenylation and methylation state of the G, subunit can also influence the interaction of the heterotrimeric G-protein with the seven-transmembrane receptor. In the case of transducin, both farnesylation and methylation are required for high-affinity binding to the receptor rhodopsin (Fukada et al., 1994), and as with the G,, and G, interaction, a farnesylated peptide corresponding to the C terminus of G, can disrupt the rhodopsin-transducin complex (Kisselevet al., 1994). Prenylation and methylation of G, is also critical for the interaction of G, with downstream effectors, as has been demonstrated in the regulation of a phospholipase CP, (Parish et al., 1995), although it is not clear if this effect is due to enhanced membrane binding of the fully processed Go, or to a specific G,,-phospholipase CP, interaction. In the case of Rab proteins, prenylation plays a role in the interaction of Rab with Rep and GDI. In addition to promoting the association of Rab with membranes (Overmeyer and Maltese, 1992), prenylation of Rab is required for binding to GDI (Musha et al., 1992).Although both monoand digeranylgeranylated Rab proteins can associate with GDI, the length of the prenyl group affects the ability of Rab to bind GDI because a Rab5 mutant with a farnesylation site in place of the geranylgeranylation sites binds weakly to GDI (Ullrich et al., 1993).Another system in which prenylation has been shown to affect protein-protein interactions is in the biosynthesis of hepatitis delta virus. Prenylation of the large antigen of hepatitis

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delta virus is required for its assembly with the hepatitis B surface antigen in the formation of hepatitis D virus particles (Hwang and Lai, 1993). XII. Role of Ras GTPase Family Members in Immunobiology: The Ras Pathway

Many of the components of the Ras signaling pathway, which were originally defined from work involving growth factor signaling in fibroblasts, have now been demonstrated in cells of the immune system. In T lymphocytes, the Ras pathway has been shown to be important in the immediate activation events triggered via the T cell antigen receptor (TCR), which leads ultimately to IL-2 secretion and upregulation of IL-2 receptors (IL2R). The Ras pathway is also involved in the proliferative events in T cells triggered by binding of IL-2 to its receptor (IL-2R) (Pastor et al., 1995). The Ras pathway is activated in other antigen receptor signaling systems related to the TCR, including the B cell antigen receptor (Cambier et al., 1994), and in mast cell activation via the high affinity receptor for IgE, FceRI (Fukamachi et al., 1993; Turner et al., 1995). The Ras pathway is involved in other aspects of lymphocyte biology, including the regulation of B cell function by the CD40 receptor (Gulbins et al., 1996a) and T cell activation via engagement of the GD3 disialoganglioside (Ortaldo et at., 1996). As is the case in growth factor receptor signaling in fibroblasts, signaling from lymphocyte antigen receptors and the IL-2R involves activation of Ras via the SOS guanine nucleotide exchange factor through a series of protein-protein interactions that initiates with tyrosine phosphorylation events (Quilliam et al., 1995). In the case of growth factor receptors, intrinsic tyrosine kinase domains in the receptor autophosphorylate receptor tyrosine residues which serve as adapter sites for the GrbYSOS complex, which in turn activates Ras. The TCR and the IL-2R lack intrinsic kinase activity but initiate the Ras pathway through receptor-associated kinases of the Src family, including ZAP-70, p56Ick,and ~ 5 9 s in " the case of the TCR, and through activation of ~ 5 6 'in ' ~the case of the IL-2R (Weiss and Littman, 1994).These kinases phosphorylate multiple substrates, including proteins that couple to the GrbYSOS complex. The phosphorylated proteins that link SOS/Grb2 to the TCR and IL-2R appear to be different. In the case of the TCR, a 36-kDa protein serves as the phosphoprotein adapter to Grb2 (Buday et al., 1994; Reif et al., 1994; Sieh et al., 1994), whereas for IL-2R, the Shc protein is phosphorylated by p56Ick,coupling the activated kinase to the GrbYSOS complex(Ravichandran and Burakoff, 1994). FcsRI activation of Ras in mast cells has also been shown to involve the GrbYSOS pathway (Turner et al., 1995); it is not clear which phospho-

PHENYLATION OF Has CTPasr PROTEINS

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protein adapter couples the Src kinases associated with this receptor to GrbWSOS. The events downstream of Ras are fairly well understood in the case of signaling via the TCR (Pastor et d., 1995). Ras activation in response to TCR activation leads ultimately to upregulation of a transcription factor that activates the IL-2 promoter, known as nuclear factor of activated T cells (NFAT). NFAT is a complex of AP-1, itself a complex of the Fos/ Jun factors, and NF-ATp, a member of the c-re1 family of transcription factors, (€320, 1994). The pathway leading to activation of NFAT by Ras most likely involves activation of the MAPK pathway. Activation of Erk2 via Raf and MEK has been demonstrated in T lymphocytes (Izquierdo et al., 1993, 1994; Franklin et d., 1994). It is likely that NFAT activation by Ras ultimately involves activation of AP-1 through the induction of the cfos gene; this could occur via activation of the Elk-1 transcription factor by the Erk2 map kinase (Marais et al., 1993).As in the T cell, IgE receptor activation in the mast cell leads to activation of NFAT via the Ras-RafMek-map kinase-Elk-1 cascade (Turner and Cantrell, 1997). Additionally, another member of the Ras superfamily, Rac-1, has been implicated in NFAT activation in mast cells (Turner and Cantrell, 1997). In granulocytes such as the neutrophil, Ras is activated in response to proinflainmatory mediators. For example, Ras and its downstream effectors, Raf and MAPK, are activated in human neutrophils in response to the chemoattractants FMLP and C5a (Buhl et al., 1994; Worthen et al., 1994). The FMLP and CSa receptors are seven-transmembrane spanning G-protein coupled receptors, and the activation of the Ras pathway in neutrophils via these receptors is sensitive to pertussis toxin. The linkage between the G-protein coupled receptors and the Ras pathway in the neutrophil has not been firmly established but appears to involve the Src-family kinase Lyn. FMLP stimulates Lyn, which in turn binds and phosphorylates Shc; this could lead to Ras activation via phosphorylated Shc binding to GrbWSOS (Ptasznik et al., 1995). There is some data to suggest that the expression of proteins in the Ras pathway can be modulated in the neutrophil in viva in response to inflammatory stimuli. Neutrophils from bum patients contain elevated levels of Ras and Ras-GAP but reduced levels of Rapl, a Ras superfamily member that regulates the NADPH oxidase (Brom et al., 1993). Neutrophils from burn patients exhibit impaired chemotactic and phagocytic function, although it is not clear what role, if any, the elevation of Ras protein levels has in this impaired function. Although Ras activation plays a growth stimulatory role in T cells, there is evidence that in some settings, it can actually transduce growth inhibitory and apoptotic signals. For example, in a recent study it was shown that Ras negatively regulates calcium-dependent immediate early gene induction in

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lymphocytes (Chen et al., 1996). Furthermore, FAS-induced apoptosis in lymphocytes involves Ras activation through a pathway involving ceramide generation via a sphingomyelin signaling pathway (Gulbins et al., 1995). This was indicated by an increase in Ras-GTP levels upon FAS stimulation of Jurkat cells and by the inhibition of FAS-induced apoptosis by a dominant-negative Ras mutant. FAS-induced events downstream of Ras involve the generation of superoxide anions (Gulbins et al., 1996b), which have also recently been implicated in signaling events downstream of Ras induced by mitogenic stimulation of NIH-3T3 fibroblasts (Irani et al., 1997). Ras activation is also involved in apoptosis induced by the cytokine, tumor necrosis factor (TNF) (Trent et al., 1996). Like FAS, TNF activates a sphingomyelin pathway leading to ceramide production, which has been shown to cause phosphorylation and activation of Raf via a CAP kinase (Yao et al., 1995). It remains to be seen whether FAS-induced apoptosis, which involves ceramide generation, also results in CAP kinase and Raf activation. It is apparent that further studies are needed to sort out the tangle of signaling pathways in lymphocytes and other cells that involve Ras, which can result in a variety of responses, including activation, growth, or cell death. XIII. The Rho/Rac Pathway and leukocyte Function

Most cells of the immune system are motile and migrate in response to specific chemotactic stimuli. Leukocyte migration involves integrindependent adhesioddeadhesion events and changes in the cell cytokeleton, including actin polymerization and membrane ruffling (Stossel, 1993). There is direct evidence from studies in immune cells that integrindependent adhesion events and changes in the cytoskeleton involve Rho proteins. The involvement of Rho proteins in chemoattractant-induced effects on integrin-dependent adhesion was illustrated in a recent paper in which a lymphoid cell line, transfected with the FMLP or IL-8 chemoattractant receptors, showed agonist-stimulated activation of nucleotide exchange on RhoA within seconds (Laudanna et al., 1996). Furthermore, in this paper it was demonstrated that Clostridium botulinurn toxin C3 ADP ribosyltransferase, an enzyme that inhibits Rho function through ADP ribosylation, blocked agonist-induced lymphocyte a 4 p l integrin-mediated adhesion to vascular cell adhesion molecule-1 and also blocked neutrophil p2 integrin-mediated adhesion to fibrinogen. The involvement of Rho proteins in cytoskeletal organization in leukocytes is further supported by the finding that the C3 ADP ribosyltransferase inhibits actin microfilament formation and chemoattractant-induced motility in neutrophils (Stasia et al., 1991). The C3 ADP ribosyltransferase also inhibits events that involve

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leukocyte cell-cell interactions, including the CD1ldCD18-dependent homotypic aggregation of B cells (Tominaga et al., 1993) and the cytolytic function of cytotoxic T cells (Lang et al., 1992). Additionally, CDC42Hs is required for the polarization of T cells toward antigen presenting cells (Stowers et al., 1995). Further evidence for the involvement of Rho family members in leukocyte cytoskeletal organization and cell motility comes from studies of the Wiskott-Aldrich syndrome (WAS).WAS is a hematopoietic disorder characterized by thrombocytopenia, recurrent infections, and eczema (Ammann and Hong, 1989).The cellular abnormalities in WAS patients include cytoskeletal defects in T cells and platelets (Molina et al., 1992) and defective neutrophil chemotaxis (Ochs et al., 1990). The genetic defect in WAS has been mapped by positional cloning (Derry et al., 1994). The involvement of the WAS protein (WASP) in regulation of actin polymerization was demonstrated by the recent finding that WASP binds to CDC42Hs (Symons ef al., 1996). Overexpression of WASP produced intracellular clusters of the protein that were highly enriched in polymerized actin; formation of these clusters was inhibited by coexpression of dominantnegative CDC42Hs-Nl7. Thus, mutation of WASP, a downstream effector of CDC42Hs, can have profound effects on immune cell functions that involve regulation of the cytoskeleton. XN. Regulation of the Neutrophil NADPH Oxidose by Roc and Rap

Activation of neutrophils by proinflammatory mediators, including the chemoattractant peptides FMLP and C5a, leads to a number of cellular responses including the generation of toxic and microbiocidal oxygen metabolites such as superoxide anion and hydrogen peroxide. This event, termed the respiratory burst, is due to activation of the multisubunit NADPH oxidase complex (Chanock et al., 1994). The oxidase consists of a heterodimeric flavocytochrome b that consists of 22- and 91-kDa transmembrane protein components (Parkos et al., 1987). The oxidase is also composed of two cytoplasmic proteins, p47P""' and p67P"", that bind tightly to the transmembrane oxidase components upon cell activation (Clark et al., 1990) . The inability to generate the respiratory burst seriously compromises the host defense system, as evidenced by individuals with chronic granulomatous disease, a hereditary condition caused by a mutation in one of the NADPH oxidase components (Dinauer, 1993). The NADPH oxidase is regulated by two geranylgeranylated GTPbinding proteins, RaplA and Rac. The involvement of these GTP-binding proteins was first suggested by the requirement for guanine nucleotides in the activation of the oxidase in a cell-free system (Gabig et al., 1987),

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and the involvement of RaplA in the NADPH oxidase system was first suggested by its association with purified neutrophil flavocytochrome b (Quinn et al., 1989).This interaction is functionally important in NADPH oxidase function because cytosol immunodepleted of RaplA is unable to reconstitute oxidase activity unless recombinant RaplA is added back (Eklund et al., 1991). Furthermore, dominant inhibitory mutants of RaplA inhibit the NADPH oxidase when expressed in differentiated HL-60 cells and EBV-transformed B cells (Gabig et al., 1995; Maly et al., 1994). Rac was first shown to play a role in regulation of the NADPH oxidase by experiments in the cell-free system (Abo d. al., 1991; Knaus et al., 1991).In unstimulated cells, Rac is present in the cytosol and is complexed with RhoGDI (Aboet al., 1994). Upon immunologic activation, Rac dissociates from RhoGDI and translocates to the plasma membrane (Abo et al., 1994; Quinn et al., 1993). Rac translocates independently of the p47PhoX and p67ptiox proteins and interacts with both the p2Wp91 flavocytochrome subunits and with p67P’’0xvia its effector domain (Diekmann et al., 1994; Heyworth et al., 1994). The role of the Rac geranylgeranyl group in activation of the NADPH oxidase has been examined. Unprenylated Racl was found to activate the oxidase in the cell-free system, but only when it was preloaded with GTPyS (Heyworth et al., 1993).This suggested that prenylation of Rac is required only in the activation of Rac itself, presumably through an interaction of Rac with a guanine nucleotide exchange protein, but that prenylation is not absolutely required for the activation of the oxidase by Rac. However, recent evidence suggests that prenylation of Rac is an important determinant in the activation of the oxidase. It was shown that prenylated Racl and Race are significantly more effective in activating the oxidase in vitro than the nonprenylated forms (Kreck et nl., 1996). Racl is a more effective activator than Race; this is likely due to the presence of a polybasic domain near the CaaX of Rac, as is found in K-Ras4B. The polybasic domain of Racl is an important determinant of membrane binding because elimination of only one of the charged residues markedly reduces its activation of the oxidase. The polybasic domain contributes to the membrane binding of Rac via electrostatic interactions. This is indicated by the finding that the activation of the oxidase by Racl but not by Rac2 is sensitive to salt concentration, and that addition of acidic phospholipids to reconstituted oxidase subunits enhances the activation by Racl but not by Rac2. The exact role that Rac plays in the regulation of the NADPH oxidase remains unclear, although it is reasonable to propose that its function may be to anchor the soluble p47Phohat the plasma membrane in proper orientation to the transmembrane oxidase components (Kreck et al., 1996).

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XV. Regulation of Phospholipase D by RhoA

There may be another level of regulation of the NADPH oxidase by a Rho protein. Phosphatidic acid, a product of the hydrolysis of phospholipids by phospholipase D (PLD),enhances oxidase activity (Agwu et al., 1991). PLD activity in human neutrophils is activated by GTPyS and this effect was suggested to be mediated by a Rho protein because Rho-GDI can inhibit the activation of PLD (Bowman et nl., 1993). Depletion of Rho from membranes with Rho-GDI, followed by add back of recombinant Rho to the membrane, showed that RhoA but not Cdc42Hs could reconstitute PLD activity in rat liver membranes and in human neutrophil membranes(Kwak et al., 1995; Malcolm et nl., 1994). Evidence for Rho protein involvement in receptor-mediated PLD activation in intact cells has been obtained through the inactivation of Rho proteins with either the Clostridiurn botulinurn C3 ADP ribosyltransferase or the Clostridiurn dificile toxin B, a Rho glucosylation enzyme (Malcolm et al., 1996; Schmidt et al., 1996). RhoA activation of PLD has also been implicated in IgE receptor-mediated mast cell activation because the C. dificile toxin B abolishes antigeninduced PLD activation and granule enzyme release (Ojio et al., 1996). However, the involvement of RhoA in the activation of PLD has been questioned by a recent study involving HL-60 cells; this study reported that depletion of RhoA from membranes with Rho-GDI had no effect on PLD activity and attributed the activation of PLD by GTPyS to the GTPbinding protein, Arf (ADP ribosylatioii factor) (Martin et al., 1996). XVI. Role of C-Terminal Methylation of Prenylated Proteins in NADPH Oxidase Regulation and Other Leukocyte Functions

It has been suggested that the C-terminal inethylation of Rac and Rap proteins is regulated and plays a role in the translocation of these proteins to the plasma membrane upon FMLP-induced activation of the NADPH oxidase in neutrophils. The amount of carboxymethylation of Ras-related proteins in neutrophils, including Rac and Rap, increases in response to FMLP or nonhydrolyzable GTP analogs, both in intact cells and in cell lysates (Philips et al., 1993). Furthermore, N-acetyl-S-trans,trans-farnesylr,-cysteine (AFC) (see Fig. 3), an inhibitor of the carboxyinethyltransferase, effectively inhibits FMLP-induced superoxide generation, whereas N acetyl-geranylcysteine, a poor inhibitor of the methylase, does not inhibit superoxide generation. In neutrophils, prenylcysteine-directed carboxymethyltrarisferase activity is localized to the plasma membrane (Pillinger et nl., 1994). This methylase activity is dependent on phosphatidic acid, a lipid that increases in concentration upon neutrophil activation. These data suggest that upon activation of neutrophils with FMLP, Rac is released

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from its interaction with the GDI protein and then translocates to the plasma membrane where it becomes carboxymethylated and participates in the activation of the NADPH oxidase. Carboxymethylation of prenylated proteins has also been suggested to play a role in other aspects of leukocyte activation. The heterotrimeric Gprotein subunit Gy2 is carboxymethylated in response to FMLP, and AFC inhibits the reaction (Philips et al., 1995). In addition to its inhibition of FMLP-mediated superoxide generation in neutrophils, AFC inhibits FMLP-mediated homotypic aggregation but enhances both the FMLPinduced upregulation of CDllbICD18 and the granule enzyme release in these cells (Philips et al., 1995). Furthermore, agonist-mediated activation of human platelets (Huzoor-Akbar et al., 1993) and the chemotaxis of mouse peritoneal macrophages toward lipopolysaccharide (LPS)-activated serum are also inhibited by AFC (Volker et al., 1991). Although AFC inhibits a variety of leukocyte activation-dependent responses, the role of the carboxymethyltransferase in these processes has been questioned (Ma et al., 1994). These authors show that several AFC analogs that are not inhibitors of the methyltransferase can nonetheless inhibit agonist-induced platelet aggregation. Furthermore, they found that the KM of farnesylcysteine for the platelet methyltransferase in vitro is -28 PM, whereas AFC inhibits platelet aggregation in the range of 1-10 PM (Huzoor-Akbar et al., 1993), suggesting that the methyltransferase is not the target of AFC in platelets. Additional studies to delineate the mechanism of inhibition of leukocyte activation by AFC are clearly necessary. XVII. Role of Rab Proteins in Membrane Transport in Leukocytes

Membrane transport functions that play particularly important roles in the biology of the immune system include endocytosis mediated via immunoglobulin Fc receptors and complement receptors. These types of receptor-mediated endocytosis are important in the clearance of foreign antigens by phagocytic cells and in the presentation of antigens via the MHC class I1 pathway by antigen presenting cells. At least four distinct Rab proteins, Rab4, Rab5, Rab7, and Rab9, play a role during various stages of endocytosis (Bottger et al., 1996; Rybin et al., 1996; Soldati et al., 1995). However, studies on the role of these proteins in the endocytic process in cells of the immune system are lacking. Another important immune system function that involves membrane transport is exocytosis, also known as degranulation or regulated secretion. For example, many inflammatory events including allergic reactions are triggered by antigen binding to high-affinity IgE receptors on mast cells, resulting in the rapid release of proinflammatory mediators from preformed

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secretory granules. Other granulocytes, including basophils, neutrophils, and eosinophils, degranulate in response to inflammatory stimuli. There is significant data from studies in leukocytes demonstrating that Rab proteins play an important role in exocytosis. The involvement of Rab proteins in exocytosis was first suggested by the ability of nonhydrolyzable GTP analogs to trigger mast cell degranulation when delivered through a patch pipette (Fernandez et al., 1984). GTP analogs have also been shown to trigger exocytosis in other granulocytes, including neutrophils (Nusse and Lindau, 1988) and eosinophils (Nusse et al., 1990). Several studies have implicated the Rab3A protein in the GTP-dependent exocytotic process. Mast cell degranulation is triggered by the injection of peptides corresponding to the effector domain of Rab3A (Oberhauser et al., 1992). Rab3A appears to play a role in regulated exocytosis in other cells because application of the Rab3A effector domain peptide to permeabilized pancreatic acini, chromafin cells, and insulinsecreting cells also triggers secretion (Nuoffer and Balch, 1994). Further pharmacological evidence for the involvement of Rab3A in exocytosis comes from the finding that prenylcysteine analogs stimulate exocytosis in permeabilized HIT-T15 cells (Regazzi et al., 1995). These authors suggest that Rab3A might normally inhibit exocytosis, and that the Rab3A effector peptide or the prenylcysteine analogs stimulate exocytosis by disrupting an inhibitory interaction between the prenylated Rab3A protein and a Rab effector protein. That Rab3A might act as a negative regulator of exocytosis is supported by the finding that microinjection of Rab3A antisense oligonucleotides enhanced exocytosis in adrenal chromaffin cells (Johannes et al., 1994). However, another study found the opposite result for a different isoform of Rab; in anterior pituitary cells microinjection of Rab3B antisense oligonucleotides inhibited regulated exocytosis but did not affect constitutive secretion or endocytosis (Liedo et al., 1993). The involvement of the Rab3 protein in exocytosis is equivocal. Although rat peritoneal mast cells express the Rab3B and Rab3D isoforms (Oberhauser et al., 1994), guinea pig eosinophils do not express any known isoforms of Rab3, even though they degranulate in response to nonhydrolyzable GTP analogs (Lacy et al., 1995). It has been suggested that other Rab proteins could play a role in exocytosis. In resting human neutrophils, Rab5A is localized to both membranes and cytosol, and upon challenge with PMA there is increased membrane association of the protein and a concomitant decrease in the cytosolic pool (Vita et al., 1996). The time course for the increased membrane association parallels the time course of exocytosis, suggesting that Rab5A might play a role in the secretory process. Alternatively, regulation of exocytosis by GTPases might involve proteins other than, or in addition to, Rab proteins because some members

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of the heterotrimeric G-protein family have been implicated in this process (Aridor et al., 1993). Although there is significant evidence to suggest a role of Rab proteins in exocytosis, further studies are required to understand their exact involvement in this process. XVIII. Regulation of Vesicular Transport by Rho Proteins

Although the Rab family of GTP-binding proteins is well known for its function in vesicular transport, there is an increasing appreciation for the involvement of Rho family members in these transport processes. For example, activated mutants of RhoA and Racl impair the formation of clathrin-coated vesicles in cells and in a reconstituted cell-free system (Lamaze et al., 1996). Cdc42Hs may also be involved in membrane transport because it is localized to the Golgi apparatus and its intracellular distribution is affected by brefeldin A, an agent that has profound effects on vesicular transport (Erickson et al., 1996).Additionally, a newly discovered Rho family member, RhoD, was shown to regulate cell morphology and endosome dynamics in a variety of mammalian cell types, including the macrophage cell line J774 (Murphy et al., 1996). Overexpression of wild-type RhoD or a GTPase-defective RhoD caused striking changes in cell morphology, including the formation of extended membrane processes that protruded from the body of the cell that were enriched in F-actin. This was accompanied by the disappearance of actin stress fibers and the disassembly of focal adhesion complexes in the cell body. Furthermore, wild-type RhoD and the RhoD mutant were localized to the plasma membrane and endosomes, and the RhoD mutant dramatically reduced the motility of endosomes in the cell. These studies indicate that RhoD regulates the movement of endosomes via a process that may depend on actin stress fibers. XIX. Other Prenylated Proteins

Several other prenylated proteins that might play roles in leukocyte function have been identified. Two interferon-? ( IFN-.)I)inducible GTPbinding proteins of unknown function have been identified in human fibroblasts (Cheng et al., 1991). One of these proteins, huGBP1, contains a C-terminal CTIS sequence predictive of modification by farnesyltransferase, whereas the other, huGBP2, contains a CNIL sequence at its C terminus that predicts modification by GGTase-I. HuGBPl and its murine homolog are induced by IFN-y and LPS in human monocytes, the human promyelocytic HL-60 cell line, and in murine macrophages, and their prenylation is sensitive to farnesyltransferase inhibitors (Nantais et al.,

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1996).Another potentially important prenylated protein is the yeast YDJl protein and its human homolog, hDJ2. The yeast YDJl protein is a farnesylated protein that functions as a molecular chaperone and is involved in cell cycle regulation (Yaglom et al., 1996).Other prenylated proteins that could be involved in immune cell function are two protein tyrosine phosphatases referred to as PTPcm (Cates et al., 1996).These phosphatases can transform human epithelial cells when overexpressed, and thus may normally play a role in regulating cell growth. XX. Prenyltransferase Inhibitors

The importance of the Ras pathway in cellular transformation and cancer, and the discovery that Ras requires farnesylation for its function, sparked the development of FTase inhibitors as potential chemotherapeutic agents. HMG-CoA reductase inhibitors such as lovastatin that inhibit prenylation of both farnesylated and geranylgeranylated proteins through their inhibition of isoprenoid synthesis, existed even before the discovery of protein prenylation and have been valuable tools for understanding the biological roles of prenylation. However, HMG-CoA reductase inhibitors have been considered unsuitable as clinically useful inhibitors of Ras function because they inhibit the biosynthesis of downstream metabolites in the mevalonate pathway, including cholesterol, dolicliol, and ubiquinone, as well as the prenylation of both farnesylated and geraiiylgeranylatedproteins. The findings that tlie CaaX motif itself is the minimal essential element for substrate recognition and catalysis by FTase and GGTase-I and that substitution of tlie second aliphatic amino acid within the CaaX with an aromatic amino acid converts the CaaX peptide substrate into a competitive inhibitor (Brown et al., 1992; Goldstein et al., 1991; Reiss et al., 1990; Schaber et al., 1990) serve as a starting point for the development of potent, cell active inhibitors of FTase. A number of CaaX peptidomimetic compounds that display excellent selectivity for FTase inhibition compared to GGTase-I inhibition have been reported, including L-731,734,BZA-SB, and BS81 (see Fig. 3) (Garcia et al., 1993;James et al., 1993; Kohl et al., 1993).These farnesyltransferase inhibitors ( FTIs) are modified CaaX peptides that lack peptide bonds and are therefore resistant to hydrolysis by proteases. Additionally, many of the first peptidomimetics were made as prodrugs, containing an esterified C-terminal carboxylate group that eliminates the charged nature of the molecule, making it permeable to cell membranes. They are prodrugs because significant activity against FTase requires generation of the free carboxylate through the action of cellular esterases. Nonpeptide mimetics that lack the C-terminal carboxylate and/or tlie sulfhydryl moeity of the

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cysteine residue in the CaaX have also been developed (Bishop et al., 1995; Hunt et al., 1996; Vogt et al., 1995;Williams et al., 1996).In addition to CaaX competitive inhibitors, compounds competitive with FPP such as manumycin, as well as bisubstrate analogs competitive with both CaaX and FPP, have been developed (Fig. 3) (Haraet al., 1993;Pate1 et al., 1995). The CaaX competitive FTIs can block the famesylation of Ras and other FTase substrates in cells (Garcia et al., 1993; James et al., 1993; Kohl et al., 1993) and, in general, are more potent in cells than FPP competitive compounds (Hara et al., 1993).The efficacy of these compounds in inhibiting farnesylation in cells is illustrated by their effects on the Ras signaling pathway. FTIs inhibit many aspects of the transformed phenotype that are induced through the introduction of oncogenic H-ras into rodent fibroblasts, including anchorage-independent cell growth, rapid growth in monolayer culture, and alterations in cell morphology (James et al., 1993; Kohl et al., 1993; Prendergast et al., 1994). FTIs inhibit the formation and growth of rodent and human xenograft tumors in nude mice (Hara et al., 1993; Kohl et al., 1994; Sun et al., 1995). Additionally, the FTI L-744,832 is efficacious in a transgenic mouse model of mammary cancer (Kohl et al., 1995). In this model, oncogenic H-ras is expressed under control of the MMTV promoter, which induces mammary and salivary carcinomas (Sinn et al., 1987). Daily treatment of tumorbearing mice with L-744,832 induced a rapid regression of the tumors, and continual treatment prevented the reappearance of new tumors (Kohl et al., 1995). Cultured cells growing under anchorage-independent conditions undergo apoptosis in response to FTI treatment, suggesting that the rapid tumor regression induced by FTI treatment in the H-ras oncomouse model might also be due to apoptosis (Lebowitz et al., 1997). No detectable toxicity has been reported in animal studies involving FTI treatment. The lack of toxicity was unanticipated, given the ubiquitous importance of Ras in cell proliferation. One explanation for the lack of global toxicity in the face of dramatic effects on tumor growth is that many of the published studies used tumors that are driven by activated H-Ras, which is a relatively poor substrate for FTase and is thus easily inhibited. For example, the &, of FTase for H-Ras, which has a CaaX where X = ser, is much higher than the K,,, of FTase for K-Ras, where X = met (James et al., 1995). Additionally, 10-fold higher concentrations of the BZA-5B FTI are required to block farnesylation of the nuclear lamins compared to those for H-Ras (Dalton et al., 1995). Another explanation for the lack of toxicity in normal tissues is that transduction of growth proliferative signals in normal cells may rely on other forms of Ras, such as K-Ras and N-Ras, or Ras-related proteins such as R-RasmC21. This is suggested by studies that showed that FTIs did not inhibit the EGF-

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stimulated activation of MAPK in nontransformed cells but did inhibit the MAPK activation induced by oncogenic H-ras (James et al., 1994a). Additionally, cross-prenylation of some farnesylated proteins by GGTaseI in the presence of an FTI blockade might explain the lack of toxicity. R-RasZflC21, which is capable of triggering malignant transformation (Graham et al., 1994), as well as K-Ras4B are prenylated by both FTase and GGTase-I in vitro (James et al., 1995; Carboni et al., 1995) and might remain functional and transduce growth signals in normal tissues treated with an FTI. The finding that K-Ras4B, which is the predominant form of mutated Ras associated with cancer (Barbacid, 1987), is prenylated by GGTase-I as well as FTase in vitro (James et al., 1995) has raised important questions concerning the development of FTIs as chemotherapeutic agents. Although K-Ras4B is found as a farnesylated protein in vivo (Casey et al., 1989),it remains prenylated in FTI-treated cells (James et al., 1996), and preliminary data suggest that this is due to cross-prenylation by GGTase-I (Lerner et al., 1997; Pai et al., 1996; Rowel1 et al., 1997). Furthermore, K-Ras4B containing an altered CaaX sequence (CVIL) that is presumed to be exclusively a GGTase-I substrate is transforming to cells (Hancock et al., 1991b; Kato et al., 1992), suggesting that geranylgeranylated K-Ras4B in FTItreated cells would be functional. Sebti, Hamilton, and coworkers have further explored the issue of K-Ras4B cross-prenylation through their development of prenylation inhibitors that are more specific for GGTase-I compared to FTase. These compounds were derived from FTI peptidomimetics by replacing the methionine residue of an FTI peptidomimetic with leucine in the X position of the CaaX (Fig.3) (Lerner et al., 1995). Although one of these compounds was reported to block K-Ras processing in NIH-3T3 cells and to inhibit MAP kinase activation, recent data suggest that a combination treatment with both an FTI and a GGTase-I inhibitor is required to effectively inhibit K-Ras4B prenylation in human tumor cell lines (Lerner et al., 1997). Although FTIs alone may not inhibit K-Ras4B prenylation, FTIs can inhibit the anchorage-independent growth of a variety of cell lines derived from human tumors including those containing K-Ras4B mutations (SeppLorenzino et al., 1995; Nagasu et al., 1995). The sensitivity of these tumor lines to growth inhibition by the FTI varied greatly and was independent of the ras mutational status of the cell. This suggests that human tumor cell proliferation can be regulated by farnesylated proteins in addition to Ras. One such protein may be RhoB, a member of the Ras superfamily of GTPases that can be both farnesylated and geranylgeranylated in vivo (Adamson et al., 1992). FTI treatment of cells disrupts the intracellular localization of this protein, and cells transformed with an FTI-resistant

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form of RhoB containing an N-myristylation site require 10-fold higher concentrations of FTI to be growth inhibited (Lebowitz et al., 1995). Growth inhibition by FTIs might involve multiple mechanisms because the processing of at least 18 cellular proteins is affected by FTI treatment (James et al., 199413). In addition to the potential of FTIs for cancer treatment, GGTase-I inhibitors might also have potential as chemotherapeutics. HMG-CoA reductase inhibitors and the GGTase-I inhibitor, GGTI-287 (Fig. 3),inhibit the proliferation of cultured cells through a mechanism that involves growth arrest in the GI phase of the cell cycle (Vogt et al., 1996). Progression of a cell from GI into S phase involves the ubiquitin-dependent degradation of the p27 cyclin-dependent kinase inhibitor (Pagan0 et al., 1995), and the HMG-CoA reductase inhibitor pravastatin prevents the elimination of p27 through a mechanism that appears to involve geranylgeranylated Rho proteins (Hirai et d., 1997). The involvement of geranylgeranylated Rho proteins in p27 elimination is suggested by the finding that in pravastatintreated cells, addition of liposomes containing the GGTase-I substrate GGPP but not the FTase substrate FPP results in a decrease in p27 protein levels and progression of the cells through GI into S. Furthermore, the Rho inactivator, C3 ADP ribosyltransferase,prevents the ability of GGPP to stimulate progression into S phase in pravastatin-treated cells. Additionally, both lovastatin and a GGTase-I inhibitor block the PDGF-induced tyrosine phosphoylation of the PDGF receptor (McGuire et al., 1996). A Rho protein could be involved in this aspect of signaling because the PDGF type B receptor has been found to associate with Rho (Zubiaur et al., 1995). The ability of GGTase-I inhibitors to induce G, arrest, to inhibit multiple aspects of signal transduction, and to block the prenylation of KRas4B in conjunction with FTI treatment suggests that these agents might also be suitable as chemotherapeutics. In this regard, preliminary studies indicate that GGTase-I inhibition can block the growth of several human tumor lines in nude mice (Sun et al., 1997). MI. Effects of Prenylation Inhibitors on Leukocyte Function

Although FTase inhibitors show little toxicity in animal models, the potential effect of these inhibitors on the function of the immune system has not been adequately addressed. The HMG-CoA reductase inhibitor lovastatin inhibits both proximal and distal signaling events in the human Jurkat T cell line and in normal human peripheral blood mononuclear cells activated through the TCR (Goldman et al., 1996). In Jurkat cells, lovastatin inhibited both the processing of Ras and the activation of MAPK. Additionally, TCR signaling events that are presumably independent of

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Ras were inhibited, including mobilization of intracellular calcium, inositol phosphate production, and tyrosine phosphorylation, suggesting the involvement of a prenylated protein other than Ras in these aspects of TCR-mediated signaling. The effect of lovastatin on these Ras-independent signaling events was specific to the T cell receptor because calcium signaling and inositol metabolism triggered by transfected type-1 muscarinic receptors were unaffected in these cells. The potential for prenylation inhibitors to affect diseases involving lymphocyte proliferation and/or differentiation is suggested by several other studies involving HMG-CoA reductase inhibitors. For example, lovastatin showed some efficacy in inhibiting chronic allograft rejection in an animal model (O’Donnell et al., 199.5). IIMG-CoA reductase inhibitors and zaragozic acid, an FTI isolated from natural products, can inhibit signaling, specifically inositol lipid metabolism, in human keratinocytes induced by inflammatory mediators such as PAF and bradykinin (Alaei et al., 1996). This suggests that prenylation inhibitors could ameliorate the symptoms of inflammatory skin diseases. Another cell of the immune system that is responsive to HMG-CoA reductase inhibition is the human macrophage; it has been shown that lovastatin inhibits the expression of the type I lipoprotein scavenger receptor gene in these cells (Umetani et al., 1996). The inhibition of lipoprotein scavenger receptor expression in niacropliages is likely not related to the efficacy of this cholesterol-lowering agent in cardiovascular disease management because the inhibition of gene expression occurred at concentrations (5-15 ~ L ) Mfar higher than the peak plasma concentration commonly achieved in patients treated with this agent. XXII. Conclusion

Members of the Ras superfainily of GTP-binding proteins regulate a wide variety of cellular processes, and many of members of this family have been shown to play an iinportant role in tlie function of irninune system cells. I expect that appreciation of the importance of these proteins in immunobiology will only continue to grow as studies of these proteins and tlie discovery of new family members progress. The C-terminal processing of the Ras superfainily of proteins, which depends on the action of prenyltransferases and other processing enzymes including the CaaX protease, the prenylcysteine-directed methyltransferase, and in some cases palmitoyltransferase, is critical to the function of these proteins. The development of specific inhibitors of these C-terminal processing enzymes, in particular, inhibitors of farnesyltransferase, has proceeded rapidly in recent years and has helped to illustrate the importance of protein prenylation in various cell functions. Studies of these prenylation inhibitors in irninune

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system function should be expanded. These studies would be valuable not only from a clinical standpoint but also to aid in our understanding of the importance of Ras superfamily members in the proper functioning of the immune system. ACKNOWLEDGMENTS I thank Dr. Jay Gibbs and Dr. Charles Omer of the Merck Research Labs for their advice and suggestions on the manuscript.

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A I W A U C h 5 IN IMMUNOLOCY VOL hH

Generation and TAP-Mediated Transport of Peptides for Major Histocompatibility Complex Class I Molecules FRANK MOMBURG AND GUNTER J. HhMERLING Deparhnent of Molecular Immunology, German Cancer Research Center (DKFZj,

69120 Heidelherg, Germany

1. Iniroduction

During evolution of the adaptive immune system as a defense against environmental pathogens two major groups of microbes had to be dealt with that differ with regard to their intracellular location: microbes replicating in the cytosol, e.g., viruses and some bacteria, and those propagating in vesicular compartments, e.g., some bacteria, or their toxic products that enter endosomal/Iysosomal compartments via the endocytic pathway. Therefore, different pathways needed to be developed for the presentation of antigens located in distinct subcellular compartments. This is achieved by the two classes of major histocompatibility complex (MHC) molecules that are specialized peptide receptors and serve to display antigenic peptides at the cell surface for recognition by T lymphocytes. Peptide antigens generated in the vesicular compartments of the endocytic pathway are usually loaded onto MHC class I1 molecules. These are targeted to endosomaVlysosoina1 loading compartments with the help of the invariant chain, which carries an endosoinal sorting signal. After binding of peptides the resulting MHC class 11-peptide complexes proceed to the cell surface for screening by CD4+ T cells. In contrast, pathogens dwelling in the cytosol will be subject to degradation by the major cytosolic proteolytic machinery, the proteasome, which is an evolutionary ancient protease that is found in eu- and archaebacteria. The immune system makes use of the peptidic degradation products generated in the cytosol by translocating them into the lumen of the endoplasmic reticulum (ER) where they assemble with newly synthesized MHC class I molecules and are transported to the cell surface for recognition by CD8+ T lymphocytes. This process allows T cells to continuously sample the cell surfaces for the presence of peptides derived from a potentially harmful intruder. Translocation of peptides into the ER is achieved by the TAP transporter (transporter associated with antigen processing), which belongs to a large family of membrane translocators containing an ATP-binding cassette (ABC). TAP appears to be a specialized ABC transporter that serves exclusively the immune system as indicated by the observation that 191

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TAP deficiency in human patients or in knockout mice seems to solely affect the immune system, in which it plays a pivotal role in the class I presentation pathway. II. TAP as the Principal Peptide Supplier for MHC Class I Molecules

A. EVIDENCE FOR PEPTIDE TRANSLOCATION BY TAP Approximately a decade ago, strongly reduced levels of cell surface class I molecules had been detected in various cell lines that had undergone radiation or chemical mutagenesis or that had been treated with class I antibodies and complement (Kavathas et al., 1980; DeMars et al., 1984, 1985; Ljunggren and Karre, 1985; Karre et al., 1986; Salter et al., 1985). By culturing the mutant cells in high concentration of class I-binding peptide, class I surface expression could be reconstituted (Townsend et al., 1989; Cerundolo et al., 1990). Unstable complexes of class I h e a y chains and &-microglobulin (P2m)were largely retained in the ER of the mutant cells (Salter and Cresswell, 1986; Ljunggren et al., 1989). Incubation at ambient temperature, which has a stabilizing effect on the dimers, was found to induce their transport to the cell surface (Ljunggren et al., 1990; Schumacher et al., 1990). Furthermore, the mutants were unable to present intracellular antigens to cytotoxic T cells, whereas exogenously added peptides or peptides introduced into the ER by a signal sequence were efficiently presented (Townsend et al., 1989; Ohkn et al., 1990a; Cerundulo et al., 1990; Hosken and Bevan, 1990; Anderson et al., 1991). Addition of peptide ligands to detergent extracts of mutant cells induced class I molecules to assemble with Pem (Townsend et al., 1990; Schumacher et al., 1990; Elliott et al., 1991). From these results it was concluded that the supply of peptides into the ER was grossly disturbed in the mutant cells. With the cloning of cDNAs coding for molecules with homology to ABC transporters, TAPl and TAP2 (see Section V), the defects in assembly and antigen presentation by class I molecules could be linked to the function of a peptide transporter in the ER membrane. The defective phenotypes of different mutants were found to be reversible by transfection of TAPl (Spies and DeMars, 1991; Spies et al., 1992), TAP2 (Powis et al., 1991a; Attaya et al., 1992; Kelly et al., 1992), or both TAPl and TAP2 cDNAs (Arnold et al., 1992; Momburg et al., 1992). The underlying defects in TAP genes have been determined as transcriptional inactivation of TAPl in human LCL721.134 B lymphoblastoid cells (Spies et al., 1990);deletion of TAPl and TAP2 in LCL721.174 (Spies et al., 1990) and its derivative, the BxT hybrid .174xCEM.T2 (T2), a frameshift mutation in TAP2 leading to a functionally defective molecule of extended length in the human B

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lymphoblastoid line BM36.1 (Kelly et a1 , 1992); and a premature stop codon early in the TAP2 sequence present in the mouse T lymphoma cell line RMA-S (Yang et al., 1992a). These transfection studies also indicated that both TAPl and TAP2 molecules need to be functional for the bulk of class I peptide loading and presentation to occur. Independent evidence for the existence of genes that influence the repertoire of class I-bound peptides came from studies with intra-MHC recombinant inbred rat strains. The antigenicity of the rat class I molecule, RTl.A', both as alloantigen and as restriction element, was modified by a locus in the MHC class I1 region termed cini (for class I modifier) existing in two allelic phenotypes, cirrio and cim" (Livingstone et al., 1989, 1991). Furthermore, the transit of RT1.Ad molecules to the cell surface was retarded in the presence of cim" but not &ma,leading to the worhng hypothesis that the cirri6 product supplied RT1.Adwith ill-suited peptides (Powis et al., 1991b).cini was demonstrated to be the TAP2 product, which has an extensive allelic polymorphism in the rat (Powis et al., 1992a; see Section VI). Transfection of TAP2l (cim")cDNA into c i d host cells restored the cim" antigenic phenotype of RT1.Ad molecules and caused a significant shift in the high-performance liquid chromatography (HPLC) spectrum of peptides extractable from RT1.Adtoward more hydrophilic peptide species (Powis et al., 1992a).This finding was fully consistent with TAP molecules acting as peptide transporters, although direct proof was still laclung.

B. TAP-DEPENDENT versus -INDEPENDENT ANTIGENPRESENTATION For avaiiety of peptides presented by classical or medial class I molecules it was shown that their presentation is abrogated in TAP deficiency mutants. Among these epitopes were Kd, Kk, Dd, and HLA-A1 and -A2-restricted peptides derived from the influenza virus nucleoprotein or matrix protein, a K"-restricted epitope from vesicular stomatitis virus (VSV) nucleocapsid protein, the minor histocompatibility antigen HA-2 presented by HLAA2, the N-formylated mitochondrial-derived peptide MTF" presented by the mouse class Ib molecule M P 3 (HMT')),and unknown allostimulatory peptides presented by Kh or Qal" (Powis et al., 1991a; Anderson et al., 1991; Aosai et a ! , 1991; Hermel et al., 1991; Kelly et d., 1992; Attaya et al., 1992; Spies et al., 1992; Momburg et al., 1992; Eisenlohr et a1 , 1992; Zhou et al., 1993a, 1994; Bacik et al., 1994).Also, Qa-2, another mouse class Ib molecule, required functional peptide transporters for stable assembly of its soluble and the glycosylphosphatidylinositol-anchoredisoforms (Tabaczewski and Stroynowski, 1994). In mice harboring a disrupted TAPl gene, class I cell surface levels were severely reduced, lymphoblasts were unable to present endogenous

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antigen, and the number of peripheral CD8+ T cell was reduced to <1% (Van Kaer et al., 1992). These findings underscore the physiological relevance of TAP-mediated peptide transport for class I-restricted antigen presentation and positive selection of cytotoxic T cells. As a consequence of laclang negative selection, cells from TAPl“ mice were, however, able to mount strong in vitro responses against syngeneic H-2‘ class I antigens loaded with TAP-dependent peptides (Aldrich et nl., 1994a). This result, and the capacity to react against allogeneic €I-2dcells, shows that positive selection is not completely abrogated in the absence of TAP. The extent of positive selection in T A P P mice could be increased by a transgeneexpressed human Pzni that partially rescued cell expression of class I molecules (van Santen et al., 1995). With regard to diversity and peptide specificity, the few remaining CD8+T cells in T A P P mice appeared quite similar to those of wild-type mice (Sandberg et al., 1996). Surprisingly, even TAPI@,m” double-mutant mice that have even lower numbers of CD8+ peripheral T cells than TAPI” mice are able to mount functional CTL responses (Ljunggren et al., 1995). Natural killer (NK) cells from TAP]’ mice were found to be normal in number but tolerant toward most syngeneic and allogeneic targets. Defective TAP1 expression was sufficient to render nontransformed target cells susceptible to N K cellmediated lysis (Ljunggren et al., 1994).On the other hand, the NK-sensitive phenotype of TAP-deficient tumor cells could be reversed by transfection of TAP (Franksson et al., 1993; Salcedo et al., 1994). Spectra of metabolically labeled peptides that have been eluted from various immunoprecipitated class I molecules (HLA-A2, -A3, H-2DP, K”, Kh/Db)are strongly depleted in the absence of functional peptide transporters (Wei and Cresswell, 1992; Anderson et al., 1993),pointing to an essential role of TAP as the principal supplier for peptides generated from freshly synthesized proteins. Depending on the antigenic system studied, the processing defect of TAP-deficient mutant cell lines may not be a complete one, in particular if peptide loading of class I molecules is read out with CTL-mediated killing$which is a tool of highest sensitivity. CTL specific for VSV, influenza virus, or Sendai virus epitopes lysed virusinfected TAP2-deficient RMA-S cells under conditions of higher virus load and longer incubation times (Esquivel et al., 1992; Hosken and Bevan, 1992; Zhou et al., 199313; Ossevoort et al., 1993).Some of these results may be explained by loading of D”/Kbmolecules in endosomal compartments or on the cell surface following endosomal processing of internalized virus (see below). The K”-restricted VSV nucleoprotein epitope was, however, also presented when it was endogenously expressed by transfection (Hosken and Bevan, 1992). Likewise, RMA-S cells presented epitopes derived from endogenous Rauscher virus (Sijts et al., 1992; Ossevoort et

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al., 1993), a proportion of Qa1”-restricted peptides recognized by Qdniindependent CTL (Hermel et d., 1991; Aldrich et al., 1992),and a subset of endogenous peptides recognized by allo-H-2(K)h reactive CTL (Ohlkn et al., 1990b; Aosai et al., 1991; Ossevoort et at., 1993). In mouse CMT 64.5 carcinoma cells that do not express detectable levels of TAPl or TAP2 molecules in the absence of IFN-y, presentation of a K”-boundVSV epitope was partially restored after stable transfection with the rat TAPl chain only (Gabathuler pt al., 1994). Although peptide transport measured in vitro is reduced to background level in RMA-S cells (see Section IX,A), it cannot be excluded that the functional TAPl subunit can form homodimers to some extent and enable a limited degree of peptide supply to the ER that is sufficient for the stimulation of CTL. As opposed to classical class I molecules, some nonclassical ones do not require TAP for efficient cell surface expression. Murine TL molecules, whose expression is restricted to intestinal epithelial cells and a few other cell types including thymocytes (Teitell et al., 1994), are expressed in normal amounts on the surface of TL-transfected TAP-deficient RMA-S cells (Holcombe et al., 1995; Rodgers et al., 1995). Stable &m-associated TL molecules were reported to be devoid of detectable peptide (Holcombe et al., 1995).Consistent with the expression of TL molecules on thymocytes of TAP1 mice, positive selection of intestinal intraepithelial lymphocytes (Sydora el al., 1996) or of natural T cells (Joyce et al., 1996) is fully functional in these mice. Similarly, expression and recognition of nonpolymorphic T10” class Ib molecules is not TAP dependent and no peptides could be eluted from thein (Kaliyaperumal et ul., 1995). In keeping with the presentation of microbial long-chain fatty acid and lipoglycan antigens by human class I-like C D l b molecules (Beckman et al., 1994; Sieling et al., 1995), CDlb-transfected T2 cells express normal surface levels of this non-MHC-encoded restriction element and present M . tuberculosisderived rnycolic acid to CD4-lCD8 T cells (Porcelli et al., 1992). The same was recently reported for the related C D l c molecules (Beckman et al., 1996). A TAP-deficient patient expressed normal levels of C D l a molecules (de la Salle et al., 1994).Also, the &m-dependent surface expression of mouse CD1.l molecules does not require peptide transporters (Brutkiewicz et al., 1995; Teitell et nl., 1997). Mouse CD1 molecules do, however, associate with long peptides of 14-24 amino acids with three hydrophobic anchor positions in a class II-like manner (Castaiio et a l , 1995). These peptides may be acquired after trafficking to endosomal/ lysosomal coinpartments because mouse CD 1 contains the same tyrosinbased internalization and endosoinal targeting signal as C D l b (Sugita et a l , 1996) or HLA-DM (Marks et al., 1995; Lindstedt et al., 1995).

-’

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Four A2-restricted and one B27-restricted viral epitopes expressed in the cytosol of TAPU2-deficient T2 cells from episome-driven minigenes were efficiently presented, whereas one additional epitope required an ER translocation signal sequence for presentation (Zweerink et al., 1993).Very hydrophobic peptides might traverse the ER membrane without a need for a specialized translocator, In this study a clear correlation between the peptides’ hydrophobicity and TAP-independent presentability could, however, not be established. Therefore, it remains entirely open as to how these cytosolically expressed peptides got access to their class I receptors. It cannot be ruled out that a transport system in the secretory pathway unrelated to TAP and not specialized for peptide transport of class Ibinding peptides might account for some residual peptide supply. Also, in the study by Zhou et al. (1995),a hydrophilic Sendai viral peptide expressed in the cytosol after prolonged infection with recombinant vaccinia virus was efficiently presented by T2.Kb cells. Presentation of this minigene product employed newly synthesized class I molecules because it was blockable by brefeldin A (BFA), a microbial compound blocking export of newly synthesized proteins from the ER (Nuchtern et al., 1989; Yewdell and Bennink, 1989). This finding suggests that a speculative TAPindependent peptide translocator may be located in the early secretory pathway. Numerous studies have worked out pathways leading to the presentation of exogenously added soluble proteins or particulate antigens by class I molecules that have recently been reviewed elsewhere (Rock, 1996; Jondal et al., 1996; Watts, 1997). Depending on the antigen and the type of presenting cell used, presentation may require TAP expression and be sensitive to BFA and to inhibitors of the proteasome (see Section II1,A) indicating conventional cytosolic processing of the antigen and loading of newly synthesized class I molecules (Kovacsovics-Bankowski and Rock, 1995; Suto and Srivastava, 1995; Norbuiy et al., 1995, 1997; Reis e Sousa and Germain, 1995; Huang et al., 1996; Liu et aE.,1995; Zhou et al., 1993a, 1995). Alternatively, an endosomal, TAP-independent pathway of class I loading may be operative during processing and presentation of exogenous antigens that can be inhibited by lysosomotropic agents such as chloroquine or ammonium chloride (Zhou et al., 1993a, 1995; Liu et al., 1995; Pfeifer et al., 1993; Harding and Song, 1994; Song and Harding, 1996; Schirmbeck et aZ., 1995a,b; Schirmbeck and Reimann, 1996; Bachrnann et al., 1995; Wick and Pfeifer, 1996; Lenz et al., 1996). Depending on the nature of the antigen used the latter pathway may involve endosomal processing of the antigen followed by peptide regurgitation and loading of class I molecules on the cell surface (Pfeifer et al., 1993; Schirmbeck et al., 1995b) or loading of class I molecules in a recycling compartment (Schirmbeck and

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Reimann, 1996). As a peculiarity, a TAP-independent and chloroquinesensitive pathway was also described for the Dh-restricted presentation of truncated SV-40 T antigen variants (Schirmbeck and Reimann, 1994). Presentation of these endogenous antigens correlated with their association with the coi~stitutivelyexpressed heat shock protein hsp73, which is known to target proteins into the endosomal compartment (Dice, 1990).

c. ANTICENPROCESSING IN THE ER It is obvious that the necessity for TAP-mediated transport is overcome if the epitope is introduced into the ER by means of a signal sequence for ER translocation that is then cleaved off by the signal peptidase. Following transfection with episoinally expressed minigene constructs (Anderson et al., 1991; Zweerink et al., 1993; Khanna et al., 1994) or infection with vaccinia virus recombinant for a signal peptide/epitope construct (Eisenlohr et al., 1992; Bacikct al., 1994; Restifoet al., 1995) the respective epitopes were readily presented by TAP-deficient cells. These experiments also highlight that loading of peptides onto class I molecules does not generally require the presence of TAP (see Section IV). Using a similar approach, TAP-independent presentation of influenza viral epitopes derived from longer precursors was demonstrated (Snyder et al., 1994; Elliott et al., 1995). Snyder et al. found that from an ERtargeted tandem array of two epitopes, the C-terminal determinant was efficiently liberated by N-terminal cleavage, whereas trimming from the C-terminus was inefficient. The C-terminal processing capacity of the ER seems to be rather low because the coexpression of a carboxypeptidase (angiotensin-converting enzyme) in the ER significantly enhanced TAPdependent presentation of C-terminally extended peptides expressed from cytosolic minigenes (Eisenlohr et aZ., 1992). Elliott et al. reported charging of D" molecules with an influenza nucleoprotein-derived epitope following targeting of a long truncation variant into the ER. For the formation of the 9-mer epitope 40 residues must have been removed from the N terminus and 124 from the C terminus of the polypeptide. Constitutively glycosylated influenza hemagglutinin constructs or ERtargeted full-length nucleoprotein, which is aberrantly glycosylated, could not sensitize target cells indicating that the glycan(s) protected the proteins from attack by ER proteases (Elliott et al., 1995).Similarly,the presentation of an B27-restricted epitope from the ER-luminal domain of measles virus F protein was found to be dependent on TAP expression indicating that the epitope can only be generated by cytosolic proteases (van Binnendijk et al., 1992). In the same line, only a subset of epitopes derived from the ectodomain of the glycoprotein HIV gp120 can be processed in the ER, whereas all epitopes are generated in the cytosol and presented in a TAP-

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dependent fashion (Hammond et al., 1993, 1995). It is conceivable that (g1yco)proteinsusually being inserted into the ER membrane or secreted into the ER lumen by means of the Sec61 translocation complex (Schatz and Dobberstein, 1996) may undergo, to varying degrees, dislocation into the cytosol for degradation by proteasomes. This retrograde route was shown to be utilized by the viral class I inhibitors US2 and US11 (see Section XIV,A) but may have a general significance as suggested by the proteasome-dependent degradation of misfolded ER proteins in yeast (Hiller et al., 1996;Werner et al., 1996),of the intergral membrane transporter protein CFTR (Ward et al., 1995; Jensen et al., 1995; see Section VII), and of class I heavy chains in Pzm- or TAP-deficient cells (Hughes et al., 1997). Alternatively, proteins of the secretory pathway may be degraded in the cytosol using defective ribosomal products as the source, as recently proposed by Yewdell et at. (1996). Peptides that are hydrophobic signal sequences that are themselves processed out of the ER lumen. Indeed, signal peptides of 9-14 residues have been isolated from HLA-A2 molecules expressed by T2 cells (Henderson et al., 1992; Wei and Cresswell, 1992) or by TAP-positive cells (Hunt et al., 1992; Engelhard, 1994a). The signal sequence can further be processed by the signal peptide peptidase leading to release of an N-terminal fragment into the cytosol (Lyko et al., 1995). Consistent with such a cleavage, the recognition of the H-2D/L leader-derived epitope 3-11 by Qdmdependent Qalb-restrictedCTL is TAP dependent (Aldrich et al., 199413). Also, a modified Dh signal peptide (26 residues) and the long signal sequence of LCMV gp33 (58 residues) may be processed by signal peptide peptidase to account for the strictly TAP-dependent presentation of T cell epitopes located at positions 2-10 and 33-41, respectively (Uger and Barber, 1997; Hombach et al., 1995). On the other hand, a modified signal sequence of hemagglutinin (17 residues), into which the A2-restricted influenza matrix protein epitope was introduced at position 3-11, or the short leader sequence of calreticulin (17residues) appear to be translocated into the ER in their uncleaved forms because the A2 epitope is presented by T2 cells (Gukguen et al., 1994) and the calreticulin peptide 1-10 was eluted from TZderived A2 molecules (Henderson et al., 1992). The topology with regard to the ER membrane also seems to determine the TAP dependence of epitopes derived from the multitopic Epstein-Barr virus LMP2 protein (Lee et al., 1996). Whereas presentation of two epitopes locating to membrane-integrated sequences was found to be TAP independent, peptide transporters were required for presentation of another epitope that maps to a cytoplasmic loop of LMP2. The observation that HLA class I alleles, such as A3, B7, B27, or B51, are expressed in low but easily detectable amounts on the surface of T2

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cells prompted Smith and Lutz (1996) to analyze TAP-independent peptides bound to HLA-B7 molecules. In contrast to HLA-A2, no metabolically labeled peptides could be eluted from HLA-B7, hut a spectrum of B7bound peptides was detected spectrophotometrically. In comparison with B7-eluted peptides from TAP-expressing control cells, TWB7-derived peptides were on average more hydrophobic (and possibly longer) but had the typical HLA-B7 peptide-binding motif. Thus, B7 and A2 seem to utilize distinct TAP-independent peptide supply mechanisms. Because T2/B7bound peptides did not detectably incorporate [ 'HILeu and ['HIPro after a 12-hr pulse period it may be speculated that they are derived from longlived cellular proteins processed in the endocytic pathway in the course of normal turnover. 111. Generation of Antigenic Peptides from Endogenous Antigens

A. EVIDENCE FOR PEPTIDE GENERATION HY PHOTEASOMES The proteasome was characterized as the source of the main proteolytic activity in the cytosol and the nucleus, thus making this particle the prime candidate for generator of peptides presented by class I molecules (reviewed by Goldberg and Rock, 1992). Experiments in which membranepermeable proteasome inhibitors were added to antigen presenting cells provided the first direct evidence for the implication of proteasomes in the generation of peptides presented by MHC class I molecules. Peptidyl aldehydes such as N-acetyl-leucyl-leucyl-norleucinal( LLn L) inhibited the proteolytic activities of proteasomes in uitro, the cellular turnover of short-lived and long-lived proteins, the presentation of endogenously synthesized or cytoplasmatically introduced antigens, and the assembly of class I molecules (Rock et al., 1994; Harding et nl., 1995; Yarig et al., 1996; Sijts et al., 1996a; Hughes et nl., 1996). Because peptidyl aldehydes do not only affect proteasomal activity but also are potent inhibitors of the cytosolic cystein protease calpain I1 (Sasaki et al., 1990) and even of unknown ER-resident protease(s) (Hughes et al., 1996), the relative contribution of proteasome-mediated processing was uncertain. Recent studies employing the bona fide proteasome-specific microbial inhibitor lactacystin (Fenteany et al., 1995) have shown that the processing and presentation of a variety of influenza virus-derived epitopes can indeed be decreased by lactacystin (Cerundolo et al., 1997).A further study in which various peptidyl aldehyde inhibitors and lactacystin were used, however, provided evidence that presentation of other epitopes derived from influenza viral proteins may not significantly be affected by any of the inhibitors (Vinitsky et al., 1997). This points to the possibility that proteases other than proteasomes may play a role in antigen presentation.

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In this line it is interesting to note that the aldehyde inhibitor MG132 did not block the processing of epitopes from cytosolic minigene products 5 17 residues, whereas the processing of larger precursor peptides was clearly affected, suggesting C-terminal trimming by nonproteasomal cytosolic or ER-luminal protease(s) of the short precursors (Yang et al., 1996).

B. NEWINSIGHTS INTO STRUCTURE, ASSEMBLY,AND FUNCTION OF

PROTEASOMES

The proteasome is a cylinder-shaped multisubunit complex of -700 kDa MW. This 20s particle represents the active proteolytic core of larger complexes (26s proteasome and PA28-proteasome complex) whose function will be discussed later. Proteasomes are composed of 14 a-type and 14 @-typesubunits of 22-30 kDa MW) that are organized in four stacked rings in the order and stoichiometry a7P7&a7, as recently reviewed elsewhere (Coux et al., 1996; Groettrup et al., 1996a). Although the ultrastructure of proteasomes is highly conserved (Peters, 1994; Koster et al., 1995) the subunit composition has become increasingly complex in the course of evolution. The unique a and @ subunits present in proteasomes of the archaeon T h e m p l a s m a acidophilum have apparently diversified into families of a-type and @-typesubunits with two members each in eubacteria (Tamura et al., 1995) and 7 members each in yeast (Hilt and Wolf, 1995). Possibly along with the development of immune systems, the number of @type subunits has further increased to 10. In man and rodents, 3 @-type subunits possess homologous replacement partners that are induced by IFN-y (see Section 111,D). The elucidation of the 3D structure of the 20s proteasome of T. acidophilum by X-ray crystallography has greatly advanced our understanding of the molecular function of this multicatalpc protease (Lowe et al., 1995). Soaking of the proteasome crystal with the inhibitor LLnL revealed particular residues in the N-terminal regions of the 14 identical f3 subunits as active sites. These are oriented toward the central cavity of the cylinder and are thus buried away from the cytosolic environment. Another remarkable structural feature is that the access to the central cavity is controlled by very narrow entrances formed by the outer a rings and the inner @ rings, respectively, requiring complete unfolding of the polypeptide substrates. Recently, the much more complex structure of the 20s particle from yeast was solved (Groll et al., 1997). The general architecture of proteasomes from yeast and T. acidophilum are similar. As opposed to the latter, it was found, however, that in yeast proteasomes there is no access to the interior from the outer a rings. Instead, narrow side windows, located at the interface between a and /3 rings, may allow the passage of

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unfolded, extended polypeptide chains. Alternatively, it is well conceivable that binding of the regulatory 19s or 11s complexes (see below) may induce conformational changes in the a rings leading to the controlled opening of entry ports at the cylinder ends in vivo. In the yeast proteasomes, the 14 different subunits of a/3 proteasomal halves occupy unique positions within the rings and halves are related by twofold rotational symmetry. As revealed by mutational and structural analysis (Seemuller et al., 1995; Lowe et al., 1995), a free NH2-terminal Thr residue essentially contributes to the catalytic system of the &type subunit in the T. acidophilum proteasome. The yeast &type subunits PRE2, PRE3, and PUP1, homologs of the IFN-y-replacable humadrodent subunits, were labeled by LLnL or lactacystin and thus marked as the primary active subunits (Groll et al., 1997). Only these &type subunits possess an N-terminal Thr in their mature forms (Groll et al., 1997; Chen and Hochstrasser, 1995; Lilley et al., 1990; Lee et al., 1990; Groettrup et al., 1996b; Hisamatsu et al., 1996). They are incorporated into preproteasome complexes as inactive proproteins followed by autocatalytical processing of the prosequence at a conserved Gly-' 1Thr'ThF consensus site (Chen and Hochstrasser, 1996; Seemiiller et al., 1996, Schmidtke et al., 1996; Frentzel et al., 1994; Yang et al., 1995). For eukaryotic but not the archaebacterial active &type subunits, the prosequence is required for incorporation into precursor complexes (Chen and Hochstrasser, 1996; Cerundolo et al., 1995; Seemuller et al., 1996). Specific proteolytic activities could be tentatively assigned to the mentioned yeast subunits (Groll et al., 1997). The observation that putatively inactive @-type subunits (Lee et al., 1990; Groll et al., 1997) as well as as mutated variants of the active subunit LMP2 (Schmidtke et al., 1996) are cleaved at locations 8-10 residues upstream of Thr' led to the conclusion that an additional unspecific endopeptidase activity may exist in the proteasome (Groll et al., 1997). BY PROTEASOMES I N Ir,mo C. PEPTIDES GENERATED

Consistent with a minimal &stance of 28 A between two catalytic sites (Lowe et al. 1995), the average length of peptides produced by T. acidophiluin proteasomes from oxidized insulin @chain or hemoglobin, respectively, had a narrow size distribution of approximately 3-15 residues with a maximum at 7 or 8 residues (Wenzel et al., 1994).Similar size distributions were published for peptide spectra generated by human or mouse proteasomes from long precursor peptides (Ehring et al., 1996; Niedermann et al., 1996). More than 50% of final proteolytic fragments derived from an ovalbumin 44-mer were in the size range of class I ligands (Niedermann et al., 1996).The in vitro generation o f antigenic epitopes out of ovalbumin

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and P-galactosidase was first demonstrated by Dick et al. (1994). Further studies employing synthetic oligopeptides instead of denatured proteins have shown that the cleavage specificity of proteasomes is strongly influenced by residues flanking the class I-binding epitope (Niedermann et al., 1995; Eggers et al., 1995). A subdominant ovalbumin epitope was preferentially destroyed by proteasomal cleavage but could partially be rescued by shuffling the epitope between the flanking sequences of the dominant epitope (Niedermann et al., 1995). Glycine or proline residues adjacent to epitopes interfered with their effective liberation (Eggers et al., 1995; Niedermann et al., 1995). A naturally occurring point mutation (Lys --+ Arg) in a viral 8-mer epitope was shown to promote destruction of the epitope by proteasoinal cleavage leading to deficient antigen presentation (Ossendorp et al., 1996). In line with the latter finding, it seems likely that unfavorable cleavage preferences of the proteasome contributed to the inefficient processing of a variety of potential class I-binding peptides in the sequence contexts of whole proteins that were noted in studies analyzing the efficiency of antigen presentation in. vivo (Del Val et al., 1991; Painer et al., 1991; Sijts et al., 1996b; Deng et al., 1997).

D. IMMUNOMODULATION OF PROTEASOME S T R U C T U R E A N D CLEAVAGE SPECIFICITY Since the discovery of the genes for two proteasome subunits, LMPB and LMP7, in the class I1 region of the MHC (Brown et al., 1991; Glynne et al., 1991; Martinez and Monaco, 1991; Kellyet al., 1991; Ortiz-Navarrete et al., 1991), much interest has focused on the potential role of these subunits in antigen presentation. The IFN-y-inducible @type subunits LMPB and LMP7 replace the highly homologous “housekeeping” subunits WMB-1 and M6, respectively (Belich et al., 1994;Frtih et al., 1994;Akiyama et al., 1994).This replacement occurs during assembly of precursor proteins into /3 rings because the composition of mature proteasomes is not further affected by IFN-y (Aki et a l , 1994) and no free processed MB-1 or 6 proteins have been detected (Friih et al., 1994). Recently, a third IFN-yinducible /3-type subunit and its counterpart were identified. MECL-1 displaces the housekeeping subunit, Z (MC14) (Hisamatsu et al., 1996; Groettrup et al., 1996b; Nandi et al., 1996). Because IFN-y does not significantly influence mRNA and protein levels of the housekeeping subunits, the replacement is believed to occur through a competitive mechanism because the IFN-y-induced subunits may have a higher affinity for the specific site of incorporation (Kuckelkorn et al., 1995) and/or they are expressed in greater amounts (Friih et al., 1994).

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Furthermore, several cell lines have been described that constitutively express LMP2 and/or LMP7 (Belich et al., 1994; Aki et al., 1994; Yang et al., 1995) and IFN-y-induced changes of tlie proteasome composition are rather slow and often inconiplete (Fruh et nl., 1994; Aki et al., 1994; Hisainatsu et al., 1996; Groettrup et al., 199617). Because incorporation of either LMP2 or LMP7 into 20s particles can occur in the absence of the other subunit (Fruh et al., 1994; Kuckelkorn et al., 1995) and the proteasome probably assembles by dimerization of cornplete a/3 halves (Yang et al., 199.5), theoretically up to 36 different proteasoine subpopul at‘ions may temporarily coexist (Nandi et al., 1996). On the other hand, cooperation in the incorporation of certain subunits was noted (Gaczynska et al., 1996), wliich is likely to reduce tlie number of actually existing proteasome entities. A series of initial studies addressing the role of LMPB and LMP7 by using mutant cell lines lacking these proteins consistently revealed no detectable contribution of the MHC-encoded subunits to the bulk of peptides bound to class I and the presentation of various viral antigens (Arnold et nl., 1992; Moinburg et al., 1992; Yewdell et al., 1994; Zhou et nl., 1994). Recently, however, it has been shown that induced expression of LMP2 or LMP7 can (partially)overcome particular presentation defects (Sibille et nl , 1995; Cerundolo et al., 1995).More irnportant, mice lacking either tlie LMP2 or LMP7 gene showed reduced a capacity to present particular antigens (Fehling et al., 1994; Van Kaer et nl., 1994), reduced class I cell surface expression (Fehling et al., 1994), or CTL precursors (Van Kaer et al., 1994). The phenotype of crosses of LMP2’ and LMP7’- mice is still undetermined. The use of short fluorogenic peptide substrates to characterize differential cleavage patterns of uninduced vs. immunomodulated proteasoines has brought about conflicting results (Driscoll et al., 1993; Gaczynska et al., 1993, 1994, 1996; Van Kaer et al., 1994; Ustrell et al., 199.5; Kuckelkorn et ul., 1995; Groettnip et al., 1995; Ehring et n l , 1996; Stohwasser et al., 1996) that have been reviewed recently (Groettrup et al., 1996a). The majority of studies reported that iinrnunoproteasoines show an enhanced cleavage after hydrophobic or basic residues, whereas cleavage after acidic residues is reduced consistent with the properties of C-terminal residues in class I-bound peptides. Also, the use of oligopeptides to assay tlie two types of proteasomes did not settle this issue. Whereas one group did not note any difference between cleavage products generated by LMPB +/ L M P F or LMPB-ILMP7- proteasoines (Ehring et al., 1996), another group reported that an antigenic epitope was less often destroyed in the presence of LMP2 and/or LMP7 (Boes et al., 1994, Kuckelkorn et al., 1995).

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E. ROLE OF PROTEASOME ACTIVATORCOMPLEXES IN ANTICENPRESENTATION The symmetrical binding of two 19s (PA700) cap complexes to the 20s proteasome gives rise to the 26s proteasome of -2 MDa MW (Koster et al., 1995; Peters, 1994). In vivo assembly of a portion of 20s proteasomes with preexisting 19s cap complexes was demonstrated in pulse-chase experiments (Yang et al., 1995). The 26s proteasome mediates the ATPdependent degradation of mostly ubiquitin-conjugated proteins. Among the -15 different subunits of the 19s cap complex 1 ubiquitin-binding protein has been identified as well as 6 different putative ATPases that are thought to catalyze the unfolding of protein substrates (reviewed by Coux et al., 1996; Jentsch and Schlenker, 1995; Rubin and Finley, 1995). The ubiquitin system was shown to rapidly eliminate abnormal proteins and control the turnover of several short-lived regulatory proteins including transcription factors, oncogene products, or cyclins (reviewed by Ciechanover, 1994; Hochstrasser, 1995). Since the initial observation by Townsend and colleagues (1988) that ubiquitin tagging can facilitate the class I-restricted presentation of an influenza virus N P eptitope, little direct evidence for the involvement of the ubiquitin pathway in antigen presentation has accumulated. For two secretory proteins with intramolecular disulfide bonds, ovalbumin and P-galactosidase, a dependence on ubiquitin conjugation for class I-restricted antigen presentation has been shown (Michalek et al., 1993, 1996; Cox et al., 1995; Grant et al., 1995). The use of mutant cells with a temperature-sensitive defect of Ub-activating enzyme E l has, however, led to conficting results (Michalek et al., 1993, 1996; Cox et al., 1995).For N-terminally modified P-galactosidasevariants, Grant et al. (1995) demonstrated a correlation between ubiquitin acceptor properties of the proteins and their capacities to be degraded by 26s proteasomes and to be presented after cytoplasmic loading. Both degradation and presentation could be blocked by peptide aldehyde inhibitors. In contrast to native ovalbumin, the presentation of chemically denatured ovalbumin (introduced into the cytoplasm by osmotic loading) or endogenously synthesized ovalbumin (expressed from recombinant vaccinia virus) was not affected by a temperature-sensitive defect of the ubiquitinactivating enzyme E 1(Michaleket al., 1996).Because ovalbumin presentation remained sensitive to LLnL these findings rather suggest a role for an ubiquitin-independent pathway and 20s proteasomes in antigen processing while leaving the relevance of 26s proteasomes uncertain. Alternatively, the 20s proteasome was shown to associate with another activator complex of -180 kDa in the absence of ATP, the ring-shaped 11s or PA28 regulator (Coux et al., 1996). PA28 enhances the cleavage of short peptide substrates in vitro but not of (ubiquinitated) proteins

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(Greottrup et nE., 1996a). Two distinct but homologous components of 27 kDa MW, PA28a and PA280, have been cloned (Realini et al., 1994; Ahn et al., 1995). In vitro studies have shown that the PA28a subunit alone can stimulate proteasome activity, but that the heterodimeric structure of the hexameric PA28 activator is required for maximal proteasome activation (Song et al., 1996). PA28a and PA28p proteins are inducible by IFN-7 suggesting a role of PA28 in antigen presentation. PA28a stably expressed in fibroblasts significantly enhanced the class I-mediated presentation of two cytomegaloviral epitopes (Groettrup et al., 1996~). A favorable modulation of the proteasomal cleavage activity appears to be the reason for this augmenting effect. In a certain range of substrate concentrations, PA28 was shown in vitro to strongly promote the generation of double-cleavage products derived from 19- to 25-mer peptides. These double-cleavage products contained significantly higher amounts of class I ligands and potential precursors (Dick et al., 1996).

F. TARGETING OF PEPTIDES TO THE PEPTIDE TRANSPORTER? It is an open question as to how peptides that are released from proteasomes reach the TAP peptide transporter located in the ER membrane (Kleijmeer et al., 1992; Russ et nl., 1995). Peptide epitopes derived from cytoplasmic proteins are either undetectable or present in very low amounts in the absence of class I molecules that specifically bind these peptides and thus protect them from final degradation (Griem et al., 1991; Pamer et aZ., 1991; Sijts et al., 1996b).This strongly suggests that peptides finding no class I receptor are fully degraded in a short time, e.g., by cytoplasmic aminopeptidases (Taylor, 1993),and invokes a role for chaperone molecules protecting peptides on their way to TAP from 26s proteasomes that are present in the cytoplasm (Yang et al., 1995). Because the 20s proteasome has been detected on the ER membrane by immunoelectron microscopy (Rivett, 1993) or associated with the microsomal subcellular fraction (Yang et al., 1995), it can, however, not be excluded that peptides are directly fed into TAP by ER-associated proteasomal subpopulations. Based on findings that the cytosolic heat shock proteins hsp70 and hsp90 bind cytosolic peptides and elicit tumor-specific immunity (Udono and Srivastava, 1993, 1994), Srivastava coined the hypothesis that heat shock proteins may generally serve to carry antigenic peptides to the peptide transporter (Srivastava et al., 1994). Direct biochemical evidence for this assumption, however, is still lacking. IV. Peptide Loading of Class I Molecules in the ER

Different aspects of this topic have been reviewed elsewhere (Jackson and Peterson, 1993; Barber and Parham, 1993; York and Rock, 1996).

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Here, we will mainly focus on novel results related to transient molecular complexes in the ER involving class I molecules and TAP. In the course of maturation in the ER, newly synthesized class I molecules are transiently associated with chaperones (reviewed by Williams and Watts, 1995; Helenius et al., 1997). Recent investigations indicated that the maturation of class I heavy chain-µglobulin-peptide heterotrimers is more complex than previously thought, involving a number of different chaperones. Unsolved discrepancies between the assembly pathways of human and mouse class I molecules add further complexity to this subject. The first chaperone class I heavy chains (HC) encounter is calnexin (p88)with which they rapidly and quantitatively associate after their biosynthesis into the ER membrane (Degen and Williams, 1991; Degen et al., 1992; Galvin et al., 1992; Hochstenbach et al., 1992). Calnexin facilitates folding and disulfide bridge formation of the nascent heavy chains and promotes assembly of heavy chains with P2m (Vassilakos et at., 1996). Regarding the latter function of calnexin, controversial results have been obtained for HLA heavy chains (Tector and Salter, 1995). Calnexin efficiently retains immature heavy chains in the ER ( Rajagopalan and Brenner, 1994; Jackson et al., 1994). However, in human mutant cells that do not express calnexin (Scott and Dawson, 1995) or in mouse cells that are deficient for glucosidase I1 (Balow et al., 1995), whose activity is required for the association of class I with calnexin, cell surface expression of class I molecules is not compromised. The data suggest that in these circumstances calnexin may be functionally replaced by the ER chaperone BiP (Balow et al., 1995),to which a subpopulation of (possibly aggregated) free heavy chains can also bind in normal human cells (NoRner and Parham, 1995). Several investigators have observed that human HC-&m heterodimers no longer associate with calnexin (Sugita and Brenner, 1994; Ortmann et al., 1994; N o h e r and Parham, 1995) whereas mouse HC remain bound to calnexin after assembly with &m (Degen et al., 1992; Jackson et al., 1994; Suli et al., 1994, 1996). One report, however, also demonstrated the presence of such ternary complexes for human cells (Carreno et al., 1995). Sadasivan et al. (1996) provided evidence that the soluble ER protein calreticulin replaces calnexin as chaperone after association of HLA HC with P2m and remains associated during the transient interaction of HLA HC-P2m with TAP (see below). In contrast, calnexin was found as part of complexes of H-2 HC-P2m and TAP (Carreno et al., 1995; Suh et al., 1996) and accompanied mouse class I molecules even after peptide loading and release from TAP (Suh et al., 1996). The distinct behavior of human and mouse class I heavy chains appeared to be due to intrinsic properties and not to the chaperone species involved because mouse heavy chains expressed in human cells still coprecipitatedwith &m and calnexin (NoRner

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and Parham, 1995; Carreno et al., 1995). In TAP-deficient human T2 and mouse RMA-S transfectants, K” or D“ molecules were released to the cell surface in greater quantities than HLA-A2, -A3, or B27 molecules suggesting that the quality control for mature mouse class I molecules may be less stringent than that for human class I (Anderson et al., 1993). In clear conflict with investigations on the mouse class I-TAP complex mentioned previously, but in line with findings in the human system, another recent study reported an association of H-2K” molecules with calreticulin (Van Leeuwen and Kearse, 1996).Calreticulin-associated class I heavy chains were mostly associated with TAP, whereas calnexin did not participate in this complex. Distinct glycoforms of Kbwere shown to interact with calnexin and calreticulin, res ectively. Deglucosylation of N-linked glycans controled the release of K molecules from both calreticulin and TAP (Van Leeuwen and Kearse, 1996). More work is required to sort out the apparent discrepancies among studies on class I assembly in the mouse and between mouse and human. The physical association of class I molecules with the TAP1-TAP2 dimer was demonstrated for the human and mouse system by coimmunoprecipitation (Ortmann et al., 1994; Suh et nl., 1994). In TAP2-deficient mutants, class I was shown to bind to the TAPl subunit suggesting that TAPl mediates the contact with class I molecules, but it was left open whether the TAP2 subunit also has the capacity to associate with class I (Suh et al., 1994; Androlewicz et al., 1994). Addition of peptide to permeabilized cells or lysates releases class I from the complex with TAP (Ortmann et al., 1994; Suh et al., 1994, 1996; Carreno et al., 1995). Consistent with the concept that peptide loading by TAP is a major checkpoint for class I maturation, addition of the proteasome inhibitor LLnL to cells significantly prolonged the association of human or mouse class I molecules with TAP (Hughes et al., 1996; Suh et al., 1996). In heavy chain-deficient 721.221 mutant cells, &m alone is able to efficiently associate with TAP, thus providing an elegant mechanism to retain the soluble &m molecule at the site of peptide loading (Solheim et al., 1997). Studies empoying the mutant lymphoblastoid cell line 721.220 implicated another molecule in the formation of the class I-TAP complex (Grandea et al., 1995). A 48-kDa glycoprotein, termed tapasin, is found in complexes with TAP, HC-&m, and calreticulin in normal cells and is absent in 220 cells (Sadivasan et al., 1996).Diverse class I alleles expressed in .220 cells fail to associate with TAP leading to defective peptide capture and cell surface expression (Greenwood et al., 1994; Grandea et al., 1995). Tapasin, which was first identified by coprecipitation with anti-TAP antibodies (Oltmann et al., 1994), appears to form a bridge between heavy chains and TAP and can independently bind to either TAP or the HC-&m-

r

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calreticulin complex (Sadasivan et al., 1996; Solheim et al., 1997). The tapasin gene itself, or gene(s) controlling the expression of tapasin, maps to chromosome 6, as shown by chromosome transfer to .220 cells, and possibly to the MHC at 6p21 (Grandea et al., 1996). Reconstitution of ,220 cells with the cloned tapasin cDNA is required to formally establish its role. Early results pointed to allele-specific variations of the deficient class surface expression present in ,220 cells (Greenwood et al., 1994), but biochemical evidence is needed to strengthen this point. The question of HLA allele specificity in complex formation with TAP has been investigated by Neisig et al. (1995) using normal human B cells and TAP2-deficient mutants. Among the many HLA-A, -B, and -C molecules analyzed, some HLA-B alleles were found to be poorly associated with TAP. B35 subtypes showed differential affinity to TAP and differed only with regard to the chemical properties of the polymorphic residue 116 located at the bottom of the peptide-binding groove. Because of this location it is difficult to envisage how this residue could directly influence the interaction with TAP (or tapasin). Poorly TAP-associated HLA-B molecules rapidly migrated to the cell surface (Neisiget al., 1995). In contrast, HLA-C molecules appear at low levels at the cell surface, while being synthesized in normal amounts, but appear to associate stably with TAP. Interestingly, HLA-C molecules needed a 10-fold higher concentration of a completely degenerated 9-mer peptide mixture than HLA-A and -B alleles to be released from TAP in vitro (Neisig et al., in press). These results suggest a correlation between the abundance of peptides binding to a particular class I allele, its retention time in the complex with TAP, and the rate of transit to the cell surface. For two mouse class I molecules, Dd and Ld,point mutations of residues 227 and 222, respectively, abrogate assembly with TAP (Carreno et al., 1995; Suh et al., 1996). These residues map to an exposed loop in the a3 domain. Furthermore, antibody epitopes in the agdomain are masked in the complex with TAP suggesting that this structure is directly involved in the interaction (Carreno et al., 1995; Suh et ul., 1996). TAP-associated Ld molecules exist in a distinct antibody-defined “open” conformation that is lost upon peptide binding and dissociation from TAP (Carreno et al., 1995). Regarding whether extracellular domains of class I are sufficient for complex formation with TAP, conflicting results have been obtained. Although the soluble naturally occurring variant of HLA-G could not be coprecipitated with TAP antisera (Lee et al., 1995), soluble Ld or Dd chimeric molecules were associated with TAP to some extent (Carreno et al., 1995; Suh et al., 1996). Studies employing HLA-A2 molecules with a point mutation (T134K) in an outside loop of the az domain have shed

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light on another structural aspect of the intricate class I assembly pathway. This mutation induced a complete failure of TAP association and TAPdependent peptide loading that could be overcome by ER-targeted minigene products (Peace-Brewer et al., 1996; Lewis et al., 1996). Unlike wildtype class I molecules that are retained in the ER of TAP-deficient mutants, empty HLA-A2(T134K)molecules are rapidly released to the cell surface. Recent experiments suggest that this deficiency is due to the inability of A2(T134K) to bind to calreticulin, thus pointing to an important role for calreticulin in the retention of unloaded class I heterodimers ( J. Frelinger, personal communication). It seems important to note, however, that TAP association is not an indispensable requirement for peptide loading. It has been demonstrated for various class I alleles that ER-targeted peptides driven from minigene constructs can stimulate CTL responses in TAP-deficient cells (see Section 11,C). The transient association of class I molecules with chaperones and TAP, however, may greatly facilitate peptide loading by maintaining class I in a peptide-receptive conformation and by providing a high local concentration of peptide. The prolonged TAP association of HLA-C molecules suggests that class I molecules are trapped in the TAP complex until a suitable peptide is eventually transported. Similarly, RT1.Aa molecules transfected into cells expressing a noncompatible TAP-B (cirnb) molecule (see Section II,A) stably bound to TAP and thereby outcompeted the normal TAP association of endogenously expressed RT1.A" molecules (Knittler and Howard, 1997). This block could be lifted by coexpression of a TAPS-A chain (see Section VI). V. TAP Genes and Their Regulation

Almost simultaneously,TAPl cDNAs were isolated from human, mouse, and rat cells (Trowsdale et al., 1990; Spies et al., 1990; Monaco et al., 1990; Deverson et al., 1990),followed by the cDNAs coding for the corresponding TAP2 proteins (Powis et al., 1991a,b; Bahram et al., 1992; Yang et al., 1992a). The search for transporter sequences was directed by the finding that in one of the class I-deficient human cell lines, LCL 721.174, almost the entire class I1 region of its hemizygous MHC (centromeric from the DPA locus and telomeric from the DRA locus) was deleted (Spies et al., 1990). Likewise, in the rat, the cim locus could be mapped between the RT1.H (equivalent to D P ) and RTI.Ba (equivalent to DQA) loci (Deverson et al., 1990; Livingstone et al., 1991). In this genomic region, a cluster of four genes was discovered comprising the transporter genes TAPl and TAP2, each being preceded by the genes coding for the proteasome P subunits, LMP2 and LMP7, respectively (Brown et al., 1991; Glynne et

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al., 1991; Martinez and Monaco, 1991; Kelly et al., 1991; Ortiz-Navarrete et al., 1991). In the MHCs of human, mouse, and, most likely, the rat, TAP and LMP genes are arranged in a colinear fashion and map to corresponding locations in the MHC (Hanson and Trowsdale, 1991, Beck et al., 1992, Carter et al., 1994). The entire region of the human MHC encompassing TAP, LMP genes, and the adjacent DOB gene has been sequenced (Beck et al., 1992). Human TAPl and TAP2 comprise 8 and 10 kbp of DNA, respectively, separated by 7 kbp in which the LMP7 gene is located. TAPl and TAP2 genes both have 11exons, 8 of which are the same size and all exodintron boundaries are identical in their boundary class (Beck et al., 1992). The organization of LMP2 and LMP7 genes is also very similar.LMP2, however, is encoded on the (-) strand, whereas LMP7 and TAP genes are encoded on the (+) strand. These structural features led to the suggestion that the array LMP2:TAPl:LMP7:TAP2 arose by duplication of a primordial unit consisting of one proteasome gene and one transporter gene followed by inversion of LMP2 (Beck et al., 1992). In the human MHC, a hotspot of recombination has been mapped within the TAP2 locus (Cullen et al., 1997; see Section XIV,D). The expression of TAP1 and TAP2 inRNAs is inducible by IFN-y (Trowsdale et al., 1990; Bahram et al., 1991; Powis et al., 1992b), IFN-P, or TNF-a (Epperson et al., 1992). As with class I, IFN-y and TNF-a act synergistically in the induction of TAPl (Epperson et al., 1992; Min et al., 1996; Johnson and Pober, 1990). Also, LMPS and LMP7 are inducible by IFN-y (Monaco and McDevitt, 1986; Glynne et al., 1991; Kelly et al., 1991; Ortiz-Navarrete et al., 1991; Friih et al., 1992, 1994; Yang et al., 1992b). This feature links these auxiliary proteins involved in antigen processing functionally to MHC class I and I1 molecules, which are induced in the course of an immune response by lymphokines (Ting and Baldwin, 1993). In the promoter regions of TAP and LMP genes, interferonstimulated response elements, gamma-activated elements, and NF-KBelements (for the response to TNF-a) are found (Beck et al., 1992; Friih et al., 1992; Wright et al., 1995; Min et al., 1996). TAP1 and LMP2 genes are divergently transcribed and coordinately regulated from a shared bidirectional promoter (Wright et aZ., 1995). Knockout mice lacking the interferon-induced DNA-binding protein IRF-1 show severely reduced levels of both LMPS and TAPl, which explains the paucity of CD8+ cells (White et al., 1996). Interestingly, TAPl mRNA was found to be more rapidly induced by IFN-y or IFN-P than class I heavy chain mRNA (Epperson et al., 1992; Min et al., 1996). This suggests that the baseline of TAP expression may not be sufficient to support increases of class I during lymphokine-driven immune responses. As far as the limited amount

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of data allow for a conclusion, TAP and class I molecules appear to be coordinately expressed in normal tissues including placenta ( Joly and Oldstone, 1992; Neumann et al., 1997; Clover et al., 1995; Rodriguez et al., 1997). Aberrant expression of TAP and LMPs in tumors will be discussed in Section XIV,C. VI. TAP Proteins in Different Species

From six species, TAP1 cDNA sequences have been cloned and entered in databases. TAPl open reading frames significantly vary in length. 748, 747 (or 748), 724, 725, 763, and 739 amino acids are predicted for human, gorilla (Gorilla gorilla), mouse ( M u s musculuslcastaneus),rat (Rattus noruegicus), hamster (Mesocricetus auratus), and salmon (Salmo salar) TAPl chains, respectively, corresponding to predicted molecular weights between 79 and 83 kDa. Alignments of these TAPl sequences shows that those from human and gorilla differ from the others by two short sequence insertions in the N-terminal part of the molecule (corresponding to exon 1). Syrian hamster TAPl and the distantly related salmon TAPl have sequence extensions at the C terminus compared with the other TAPl species. It is unknown whether these variations in TAPl protein length imply functional differences with regard to TAP-associated molecules or substrate specificity in the involved species. Human TAPl can form functional heterodimers with mouse or rat TAP2 subunits (Armandola et al., 1996). TAP2 cDNAs code for proteins of 703 (or 687; see below) amino acids in man, 703 amino acids in gorilla, and 702, 703, and 704 amino acids in mouse, rat, and hamster, respectively, with a predicted molecular weight of 77.5 kDa (or 75.5 kDa). The distantly related salmon TAP2 sequence containing 724 residues shows insertions in the N- and C-terminal ends. The analysis of cross-species homologies of the previously mentioned TAPl and TAP2 sequences depicts the expected phylogenetic distances between hominoids, rodents, and fish. Using the Clustal alignment (Higgins and Sharp, 1989), TAPl sequences from hominoids and rodents possess between 69.2% (human vs hamster) and 98.8% (human vs gorilla) identical amino acids (F. Momburg, unpublished data). TAP2 proteins are are even less divergent, with similarities of 74.6-77.0% between hominoid and rodent sequences. Although TAPl and TAP2 are closely related in their predicted structure (see Section VIII), they share only between 35.5% (human) and 36.6% (mouse) of their amino acids. The recently cloned TAPl and TAP2 sequences of the Atlantic salmon (Grimholt. 1997) are at the far end of the currently known TAP phylogeny. Although SasaTAP1 and SasaTAP2A share only -40-42% of their residues with the equivalent homoinoid or rodent TAPl and TAP2 proteins, respectively, the differ-

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ence between the TAP units of salmon is conserved (36.1% homology). This suggests that TAPl and TAP2 genes arose by duplication from a putative ancestral transporter gene before the speciation of vertebrates. By PCR techniques, various allelic variants of human TAPl and TAP2 genes have been detected that entail only a limited extent of sequence variation in the corresponding proteins. Human TAPl was reported to be dimorphic at positions 333 (IleNal), 370 (AlaNal), 458 (Val/Leu), 637 (Asp/Gly),648 (Arg/Gln),and 659 (Arg/Gln),and human TAP2 at positions 379 (Val/Ile), 565 ( A l n h r ) , 577 (MetNal), 651 (Arg/Cys), 665 (Thr/Ala), and 687 (Stop/Gln) (Colonna et al., 1992; Powis et al., 1992b, 1993; Jackson and Capra, 1993, 1995; Carrington et al., 1993; Aoki et al., 1994; MoinsTeisserenc et al., 1994; Kellar-Wood et al., 1994; Szafer et al., 1994; Can0 and Baxter-Lowe, 1995; Saji et al., 1996; Chen et al., 1996; Cesari et al., 1997). The TAP2 dimorphism at residue 687 results in variant proteins that differ in length by 16 amino acids. In heterozygous cells, short and long TAP2 variants are incorporated into TAP1-TAP2 heterodimers with equal efficiencies (Spies et al., 1992; Kelly et al., 1992). The mentioned amino acid dimorphisms (and additional silent nucleotide exchanges) combine to at least five allelic variants of TAP1 and at least seven allotypes of TAP2 that have been classified by the overlapping nomenclatures of the WHO Nomenclature Committee and by Powis et al. (1993). Possible functional consequences of human TAP polymorphism with regard to substrate specificity and linkage to autoimmune diseases are discussed in Section X,D). The three known gorilla TAPl alleles (GogoTAPla-c) concordantly differ from human TAPl allotypes in five residues, in addition to four dimorphic residues that occur only in gorilla sequences (Laud et al., 1996). The four gorilla TAP2 sequences (GogoTAP2a-d) collectively vary at two positions from all human TAP2 allotypes and contain four additional residues with gorilla-specific allelic dimorphisms (Loflin et al., 1996). The absence of ancestral polymorphisms suggest that gorilla and human TAPl and TAP2 genes have diversified since the divergence of the hominoid lineages. TAPl [b, d, J k, g7(NOD), cas, and N O N ] and TAP2 allelelic variants (b,d,J k, g7, cas, and sw)of mouse inbred strains are dimorphic at seven independent sequence positions each (Pearce et al., 1993; Marusina et al., 1997). Likewise, TAPl and TAP2 cDNAs cloned from Syrian hamster FF and BHK cells code for six amino acid exchanges between TAPl sequences and 6 between TAP2 sequences (Lobigs et al., 1995, M. Lobigs et al., submitted for publication). A slightly higher degree of polymorphism has been noted for TAPl sequences derived from rat inbred strains ( a d , c, d o l , k, I, n, and u ) .Twelve positions with allelic dimorphism

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occur in these seven sequences (E. V. Deverson, submitted to EMBL database). With very few exceptions, amino acids involved in allelic dimorphism of hominoid or rodent TAPl and TAP2 sequences are all located at distinct species-specific positions, suggesting that at least some of them may have resulted from an intraspecies genetic drift that may not necessarily be linked with functional differences. Rat TAP2 appears to be a remarkable exception from other TAP subunits because a more significant allelic polymorphism was found that is the basis of the cini phenomenon mentioned previously (Powis et al., 1992a). Of the four available allelic forms of rat TAP2, two are TAP2-A (cim')haplotypes (av2 and 1) and two are TAP2-B ( c i d ) haplotypes (c and u ) . The consensus sequences of TAP2-A and TAP2-B alleles show 25 amino acid exchanges that are more frequent in the N-terminal half. (There are only two conserved substitutions between TAPP"' and TAP2' and one conserved exchange between TAP2' and TAP2.) By genomic typing of 14 RT1 standard haplotypes, 7 were found to belong to the TAP2-A group and 7 to the TAP2-B group (Joly et al., 1994). Based on a intron length polymorphism the 7 TAP2-B sequences can be further split into two phylogenetic groups (Joly et al., 1994). VII. TAP as a Member of the ABC Transporter Superfamily

TAPl and TAP2 sequences were assigned to the ABC superfamily of transporter due to the presence of highly conserved nucleotide-binding domains containing the Walker A and Walker B consensus motifs of ATPbinding proteins (Monaco et al., 1990; Deverson et al., 1990; Trowsdale et al., 1990; Spies et al., 1990; Bahram et al., 1991; Powis et al., 1992b; Walker et al., 1982). Several hundred ABC transporter sequences have been identified in species ranging from bacteria to human (reviewed by Higgins, 1992; Doige and Ames, 1993). As a common structural feature, they all contain a functional core of two multimembrane-spanning and two nucleotide-binding domains that can be expressed as four noncovalently linked polypeptides or fused to one to three proteins depending on the ABC transporter. To these core domains are added regulatory domains in the large mammalian ABC transporters (e.g., in CFTR and MDR1) or separate periplasmic-binding proteins that deliver substrate to the membrane-bound components of bacterial ABC transporters (e.g., the oligopeptide- or the maltose-binding protein). Energy in the form of triphosphate nucleotides is required for substrate translocation, but the exact mechanism(s) is still largely unknown. ABC transporters translocate a wide range of substances, including ions, sugars, polysaccharides, hydrophobic

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compounds, amino acids, oligopeptides, and large polypeptides, over membranes. The oligopeptide permease of Salmonella typhimurium, translocating peptides of two to five amino acids (Hiles et al., 1987);the STE6 transporter of Saccharomyces cereuisiue, which catalyzes the secretion of the mating pheromone a-factor, a prenylated and methylated 12-mer peptide (Kuchler et al., 1989);and a variety of bacterial ABC transporters that export proteins ranging in size from 3.3 to 177 kDa (reviewed by Kuchler, 1993) resemble TAP with regard to substrate specificity. Examples for ABC transporters in vertebrates are the cystic fibrosis transmembrane conductance regulator (CFTR) that is a chloride channel (reviwed by Riordan, 1993; Welsh and Smith, 1993; Gadsby et al., 1995), the peroxisomal membrane protein 70 and the adrenoleukodystrophy protein of unknown specificity (Kamijo et al., 1990; Mosser et al., 1993), the multidrug resistance-related protein transporting a broad range of anionic organic compounds (reviewed by Lautier et al., 1996; Loe et al., 1996), and the P-glycoproteins MDRl and MDR3 that actively translocate various hydrophobic drugs or short-chain lipids and phospholipids, respectively (van Helvoort et al., 1996; reviewed by Gottesman and Pastan, 1993; Borst et al., 1993). The P-glycoproteins are the ABC transporters with the greatest sequence homology to TAP (Powis et al., 1992b; Hughes, 1994). To date, TAP is the only ABC transporter dedicated to a specialized function in the immune system. A recent search for Walker A and Walker B motifs in databases containing expressed sequence tags revealed the presence of 30-50 unknown ABC transporters in the human genome (F. Momburg, unpublished results). It can be speculated that some of them may be involved in immune functions. VIII. Structure of TAP Molecules

TAPl and TAP2 proteins both contain a N-terminal hydrophobic domain (-60% of the sequence) that is predicted to cross the ER membrane several times and a C-terminal hydrophilic domain harboring the nucleotide-binding fold. The subcellular location of TAP molecules is restricted to the ER as indicated by nearly perfect colocalization with the ER marker BiP (Kleijmeer et al., 1992; Russ et al., 1995; Hengel et al., 1997), and to cisternae of the cis-Golgi as revealed by immunoelectron microscopy (Kleijmeer et al., 1992). The cytosolic orientation of the ATPbinding domain was concluded from the finding that immunogold labeling occurred predominantly at the cytosolic side of membranes using an antibody specific for the C terminus of TAPl (Kleijmeer et al., 1992). Also in microsomes. the C-terminal domains of TAPl and TAP2 were accessible

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to antibodies leading to inhibition of peptide transport (Schumacher et al., 1994a; van Endert et al., 1994a). Because other ABC transporters, sucli as Pgp or CFTR, are expressed on the plasma membrane, TAP seems to be retained in the ER by an as yet unknown mechanism. The previously mentioned common structure of ABC transporters suggested that TAPl and TAP2 might function as heterodimers. Coinimunoprecipitatioii of human (or mouse) TAPl and TAP2 molecules confirmed this assumption (Kelly et nl., 1992; Spies et al., 1992; Ortmann et al., 1994; Suh et al., 1994; van Endert et al., 1994a; Russ et al., 1995) but left open the possibility that multiple copies of each subunit may be present in TAPI-TAP2 complexes. TAPl and TAP2 subunits seem to assemble extremely rapidly after biosynthesis (Russ el al., 1995).A minor subpopulation of human TAP1, but not TAPS, was found to be glycosylated (Russ et al., 1995). The membrane topology of the hydrophobic domains of TAPl and TAP2 is only vaguely known. By using different algorithms, 6-10 potential transmembrane regions have been predicted for TAPl (Trowsdale et al., 1990; Monaco et al., 1990; Moinburg et ul., 1996) and TAP2 (Bahrain et al., 1991; Powis et nl., 1992b; Momburg et al., 1996). Like other ABC transporters, TAPl and TAP2 proteins lack a typical N-terminal leader sequence. By employing in uitro translation in the presence of TAP-deficient microsornes, we are currently analyzing the inenibrane topology of COOH terininally truncated variants of rat TAP2'. Truncation products are tagged at the new COOH tenninus with a peptide containing niultiple glycosylation sites and repeated epitopes for recognition by a monoclonal antibody. Preliminary data suggest that the previously proposed location of mernbrane-spanning segments and orientation of interconnecting loops (Momburg et al., 1996) may essentially be correct ( J . Koopmann and F. Momburg, unpublished results). This model predicted three or four transmembrane segments in the immediate N-terminal part followed by three pairs of transmernbrane segments that are separated by relatively long cytoplasmic loops, whereas ER-located sequences would be very short. This model is consistent with the cytoplasmic orientation of genetically defined residues determining transport specificity (Momburg et al., 1996; Armandola et al., 1996) and of sequence stretches identified by photoaffinity labeling with peptides (Nijenhuis et nl., 1996; Nijenhuis and Hammerling, 1997; (see Section XI1,B). IX. In Vitro Assays for Peptide Binding and Transport by TAP

A. PEPTIDE GLYCOSYLATION. CI.ASS 1 CAPTURE, A N D BINDING ASSAYS Because of their intricate membrane-integrated structure TAP heterodimers cannot easily be purified for investigation of their biochemical

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properties. The development of in vitro assays based on cells selectively permeabilized at the plasma membrane with the bacterial toxin streptolysin 0 (SLO) (Neefjes et al., 1993; Androlewicz et al., 1993) or based on microsomes prepared from TAP expressing cells (Shepherd et al., 1993; Heemels et al., 1993) established our current understanding of the molecular function of this membrane translocator. Using 3H- or lZ5I-labeledclass I ligands Shepherd et al. (1993) and Androlewicz et al. (1993) demonstrated TAP- and ATP-dependent peptide accumulation in the ER by immunoprecipitation of the peptide-binding HLA-A3 or Kb class I molecule. When SLO-permeabilized cells were reconstituted with cytosol to render the secretory pathway functional, loading with transported peptide induced release of class I molecules from the ER to the Golgi (Androlewiczet al., 1993). In order to monitor peptide translocation into the ER independent of subsequent binding to class I molecules, model peptides have been employed that are equipped with a Tyr residue for radioiodination and a AsnXaa-Thr/Ser glycosylation consensus signal (Neefjes et al., 1993; Heemels et al., 1993). Following translocation into the ER, the oligosaccharyltransferase complex will conjugate an N-linked glycan to the peptide. After detergent lysis this ER-specific label can be used to recover the translocatedlglycosylated fraction from the bulk of nontransported radiolabeled input peptide by means of the lectin concanavalin A immobilized on Sepharose beads. The N-linked glycan also serves as a retention device that prevents efflux of transported peptides from the ER (see Section XIII) (Heemels et al., 1993; Momburg et al., 1994a; Roelse et al., 1994).Glycosylated, ER-retained peptides have been found to be remarkably stable, whereas nontransported peptides that are in contact with permeabilized cells or the outside of microsomes undergo degradation due to the activity of associated proteases. These studies clearly confirmed that both TAPl and TAP2 molecules need to be expressed for peptide transport suggesting that functional transporters consist of at least one TAPl and one TAP2 subunit. Cells expressingonly TAPl (RMA-Sand .174/B6) or only TAP2 (TAPl-’- splenocytes and .174/J4) do not exhibit significant peptide translocation in vitro (Androlewicz et al., 1993, 1994; Shepherd et al., 1993; Momburg et al., 199413).Likewise, infection of Sf9 insect cells with both recombinant baculoviruses coding for TAPl and TAP2 reconstituted peptide transport, but infection with the individual baculoviruses alone did not (Meyer et al., 1994; Nijenhuis et al., 1996). Collectively, these studies disproved earlier works describing efficient ATP-independent peptide entry into the ER ( L 6 y et al., 1991; Koppelman et al., 1992).

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To monitor the initial step of peptide interaction with TAP, binding assays have been developed on the basis of microsomes derived from TAPexpressing insect cells (van Endert et al., 1994a; Uebel et al., 1995). AS opposed to the actual membrane translocation, binding of peptide to cytosolic structures of the TAP1-TAP2 complex does not require ATP and was found to even be enhanced at low temperatures (van Endert et al., 1994a). Microsomes containing only human TAP1 or human TAP2 did not show specific peptide binding (van Endert et al., 1994a; Uebel et al., 1995).

B. DIRECT TAP ASSAYSVERSUS COMPETITION ASSAYS We will add some technical comments that apply to the investigation of the substrate specificity of peptide transport. In several studies unlabeled peptides have been utilized as competitors for ATP-dependent translocation/ glycosylation of a radiolabeled reporter peptide, for class Ibinding subsequent to TAP-mediated transport, or for the ATP-independent binding to TAP. Competitor peptides are compared by means of their concentrations to inhibit translocation of the reporter by half ( ICso values). However, competition assays should be interpreted with some caution because competing peptides may interfere with transport of the reporter without being transportable themselves. Further complexity arises when both the reporter and the competitor peptide contain a glycosylation site leading to competition for both transport and glycosylation (van Endert et al., 1994a; Neefjes et al., 1995). In addition, the poorly understood phenomenon of retrograde transport might affect the outcome of competition experiments. If the competitor has a higher affinity for the putative exporter it might increase ER accumulation of the reporter peptide. Another caution concerns the action of proteases that can hardly be fully removed from semiintact cells or microsomal preparations. Partial degradation of competitor peptides 2 12 residues may give rise to products that possess a higher affinity for TAP because of the preference of TAP for slightly shorter substrates (see Section X,A). Truncated variants will end or begin with a new amino acid and put internal residues into a new register. Because of the complex sequence specificity of different TAP molecules (see Section X,B) this might confer an altered affinity for TAP. For similar reasons, assays measuring direct binding or competition for binding carried out at 4°C do not necessarily reflect specificities determining the actual translocation of substrate. A limited number of comparative studies, however, suggest a sufficiently high concurrence between rank orders of peptides in translocation (competition) and binding

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(competition) assays (van Endert et al., 1995; Koopmann et ul., 1996; Ahn et al., 1996a). X. Subsirate Specificity of Peptide Transport

A. LENGTH SEL~ECTIVITY OF PEPTIDE TRANSPORT The majority of MHC class I molecules associate with peptide ligands of 8-10 amino acids, whereas some alleles (e.g., HLA-A*1101 or HLAA*6801) can also bind slightly longer peptides of 11 or 12 amino acids (reviewed in Raminensee et al., 1993,1995; Engelhard, 1994a,b).In regard to the relatively strict length requirements imposed on class I ligands it was an important question whether peptide transporters preselect peptides of approximately 9 amino acids or whether longer precursor peptides are also translocated into the ER that thereafter may be processed to their final length. In a number of initial investigations, the question of length selectivity was addressed by using peptides ranging from 6 to 40 residues as competitors for radiolabeled reporter peptides in in vitro transport assays (Shepherd et al., 1993; Heemels et al., 1993; Momburg et al., 1994a; Schumacher et al., 1994a; Androlewicz and Cresswell, 1994). Collectively, peptides of 9 or 10 amino acids were the most efficient competitors, but examples of much longer peptides were also reported, e.g., a 20-mer (Momburg et al., 1994a) or a 24-mer (Androlewicz and Cresswell, 1994), that inhibited translocation of a 9-mer proband peptide with moderate efficacies. The interpretation of these findings is problematic because significant proteolytic activities are associated with SLO-permeabilized cells (Momburget al., 1994a)as well as microsomal preparations (Heemels and Ploegh, 1994; Obst et al., 1995). The degradation of a 16-mer was shown to likely occur on the cytosolic side of the ER membrane (Heemels and Ploegh, 1994). As a consequence of partial peptide degradation (and possibly as a consequence of selection by TAP), input peptides of 16, 20, or 40 amino acids were glycosylated after TAP-mediated transport as truncated variants of approximately 12 residues. Likewise, a 13-mer peptide was retrieved from the ER in intact form but also as truncated 10-or 12-mers, respectively (Momburg et al., 1994a). In the previously mentioned competition studies proteolysis was usually not controlled for; thus, it is uncertain whether degradation products contributed to the inhibitory capacity of a given competitor. The use of peptides that are tagged with a tyrosine residue for radioiodination and a glycosylation signal at opposite ends circumvented these problems and allowed to approach the lower and upper limits of size selection

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by TAP. Proteolytic degradation of so tagged peptides would render the products undetectable in the transportlglycosylation assay through loss of either the radiolabel or the carbohydrate moiety. Using this approach Heemels and Ploegh (1994) demonstrated the transport of peptides up to 16 residues long, whereas a 9-mer was translocated with the highest efficiency. Two series of length variants with defined sequences between 6 and 30 amino acids and one series of partially randomized peptide libraries between 10 and 40 amino acids have been studied by Koopniann et al. (1996). Rat TAPl +2" (cinf),rat TAPl +2" (cim"),and human TAP translocated the 10-iner peptides of the three series with the highest efficiencies, followed by the 8-mer and 12-mer variants. That the superior transport of the 10-mers was not due to their more efficient glycosylation or higher resistance to degradation was controlled for. In contrast to the previous study describing a more permissive behavior of rat cini' TAP than of rat cim" TAP for 12- to 16-nier peptides (Heemels and Ploegh, 1994), we found a more relaxed length selection for long peptides by rat TAP1+2& (Koopmann et al., 1996). Transport of two 6-mers was low but clearly detectable above background. Six amino acids likely represents the lower length limit for TAP substrates because we failed to translocate a 5-mer in a previous study (Momburg et al., 1994a). However, at comparably low efficiencies, transport of 20-, 25-, 30-, and 40-men was detected and their glycosvlation confirmed by biochemical analysis. The 40-mer library represents the longest peptide whose transport by TAP has been reported to date. In conclusion, in vitro studies have shown that TAP molecules from different species prefer peptides of a length that js suited for direct binding to class I molecules, but also much longer (and slightly shorter) peptides can be translocated. In another approach, the peptide-binding assay has been employed to analyze the peptide length preference of TAP. Using fully randomized peptide libraries between 'iand 18 residues, van Endert et a1 (1994a) found that 8- through 16-mers efficiently competed with the binding of radiolabeled 9-mers to hTAP in microsomes, whereas the 7-mer and the 18-mer library were significantly less potent inhibitors. No length selection was concluded from another binding study using competitors in the range of 9-15-mers (Uebel et al., 1995). We assessed the binding to rTAP1+2" microsomes of radiolabeled peptides for a series of length variants and compared them to their ATP-dependent transport (Koopniann et al., 1996). The 10-mer showed the highest binding affinity, the 14- and 18-mer peptides bound moderately to TAP, and no significant binding was detected for the 6- and 30-mer peptides. Thus, the direct binding capacities of these peptides agree well with their ranking in the transportlglycmylation assay.

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Peptides longer than 12 amino acids will be unable to stably associate with class I molecules in the usual way with their free amino and carboqd termini being fixed in the binding groove. Such peptides may be subject to different pathways following translocation into the ER. Some long peptides may bind to certain class I variants in an uncommon fashion and even be carried to the cell surface. A prominent self-peptide eluted from HLA-B27 molecules was described to contain 16 residues (Frumento et al., 1993). A subset of HLA-B"2705 molecules (5-20%) was reported to contain long peptides of up to approximately 33 residues (Urban et al., 1994). By means of a monoclonal antibody the HLA-B27 subpopulation associated with long peptides can be detected on the cell surface. Therefore, it seems possible that long peptides are presented to T cells. Detection of such complexes on the cell surface required expression of TAP (Urban et al., 1994), thus providing in vivo evidence for TAP-mediated translocation of long peptides. Another study described a subpopulation of Kb molecules derived from normal mouse cell lines that was not associated with µglobulin and was complexed with peptides of >15 amino acids (Joyce et al., 1994).The long peptides bound to HLA-B27 or Kb did not show the typical spacing of anchor residues and can be anticipated to extend out of the closed class I-binding groove at one or both ends. Signal sequence-derived peptides that exceeded the canonical length of ligands (9-mers) by 1-5 amino acids have been eluted from HLA-A2 molecules (Henderson et al., 1992, Wei and Cresswell, 1992). By crystallography, a 10-mer peptide bound to HLA-A2.1 was shown to protrude out of the binding site with the C-terminal residue (Collins et al., 1994), and this mechanism is likely to apply to an 18-mer peptide bound to Kk(Olsen et al., 1994). Alternatively, peptides may be exported into the cytosol by the unknown efflux machinery described in Section XI11 for further degradation and possible reimport. Other long peptides may be trimmed by ER proteases, either in solution or after loose association of the C-terminal end with the class I binding cleft as suggested by Rammensee et al., (1993).

B. TAP SPECIFICITY FOR PEPTIDE SEQUENCES The specificity with regard to the peptide sequence was investigated by us and other laboratories for TAP molecules from various species. Using glycosylatable model peptides that were substituted at different positions within the sequence we systematically established specificity patterns for allelic variants of rat TAP, mouse TAP, and human TAP. Because the Cterminal amino acid is very often used to anchor peptides in the binding groove of class I molecules and the repertoire of C-terminal anchor residues is different for human and mouse class I-binding peptides (reviewed in Rammensee et al., 1993, 1995; Engelhard, 1994a, 1994b), it was of prime

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interest to investigate the specificities of TAPS from different species with regard to the C-terminal residue. Two major patterns of TAP specificity became evident, one being selective for peptides with hydrophobic amino acids at the C-terminal position and the other being permissive for nonhydrophobic amino acids. When using the peptide series RYWANATRSX, with X being 1of the 20 common amino acids (Momburg et al., 1994b), mouse TAP1/2 (expressed by RMA cells) and rat TAP1/2' by transfection into TAPU2-negative T2 cells preferred RYWANATRSX peptides with hydrophobic C-terminal residues over those peptides with C-terminal charged or polar or small residues. Human TAP (expressed in LCL721 cells) and rat TAPIIBa (expressed in T2 cells), however, transported the whole set of RYWANATRSX exchange variants with similar efficiencies. The strong selection of mouse TAP and rat TAPU2" against charged C-terminal residues was confirmed using two additonal sets of peptides with the consensus sequences TVDNKTRYX and TNKTRIDGQYX (Momburg et al., 1994b; Obst et al., 1995). In these experiments, some selection by human TAP against acidic and certain small (polar) C-terminal residues was noted while the efficient transport of peptides with positively charged and hydrophobic C-terminal residues was confirmed. Rat TAPUP showed a fully nonselective phenotype for the latter series of C-terminal variant peptides. Using slightly different approaches, other investigators obtained similar results. Heemels et al. (1993)also clearly demonstrated a permissive phenotype of rat TAPlI2-A and a restrictive phenotype of rat TAP1/2-B with respect to the C-terminal peptide residue by studying the transport characteristics of microsomes from various cim" and cimbrat strains. cimabut not cim' microsomes accumulated glycosylatable peptide libraries with basic C-terminal residues. In transport competition experiments, TYQRTRAU variants with charged C-terminal residues efficientlyinhibited translocation of reporter peptides into cim" but were ineffective when cim' microsomes were used. Furthermore, ATP-dependent uptake of radiolabeled proband peptide into mouse microsomes was effectively outcompeted by only those FAPGNYPAX variants with aromatic or aliphatic X residues showing the selective phenotype of mouse TAP (Schumacher et al., 1994a). At variance with these results, Androlewicz et al. (1993) reported transport and subsequent loading onto HLA-A3 molecules of the nef7B peptide QVPLRPMTYK using SLO-permeabilized RMA.A3 transfectant cells. This finding illustrates that the selection by mouse TAP against peptides terminating on basic amino acids may not be an all-or-nothing feature. It is conceivable that for some peptides, but not others, internal (e.g., the penultimate) residues can override the effect of the C-terminal residue.

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For instance, the acceptance by human TAP of peptides with glutamic acid at the C-terminal position appears to be influenced by the whole peptide sequence (Androlewicz and Cresswell, 1994; Momburg et al., 1994b). As opposed to peptide series having a variable C-terminal position, no major differences between the specificities of rat TAPd,rat TAP", human TAP, and mouse TAP were found when 86 model peptides with systematic exchanges at positions 1-8 (of 9-mers) were assayed (Momburg et al., 1994b; Neefjes et al., 1995). Using a 384-membered peptide library with variations at positions 1, 4, 6, 7 , and 8, but with the C-terminal position 9 fixed, Heemels et al. (1993) noted that the bulk of peptides that accumulated in rat cim" TAP microsomes were slightly more hydrophilic than the bulk of peptides accumulated in cimhTAP microsomes. The same authors constructed smaller libraries in which each position of a 9-mer was individually replaced by a mixture of seven amino acids. By probing these peptide mixtures side by side on cim" and cimhmicrosomes, a consistent difference in transport specificity of dimorphic rat TAPs was only evident for the mixture with the variable residue at position 9 (Heemels and Ploegh, 1994). No species- or allele-specific differences between rat cim", rat cim", human, or mouse TAP specificities were conspicuous from our investigations (Momburg et al., 199413: Neefjes et al., 1995). Many of the internal amino acid substitutions, however, clearly affected the relative efficiencies of transport by all tested TAPs, e.g., proline at positions 2 or 3 negatively influenced the transport, whereas glutamic acid at positons 6 or 7 enhanced transport. The relevance of internal residues for TAP recognition is substantiated by a study in which Kb- or Db-binding virus-derived epitopes were tested as competitors for transport by TAP (Neisig et al., 1995). The four peptides that competed with the lowest efficiencies all contained proline at position 3. Replacement of proline by alanine rendered these peptides modest to good competitors. Furthermore, affinities could be drastically improved by addition of natural flanking residues directly surrounding the sequence of the poorly transported viral peptides with proline at position 3. This finding suggests that some immunodominant epitopes may be translocated as slightly extended precursors to circumvent structural constraints imposed by certain internal amino acids. In another study analyzing the transport and presentation of a Kd-derivedself-peptide and analogous sequences from other class I molecules, a strong inhibitory influence on TAP-mediated transport of leucine vs asparagine at position 5 of the 10mer epitope was noted (Gournier et al., 1995). In a number of additional studies, the specificity of human or mouse TAP for peptide sequences was addressed by using naturally processed class I ligands (or precursor peptides or sequence variants thereof) as

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competitors for the binding or translocation of different radiolabeled re1993;Androlewicz et d.,1993;van Endert porter peptides (Shepherdet d., et aZ., 1994a; Chang et nl , 1996). Because of the small number and the unsystematic sequence variation in the examined peptides no conclusions have been drawn with regard to the sequence specificity of TAP. Investigating the requirements of the interaction of TAP with its substrates, Androlewicz and Cresswell (1994) constructed polyalanine 9-mer or 10-mer peptides containing one or two substitutions with other amino acids. Most of these “minimal” peptides still exhibited a surprisingly high affinity for TAP. From the analysis of a large number of substituted polyalanine peptides and natural class I ligands in binding competition assays, van Endert et al. (1995) derived a relaxed motif for peptide binding to human TAP. This motif does not comprise mandatory amino acids at defined positions, as it was described for class I-binding motifs, but rather provides information about Favorable and deleterious residues at the N-terminal three and the C-terminal positions. Investigating a fully randomized 9-mer peptide library in comparison with sublibraries containing one conserved residue (from the sequence RRYNASTEL), Uebel et al. (1995) found that the N-terminal three residues of the sequence contributed the most to the binding affinity. Remarkably, the affinity for human TAP of the high-affinity binder RRYNASTEL ( K , = 310 mi)was only 11 times higher than the affinity of the fully randomized (X), library. This finding highlights the relatively high degree of sequence promiscuity of peptide transporters.

C CIIEMICALLY MODIFIEDPEPTIDE SUHSTHATES Chemical modification of the peptide ligand by methylation or acetylation of the NH2 group or amidation of the COOH group significantly affected the capability of a peptide to be transported or to act as a competitor for transport (Momburg et nl., 1994b; Schumacher et al., 1994a).These findings suggest that charged amino andlor carboxyl groups of the peptide ligand may facilitate interaction with the transporter. A free NHQterminus, however, cannot be an absolute requirement for transport because the N fonnylated mitochondria1 peptide MTF” depends on TAP expression for presentation by H-2M3 molecuIes (Attaya et al., 1992). To analyze the stereospecificityof substrate recognition by TAP, peptides containing one or two D amino acids were tested for transport (or inhibition of transport) (Gromm6 et al., 1997). Introduction of a D amino acid at the C-terminal or the penultimate position reduced peptide transport significantly. This decreased affinity was even more pronounced for D amino acid substitutions at the N-terminal and second positions. Two D amino acids at opposite ends completely abrogated transport. Also, a pep-

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tide consisting entirely of D amino acids was a very poor substrate for TAP. These findings indicate that the proper orientation of peptide side chains toward the backbone is of great importance for ligand interaction with TAP, suggesting both the backbone and side chains contact a putative binding pocket. Remarkably, isosteric peptide bonds that maintain the conformation of the backbone, but fail to form hydrogen bonds, even enhanced peptide translocation. Peptides containing one amino acid conjugated with a bulky hydrophobic group, such as dansyl or anthranyl, were efficiently transported (Uebel et al., 1995; Gromm6 et al., 1997; Nijenhuis et al., 1996). At almost all sequence positions, introduction of such a bulky amino acid improved peptide affinity (Uebel et al., 1995).Extremely extended side chains corresponding to a peptide of -21 amino acids were found to be tolerated by TAP without loss of translocation efficiency. This suggests that peptide side chains do not have to fit into small pockets present in a putative binding fold of TAP, but that side chains may rather contact the walls of an open tunnel-like structure. Interestingly, the peptide modifications with very long branches were still able to compete with peptide transport quite efficiently while hardly being translocated themselves. Thus, they represent the first rationally designed inhibitors of TAP. An unexpected additional panel of substrates for TAP was suggested by a recent study showing overexpression of TAP in a number of multidrugresistant carcinoma and leukemia lines (Izquierdo et al., 1996). TAP transfection into T2 cells conferred mild resistance to etoposide, vincristine and, doxorubicin. Furthermore, etoposide and, vincristine were able to compete with the translocation of a radiolabeled reporter peptide. Thus, TAP may modestly contribute to the MDR phenotype. In conclusion, available evidence indicates that species- or allele-specific differences in the specificities of peptide transporters are confined to the C-terminal residue. The selective phenotypes seem to be fully included in the permissive phenotypes. Peptide transport and peptide-binding experiments have shown that the N-terminal and adjacent amino acids are also involved in the peptide recognition by TAP. Although largely extended side chains are tolerated, proper stereochemical conformation as well as free amino and carboxyl groups are important for the interaction of TAP with peptides substrates. Because TAP can handle peptides that range from 6 to approximately 40 residues, it is difficult to envisage how the backbones of short and very long peptides can be accommodated in a common binding fold. We rather assume that long peptides may contact a putative binding fold with their extremities while protruding out in the middle.

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D. PEPTIUE SUPPLY BY POLYMORPHIC TAPS I N DIFFERENT SPECIES

The specificities of peptide transporters from different species appear to be in good agreement with the characteristics of peptides that are bound by class I molecules expressed in cis suggesting convergent evolution of the two specificities. As far as is known, allelic forms of H-2 molecules exclusively associate with peptides having a hydrophobic C-terminal residue and this residue always serves as a C-terminal anchor (reviewed by Rammensee et al., 1995). In contrast, there are several HLA-A, -B, and -C allotypes that predominantly or exclusively bind peptides with Cterminal Lys or Arg (e.g., HLA-A"0301, A"1101, A*6801, and B*2705), which is consistent with human TAP efficiently transporting peptides that terminate with basic residues. No human class I molecules have been described that prefer binding peptides containing C-terminal amino acids with small side chains (Gly, Ala, Ser, Cys, Thr, or Pro), acid amide (Gln or Asn), or acidic (Glu or Asp) residues. Nevertheless, these residues are not selected against by human TAP, with the exception of acidic residues in some sequence contexts (Momburg et al., 1994b; Androlewicz and Cresswell, 1994; Obst et al., 1995; van Endert et al., 1995; see Section X,B). It may be speculated that permissiveness for these residues was a necessary price evolution had to pay to enable efficient transport of Cterminal Arg and Lys by human TAP and the same argument may apply for the even more pronounced permissiveness of rat TAPd. [Only for peptides binding to the chicken class I molecule B-F4 has the occurrence of Glu as a C-terminal anchor residue been reported (Kaufman et al., 1995).] Interestingly, all human class I alleles that bind basic C-terminal peptide residues possess Asp at positions 116 and 77, two amino acids lining the F pocket of the binding groove that accommodates the Cterminal side chain (reviewed by Barber and Parham, 1993; Madden, 1995),whereas such a constellation is not found in mouse class I molecules. Because human TAP molecules have the capacity to efficiently translocate peptides with hydrophobic C-terminal residues, it is not surprising that human TAP1-TAP2 dimers (or mTAP1-TAP2 expressed in RMA-S cells following transfection with human TAPB) are able to supply mouse Kk, Kd, Dd, Db,and Kb molecules with epitopes derived from different viral proteins (Yewdell et al., 1993). Another study revealed that rat, Syrian hamster, monkey, and human TAP can substitute for mouse TAP in supplying Kd molecules with peptides for recognition by vaccinia-specific CTL (Lobigs and Mullbacher, 1993).This finding suggests that a certain degree of promiscuity of TAP for peptide sequences may be a conserved feature among mammalian species. As described in Section VI, a limited sequence polymorphism is found in allelic variants of human and mouse TAP1 and TAP2 proteins. Transport

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specificities of allelic forms of TAP in these species have been scrutinized because of potential implications for the induction of autoimmune diseases in these species. A detailed analysis of human lymphoblastoid cell lines with typed TAP1 and TAP2 alleles showed that the sequence polymorphism of human TAPl/TAP2 molecules does not entail significant alterations of TAP-mediated transport of peptides with variable C-terminal residues (Obst et al., 1995). Likewise, lack of functional polymorphism of human TAP alleles, with regard to affinity and specificity for peptides of various lengths sequences, was observed in a peptide-binding study (Daniel et al., 1996). Also, no differences in the kinetics of peptide transport were noted in the latter study. The specificities of various inbred mouse strains was analyzed using a 2304-member library of glycosylatablepeptides with variations at positions 1, 4, 6, 7 , 8, and 9 (Schurnacher et al., 1994b). HPLC profiles of ERaccumulated library members were virtually identical for the five mouse TAP haplotypes tested, and similar to the HPLC profile of cim" rat TAP, thus confirming the selective transport specificity of mouse TAP alleles. Also, another study utilizing individual peptide sequences in transport/ glycosylation assays did not reveal significant differences between the TAP specificities of mouse H-2d, H-2k, or H-2'hkcelllines (Obst et al., 1995). However, due to technical limitations of the in vitro assays used it cannot be excluded that the minor sequence polymorphism of human and mouse TAP alleles may select for (or against) certain peptides that are important in some types of immune response. Thus, it remains unknown whether this minor polymorphism reflects genetic drift or Darwinian evolution. Also in the rat, a concordance appears to exist between transport specificities and requirements of concomitantly expressed class I allotypes. Two TAP specificities associated with cim* and cim' phenotypes of RT1.Ad class I molecules, respectively, have been characterized that are equally distributed among rat inbred strains (Joly et al., 1994; see Section VI). Rat RT1.Ad molecules that are, except in MHC congenic inbred strains, coexpressed with the permissive TAPBdhave a strong preference for peptides with a C-terminal Arg residue consistent with three negative charges in residues lining the C-terminal class I binding pocket (Powis et al., 1996). If this class I molecules is coexpressed with the selective rat TAP2' molecule, the dominance of Arg at the C-terminal position of RTl.Adbound 9-mer peptides fully disappears and a new dominant residue is found at position 7 . This indicates that this selective rat TAP allotype tightly excludes peptides with C-terminal Arg leading to loading of RT1.A' with ill-suited peptides and subsequent ER retention. Likewise, the functional polymorphism of rat TAPS was shown to significantly affect the hydrophobicity profiles of peptides eluted from HLA-B27 molecules in

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B*2705 transgenic rats as well as presentation of the cim-dependent H-Y minor histocompatibility antigen (Simmons et al., 1996).Mutation of either Asp77 or Glu97 of RT1.Adinto the respective RT1.A residues relieves the retention phenotype in cimbbcells (Powis et al., 1996). This fact, and the absence of two of three negatively charges F pocket residues in RT1.A" molecules, strongly suggests that peptides binding to RT1.A" may well comply with the stringent selection of TAP" for peptides with hydrophobic C-terminal residues. Cells lines derived from Syrian hamster were functionally polymorphic with regard to supplying certain transfected mouse class I molecules with viral or self-peptides (Lobigs et al., 1995). Peptide loading of Kkor K ' could be reconstituted by expression of rat TAPl.'-TAPS" in the presentationdeficient cell lines BHK and NIL-2, pointing to a pivotal role of TAP specificity for this phenomenon (Lobigs and Miillbacher, 1995; Lobigs et al., 1995). The Syrian hamster expresses two class I molecules for which no allelic polymorphism was detected in several Hm-1 allodisparate strains (Darden and Streilein, 1984).In R. noruegicus possessing only one classical MHC class I locus (RT2.A)class I polymorphism is significantlylower than in M . musculus or Homo sapiens. Thus, there seems to be a correlation between low class I polymorphism and the presence of functionally polymorphic peptide transporters in these four species. At first look, this suggests that polymorphic TAP function may have compensated during evolution for a limited peptide repertoire presented by class I molecules. The restrictive mouse TAP molecules supply the numerous H-2 class I isoand allotypes with peptides that preferentially contain hydrophobic Cterminal residues, whereas in line with the binding properties of HLA class I molecules, human TAP has a broader specificity. There is no reason to assume that restricted peptide transport in the mouse does entail any disadvantage for the defense against pathogens. Notably, the selection for peptides ending on hydrophobic amino acids is first imposed at the level of TAP because mouse proteasomes are able to cleave C terminal of basic amino acids as recently shown with a synthetic ovalbumin peptide (Niedermann et al., 1995). In the rat, the two major TAP specificities coexist. Current knowledge indicates that peptide binding properties of allelic forms of rat class I molecules that are linked in cis to a given TAP allele are adapted to its transport specificity or vice versa. The selective TAP specificity in vitro is included in the nonselective one, and consistently the cim" phenotype is dominant in the heterozygous situation in vivo (Livingstone et al., 1989, 1991). Currently, we can only speculate about the functional significance for the rat to develop and to maintain a restricted TAP phenotype in a recessive fashion. It may have provided a selective advantage to avoid certain CTL responses against virus-derived or self-peptides terminating

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on charged C-terminal amino acids. First results indicate that among Syrian hamster TAP allotypes permissive, intermediate, and selective specificities for C-terminal residues exist that are controlled by the TAP1 subunit (Momburg and Labigs, unpublished results), In viva, the significance of this functional polymorphism of hamster TAPs measured in vitro is, however, unclear. Thus, in the Muridae species mouse, rat, and hamster the immune system has evolved quite divergent ways to bring about antigen presentation by class I molecules. It will be interesting to study the in vitro substrate specificity of recently cloned TAP molecules from salmon that diverged from primate and rodent TAPs a long time ago. XI. Biochemical Characteristics of Peptide Transport

A. QUANTITATIVE PEPTIDEBINDINGSTUDIES Microsomes prepared from Sf9 insect cells that overexpress human TAP after infection with recombinant baculovirus (van Endert et al., 1994a; Uebel et al., 1995) or microsomes prepared from rTAP-transfected T2 cells have successfully been used for quantitative binding studies (Koopmann et al., 1996).Linear Scatchard plots of the binding values of different peptides indicate a noncooperative ligand-receptor interaction with identical, independent binding sites (van Endert et al., 1994a; Uebel et al., 1995; Koopmann et al., 1996; J. Koopmann and F. Momburg, unpublished results). Affinity constants of 0.4, 0.6, and 1.6 p , have ~ been reported for a 9-mer (van Endert et al., 1994a; Uebel et al., 1995>,a 10-mer, and a 14-mer peptide (Koopmann et al., 1996), respectively. ATP did not influence the affinity constant or saturation value for peptide binding (Uebel et al., 1995). For microsomes from T2.rTAP1/2a cells, approkimately lo5 binding sites per cell equivalent have been calculated, providing an estimate for the number of TAP heterodimers expressed in these transfectants (Koopmann et al., 1996). It is likely that normal cells express fewer TAP molecules, but precise estimations are lacking. OF PEPTIDE TRANSPORT B. KINETICPARAMETERS With different experimental systems employing permeabilized cells or microsomes, it was established that ATP-dependent peptide uptake into the ER and subsequent glycosylation (or class I binding) is quite rapid, reaching saturation within approximately 5-10 min of incubation at 37°C (Neefjes et al., 1993; Shepherd et al., 1993; Heemels et al., 1993; Androlewicz et al., 1993; Momburg et al., 1994a). Peptide translocation was reported to be efficient at 23°C and, although with clearly retarded kinetics, to occur at 10 or even 4°C (Shepherd et al., 1993; Heemels et al., 1993).

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Recently, data became available addressing kinetic features of TAPmediated peptide transport. Measuring accumulation of titrated amounts of a 9-mer proband peptide (TYNRTRALI) in mouse microsomes, Yang and Braciale (1995) found that transport followed Michaelis-Menten kinetics with a K,,, value of 0.7 PM. For a variety of natural class I ligands we have determined K,,, values between 0.4 and 20 PM ( J . Koopmann and F. Momburg, unpublished results). It is currently unknown how these relatively high K,,, values measured in vitro fit with cytosolic concentrations of processed peptide that are assumed to be <2 X M (Yang and Braciale, 1995). At effective concentrations below K,,, the velocity of peptide transport increases proportionally to the peptide concentration. Changes in cytosolic peptide concentration occurring, e.g., during viral infections should therefore immediately affect the availability of these peptides for transport and subsequent class I loading. If, however, peptide-carrying chaperones, such as hsp70, were to feed their peptide load directly into TAP transporters, then K,,, values measured for peptides in solution and in the absence of cytosol may not be the actual parameters governing the translocation reaction in vivo. The use of glycosylatable peptides that are retained in the ER could overcome the problem of peptide export antagonizing peptide import by TAP. Recent studies have shown, however, that a fraction of some, but not all, glycosylatable peptides may be subject to rapid retrograde transport before the peptide is conjugated with an N-linked glycan for retention (see Section IX,A). Therefore, as long as peptide import and export are not separated experimentally, only apparent initial velocities and Michaelis constants for transport can be determined, which may underestimate the actual values describing the function of TAP. Another question of interest is whether the velocity of transport and saturating peptide concentrations may be regulated by molecules transiently associated with TAP (see Section IV). Deficiency mutants lacking tapasin or class I molecules should be used to address this point. Certain ABC transporters, e.g., P-glycoprotein or CFTR, contain a hydrophilic regulatory domain that controls the activity of the transporter through reversible phosphorylation states, but TAPl-TAP2 dimers lack such a regulatory region. In addition, we have been unable to detect phosphorylated isoforms of TAP subunits by 2D gel electrophoresis ( J . Koopmann and F. Momburg, unpublished results).

C. NUCLEOTIDEUSAGEBY TAP TAP1 and TAP2 proteins belong to the ABC transporter family and each possesses a nucleotide binding domain (NBD). Therefore, it could be anticipated that peptides are translocated under consumption of ATP.

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Transport assays with semiiritact cells (Neefjes et nl., 1993; Androlewicz et al., 1993) or microsornes (Shepherd et a!., 1993; Meyer et ul., 1994) indeed proved the strict requirement for ATP, which can be supplied in a regenerating system (Shepherd et al., 1993; Androlewicz et al., 1993) or by addition of saturating amounts (10 n m ) (Neefjeset al., 1993; Meier et al., 1994). Conversely, nucleotide depletion by apyrase completely abrogated transport (Androlewicz et nl., 1993). Moreover, no peptide transport was observed when nonhydrolyzable analogs (ATP-$3, AMP-PNP, or AMPPCP) were used instead of ATP (Neefjes et al., 1993; Shepherd et al., 1993; Meyer et al., 1994),indicating that substrate translocation is powered by hydrolysis of ATP, whereas mere binding of nucleotide is not sufficient for this process. Also, GTP and UTP have been reported to be facilitators of peptide translocation (Androlewicz et nl., 1993; Hill et al., 1995). From studies on the ABC transporters, the P-glycoprotein and CFTR transporter (Senior et al., 1995; Gadsby et al., 1995), it was concluded that substrate translocation involves a catalytic cycle in which the two NBDs mediate hydrolysis of ATP in an alternating but strongly cooperative manner that may further be regulated by phosphorylation states of the regulatory doniain (Hwang et al., 1994). Such advanced biochemical studies addressing the catalytic cycle of the NBDs in intact TAPl and TAP2 subunits are entirely missing. An initial study reported that peptide bound to the transporter at 4°C was expelled by ATP suggesting that peptide binding might be a step preceding iiucleotide binding (van Endert et al., 1994a). These results, however, were not confirmed by another study (Uebel et al., 1995) and our own unpublished data. In conclusion, the molecular mechanism underlying peptide translocation by TAP is unclear and awaits future experirnentation that might employ directed blockade of TAPlRAP2 NBDs by chemical modification, photolabile nucleotides analogs, or the phosphate analog vanadate (Baukrowitz et al., 1994; Urbatsch et al., 1995). Two groups have assessed the in vitro nucleotide binding properties of isolated C-terminal domains froin TAPl and TAP2 expressed as recombinant proteins in Escherichia coli or drosophila cells, respectively (Muller et al., 1994; Wang et al., 1994). Photoaffinity labeling of purified human or mouse C-terminal domains with 8-azido-ATP was inhibited by cold nucleotides in the order ATP > GTP > CTP > ITP > UTP, indicating the rank order of affinities. No significant difference was noted in the binding properties of NBDs from mouse TAPl and TAP2 (Wang et al., 1994). ATP hydrolysis by the NBD of hTAP1, however, was not detected in this artificial system (Muller et al., 1994). Closer to the physiological situation, the affinity of nucleotides for intact TAP heterodimers was monitored using extracts of fibroblasts infected with vaccinia viruses recombi-

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nant for TAP1 or TAP2 (Kuss et al., 1995). These experiments confirnied the superior affinity of ATP, whereas GTP was found to be of lesser affinity than CTP or UTP. We have recently determined the kinetic parameters for peptide transport by microsonies in the presence of ATP. ATP was found to support half-maximal translocation at a concentration of 94 PM ( J . Koopniaiin arid F. Momburg, unpublished results). A similar value was found for GTP. These results show that the NBDs of TAP have a relative low affinity for nucleotides with K,;,,valuesbeing one or two logs higher than the K,,,values for peptides. Furtherinore, the specificity for ATP does not appear to be pronounced. Because GTP is present in the cytosol in high concentrations similar to those of ATP (=1 I n M ) it is likely that GTP serves as substrate for TAP as well. X11. tinking TAP Structure and Function

A STUDIES EMPLOYING CHIMERIC TAP S u n u ~ i ~ s The pronounced difference in transport specificities of rat TAP lA-TAP2" and TAPl"-TAP2 prompted the analysis of which of the 25 amino acids at variance between the TAP2 chains involved may determine the transport specificities. Fortunately, TAP2fl and TAP2" cDNAs were similar enough to use shared sites for restriction enzymes for the construction of ?everdl chimeric niolecules in which parts of the sequence were exchanged in a criss-cross fashion (Momburg et nl , 1996). TAP2d" chimeras were coexpressed with rat TAPl,' in T2 cells and an'ilyzed with the two series of C-terminally substituted peptides. The capacity of rat TAP2 to admit transport peptides with charged, polar, or small C-terminal side chains could be mapped to two short sequence stretches encompassing residues 217/218 and 374380, respectively, whereas the remaining 21 polymorphic residues had either no or only a very subtle influence 011 substrate specificity. These two pairs of amino acids appeared to contribute to different aspects of the specificity independent of each other, with 374380 of TAPB" facilitating transport of sniall (polar) C-terminal residues and 217/218 of TAPSJ facilitating transport of positively charged C-ter~ninal residues (Momburg et nl.. 1996). Consistent with these data, a point mutation introduced at position 374 of hiinian TAP2 (A + D) decreased the capacity to translocate a peptide ending on A h . whereas the transport of peptides encling on Arg or Phe was unaffected (Armandola et a1 , 1996).

B STlI DIE5 WITH PHOT0REACI"E P E l T I D E 5 TAP inolecules could be photoaffiriity labeled with radioiodinated photoreactive peptides in the absence of ATP m d at 4"C, which is consistent with

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the binding assay (Androlewiczand Cresswell, 1994; Nijenhuis et al., 1996; Wang et al., 1996). Photo-cross-linking experiments with deficiency mutants or insect cells expressing only TAPl or TAP2 revealed that both TAP subunits are required to constitute the binding site for photoconjugated peptides (Androlewiczet al., 1994; Nijenhuis et al., 1996). Collectively, 10 8- to ll-mer photopeptides have been studied that contained the photolabile amino acid derivative at different positions within the sequence. For the majority of peptides, cross-linkingof a peptide to the TAP heterodimer resulted in labeling of both human TAP subunits; however, the ratio between the label incorporated into each subunit strongly differed. Three peptides were reported to exclusively label hTAP2 (Wang et al., 1996). No correlation of the proximity of the photoreactive group to one of the peptide termini with the predominantly cross-linked TAP subunit could be established. Nijenhuis et al. (1996) studied photolabeling heterologous TAPSconsisting of hTAPl and either rTAP2a or rTAP2". Two photopeptides shown to be inefficiently transported by hTAP l/rTAP2" also poorly cross-linked subunits of this TAP dimer. When the same peptides were reacted with hTAPl/rTAP2d, not only was the rTAP2dclearly labeled but also the label of hTAP1 was enhanced. This, together with the linear Scatchard plots of binding curves obtained with various peptides (see Section XI,A), suggests that sequences of both TAPl and TAP2 cooperatively form a unique binding site for a single peptide molecule. Covalent dimerization of TAPl and TAP2 molecules by means of a peptide containing two UV-labile residues is required to prove this assumption. From the cross-linking studies discussed previously it can be concluded that both the TAPl and the TAP2 parts of the peptide transporter are in some way involved in substrate binding. The extent to which each subunit contributes to substrate specificity may, however, vary between TAP species. By further elaborating on the photo-cross-linking approach, Nijenhuis et al. identified sequence sections in human TAPl and TAP2 molecules that are involved in peptide binding. Following labeling with photoreactive peptide, TAP subunits were immunoprecipitated, the precipitate was partially digested with trypsin or cyanogen bromide, and fragments were reprecipitated with antisera specific for short epitopes being part of the sequences of the membrane-spanning domains of TAPl or TAP2 (Nijenhuis et al., 1996; Nijenhuis and Hammerling, 1996). In both human TAPl and TAPB, sequence stretches were labeled that are located C terminal and N terminal of the (most C-terminally located) pair of putative membraneintegrated segments, which is followed by the hydrophilic nucleotidebinding domain. It is satisfyingto note that in the TAP2 subunit, the photocross-linked part of the molecule encompassed residues 374/380, which

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controlled substrate specificity in rat TAP2 chimeras (see Section XII,A). At variance with the data obtained with rat TAP chimeras, the putative cytoplasmic loop containing residues 217/218 was not identified by crosslinking. If human TAP2 and rat TAP2 chains do not significantly differ in their folding, which we consider quite unlikely, it seems possible that the effect of residues 217/218 on substrate specificities of rat TAP2"" may rather be exerted by affecting the structure of sequence parts further Cterminal than by directly contributing to the peptide binding site.

C CONTRIBUTION OF TAPl A N D TAP2 To SIJBSTRATE SPECIFICITY In the rat, the pronounced difference between the transport specificities of cim" and cim" allelic variants of TAP appears to be caused solely by the considerable variation between TAP2-A (cim")sequences on the one hand and TAPS-B (cim")sequences on the other hand (Powis et al., 1992a). Preferential transport in vitro of peptides with hydrophobic C termini by cells expressing TAPl"-TAPB" or other homozygous cim' TAP1-TAP2 diniers (Heemels et ul., 1993) was exactly found in experiments using T2 transfectants expressing rat TAPl"-TAP2" (Momburg et al., 1994b). Sensitive CTL assays, however, suggested that the spectrum of peptides translocated by TAP1"-TAPBd may not be fully identical to the spectrum translocated by TAPl"-TAP2 dimers, pointing to a minor influence of polymorphic rat TAPl chains (Powis et al., 1992a). The analysis of combinations of TAPl and TAP2 chains from rat cim", rat cim', mouse, or human also suggested that the TAP2 subunit may be more significant than the TAPl chain in controlling the transport specificity with regard to the C-terminal peptide residue (Armandola et al., 1996). In T2 cells expressing human TAPl or mouse TAPl together with either the permissive rat TAP2*or the selective rat TAPB", the transport specificity was governed by the characteristics of the TAP2 molecule. Similarly, the pairs hTAP1-mTAP2 and rTAP1-mTAP2 showed the selective phenotype. However, when rat TAPl was combined with human TAP2, transport of peptides ending on small (polar) residues was reduced, and the mTAP1hTAP2 hybrid additionally failed to transport C-terminally charged peptides leading to a typical selective pattern. These findings suggest that the TAPl subunit may actively influence substrate specificity of peptide transport. XIII. Export of Peptides from the ER

Peptides containing no consensus site for glycosylation or peptides for which the glycosylation was inhibited by tunicamycin were found to only scarcely accumulate in the ER (Heemels et ul., 1993) or to be released

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from the ER subsequent to an initial accumulation (Shepherd et al., 1993; Momburg et al., 1994a; Schumacher et al., 1994a; Roelse et al., 1994). This efflux of peptide was shown to be an ATP-dependent and temperaturesensitive step (Shepherd et al., 1993; Schumacher et al., 1994; Roelse et al., 1994). In one study peptide could be recovered after export from the ER and subsequently used again for TAP-dependent translocation into microsomes (Roelse et al., 1994). There is preliminary evidence that TAP does not mediate the retrograde transport itself (Roelse et al., 1994). The main function of such a peptide exporter may be the maintenance of low steady-state peptide concentrations in the ER to favor loading of class I molecules with freshly transported high-affinity ligands. Peptides that are too long for stable binding to class I could be reintroduced to the potent processing machinery of the cytosol before being recycled into the ER. Removal of unbound peptide from the ER and fast degradation in the cytosol would explain the absence of antigenic peptides from extracts of tissues that do not express sufficient levels of class I molecules able to specifically bind and protect these peptides (Griem et al., 1991). Alternatively, translocated peptides may bind to ER-resident proteins. Recently it was shown that TAP-transported peptides can be cross-linked to the stress protein gp96 (Lammert et al., 1997; Marusina et al., 1997), a heat shock protein that is able to elicit tumor- or virus-specific CTL responses across allogeneic barriers (Udono et al., 1994b; Suto and Srivastaw, 1995; Arnold et al., 1995) and that carries immunogenic peptides (Nieland et al., 1996).Other unidentified ER proteins have been labeled by Marusina et al. (1997). XIV. Involvement of TAP in Diseases

A. INHIBITION OF PEPTIDE TRANSPORT BY VIRALPROTEINS Viruses have evolved a plethora of strategies to evade cellular immune responses elicited by class I molecules (reviewed by Hill and Ploegh, 1995; Spi-iggs, 1996). Two members of the human herpesvirus family evolved proteins that specifically inhibit the transport function of TAP. The protein ICP47 expressed by herpes simplex virus (HSV) types 1 and 2 during the immediate early phase of infection leads to retention of class I molecules in the ER and renders fibroblasts resistant to lysis by CD8+ CTL (York et al., 1994; Hill et al., 1994).The 9-kDa soluble protein ICP47 was shown to inhibit peptide transport by stable binding to the cytosolic face of TAP and thus outcompeting class I-binding peptides (Hill et al., 1995; Friih et al., 1995; Ahn et al., 1996a; Tomazin et al., 1996).In HSV-infected placentally derived human cells the intracellular transport of HLA-G molecules was blocked through the action of ICP47. This result may provide a link be-

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tween HSV infection and spontaneous fetal loss because I-ILA-G is thought to silence NK cells attacking this HLA-A and -B negative tissue (Schust et al., 1996). In fibroblasts infected with human cytomegalovirus (HCMV) the forination of ternary HC-&in-peptide coniplexes is drastically reduced during the early and late phase of infection (Beersma et nl., 1993; Yamashita et al., 1993; Warren et al., 1994). Permissive infection of fibroblasts with HCMV is characterized by a continuous decline in the capacity to translocate peptides in an ATP-dependent manner into the ER (Hengel et al., 1996).This inactivation of peptide transport is operative despite augmented levels of TAP expression during HCMV infection. Recently, the US6 gene product was identified as the responsible protein that interferes with peptide transport by an as yet unknown niechanism involving its ER-lurninal domain (Hengel et al., 1997; Ahn et al., 1997). US6 codes for a 21-kDa glycoprotein whose subcellular localization is restricted to the ER and whose expression kinetics correlates with the inhibition of peptide transport during the early and late phase of infection (Hengel et al., 1997). The US6 protein associates with the transient assembly complex composed of TAP1-TAPS, class I heavy chain, &in, calreticulin, and tapasin (see Section IV). In this context it is worthwhile to note that HCMV expresses a cascade of consecutive functions mediated by different US genes that subvert immune responses by CTL. The US3-encoded gly~vprotein,that is expressed at the very beginning of infection, retains peptide-loaded class I molecules in the ER (Ahn et nl., 1996b; Jones et a[., 1996). The US2 and US11 gene products dislocate nascent class I heavy chains from the Sec61 translocation complex to the cytosol where they are rapidly degraded by the proteasome (Wiertz et nl., 1996a,b; Machold et d.,1997). US2 and US11 glycoproteins are abundantly expressed up to 24 hr postinfection, whereas gpUS6 is maximally expressed 48-96 hr postinfection. However, the NK-mediated immune surveillance is also targeted by cytomegalovirus. A &in-associated, peptide-binding class I homolog, UL18, was recently demonstrated to inhibit attack by natural killer cells (Browne et al., 1990; Fahnestock et at., 1995; Reyburn et al., 1997; Farrell et ul., 1997).

B CONGENITAL HUMAN TAP DEFICIENCY Currently, the only report on a clinical case of congenital TAP deficiency was provided by de la Salle et n2. (1994). Two siblings from a Moroccon family were described with a homozygous for mutation in the TAP2 gene leading to a premature stop codon at amino acid 253. The class I cell surface expression on peripheral blood mononuclear cells was very low (1-3% of normal levels) and could be partially rescued by exogenous A3-

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binding peptides. CD l a molecules were normally expressed on epidermal Langerhans cells and fully inducible on dendritic cells consistent with the TAP-independent expression of this class I-like molecule (see Section 11,B). Although normal numbers of CD4+CD8- T cells were observed, CD8+CD4- crp T cells were present in reduced but significant numbers in the siblings and were able to mount allogeneic reponses in the most severely affected child, who also showed expanded populations of CD4’CD8+ T cells and of CD4-CD8- y6 T cells. The number of natural killer cells was not affected, but their cytotoxic activity was reduced. The children presented with chronic bacterial sinobronchial infections. High antibody titers against various viruses were monitored. Thus, inherited TAP deficiency leads to an immune status similar to that of TAPl” mice (see Section 11,B) but seems to be less pronounced probably because CD8’ T cell populations in the patients had been more expanded in response to a more extensive exposure to pathogens. C. Loss OF TAP EXPHESSION IN TUMORS

A variety of human tumor cell lines and tumor tissues have been analyzed for the status of TAP expression in comparison with class I molecules and MHC-encoded proteasome subunits. In a survey of in vitro tumor cell lines three small cell lung carcinoma lines were found that exhibited deficient presentation of endogenous antigens (Restifo et al., 1993). In these cell lines, transiently expressed Kd molecules were not transported through the Golgi, and TAPl, TAP2, LMP2, and L M P 7 mRNAs were not expressed in detectable levels. Treatment with IFN-y reversed the defects in TAP/LMP expression and antigen processing. A similar screening of solid human tumors and cell lines revealed a human small cell lung cancer line harboring a novel TAPl sequence variation (R659Q)that seemingly entails a defective peptide transport function (Chen et al., 1996). Another study provided evidence for a loss of heavy chain, &m, TAPl, and L M P 2 expression, whereas expression of TAP2 and L M P 7 was maintained (Singal et al., 1996). This phenotype was observed for various small cell lung cancer, hepatocellular carcinoma, and colon adenocarcinoma lines as well as for basophilic leukemia and B cell lymphoma lines. A mostly synchronous downmodulation of class I heavy chain, &m, TAPlI2, and L M P 2 / 7 mRNA and protein levels as well as TAP-mediated peptide transport was noted for renal carcinomas, one in comparison with the corresponding normal kidney tissue (Seliger et al., 1996a,b).This downmodulation was even more pronounced in a corresponding lymph node metastasis suggesting tumor escape from immune surveillance. The expression of these proteins could be reconstituted in a coordinated fashion by IFN-y (Seliger et al., 1996a). Similar results were reported for a sarcoma line (Heike et al., 1996).

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Concurrent loss of TAPlI2 mRNA expression was also reported for a Burkitt lymphoma line whose antigenicity could be restored with an ERtargeted CTL epitope expressed from a minigene (Khanna et al., 1994). In hepatocellular carcinoma lines a selective reduction of HLA-B, and -C surface expression was partially caused by reduction of TAP expression (Kurohkohchi et al., 1996). Sanda et al. (1995) noted a selective underexpression of TAP2 mRNA in a prostate carcinoma line, whereas heavy chain and Pzm mKNAs were clearly detectable. A progressive loss of tumorassociated antigens and TAPl expression in the course of malignancy was monitored for a case of recurrent metastatic melanoma (Maeurer et al., 1996). Investigating a large panel of melanoma cell lines, another group noted that loss of TAPl, TAP2, LMP2, or LMP7 expression does not appear to be a frequent event in malignant melanoma (thor Straten et al., 1997). Similar to the human small cell lung cancer lines mentioned previously, the murine carcinoma cell line CMT 64.5 expressed large amounts of TAP proteins only upon induction by IFN-y, consecutively assembled class I molecules, and presented class I-restricted antigens (Klar and Hammerling, 1989; Sibille et al., 1992; Gabathuler et al., 1994). A similar phenotype was described for BC2 and other mouse fibrosarcoma lines and for mouse embryonic cell lines (Sibille et al., 1992; Kuroda et al., 1995; Bikoff et al., 1991). Other laboratories have analyzed TAP expression in situ by immunohistology. In the studies by Cromme and associates cervical carcinoma specimens were screened for TAPl and for class I expression (Cromme et al., 1994a). In approximately half of the 76 cases, neoplastic cells showed loss of TAP]. In most cases the lack of staining for TAPl was coincident with a lack of staining for HLA-A, -B, and -C. The loss of TAPl/class I expression was more pronounced in lymph node metastases than in primary tumors (Cromme et al., 1994b). In another immunohistological survey of 100 cervical carcinomas, TAP expression was lost in approximately 30%of the tumors along with a more frequent downmodulation of HLA-A and -B locus products (Keating et al., 1995). In a subset of colorectal carcinomas (14%), but never in adenomas and samples of nonneoplastic mucosa, TAP expression was lost mostly in parallel with class I heavy chains and Pzm (Kaklamanis et al., 1994). Loss of TAPl expression in concert with loss of Pzm and/or heavy chain is also quite frequent in non-small cell lung carcinomas (Korkolopoulou et al., 1996). In this study no relationship was found, however, between the expressional status of class I and TAPl expression on the one hand and histopathological tumor parameters or survival on the other. Finally, a concordant loss of heavy chain, &m, and TAPl expression was observed in breast carcinoma specimens (Kaklamaniset al., 1995).

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This loss was more frequent in lymph node metastases than in primary lesions. In conclusion, the loss of TAP expression by malignant tumors seems to be a frequent phenomenon. In addition to the previously much noticed expressional loss of class I heavy chains and Pzm (reviewed by Mijller and Hammerling, 1992),it represents yet another efficient mechanism by which tumor cells downmodulate peptide-presenting class I molecules from the cell surface in order to escape immune attack by cytotoxic T cells. D POSSIBLE ROLE OF TAP I N AUTOIMMUNE DISEASES Several groups have conducted genetic studies to reveal correlations between the presence of human TAPl or TAP2 allotypes and the susceptibility to or protection from HLA-associated autoimmune diseases. Patients with ankylosing spondylitis (Colonna et al., 1992; Burney et al., 1994; Maksymowych et al., 1995; Westrnan et al., 1995),atopic dermatitis (Kuwata et al., 1994, 1995), Behcet’s disease (Gonzalez-Escribano et al., 1995; Ishihara et al., 1996a), celiac disease (Colonna et ul., 1992; Powis et al., 1993; Djilali-Saiah et al., 1994; Tighe et al., 1994; Meddeb-Garnaoui et al., 1995), Crohn’s disease (Heresbach et al., 1996), dermatitis herpetiformis (Hall et al., 1996), insulin-dependent diabetes mellitus (IDDM; Colonna et al., 1992; Caillat-Zucinan et al., 1993, 1995; Jackson and Capra, 1993, 1995; Ronningen et al., 1993; van Endert et al., 1994b; Kawaguchi et al., 1994; Nakanishi et al., 1994; Martinez-Laso et al., 1994; Esposito et al., 1995; Maugendre et al., 1996), juvenile chronic arthritis (Donn et al., 1994; Ploski et al., 1994),multiple sclerosis (Liblau et al., 1993; Vandevyver et al., 1994; Middleton et al., 1994; Kellar-Wood et al., 1994; Bennetts et al., 1995; Bell and Hamachandran, 1995; Moins-Teisserenc et al., 1995), primary biliary cirrhosis (Gregory et al., 1994), psoriasis (Fakler et al., 1994; Hijhler et a l , 1996), Reiter’s syndrome (Barron et al., 1995), rheumatoid arthritis (Wordsworth et al., 1993; Singal et a1 , 1994; Vandevyver et al., 1995; Hillarby et al., 1996), sarcoidosis (Ishihara et al., 1996b); systemic lupus erythematosus (Davies et al., 1994; Ocal et al., 1996; Takeuchi et al., 1996a), and systemic sclerosis (Takeuchi et al., 199613) were typed for their TAPl and TAP2 alleles. Although a linkage disequilibrium between TAP2 alleles and HLA-DR alleles was noted by several authors (van Endert et al., 1992, 1994b; Ronningen et al., 1993; Eiermann et al., 1993; Bennetts et al., 1995; CaillatZucman et al., 1995; Esposito et al., 1995; Gonzalez-Escribano et al., 1995; Jackson and Capra, 1995; Meddeb-Garnaoui et al., 1995; Ishihara et al., 1996; Takeuchi et al., 1996b; Hall et al., 1996; Djilali-Saiah et al., 1996; Thomsen et nl., 1996; Cullen et aE., 1997), almost all recent studies concluded that there is no primary association between TAP alleles and the

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mentioned autoimmune diseases. Only for Reiter's syndrome and rheumatoid arthritislFelty's syndrome is discussed whether TAP2 may be an independent predisposing factor (Rarron et nl , 1995; Hillarby et nl., 1996). With regard to these results it is not surprising that the study by Obst et nl. (1995) did not reveal significant differences in the peptide transport patterns of human B cell lines expressing different TAP alleles (see Section X,D). The observation by some authors that the TAPlK' allele in nonobese diabetic (NOD) mice is expressed at abnormally low levels and thereby may contribute to the generation of autoimmune diabetes (Faustman et a1 , 1991; Fu et al., 1993;Li, 1994; Huanget al., 1995)is controversial. Other authors have shown normal constitutive and cytokine-regulated levels of TAP and class I expression in cell lines and normal splenocytes from NOD mice (Gaskins et nl., 1992; Pearce et d . , 199s) and unaltered peptide transport capacity and specificity of NOD lymphocytes (Schumacher et a1 , 1994b). Furthermore, defective presentation of the H47a minor histocompatibility antigen by the H-2" haplotype was found to be TAP independent (Serreze et al., 1996). Also, studies on TAP expression in €3 cells of human IDDM patients (Wang et al., 1995) or in IDDM islets (Vives-Pi et al., 1996) suggested TAP dysfunctions in this disease. XV. Concluding Remarks

The peptide transporter TAP is now firmly established as a crucial link between the peptide generation machinery in the cytosol, the proteasome, and the peptide-presenting MHC class I molecule in the lumen of the ER. The peptide translocation inechanism enables T lymphocytes to continuously probe for foreign microbes that have invaded the cell and propagate in the cytosol. At the same time, the endogenous peptides presented in the thymus are pivotal in the course of negative and positive selection and thereby determine antigens to which we will be tolerant. TAP will also allow the immune system to sense the presence of peptides generated by mutational events in the course of tumorigenesis. Most likely, environmental pathogens have been the major evolutionary force driving the development of the antigen presentation pathway because most tumors arise rather late in life and do not affect a whole population, as in the case of infectious agents. Within a few years since the discovery of TAP much knowledge has been accumulated concerning peptide selection, with the most important finding being that TAP appears to preselect peptides according to the requirements of the binding groove of class I molecules. These data equal or surpass what is known about the substrate specificity of many of the

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other ABC transporters but, as it is the case with other well-studied ABC transporters, very little is known about the mechanism of substrate translocation. In the future, studies addressing the precise topology of TAP and the transport mechanism will be of prime importance. It is hoped that one day the extreme difficulties in crystallizing such complex multi-membranespanning structures will be overcome and that the crystollographic structure will provide us with hints about how the ABC transporters work. It is also unresolved how the peptides reach TAP after they have been generated by the proteasome. Does this work by diffusion or is there a specific peptide shuttle (heat shock proteins?) that would also protect the peptides from proteolysis in the cytosol. In the E R a peptide shuttle may not be required because the class I molecules are associated with TAP via tapasin so that peptides may be directly fed into class I molecules. However, it is not fully clear whether this association is of the same importance for all class I alleles. It is interesting to note that certain viruses, such as HSV, HCMV, and possibly others, are very efficient in inhibiting TAP. This raises the hope that elucidation of the respective inhibitory processes will also yield insight into the transport mechanism of TAP. Therapeutic interference with the viral TAP inhibitors may also provide a way to enhance the immune response against these viruses. Likewise, investigation of these inhibitors, and of TAP transport in general, may also lead to development of drugs that downregulate immune responses, e.g., in transplant rejections. In summary, much progress has been made in our understanding of the function of TAP, but many challenging questions are still awaiting answers.

REFERENCES Ahn, J. Y., Tanahashi, N., Akiyama, K., Hisamatsu, H., Noda, C., Tanaka, K., Chung, C. H., Shibmara, N., Willy, P. J.. Mott, J. D., Slaughter, C. A., and DeMartino, G. N. (1995). FEBS Lett. 366, 37-42. Ahn, K., Meyer, T. H., Uebel, S., SempB, P., Djahallah, H., Yang, Y., Peterson, P. A., Frtih, K., and Tamp& R. (1996a). EMBO J. 15, 3247-3255. Ahn, K., Angulo, A., Ghazal, P., Peterson, P. A., Yang, Y., and Frtih, K. (199613).Proc. Natl. Acud. Sci. USA 93, 10990-10995. Ahn, K., Gruhler, A,, Galocha, B., Jones, T. R., Wiertz, E. J. H. J., Ploegh, H. L., Peterson, P. A., Yang, Y., and Fnlih, K. (1997). Zmrnunity 6, 613-621. A h , M., Shimbara, N., Takashima, M., Alo'yama, K., Kagawa, S., Tamura, T., Tanahashi, N., Yoshimura, T., Tanaka, K., and Ichihara, A. (1994).J. Biochern. 115, 257-269. Akiyama, K.-y., Yokota, K.-y., Kagawa, S., Shimbara, N., Tamura, T., Akioka, H., Nothwang, H. G., Noda, C., Tanaka, K., and Ichihara, A. (1994). Science 265, 1231-1234. Aldrich, C. J., Waltrip, R., Hermel, E., Attaya, M., Fischer Lindahl, K., Monaco, J. J., and Forman, J. (1992). j . Immunol. 149,3773-3777. Aldrich, C. J., Ljunggren, H.-G., Van Kaer, L., Ashton-Rickardt, P. G., Tonegawa, S., and Forman, J. (1994a). Proc. Natl. Acad. Sci. USA 91,6525-6528.

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Yang, Y.,Waters, J. B., Frtih, K., and Peterson, P. A. (1992b). Proc Nutl. Acud. Sci USA 89,4928-4932. Yang, Y., Friih, K., Ahn, K., and Peterson, P. A. (1995).J. Biol. Chem. 270,27687-27694. Yewdell, J. W., and Bennink, J. R. (1989). Science 244, 1072-1075. Yewdell, J. W., Esquivel, F., Arnold, D., Spies, T., Eisenlohr, L. C., and Bennink, J. R. (1993).J. Exp. Med. 17'7, 178551790, Yewdell, J., Lapham, C., Bacik, I., Spies, T., and Bennink, J. (1994).J.Zmmunol. 152,11631170. Yewdell, J. W., A n t h , L. C., and Bennink, J. R. (1996).J. Immunol. 157, 1823-1826. York, I. A,, and Rock, K. L. (1996). Annu. Rev. Zmmunol. 14, 369-396. York, I. A., Roop, C . , Andrews, D. W., Riddell, S. R., Graham, F. L., and Johnson, D. C . (1994). Cell 77, 525-535. Zhou, X., Glas, R., Liu, T., Ljunggren, H.-C., and Jondal, M. (1993a). Eur. J. Zmmunol. 23, 1802-1808. Zhou, X.,Clas, R., Momburg, F., Hammerling, G. J., Jondal, M., and Ljunggren, H.-C. (1993b). Eur. J. Immunol. 23, 1796-1801. Zhou, X., Momburg, F., Liu, T., Abdel Motal, U. M., Jondal, M., Hammerling, C. J., and Ljunggren, H.-C. (1994). Eur. J. Zmmunol. 24, 1863-1868. Zhou, X., Liu, T., Franksson, L., Lederer, E., Ljunggren, H.-G., and Jondal, M. (1995). Scund. J Zmmunol. 42, 66-75. Zweerink, H. J., Gammon, M. C., Utz, U., Sauma, S. Y., Harrer, T., Hawldns, J. C., Johnson, R. P., Sirotina, A., Hermes, J. D., Walker, B. D., and Biddison, W. E. (1993).]. Immunol. 150, 1763-1771.

ADVANCES IN IMMUNOLOGY VOL 6H

Adoptive Tumor Immunity Mediated by Lymphocytes Bearing Modified Antigen-Specific Receptors

m o w BROCKER

AND KLAUSKARJALAINEN

B a d Inshie for Immunology, CH-4005 Easel, Swifzdand

1. Adoptive Tumor Therapy

Immunogenicity of tumors is dependent on the expression of tumor antigens that can be recognized by the host immune system as foreign. Ideally, for the generation of a specific antitumor response, these novel antigens should not be found on normal cells. They also should be presented in an appropriate manner (by the tumor or professional antigen presenting cells) to the immune system of the host. In cases in which these requirements have been fulfilled, adoptive immunotherapy experiments using tumor-specific T lymphocytes have shown that a successful treatment of tumor-bearing animals is possible (Cheever et al., 1984). In addition, these animals developed long-lasting systemic immunity against the tumors ( Fass and Fever, 1972; Colombo et al., 1985) and, therefore, these experiments draw attention to T cells as potential mediators of stable and long-lasting tumor eradication (reviewed in Greenberg, 1991). However, potential pitfalls for an adoptive tumor therapy based on T cells are also well described: ( 1)the ineffective presentation of tumor-associated antigen by downregulation of major histocompatibility complex (MHC) on tumor cells (Refisto et al., 1993), (2)the presentation of antigen in the absence of costimulatory molecules leading to T cell anergy (reviewed in Muller et al., 1989; Allison et al., 1995), or ( 3 )the inability of the transferred effector T cells to keep up with an extremely rapidly proliferating tumor (De Boer et al., 1985; De Boer and Hogeweg, 1986) may lead to tumor outgrowth. The discovery of the lymphokine-activated killer (LAK) cell phenomenon (Grimm et al., 1982; reviewed in Rosenberg, 1988) seemed to provide researchers with a potent alternative method to generate a T lymphocyte response against tumor-associated antigens. These LAK cells were easy to obtain in large quantities and showed broad reactivity to a wide range of tumors (Grimm et nl., 1982; reviewed in Rosenberg, 1988). Again, the initial benefits of a LAK cell-based immunotherapy (reviewed in Rosenberg and Lotze, 1986) were not always observed. Apparently no particular features of cancer patients could be described that would predict a therapeutic outcome, nor would all types of cancer or all patients with one cancer type respond to LAK cell-based immunotherapy (Stoter et al., 1992; Parmiani, 1990). In 257

Copynglit 9 1998 by Acadeinii Prmr ~nm y farm resewed 0065-277mn $25 on

AII nghts of repduction

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THOMAS BHOCKER A N D KLAUS KARJALAINEN

addition to severe toxicity, LAK cells could not necessarily kill tumor cells in vivo, despite successful and potent tumor lysis in vitro (Grimm et al., 1982). These disappointments encountered with LAK cell therapies were followed by a third promising type of tumor reactive killer cells, the tumorinfiltrating lymphocytes (TIL). These lymphocytes derived from tumor infiltrate, contain tumor-specific T cells, and are 50-100 times more potent than LAK cells in eradication of small metastases but have virtually no detectable antitumor activity against large tumor inasses (Rosenberg et al., 1986, 1988). Furthermore, their use is limited to only a few malignancies from which TILs can be derived (Rosenberg et al., 1994). II. Single-Chain Fv Receptors

A. I N VITRO STUDIES A general limitation in the use of lymphocytes for adoptive immunotherapy is the difficulty of obtaining large numbers of lymphocytes that are specific for tumor cells. Monoclonal antibodies (mAbs),however, can occasionally be raised against antigens that are associated with tumors (Waldmann, 1991).One approach, merging T cell functions with antibody specificities, was to create engineered T cell receptors composed of single-chain variable domains of mAbs (sFv) fused to signaling components of the T cell receptor/CD3 complex (TCRl chain; Brocker et al., 1993; Eshhar et al., 1993; Moritz et al., 1994) or the low-affinity receptor for IgG, FcyRIII(CD16) ( y chain; Eshhar et al., 1993). In these initial studies in which the the fusion proteins (Fv-l and Fv-y) were stably introduced via protoplast fusion or electroporation into hybridomas and cell surface expression of the chimeric receptors demonstrated by FACS analysis, the transfectants were able to respond to an antigen-specific trigger via these receptors by IL-2 production or target cell lysis (Brocker et al., 1993; Eshhar et al., 1993; Stancovskiet al., 1993).Additional experiments showed that similar results could be obtained with human TILs from tumor biopsies, which after retroviral transfection would express chimeric Fv-y receptors, with an Fv portion specific for ovarian carcinoma antigen (Hwu et al., 1993).The Fv approach was further extended by rendering T lymphocytes specific for HIV by transfecting CD8' T cells from human PBMCs with Fv-5 or CD4-5 receptors, which specifically recognized the HIV protein gp41 (Roberts et al., 1994).When activated, human T lymphocytes derived from PBL of different donors were retrovirally transfected in order to express a Fv-y receptor, they showed lytic capacity against tumors, and produced tumor necrosis factor-a and GM-CSF in vitro (Weijtens et al., 1996). Although promising results were obtained with the previous in

ADOPTIVE TUMOR IMMUNITY

259

vitro studies, none of these reports has demonstrated an effective in vivo antitumor function of the Fv receptor expressing T cells.

B. rjv vrvo STUDIES 1. Fv-c Expressing T Cells It had previously been described that chimeric receptors, in which cy7,CD3q and FcgRIy chains were fused toplamic domains of the to other extracellular domains (Irving and Weiss, 1991; Letourneur and Klausner, 1992; Romeo and Seed, 1991; Wegener et d.,1992), can transduce signals into the transfected cells and trigger effector functions when cross-linked via mAbs. Therefore, the results obtained with Fv-y or Fv-5 receptors were, to a certain extent, anticipated. However, similar to the Fv-5 or Fv-y studies mentioned previously, the initial signal transduction studies (Irving and Weiss, 1991; Letourneur and Klausner, 1992; Romeo and Seed, 1991; Wegener et al., 1992) had been performed with hybridomas, cell lines, or tumors representing activated, fully differentiated T cells. In order to create an in vivo model for adoptive immunotherapy with “normal” lymphocytes expressing chimeric Fv receptors, we created transgenic mice, expressing an Fv-[receptor under the control of T-cell specific regulatory elements (human CD2 enhancer and mouse TCRa promoter; Brocker and Karjalainen, 1995).In these mice aII T cells (see Fig. I), as well as natural killer (NK) cells, expressed the Fv-l receptor. Furthermore, all LAK cell subpopulations derived from these mice express the chimeric receptors (see below) and enabled us to test the efficacy of this cell type when rendered tumor specific. In initial experiments syngeneic tumor cells (EL4 lymphoma) were directly injected into these transgenic C57BW6 mice. The tumor cells were transfected with the antigen (human CD3.9) recognized by the transgenic Fv-c receptor as described earlier (Brocker et al., 1993). Because all the T cells as well as NK cells in the transgenic mice expressed the tumorspecific receptor, it was surprising that a complete protective effect was not observed (Fig. 1). Only 20% of the transgenic mice survived the ipinjected EL4 inoculation, whereas all the others had to be sacrificed due to tumor outgrowth. A possible explanation for this failure was discovered when we tested the function of the chimeric transgenic receptor in vitro. Once the T cells were activated via their endogeneous TCWCD3 complexes (by anti-TCR antibodies or Con A), they were able to kill with high specificity and efficacy via their Fv-[ receptor, the EL4 tumor target expressing the antigen (Brocker and Karjalainen, 1995). In contrast, resting T cells could not be triggered via the chimeric receptor to display any effector

anti-Fv

:At&\ Tg

n-Tg

140-

.

anti-CD3

0100

10'

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+ Fvc-transgenic

C57BU6-recipients non-transgenic C57BU6-recipients

60

4 200 1

I w,

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I

I

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80

1

> 100

days post injection

FIG.1. (A) T cells from Fv-5 transgenic mice express the modified antigen receptor on all T cells. Splenocytes from transgenic and nontransgenic animals were analyzed by twocolor flow cytometry. Single-color histograms were obtained by gating on the CD3 population in two-color stainings, using 2C11-FITC and the Fv-specific mAb A1.3 biotin plus streptavidin PE. The top histogram shows the Fv-5 expression level and the lower panel compares TCWCDS expression of transgenic (Tg) vs. nontransgenic (n-Tg) lymphocytes. (B) Two groups (n = 5 per group) of C57BV6 mice were injected intraperitoneally with lo6 EL4 tumor cells ( lo4x lethal dose), previously transfected with human C D ~ Ethe , native antigen recognized by the Fv-5 receptor. 0,nontransgenic mice; H,Fv-6 receptor transgenic mice.

ADOPTIVE TUMOR IMMUNITY

26 1

functions even in the presence of costimulus via CD28 or CD4 (Brocker and Karjalainen, 1995). These results were in contrast to those obtained with hybridomas and cell lines expressing Fv receptors and suggested that the triggering requirements for effector functions seem to be different in resting cells compared to activated T cells. The surviving transgenic mice from the previous tumor experiment might have had a high percentage of otherwise preactivated lymphocytes, which were now also able to lyse the tumor via the chimeric Fv-J receptor, whereas the T cells in the transgenic mice dying of tumor burden might have been in a resting state and failed to fight the tumor. Therefore, if Fv receptor expressing T cells were to be used in an adoptive tumor therapy, they would need to already be activated. To further investigate this hypothesis we isolated T cells from FV-Jtransgenic mice, activated them in vitro with Con A, expanded them for up to 5 days with low doses of IL-2 to increase cell numbers, and coinjected them together with the EL4 tumor into syngeneic nude mice ( lo5EL4 tumor cells = lOOOX lethal dose, plus varying amounts of effector cells). These experiments were repeated several times with similar results. When low doses of recombinant IL-2 were given to the T cell-treated animals, the transferred tumor-specific T cells showed a clear effect: With increasing numbers of given T cells expressing the Fv-J receptor, the survival of the experimental group was prolonged (Fig. 2). This was in contrast to the control groups that received normal T cells (not expressing the Fv-J receptor) plus IL-2, in which no effect could be observed. The prolonged survival of the experimental group was proportional to the amount of T cells given. Furthermore, when the outgrowing tumors were analyzed for expression of tumor antigen, they showed in all cases either an extreme downregulation or a complete loss of the antigen, indicative of a negative selective pressure via the FV-Yreceptor toward its specific antigen. Remarkably, when activated Fv-5 receptor expressing T cells were administered without exogeneous IL-2, similar to the groups that received nonspecific T cells or no T cells at all, no retarded onset of tumor growth was observed (Fig. 2). This indicated that signaling through the chimeric Fv-( receptor on activated lymphocytes was not qualitatively identical to signaling through the very same receptor expressed in hybridomas. Indeed, although in hybridomas after Fv-l signaling IL-2 production was observed (Irving and Weiss, 1991; Wegener et al., 1992; Brocker et al., 1993), in activated lymphocytes no or very little IL-2 could be detected after Fv-6 triggering (T. Brocker and K. Karjalainen, unpublished observations). Taken together, these results indicated that the transferred T cells were most likely unable to produce sufficient amounts of IL-2 for survival upon encountering the tumor antigen. Thus, without the administration of exogeneous IL-2, activated F v - c T cells are most likely short lived. In

262

TIIOMAS BROCKER A N D KLAUS KARJALAINEN

0

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30

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days post injection + 105 tumor cells

-

7 lo5 tumor cells + 10 T cells

I

I 105 tumorcells+4x10 Tcells

I

105 tumor cells + 4x10 Fv-Ci T cells

105 tumor cells i10 Fv-S* T cells

Fic:. 2. Five C57BWf.5 nude mice per group were injected subciitaneonsly with loi EL4 tumor cells (= lo" X lethal dose) expressing the antigen recognized by the Fv-6 receptor. All mice received during the first week of the experimental period 80000 U IL-2 per day ip. In addition, they were coinjected sc at the same time they received the tumor with the no T cells; 0, 10' nonnal following amounts of the indicated T lyn~phocytepopulations: 0, activated T cells; 0 , 10' trunsgenic, Fv-6 receptor expressing, activated T cells: A, 4 X 10' normal activated T cells; A, 4 X 10' transgenic, Fv-5 receptor expressing activated T cells.

contrast, when the same Fv-r T cells were injected together with exogeneous administration of IL-2, they had a clear effect on the tumor (Fig. 2). 2. Fu-5 Expressing LAK Cells To induce LAK cells in uitro, we incubated B cell-depleted transgenic and normal splenocytes with 1000 U/ml of recombinant IL-2 for 5 days as previously described (Mule et al., 1984; Lafreniere and Rosenberg, 1985a). In subsequent three-color FACS analysis we analyzed the culture for Fv receptor expression in the different subsets of LAK cells. IL-2cultured LAK cells can be divided into three major subpopulations based on expression of NK1.1, CD8a, or both (Ballas and Rasmussen, 1987,

263

ADOPTII'E TUMOR I.MMUN"lY

1990, 1993). All three populations expressed the Fv-( receptor at similar levels on their surface (Fig. 3 ) . These same LAK populations from transgenic and control animals were sorted and tested for their capacity to lyse tumor cells in vitro via their Fv-( receptor. As shown in Fig. 4, all three populations of the transgenic LAK cells expressing the Fv-5 receptor were able to efficiently lyse ELA tumor cells expressing the transfected antigen, whereas nontransgenic LAK cells lacked lytic capacity. Only NK1.1' LAK subpopulations lysed control YAC cell targets efficiently, which is a sign of natural killer cell capacity (Ballas and Rasmussen, 1990). None of the tested groups lysed untransfected EL4 cells over background levels. Clearly, although the nontransgenic LAK cells could lyse neither transfected EL4 nor untransfected E M , the F v - c LAK cells of all subpopulazO1

NK1.1+ CD8a'

I1

7

NK1.1'

CDSa'

CD8a

anti-Fv Fir;. 3. Three-color FACS analysis of a S-day I A K ciilture frc)iii B cell-depleted splrnocytes of transgenic and nontransgenic inicr. NK1.l-PE and CDHa FITC were used to perform analysis of LAK subpopiilatioiis. We then gated on the indicated populations t o analyze expression of the Fv-5 receptor with a biotinylated rnAb specific for the Fv portion of the receptor (Brocker ef a[., 1993) followed by streptiavidin Red613. The histograms are displayed as overlays of the identical subpopulations from nontransgenic and transgenic origin

264

T H O M A S BROCKER A N D KLAUS KARJALAINEN

so

I NKl.1'

CDSa+

NK1.1- CDSa+

30

0

3:1

NK1.1'

6:l 18:l 54:l

I

I

I

I

3:l 6:l l8:l 54:l

40

CDSa'

NK1.1- CDSa+

P 3:1

6:1 18:l 54:l

effector : target ratio -0-

ELAwildtype

+ EL4 transfected

-I)-

YAC

FIG.4. The LAK cell subpopulations from Fig. 3 were sorted according to expression of NK1.1, CD8a, or both and tested separately for their capacity to lyse different target EL4 wild cells in a standard 5-hr 5'Cr-release assay. The target cells were YAC cells type (0).and EL4 transfected with antigen recognized by the Fv-5 receptor (0).

(a),

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ADOPTIVE TUMOR IMMUNITY

tions efficiently lysed the transfected EL4, recognized via their Fv-6 receptor. Previous studies using LAK cells in adoptive immunotherapies have demonstrated that successful therapies require the adoptive transfer of both LAK cells and 1L-2 (Mazumder and Rosenberg, 1984; Mule et al., 1984; Lafreniere and Rosenberg, 1985b).We therefore coinjected in vitrogenerated LAK cells together with lo5 EL4 tumor cells (= lO0OX lethal dose) sc into syngeneic nude mice. All groups received IL-2 injections twice daily (total 80,000 U IL-2 per mouse daily) during the first week postinjection. Wild-type LAK cells not expressing the Fv-5 receptor had no effect on the tumor growth compared to the experimental group that received the tumor only plus IL-2 (Fig. 5 ) . In contrast, the trangenic LAK cells that expressed the Fv-6 receptor were able to inhibit tumor growth completely (Fig. 5). In this group of mice, even after 18 weeks no tumor growth was observed. LAK cells rendered specific for the tumor by expression of Fv-creceptors were able to efficiently lyse tumor cells in vitro and in viuo. A tumor resistant to wild-type LAK cell lysis (Fig. 4, bottom), and therefore able to grow in mice treated with normal LAK cells, is recognized and eradicated

IT

* 105tumor cells + 10 I LAK cells

A10

+

0

25

105 tumor cells + 10

I

FV-c

+

LAK cells

50

days post injection FIG.5. The same experimental protocol as in Fig. 2 was utilized, but the injected cells were tumor cells alone (IJ), tumor cells plus 10' nontransgenic LAK cells (0). and tumor cells plus 107 transgenic LAK cells expressing the Fv-6 receptor ( 0 ) .

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THOMAS BROCKEH AND KLAUS KARJALAINEN

specificallyby LAK cells expressing Fv-{ receptors. When the same tumor cells were reinjected into the surviving mice (in Week 18 after the first injection), the initial LAK injection did not show an additional protective effect indicating that LAK cells are not able to establish long-lasting systemic immunity against the tumors (data not shown). These experiments demonstrate that Fv-5 receptors are a possible way to render LAK cells specifically cytotoxic to tumors expressing defined antigens. In addition, LAK cells are more potent killers compared to normal T lymphocytes that express the very same receptor, as indicated by the different efficiencies of tumor eradication displayed by the two types of cells when injected with the same numbers of cells (Figs. 2 and ,5, lo7 effector cells). Because of the higher cytotoxic efficiency of LAK cells, no antigen escape variants were observed in the mice that received LAK cells in contrast to mice receiving T cells. 111. New Approaches

From our previous experiments with normal lymphocytes expressing Fv-

C receptors, it became clear that the TCRS chain signaling module does not mediate all the functions of the full native TCWCD3 signaling complex. First, resting Fv-bearing T cells could not be activated by tumors even in the presence of costirnulatory anti-CDZ8 antibodies. Second, previously activated cells were unable to produce cytokines upon tumor recognition. Because modification of T cell specificity could potentially circumvent natural T cell unresponsiveness against certain antigens, we searched for another way to involve the complete TCWCD3 complex in recognition of native antigen. With knowledge gained from the resolved crystal structure of the TCRP chain (Bentley et at., 1995),we introduced a single-chain Fv portion derived from an Ag-specific mAb into the TCWCD3 complex (Brocker et al., 1996) by fusing it with the N terminus of the TCRP chain (Fig. 6, see color plate). We demonstrated that T cells obtained from a single transfection step showed acquisition of specificity for native antigen determined by the Fv portion of the construct. When tested in T hybridomas, the FV-TCRP chain could compete with endogeneous TCRP chain for expression within the complete TCFUCD3 signaling complex: Transfected T cells recognized the native antigen in addition to peptide/MHC complexes. To further extend the studies with this chimeric FV-TCRP chain, transgenic mice expressing this protein on normal T lymphocytes are being generated. These mice will eventually tell us if this new approach of manipulating T cell specificity enables us to exploit norrnal T cell physiology more broadly by affecting cytokine production, migration, and memory.

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Here we have reviewed our experience with a very specific approach for adoptive tumor immunity. N o doubt our approach, combined with other methods of irninune modulation such as increasing the costiinulatory properties of tumors and/or anti-CTLA treatment, could evolve to therapeutic treatment protocols that will prove to be greatly beneficial (Townsend and Allison, 1993; Pardoll, 1993; Allison et al., 1995). ACKNOWLEDGMENTS The authors thank Drs. K. Cainpbell and R. Torres for carefully reading the manuscript. The Basel Institute for Iniiniinology w a s founded and is supported by Hoffinami-La Roche, Ltd.. Basel, Switzerland.

REFERENCES Allison, J. P., Huiwitz, A. A,, and Leach, 13. R. (199Fi).Manipulation ofcostiinulatory signals to enhance antitumor T-cell responses. Cum. Opin. Ziwiunol. 7, 682-686. Bailas, Z. K., and Hasinussen,W. (1987). Lyinphokine-activatedkiller (LAK)cells. 1II.Characteiization of LAK precursors and susceptible target cells within the murine thymus. I. I r ? i i n u r i o / 139, 3542-3549. Ballas, Z. K.. and Kasmusseii, MI. (1990). Lymphokine-activated killer (LAK) cells. I\’. Characterization of inttrine LAK effvctor subpopulations. I. Imrnrmol. 144, 386-395. Ballas, Z. K., and Rasmussen,W. (1993).Lympliokine-activatedkiller cells. VII.IL-4 induces an N K 1 . l +CD8 alplia+beta- TCH-alpha beta B220+ lyinphokine-activatedkiller subset. I. Irnrnrrnol. 150, 17-30. Bentley, G. A,, Boulot, G., Karjalainen, K.. and Mariuzza, R. A. (1995). C~ystalstructure of the beta chain of a T cell antigen receptor. Scierice 267, 1984-1987. Brocker, T., ;ind Karjalainen, K. (19Y.5). Signals through T cell receptor-zeta chain alone are insufficient to prinie resting T lymphocytes. 1. Eip. Med. 181, 1653-1659. Brocker, T., Peter, A,, Traiineckvr. A,. antl Karjalainen, K. (1993). New simplified niolecular . 143351439, design for functional T cell receptor. Eur. /. Z r i t m m ~ l 23, Brocker, T., Riedinger. M., and Karj;ilainen, K. (1996). Redirecting the complete T cell receptor/CD3 signaling nladiinery towards native antigen via modified T cell receptor. E r r . I. Ftnrrwtnol. 26, 1770-1774. Chewer, M. .4,, Greenberg, P. D., and Fefer, A. (1984). Potentid for specific cancer therapy with iininiine T lyinphocytes.I. B i d Response Mod$ 3, 113-127. Colombo, M. P., Parenza, M., and Parmiani, G. (1985). Adoptive inininnotherapy of a BALB/c lympl~oniaby syngeneic anti-DBNZ immune lyinphoid cells: Characterization of the effector population and evidence for tlie role of tlie host’s non-T cells. Canccr Iinmunol. Imtnr/tzot/ier. 20, 198-204. De Boer, R. I.. antl Hogeweg, P. (1986). Interactions between niacrophages and Tlymphocytes: Tinnor sneaking through intrinsic to helper T cell dynamics.I. Theor. Biol. 120, 3331-3Sl. De Boer, R. J., Hogeweg, P., Dullens, H. F., De Weger, R. A., and Den Otter, W. (1985). Macrophage T lymphocyte interactions i n the anti-tumor iminrtne response: A mathematical model. /. Ztni~umil.134, 2748-2758. Demotz, S . , Sette, A , , Sakaguchi, K., Bucliiier, R., Appella, E., and Grey, H. M. (1991). Self peptide recpirenient for class 11 major histocompatibility complex allorecognition. Proc. Nut! A c d . Sci. USA 88, 8730-8734.

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Eshhar, Z., Waks, T., Gross, C., and Schindler, D. G. (1993).Specificactivation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc. Natl. Acad. Sci. USA 90, 720-724. Fass, L., and Fefer, A. (1972).Factors related to therapeutic efficacyin adoptive chemoimmunotherapy of a Friend virus-induced lymphoma. Cancer Res. 32,2427-2433. Greenberg, P. D. (1991). Adoptive T cell therapy of tumors: Mechanisms operative in the recognition and elimination of tumor cells. Ado. Immunol. 49, 281-355. Grimm, E. A,, Mazumder, A,, Zhang, H. Z., and Rosenberg, S. A. (1982). Lymphokineactivated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.1. Exp. Med. 155, 1823-1841. Hwu, P., Shafer, G. E., Treisman, J., Schindler, D. G., Gross, C., Cowherd, R., Rosenberg, S. A,, and Eshhar, Z. (1993).Lysis ofovarian cancer cells by human lymphocytesredirected with a chimeric gene composed of an antibody variable region and the Fc receptor gamma chain.]. Exp. Med. 178, 361-366. Ining, B. A,, and Weiss, A. (1991). The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 64, 891-901. Lafreniere, R., and Rosenberg, S. A. (19854. Successful immunotherapy of murine experimental hepatic metastases with lymphokine-activatedkiller cells and recombinant interleukin 2 . Cancer Res. 45, 3735-3741. Lafreniere, R., and Rosenberg, S. A. (1985b). Adoptive immunotherapy of murine hepatic metastases with lymphokine activated killer (LAK) cells and recombinant interleukin 2 (RIL 2) can mediate the regression of both immunogenic and nonimmunogenic sarcomas and an adenocarcinoma. 1.Immunol. 135, 4273-4280. Letourneur, F., and Klausner, R. D. (1992). Activation of T cells by a tyrosine kinase activation domain in the cytoplasmic tail of CD3 epsilon. Science 255, 79-82. Mazumder, A,, and Rosenberg, S. A. (1984). Successful immunotherapy of natural killerresistant established pulmonary melanoma metastases by the intravenous adoptive transfer of syngeneic lymphocytes activated in vitro by interleukin 2. J. Exp. Med. 159,495-507. Mazumder, A,, Eberlein, T. J., Grimm, E. A., Wilson, D. J., Keenan, A. M., Aamodt, R., and Rosenberg, S. A. (1984). Phase I study of the adoptive immunotherapy of human cancer with lectin activated autologous mononuclear cells. Cancer 53, 896-905. Moritz, D., Wels, W., Mattern, F., and Groner, B. (1994). Cytotoxic T lymphocytes with a grafted recognition specificity for ERBB2-expressing tumor cells. Proc. Natl. Acad. Sci. USA 91, 4318-4322. Mule, J. J,, Shu, S., Schwarz, S. L., and Rosenberg, S. A. (1984). Adoptive immunotherapy of established pulmonary metastases with LAK cells and recombinant interleukin-2. Science 225, 1487-1489. Muller, D. L., Jenkins, M. K., and Schwartz, R. H. (1989).Clonal expansionversus functional clonal inactivation: A costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu. Reo. Immunol. 7, 445-480. Pardoll, D. M. (1993). New strategies for enhancing the immunogenicity of tumors. Curr. +in. Immunol. 5, 719-725. Pariniani, G. (1990). An explanation of the variable clinical response to interleukin 2 and LAK cells. Immunol. Today 11, 113-115. Refisto, N. P., Kawakami, Y., Marincola, F., Shamamian, P., Taggarse, A,, Esquivel, F., and Rosenberg, S.A. (1993). Molecular mechanisms used by tumors to escape immune

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recognition: Immunogene therapy and the cell biology of major histocompatibilitycomplex class I. I. Znimzinother. 14, 182-190. Roberts, M. R., Qin, L., Zhang, D., Smith, D. H., Tran, A. C., Dull, T. J., Croopman, J. E., Capon. D. J., Bym, R. A,, and Finer, M. H. (1994).Targeting of human immunodeficiency virus-infected cells by CD8+ T lymphocytes armed with universal T-cell receptors. Blood 84, 2878-2889. Romeo, C., and Seed, B. (1991). Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides. Cell 64, 1037-1046. Rosenberg, S. A. (1988). Immunotherapy of cancer using interleukin 2: Current status and future prospects. Immunol. Toduy 9, 58-62. Rosenberg, S. A,, and Lotze, M. T. (1986). Cancer immunotherapy using interleukin-2 and interleukin-%activated lymphocytes. Annu. Rev. Immunol. 4, 681-709. Rosenberg, S. A,, Spiess, P. J., and Lafreniere, R. (1986). A new approach to the adoptive immunotherapy of cancer with tumor infiltrating lymphocytes. Science 233, 1318-1321. Rosenberg, S. A,, Packard, B. S., Aebersold, P. M., Solomon, D., Topalian, S. L., Lotze, M. T.,Yang,J. C., Seipp, C. A,, and Carter, C. (1988).Use oftumor-infiltratinglymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. N . En& 1.Med. 319, 1676-1680. Rosenberg, S. A,, Yannelli, J. R., Yang, J. C., Topalian, S. L., Schwartzentruber, D. J., Weber, J. S., Parkinson, D. R., Seipp, C. A., Einhorn, J. H., and White, D.E. (1994). Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2. J. Nutl. Cancer Inst. 86, 1159-1166. Stancovski, I., Schindler, D. G., Waks, T., Yarden, Y., Sela, M., and Eshhar, Z . (1993). Targeting of T lymphocytes to Ne1dHER2-expressing cells using chimeric single chain Fv receptors. /. Immunol. 151, 6577-6582. Stoter, G., Goey, S. H., Batchelor, D., Eggermont, A. M., Lamers, C., Gratama, J. W., and Bolhuis, R. L. (1992). Treatment of disseminated renal cell cancer with combinations of interleukin-2, lymphokine-activated killer cells, and alpha-interferon. Prog. Clin. Biol. Res. 378, 225-233. Townsend, S. E., and Allison, J. P. (1993). Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected Melanoma cells. Science 259, 368. Waldmann, T. A. (1991). Monoclonal antibodies in diagnosis and therapy. Science 252,16571662. Wegener, A. M., Letourneur, F., Hoeveler, A., Brocker, T., Luton, F., and Malissen, B. (1992). The T cell receptorKD3 complex is composed of at least two autonomous transduction modules. Cell 68, 83-95. Weijtens, M. E., Willemsen, R. A,, Valerio, D., Stam, K., and Bolhuis, R. L. (1996). Single chain Idgamma gene-redirected human T lymphocytes produce cytokines, specifically lyse tumor cells, and recycle lytic capacity. J. Immunol. 157, 836-843. This chapter was accepted for publication on July 2, 1997.

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Membrane Molecules as DifferentiationAntigens of Murine Macrophages ANDREW J. MCKNIGHT AND SIAMON GORDON Sir Wiliam Dunn School of Pafhoiogy, University of Oxford, Oxford OX 1 3UE, Unitad Kingdom

1. Introduction

The development of monoclonal antibodies directed against membrane molecules of mature murine macrophages (Springer et al., 1978, 1979; Austyn and Gordon, 1981) provided early markers to study the distribution of cells in tissues and their differentiation in uitru. In some cases the functions of novel molecules were soon discovered (Beller et al., 1982); many antigens still have no known functions, whereas other antibodies that blocked selective functions such as adhesion were found to be directed against known receptors (e.g.,class A scavenger receptor) but nevertheless provided useful reagents for biochemical characterization ( Fraser et al., 1993). Knowledge of the range of plasma membrane antigens expressed by macrophages has lagged compared with that of T lymphocytes in several species but has accrued gradudy in man (mostly on blood monocytes) and mouse (mainly tissue macrophages). A panel of reagents is now available to analyze heterogeneity of various murine macrophage subpopulations and closely related myeloid dendritic cells and to define their roles in immunologic responses. It is possible to localize macrophages during development and in the adult animal and to characterize the phenotype of cells in different anatomic sites and in response to particular inflammatory, infectious, and immunologic stimuli. The increasing production of transgenic and gene knockout mice has accelerated the application of antibodies to analysis of macrophage-specific functions as well as the contributions of macrophages to alterations in other cell types. It is therefore timely to review current knowledge of selected antigenic markers of particular interest to experimental irninunologists.We shall emphasize structural information and selectivityof expression. Recent compilations of antigens expressed by rodent (Leenen et al., 1996) and human macrophages and other myeloid cells (Garni-Wagner and Todd, 1996)can be found elsewhere. After considering individual molecules in some detail, we show how a small group of reagents can be utilized to characterize macrophages in selected situations. II. Differentiation Antigens Expressed by Murine Monocytes and Macrophages

The following extensive, but not exhaustive, list of differentiation antigens expressed by murine monocytes and macrophages attempts to catego271

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rize these molecules on a structural level. In most cases these antigens are not macrophage restricted (perhaps F480 and sialoadhesin are the best examples of those that are), but rather are expressed by other hematopoietic cell types (e.g., CR3KDllbCD18) and nonhematopoietic cells (e.g., mannose receptor). We have focused on the expression, structure, and function of these molecules in relation to the mouse system, paying reference to species homologs at relevant points. Structural diagrams, used for a number of molecules, are taken from Barclay et al. (1997), which in conjunction with Fraser and Gordon (1993), should be used as a reference point for the tissue expression, structure, and function of other antigens expressed by macrophages but not covered in this review. A. EGF-TM7 ANTIGENS The members of this novel family of seven transmembrane-spanning cell surface molecules demonstrate a predominantly leukocyte-restricted expression pattern. To date, a physiologic function for these antigens has yet to be defined. 1. F4/80

The F480 monoclonal antibody ( IgGZbisotype) was raised in rats immunized with thioglycollate-elicited mouse peritoneal macrophages (Austyn and Gordon, 1981). As one of the earliest macrophage-specific reagents available, this antibody has been extensively used in conjunction with a rabbit polyclonal antiserum raised against affinity-purified F4/80 antigen for the identification of macrophage populations in mouse tissues by immunohistochemical techniques (Hume and Gordon, 1985; Gordon et al., 1992). These analyses were aided greatly by the resistant nature of the F480 antigen to glutaraldehyde and paraformaldehyde fixation. The F 4 80 molecule is a 160-kDa cell surface glycoprotein (Austyn and Gordon, 1981; Starkey et al., 1987) expressed at low levels on circulating blood monocytes and at higher levels on a range of tissue macrophages, including Kupffer cells (liver), splenic red pulp macrophages, brain microglia, gut lamina propria macrophages, and Langerhans cells (skin) (Hume and Gordon, 1985; Gordon et al., 1992; McKnight and Gordon, 1996). F480 is absent from the progenitor cell for macrophages and neutrophils (CFUc) and in the bone marrow the antigen is first detected on a nonadherent precursor with expression maintained on differentiated, adherent macrophages (Hirsch et al., 1981).At relatively early time points during ontogeny, F4/80+" macrophages appear in the yolk sac proceeded by the fetal liver, where they are found in erythroblastic islands (Morris et al., 1991a). The F480 antibody does not react with other leukocytes, including mature dendritic cells and monocyte-derived osteoclasts, or nonhematopoietic cell

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types. Through a quantitative indirect binding assay the richest sources of tissue F480 antigen were determined to be bone marrow, spleen, lymph nodes, gastointestinal tract, liver, and kidneys (Leeet al., 1985). Langerhans cells in the epidermis of skin are F4/80tV', although the molecule is somewhat intriguingly lost from the surface of these cells as they migrate to draining lymph nodes following their exposure to foreign antigens (Hume et al., 1983). F480 expression remains undetectable on interdigitating cells in T lymphocyte-dependent areas of secondary lymphoid organs, into which these migratory Langerhans cells develop. Macrophages localized to T cell areas of the spleen (white pulp), lymph nodes (paracortex), and Peyer's patches are also consistently F4/80-" in normal mice, although these cells can be detected through their expression of other macrophage markers such as macrosialin (Rabinowitz and Gordon, 1991). The isolation of cDNA clones encoding F480 has led to the description of a novel subfamily of seven transmembrane-spanning molecules that also includes human CD97 and EGF module-containing mucin-like hormone receptor 1 (EMRl), the probable human homolog of F480 that shows 68% overall amino acid identity to the mouse molecule (McKnight et al., 1996; Lin et al., 1997; Hamann et nl., 1995; Gray et al., 1996; Baud et al., 1995). These leukocyte antigens are characterized by their novel hybrid structure: the seven transmembrane regions are fused, via a spacer region of approximately 300 amino acids, to a variable number of extracellular EGF-like domains, which has resulted in their EGF-TM7 nomenclature (Fig. 1)(McKnight and Gordon, 1996). CD97 is a 75- to 85-kDa glycoprotein constitutively expressed on human monocytes, granulocytes, and at low levels on resting T and B lymphocytes, with expression levels rapidly upregulated following lymphocyte activation (Eichler et al., 1994). CD55 (decay accelerating factor) has been identified as a cellular ligand for CD97 (Hamann et al., 1996), although the functional significance of this interaction remains to be determined. The tissue distribution of EMRl has not yet been determined by immunohistochemical techniques; however, RT-PCR analysis suggests that its pattern of expression is not as tightly regulated as F480 in mice (Baud et al., 1995).The genes for human CD97 and EMRl have been mapped to the short arm of chromosome 19 (19~13.12-13.UCD97and 19p13.3/EMR2), whereas the F480 gene has been localized to the syntenic region on mouse chromosome 17 (Hamann et al., 1995; Baud et al., 1995; Lin et al., 1997; McKnight et al., 1997). F480 cDNA encodes a 931-amino acid precursor with a predicted signal sequence of 27 amino acids, resulting in a mature polypeptide of 904 amino acids with a predicted mass of 98.9 kDa, containing 10 potential N-glycosylation sites and a membrane-proximal region rich in Ser and Thr residues (McKnight et al., 1996). Biochemical studies have established

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F4/80

7 r Human EMRl

Human CD97

FIG.1. Diagrammatic representation of the known members of the EGF-TM7 family of cell surface molecules. Triangles denote EGF domains, closed circles denote N-linked glycosylation sites, and ss represents postulated disulfide bonds. The glycosaminoglycan attachment demonstrated for F4/80 is identified by GAG, whereas the 0-linked sugars are depicted in the Sermhr-rich spacer region.

the presence of extensive N-linked and moderate 0-linked carbohydrate modification of the F480 antigen, as well as the presence of a chondroitin sulfate glycosaminoglycan attachment possibly within the fourth EGF-like domain at position Ser207 within the motif Ser-Gly-Xaa-Gly, a common glycosaminoglycan attachment site (Haidl and Jefferies, 1996). It remains to be determined whether the glycosaminoglycan moiety participates in the functions predicted for F480, such as adhesion to components of the extracellular matrix. The presence of a consensus sequence for the chelation of Ca'+ ions in EGF-like domains 3-7 of F480 further suggests that this region of the molecule might participate in adhesion between F480+" macrophages and other cell types or matrix proteins. Both F480 and CD97

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contain an Arg-Gly-Asp motif in their extracellular regions (although not found in EMR1) that might serve as a recognition sequence for the binding of certain integrins to these members of the EGF-TM7 family. However, the hybrid nature of the F480 structure suggests that it also serves as a signaling receptor through its coupling to cytoplasmic G-protein complexes. The transmembrane-spanning regions of the EGF-TM7 members show significant homology to the glucagodvasoactive intestinal peptide/calcitonin peptide hormone receptor family (previouslytermed the secretin receptor family of Tm7 molecules) but are otherwise unrelated to the increasingly large number of Tm7 molecules that have been cloned and sequenced (Segre and Goldring, 1993).

B IGSF ANTICENS The IgSF molecules expressed by macrophages are involved in cellular interactions, migration, and phagocytosis. Sialoadhesin is included here in terms of its structural properties, although it is known to function as a macrophage-restricted lectin, whereas M-CSFR is included in the section covering cytokine receptors.

1. Sialoadhesin Sialoadhesin (recognized by the SER-4,3D6, and MOMA-1 monoclonal antibodies) is a nonphagocytic 185-kDa cell surface receptor expressed at high levels on stromal macrophages in the bone marrow, marginal zone metallophils in the spleen, and subcapsular and medullary lymph node macrophages (Crocker and Gordon, 1989). A lower and more variable level of sialoadhesin expression is found on macrophages in the red pulp of the spleen, Kupffer cells, alveolar macrophages, as well as cells in the gut and uterus. During embryogenesis, sialoadhesin expression appears later than F480 and is detected on macrophages in the fetal liver at approximately Day 1S or 16 of development and subsequently in spleen and bone marrow before birth (Morris et al., 1992). Electron microscopy studies have demonstrated a concentration of cell surface sialoadhesin at the point of contact between sialoadhesin+" macrophages in the bone marrow and developing neutrophils and eosinophils (Crocker et al., 1990). Monocytes are sialoadhesin-"' and peritoneal macrophages express sialoadhesin at low levels; however, the antigen can be induced on resident or elicited peritoneal macrophages following in vitro treatment with mouse serum, which contains a potent sialoadhesin-inducing component (Crocker et al., 1989). This activity can be blocked by the addition of IL-4, IL-13, IFN-7, or blocking antibodies specific for IFN-/3 (McWilliam et al., 1992; Doyle et nl., 1994; P. Tree et al., unpublished results). A number of

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inflammatory macrophages have also been shown to express sialoadhesin in uiuo. Initially termed sheep erythrocyte receptor (SER), through its ability to agglutinate unopsonized sheep erythrocytes, sialoadhesin recognizes glycoprotein and glycolipid ligands that terminate with the oligosaccharide sequence Neu5Accr2 + 3GalB1 + 3GalNac, which is found at high levels on neutrophils (Crocker et al., 1991, 1995). Sialoadhesin was also found to mediate macrophage adhesion to the mouse T lymphoblastic cell line TK-1 and to subpopulations of normal mouse thymocytes, T cells and B cells, which presumably express the sialoadhesin recognition sequence (van den Berg et al., 1992). These adhesion events are inhibited by the antibodies SER-4 and 3D6 but not by MOMA-1. Purification of sialoadhesin from mouse spleen by immunoaffinity chromatography permitted NH2-terminal and internal peptide sequencing of the antigen and design of degenerate oligonucleotides. The resulting isolation and sequencing of sialoadhesin cDNA clones identified this receptor as a type I transmembrane glycoprotein comprising 17 IgSF domains (Fig. 2) (Crocker et al., 1994). Sequence analysis has shown sialoadhesin to be closely related to a subgroup of IgSF molecules, namely CD22, CD33, myelin-associated glycoprotein (MAG), and Schwann cell myelin protein (Kelm et al., 1994). The structural features of sialoadhesin and its demonstrated recognition of sialylated ligands has led to its description as an Itype lectin (Powell and Varki, 1995). The sialic acid recognition domain of sialoadhesin has been mapped to the NH2-terminal V-set IgSF domain, which is both necessary and sufficient for ligand binding (Nath et al., 1995). The sialoadhesin gene (Sn) has been mapped to mouse chromosome 2 using in situ hybridization, whereas the genes for CD22 and MAG have been localized to the short arm of mouse chromosome 7. Similarly, the human sialoadhesin gene maps to chromosome 2 0 ~ 1 3and is not linked to the genes for CD22, CD33, or MAG, which map to human chromosome 19 (MucMow et al., 1995). 2. FcyRI (‘CD64) FcyRI is a 70-kDa high-affinity receptor for IgG and a member of the IgSF, comprising three extracellular C2-set IgSF domains (Fig. 2) (Sears et al., 1990; Quilliam et al., 1993).This receptor is the only IgG receptor for which binding to monomeric ligand (in particular IgG2,) can be measured directly (Fridman, 1991; Ravetch and Kinet, 1991).A monoclonal antibody specific for mouse FcyRI has not been reported; however, based on ligand binding and mRNA expression patterns the FcyRI antigen appears to be highly expressed by mouse macrophages. FcyRI expression on macrophages is further increased in response to IFN--y, and this is enhanced by

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glucocorticoids (Sivo et al., 1993).FcyRI plays a role in the phagocytosis of IgG-containing immune complexes by macrophages as well as antibodydependent cellular cytotoxicity (ADCC),cytokine synthesis, and the induction of the respiratory burst in these cells. The association of FcyRI with the y chain of FcsRI is required for its signal transduction activity.

3. FcyRrr ( C D ~ ~ I F ~ ~(CDIS) RIII The monoclonal antibody 2.4G2 (rat IgGZbisotype) recognizes both types I1 and I11 Fc receptors for mouse IgG ( FcyRII and FcyRIII) by reacting with a common nonpolyniorphic epitope on the extracellular region of both receptors (Unkeless, 1979). FcyRII is a low-affinity receptor that binds to immune complexes containing IgGL,IgGzd,or IgG2b, but not to monomeric IgG, and is expressed on monocytes and macrophages, as well as granulocytes, mast cells, B cells, and T cells (Ravetch and Kinet, 1991). FcyRII is a 40- to 60-kDa single-chain glycoprotein, consisting of two extracellular C2-set IgSF domains (Ravetch et al., 1986), and is expressed at greater than 1 X lo5 molecules on the macrophage cell surface (Fig. 2). Alternative splicing generates two distinct mRNA species encoding mouse FcyRII, termed b l and b2, that differ by the in-frame insertion of 47 amino acids in the cytoplasmic tail of the b l isoform (Qiu et al., 1990). The b l form of FcyRII is preferentially expressed by lymphocytes and myeloid precursors, whereas the b2 form is predominantly expressed by mature myeloid cells. Transfected fibroblasts expressing the FcyRII b2 form internalize immune complexes into coated pits leading to ligand transport to lysosomes, presumably resulting in their degradation, whereas the FcyRII b l form fails to mediate endocytosis of immune complexes. This discrepancy suggests that the inserted sequence within the cytoplasmic tail of FcyRII b l in some way inhibits receptor-mediated uptake of immune complexes, possibly as a result of Ser phosphorylation. Following its ligation, FcyRII functions to inhibit activation signals generated by membrane immunoglobulin on B cells partly through its recruitment and activation of protein tyrosine phosphatases. On myeloid cells, in addition to its role in phagocytosis, FcyRII cross-linking by immune complexes triggers ADCC and release of inflammatory mediators. FcyRIII is a low-affinity receptor for IgG that, like FcyRII, displays preferential binding to immune complexes of IgG,, IgGz,, and IgGzb. As a result of its reactivity with the 2.4G2 antibody, murine FcyRIII was initially classified as an FcyRII molecule (Weinshank et al., 1988). This cross-reactivity undoubtedly results from the extremely high level of sequence homology between the extracellular domains of FcyRII and FcyRIII. FcyRIII is expressed by macrophages, myeloid precursors, granulocytes, mast cells, and, unlike FcyRII, NK cells (Ravetch et al., 1986; Weinshank et al., 1988). The mouse FcyRIII molecule is a multimeric

CH. 5 , FIG.6. Model of the Fv-TCRP chain. Shown are the TCHP chain protein in red and the single chain Fv protein in yellow. Both are connected \.id a 3-amino-acid linker (short green line), whereas the V, and V, chain doniains of the mAb are connected via a linker region (long green line) as described in Brocker et nl. (1996). Indicated are the areas that interact with either MHUpeptide or native antigen.

A

B

Ca. 7 , FIG.1. Structural model representation of DRPpeptide complexes associated with RA. The 3ibbon” diagrams illustrate the complex with peptide bound in the class I1 groove from the “top”orientation (A) and the “side” orientation (B). The key polymorphic residues within the shared epitope sequence of the DRP chain are depicted as in the DRBl”0401-encoded inolecule. The side chain of residue 71 is positioned to conkact peptide and influences the specificityof interactions with peptide residue 4. In addition, each of the DRP side chains illustrated potentially form direct contacts with the TCR engaged in T-cell recognition. As discussed in the text, these dual functions predict complex inechanislns to account for the genetic association of this region of the molecule with RA. Figure was generated by Carol DeWeese, Departrnent of Bioengineering, University of Washington School of Medicine, using MolScript (Kraulis, 1991).

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complex formed through the association of the FcyRIIIa chain, a 40- to 60-kDa glycoprotein consisting of two extracellular C2-set IgSF domains (Ravetch et al., 1986), with the y chain of FccRI, which is necessary for surface exprrssion of FcyRIII on macrophages, neutrophils, and mast cells (Fig. 2 ) (Ra et al., 1989; Kurosaki and Ravetch, 1989). The lchain of the T cell receptor may be utilized in the formation of y - l heterodimers, which can substitute for y-y homodimers within the FcyRIII complex expressed by N K cells. Effector responses resulting from FcyRIII crosslinking follow signal transduction by y-y homodimers, which also function to prevent degradation of FcyRIIIa chains within the endoplasmic reticulum. Macrophage phagocytosis mediated through Fc receptors is a tyrosine kinase-dependent event. A number of protein substrates are found to be Tyr phosphorjlated during phagocytosis, including the y subunit of Fc-yRI and FcyRIII, by the Syk tyrosine hnase (Greenberg et al., 1994, 1996) Both FcyRII and FcyRIII on macrophages are differentially modulated by IFN-y. Resident peritoneal macrophages and the macrophage cell line, P388D1, constitutively express high levels of both FcyRII and FcyRIII and actively phagocytose IgG-opsonized particles. Following treatment with IFN-y, peritoneal macrophages downregulate Fcy RII gene expression but continue to express FcyRIII. In contrast the RAW 264.7 and J774a macrophage cell lines respond to IFN-?/ by dramatically increasing their ability to phagocytose IgG-opsonized particles, which correlates with the induction of FcyRIII gene transcription and cell surface expression ( Weinshank et al., 1988). Thioglycollate-elicited peritoneal macrophages express high levels of F q R I I I and very low levels of FcyRII. FcyRII-deficient mice have revealed an inhibitory role for the molecule on B cells and mast cells in vivo (Takai et d., 1996). FcyRII-’- mice display augmented B cell responses to rabbit anti-mouse IgM (p-specific) in vitro and increased antibody titers following in vivo challenge with either T-dependent or T-independent antigens. Mast cells from FcyRII-’- mice were shown to degranulate and release serotonin following cross-linking of FcyRIII, in contrast to cells from normal animals that could, howwer, be activated through FccRI. In vivo mast cell responses were also elevated in FcyRII-’- mice as demonstrated by an enhanced passive cutaneous anaphylactic response. In contrast, mice lacking FcyRIII and FcERI expression as well as functional FcyRI were generated through targeted disruption of the shared y subunit locus (Takai et al., 1994).These animals are found to be immunocompromised as a result of a loss of FcR-mediated effector functions in macrophages, NK cells, and mast cells.

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4. B7-1 (CD8O)/B7-2 (CD86) B7-1 and B7-2 are costimulatory molecules expressed on antigenpresenting cells that interact with both CD28 and CTLA-4 (CD152) expressed primarily as coreceptors on T cells. Binding of B7-1 or B7-2 to their coreceptors on T cells delivers a second signal that, in conjunction with T cell receptor/major histocompatibility complex (MHC)-peptide ligation, leads to T cell activation. B7-1 and B7-2 show 25% amino acid sequence identity and each contains two extracellular IgSF domains, a transmembrane domain and cytoplasmic tail (Fig. 2). Mouse B7-1 is recognized by the monoclonal antibodies 16-10A1 (hamster IgG) and l G l 0 (rat IgGz, isotype), whereas B7-2 is recognized by the monoclonal antibodies GL-1 (rat IgGzaisotype) and 2D10 (rat IgGzb isotype). B7-1 and B7-2 are expressed by a variety of antigen-presenting cell types including dendritic cells, Langerhans cells, activated monocytes and macrophages, activated B cells, and activated T cells (Lenschow et al., 1996). Generally, B7-2 is expressed earlier than B7-1 following stimulation and is thus predicted to play a dominant role in the primary immune response. B7-2-deficient mice display a more severe phenotype in terms of defective T celldependent immune responses than B7-l-deficient mice, which display near-normal Thl- and Th2-type responses (Lenschowet al., 1996; Freeman et al., 1993). Mouse peripheral blood monocytes are B7-1-", although expression can be induced by IFN-y. In comparison, thioglycollate-elicited peritoneal macrophages express low levels of B7-1 and B7-2, which are upregulated in response to lipopolysaccharide (LPS); paradoxically, IFN-7 decreases B7-1 expression and increases B7-2 expression on this population of cells. IL-10 blocks the induction of B7-1 and B7-2 on peritoneal macrophages, possibly as a function of its immunosuppressive activity in the course of downregulating cell-mediated immune responses. A detailed study has yet to establish the in vivo expression patterns of B7-1 and B7-2 on professional antigen-presenting cells. As such, the distribution of these key costimulatory molecules on macrophage subsets, within lymphoid and nonlymphoid organs in normal and disease states, remains to be established.

5. CD31 CD31 (also termed PECAM-1; platelet/endothelial cell adhesion molecule-1) is a cell adhesion molecule comprising six extracellular C2set IgSF domains (Fig. 2). The molecule is highly expressed by endothelial cells, especially at the junctions between neighboring cells, and is also detected on the surface of monocytes, certain populations of macrophages, granulocytes, platelets, and a subset of circulating lymphocytes. CD31

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interacts both homotypically, with several IgSF domains seemingly involved, and heterotypically with the cwP3 integrin and glycosaminoglycans. Mouse CD31 is recognized by the monoclonal antibody2H8 (hamster IgG), the in vivo administration of which can block transendothelial migration of leukocytes, particularly neutrophils, into the peritoneum in response to injection of thioglycollate broth (Bogen et al., 1992, 1994). As a result, it has been suggested that CD31 plays a role in diapedesis, leading to extravazation of inflammatory cells into tissues.

6. MHC Class 1 and Class 11 ( l a ) In common with most cell types, monocytes and tissue macrophages constitutively express class I MHC molecules. Class I1 MHC is expressed at low levels by circulating monocytes and resident tissue macrophages, in contrast to the high levels constitutively expressed by dendritic cells. IFN-y, IL-4, or IL-13 treatment induces increased surface expression of MHC class I1 on macrophages. A wide range of monoclonal antibodies are available that detect either polymorphic or common determinants on both classes of MHC molecules. The crucial roles played by both MHC molecules in the presentation of endogenously derived peptides (class I ) and processed exogenous antigens (class 11) to CD8+" and CD4'" T cells, respectively, have been well documented. C. LECTINS Macrophages express many types of cell surface lectins. The antigens discussed in this section are highly selective and do not include the I-type lectin family, to which sialoadhesin belongs (see IgSF antigens).

1 . Mannose Receptor The mannose receptor [previously described as the mannosyl fucosyl receptor] is a 175-kDa type I membrane glycoprotein expressed at the surface of mature tissue macrophages and hepatic endothelial cells but absent from circulating monocytes (Pontow et al., 1992). Expression of mannose receptor on mouse macrophages is regulated by Thl- and Th2type cytokines: IL-4 and IL-13 both enhance mannose receptor expression, whereas IFN--y decreases total cellular mannose receptor levels and its uptake of niannosylated ligands (Stein et al., 1992; Doyle et al., 1994). To date, monoclonal antibodies specific for the murine mannose receptor are unavailable, although a rabbit anti-mouse mannose receptor antiserum has been generated (P. D. Stahl, unpublished results). A number of monoclonal antibodies have been developed that recognize the human mannose receptor; however, there has thus far been a lack of immunohistochemical analysis of tissues performed with these reagents. In addition to macro-

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phages, human dendritic cells express the mannose receptor at high levels, where it has been found to participate in antigen uptake and presentation (Sallusto et a!., 1995). Similarly, a glycan receptor expressed by mouse Langerhans cells is partly responsible for the internalization of zymosan particles by these cells (Reis e Sousa et al., 1993).However, the relationship between this receptor, which can be blocked with mannan or P-glucan, and the mannose receptor has yet to be established. The extracellular region of the mannose receptor comprises a 136-residue NH2-terminal cysteine-rich domain, containing six Cys residues, that shows sequence similarity to the B subunit of the plant toxin Ricin D, followed by a fibronectin type I1 domain and eight C-type lectin domains [carbohydrate recognition domains (CRDs)] (Fig. 3) (Harris et al., 1992, 1994). The structural organization of the mannose receptor closely resembles its human homolog (82% identity at the amino acid level), as well as DEC-205 (Fig. 3),the M-type receptor for secretory phospholipases A2, and a fourth member of this lectin family detected by Northern blot analysis in a wide range of mouse tissues (Taylor et al., 1990; Ezekowitz et al., 1990; Jiang et al., 1995; Higashino et al., 1994; Wu et al., 1996). The gene for the mannose receptor maps to the short arm of mouse chromosome 2 and human chromosome 10~13; both genes contain 30 exons and 29 introns (Harris et al., 1994; Eichbaum et al., 1994; Kim et al., 1992). Mannose receptor binds to oligomannose and oligofucose-containing carbohydrate polymers and can contribute to phagocpc uptake of foreign microorganisms such as Candida nlbicans (Mar6di et al., 1991),Pneumocystis carinii (Ezekowitz et al., 1991), and Mycobacterium tuberculosis (Schlesinger, 1997). As such, the molecule is thought to play a key role in the first line of defense against infectious agents. Notably, alveolar macrophages express relatively high levels of mannose receptor that could function in their key role in innate immunity against airborne pathogens. This suggestion is further strengthened by the observation that mannose receptor ligation leads to the secretion of an array of inflammatory mediators such as reactive oxygen intermediates and neutral proteinases. Neither the NH2terminal cysteine-rich domain nor the fibronectin type 2 domain are necessary for mannose receptor-mediated endocytosis of mannosylated ligands. In contrast, three CRDs (4,5, and 7) are required for high-affinity binding and internalization of multivalent glycoconjugates (Taylor et al., 1992). CRD4 of the mannose receptor shows the highest conservation between mouse and human (92% identity), which is consistent with the suggestion that this domain of the molecule defines both the carbohydrate specificity and binding affinity of the intact receptor. A recombinant chimera consisting of the cysteine-rich domain from mouse mannose receptor fused to the Fc portion of human IgG, (CR-Fc)

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2

COOH

mannose receptor

DEC-205

MMGL

FK:.3. Deduced structures for the inannuse receptor, DEC-205, and MMGL (inonomer). C-type lectin (CL)domains, fibronectin type 11 (F2) domains, and N-linked glycosylation sites are shown.

has been used successfully to search for alternative inannose receptor cellular ligands (Martinez-Pomares et al., 1996). The CR-Fc construct identifies a population of inacrophages in the marginal zone of the spleen (inetallopliilicmacrophages) and subcapsular sinus of lymph nodes. Following immunization, cells binding to CR-Fc are detected in germinal centers, splenic white pulp, and follicular areas of the draining lymph nodes. This

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study suggests that mannose receptor+vemacrophages might interact with a population of specialized antigen-presenting or antigen-transporting cells within lymphoid tissues, through the cysteine-rich domain of mannose receptor and an as yet uncharacterized ligand. Such a mechanism would facilitate the initiation of immune responses to antigens recognized through the C-type lectin domains of mannose receptor. 2. Macrophage GalactoselN-Acetyl-Galactosamine- Specijk Lectin Macrophage galactoselN-acetyl-galactosamine-specificlectin (MMGL) was first identified as a macrophage cell surface lectin that participates in the binding of macrophages to tumor cells through its recognition of terminal galactose and N-acetyl-galactosamine structures (Oda et al., 1989). The amino acid sequence of MMGL deduced from a cDNA clone indicates that the molecule is a type I1 membrane protein with a predicted molecular weight of 34.5 kDa, comprising a single extracellular C-type lectin domain and three membrane-proximal leucine-repeating a-helices that may serve in the formation of homodimers (Sat0 et al., 1992). During the course of studies aimed at elucidating the molecular nature of the macrophage marker ER-MP23, an antibody that labels most connective tissue macrophages and macrophages within T cell areas of lymphoid organs, it was found that ER-MP23 recognizes MMGL (Voerman et al., 1995). Northern blot analysis of MMGL mRNA expression demonstrates that the molecule is absent from resident peritoneal macrophages but is strongly induced by inflammatory stimuli such as thioglycollate broth and Streptococcus pyogenes. In addition to the staining pattern with ER-MP23, immunohistochemical analyses of normal mouse tissues have been performed using another anti-MMGL monoclonal antibody (LOM-14; rat IgGzbisotype) (Imai et al., 1995; Mizuochi et al., 1997). MMGL+" macrophages were detected in the dermis and subcutaneous layer of skin and in the heart, skeletal muscle, and lung. Species homologs of MMGL have been characterized in human [human macrophage lectin-l(HML-l)] and rat (asialoglycoprotein binding protein) that display similar carbohydrate specificity as MMGL (Suzuki et al., 1996; Ii et al., 1990).A recombinant form of the human molecule, HML-1, shows binding to a human carcinomaassociated antigen (Tn antigen) that contains clusters of terminal N-acetylgalactosamine linked to Ser or Thr residues, further suggesting that this macrophage-restricted lectin may contribute to the recognition of malignant cells by tumoricidal macrophages (Suzuki et al., 1996). 3. CD62L CD62L (L-selectin) was initially described as a peripheral lymph node homing receptor recognized by the monoclonal antibody MEL-14 (rat

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IgGe, isotype) (Gallatin et al., 1983) that blocks in vitro binding of lymphocytes to peripheral lymph node HEV and lymphocyte extravasation into peripheral lymph nodes in vivo (Butcher and Picker, 1996). CD62L is a member of the selectin family of molecules, which also includes the related E-selectin (CD62E) and P-selectin (CD62P) cell surface antigens, and consists of an NH2-terminal C-type lectin domain, an EGF-like domain, and two complement control protein (CCP) domains, followed by a short spacer containing a proteolytic cleavage site, transmembrane, and cytoplasmic regions (Lasky, 1995; Tedder et al., 1995). The selectins mediate leukocyte tethering and rolling on endothelium prior to extravasation from the blood into tissues, for example at inflammatory sites. Specifically, CD62L participates in lymphocyte homing via high-endothelial venules into peripheral lymph nodes. CD62L is expressed by mouse monocytes, granulocytes, and peripheral lymphocytes and is rapidly lost from the surface of activated cells as a result of proteolytic cleavage. Nahe T cells express higher levels of CD62L, which partly explains their preferential homing to peripheral lymph nodes compared with CD62L'" memory T cells. Similar to CD62E and CD62P, the C-type lectin domain of CD62L mediates low-affinity binding to oligosaccharide sequences related to sialyl Lewis x on molecules such as CD34, glyCAM-1, and MAdCAM-1 (Lasky, 1995; Tedder et al., 1995). 4 . Mac-2 (Galectin-3)

Mac-2 is a 30- to 35-kDa antigen detected at high levels on thioglycollateelicited peritoneal macrophages, using the monoclonal antibodies M3/31 and M3/38 (Ho and Springer, 1982), and is also expressed by mast cells, basophils, dendritic cells, osteoclasts, epithelial cells, fibroblasts, and endothelia, with the majority of the protein localized intracellularly (Hughes, 1994). Mac-2 lacks both a signal peptide sequence and a hydrophobic transmembrane domain and is secreted through an as yet uncharacterized mechanism (Cherayil et al., 1989). As such, cell surface detection of Mac2, which may form non-disulfide-linked oligomers, reflects its recognition of other membrane glycoproteins. These recognition events are mediated through the COOH-terminal CRD of Mac-2 and can be blocked by certain sugars, including galactose, resulting in reduced levels of cell surface detection, Mac-2 also binds to Mac-2-binding protein, found at high levels on the surface of activated macrophages as well as in a soluble secreted form (Friedman et al., 1993; Chicheportiche and Vassalli, 1994). Secreted Mac-2 might function as an antiadhesive agent by binding to laminin and thereby preventing inflammatory macrophages from anchoring to this component of basement membranes.

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D. COLLAGENOUS RECEPTORS There are a number of molecules that function as scavenger receptors, several of which lack collagenous domains (Pearson, 1996).In this section we concentrate on two well-characterized antigens that appear to play crucial roles in macrophage biology. 1. Scavenger Receptor

The class A scavenger receptor exists in two forms generated through alternative splicing of RNA transcripts, designated type I (SRA-I) and type I1 (SRA-11) (Krieger and Herz, 1994). Both isoforms of mouse scavenger receptor are recognized by the 2F8 monoclonal antibody (rat IgGeb isotype), which was isolated through its ability to inhibit the EDTA-resistant adhesion of RAW 264.7 cells (a macrophage cell line) to serum-coated tissue culture plastic (Fraser et al., 1993). The 2F8 antibody has been used to detect macrophage populations in lymphoid and nonlymphoid organs of normal adult mice. Scavenger receptor+"'cells are located in the spleen (red pulp and marginal zone), lymph node (subcapsular region and medulla), peritoneum, thymic medulla, liver (Kupffer cells and sinusoidal endothelial cells), lung (alveolar macrophages), and gut (lamina propria) (Hughes et nl., 1995). 2F8 staining is, therefore, generally accepted as a maturation marker of mouse macrophages because monocytes are scavenger receptor-"'. The pattern of distribution of scavenger receptor+" cells suggests a key role for the molecule in innate immune response mechanisms, particularly in the lung and gut, of normal animals. In addition, the scavenger receptor has been shown to participate in the uptake of apoptotic thymocytes by peritoneal macrophages in vitro through a clearance mechanism that can be partially blocked by the 2F8 antibody (Platt et al., 1996). Both SRA-I and SRA-I1 are type I1 glycoproteins expressed as trimers on the cell surface, although both isoforms are predominantly located within intracellular pools. The extracellular region of SRA-I is composed of a COOH-terminal scavenger receptor cysteine-rich domain, a collagenous domain, and an a-helical coiled coil structure (Fig. 4). SRA-I1 is identical to SRA-I up to the end of the collagenous domain, but then it lacks the cysteine-rich domain. It is possible that scavenger receptors at the cell surface are composed of homotrimers or heterotrimers of SRA-I or SRA-I1 polypeptides. Both SRA-I and SRA-I1 bind avery wide range of polyanionic ligands including lipoteichoic acid, a cell wall component of gram-positive bacteria, and LPS, further suggesting that the molecule plays a role in host defense through the recognition and endocytosis of pathogens. This contrasts with the injurious activity of the scavenger receptor during athero-

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FIG.4. Collagenous scavenger receptor fainily members expressed on macrophages. The cysteinc-rich scavenger receptor domain in SRA-I and M A R C 0 are shown as a box (S) and N-linked sugars are depicted AS closed circles.

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genesis (Brown and Goldstein, 1983; Suzuki et al., 1997). The scavenger receptor binds to modified low-density lipoproteins (LDLs),such as acetylated and oxidized LDL, and through their internalization results in the generation of foam cells, a population of LDL-cholesterol-filled macrophages within atheromatous plaques. The expression and function of the scavenger receptor is enhanced by macrophage-colony stimulating factor (M-CSF), which may be an important variable in the scavenger receptormediated uptake of LDL-cholesterol in vivo and the development of atherosclerotic lesions (de Villiers et al., 1994). SRA-I and SRA-I1 also bind to advanced glycation end product (AGE)-modified proteins, which may have significance in the internalization and presentation of modified selfantigens by MHC class IIcvecells and the initiation of autoimmune responses. Macrophages from SRA-'- mice, generated through homologous recombination, exhibit reduced internalization and degradation of AGEmodified BSA and a marked decrease in acetylated and oxidized LDL uptake in vitro (Suzuki et al., 1997). Strikingly, SRA+ X apolipoprotein (apo) E+ double knockout mice have significantly smaller atherosclerotic lesions than those found in single knockout apo E-I- littermates, which are known to develop spontaneous hypercholesterolemia and severe atherosclerotic lesions (Suzuki et al., 1997; Zhang et al., 1992; Plump et al., 1992). SRA+ animals also show increased susceptibility to infection by Listeria monocytogenes and herpes simplex virus type-1, confirming the predicted role of the molecule in host defense against pathogens. 2. MARCO MARCO is a type I1 cell surface receptor with a collagenous structure originally cloned from a mouse macrophage cDNA library using a human type XI11 collagen cDNA probe (Elomaa et al., 1995). The molecule is a disulfide-linked trimer with an extracellular collagenous sequence characterized by 89 tandemly repeated Gly-Xaa-Yaa triplets, interrupted once by Ala-Glu-Lys, and a COOH-terminal scavenger receptor cysteine-rich domain that shows 48.9% amino acid identity with the COOH-terminal domain of SRA-I (Fig. 4). In situ hybridization using MARCO-specific RNA probes and immunohistochemical staining with rabbit anti-MARC0 antiserum have demonstrated macrophage-restricted expression of MARCO mRNA and protein. In normal mice MARCO is expressed by macrophages in the marginal zone of the spleen and the lymph node medullary region. The normal liver and lungs do not contain MARCO'" macrophages, which contrasts with 2F8 staining demonstrating the presence of scavenger receptortve macrophages in these organs. Similar to SRA-I and SRA-I1 expressed by macrophages, COS-7 cells transfected with MARCO cDNA bound fluorescein-labeledEscherichia coli and Staph-

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ylococcus aureus bacteria as well as acetylated LDL. In terms of its expression by specialized subpopulations of macrophages that are predicted to bind and phagocytose bacterial polysaccharides, MARC0 is believed to play a role in host defense and homeostasis.

E. MUCINS 1. Macrosiah Macrosialin is described as a pan-macrophage marker, originally identified by the FAA1 monoclonal antibody (rat IgGZdisotype) raised against concanavalin A affinity-purified glycoproteins from the P815 mastocytoma cell line (Smith and Koch, 1987). Macrosialin is a heavily glycosylated antigen of 85-115 kDa abundantly expressed by a wide range of mouse tissue macrophages and, at lower levels, dendritic cells. Circulating monocytes are macrosialin+ve, whereas other populations of leukocytes are macrosialin-ve.Splenic macrophages in both the red and the white pulp express high levels of macrosialin, as do macrophages within the thymic medulla. Expression levels are low on resident peritoneal macrophages but strongly upregulated (up to a l7-fold increase) in response to inflammatory stimuli such as thioglycollate broth (Rabinowitz and Gordon, 1991).The molecule is predominantly located intracellularly within late endosomes or lysosomes, with only a small fraction expressed at the cell surface. The isolation of a macrosialin cDNA clone, by transient expression cloning in COS-7 cells, has identified the molecule as the murine homolog of the human CD68 antigen (72% amino acid identity) (Holness et al., 1993; Holness and Simmons, 1993). Immunohistochemical analysis of human CD68 expression indicates a less restricted staining pattern with the antigen detected in neutrophils, basophils, and certain lymphocytes, in addition to monocytes and macrophages (Pulford et al., 1990). The deduced amino acid sequence of macrosialin consists of a 318-residue precursor with a predicted signal sequence of 20 amino acids. The mature polypeptide therefore comprises a 271-residue extracellular region, a transmembrane region, anti a short cytoplasmic tail (10 amino acids). The predicted mass of the core polypeptide is only 33 kDa; however, extensive posttranslational addition of N - and O-linked carbohydrates accounts for approximately twothirds of the mass of the mature glycoprotein (Fig. 5 ) . Resident and exudate populations of peritoneal macrophages also express differentially glycosylated forms of macrosialin, as determined by binding of ['251]wlieatgerm agglutinin, which shows 29-fold higher binding to the exudate cell glycoform (Rabinowitz and Gordon, 1991). On activated macrophages the antigen acquires poly-N-acetyllactosamine structures and numerous terminal sialic acid residues. A further glycoform of macrosialin

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macrosialin FIG. 5 . Diagrammatic representation of macrosiahn, a mucin structure expressed by macrophages. The membrane-proximal lamp-related domain is labeled "?' because the structure of this domain has not yet been solved by X-ray crystallography or NMR.

is induced through phagocytic uptake of zymosan particles in vitro (da Silva and Gordon, 1997).This form of the molecule demonstrates elongated, and more complex, O-linked carbohydrate structures. The functional significance of these macrosialin glycoforms in terms of macrophage physiology has yet to be determined. Protein sequence analysis has identified a significant degree of homology between macrosialin (CD68) and members of the ubiquitously expressed lamp (lysosomal-associated membrane protein) family of acidic, highly glycosylated lysosomal molecules, including lamp-1 (CD107a) and lamp2 (CD107b) (Holness et al., 1993; Fukuda, 1991).The extracellular region of the lamps is partitioned into two related subdomains by a short linker sequence rich in Ser, Thr, and Pro residues. In macrosialin there is only one lamp-related domain located adjacent to the membrane, whereas the Ser- and Thr-rich NH2-terminal region of macrosialin shows no homology

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to the lamps. This NH2-terniinal mucin-like region of macrosialin comprises 43% Ser/Thr residues and is characteristic of mucin-like molecules, which bear abundant O-linked glycan chains, including CD43 (a widely distributed leukocyte antigen), CD34 (a marker for bone marrow stem cells), and glyCAM-1 (a HEV ligand for CD62L). The structural similarities between niacrosialin and the lamps suggest that macrosialin might function within macrophage lysosomes to protect the lysosomal membrane from damage by acid hydrolases following ingestion and phagosome-lysosome fusion. Recently, it has been demonstrated that niacrosialin from lysates of mouse peritoneal macrophages, macrophage cell lines, and rat Kupffer cells can bind to oxidized low-density lipoprotein ( Ramprasad et al., 1995; van Velzen et al., 1997).Whether this recognition participates in the uptake of oxidized LDL or occurs followinginternalization of the modified lipoprotein remains to be determined. F INTEGHINS 1. Coniplement Receptor 3 K D 1 1bCD18 Complement receptor 3 (CR3) is a member of the P2 integrin family of adhesion molecules, which also includes LFA-1 (CDllaCD18),p150,95 (CDllcCD18), and aDP2. Monoclonal antibodies have been developed that specifically recognize the CD1l b subunit (a" chain) of mouse CR3 (including MU70 and 5C6) (Springer et al., 1978, 1979; Rosen and Gordon, 1987). The MU70 antibody was initially shown to inhibit binding of iC3bopsonized sheep erythrocytes to mouse leukocytes. CR3 expression is not, however, restricted to macrophages and it is detected on circulating monocytes, neutrophils, NK cells, B cells in the thymus, and activated or memory CD8+" T cells. Indeed, CR3 expression on normal murine tissue macrophages is heterogeneous; resident peritoneal macrophages, splenic marginal zone macrophages, and microglia in the brain express high levels of CR3, whereas most other populations of tissue macrophages, including Kupffer cells and cells in the bone marrow stroma, display a CR3'"" phenotype. The CR3 molecule is absent from the surface of resident alveolar macrophages but is expressed by newly recruited monocyteshacrophages. C D l l b and CD18 are noncovalently associated at the cell surface and mediate ligand binding in a divalent cation-dependent fashion. C D l l b contains an NHz-terminal region of approximately 200 amino acids (the I domain) that displays inany of the binding functions of the intact CR3 molecule (Lee et al., 1995; Zhang and Plow, 1996). CD18 interacts with several cytoskeletal proteins, including a-actinin, filamin, and cytohesin1, and might regulate CR3 ligand binding. In addition to iC3b, CR3 interacts with extracellular matrix proteins such as fibrinogen and the

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cell surface molecules ICAM-1 (CD54) and CD23. In uitro, macrophage adhesion to bacteriological plastic in the presence of serum is mediated by CR3, since the 5C6 antibody or EDTA can inhibit this binding. In viuo administration of the 5C6 antibody blocks monocyte and neutrophil recruitment to sites of inflammation and consequently potentiates Listeria monocytogenes infection by interfering with granuloma formation and thereby preventing containment of the pathogen (Rosen et al., 1989).5C6, unlike the vast majority of immunoglobulins, also has the ability to cross the blood-brain barrier, resulting in the induction of microglia DNA synthesis and apoptosis (Reid et al., 1993). CRSdeficient mice have been generated through targeting of the C D l l b gene, resulting in loss of CD11bCD18 surface expression as detected by anti-CDllb staining (Coxon et al., 1996). CR3-’- mice display reduced leukotriene B,-induced leukocyte adhesion to vascular endothelium in vivo, in accordance with a role for CR3 in cell adhesion events leading to leukocyte extravasation. Surprisingly, neutrophil accumulation in the peritoneal cavity of CR3-I- mice was increased in response to thioglycollate broth. This accumulation of neutrophils appeared to be associated‘withdecreased apoptosis and clearance of recruited cells, which is therefore predicted to result from CDllbCD18mediated phagocytosis and the resultant respiratory burst, an unexpected role for CR3 in regulating inflammatory cell numbers. A second CR3deficient strain of mice has been described, similarly generated through a targeted disruption of the C D l l b gene, that also demonstrates normal neutrophil accumulation in the peritoneal cavity in response to thioglycollate broth (Lu et al., 1997).These studies with CR3-’- animals suggest that previous anti-CR3 antibody blocking studies in vivo may have indirectly interfered with cell migration or activation (Plow and Zhang, 1997). 2. LFA-1 (CDllaCD18) and p150,95 ( C D l l c C D l 8 ) LFA-1 is expressed on a wide range of leukocytes, including monocytes and macrophages, with the level of LFA-1 expression on macrophages increased in response to IFN-.)I treatment (Strassmann et al., 1986). As well as its expression on dendritic cells (Metlay et al., 1990), CDllcCD18 can be detected on a large number of tissue macrophages using the N418 monoclonal antibody. 3. pl@3 Integrins Monocytes and macrophages express a range of pl and p3 integrins, which play a role in their adhesion to components of the extracellular matrix and interactions with other cell types (Hemler, 1990; Leenen et al., 1996).Conversely, it has been demonstrated that VCAM-1 is expressed on macrophages within erythroblastic islands, whereas its ligand VLA-4

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(a4P1,CD49dCD29) is expressed by the surrounding erythroblasts (Sadahira et al., 1995). Monoclonal antibodies specific for either VCAM-1 (M/ K-1) or the a chain of VLA-4 (PS/2) disrupted erythroblastic islands cultured in zitro. This suggests that VCAM-1 expressed on macrophages represents the previously described divalent cation-dependent erythroblast adhesion receptor (Morris et al., 1988, 1991b). G. CYTOKINE RECEPTORS Monocytes and macrophages are known to express a number of cytokine and chemokine receptors, which play an important role in the generation, differentiation, migration, and activation of these cells. These include the receptors for IFN-y, M-CSF, IL-1-IL-7, IL-10, IL-13, and a number of C-C and C-X-C chemokines. The expression patterns and structures of these receptors have been described in detail elsewhere (Callard and Gearing, 1994; Leenen et al., 1996; Barclay et al., 1997; Vaddi et al., 1997). The IFN-yR and M-CSFR are reviewed here due to their central roles in macrophage development and activation. The M-CSFR is included here although its extracellular region comprises five IgSF domains. 1 . ZFN-yR

IFN-y, initially described as macrophage activation factor, plays a critical role in the process of macrophage activation resulting in microbicidal activity and enhanced antigen presentation. The mouse IFN-yR is widely expressed by both hematopoietic and nonhematopoietic cell types, with the exception of mature erythrocytes, and can be detected by the GR-20 monoclonal antibody (rat IgGZdisotype), which reacts with the a-chain subunit (CD119)of the functional IFN-yR (LeClaire et al., 1992).A second species-specific component of the IFN-yR, termed IFN-yR P-chain or accessory factor-1, is known to be crucial for generating biological activity following IFN-y binding (Fig. 6 ) (Hemmi et al., 1994). Mice lacking the IFN-yR a-chain show defects in their ability to generate innate immune responses to infection with intracellular pathogens (Huang et al., 1993). 2. M-CSFR The M-CSFR (CD115) is the product of the c-fms protooncogene. MCSFR is a member of both the IgSF and subclass I11 of the growth factor receptor family with tyrosine kinase activity that also includes c-kit (CD117) and platelet-derived growth factor receptor (CD140ah) (Fig. 6). The receptor is expressed by monocytes and macrophages, as well as their precursors, and mediates survival, proliferation, and differentiation of these cells following M-CSF ligation. Expression of M-CSFR can be detected using

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CD119

IFNrAF-1

x

PPPPPPPVP? PPPPP Ibbbbbbbbib bbbbb COOH

U COOH

FIG.6. The IFN-yR and M-CSFR expressed by inonocytes and macrophages. IgSF domains, fibronectiri type I11 (F3)domains, and N-linked sugars are shown. The cytoplas~nic protein tyrosine kinase domain in the M-CSFR is also shown (K).

the 2E-11 monoclonal antibody, which has also been found to block MCSF binding and block M-CSF-induced colony formation (Shadduck et al., 1993). Op/op mice lack M-CSF as a result of a single point mutation in the M-csfgene (Yoshidaet al., 1990).These mice have a severe deficiency in monocyte-derived osteoclasts and develop osteopetrosis due to impaired bone remodeling (Kodama et al., 1991). Interestingly, in addition to osteoclasts only certain other populations of macrophages are missing from these M-CSF-deficient animals including peritoneal macrophages, splenic marginal zone metallophils, and lymph node subcapsular sinus macrophages (Witmer-Pack et al., 1993). Other populations of macrophages reach substantial levels in a range of tissues, as do dendritic cells, and as a consequence would appear to be less M-CSF dependent.

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CELLSURFACE ANTIGENS H MISCELLANEOUS MACHOPHAGE 1. CD14 CD14 is a 55-kDa glycoprotein expressed at the cell surface of monocytes, macrophages, and neutrophils via a glycosyl-phosphatidylinositol anchor. The monoclonal antibody rmC5-3 (rat IgG, isotype) has been used to detect a small number of CD14+"' Kupffer cells in the liver of normal mice, which increase in number following intraperitoneal administration of LPS (hlatsuura et al., 1994). CD14 functions as a receptor for the complex formed between LPS and the LPS-binding protein, a 60-kDa acute-phase protein that binds to the lipid A moiety of LPS. The observation that transgenic mice overexpressingCD14 are hypersensitive to LPS parallels the finding that CD14-deficient mice are extremely resistant to endotoxin shock, induced by either LPS or gram-negative bacteria (Ferrero et al., 1993; I-Iaziot et al., 1996). 2. 714 The 7/4 monoclonal antibody (rat IgGz, isotype) is often used to detect activated macrophages. The antigen recognized by 7/4 is absent from resident or unstimulated macrophage populations, but is induced at the cell surface following intraperitoneal infection with agents such as BCG or C. pamum and on macrophages within granulomata. The 7/4 antigen is also constitutively expressed by neutrophils and has proved extremely useful in the depletion of neutrophils from cell sources such as the bone marrow (Bertoncello et al., 1991). Treatment of mice with steroids leads to reduced binding of the 7/4 antibody to neutrophils (P. Tree et al., unpublished results), The antibody defines a polymorphic determinant expressed by an approximately40-kDa membrane protein on certain inbred strains, such as C57/BL6, AKR, and 129/Sv mice, but not on others, including BALB/c (Hirsch and Gordon, 1983). 7/4 cDNA clones have not yet been isolated.

3. CD44 CD44, previously termed phagocyte glycoprotein-1 (Pgp-1), Ly-24, and Hermes, is a 90- to 100-kDa heavily glycosylated cell surface glycoprotein (Lesley et d., 1993).A number of monoclonal antibodies specific for mouse CD44 have been generated, including IM7 (rat IgGZbisotype), I42/5 (rat IgGZdisotype), and 8D2 (rat IgG), that recognize nonpolymorphic determinants of the CD44 antigen, whereas RAMBM44 (anti-Pgp-1.1) and C71/ 26 (anti-Pgp-1.2) react with allelic forms of the molecule (Trowbiidge et d., 1982; Lesley and Trowbridge, 1982).A large number of CD44 isoforms

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are generated through alternative splicing of the CD44 gene. The standard form of the molecule that lacks variably spliced exons, designated CD44H, is expressed by a wide range of hematopoietic and nonhematopoietic cell types including monocytes and macrophages, lymphocytes, granulocytes, fibroblasts, epithelial cells, and cells within the central nervous system. In many nonlymphoid organs (e.g., liver) CD44 is an excellent marker for resident and recruited macrophages. The form of CD44 expressed on resident peritoneal macrophages differs from the form of the molecule expressed on thioglycollate broth-elicited macrophages (Camp et al., 1991). On elicited macrophages CD44 is more heterogeneous in structure, reflecting an increase in N-linked glycosylation of the molecule. In addition, CD44 on these cells is not associated with the cytoskeleton, whereas on the resident macrophage population both nonphosphorylated (cytoskeleton-associated)and phosphorylated (unassociated) forms of the antigen are expressed. Although previously shown to be increased on activated or memory T cells, CD44 expression by peripheral T cells and single-positive thymocytes (CD4+veCD8-ve or CD4-"CD8+") is strain dependent, with high numbers of CD44+" T cells in Pgp-1.1 mice (BALB/c and CBA/J) but lower numbers in Pgp-1.2 strains (C57BU6, AKR, and C3H/He) (Lynch and Ceredig, 1989). CD44 functions as the principal receptor for hyaluronate, a component of the extracellular matrix, and has also been reported to bind to collagen, fibronectin, and laminin. As such, CD44 is probably involved in macrophage adhesion within tissues although its functional role on monocytes and macrophages has yet to be clearly defined. In contrast, a key role for CD44 has been identified in terms of lymphocyte homing and recruitment to sites of inflammation. 4. CD45 The CD45 molecule [previously termed leukocyte-common antigen (LCA) and Ly-51 is an abundant cell surface glycoprotein expressed by all hematopoietic cell types, including monocytes and macrophages, but not by erythrocytes (Thomas, 1989;Trowbridge and Thomas, 1994). The CD45 antigen is heavily N - and O-glycosylated and contains a large cytoplasmic tail with two phosphotyrosine phosphatase domains. Alternative splicing of three exons that are inserted following a common NH2-terminalregion of seven amino acids generates eight possible combinations of CD45 mRNA transcripts. These CD45 isoforms, classified through the epitopes encoded by their relevant exons designated CD45R0, CD45RA, CD45RB, and CD45RC, are differentially expressed by subsets of naive and memory CD4'" T cells. A wide range of monoclonal antibodies against common

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determinants on the mouse CD45 antigen, as well as CD4.5 isoform-specific antibodies (CD45R antibodies), are available. However, CD45 isoforin expression by monocytes and populations of tissue macrophages have not been studied.

5. Transferrin Receptor (CD71) The transferrin receptor (TfR) plays a key role in the internalization of iron (Fez+)-chelated transferrin by proliferating cells. The molecule is expressed at the cell surface as a homodimer of 95-kDa monomers. Jnterestingly, mature tissue macrophages constitutively express TfR in the absence of cell activation or proliferation, whereas circulating monocytes are TfR-". This pattern of expression identifies the TfR as a marker of macrophage maturation, detected by a panel of TfR-specific monoclonal antibodies including H129.121, ER-MP21, and C2F2. TfR expression is decreased following macrophage treatment with IFN-7. 6. Other Corriplement Receptors

Mouse complement receptor-1 (CRI) and CR2 (CD21) derive from alternative splicing of the Cr2 gene. Mouse CR1 comprises 21 CCP domains, whereas the CR2 molecule lacks 6 NHz-terminal CCP domains and consists of 15 in total. CR1 can be detected by the monoclonal antibody 8C12 (rat IgGz, isotype), which reacts with a CR1-restricted epitope and does not hind to the CR2 antigen. A second antibody, 7G6 (rat IgG2,, isotype) recognizes both CR1 and CR2. Using the 8C12 reagent, CR1 has been reported to be expressed on the majority of mouse peritoneal macrophages, follicular dendritic cells, peripheral B cells, and f-Met-LeuPhe-stimulated granulocytes (Kinoshita et al., 1988). A separate study, however, reports that both CR1 and CR2 are absent from the surface of thioglycollate-elicited mouse peritoneal macrophages (Martin and Weis, 1993).A related mouse gene Crnj encodes a 65-kDa cell surface molecule (Crry/p65), comprising 5 CCP domains, that is widely expressed by both hematopoietic, including macrophages, and nonhematopoietic cell types (Molina et al., 1992; Li et al., 1993). Both CR1 and Crry bind to C3b and C4b. CR1 presumably participates in the engulfment of opsonized particles coated with these complement components by phagocytic cells and also manifests cofactor activity for factor I-mediated cleavage of mouse C3b; Crry displays both decay-accelerating factor activity (like CD55) and cofactor activrity for factor I-mediated cleavage of both mouse C3b and C4b (like CD46) (Kim et al., 1995). As a result, Crry is considered to be the primary complement regulatory protein at the surface of mouse cells.

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7. Other Antibodies Used to Detect Mcicrophage Populations The expression patterns of the antigens recognized by ER-TR9, ER-HR3, the ER-MP series, and the MTS series of monoclonal antibodies are reviewed in detail by Leenen et al. (1996). With the exception of ER-MP23, which was shown to detect MMGL (Voerman et al., 1995), there is limited biochemical data for the molecules recognized by these reagents. 111. Use of Differentiation Antigens to Characterize Macrophages in Sib and in Vitro

The markers described in the previous section vary in their usefulness to characterize the macrophage phenotype in uiuo, depending on specificity of expression, the stability of particular antigens to handling of tissues and fixation, and the quality of the antibodies available. Some general aspects of macrophage heterogeneity will be summarized briefly, and the differential expression of macrophage antigens will be considered as a function of tissue localization and cellular activity. For further details, consult more general treatments of macrophage immunobiology (Gordon, 1996a,b) and of immunocytochemical methods (Gordon et al., 1992).

A HETEROGENEITY OF MACROPHAGES Mature macrophages can be detected in murine embryonic tissues from approximately Day 9 of development in yolk sac and subsequently in hematopoietic organs (liver, then spleen and bone marrow), as well as throughout mesenchymal tissues and developing organs such as the brain (Morns et nl., 1991a; Perry et al., 1985). Sites of tissue remodeling, for example, the limb buds, contain prominent macrophages with evidence of active phagocytic reinoval of apoptotic cells ( Hopkinson-Woolley et al., 1994). Fetal liver macrophages (Days 14-16) are particularly abundant and contribute to definitive erythropoiesis through cluster formation with developing erythroblasts (Morris et al., 1988)while also ingesting discarded erythrocyte nuclei (Fig. 7). Some maturation of macrophage populations, in lymphoid-hematopoietic organs and brain, for example, continues after birth. In the normal young adult mouse there are many well-characterized populations of resi-

Fic:. 7 . Phenotpe of macrophages in fetal and adult niurine liver in the absence or neutrophil: presence of graniiloina forination. M 4, ~nacrophage:PMN, ~~olyrno~~lii~nucle,ir Ia, class I1 MHC; Sn, sialoadhesin; SR-A, class A scavenger receptor; CR3, coinplrinent receptor type 3; EbR, divalent cation-dependent adhesion receptor for erythrohlasts.

endothelial cell

Kupffer cell F4/80+ F A / l 1 + S n+l- %-A+ cD44+ cR3714- Ia-

F4/80+ FA11 I + Sn+ SR-A+ CD44t CR3+ (also on PMN) 714+ (also on PMN) Ia+

T cell

sinusoid BCG-induced granuloma normal adult liver

monocyte CR3+ F4/80+/-

foetal liver

stromal Mg FAI11+ Sn-(d14)/+(d16) EbR+

300

ANDREW J. MCKNIGHT AND SIAMON GORDON

dent macrophages in most tissues, in the absence of any known inflammatory signal. These cells are present at portals of entry, such as skin, gut, and lung, but cells not obviously involved in defense functions are also widely distributed throughout the interstitium and parenchyma of most organs. Cells within the neuropil (microglia) and within epithelia (e.g., Langerhans cells) are highly ramified compared with Kupffer cells (liver) and less adherent macrophages (e.g., alveolar and serosal macrophages). There is considerable microheterogeneity of phenotype of these macrophage subpopulations (Fig. 8; Table I), reflecting their different life history and interactions with other cells in their microenvironment. Within organs such as spleen (the site of hematopoiesis, immune functions, and clearance of senescent blood cells), macrophages are markedly heterogeneous even within the same tissue (Fig. 8). For example, it is possible to distinguish distinct macrophage populations in red pulp, the marginal zone, and white pulp. The macrophage lineage shares precursors with myeloid dendritic cells in lymphoid organs and osteoclasts, but each of these lineages differentiates terminally into specialized cells (Fig. 9). Further information on the properties and functions of these cells is available elsewhere (Steinman, 1991, 1997); here it should be emphasized that although specialized antigenpresenting cells (mature dendritic cells) can be differentiated from classical macrophage subpopulations there are overlapping phenotypes of cells in situ that cannot be easily classified as belonging to one or the other lineage. Progressive differentiation in vivo and in cytokine-supplemented culture systems yields cells that are no longer modulatable by environmental and cytokine stimuli (Inaba et al., 1992; Sallusto and Lanzavecchia, 1994). In response to an inflammatory or immunologic stimulus, local resident macrophages can undergo “reactivation” and upregulate expression of antigen markers that are normally undetectable or present at low levels. In addition, newly recruited monocytes enter local sites and differentiate either into “elicited’ nonspecifically stimulated macrophages or, under the influence of cytokines such as IFN-7, acquire a range of properties associated with immunologically activated macrophages, including enhanced antimicrobial activity as well as MHC class I1 expression. Immune activation covers a spectrum of changes depending on the cytokine influence (activation, alternative activation, or deactivation), as illustrated in Fig. 10. In some microbial infections, activated macrophages interact with T lymphocytes, fibroblasts, and other myeloid cells, including dendritic cells, to form hypersensitivity granulomata; these can be distinguished from foreign body granulomata, which do not involve antigen-specific activation of T lymphocytes.

marginal zonelmetallophilic M$ CR-FCU Sn++ SR-A+ CR3+

red pulp M$ F4/80+ Sn+l- FA/11+

S n u SR-A+ FA11 1+ F4/80+/-

fol germinal centre CR-Fc+

tingible body M$ CR-Fc+ FA/ll+

subcapsular sinus M$ F4/80- S n u FA/ll+ CR-Fc++

I

follicular dendritic cell region CR-Fc+

spleen

white pulp M$ ~ ~ 1 (also 1 + DC)

lymph node (immune)

8. Macrophage heterogeneity in secondary lymphoid organs. M 4 macrophage; DC, dendritic cell; PMN, polymorphonuclear neutroPALS, penarteriolar lymphoid sheath; Sn, sialoadhesin; SR-A, class A scavenger receptor; CR3, complement receptor we 3; C R - F ~ , cells expressing a ligand for a mannose receptor cysteine-rich domain FC chimera (for details see Martinez-Pomares et al.,19%). FIG.

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TABLE I SELECTEDMEMBRANE ANTIGENSOF MURINEMACROPHAGES inAb

Antigen

Expression

Regulation/Comments

M4

Maturation marker, absent from T cell areas Phagocytosis and inflammatory stimuli induce altered glycoform s Cytokines, serum factor

F480

F480

Selected

FN11

Macrosialin (mouse CD68)

Pan

SER-4 3D6 MOMA-1 2F8

Sialoadhesin

Selected

M4 and DC

M4

2.4G2 5C6 M 1/70 714

Selected M4 and liver sinusoidal endothelium FcyRIIflII M4, PMN, B cells CR3 PMN, some M4, NK (CDllbCD18) cells 7/4 PMN and activated

8D2

CD44

Scavenger receptor A

M4 M4, T cells, fibroblasts

TIB120

MHC class I1 (Id

DC, M4, others

M-CSF

Selected M 4 (e.g., central nervous system) Polymorphic, innnune activation Good M4 marker in some nonlymphoid organs (e.g., liver) Upregulation by IFN-y, IL-4, and IL-13

Note. For references Fee text, M4, macrophage: DC, dendritic cell; PMN, neutrophil.

Once isolated from the animal, even with due care to recover embedded macrophages from tissues, for example, by collagenase digestion, macrophages rapidly adapt to their artificial culture environment and lose their specialized properties. This results in marked changes in antigen profile (loss of markers as well as upregulation of previously repressed markers). FACS sorting of bone marrow progenitors followed by in vitro culture with specific growth factors, such as M-CSF and GM-CSF, can provide useful information about precursor-product relationships and microheterogeneity among independent colonies (Hirsch et al., 1981). Specialized culture systems are available to induce dendritic cell (Inaba et al., 1992)or osteoclast (Tondravi et al., 1997) differentiation in vitro compared with macrophages. Cell lines, apart from their proliferation, often lack specialized macrophage molecules, whereas some receptors and antigens may be retained or can be induced, for example, by PMA-induced differentiation in vitro. One of the best examples of useful antigen expression retained during differentiation in vitro is that of the appearance of the F4/80 antigen on embryonic stem cells forming embryoid bodies and

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303

F I ~ :9.. Mononuclear cell differenti,ition. "Not clear if circulating precursors are alre,idy distinct. ""hlyeloid origin: lymphoid origin dendritic cells are not shown,

macrophages in uitr-u in specialized culture conditions (Wiles and Keller, 1991). IV. Conclusion

Membrane molecules play an important role in all aspects of macrophage immunobiology, as receptors for growth and differentiation factors, chemokines and lymphokines, recognition and adhesion molecules for cellular and microbial ligands, and as regulators of cell signaling and gene expression. They determine and modulate homeostatic interactions with the host, contribute to innate and adaptive immunity, and mediate many of the pathological consequences of deficient or excessive activation. During the past 20 years there has been rapid progress in characterizing macrophage plasma membrane molecules that underlie the induction and effector phases of humoral and cellular immunity, and their unique endocytic and phagocytic activities. As genetic and immunochemical targeting of macrophage antigens gains nioinentuin, there will be confirmation and

@Jkm-

PRXMEDIACTNATED

Respiratory Burst (RB)

ALTERNATIVE ACTIVATION

DEACTIVATED

FIG.10. Spectrum of macrophage activation, Ia, class I1 MHC; MR, mannose receptor; iNOS, inducible nitric oxide synthase.

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305

extension of current concepts of macrophage differentiation and functions, along with discovery of new molecules relevant to pathogenesis and therapy.

NOTEADDED IN PROOF A number of monoclonal antibodies specific for mouse FcyRI have been developed in conjunction with FcyRI knockout mice (P. M. Hogarth, personal communication). F c y R I P mice demonstrate reduced type 111 hypersensitivity responses in duo, reduced ADCC killing in uitro, but enhanced primary antihapten responses (all IgC subclasses) in oioo. It has also been reported recently that a group of inhibitory receptors, analogous to killer inhibitory receptors (KIRs) expressed by NK cells, are expressed on myelomonocytic cells (Cosman et al., (1997). Immunity 7, 273-282; M. Colonna, personal communication). The cytoplasmic tails of these receptors contain putative immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and are predicted to participate in the regulation of macrophagemediated cytotoxicity.

ACKNOWLEDGMENTS The authors are funded by the Medical Research Council, United Kingdom. We thank the authors of “The Leukocyte Antigen FactsBook,” 2nd edition (Academic Press, London) for permission to use their structural diagrams.

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Major Histocompatibility Complex-Directed Susceptibility to Rheumatoid Arthritis GERALD T. NEPOM Virginia Mason Research Cenkr and Depariment of immunolagy, Universi& of Washington Schaal of Medicine, %Me, Washington 98 101

1. Introduction

Genetic associations between rheumatoid arthritis (RA) and specific genes in the human major histocompatibility complex (MHC) on chromosome 6 (6~21.3)provide a guidepost for understanding mechanisms of dsease susceptibility and immunologic events in RA. The human MHC region includes at least 6 HLA class I genes and 14 HLA class I1 loci within a 4-megabase regon, as well as several other genes functionally related to pathways of antigen presentation and processing (Ragoussis et nl., 1991; Kelly and Trowsdale, 1994). One of these loci, HLA-DRB1, accounts for the strong MHC associations with RA, and detailed genetic, structural, and functional studies of the genes and gene products encoded at this locus have greatly contributed to evolving concepts of disease heterogeneity, prognosis, and pathogenesis. HLA-DRB1 is a highly polymorphic class I1 locus. Well over 150 allelic variants arc: known, which differ from each other, sometimes by as few as one or two nucleotide substitutions. This extreme polymorphism has provided the essential technical means for unraveling the complexity of HLA associations with RA because only a few of the specific DRBl alleles are increased in frequency among patients. In Caucasians, DRBl"0401, "0404, and "0101 are all associated with rheumatoid arthritis. DRBl"0401 and DRBl"0404 are both subtypes of HLA-DR4, a common HLA-DR specificity. In various studies of adults with rheumatoid arthritis, DRBl"0401 is present in from 50 to 60% of patients compared to 15-20% of controls; DRBl"0404 is present in 2535% of patients compared to 5-7% of controls (Nepom et al., 1989; Gao et al., 1990; Ronningen et al., 1990;Wordsworth et al., 1989). DRBl"0101, on the other hand, is present in approximately 20% of patients as well as 20% of controls. Interestingly, however, DRBl"0101 is present in approximately half of all patients who lack HLA-DR4. Table I summarizes risk estimates for these three RA susceptibility alleles based on a projected disease frequency of approximately 1%in the Caucasian population. Because the same genes are also prevalent in the unaffected population, the predictive value of these genes is rather low. DRBl"0401 confers an 31.5

Copyright 0 1998 Iry Academic Prpss r\ll rights ol' rrprrrluction in any form reserved. (XE5-2776WX $25 (XI

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RISK

Allele DRBI"0401 DRBl'0404 DRBl*OlOl '0401/"0404

TABLE I ESTIMATES F O R HLA GENES ASSOCIATED WITH RA I N CAUCASIANS Previous Nomenclature Dw4 Dw14 DR1 Dw414

Relative Risk" 6 5 1

-100

Absolute Riskh

1 in 35 1 in 20 1 in 80 1 in 7

Relntive r i y k : The odds ratio for an individual with the risk allele compared to individuals without the risk allele. "Ahsolute risk: The likelihood that an individual with the risk allele(s) will have RA. I'

absolute risk for RA of 1 in 35 and DRBl"0404 a risk of 1 in 20. The highest absolute risk for susceptibility to RA comes in individuals who carry both a DRBl"0401 and a DRBl"0404 gene; however, even in these "at-risk' heterozygotes, only approximately 1 in 7 will develop RA. Gene dosage also may correlate with the degree of disease severity (Combe et al., 1995;Weyand et al., 1992;Wordsworth et aZ., 1992)or with extraarticular disease manifestations including rheumatoid vasculitis or Felty's syndrome (Starkebaum et al., 1997). There have been extensive studies to determine whether differences between the DRBl"0401, "0404, and "0101 alleles influence the degree of susceptibility or clinical course (see "Rheumatic Disease Clinics of North America," 1992), and articles therein; Moreno et al., 1997; Wagner et al., 1997; Reveille et al., 1996; Weyand et al., 1995; Gough et al., 1994). In general, the DR4-positive RA-associated alleles DRBl"0401 and "0404 are consistently correlated with severe, erosive disease, whereas the DRBl"0101 is associated with milder or seronegative disease (Vehe et al., 1993; Nepom and Nepom, 1992; Nepom et al., 1989). In addition to DRBl"0401, "0404, and "0101, which are associated with RA in Caucasian populations, DRBl"0405 and DRBl"1402 are also RA susceptibility genes in their different ethnic populations (Ollier and Thompson, 1992). These latter alleles are predominantly associated with RA in populations with a low prevalence of the DRBl"0401 and DRBl"0404 genes; that is, in Japan and Korea, DRBl"0405 is associated with RA, and in Native Americans the RA-associated DRBl allele is "1402 (Willkens et al., 1991). There is a remarkable structural relationship among these different RA susceptibility genes. Although these different RA-associated alleles vary tremendously in some areas of their sequence, they nevertheless all encode a similar short sequence of key amino acids at one site in the HLA class

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I1 molecule, which has been termed the "shared epitope" (Gregersen et al., 1987).The RA-associated shared epitope consists of the sequence LeuLeu-Glu-G1n-Arg-Arg-Ala-Ala, which occurs from residues 67 to 74 of the DRBl polypeptide chain. This sequence occurs in each of the RAassociated alleles-DRBl"0101, "0404, *0405, and "1402-and occurs with a single conservative change, Arg + Lys at residue 71, in the DRBl"0401 susceptibility allele. Because all DRBl alleles that contain this sequence are found associated with RA in different ethnic populations, and there are no DRBl alleles associated with RA that do not carry this sequence, it appears that the primary unit of disease susceptibility encoded in the MHC is not a specific allele but is in fact this specific sequence segment contained within several DRBl genes. Studies of the evolutionary history of DRBl genes have highlighted the unique character of this shared epitope sequence. Although the DRB 1 structural gene is generally highly conserved among species and individuals, there are clusters of polymorphic segments within the second exon that show substantial variation. One of the features of MHC evolution is that these clustered variable sequences occur in multiple distinct species and allelic lineages, indicating a shuffling or intermixing of conserved sequence motifs. The shared epitope region associated with RA occurs in such a conserved motif region. The particular clusters of amino acids in this region represent a fixed sequence combination that has a distinct trans-species and trans-allelic evolutionary history, recurring on multiple different genes. Such a strong segmental motif conservation occurring on multiple alleles is consistent with a direct functional role for this sequence, in that selection mechanism!; maintaining such a sequence pattern act on the expressed sequence. There is also presumably an interesting genetic exchange mechanism to mantain and distribute such a successful sequence motif among multiple genes (Gaur and Nepom, 1996; Gyllensten et al., 1991; Sigurdardottir et al., 1992; Gaur et al., 1997). The primary association of RA with the shared epitope, rather than with a single specific gene, and the strong evolutionary conservation of this segmental sequence element support several conclusions. First, RAassociated disease susceptibility encoded by the MHC is primarily due to the function of the DRBl gene itself, rather than to any linked sequences within the MHC, because these linked sequences are very disparate on the different RA-associated haplotypes. Second, the sites within the shared epitope sequence that differ from alleles not associated with RA provide a direct structural clue to mechanisms of disease susceptibility. Third, the strong selective pressure to generate and maintain this polymorphic region on the DRHl molecule implies an important role in specific HLA molecular function.

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II. Mechanisms to Account for the Association of the Shared Epitope with RA

HLA-DRB1 molecules, like all class I1 MHC molecules, bind antigenic peptides and present these peptides for recognition to antigen-specific T lymphocytes. HLA-DR1, -DR3, and -DR4 molecules have been structurally resolved by crystallography,and all contain a deep cleft where peptides are bound (Stern et al., 1994; Ghosh et al., 1995). Side chain interactions from the antigenic peptide are responsible for specific interactions with discrete pockets deep in the groove, where the amino acids that form the pockets within the class I1 molecule are encoded by polymorphic segments of the class I1 genes. The shared epitope sequence encodes a portion of the DRBl molecule that forms one of these peptide-binding pockets, and thus it is plausible to consider the potential role of the shared epitope in selecting the types of antigenic peptides that can bind to HLA-DR molecules. On the other hand, although each RA-associated DRBl allele encodes the shared epitope sequence, they differ in other sequences encoding several other important peptide-binding pockets. The molecular perspective provided by this structural view of RA-associated DR molecules is thus not entirely straightforward, and several different molecular mechanisms can be envisioned to account for the association between RA and the specific shared epitope sequence on DRB class I1 susceptibility molecules. These are listed in Table 11, and discussed below.

A. MODEL^ The HLA-DR molecules associated with RA are presumed to bind a specific (as yet unidentified) pathogenic peptide. This model gets its impetus from a detailed consideration of the way in which HLA-encoded variation in the class I1 molecule has a major role in selecting specific peptides for binding and presentation. Many studies have analyzed peptides that are bound by DR4 molecules (Friede et al., 1996; Rothbard et al., 1994; Fu et al., 1995; Sette et al., 1993; Hammer et al., 1994; Kirschmann et al., 1995; McNicholl et d., 1995; Woulfe et al., 1995; Davenport et al., TABLE I1

MODELSTO ACCOUNTFOR THE SHAHEDEPITOPEASSOCIATION WITH RA

1. A specific aithritogenic peptide is bound by shared epitope-positive alleles; 2. The shared epitope is itself an antigenic peptide bound by other MHC molecules: 3. There is direct T cell recognition of the shared epitope-positive p chain a-helix on the class 11 molecule that dominates over peptide-specific recognition;

4. Mimicry mechanisms account for recognition of the shared epitope-positive class 11 molecule.

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1997).These peptides contain a particular motif of amino acid side chains, at relative positions 1,4,6, and 9. These four positions, in turn, correspond to four separate pockets, located within the class I1 peptide-binding groove. Figure 1 isee color plate) presents a model of the HLA-DRBl"0401 molecule containing the shared epitope region illustrating the relationship to a bound peptide. As with all HLA-DR molecules, the RA-associated binding motif for pocket 1 prefers a peptide side chain residue 1 that is hydrophobic (usually W, Y, F, L, V, or I). Within this set of permitted hydrophobic amino acids, the size of the residue at position 1 is influenced by HLA polymorphism around pocket 1; a glycinehaline polymorphism at P chain codon 86 dictates binding of large (F, Y, W) or small (I, L, V ) peptide residues (Matsushita et al., 1994; Demotz et al., 1996; Davenport et ul., 1995). However, this distinction at pocket 1 is of doubtful disease significance because both 86-Gly and 86-Val variants are associated with RA susceptibility. The RA-associated binding motif for peptide residue 4 is potentially much more selective. This is the position influenced by the shared epitope region polymorphisms. Changes in amino acids 67, 70, 71, and 74 are sufficient to alter disease susceptibility, and these amino acid residues form a portion of the P chain a-helix that is positioned to contact residue 4 of the bound peptide. Antigenic peptides with negatively charged residues in relative position 4 (Glu, Asp) are efficiently bound by alleles containing the Arg or Lys residues at DRP position 71. On the other hand, peptides that contain positively charged residues in position 4 (Arg, Lys) are not bound by shared epitope-positive molecules, presumably due to direct charge incompatibilities. Positively charged residues at peptide residue 4 are permitted for binding to other closely related DR molecules that differ from DR131"0404 only in the shared epitope region, i.e., DRBl"0402 and "0407 (Hammer et al., 1995; Davenport et al., 1997; Rammensee et al., 1995). It should be noted, however, that the ability of shared epitope-positive molecules to bind peptides with negatively charged amino acids at residue 4 represents a preference for high-avidity binding interactions. Peptides with neutral and small amino acids at residue 4 are also capable of binding to the same class I1 molecules, albeit with lower avidity. Thus, the restricted motif for peptide residue 4 may be favored, but is not obligatory, for antigens bound by RA-associated class I1 molecules. Additional allelic differences are highlighted by the binding motif preference for peptide residues at position 6. The DR4 binding motif at this position prefers polar residues such as threonine or serine; this is the same for €\A-associatedalleles ("0401 and "0404) and nonassociated alleles ("0402 and "0403) (Rammensee et al., 1995). Other non-DR4 alleles, including the RA-associated DRBl"0101, and DRBl"l402-encoded mole-

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cules do not show this preference. Thus, although it is possible to derive a fairly specific preferential motif for the DR4 molecules associated with RA by a combination of pockets 4 and 6, this motif is not shared by the non-DR4 molecules associated with RA. At the binding pocket for residue 9, the association with RA is also not clear. The preferential peptide residue binding in pocket 9 is largely determined by the charge of the polymorphic residue on the DRP chain at position 57 (B. Nepom et al., 1996; Kwok et al., 1996). In the absence of Asp57, peptides that carry a negatively charged residue at position 9 are preferred for binding, whereas alleles that carry an Asp57 do not allow this interaction. Within the RA-associated family of alleles, DRB 1variation at codon 57 is found. For example, DRBl"0401 and DRBl"0404 carry Asp57; DRBl"0405 does not. Overall, there is no single RA-associated peptide motif. Within the different DR alleles associated with RA, DRBl"0401 and "0404 share a common motif, which has been suggested as a blueprint for an arthritogenic peptide (Table 111). However, as detailed previously, this motif fails to adequately explain the shared epitope association with RA on multiple allelic backgrounds, leaving open the distinct possibility that mechanisms other than binding a specific arthritogenic peptide are actually the basis for the HLA-encoded genetic susceptibility. B. MODEL^ Model 2, listed in Table 11, suggests that the basis for the shared epitope association with RA is that the DRBl molecule itself is an antigen, in which proteolysis leads to presentation of peptides derived from the DRB1encoded protein. If one of these processed peptides contained the shared epitope sequence, it could be a potential antigen in common among the TABLE 111 THEPEPTIDE-BINDING MOTIFFOR HLADR4 MOLECULESASSOCIATED WITH RA Residue Allele

1

4

6

DRB1'0401

W, F, Y, I, L, V

T, S preferred"

not E, D

DRBl"0404

W, F, Y, I, L, V

T, S preferred"

not E, D

DRBl"0405

W, F, Y, I, L, V

not R, K; E, D allowed" not R, K; E, D allowed" not R , K; E, D allowed"

T, S preferred"

E, D allowed

~

' Others permitted, but with lower avidity binding.

~ _ _ _ _ _

9

MHC-DIRECTED SUSCEPTIBI1,ITY

32 1

different RA-associated alleles. To date, there is a lack of direct evidence for this model. However, there are a number of observations that suggest that presentation of a self-MHC peptide such as the shared epitope may indeed occur. When endogenous peptides are eluted from cell surface class I1 molecules and analyzed, a common observation is the detection of MHCderived peptides. For example, peptides eluted from multiple DR molecules include various HLA-A, HLA-B, and even other class II-derived sequences (Chicz et nl., 1993). Many of these peptides are derived from conserved sequences; however, some peptides from polymorphic regions of the MHC molecules have also been described (Chicz et al., 1993; Rammensee et al., 1995), although the shared epitope sequence has not yet been among those identified. Indeed, analogous to "minor histocompatibility antigens," which represent polymorphic peptides present in different individuals, self-peptides derived from the polymorphic regions of HLA molecules may create important antigenic targets for T cell recognition. In this case, of course, genetic studies would show linkage and association with the MHC because the polymorphic antigen is encoded by the DRBl locus. In a recent study of autoreactive T cells derived from autoimmune patients, Miller et al. (1993) described a human T cell clone with an interesting specificity: This CD4' T cell was specific for DRBl"0401containing cells but appeared to be restricted by the DRB4 locus. One interpretation of these data is that a peptide derived from DRBl"0401 was being presented to T cells by a class I1 molecule encoded by the DRB4 locus. Because DRBl"0401 differs from DRBl"0402 only in the shared epitope region, and this clone did not react to DRBl"0402 in the presence of the same DRB4 molecule, it is possible that presentation of the shared epitope as a self-antigen was in fact observed. Other recent studies using mice transgenic for HLA class I1 molecules have also shown that peptide antigens corresponding to sequences homologous to the shared epitope region are sometimes immunogenic in these mice, indicating the potential, at least in overtly immunized situations, for self-peptide presentation (Zanelli et d., 1996). Zanelli et al. have found that presentation of Eb peptides by H-2A molecules produces a protective effect in a mouse model of collageninduced arthritis (Zanelli et al., 1995). They have also hypothesized that DR-derived peptides may influence T cell selection in the same model (Zanelliet al., 1997).Evidence for recognition of polymorphic self-peptides in human:; has been reported by Salvat et al. (1994), who described in vitro T cell reactivity to peptides derived from HLA-DRB1-encoded proteins. Thus, evidence is accumulating to support the potential role of a

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shared epitope self-peptide in immune recognition, but it is not yet clear how this mechanism would directly account for RA susceptibility. C. M O U E L ~ Model 3 for the shared epitope association in RA posits that there is direct T cell recognition of the DRP amino acids encoded by the shared epitope, recognized in the context of the interaction between class I1 molecules and antigen-specific T cell receptors. This model is based on a number of experimental observations and molecular modeling insights that indicate that several of the key polymorphic residues within the shared epitope region, at positions 67, 70, and 74, all likely play a direct role by contacting the T cell receptor (TCR),relatively independent of the nature of the specific peptide bound in the class I1 groove. Structurally, polymorphic amino acid residues on the a-helical loop of the P chain in the DR structure contact peptide or contact TCR or both. Site-directed mutagenesis experiments using individual amino acid substitutions on the DRBl”0404 molecule indicate that alterations at any of these key polymorphic sites within the shared epitope are sufficient to ablate recognition by allospecific human T cell clones (Hiraiwa et al., 1990). Indeed, this recognition is specific for the shared epitope even when the antigen presenting cells are defective in HLA-DM and are therefore inefficiently loaded with normal peptides (Penzotti et al., 1996). In the analysis of a CD4+ human T cell clone recognizing a rubelladerived peptide in the context of DR4 molecules, an intriguing observation also points to the direct role of residues within the shared epitope sequence as TCR contact sites. In these experiments, peptide variants were recognized by an antigen-specific T ceIl when presented on different DR4 restriction elements that differed at codon 74, within the shared epitope region. Specifically, peptides with a Glu substitution at position 5, a TCR contact site, were presented by DR4 molecules with an alanine at residue 74; peptides with a valine substitution were presented by DR4 molecules with a Glu at residue 74 (Nepom et al., 1996b). This simple structural complementarity implies that the same TCR recognized the Glu residue required for activation whether it came from the peptide or from the MHC molecule. This functional equivalence, in turn, suggests that direct TCR recognition of the MHC polymorphic residue was permissive for agonist function and full activation. This type of direct TCR recognition of residues within the shared epitope sequence element may be the basis for numerous observations of T cell “promiscuity” with respect to the MHC restriction. These polyreactive patterns of T cell activation have been attributed to HLA-DR “super types” that correspond to recognition of residues 67-74 on both RA-associated

MHC-DIRECTED SUSCEPTIBILITY

323

and multiple non-RA-associated alleles (Nepom et al., 1996b; Berte et al., 1988; Ou et al., 1997). As discussed previously, studies of the evolution of MHC sequence polymorphisms suggest a similar conclusion. It appears likely that sequences within the codon 67-74 region represent conserved motifs that have become evolutionarily dispersed throughout multiple alleles but that nevertheless have been maintained, even in different species and loci, implying functional selection (Gaur et al., 1996, 1997; Lundberg and McDevitt, 1992; Gyllensten et al., 1991).A plausible unifylng hypothesis is that the conserved sequence elements that persist in modem humans, including the shared epitope, perform an important essential function in T cell selection and recognition and are being maintained for that purpose. This then leads to immunologic consequences in humans, such as a high degree of alloreactivity, frequent promiscuous MHC restriction, and, in the case of rheumatoid arthritis, multiple alleles that differ at other polymorphic sites but that share the conserved shared epitope sequence element. There are additional structural and functional implications of the model in which direct TCR recognition of the DRBl shared epitope is the important mechanism accounting for disease susceptibility. A detailed molecular model of the TCR-peptide-MHC trimolecular complex has been recently reported i n which homology model building techniques and interactive graphics were used to orient the TCR on the DRBl"0404 molecule (Penzotti et al , 1997).Among numerous contacts predicted between the MHCpeptide complex and the TCR in the trimolecular model, most notable are specific interactions with DRBl"0404 residues 67-74. The Arg at position 71 is part of an extended hydrogen bonding network in the shared epitope region that potentially creates a specific conformational unit recognized by the TCR. As part of this hydrogen bonding network, shared epitope residues establish direct interactions with TCR residues in the CDR elements, as would be expected for recognition sites involved in specific activation. An additional intriguing observation in this modeling study was the correlation between predicted contact sites and specific V@ TCR elements known to be present in oligoclonal T cell populations in RA patients. Thus, this model proposes a role for the shared epitope in which the RA susceptibility molecule participates directly as a dominant selection element in direct TCR recognition for activation and amplification, possibly contributing toward the TCR bias observed in oligoclonal T cell expansion. Because disease pathogenesis in RA likely involves triggering of autoreactive T cells, this model suggests a mechanism whereby direct recognition of the shared epitope selects for a high frequency of potentially self-reactive T cells in the susceptible individual. The transition from susceptibility to

324

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T. NEPOM

actual disease reflects activation of this self-reactive pool in the context of additional tissue-specific triggering events. D. MODEL^ Proposed to account for shared epitope associations in RA, model 4 considers the role of the shared epitope sequence as a potential target for molecular mimicry. Database comparisons have found sequences similar to the shared epitope in numerous proteins, including an Epstein-Barr virus g p l l 0 and an Escherichia coli DNA J protein (Roudier et al., 1989; Albani et al., 1992). This homology was initially interpreted in the context of antigenic cross-reactivity, although it has more recently been proposed that intermolecular mimicry may occur, in which heat shock proteins bind class I1 molecules that contain the shared epitope (Auger et al., 1996). Although it is premature to speculate on the specific disease-promoting mechanisms such an interaction might provoke, this is an intriguing suggestion that may lead to plausible models of disease susceptibility. It should be noted, however, that the specificity of heat shock proteins for the shared epitope is apparently restricted to some, but not all RA-associated DRBl alleles (Auger et aZ., 1996). Can these alternative models be reconciled with each other? As illustrated in Fig. 2, the different models potentially implicate different steps in a HLA-directed pathway to RA. The gene dosage effects discussed previously, *andin particular the association of heterozygous DRB 1*0401/ "0404 genotypes with RA, suggest the possibility of significantcontributions to susceptibility at the level of positive selection during T cell development. There is evidently some functional synergy between DRBl"0401 and DRBl"0404 in that the risk in heterozygotes is considerably higher than the simple additive risk of each of these two alleles alone. Because DRBl"0401 and "0404 differ from each other only by a single Lys-Arg substitution at residue 71, it is possible that positive selection on one of these restriction elements predisposes for a high degree of autoreactivity on the other. In any event, dominant T cell recognition properties of the conserved shared epitope sequence motif (model 3) are likely to be a major factor in selecting T cells that mature and become established in the peripheral lymphoid compartment. Because such positive selection events are strongly influenced by peptides presented during thymic development, there is likely to be some contribution from specific self-peptides in this step, potentially integrating elements of models 1 and 2 with this pathway as well. This pathway predicts that the predominant functional contribution of the susceptible class I1 molecule is to establish a high frequency of potentially autoreactive cells in the periphery of individuals with the RA-

MHC-DIRECTED SUSCEPTIBILITY

325

FIG.2. The HLA-directed pathway to RA.

susceptible genotype. Because most individuals who carry this genotype do not get RA, it is evident that the specific triggering events are relatively infrequent or, alternatively, if frequent, that regulatory mechanisms usually prevent disease progression. The nature of the specific triggering events in the synovial compartment is completely unknown. On the one hand, a specific pathogen could be involved, eliciting an antigen-specific response that subsequently spreads to include synovial autoantigens. If this antigenspecific T cell response includes a subset of the TCR selected and amplified by virtue of recognition of the shared epitope, the threshold for activating an autoimmune disease in an individual with the RA-associated susceptibility genes may well be much lower than that in individuals who do not carry these genes. On the other hand, disease-triggering events may be relatively nonspecific if the key immune recognition specificity is for self-antigens (Thomas and Lipsky, 1996).Disparate pathogenic insults may converge on a common pathway in which self-antigens, possibly including the shared epitope itself, become presented at high density in the synovial compartment. A high local antigen concentration in association with a high peripheral precursor frequency of autoreactive cells may be sufficient to trigger disease. Indeed,

326

GERALD T. NEPOM

if self-antigen presentation in the synovium of susceptible individuals is pathogenic, it may be that DRBl"0401 interactions with heat shock proteins, as suggested by model 4, contribute to a subsequent autoimmune d'isease course.

111. Clinical Applications

The characterization of the HLA class I1 genes associated with rheumatoid arthritis has clinical utility for patient prognosis, for therapeutic management, and for the design of specific immunomodulators. Table IV summarizes the prognostic value of assessing HLA susceptibility genes in a patient with rheumatoid arthritis. As discussed previously (see Table I), the HLA-DRB1 susceptibility alleles are prevalent in the normal population. Although inheritance of these alleles confers a relative risk of rheumatoid arthritis, the absolute risk of disease for an individual is nevertheless only 3-15%. For this reason, widespread population screening using genetic typing to predict RA in the general population is not useful. However, there is one important aspect of typing for HLA susceptibility genes in rheumatoid arthritis that gives it clinical utility. This is the observation that the DRBl"0401 and DRBl"0404 genes are associatedwith severe erosive forms of disease (Nepom and Nepom, 1993; Weyand et al., 1995; Gough et al., 1994; Wagner et al., 1997; Moreno et al., 1997; Calin et al., 1989; van Zeben et al., 1991; Olsen et al., 1988). Although the predictive value for RA in the general population is quite low, the predictive value for early erosive disease in patients is quite high (Nepom et al., 1996a). Use of HLA-DRB1 genotyping in the early arthritis patient, during the initial clinical and laboratory assessment, should be a useful tool to identify a subset of patients with a high risk of early joint erosions and consequent poor clinical course. The anticipated corollary of a prognostic indicator, of course, is improved clinical management. In rheumatoid arthritis, aggressive treatment, including combination drugs, has been proposed to improve patient outcomes (O'Dell et nl., 1996a; Wilske and Yocum, 1996; Breedveld, 1995; Tugwell et al., 1995).Therefore, the most promising clinical utility for HLA-DRB1 TABLE IV PHOCNOSTIC VALNE OF HLA-DRB1 TESTING Marker of erosive disease Identifies candidates for early aggressive theripy Sensitivity and specificity will depend on the clinical demographics and genetic background of the patient population being tested

MIIC-DIRECTED SUSCEYTIBILITY

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typing is to help in the assessment of which patients are candidates for aggressive forms of therapies (O’Dell et al., 1996b). The sensitivity of this type of HLA testing for prediction of early erosive disease will depend on the demographics of the patient population tested. In North American Caucasians, it is estimated to be approximately 70-80%; in American blacks and Hispanics it will be substantially less (McDaniel et al., 1995; Boki et al., 1992; Reveille et al., 1996). The specificity of HLA testing will also vary greatly, depending on a variety of other clinical parameters. For example, in very early forms of arthritis, when the differential diagnosis may include not only rheumatoid arthritis but also reactive arthritis, gout, and self-limited forms of disease, the specificity is likely to be low. For this reason, a combination assessment of HLA-DRB1 typing and rheumatoid factor may be most efficient (Gough et nl., 1994; Nepom et al., 1996a). One of the interesting implications of this disease stratification based on HLA is that the nonerosive forms of rheumatoid arthritis are genetically distinct from the erosive phenotype. Indeed, there is no specific HLA allelic association with nonerosive forms of rheumatoid arthritis (Nepom et al., 1989; Weyand et al., 1995).In some populations, nonerosive forms of disease represent from one-third to one-half of all patients, and this genetic distinction likely implies a distinct pathway of disease. As disease mechanisms in HLA-associated RA are further elucidated, it may well become evident that nonerosive RA is a separate disease. The importance of this distinction is reflected in the prospects for more specific forms of immunotherapy for RA. A large number of novel drugs that target T cell activation are currently under development, and peptide-based therapies designed to interfere with the HLA-peptide-TCR interaction are also envisioned. Rational use of such specific immunomodulators in RA will be facilitated 1 ~ progress y in our mechanistic understanding of the interactions between the synovial compartment, the peripheral T cell compartment, and the HLA gene expression that characterize the RA patient. REFEHENCES Albani, S.,Tuckwell,J. E., Esparza, L., Carson, D. A., and Roudier, J. (1992).The susceptibility sequence to rheumatoid arthritis is a cross-reactive B cell epitope shared by the Escherichia coli heat shock protein dnaJ and the histocompatibility leukocyte antigen DRB10401 molecule. J. Clin. Invest. 89, 327-331. Auger, I., Escola, J. M., Gorvel, J. P., and Roudier, J. (1996). HLA-DR4 and HLA-DR10 motifs that carry susceptibility to rheumatoid arthritis bind 70-kD heat shock proteins. Nat. Med. 2, 306-310. Berte, C. C., Tanigaki, N., Tosi, R., Gorski, J.. and Mach, B. (1988). Serologcal recognition of HLA-DH allodeterminant corresponding to DNA sequence involved in gene conversion. liriiriunogenetics 27, 167-173. Boki, K. A., Panayi, G. S., Vaughan, R. W., Drosos, A. A., Moutsopoulos, H. M., and Lanchbury, J. S. (1992). HLA class I1 sequence polymorphisms and susceptibility to

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rheumatoid arthritis in Greeks. The HLA-DR beta shared-epitope hypothesis accounts for the disease in only a minority of Greek patients. Arthritis Rheum. 37, 749-755. Breedveld, F. C. (1995). New perspectives on treating rheumatoid arthritis. N . En& J. Med. 333, 183-184. Calin, A,, Elswood, J,, and Klouda, P. T. (1989). Destructive arthritis, rheumatoid factor, and HLA-DR4. Arthritis Rheum. 32, 1221-1225. Chicz, R. M., Urban, R. G., Gorga, J. C., Vigndi, D. A. A., Lane, W. S., and Strominger, J. L. (1993). Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles. J. Exp. Med. 178, 27-47. Combe, B., Eliaou, J.-F., Daures, J.-P., Meyer, O., Clot, J., and Sany, J. (1995). Prognostic factors in rheumatoid arthritis. Comparative study of two subsets of patients according to severity of articular damage. Br. J . Rheum. 34, 529-534. Davenport, M. P., Quinn, C. L., Chicz, R. M., Green, B. N., Willis, A. C., Lane, W. S., Bell, J. I., and Hill, A. V. S. (1995). Naturally processed peptides from two diseaseresistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DRP chain. Proc. Natl. Acad. Sci. USA 92,6567-6571. Davenport, M. P., Godkin, A,, Friede, T., Stevanovic, S., Willis, A. C., Hill, A. V. S., and Rammensee, H. G. (1997). A distinctive peptide binding motif for HLA-DRBl"0407, an HLA-DR4 subtype not associatedwith rheumatoid arthritis. 1mmunogenetic.s45,229-232. Demotz, S., Barbey, C., Corradin, G., Amoroso, A,, and Lanzavecchia, A. (1996). The set of naturally processed peptides displayed by DR molecules is tuned by polymorphism of residue 86. Eur. J. Immunol. 23, 425-432. Friede, T., Gnau, V., Jung, G., Keilholz, W., Stevanovic, S., and Rammensee, H. G. (1996). Naturd ligand motifs of closely related HLA-DR4 molecules predict features of rheumatoid arthritis associated peptides. Biockim. Biophys. Acta 1316, 85-101. Fu, X., Bono, C. P., Woulfe, S. L., Swearingen, C., Summers, N. L., Sinigaglia, F., Sette, A,, Schwartz,B. D., and Karr, R. W. (1995).Pocket 4 ofthe HLA-DR(a,/3l00401)molecule is a major determinant of T cell recognition of peptide. J. Exp. Med. 181, 915-926. Gao, X., Olsen, N. J., Pincus, T., and Stastny, P. (1990). HLA-DR alleles with naturally occurring amino acid substitutions and risk for development of rheumatoid arthritis. Arthritis Rheum. 33,939-946. Gaur, L. K., and Nepom, G. T. (1996). Ancestrd major histocompatibility complex DRB genes beget conserved patterns of localized polymorphisms. Proc. Natl. Acad. Sci. USA 93,5380-5383. Gaur, L. K., Nepom, G. T., Snyder, K. E., Anderson, J., Pradarpurkar, M., Yadock, W., and Heise, E. R. (1997). MHC-DRB allelic sequences incorporate distinct intragenic trans-specific segments. Tissue Antigens 49, 342-355. Ghosh, P., Amaya, M., Mellins, E., andwiley, D. C. (1995).The structure of an intermediate in class I1 MHC maturation: CLIP bound to HLA-DR3. Nature 378, 457-462. Cough, A., Faint, J., Salmon, M., Hassell, A,, Wordsworth, P., Pilling, D., Birley, A., and Emery, P. (1994). Genetic typing of patients with inflammatory arthritis at presentation can be used to predict outcome. Arthritis Rheum. 37, 1166-1170. Gregersen, P. K., Silver, J., and Winchester, R. J. (1987). The shared epitope hypothesis: An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum. 30, 1205-1213. Gyllensten, U. B., Sundvall, M., and Erlich, H. A. (1991). Allelic diversity is generated by intraexon sequence exchange at the DRBl locus of primates. Proc. Natl. Acad. Sci. USA 88,3686-3690.

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Hammer, J., Bono, E., Gallazzi, F., Belunis, C., Nagy, Z., and Sinigaglia, F. (1994). Precise prediction of major histocompatibility complex class 11-peptide interaction based 011 peptide side chain scanning. J. Exp. Med. 180, 2353-2358. Hammer, J., Gallazzi, F., Bono, E., Karr. K. W., Cuenot, J., Vdsasnini, P., Nagy, Z. A,, and Sinigaglia, F. (1995). Peptide binding specificityof HLA-DR4 molecules: Correlation with rheumatoid arthritis association. 1. Exp. Med. 181, 1847-18.55. Hiraiwa, A., Yamanaka, K., Kwok, W. W., Mickelson, E. M., Masewicz, S., Hansen, J. A., Radka, S. F., and Nepom, G. T. (1990). Structural requirements for recognition of the HLA-Dw14 class I1 epitope: A key HLA determinant associated with rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 87, 8051-8055. Kelly, A,, and Trowsdale, J. (1994). Novel genes in the human major histocompatibility complex class I1 region. Int. Arch. Allergy bnmunol. 103, 11-15. Kirschmann, D. A,, Duffin, K. L., Smith, C. E., Welply, J. K., Howard, S. C., Schwartz, B. D., and Wonlfe, S. L. (1995). Naturally processed peptides from rheumatoid arthritis associated and non-associated HLA-DR alleles. /. lmmunol. 155, 5655-5662. Kraulis, P. J . (1991). MOLSCRIFT: A program to produce both detailed and schematic plots of protein structures. J . Appl. Crystallogr. 24, 946-950. Domeier, M. E., Johnson, M. L., Nepom, G. T., and Koelle, D. M. (1996). Kwok, W. W., HLA-DQB1 codon S7 is critical for peptide binding and recognition. J. Exp. Med. 183, 1253-1258. Lundberg, A . S., and McDevitt. H. 0.(1992).Evolution of major histocompatibility complex class I1 dlelic diversity: Direct descent in mice and humans. Proc. Natl. Acad Sci. USA 89, 6545-6549. Matsushita, S., Takahashi, K., Motoki, M., Komoriya, K., Ikagdwa, S., and Nishimura, Y. (1994).Allele specificityof structural requirement for peptides bound to HLA-DRBl”0405 and -DR131”0406 complexes: Implication for the HLA-associated susceptibility to methimazole-induced insulin autoimmune syndrome. J. Exp. Med. 180, 873-883. McDaniel, I). 0.. Alarcon, G. S., Pratt, P. W., and Reveille, J. D. (1995). Most AfricanAmerican patients with rheumatoid arthritis do not have the rheumatoid antigenic determinant (epitope).Ann. Intern. Med. 123, 181-187. McNicholl, J . M., Whitworth, W. C., Oftung, F., Fu, X., Shinnick, T., Jensen, P. E., Simon, M., Wohlliueter, R. M., and Karr, R. W. (1995). Structural requirements of peptide and MHC for DR(a,/31”040l)-restrictedT cell antigen recognition. I. Immunol. 155, 19511963. Miller, G., Nepom, G. T., Reich, M. B., and Thomas, J. W. (1993). Autoreactive T cells from a type I diabetic recognize multiple class 11products. Hutri. Imtnunol. 36,219-226. Moreno, I., Valenzuela, A,, Garcia, A., Yelamos, J., Sanchez, B., and Hernanz, W. (1997). Association of the shared epitope with radiological severity of rheumatoid arthritis. J. Rheumotol. 23, 6-9. Nepom, B. S,, and Nepom, G. T. (1993). Immunogenetics and the rheumatic diseases. In “Textbook. of Rhenmatology” (W. N. Kelley, E. D. Harris, Jr., S . Ruddy, and C. B. Sledge, Eds.), 4th ed., Vol. I, pp. 89-107. Saunders, Philadelphia. Nepom, B. !;,, Nepom, G. T., Coleman. M., and Kwok, W. W. (1996). Critical contribution of /3 chain residue 57 in peptide binding ability of both HLA DR and DQ molecules. Proc. Nati. Acad. Sci. USA 93, 7202-7206. Nepom, G. T., and Nepoin, B. S. (1992). Prediction of susceptibility to rheumatoid arthritis by human leukocyte antigen genotyping. In “Rheumatic Disease Clinics of North America” (G. T. Nepom, Ed.), Val. 18, pp. 785-792. Saunders, Philadelphia. Nepotn, G . ‘T,, Byers, P., Seyfried, C.. Healey, L. A,, Wilske. K. R., Stage, D., and Nepom, B. S.(1989).HLA genes associated with rheumatoid arthritis. Arthritis Rheum. 32,lS-21.

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Nepom, G. T., Gersuk, V., and Nepotn, B. S. (l996a). Prognostic implications of HLA genotyping in the early assessment of patients with rheumatoid arthritis. J. Rheumutol. 23, 5-9. Nepom, G. T., 011,D., Lybrand, T., DeWeese, C., Domeier, M., Buckner, J., Mitchell, L. A., and Tingle, A. J. (1996b). Recognition of altered self-MHC molecules modulated by specific peptide interactions. Eur. J. Iminunol. 26, 949-952. O'Dell, J. R., Haire, C. E., Erikson, N., Drymalski,W., Palmer, W., Eckhoff, P. J., Garwood, V., Maloley, P., Klassen, L. W., Wees, S., Klein, H., and Moore, G. F. (19964. Treatment of rheumatoid arthritis with methotrexate alone, sulfasalazine and hydroxychloroquine, or a combination of all three medications. N . Engl. J. Med. 334, 1287-1291. O'Dell, J., Nepom, B., Haire, C., Drymalski, W., Palmer, W., Eckhoff. J.. Klassen, L., Wees, S., Thiele, G., Moore, C.,and Nepom, G. T. (1996b).DRBl typing in rheumatoid arthritis (RA) is useful in predicting response to specific therapy. J. Rheum., in press. Ollier, W., and Thomson, W. (1992). Population genetics of rheumatoid arthritis. In "Rheumatic Disease Clinics of North America" (G. T. Nepom, Ed.), Vol. 18, pp. 741-759. Saunders, Philadelphia. Olsen, N. J,, Callahan, L. F., Brooks, R. H., Nance, E. P., Kaye, J. J., Stastny, P., and Pincus, T. (1988). Associations of HLA-DR4 with rheumatoid factor and radiographic severity in rheumatoid arthritis. Am. J. Med, 84, 257-264. Ou, D., Mitchell, L. A., and Tingle, A. J. (1997). HLA-DR restrictive supertypes dominate promiscuous T cell recognition: Association of multiple HLA-DR molecules with susceptibility to autoimmune diseases. J. Rheumutol. 24, 253-261. Penzotti, J. E., Doherty, D., Lybrand, T. P., and Nepom, G. T. (1996). A structural model for TCR recognition of the HLA class I1 shared epitope sequence implicated in susceptibility to rheumatoid arthritis. I. Autoirntnun. 9, 287-293. Penzotti, J. E., Nepom, G. T., and Lybrand, T. P. (1997). TCWHLA-DRBl"04 molecular modeling predicts site-specific interactions for the DR shared epitope associated with rheumatoid arthritis. Arthritis Rheum 40, 1316-1325. Ragoussis, J., Monaco, A,, Mockridge, I., Kendall, E., Campbell, R. D., and Trowsdale, J. (1991). Cloning of the HLA class I1 region in yeast artificial chromosomes. Proc. Nutl. Acud. Sci. USA 88, 3753-3757. Rammensee, H. G., Friede, T., and Stevanoviic, S. (1995). MHC ligands and peptide motifs: First listing. Zmrnunogenetics 41, 178-228. Reveille, J. D., Alarcon, G. S., Fowler, S. E., Pillemer, S. K., Neuner, R., Clegg, D. O., Mikhail, I. S., Trentham, 13. E., Leisen, J. C. C., Gluhm, G., Cooper, S. M., Duncan, H., Tuttleman, M., Heyse, S. P., Sharp, J. T., and Tilley, B. (1996). HLA-DRB1 genes ~ . 1802-1807. and disease severity in rheumatoid arthritis. Arthritis R ~ c x T39, Ronningen, K. A,, Spurkland, A,, Egeland, T., Iwe, T., Munthe, E., Vartdal, E., and Thorsby, E. (1990). Rheumatoid arthritis may be primarily associated with HLA-DR4 molecules sharing a particular sequence at residues 67-74. Tissue Antigens 36, 235-240. Rothbard, J. B., Marshall, K.. Wilson, K. J., Fugger, L., and Zaller, D. (1994). Prediction of peptide affinity to HLA DRBl"0401. lnt. Arch. Allergy 1nLmunol. 105, 1-7. Roudier, J., Petersen, J., Rhodes, G. H., Luka, J., and Carson, D. A. (1989). Susceptibility to rheumatoid arthritis maps to a T-cell epitope shared by the HLA-Dw4 DR p-1 chain and the Epstein-Barrvirus glycoproteingpll0. Proc. Nutl. Acud. Sci. USA 86,5104-5108. Salvat, S., Auger, I., Rochelle, L., Begovich, A,, Geburher, L., Sette, A,, and Roudier, J. (1994). Tolerance to a self-peptide from the third hypervariable region of HLA DRB1'0401 in rheumatoid arthritis patients and normal subjects. J. Immunol. 153,53215329.

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Sette, A,, Sidney, J., Oseroff. C.. del Cuercio, M., Sonthwood, S.. Arrhenius, T., Powell, M. F., Colon. S . M., Gaeta. F. C. A,, and Grey, H. M. (1993). HLA-DR4Dw4-binding motifs illustrate the biochemical basis of degeneracy and specificityin peptide- DR interactions. J . Z n t t t i i t t i d . 151, 3163-3170. Sigurtlardottir, S., Borsch, C., Gustafsson, K., and Andersson. L. (1992). Exon encoding the antigeri-binding site of MHC class I1 &chains is divided into two subregions with 148, 968-973. different evolutionq histories. J . Z~~miurtol. Starkebaum, G., Loughran, T. P., Jr,, Caur, L. K.. Davis, P., and Nepoin, B. S. (1997). Immunogenetic siinilarities between patients with Felty's Syndrome and those with clonal expansions of large granular lymphocytes in rheumatoid arthritis. Arthritis Rheiou. 40, 824-626. Stern, L. J., Brown, J. H., Jardetzky, T. S., Gorga, J. C.. Urban, R. G., Strominger, J. L., and W h y , D. C. (1994). Crystal structure of the human class I1 MHC protein HLADR1 coinplexed with an influenza virus peptide. Nature, 368, 215-221, Thomas. R.. and Lipsky, P. E. (1996).Presentation of self peptides by dendritic cells: Possible implicatioi-isfor the pathogenesis of rheumatoid arthritis. Arthritis Rlzetirrt. 39, 183-190. Tugwell, P., Pincus, T., Yocuin, D.. Stein, M., Cluck. O., Kraag, G., McKendry, R . , Tesser, J., Baker, F., and Wells, G.. for the Methotrexate-Cyclosporine Combination Study Group (1995). Combination therapy with cyclosporinr and methotrexate in severe rheumatoid arthritis. h . Engl. J. Med. 333, 1,37-141. van Zeben, D., Hazes, J. M. W., Zwindcrnian, A. H., Cats, A., Schreuder, G. M. T., D'Amaro, J.. ;inti Breedveld. F. C. (1991).Association of HLA-DR4 with a inore progressive disease course in patients with rheumatoid arthritis. A r t h i t i s Rheum. 34, 822-830. L'ehe, R. K., Nepoin, G . T., Wilske, K. R., Healey, L. A., Stage, D., Begovich, A. B., and Nepom, B. S. (1993). Erosive rheumatoid factor positive and negative rheumatoid arthritis are imriiuriogenetically similar. J. Rltettrrtatol. 21, 194-196. Wagner, U . , Kaltenhauser, S., Sauer, H., Arnold, S . , Seidel, W., Hantzschel, H., Kalden, J. R., and Wassmuth, R. (1997'). HLA markers and prediction of clinical course and oiitcome in rheiiinatoid arthritis. Arthriti.r R/zeron. 40, 341-351. Weyand, C. M.. Hicok, K . C., Conn, D. L., and G o r o n i , J. J. (1992). The influence of HLA-DRB1 genes on disease severity in rheiimatoid arthritis. Ann. Intent. Med. 117,801 -806. Weyand, C. M., McCarthy, T. G., and Goronzy, J. J. (1995). Correlations between disease phenotype and genetic heterogeneity in rheumatoid aithritis. J. Clin. Innest. 95, 21202126. Wlkens, R. F., Nepom, G. T., Marks, C. R., Nettles. J. W., and Nepom, B. S. (1991).The association of HLA-DwlG with rheiiinatoid arthritis in Yakima Indians: Further ebiidence for the "sliared epitope" hypothesis. Arthritis Rhen~tt.34, 43-47. Wilske, K. R., and Yocuin, D. E. (1996). RA: The status and future of combination therapy. J. Rheioiu~tol.23, 1. Wordsworth, B., Lanchbury, J. S. S., Sakkas, L. I.. Welsh, K. I., Panap, G. S., and Bell, J. I. (1989).HLA-DR4 subtype frequencies in rheumatoid nrthritis indicate that DRBl is the major susceptibility locus within the HLA class I1 region. P ~ C J C N . d Acad Sci. USA 86, 10049-10053. Wordsworth. P., Pile, K . D., Buckley, J. D.. Lanchbury, J. S. S., Ollier, B., Lathrop, M., and Bell, J. I. (1992). HLA hetercqgosity contributes to susceptibility to rheuinatoid arthritis. P m . J , Hitrrt. Genet. 51, 585-591. Woulfe. S . L., Bono, C. P., Zacheis, M. L., Kirschmann. D. A,, Baudino, T. A,, Swearingen, C., Karr, FL W., and Schwartz, B. D. (1995). Negatively charged residues interacting with the p4 pocket confer binding specificity to DRBl"O40l. Arthritis Rheum 38,1744-1753.

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Zanelli, E., Gonzdez-Gay, M. A,, and David, C. S. (1995). Could HLA-DRB1 be the protective locus in rheumatoid arthritis? Zmmirnol. Today 16, 274-278. Zanelli, E., Krco, C. J., flaisch, J. M., Cheng, S., and David, C. S. (1996). Immune response of HLA-DQ8 transgenic mice to peptides from the third hypervariable region of HLADRBl correlates with predisposition to rheumatoid arthritis. .Proc. Natl. Acad. Sci. USA 93, 1814-1819. Zanelli, E., Krco, C. J., and David, C. S. (1997). Critical residues on HLA-DRB1'0402 HV3 peptide for HLA-DQ8-restricted iinmunogenicity. Implications for rheuinatoid arthritis predisposition. J . Irnrnunol. 158, 3545-3551, This chapter was accepted for publication on June 16, 1997

ADVANCES IN IMMUNOLOGY VOL fix

immunological Treatment of Autoimmune Diseases J. R. W E N , ' F. C. BREEDVELD,' H. BURKHARDT,' A N D G. R. BUMESTER' Deportment of lnthrnol Medicine 111 and lnsrifuk for Clinicol Immunobgy, Universify Hospital ErlangenNumberg, Germny; Dspotiment of Rheumatology, leiden Universify Hospital, leiden, The Nehedods; ond Dtparhrrent of Internal Medicine 111, Medical Faculty of h e Humboldt University, Berlin, Germany

'

1. Introduction

During the past decade much progress has been made with regard to mechanisms leading to tissue destruction in autoimmune diseases. Based on this increasing knowledge new therapeutic principles have been developed with novel pharmacological and biological agents that are acting more specifically by interfering with ongoing immune processes than did treatment principles that had been available in the past. Table I gives an overview of currently investigated or already clinically tested new therapeutic approaches in autoimmune diseases. New development in the area of immunosuppressive drugs can be divided into agents inhibiting lymphokine synthesis, such as cyclosporin A or FK506; agents suppressing lymphokine signal transduction, including rapamycin and the isoxazol derivative leflunomide; compounds inhibiting nucleoside synthesis, such as mizorbine, brequinar, and mycophenolic acid; and agents affecting lymphocyte differentiation, such as 15-deoxyspergualin (Thompson et nl., 1993). Most of these new pharmacological compounds have to date been tested in the situation of organ transplantation with the exception of cyclosporin A, which has already been shown to be an efficient immunosuppressive agent in different autoimmune diseases. Furthermore, new nitrogenoxide synthase inhibitors such as N-monomethyl-2-argeninine have successfully been applied in, for example, the streptococcal cell wallinduced arthritis (McCartney-Francis et al., 1993). Studies on the clinical efficacy of biological agents in the treatment of experimental and human autoimmune diseases mainly include the use of monoclonal antibodies interfering with activation processes between antigen presenting and CD4+ helper T cells (Fig. 1),the application of antiinflammatory cytokines, and treatment principles that inhibit proinflammatory cytold'nes. The targets for these treatment approaches are cell surface molecules on CD4' T cells or cytokines as messenger molecules that control cellular function, cell differentiation, and intercellular cooperation. Furthermore, recently the possibility has been discussed of treating human autoimmune diseases by manipulating the Thl-The cell balance, an approach based on the increasing knowledge of the different role of CD4

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TABLE I NEW DEVELOPMENTS AND APPROACHESI N IMMUNOINTERVENTION IN AUTOIMMUNEDISEASES New immunosuppressive dnigs Cyclosporin A, FK506, rapamycin, brequinar, leflunomid, mycophenolic acid, 15-deoxyspergualin High-dose iv immunoglobulins Cytokine-directed therapy anti-TNF-a [mAb, TNF-a receptor constructs (p55, p75)] IL-1 receptor antagonist Antiinflammatory cytokines ( IL-4, IL-10, IL-13) Cell surface inolecule-directed therapy Antiadhesion molecule therapy (anti-ICAM-1) Nondepleting anti-CD4 mAb anti-CD28 mAb anti-CD5 mAh IL-2 fusion protein Application of autoantigens Collagen 11 Myelin basic protein Application of peptides (to date, only in experimental animal models) T cell vaccination (to date, only in experimental animal models) Changes in Thl-The balance (still rather hypothetical) Combination therapy Cyclosporin Nmethotrexate anti-TNF-a mAb/methotrexate anti-TNF-a mAb/anti-CD4 (in collagen II-induced arthritis) Gene therapy Application of antisense oligonucleotides

subsets in pathogenic mechanisms in autoimmune diseases (Adorini et

al., 1996). With our increasing knowledge about mechanisms underlying the pathogenesis of autoimmune diseases, the complexities of treatment possibilities for these disease entities have also been disclosed. Thus, in a study by Genain et al. (1996), it was shown that experimental allergic encephalomyelitis induced in marmosets by immunization with myelin oligodendrocyte glycoprotein (MOG) can be delayed by intraperitoneal treatment with MOG. However, treated animals subsequently developed a hyperacute form of this disease, Blanas et al. (1996), noted that the administration of large amounts of oral albumin (OVA)can result in the generation of CD8' cytotoxic T cells and that these T cells can promote the disease process by the administration of OVA in a murine model of insuline-dependent diabetes mellitus. The induction of oral tolerance which has been successfully used in a number of animal models for autoimmune diseases (Hafler

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antl-CDS

APC

I

I

T Cell

I

y--rTu!

anti-AAM1

11-2 fuslon protaln antl-IL-2-R

CAMPATH-1

-

Fic:. 1. Known receptor ligand systrms between CD4' T cells and antigen presenting cells of the inacrophiige-inoiiocyte linage and B cells. Different approaches for blockades of the interxtion hetween T and B cells and niacropliage-monocyte, respectively, by inonoclonal :.intibodiesand fiisioii proteins are indicated. In the huinan, situation antibodies against the CD4, CD7, and sICAM-1 inolecules have been explored in clinical trials, mostly in ~iiitoinini~ine rheumatic diseases. The same is true for a diphtheria and IL-2 fusion toxin.

and Weiner, 1996), was not confirmed in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. In both disease situations it appeares that a subgroup of patients might respond to this type of treatment as shown in a double-blind, placebo-controlled randomized trial of 90 patients with rheumatoid arthritis (Sieper et al., 1996). In this study no significant difference in the response to treatment between three groups having received different amounts of collagen I1 was seen, however, a higher prevalence of response in the type I1 collagen-treated group was observed compared to placebo-treated patients. Similarly,when 10 patients with juvenile rheumatoid arthritis were enrolled in a pilot trial that also used orally administered type I1 collagen, 8 patients revealed a reduction of both swollen and tender joint counts after 3 months of treatment (Bamett et nl., 1996). Further investigations, including controlled double-blind trials, are necessary to substantiate the reported treatment effects. Furtherinore, the already mentioned side effects, including an enhancement of autoimmune diseases, have to be considered (Blanas et al., 1996). In this review not all of the new developments and approaches for immune intervention in autoimmune diseases listed in Table I can be

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discussed. The review will concentrate on four major areas: (1)the use of biologics directed to the CD4 molecule on T helper cells; (2) the use of biological agents interfering with other cell surface molecules on T cells; ( 3 )principles that act by the blockade of proinflammatory cytokines or the use of antiinflammatory cytokines, respectively; and (4) on possibilities to treat different experimental animal models by biologic agents. Not included will be treatment principles such as the use of high-dose intravenous immunoglobuline treatment. Infused immunoglobulines may regulate autoimmunity through the idiotype/antiidiotype network depending on their variable (V) region reactivities with natural and disease-related autoantibodies, with surface immunoglobulines of B cells, or by inhibiting T cell-dependent B cell differentiation (Kalden and Manger, 1997). Placebocontrolled, double-blind studies are still missing. Furthermore, the envisaged possibility that manipulation of the idiotype network might contribute not only to insights on the pathogenesis of autoimmune diseases but might also provide a new avenue for the management of autoimmune disorders by modulation with antibodies or peptides is still an unsolved problem (Zouali et al., 1996). The finding that adhesion molecules undoubtedly play a pivotal role in different types of autoimmune diseases, especially rheumatoid arthritis (RA), has inspired trials with a monoclonal antibody (mAb) against the adhesion molecule ICAM-1 (Oppenheimer-Marks and Lipsky. 1996).The expression of adhesion molecules on endothelial cells and complementary ligands on the surfaces of circulating leukocytes and lymphocytes appears to play a critical role in cell migration to inflammatory tissue lesions. In an open-labeled study 32 RA patients were treated with different doses of a murine anti-ICAM-1 mAb (Kavanaugh et al., 1994). An improvement of arthritis was noted in approximately 50% of the patients who were treated with a higher dose of mAb. Treatment with this mAb induced T cell hyperresponsiveness that correlated with clinical benefit (Davis et al., 1995), however, when patients were retreated with this agent immune complex-mediated side effects, including uticaria, angioedema, and serum complement protein consumption, were noted (Kavanaugh et al., 1995). If this type of treatment will be of any beneficial effect in autoimmune diseases, especially in autoimmune rheumatic diseases, it has to be substantiated by further trials. From experimental animal studies the conclusion was drawn that natural occurring genetic variations in the expression of ICAM-1 or related adhesion molecules might also influence the susceptibility of RA,indicating that pharmacological approaches to reduce the expressional function of ICAM-1 might indeed be of therapeutic value (Bullard et al., 1996).

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Recently, two other new therapeutic avenues, gene therapy and the induction of apoptosis, have begun to be explored as treatment principles in autoimmune diseases. In the case of rheumatoid arthritis, candidate molecules for gene therapeutic approaches include cytokine antagonists such as IL-1 receptor antagonist (IL-lra), sTNFR, IL-10, IL-4, and sIL1R. The feasibility of this gene therapeutic approach has been tested in animal models using IL-lra (Roessler et al., 1993). In direct approaches the complementary DNA for IL-lra was inserted into an adenoviral vector and the vector construct was injected into rabbit knees (Roessler et al., 1993). A second approach included the insertion of the IL-lra cDNA in a retroviral vector and transfection of isolated rabbit synovial cells in vitrn (Bandara r’t al., 1993). The transfected cells were then placed in the contralateral knee of the same rabbit. Using this approach a suppression of antigen-induced arthritis in rabbits by ex vivo gene therapy was observed (Otani et ul., 1996). However, gene therapeutic approaches have not yet reached the level of clinical studies. Several problems have not yet been solved; for example, the identification of suitable vectors for in vivo gene delivery (E:vans and Robbins, 1995). In a murine model of systemic lupus erythematosus it was recently demonstrated that disease activity, including survival, decreased antichromatide antibodies, rheumatoid factors, and total IgG, could be modulated by cytokine gene delivery (Raz et al., 1995). A second rather new avenue is related to our increasing knowledge of apoptosis and its role in autoimmune processes. Thus, the finding that anti-Fas mAb administered intraarticulary to human T cell leukeinia virus type I tax transgenic mice induced apoptosis associated with a significant improvement of disease-associated arthritis (Fujisawa et al., 1996) has provided the base for the discussion to use this type of treatment principle in immunologically induced disease entities. However, for both new directions of the development of new therapeutics, further experimental and clinical studies are necessary to prove that these approaches will also be of value for clinical use. 11. Cytokines and Anticytokine-Related Treatment Principles in Autoimmune Diseases

A. INTHOIXJCTION

During the past decade antiinflammatory cytokines, cytokineneutralizing agents, receptor antagonists, monoclonal antibodies directed against cytokines, and cytokine receptors have been explored as therapeutic agents for treating autoimmune diseases. Cytolanes involved in the patho-

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el cil.

genesis of autoimmune diseases can be classified according to their function in three main classes: 1. Proinflammatory cytokines as produced in access, including interleukin-1, tumor necrosis factor-a (TNF-a),GM-CSF, and chemokines such as Rantes and MIP. 2. Anti-inflammatory cytokines mainly represented by IL-4 and IL-10, which might be produced in insufficient quantities to suppress inflammatory reactions in certain autoimmune diseases. 3. Molecules that block the effect of proinflainmatory cytokines such as a IL-lra. With regard to treatment approaches based on the inhibition of cytokine synthesis in autoimmune diseases, it was recently demonstrated that two antisense oligonucleotides complementary to the mRNAs of IL-2 and ICAM-1 effectively inhibit the immune responses, both in vivo and in witro (Stepkowski et nl., 1994). In applying antiinflainmatory cytokines or proinflammatory cytokine inhibiting principles, one has to be aware, first, that we do not yet know side effects that might appear during a longer course of treatment. Second, it has to be considered that different cytohnes might have different effects in different autoimmune diseases. Thus, TNF-a plays a central role as a proinflammatory cytokine in the pathogenesis of RA, in contrast to systemic lupus erythematosus in which TNF-a seems to have a protective effect. The same is true for IL-10, which has been shown to exhibit antiinflammatory effects in RA, in contrast to SLE in which IL-10 s e e m to enhance disease activity.

B. ANTI-TNF-~ TREATMENT PRINCIPLES Principles blocking TNF-a have been thus far explored in placebocontrolled, double-blind studies in RA and Crohn’s disease. 1. Anti-TNF-a nzAb Treatment in RA

In RA many proinflammatory cytokines were demonstrated to be present in inflamed joints in high concentrations and to be expressed in high copy numbers in synovial tissue. Among the cytokines, TNF-a and IL-1 attracted special attention because of their proinflammatory properties on a variety of cells known to be involved in the process of tissue destruction in RA. Both cytokines are potent activators of endothelial cells with the promotion of endothelial adhesion molecule expression and subsequent leukocyte transmigration into the adjacent tissue. In addition, both cytokines enhance phagocytic and secretory functions of granulocytes, fibroblast responsed

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with an increased growth, and cytohne production. Possibly the most important property of TNF-a and IL-1 in the pathogenesis of joint inflammation in KA is their capacity to promote cartilage and bone resorption and destruction, both by stimulation of metalloproteinase production from fibroblast and synovial lining cells and through the suppression of synthesis of matrix components by other connective tissue cells (Firestein and Paine, 1992; Shingu et al., 1993). In further support of a pivotal role of TNF-a in the development of synovitis in RA are experiments showing that mice, transgenic for a 3' modified human TNF-a gene, develop a chronic arthritis resembling RA, which can be prevented by the administration of monoclonal antibodies to TNF-a (Keffer et al., 1991). Likewise, in another murine model of RA, the collagen II-induced arthritis, administration of TNF-a mz4b induced a significant improvement of clinical parameters even after the onset of the disease (Williams et aZ., 1992). Based on this experience with an anti-TNF-a blocking therapy in animal models, based on data demonstrating that TNF-a and its two receptors (p55 and p75) are upregulated at many cites within the synovial meinbrane including the cartilage/panus junction (Husby and Williams, 1988; Deleuran et al., 1992), and based on the demonstration of elevated levels of TNF-a and the soluble forms of the two receptors in the synovial fluid of RA patients (Tetta et al., 1990), TNF-a blocking agents were introduced to the treatment of RA. The b1,ockade of TNF-a was tested, using either chimeric mAb against TNF-a or molecules consisting of TNF-a receptors, genetically fused to the Fc part of the human IgG protein. All clinical trials performed thus far with one of these agents have shown that in uiuo blockade of TNF-a leads to a significant improvement of clinical parameters in RA. Applying a chimeric human neutralizing TNF-a mAb cA2 in an open pilot study, 20 patients with moderately active, refractory RA showed clinical response with rapid improvement in each of the standard clinical assessments as well as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) (Elliott et nl., 1993).The duration of clinical response lasted for a median of 12 weeks, with all patients finally demonstrating clinical relapse. Based on this encouraging data a multicenter, placebo-controlled, double-blind clinical trial was initiated. Seventy-three patients with active disease, o'n stable doses of NSAIDs and corticoid steroids (10 mg/day), were treated with a single dose of placebo or 1 or 10 mgkg cA2 (Elliott et al., 1994a). The analysis of clinical parameters revealed a highly significant improvement over placebo in the verum groups, with a longer duration of improvement in the high dose-treated cohort. All cA2-treated patients fulfilled the Paulus criteria for 25% remission 4 weeks after treatment, and a majority of the patients still satisfied Paulus criteria for 50%remission. The onset of clinical response was rapid, in some cases with an impressive

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overnight improvement of clinical measures. After the double-blind phase of the study, at the time of clinical relapse, 1-6 months after initial treatment all patients received a second cA2 infusion at a concentration of 3 or 10 mg/kg. Clinical responses were similar to the improvement seen after the first cA2 application. This was also observed in a small clinical study in which 8 patients of the original open-labeled study received 4 infusions of cA2, however, the intervals of beneficial effect shortened with increasing infusions of the monoclonal antibody against TNF-a, indicating the induction of anti-cA2 antibodies (Elliott et al., 199413).With regard to the possible action of the anti-TNF-a! treatment in RA, Lorenz et al. (1996) were able to demonstrate that serum concentrations of IL-lp, IL-6, and soluble CD 14 were significantly diminished. In addition, a significant decrease in soluble ICAM-1 molecules in the serum of verum-treated patients was observed. This indicates that this type of treatment principle interferes with cell migration. No relevant changes were found in granulocyte or T cell functions. Absolute numbers of T cells increased only slightly and transiently after infusion of the mAb. Similar data were observed by Paleolog et al. (1996) that also demonstrated decreased serum levels of adhesion molecules after TNF-a! treatment. Based on these findings the hypothesis can be put forward that a diminished activation of endothelial cells by anti-TNF-a might interfere with migration of leukocytes into inflamed joints. In further support of this hypothesis is the observation by Tak et al. (1996), who reported a reduced expression of adhesion molecules and a decrease in cellularity of RA synovial tissue after TNF-a! blockade. This observation also supports the hypothesis that antiinflammatory effects of anti-TNF-a therapy might partly be explained by a downregulation of cytokine-induced vascular adhesion molecules and thus interfere with cell trafficking. In this context it is of interest that Edwards et al. (1996) presented evidence that staphylococcal enterotoxin B-an extremely potent macrophage activating factor in vitro and in vivo-enhances several aspects of autoimmune diseases in MLWLPR mice. Applying a transcriptional inhibitor of TNF-a, the authors were able to show that anti-TNFa therapy could be of potential use in the treatment of this arthritis model. Combining methotrexate and repeated applications of the anti-TNF-a mAb cA2, Kavanaugh et al. (1996) reported that this type of combination therapy was especially effective in those RA patients in whom the disease control by methotrexate (MTX) alone was incomplete. The clinical response achieved sustained for more than 12 weeks. However, further studies are necessary to find out what the optimal combination therapy will be in RA as well as in other autoimmune diseases. Interestingly, in the model of collagen II-induced arthritis a synergistic effect of anti-TNFa! mAb of anti-CD4 mAb was reported (Williams et al., 1994).

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34 1

When the effect of anti-TNF-a cA2 mAb therapy in RA was monitored by nuclear magnetic resonance, it could be demonstrated that patients given 10 mg/kg cA2 demonstrated a significant improvement in all clinical manifestations of the disease, associated with a highly significant reduction in gadolinium uptake in the synovium (Kalden-Nemeth et al., 1997). This finding indicates that this type of therapy primarily has an antiinflammatory effect. Further studies are necessary to see if TNF-a blocking agents will also prevent cartilage and bone destruction. In this context it is of interest that in the collagen II-induced arthritis anti-TNF-a treatment primarily prevented synovitis, whereas blocking of IL-1 was shown to be more effective in preventing cartilage destruction ( Joosten et al., 1996). A different TNF-a neutralizing mAb, the engineered human IgG-3 antibody CDP57-1, was also applied in clinical studies using doses of 0.1, 1.0, or 10 mg/kg in 24 patients with active RA (Rankin et ul., 1995). The effectiveness was compared in a double-blind fashion to placebo given to 12 patients. Treatment with this antibody also caused a significant reduction in disease activity markers such as ESR or CRP. Clinical parameters were improved over placebo with statistical significance at Weeks 1 and 2 after treatment . The best effects were seen in patients receiving the high dose of mAb. Adverse effects were reported to be miId.

2. TNF-a Receptor Fusion Proteins for the Treatment of KA Using the soluble TNF receptor p55 construct LENERCEPT, a glycosylated fused protein consisting of two human p55 TNF receptors linked to human IgG-1 Fc, reports on a double-blind, placebo-controlled study are available. One hundred patients with longstanding RA were treated with monthly intravenous injections of placebo or the receptor construct in concentrations of 0.05, 0.2, or 0.5 mg/kg (Sander et al., 1996). Maximal efficacy occurred 2 weeks after the first injection with a significant effect already obvious after 24 hr. Clinical parameters such as the Paulus criteria improved 32-50% with best results seen in the high-dose group. Similar data were reported when 50 or 10 mg of the receptor construct were infused monthly over a period of 3 months (Cutolo et al., 1996). In an extension trial 36 patients were injected monthly with 20 mg/kg for up to 1year. During the observation period 30 patients withdrew from the study, 21 for insufficient efficacy (Hasler et ul., 1996). For the 33 completers an improvement of 30% of the Paulus criteria was obvious. One case of allergic reaction and 17 cases of possibly RA-related musculus skeletal adverse reactions were listed. There were no data on the incidence of anti-dsDNA antibodies. Using a different manufktured LENERCEPT in a doubleblind, placebo-controlled trial, enrolling 118 RA patients with monthly injections of placebo or LENERCEPT concentrations of up to 0.5 mg/kg

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Pt

d

over 3 months, a total of 62 patients dropped out of the study, most of them because of a lack of efficacy. In the remaining responders an improvement of 25-50% of the Paulus criteria was reported (Furst et al., 1996). Applying a fusion protein of the Fc part of the human IgG-1 molecule and the TNF-a p80 receptor, in a double-blind, placebo-controlled study 180 patients with active RA were treated for 3 months with placebo or the fusion protein at concentrations of 0.05,2.0, or 10 mg/m2 body surface area. Injections were given subcutaneously twice a week. Concomitant medication was allowed with NSAIDs or corticosteroids up to 10 mg/day. ACR response with 20% remission was achieved in 14% of placebo-treated and in 32, 46, and 75% of the verum-treated patients, respectively. The most common adverse events reported were reactions at the site of injections and within the upper respiratory tract. Data on the appearance of anti-dsDNA antibodies are still missing. More results on the trials using TNF-a receptor fusion proteins will be available in the near future.

c. TNF-a RECEPTOR FUSIONPROTEINFOR THE TREATMENT OF CKOHN’S DISEASE As in RA, in Crohn’s disease increasing evidence points to an important role for inflammatory cytokines for the pathogenesis. Thus, consistently raised TNF-a, IL-1/3, and IL-6 secretion by normal appearing mucosa from patients with Crohn’s diseases provides evidence for a sustained immune stimulation even in the absence of obvious inflammation (Reimund et al., 1996). Woywodt et al. (1994) demonstrated by in situ hybridization mRNA for interleukin- lp, interleukin-6, and TNF-a in mucosa biopsies from Crohn’s patients. Based on these findings a trial with the monoclonal antibody cA2 was performed in Crohn’s disease. Preliminary data showed a significant decrease in inflammatory activity and clinical symptoms after a single infusion of 2, 5, or 10 mg/kg (Targan et al., 1997). D. INTERFEHON-/3 TREATMENT OF MULTIPLESCLEROSIS In a pilot study undertaken to test the safety and to establish possible side effects of the treatment with recombinant human interferon-plb ( IFN-Plb) in patients with relapsing-remitting multiple sclerosis, five groups of 6 patients were treated by subcutaneous injections three times each week with different units of /3 serum or placebo. Although some side effects were noted in all the groups, a dose-related trend in reduction of exacerbation frequency was noted. Based on this pilot study, a further study using 8 mill. units for the treatment of 15 patients had an observation period of over 6 years. Side effects abated over time. Neutralizing antibod-

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ies developed in most patients, but titers were variable, fluctuated independently of clinical course, and tended to fall with prolonged treatment. This pilot study was able to show responsivness to betaseron, and based on the data obtained, guidelines for dosing regime to evaluate optimizing the efficacy of IFN-Pl treatment in relapsing-remitting multiple sclerosiswere obtained (Knobler et al., 1993).In a subsequent study by Brod et al. (1996), proliferation and cytokine secretion of mononuclear cells after stimulation with anti-CD3 or concanavalin A in subjects with stable or relapsing and remitting multiple sclerosis ( M S) before and after comenceinent of INFP l b treatment was investigated. The most significant finding of this study was a significantlyincreased betaseron-induced secretion of TNF-a, ( IFNy ) , IL-2, IL,-6, and IL-10 and a decreased IL-4 secretion in on-treatment compared to pretreatment in peripheral blood mononuclear cell samples. However, on-treatment anti-CD3-mediated secretion of TNF-a was significantly decreased and IL-6 secretion was increased compared to pretreatment values. The authors conclude from the data that IFN-01 decreases anti-CD3-mediated TNF-a secretion but increases another inflammatory cytokine, IL-6, that could potently counteract its beneficial inimunomodulatory effects. Miller et nl. (1996), investigating the irnmunoregulatory effects of IFN-/3 treatment and interacting cytokines and human vascular endothelial cells (ECs) in MS patients, demonstrated that, using umbilical endothelial cells, the basal expression of ICAM-1 molecules was enhanced by TNF-a and to a lesser extend by IFN-P, with no effect seen by IFN-y. IVIHC class I expression on ECs was enhanced by IFN-P, IFNy and TNF-a Incubation of ECs with IFN-y but not with IFN-j3 induced class I1 expression in a dose-dependent manner. The coincubation of ECs with IFN-13 and IFN-y resulted in a significant downregulation of class 11 molecule expression dependent directly on the IFN-P concentration. Together, these studies demonstrated that IFN-P and interacting cytokines exert an complex iinniunoregulatory effect on endothelial cells including different differential modulatory capacities on various cell surface markers. From these studies, Miller et nl. (1996) concluded that IFN-P might have important implications for cytolane-based strategies in the treatment of inflammatory autoimmune diseases including multiple sclerosis. Finally, when the effect of IFN-P on blood-brain barrier disruptions was investigated by contrast-enhanced magnetic resonance imaging in relapsingremitting multiple sclerosis (Stone et nl., 199S),data were obtained suggesting that IFN-P at least temporarily inhibits the opening of the blood-brain barrier. Further studies, currently being conducted at different centers, are necessary to define the exact place of IFN-j3 in the treatment repertoire for relapsing-remitting multiple sclerosis.

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1. R. KALDEN

et nl

E. IL-1 BLOCKADE The expression and biological action of IL-1 is, as other cytokines, regulated within a complex system of several interacting proteins, such as the IL-1 converting enzyme, the IL-ha, and the two IL-1 receptors type I and type 11. IL-lra binds to IL-1 receptors as a competitive inhibitor of IL-1 binding. Treatment effects of IL-lra in murine models of RA proved to be successful in the collagen II-induced arthritis (Joosten et al., 1996) and with various results in antigen-induced arthritis in rabbits (Otani et al., 1996; Lewthwaite et al., 1994).When a mAb against IL-1 was employed in the collagen II-induced arthritis model similar beneficial effects with an effective control of disease activity were demonstrated (Geiger et al., 1993). Based on the data from the experimental animal studies 175 RA patients were enrolled in a double-blind, placebo-controlled multicenter, clinical trial. Patients received recombinant human IL-1-lra subcutaneously in different time and dose regimens, ranging up to 200 mg daily for 3 weeks, followed by weekly maintenance for 4 weeks (Campion et al., 1996). The most frequent side effects were reactions at the site of injection in 62% of patients. At the end of the 3-week treatment, daily dosing appeared more effective as assessed by the number of swollen joints, the investigator and patient assessments of disease activity, pain score, and CRP levels. In a follow-up study 472 patients with early active RA were subcutaneously treated with either placebo or rhIL-lra at concentrations of 30, 75, or 150 mg/day for 24 weeks. In the 150 mg/day group, a significant improvement of clinical parameters ranging from 20 to 35%was reported (Bresnihan et al., 1996). In addition, preliminary data on the evaluation of X-rays of treated patients are suggestive that a significant slowing in the progression of the disease with regard to cartilage and bone destruction was obvious in the verum-treated group (Watt and Cobby, 1996). Studies on synovium biopsies demonstrated a decrease in the CD3+ cell count by a mean of 55% in patients receiving 100 mg rhIL-Irdday (Cunnane et al., 1996). Given the soluble recombinant human IL-1 receptor antagonist intrarticularly (Drevlow et al., 1993) a dose-related reduction of the circumference was noted 24 hr after administration. Data on therapeutic trials blocking IL-1 in other autoimmune diseases are not yet available. F. INTERLEUKIN-6 MONOCLONAL ANTIBODY IL-6, like TNF-(Yand IL-1, is produced in high concentrations in the synovial fluid and synovial tissue. Serum levels of this proinflammatory cytokine have been shown to correlate with the disease activity. IL-6 has diverse activating effects on a variety of cells. In hepatocytes IL-6 regulates

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the switch in hepatic biosynthetic function to acute phase response with an increased production of proteins such as CRP. In a pilot study, when 5 RA patients were treated with 150 mg of a murine monoclonal anti-IL6 antibody (B-E8) over a 10-day period, CRP and ESR values were shown to decrease during the course of the treatment. The clinical evaluation showed a significant improvement in all patients, lasting for a medium of 2 months as indicated by the Ritchie Articular Index, duration of morning stiffness, and number of tender and swollen joints. The treatment was generally well tolerated with no side effects reported (Wendling et al., 1993). However, a randomized, placebo-controlled trial is necessary to confirm these encouraging results.

G. ANTI-INFLAMMATORY CYTOKINES Of interest are recent in vitro studies on the action of IL-10 and IL-4 on mononuclear cells from synovial fluid and peripheral blood from RA patients stimulated with bacterial antigens. As demonstrated in this experiment, IL- LO reverses the cartilage degradation induced by antigenstimulated mononuclear cells and IL-4 had an additive effect. IL-10 was additionally shown to have a direct stimulatory effect on prostaglandin synthesis and IL-4 as a growth factor for CD4' Th2 cells. These data have led to the discussion of applying both antiinflammatory cytokines alone or in combination as treatment possibilities for rheumatoid arthritis (van Roon et al., 1996). Supporting the possible efficacy of both antiinflammatory cytokines in situations such as RA are data indicating that IL-4 inhibits prostaglandin production by freshly prepared adherent rheumatoid synovial cells via inhibition of the biosynthesis and gene expression of cyclooxygenase I1 but not of cyclooxygenase I (Sugiyama et al., 1996). Currently, clinical studies have been started to test both antiinflammatory cytokines for their clinical efficacy in RA patients. The demonstration that IL-13 is consistently present in rheumatoid synovium, and that exogenous IL-13 has the capability to decrease the production of proinflammatory cytokines by synovial fluid mononuclear cells, suggests that IL-13 might also have a therapeutic potential for treating RA patients (Isomaki et al., 1996). This possibility gains support from observations on the collagen-induced arthritis in mice demonstrating that the systemic administration of IL-13 as well as IL-4, applying vector cells engineered to secrete these proteins, is a powerful method for delivering those cytokines in vivo and attenuating significantly the progression of joint inflammation in terms of both clinical symptoms and histological changes (Bessis et al., 1996).Again, no data are yet available on treating human autoimmune diseases with IL-13.

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J. W. KALDEN of nl.

H. IFN-y When IFN-y was tested as a therapeutic principle in patients with rheumatoid arthritis, a clinical efficacy of subcutaneous IFN-y treatment was initially suggested irl open studies and in a small double-blind study in which two doses of IFN-y, 10 and 100 mg, were compared (Lemmel et al., 1988). Subsequently, four placebo-controlled, double-blind studies were conducted giving no clear-cut data with regard to the clinical benefit of this principle (Lemmel et al., 1991; Cannon et al., 1989; Machold et al., 1992). In a recently published, randomized double-blind study comparing a 24-week treatment with recombinant IFN-y versus placebo in RA patients, IFN-.)I proved to be no more effective than placebo. IFN-y was well tolerated without increased toxicity compared with placebo (Veys et al., 1997). The authors conclude that the logic for the use of IFN in RA must be questioned, with the proviso that the optimal schedule and dosage of this compound are still unknown. Furthermore, when using IFN-.)I as a treatment principle for autoimmune diseases, one has to be aware of the possibility that a disease exacerbation might occur. I. AUTOIMMUNE EFFECTS OF CYTOKINE THERAPY Thyroid autoimmunity and clinical thyroid disease were reported in patients undergoing INF-a therapy with different malignant diseases, including myeloproliferativeand myelodysplastic syndromes or chronic hepatitis, with a frequency ranging from 13 to 50% (Kausman and Isenberg, 1994). Furthermore, the induction and/or exacerbation of inflammatory arthropathy was seen during treatment with IFN-a (Conlon et al., 1990). In addition, medication of both IFN-a and IFN-y has been described to be associated with the development of antinuclear and anti-DNA antibodies in a variety of clinical conditions including hematological malignancies and chronic hepatitis B (Wand1 et al., 1992; Ehrenstein et al., 1993; Fattovich et al., 1992). In general the autoantibodies disappeared after cessation of therapy (Ehrenstein et al., 1993; Fattovich et al., 1992). In addition, a variety of other autoimmune phenomena were noted in case reports or in treatment series of patients receiving IFN-a, such as pernicious anemia, vasculitis syndromes, immuno hemolytic anemia, and thrombocytopenia (Kausmann and Isenberg, 1994). In this context it is also of interest that RA patients who were treated with the cA2 mAb against TNF-a developed up to 7% antinuclear and anti-dsDNA antibodies of IgM and IgG isotypes (Charles et al., 1995).As in IFN-a- and IFN-y-treated patients, the antinuclear antibodies in patients receiving anti-TNF-a mAb disappeared with the cessation of the therapy. It will be of interest to determine if patients who were treated with TNFa receptor constructs also develop antinuclear antibodies.

IMMlINOI,O(:ICAL

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J FUTURE PROSPECTS Without any doubt, especially in rheumatoid arthritis, our knowledge with regard to disease-underlyng mechanisms has allowed the development of new therapeutic avenues, which very effectively interfere with ongoing inAammatory mechanisms. Major (piestions that still have not been answered are (1)How frequently can TNF-a or IL-1 blocking agents be given to patients? (2) What type of combination therapy would be feasible (such as the combination of anti-TNF-a and methotrexate or even a combination of TNF-a and IL-1 blocking principle)? and (3)What could be the long-term effect, including adverse reactions, of patients being on a long-term treatment scala with cytokine inhibitory agents. 111. AntLCD4 mAb in the Treatment of Autoimmune Diseases

At least nine different CD4 mAbs have been investigated in the treatment of autoimInune diseases. Early studies einployed murine antibodies, whereas in recent studies chimerized, primatized, and humanized antibodies were employed. With a few exceptions the data discussed in this section were mainly generated in patients with RA.

A IMMLJNOPIIARMACOLCXICAL ASITCTS 1. Kinetic.s and Distribution of CD4 Monoclonal Antibodies The half-life of unbound CD4 monoclonal antibodies was found to vary considerably depending on the dose and the individual antibody used. After infusion of 20 mg of the murine CD4 mAb MAX 16H5 (Horneff et nl., 1991) the half-life (ti) was found to be 3 and 12 hr after the infusion (mAbs were no longer detectable). The t i of chimerized antibodies was found to be 15 hr (Van der Lubbe et d ,1994). The apparent ti of humanized CD4 mAb is longer and increases with the dose to a mean value of 80 hr at 10 mg/kg (Moreland et al., 1995a). The ti of CD4 mAb seems to be considerably shorter than that of mAb that bind to antigens in the extravascular space. This suggests that CD4 mAb meets continuously with unoccupied antigens in the circulation. Saturation of CD4 binding sites in the circulation occurs with dosages above 10 mg. With higher dosages there was an incremental increase in the duration of CD4 saturation. Specific enrichment of CD4 mAb was demonstrated in organs with a high number of CD4' cells and in arthritic joints (Kinne et al., 1993). Higher dosages also led to more intense coating of lymphocytes in the arthritic joint (Choy et al., 1996). Several studies reported that the percentage of peripheral blood and synovial fluid lymphocytes coated with CD4 mAb correlated with the clinical response seen in patients (Van der Lubbe

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et al., 1994; Choy et al., 1996). This suggests that a careful study of pharmacodynamics is necessary to determine the appropriate dose of mAb for optimal clinical effect. 2. Effect on T Cells The most impressive immediate effect seen during the first trials of CD4 mAb administration is the clearance of CD4' lymphocytes from the circulation. After application of several murine CD4 mAbs, the number of CD4t lymphocytes reached pretreatment levels within 24 hr but others, such as the MAX 16H5 and in particular the chimeric cM-T412, induced a prolonged depletion of CD4" lymphocytes that lasted for several years. To date, the mechanisms responsible for the prolonged depletion of circulating CD4" T cells have not been elucidated. The most obvious explanation is that the depletion is due to trapping of antibody-coated cells in the mononuclear phagocyte system. Evidence for direct complement-mediated lysis was not found (Van der Lubbe et al., 1994). Recent studies with primatized and humanized CD4 mAbs of the IgGl and IgG4 class induced no lymphocytopenia or only transient lymphocytopenia in a limited number of patients. Evidence was provided that CD4 mAb selectively eliminates the resting naive CD4+ population. Cells expressing HLA molecules, IL-2 receptors, CD27, and the CD45RO' subset are relatively spared from depletion following CD4 mAb therapy (Jamali et al., 1992). Observations in RA patients (Van der Lubbe et al., 1997) did not confirm those in mice that CD4 mAb increases IL4 and decreases IFN-y production by mononuclear cells (Field et al., 1992). Binding of the mAb to CD4 may also lead to a downmodulation of CD4 surface molecules. However, such a modulation was only reported on administration of MAX 16H5 (Horneff et al., 1991) and not with other CD4 mAbs investigated. The effect on T cells in synovial tissue was studied during treatment with a depleting chimeric CD4 mAb (Tak et al., 1995). Repeated synovial biopsies revealed a decrease in not only the scores for CD3' and CD4' T cells but also CD8" cells, plasma cells, macrophages, type B synoviocytes, and in the expression of adhesion molecules. This decline in the presence of various inflammatory cells and adhesion molecules after CD4" cell depletion was interpreted as support for the view that CD4' T cells orchestrate the local cellular infiltration.

3. Effect on the Immune System Despite the low number of circulating T-helper cells induced during several studies or the persistent saturation of CD4 binding sites, no overt clinical signs of immune suppression have been observed. No or only transient inhibition was observed of delayed-type hypersensitivityskin reac-

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tions or of lymphocyte proliferation tests (Connolly et al., 1996; Van der Lubbe et al., 1994). The majority of individuals treated repeatedly with murine and chimeric CD4 mAbs develop human anti-mouse antibodies (HAMA). Interestingly, HAMA formation generally did not influence the T cell-depleting effect of additional injections and did not induce allergic reactions (Riethmuller et al., 1992). No HAMA formation was detected in the 3 months following treatment with high dosages of a humanized CD4 mAb.

B TREATMENT OF RHEUMATOID ARTHRITIS 1. Study Design CD4 mA'b treatment was mainly studied in RA patients refractory to conventional therapeutic regimens. The initial trials were of an open uncontrolled design, with the primary aim of assessing safety and objective biological effects. These studies used murine and chimeric mAbs. Recent studies had a placebo-controlled design. To decrease the immunogenicity and lymphocytopenia nondepleting primatized as well as humanized antibodies were applied. Some details of clinical trials of CD4 mAb treatment of RA patients are summarized in Table 11. Interestingly, the doses of CD4 mAb used in RA were generally lower and the treatment durations shorter than those used in animal models. In a rat model of organ allograft tolerance induction, 5 mg antibody per kilogram body weight allows engraftment, whereas a dose of 2 m,qkg or less was ineffective (Shizuru et al., 1990).Furthermore, studies on spontaneously occurring autoimmune disease models showed that short courses of CD4 mAb given over a period of approximately 1 week did not prevent disease progression (Shizuru et al., 1988; Wofsy and Seaman, 1987). The differences in the design of the interventions in the clinical and experimental situation may explain some of the apparent discrepancies in efficacy.

2. Clinical Evaluation a. Side Effects. CD4 mAb administration in dosages between 10 and 700 mg/week was not associated with serious toxicity. Adverse effects at the time of the CD4 mAb treatment included fever, which was sometimes preceded by rigors, headache, and occasional hernodynamic disturbances. Allergic reactions as a result of repeated treatment were reported in only two murine CD4 mAb-treated patients and in none of the chimeric, primatized, or humanized CD4 mAb-treated patients (Horneff et al, 1991; Reiter et al., 1991). Leukocytoclastic vasculitis of the skin was observed in RA patients treated with 2 X 140 mg/week of a primatized CD4 mAb (Levy et al., 1996).

TABLE I1 STUDIESON CD4 MONOCLONALANTIBODY THERAPY IN RHEUMATOID ARTHRITIS Response Total Dosage Reference

Antibody

TYFJe

(In%)

Homeff et al. (1991) Reiter et d.(1991) Goldberg et al. (1991) Wending et al. (1992) Moreland et al. (1993) Van der Lubbe et al. (1993) Choy et al. (1993) Kaine et al. (1995) Moreland et al. (1995) Panayi et al. (1996)

VIT4 MT-151 MAX.16H5 MT-151 BL4 B-F5 cM-T412 cM-T412 cM-T412 IDEC-CE9.1 OKTcdr4a 4 162W94

Studies with an open design 70 mIgG2a mIgG2a 70 mIgGl 105-210 nilgC2a 140 mIgG2a 340-400 mIgGl 100-500 cIgGl 10-700 CIgGl 70-700 cIgGl 500 hIgGl 100-500 0.2-10 mg/kg hIgG4 hIgGl 50-1500

Choy et al. (1992) Moreland et al. (1995) Van der Lubbe et al. (1995) Wendling et al. (1996) Levy et al. (1996)

cM-T412 cM-T412 cM-T412 B-F5 IDEC-CE9.1

Studies with a controlled design cIgGl 0-200 cIgGl 0-150 cIgGl 0-675 0-200 cIgGl mIgGl 0-1120

Herzog et al. (1989)

Clinical

Acute Phase

HAMA 618

3/3” 5/5 719 7/10 6/6 23/25 20/25 19/29 6/12 19/40 8/18 3/6. 515

013 0/5 519 No change 016 Reduced No change No change NA NA NA Reduced

6/10 6/10 46 6/25 2/25 22/29 NA NA 0% NA

-

None None None None Reduced

NA NA 33% NA NA

17/36, 10/13

Note. Data are derived from papers describing the treatment of RA patients with murine (m), humdmurine chimeric (c). and primatized or humanized (h) monoclonal anti-CD4 antibodies. Number of patients responding favorably, acmrding to predefined response criteria from improvment of arthritis activiq, over total nnmber treated. NA, not available.

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One patient died 6 months after infusion of 100 mg CD4 mAb as a result of E’neurwcystis carinii and Staphylococcus aureus pneumonia in association with cardiovascular failure while being treated concomitantly with methotrexate and high-dose prednisolone (Moreland et al., 1993). Despite tlie induction of significant lymphocytopenia in some studies no increase i n the number of infective episodes or lymphomas has been recorded ( Moreland et al., 1996).

b. Therqeutic Eficacy. Direct comparison of the results of the different studies is hampered by differences in study design and definitions used to define a clinical response. Without exception, all open studies applying multiple-dosage regimens of CD4 mAb reported a direct amelioration of arthritis activity lasting for several months. In most studies this was not accompanied by a consistent improvement in laboratory measures of disease activity such as acute phase proteins (Table 11). Placebo-controlled, double-blind studies (Table 11) were initially performed with cM-T412, a chimerized IgGl mAb. In the first study singledose infusions with placebo or 5 , 10, or 50 mg CD4 mAb were gwen in 3 consecutive months in 64 methotrexate-treated RA patients (Moreland et nl., 1995b). In another study 2 patients were randomized to placebo and 7 to single-dose infusions with 50 mg cM-T412 (Choy et al., 1992). In the third study 60 RA patients with less than 1 year disease duration received five daily infusions of placebo or 10, 25, or FjO mg cM-T412. Thirty patients entered a 9-month continuation phase in which single monthly doses of 50 mg of cM-T412 were given (Van der Lubbe et al., 1995). Clinical responses showed no difference between active and placebo-treated groups in these three studies. A correlation between the clinicdl response and the changes in circulating CD4’ cells was not found. In two studies of which the results were presented in abstract form, RA patients received multiple dosages of nondepleting CD4 mAb. During 4 weeks, patients with active RA (n = 136) received twice weekly placebo or 40,80, or 140 mg of IDEC-CE9.1. Seventy-sevenpercent of the patients in the 140-mg cohort, 47 and 42% in the 80- and 40-mg dose cohort, respectively, and 20% in the placebo group met the predefined response criteria (Levy et al., 1996). In the second study, with an open design, 24 RA patients received on 5 consecutive days 10, 30, 100, and 300 mg/day of the CP)4 inAb 4162W94. In contrast to the patients receiving the lower dosages, :U6 and 5/5 in the 100- and 300-mg cohorts fulfilled the response criteria, respectively, beginning as early as Day 7 and lasting in some patients lor more than 3 months (Panayi et al., 1996). The therapeutic efficacy of the most recent studies with relatively non-T cell-depleting

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antibodies applied in high dosages suggests that previous studies did not use the appropriate dosage or duration of treatment. This conclusion is supported by observations of synovial fluid-derived CD4' T cells during treatment with CD4 mAb. The percentage of antibody coating of these T cells gradually increases in the first 5 days of treatment and the coating intensity at that time correlates with the degree of clinical improvement (Choy et al., 1996). DISEASES TREATED WITH CD4 C. OTHERAUTOIMMUNE ANTIBODY THERAPY The notable absence of a long-term immunosuppression and the paucity of immediate side effects encouraged several investigators to test the therapeutic efficacy of CD4 mAb in various conditions. Controlled studies have not yet been reported. One of the earliest treatments with CD4 mAb was studied in patients with multiple sclerosis (Hafler et al., 1988).The clinical effects were unequivocal. Four patients with generalized psoriasis experienced remissions of variable duration upon CD4 mAb treatment (Prinz et al., 1991, Nicolas et al., 1991). Potential efficacy of CD4 mAb treatment was also reported by investigators who treated 12 patients with Crohn's disease with 70-700 mg cM-T412 (Radema et al., 1995). A similar treatment in 14 patients with chronic autoimmune hepatitis resulted in a clinical response for 2 months or longer in 50% of the patients (Kakavand et nl., 1992). Efficacy of CD4 mAb therapy was also reported in individual cases with more rare diseases. Patients with polychondritis (Van der Lubbe et al., 1991), vasculitis (Mathieson et al., 1990), juvenile arthritis (Horneff et al., 1995), and systemic lupus erythematosus were found to respond favorably to CD4 mAb treatment (Waldman, 1989; Prinz et al., 1996). PROSPECTS D. FUTURE The results of a rapidly increasing number of clinical trials investigating the clinical effects of different CD4 mAbs have become available. Following the disappointing results of two controlled studies with the chimeric CD4 mAb cM-T412 in RA, favorable results of two dose-finding studies with high dosages of relatively nondepleting CD4 mAb were recently presented. It is not yet clear which characteristics of the antibody are important to achieve this clinical effect. Variations in the antigen-binding characteristics, the constant regions of the immunoglobulin, as well as variations in the dose and timing of CD4 mAb may be essential to obtain clinical efficacy. Because clinical efficacy can only be judged in relatively large clinical trials, only a limited number of observations will be possible. In order to allow the best choice between mAbs to be used in patients

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future studies should also focus on the relationship between antiarthritic and inirnunologic effects. Such observations will not only support the development of this therapeutic approach but also supply important information about the pathogenesis of the disease. IV. Monoclonal Antibody Treatment against Cell Surface Antigen of T Cells (with the Exception of Anti-CD4)

A. ANTICD5 TREATMENT 1. Treatment Rationale

As is the case with the anti-CD4 reagents, monoclonal antibodies directed against other cell surface antigens of T cells have been primarily employed in patients with rheumatoid arthritis (for an overview, see Table 111). This is particularly true for the anti-CD5 reagent CD51C, which was, developed in parallel with anti-CD4 reagents (reviewed by Cush, 1997). CD5 is the human homolog to the murine LYT-1 surface molecule (Hardy, 1993). In the human situation, CD5 is present on more than 90% of mature T cells and up to 10% of all circulating B cells, which represents a subset of activated memory type B cells potentially important for the production of autoantibodies (Lydyard d al., 1993). On proliferating T cells, CD5 serves as a costimulatory molecule ( Fishwild et al., 1991). The rationale behind using anti-CD5 treatment in patients with rheumatoid arthritis was, therefore, to eliminate the majority of T cells, but it also eliminated a potentially important B cell subset. In order to enhance the depleting capacity of this agent, it was coupled to the ricin A-chain, resulting in an immunoconjugate (Fishwild and Strand, 1994) (Fig. 2). The ricin system was chosen because the poisonous portion of the ricin molecule, the A-chain, is not active extracellularly; however, intracellularly it inactivates ribosomes and thereby kills the respective cells (Fig. 3). In contrast, the B-chain has a cell-binding capacity by using glycoprotein receptors. Therefore, in order to enter the cell the ricin-A molecule always needs an additional molecule that serves as an entering vehicle. PrecIinical work had demonstrated that CD5IC treatment of cells resulted in significant reductions of mitogen and allogeneic-induced T cell proliferation and was subsequently shown to rapidly reduce the number of circulating CD5' T cells. Of special interest, anti-CD5 monoclonal antibodies without the ricin A-chain had been shown to enhance IL-2induced proliferation of circulating peripheral blood T cells; however, to suppress the proliferation of synovial fluid T cells (Fishwild et al., 1991).In animal experiments it had been established that anti-CD5 mAb treatment

CELL

Target Molecule

CD2 CD3

TABLE 111 SURFACE MOLECULES USED AS TARGETS FOR THERAPEUTIC MONOCLONALANTIBODIES IN AUTOIMMUNE DISEASES Biochemistry

CD4

m, gp50 m, gp26(g);in, gp20(d); m, p20(e); hod, p16(z) in, gp60

CD5

m, gp6i

CD7 CD25

in, gp40 in, gp55'

CD52

gp21-28

Main Cellular Reactivity

Disease Multiple sclerosis M yocarditis

Hafler et al. (1988) Gilbert et al. (1988)

T cell subset, monocytes, macrophages, dendritic cells T cells, B cell subset

Several

see Section 111

Rheumatoid arthritis, SLE

T cells Activated T cells, activated B cells, activated monocytes Leukocytes

Rheumatoid arthritis Rheumatoid arthritis

Strand et al. (1993)" Wachholz and Lipsky (1992) Kirkhani et nl. (1988) Kyle et al. (1989)

Rheumatoid arthritis, Vasculitis,' Multiple sclerosis

Isaacs et al. (1992) Mathieson et al. (1990) Moreau et al. (1994)

T cells

T cells

Note. Abbreviations used: m, monomer: hod. lieterodimer: gp, glycoprotein; p. phosphoprotein. Reagent CD5IC. inAb coupled to the riciii-A chain. "part of the IL-ZR complex. ' Used prior to mAb treatment with anti-CD4 mAb.

"

Reference

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Ftc;. 2. A schematic diagram of the regent CDSlC.

FK:.3. Iiinding (a) a n d internalization (b)of ;in iininunotoxin as examplified by the antiCD5 irnmunoconjiigate.

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resulted in a reduction of synovitis and disease severity in a collagen type II-induced arthritis (Plater-Zyberk et al., 1994). It is important to realize that the initial clinical trials using monoclonal antibodies were based on the strategy of maximally reducing circulating T cells and the subsequent data showing that there was no correlation between clinical improvement and T cell depletion were not available. Therefore, the approach using anti-CD5 coupled to ricin made good sense at that time.

2. Clinical Trials Using CD5ZC in Rheumatoid Arthritis The pilot trial using CD5IC included 16 patients who had been in a phase I, open-label, dose-escalating trial for 5-9 days (Byers et al., 1989). It was shown that the depletion of CD5+ T cells was accompanied by a clinical improvement in nearly half of the patients and a quarter showed a continuing clinical response for the subsequent 3 months. This initial trial, showing both efficacy and only minor toxicity, led to subsequent phase I1 open-label trials in rheumatoid arthritis. In these studies 79 patients recieved CD5IC in a dosage of either 0.20 or 0.33 mg/kg/day for 5 subsequent days (Strand et al., 1993). Among the patient population, 25 patients had early disease of less than 2 years duration, whereas the remaining patients included 54 individuals with a long-standing disease of a mean duration of 11 years. Of special interest, 32 patients were continuing DMARD treatment with methotrexate or azathioprine. Clinical responses were determined using the Paulus criteria in which at least a 20%improvement in at least four of six variables was necessary to signify a clinical response. During the first month postinfusion, approximately 55% of patients improved during the first week after treatment; however, subsequently a rapid decline in response rates was noted decreasing from 32 to 15%after 12 months. Approximately one-third of patients showed a 50% reduction in ESR, C-reactive protein, and rheumatoid factors; however, there was no consistent effect. There was a significant lymphopenia and a median depletion of CD4+ T cells to 21% of the initial value between Days 2 and 5 of the infusion period. One month after treatment, CD3+T cell numbers reached more than 50% of pretreatment values in the majority of patients. Circulating CD5+ B cells were also depleted; however, they returned to normal after 1 month. In contrast, neutrophils and monocytes did not change during therapy. All T cell subsets declined without showing a disproportional decrease of individual subpopulations, especially with regard to memory and naive T cells. Interestingly, the clinical results were not influenced by a parallel treatment with MTX or azathioprin.

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In these trials it had been shown that retreatment schedules with CD5IC were possible in 15 patients (Strand et al., 1993). In a single patient retreatment was performed twice and resulted in acute urticaria, pruritus, chest tightness, and increased synovitis. In this individual, there were high levels of antibodies toward CD5IC and resolution of symptoms was reached using intravenous (iv) cortisone treatment. Subsequently, in a multicenter double-blind, multiple-dose, placebocontrolled trial up to 8 mg/m2/day of CD5IC was infused for 4 days in 1 month (Olsen et al., 1996).There was no marked prolonged T cell depletion and treatment was not more effective than placebo. Of special interest, in this study a very high placebo response was noted in 48% of the patients. In general, treatment was well tolerated. The authors concluded that at this dose CD51C was ineffective for treating rheumatoid arthritis. Again, it was noted that there was no correlation between clinical response and C3’ T cell depletion, which was highest in the 8.0 mg/m2/day group with less than 50% of baseline values compared to the other groups.

3. Clinical Trials Using CDSZC in Other Autoimmunities Limited studies have been performed in graft-versus-host disease in patients with systemic lupus erythematosus, type I diabetes mellitus, inflammatoiy bowel disease, and psoriasis; however, the studies were not followed up by subsequent larger trials (Wachholz and Lipsky, 1992; Skyler et al., 1993; Kernan et al., 1988). 4. Adverse Efiects The majority of patients experienced constitutional complaints including fever, fatigue, malaise, nausea, or a “flu-like” syndrome during or immediately after the infusion period (Cush, 1997).In several patients, self-limiting edema, weight gain, and hypoalbuminemia were often observed during the subsequent days. These symptoms suggested a capillary leak syndrome resulting from the administration of the study drug. Rare serious complications of treatment included pulmonary edema, anaphylactic reactions, vasculitis-like phenomena, and even rhabdomyolysis. Overall, there was a dose-related increase of adverse effects, especially common in the group receiving 0.33 mg/kg/day. Interestingly, this toxicitiy could not be correlated to retreatment, the presence of HAMAS, or the accompanying drug regimens. In contrast, patients undergoing a second course of therapy demonstrated less toxicity, suggesting that the presence of human antibodies toward this compound may have reduced the spreading of CD5IC into the human tissue thereby limiting the adverse events. Patients receiving a second or third course of CD5IC developed very high human antimouse titers and antibodies toward the immunoconjugate. Anti-idiotypic

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antibodies, which blocked the action of CD5IC, were also present upon retreatment. However, even high levels of antibodies toward either the murine original antibody or the immunonoconjugate did not influence retreatment. CD5IC has a short half-life of 3.5 hr, with a decline in patients being retreated who exhibited markedly lower peak drug levels apparently caused by elevated HAMA titers. In summary, despite the intriguing approach of combining an anti-T cell reagent with a toxin that not only addresses T lymphocytes but also a subset of B cells, the trials with CD5IC have been disappointing. The initial hypothesis that a maximum depletion of T cells would result in an induction of tolerance was not supported by the subsequent clinical data, which demonstrated that there was no correlation between depletion and clinical success and that no sustained benefits could be reached even in those patients reaching maximum T cell depletion. Therefore, it became rapidly evident that retreatment was necessary. Here, however, the considerable levels of toxicity using higher dosages as well as the high levels of antibodies against the original antibody and the immmunoconjugate would eventually make extended retreatment periods impossible. The final data resulting from the double-blind placebo-controlled trial were particularly disappointing because no clinical benefits could be demonstrated in the light of a very high background of placebo response. Overall, the interesting concept of targeting autoimmunity-inducing cells with an immunoconjugate has suffered a severe blow because depletion or modulation of target cells has been shown to be readily possible using pure humanized reagents that do not have the problem of potential toxicity and immunogenicity of the fusion compound. Therefore, the experience with CD5IC raises severe doubts on the success of future developments of immunoconjugates for therapy of autoimmune diseases.

B. ANTI-CD7 TREATMENT Small cohorts of patients have been treated using the murine anti-CD7 mAb R F T2 or the chimeric version of the latter reagent (Kirkham et al., 1991, 1992). Using the murine antibody, daily treatment for 15 days led to disease improvement in only two of six patients and lasted for a very short period of 7-14 days despite a decrease of T cell numbers and an absent expression of the CD7 antigen on the patient’s T cells. A subsequent trial using the chimeric version of CD7 mAb demonstrated modest improvements in disease activity; however, it showed frequent adverse effects of fever, nausea, and malaise. Again, T cell numbers fell by 50% and CD7 expression was virtually absent from circulating T cells. Due to the lack of a significant clinical efficacy and to pronounced adverse effects of the

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compound. there was no further development of anti-CD7 treatment in patients with autoimmune diseases.

c

INTEHLEUKIN-2 RECEPTORTARGETED APPROACHESANTI-CD25 TREATMENT A N D TIIERAPY USINGTHE IL-2 FUSION TOXIN

1. Anti-CD25 Treatment In contr,ist to the wide availibility of many surface molecules targeted by the monoclonal antibodies mentioned so Far, the IL-2 receptor (IL2R) is present only on activated T cells, B cells, and certain monocytes, all of which appear to be particularly important in maintaining autoimmunity. The >(electivetargeting of this receptor would, therefore, spare the other cell populations potentially resulting in a lesser degree of nonspecific immunosuppression. Because IL-2 receptor (CD25) expression has been shown on both peripheral blood and synovial T cells in patients with rheumatoid arthritis (Burmester et al., 1984) and IL-2 treatment in vitro resulted in a significant T cell proliferation (Hain et al., 1990), the IL-2 receptor appears to be an important target in rheumatoid arthritis. Therefore, CAMPATH 6, a rat monoclonal antibody to the IL1-2R, was investigated in three patients with active rheumatoid arthritis (Kyle et al., 1989). In this study, two patients were described as having an excellent clinical response with long-lasting significant changes in pain score, morning stiffness, and ithe Ritchie articular index. In another individual, an initial response was noted; however, a relapse occurred after 1 month. Adverse effects included fever in two patients and an elevation of CRP levels. Despite these interesting results, the data were not followed up in subsequent larger trials. 2. IL-2 Fusion Toxin in Autoimrnune Diseases Another approach to target the IL-2R was generating fusion toxin products coupling the toxic region of the diphtheria toxin to human IL-2, thus creating a recombinant protein-killing activated IL-2R bearing lymphocytes already at very low concentrations (reviewed by Thasia et al., 1997; Williams et al., 1987).Two fusion proteins were subsequently used, termed DAB4861L-2 and subsequently DABTH91L-2 (which is lo-fold more potent). Compared to monoclonal antibodies, these agents had considerable less molecular weights of 67 or 58 kDa, respectively. In contrast to CD5IC, which is slowly internalized, the IL-2 fusion toxin rapidly enters the cell using an endocytic vesicle as soon as it is bound to its receptor. Cytotoxicity occurs within 5-15 min, resulting in a subsequent cell death after approximately 1-.3 days. Initial safety and pharmacogenetic data had been obtained in patients with hematologic malignancies, and these data served as a basis for the treatment regimens in patients with RA and recent-onset insulin-

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dependent diabetes mellitus (IDDM). To date, most of the available data have been published only in abstract form or in a recent review (Thasia et al., 1997). Larger clinical trials were performed in patients with rheumatoid arthritis using compound DAB4861L-2. An open-label, uncontrolled, dose-escalation study was performed in 19 patients and subsequently a double-blind, placebo-controlled, one-dose level trial was performed in 45 patients (Thasia et al., 1997; Moreland et al., 1995~).Using compound DAB3891L2 a double-blind, placebo-controlled, three-dose level and an open, uncontrolled, two-dose level trial were undertaken in 55 and 20 patients, respectively, Patients with RA of long durations (10-20 years) were investigated. All patients had received various DMARD regimens and were considered as refractory individuals who had failed conventional treatment schedules. They were of high activity with approximately 20-30 tender joints. One to five treatment courses were performed, in general in 4-week intervals. The criteria for responses was an improvement of more than 25% in a modified Paulus index. Although these compounds were in general reasonably well tolerated, adverse effects included transiently elevated liver enzymes as well as constitutional symptoms with chills, fever, nausea, and vomiting. The maximum tolerated dose appeared to be 0.1 mg/kg/day. No general immunosuppressive effects were seen with infections occurring equally in both the placebo and the active patient group. Lymphocyte depletion was not observed and the number of IL-2R-expressing CD4+ T cells did not consistently change suggesting that the evaluable peripheral blood lymphocytes do not directly reflect the changes in activity in individual joints. Comparing all available information, it appears that a clinical response occurred in a minority of patients, and striking clinical benefits were rarely noted. In a trial using patients with active MTX-refractory RA, a doseranging evaluation of DABSRgIL-2did not show a significant difference in the number of responders including a placebo group. Even though not all information is available and several trials have not been fully evaluated, the results published so far are not encouraging, at least in patients with long-standing and treatment refractory disease. Therefore, the investigators conclude that early patients should be included into the assessment of the efficacy of the IL-2 fusion toxin.

3. Studies in Recent-Onset IDDM Based on the safety data and clinical benefits observed in at least a subgroup of RA patients the diphtheria fusion toxin was studied in patients with recent-onset IDDM using an induction phase with DAB,,IL-2 administering 0.025-0.07 mg/kg/day as a l-hr daily infusion for 4 or 7

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days (Thasia et al., 1997). Subsequently, patients entered a maintenance protocol in which 5 rng/kg/day of cyclosporine A (CsA) was administered. Response was assessed by decreased insulin requirements, and patients were followed up monthly for the duration of response. In total, 43 patients, including 20 females and 23 males, were included with a mean age of 25 years. The compound was well tolerated at all doses with occasional constitutional syrnptoins or slight elevations of liver enzymes. Also, the CsA administration did not cause major side effects. Seventy-one percent of patients experienced a partial remission with 41% entering complete remission during the study. After 1 year 34%still had a reduced insulin requirement. These dati3 suggest that a combined inductioidmaintanance strategy using IL-2 fusion toxin and CsA is well tolerated in type I diabetes and leads to an enhanced metabolic control. Therefore, a placebo controlled trial was initiated.

4. DAB3xg IL-2 in Psorinsi,s Ten patients with recalcitrant psoriasis were evaluated using DAB3hyIL2 at two dose levels (reviewed by Thasia et nl., 1997). These treatment courses consisted of daily infusions of the compound for 5 days with followup studies during the subsequent 3 weeks, resulting in a 28-day observation period. Skin lesions were followed clinically and by histological assessment of biopsy specimens taken prior to treatment, during, and at the end of each treatment cycle. In general, treatment was well tolerated. Histologically, 8 of 10 patients showed a decrease in acanthotic epidermis in psoriatic lesions following DABjx,IL-2treatment. CD8’ lymphocytes were also reduced in the same patients. In these individuals, a complete resolution of psoriatic epidermal pathology was found. These pathological changes were accompanied by clinical improvements, and 4 patients had an almost complete clearing of the psoriasis. Therefore, a new study was initiated that included patients to be treated with higher dose levels. In summary, the concept of fusing a toxin to an immunologically important reagent is of great interest. However, as in the situation of CDSIC, rather high levels of antibodies toward the whole compound and the diphtheria toxin developed. Because most studies using biological agents suggest that a continuing treatment will be necessary over a period of possibly several years, eventually these compounds will have the problem of high iinmunogenicity. This would very likely preclude a prolonged treatment. However, they may be useful in short periods of flare up or in diseases in which a “single shot” may be sufficient to suppress disease activity for a long period.

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D A N T I - C DTREATMENT ~~~ 1. Treatment Rationale

The CD52 molecule appears to be an interesting target to modulate the immune system because this surface molecule is present on all human B and T cells at a high density of approximately 400,000 molecules per cell and at a lower density on human monocytes/macrophages. In contrast, it is not expressed by other myeloid cell types or CD34+ pluripotent stein cells (Hale et al., 1990). Apart from lymphoid cells, only mature, but not testicular spermatocoa bear this molecule (Hirsch et al., 1989). Its function is currently unknown. The reagent designed to target the CD52 molecule is CAMPATH-lH, which has a human IgCl constant region and is efficient at coinplement- and cell-mediated lysis of human lymphocytes. It was derived by humanization of the rodent antibody CAMPATH-1G. Subsequently, CAMPATH-1H was used to treat patients with rheumatoid arthritis, systemic vasculitis, and MS (reviewed by Watts and Isaacs, 1997;Yocuin and Johnston, 1997). CAMPATH-1H can be administered by either intravenous or subcutaneous routes and leads to a rapid fall in peripheral blood lymphocyte (PBL) count in all patients after a single intravenous infusion within a few hours. This suppression of PBL count lasts for at least 20 months following a single infusion and for at least 32 months after multiple infusions (Brett et al., 1996). Of special interest, CD8' T cells return more quickly to peripheral circulation than CD4+ T cells, whereas peripheral blood B cells approach baseline values by 60 days after treatment. Whereas NK cells are not affected by this reagent, monocyte numbers are transiently reduced after treatment but return to baseline values within a few weeks. The same high degree of lymphocyte depletion can be reached by subcutaneous adminstration of the reagent.

2. Clinical Trials in Rheumatoid Arthritis The initial trial using CAMPATH-1H in rheumatoid arthritis included patients who received a total dose of 60 mg intravenously over 10 days (Issacs et al., 1992). In seven of eight patients there was a significant clinical improvement with a maximal response of a 53% reduction of the Ritchie articular index and 71% of the joint score. An increased dose did not result in a greater clinical response; however, it was associated with a greater frequency of adverse effects. As had been seen with the other antiT cell reagents, there was no correlation between PBL counts and clinical response. In contrast to the profound lyinphopenia, C-reactive protein levels fell modestly and ESR did not change at all. Subsequently, several multicenter trials were performed as uncontrolled, phase I studies (reviewed by Johnston and Speer, 1994).In general, patients

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with severe RA of long duration refractory to several treatment regimens were investigated. In a multicenter trial including 41 patients receiving total dosages of 100, 2.50, or 400 mg of CAMPATH-1H over 5 or 10 days, significant improvements in the number of painful and swollen joints were seen after 3 months of treatment (Isaacs et al , 1993). Nearly 50% of patients reached a 50% Paulus criteria response at 1 month, which fell to a 20% response rate within 6 months. It appeared that higher doses resulted in a longer response. Similar results were obtained in a trial using singledose subcutaneous CAMPATH-1H (30 mg) ( Johnston et d., 1992).Parallel investigations of synovial tissue specimens demonstrated that a significant lymphocyte infiltration of the synovial tissue remained despite extremely low levels in peripheral blood showing that this reagent apparently did not reach tissue sites sufficiently (Ruderman et al., 1995). Simultaneously, clinical trials were perfomed in North America, the Netherlands, and the United Kingdom in a collaborative study. A total of 40 patient:; were enrolled in a single-dose, dose-escalating iv study (Weinblatt et al., 1995). Investigating 33 evaluable patients, the overall response rate according to the modified Paulus criteria was 61%. These responses were achieved within 3 days postinfusion, hoewever, the duration of the response lasted a median of only 2 weeks. There was a rapid fall in absolute lymphocyte counts to as low as 8% of the initial values. In a subsequent U.S. study 30 patients were treated subcutaneously for 10 days (Matteson et aZ., 1995). Again, there was a dramatic fall in absolute lymphocyte counts to less than 5% of the predose level with only a slow recovery. Parallel in vitru studies demonstrated a decline of lymphocyte responses to both initogens and recall antigens. Clinical improvement was noted in 56% of patients; liowever, it lasted a median of only 1 month. Reconstitution of the CD4' cell coinpartrnent was lowest with a preferential decrease of the naive T cell subset (Jendro et al., 1995). Finally, 41 patients were treated using repetitive dosing with 100400 mg of CAMPATH-1H over 5 or 10 days resulting in a nearly absolute lymphopenia (Isaacs et d.,1996). Eighty-six percent of patients demonstrated Significant clinical improvements, and 20% of patients maintained a 50% Paulus response at 6 months. Patients responding best were in the higher dose cohorts. Approximately one-third of patients developed antiglobulins to CAMPATH-lH, which did not correlate to clinical response. Of all itlonoclonal antibodies used, CAMPATH-1H appeared to have the most significant side effect profile. This included severe headache, fever, chills, nausea, and vomiting in the majority of patients and occurred shortly after the infusion resolving within 24 hr. In some patients, hypotension was noted. These data suggest that a severe cytokine release syndrome

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occurs, apparently caused by a complement-mediated lysis of the majority of circulating lymphocytes. In addition, an increased incident of infection predominantly of the herpes virus group was seen in patients with early lymphopenia and two infection-related deaths occurred in the CAMPATH1H trials. Therefore, the investigators concluded that CAMPATH-1H may have a place in a combination therapy with other biological agents, particularly using lower, repetitive doses.

3. Systemic Vasculitis and Multiple Sclerosis CAMPATH-1H was used to treat patients with systemicvasculitis refractory to conventional treatment with steroids and cytotoxic drugs. The patient population included two patients with microscopicpolyarteritis, one patient with Behcet’s syndrome, and one patient with Sjogren’s syndrome (Lockwood et al., 1993). A total dose of 40 mg was administered. Three patients also received an anti-CD4 monoclonal antibody. The duration of response was up to 54 months with a minimum of 3 months. This response was rapid (within 72 hr). In addition, four patients with Wegener’s granulomatosis and two patients with microscopic polyarteritis were treated (Lockwood, 1994). In four of these a combination therapy was performed with anti-CD4. In five of six patients a good clinical response was noted with remissions lasting up to 6 months. Nevertheless, two patients remained on dialysis, and one patient died at Day 7 due to a cerebrovascular event. Improvement was associated with a fall in ANCA titers and a resolution of radiological and skin signs. Therefore, CAMPATH-1H may be an interesting agent in treatment refractory vasculitis. In an open-label trial seven patients with multiple sclerosis were treated and assessed using repeated magnetic resonance imaging of the central nervous system (Moreau et al., 1994).CAMPATH-1H (120 mg) was administered intravenously over 10 days. There was a decrease in the appearance of new lesions; however, CAMPATH-1H was not detectable in the cerebrospinal fluid, again suggesting that this agent has a poor penetration into tissue sites. 4. Summary of Anti-CD52 Treatment and Future Prospects

Currently, most investigators believe that CAMPATH-lH, although an interesting agent with a potent biological action, will not be a preferential tool in treating autoimmune diseases. This is because of severe and sometimes serious adverse effects, including the pronounced cytokine release syndrome, harmful infections, and the long-lasting lymphopenia which takes long periods to resolve. Apparently, these disadvantages are not overcome by the only limited and short-lasting clinical response rate noted

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so far. It appears doubtful that some of the discussed agents will reach routine clinical application. V. lmmunologicol Treatment Principles in Animal Models of Autoimmune Disease

A. INTRODUCTION Animal models of autoimmunity are very useful to study diverse immunopathogenetic processes in well-defined experimental conditions and might help to develop rational immunotherapeutic strategies.

B. IMMUNOTHERAPY OF ANIMAL MODELSFOR AUTOIMMUNE DISEASES Biological therapies are based on attempts to modulate adverse immune responses by the use of regulatory molecules produced by cells of the immune system as well as of derivative and recombinant forms of such molecules. Under this operational definition Fall mAbs, soluble forms of cell surface receptors, cytokines and their naturally occurring antagonists, antigenic peptides, and lymphocyte or DNA vaccines. Because T cells play a pivotal role in autoimmune disesases, a variety of biological treatments are being devised that target different aspects of T cell activation. These strategies are aimed at removing or tolerizing activated or autoantigenspecific T cells, at preventing recruitment and traffic of activated T cells and monocytes, or at neutralizing the effects of proinflammatory cytokines. An overview of sequential aspects in the development of experimental models of autoimmune diseases as a basis for the development of new therapeutics principles is given in Fig. 4. In more detail than shown in Fig. 1, Fig. 5 shows the effector arm of autoimmune responses as potential target cells. Possible therapeutic interventions can interfere with the development of immune responses on different levels, including the priming of specific immune cells, migration processes, recruitment facilities within the inflamed tissue, and by alternating the function of the effector mechanism leading to tissue destruction. OF ADHESIONMOLECULES C. BLOCKING 1. Experimental Allergic Encephalomyelitis Because lymphocyte extravasation is a most crucial event at a tight site such as the blood-brain barrier, experimental allergic encephalomyelitis (EAE) seems an appropriate model for the study of therapeutic interference with leukocyte adhesion to the endothelium. Studies on the use of anti-CD11a (LFA-1)antibodies in this model provide contradictory results, varying from complete prevention or suppression of the established disease to inefficacy or even clinical aggravation (Gordon et al., 1995; Welsh et

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FIG.4. An overview of sequential aspects in the development of experimental models of autoimmune diseases. Emphasis is given to the time overlap of the different aspects and the way they relate to acute or chronic phases of the disorders (middle); individual features of single models, as well as the progressive features of the shift from one phase into another, are neglected for clarity of exposition. Following immunization, the afferent limb of the immune system (i.e., APC)is activated in iinmunopathogenetically relevant organs. Antigenspecific cells with autoaggressive potential, but also disease-controlling cells, are generated in this preclinical phase, which represents the time window for so-called preventive immunotherapies. At the outbreak of the clinical signs, the inflammatory aspects prevail in the primary orgardtissues of pathology, while the generation of disease-promoting and diseasecontrolling cells continues. If suppressor mechanisms are sufficient, the acute flare will fade in a self-limiting fashion after its peak; this is usually accompanied by development of natural protection. The predominance of disease-promoting factors and/or the insufficiency of suppressor mechanisms leads instead to chronicity (either taking a relapsing or a continuously progressive course) in which the autoreactive response amplifies to a number of tissue autoantigens (spreading). The effector phase of the immune response begins, typically leading to damage/destruction of the tissue, with susequent loss of orgadtissue function. This established phase of disease is the obvious domain of curative protocols.

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al., 1993). The preparation of different antibodies, with regard to their purity, epitope specificity, and affinity, may certainly play a role in these cases (Gordon et al., 1995). In EAE: adoptively induced by MBP1-11 peptide-specific CD4+ T cell lines, treatment with anti-CDllb (MAC-1) antibodies does not block the disease (as does anti-LFA-1), although it delays the onset of the disease and diminishes its severity when performed at the outbreak of the first clinical signs (Gordon et al., 1995).Treatments targeting the countereceptor (ICAM-1) of LFA-1 and MAC-1 do not provide a uniform picture either, although EAE induced by direct immunization appears more sensitive than adoptively transferred disease (Archelos et nl., 1993; Willenborg et al., 1993). Because the adoptive transfer models are particularly suitable to draw conclusions on the therapeutic efficacy of blocking transendothelial migration, these studies indicate that, rather than exerting the expected influence on leukocyte immigration, anti-ICAM-1 treatment in EAE seems to interfere with T cell activation, for example, through blockade of ligation with LFA-1 on the antigen presenting cells (Fig. 1) (Archelos et al., 1993). On the other hand, antibody treatment targeting a 4 P l (VLA-4), which binds to fibronectin and to VCAM-1 on endothelial cells, not only effectively blocks the accumulation of leukocytes in the CNS but also prevents the development of adoptively transferred EAE (Yednock et al., 1992). Future studies in EAE may benefit from the knowledge that adhesion molecules, such as ICAM-1, VCAM-1, and the vascular mucin MAdCAM1, are upregulated on epithelial cells of the choroid plexus but not on the endotheli a of fenestrated choroid plexus; this finding indicates that, at the blood-cerobrospinal fluid barrier, epithelial cells may play an important role for the immunosurveillance of the CNS (Steffen et al., 1996). 2. IDDrM In ID DM, anti-LFA-1 and anti-ICAM-1 treatments exert beneficial effects both in the spontaneous NOD mouse model and in the adoptively transferred insulitis, with highest efficacy warranted by a combined approach (Hasegawa et al., 1994). Also, a combined treatment of NOD mice with anti-LFA-1 and anti-ICAM-1 at a critical period of IDDM development (from 6 days to 2 weeks of age) induces long-lastingtolerance, i.e., the animals become resistant to subsequent attempts to adoptively transfer insulitis (Moriyama et al., 1996). The dramatic effects of the transient blockade of the LFA-l/ICAM-1 pathway most likely relate to its influence on the costimulation during T cell activation. Anti-L,-selectin and anti-VLA-4 treatment inhibits the development of insulitis and prevents IDDM in NOD mice by interfering with homing of

FIG.5. The effector arm of the autoimmune response as potential target of therapeutic intervention. This schematic overviewbased on experimental models of autoimmunity shows how antigen-driven responses generated by peripheral immunization (A) can lead, via priming and migration of antigen-specific T cells (B), to final destruction in target tissues of autoaggression(C).To emphasize how disease-promoting processes can be therapeutically modulated along this path, the effector mechanisms are simplified to a Thl-polarized T cell response, although it must be considered that regulatory mechanisms (whether Th2 polarized or in different constellation) may coexist. The trimolecular complex, consisting of the MHC-11, the antigenic peptide(s), and the T cell receptor complex (TCR), the costimulation cascade, as well as the IL-%dependent pathway of clonal expansion, can be targeted to suppress tissue-specific autoimmunity in the lymphoid system (A) as well as in

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lymphocyte to the islets, as well as with their trafficking to the lymph nodes (Yang et a/., 1993), excluding in this particular case a parallel influence on the activation of autoreactive T cells. The importance of the interaction of VLA-@CAM-1 for homing and adhesion of disease-relevant T cells is supported by the findings that blocking either one of the molecules reduces the degree of lymphocyte infiltration of the islets, diminishes the incidence of the disease, and delays the onset of IDDM (Baron et al., 1994). The latter study also provides evidence that the transendothelial transit of CD4' T cells into the islets is preferential in comparision with that of CD8' T cells.

3. Collagen-induced Arthritis The outcomes of different treatment trials in collagen-induced arthritis (CIA) using antibodies directed to LFA-1 and ICAM-I are at least as contradictory as those in EAE and IDDM, probably reflecting the complexity of the pharmacology of antibody therapy and its differential influence on various parameters of the model. For example, although a study reports prevention of CIA, most likely due to a pronounced inhibitory effect on cell-mediated but not humoral immunity (Kakimoto et nl., 1992), another study reports no therapeutic benefits of the respective antibodies (Zeidler et nl., 1995). On the other hand, ICAM-l-deficient mice show reduced susceptibility to CIA (Bullard et al., 1996), encouraging the further pursuit of therapeutic modulation of ICAM-1. In homozygous ICAM-1 mutant mice, the reduced incidence of CIA is not associated with lack of immunity to collagen 11, supporting the theory that impaired homing of otherwise activated lymphocytes, rather than activation processes themselves, is influenced by the genetic defect. Anti-CD18 treatment, which targets ICAM-1, MAC-1, and p150/95 (CD1lc/CD18) on leukocytes, significantly reduces joint swelling,

the tissue (C). After leaving the peripheral lymphatics and entering into the blood stream, the immigration of primed lymphocytes into the tissue can be hindered by disturbance of their adhesion mechanisms to the tissue endothelium (8).Because the recruitment of granulocytes (PMN) and inonocytes (Mono) into the inflamed tissue, as well as their inaturatiodactivation as effector cells, requires the concerted action of cytokines [e.g., tumor necrosis factors (TNF-a, TNF-P)], interferons ( IFN-.)I), interleukines (IL-1, IL-12), and chemoldnea [macrophage inflammatory protein-la and -2 (MIP-la and MIP-2), the latter a rodent homolog of IL-81, the cytokine network offers various possibilities for therapeutic interactions. The effector phase of tissue destruction (C),in turn, is mediated by proteolytic enzymes, nitric oxide (NO), and oxygen radicals liberated by resident macrophages ( M 4 ) and PMN, which can be directly enhanced by T N F - a and/or IL-ID. Thus, inhibition of proteolytic enzymes or NO synthetase, possibly accompanied by TNF-a and IL-lP neutralization, promises to halt the terminal effects of the whole pathogenic cascade.

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Inhibition

FIG.6. TI11 and Th2 antagonism in the pathogenesis of organ-specific autoimmune diseases: the example of insulin-dependent diabetes mellitus (IDDM). Activated macrophages (MQ)or dendritic cells can secrete different sets of cytokines with opposing effects (IL-12 vs IL-10). The predominance of IL-12 results in preferential induction of Thl cells (A). The Thl cytokine, IFN-y, stimulates effector macrophages to release reactive oxygen species, nitric oxide (NO), and cytokines potentially toxic for pancreatic P cells, such as TNF-a and IL-lP. I n addition, IFN-y and IL-2 activate CD8+ T cells, which not only exert a direct cytotoxic effect on the islet cells, but also secrete TNF-P and additional IFN-y. The cytokines produced by macrophages and CD8+ T cells damage the /3 cells directly or via induction of NO synthetase. Destruction of P cells results in release of autoantigenic material, such as glutamate decarboxylase (GAD) or insulin, which can further fuel autoaggression and islet destruction. Conversely, IL-10, alone or in concert with IL-4, can divert the T cell response into a Th2 pathway (B),on the one hand by downregulating B7-mediated costimulation and/or inhibiting IL-12 production, and on the other hand by cross-inhibiting Thl development as well as macrophage and CD8+ T cell activation. The end result of activation of this Th2 pathway, interestingly, seems to be a form of insulitis characterized by mononuclear infiltration, but with no signs of islet destruction (modified from Trembleau et al., 1995)

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leukocyte infiltration, and joint destruction not only in CIA but also in proteoglycan-induced arthritis in BALB/c mice (Mikecz et al., 1995). AntiMAC-1 therapy successfully prevents CIA and also protects against adoptive transfkr of the disease into SCID mice; notably, a delay of antibody administration until 10 days after transfer of spleen cells fails to exert any beneficial effects (Taylor et al., 1996). Treatment of mice with anti-VLA-4 antibodies (Zeidler et al., 1995)or anti-CD44 efficiently reduces incidence, severity, and tissue destruction in CIA (Verdrengh et al., 1995; Mikecz et al., 1995; Zeidler et al., 1995) and proteoglycan-induced arthritis (Mikecz et al., 1995). Anti-L-selectin antibodies, in contrast, do not appear to influence incidence or severity of CIA (Zeidler et al., 199s). 4 . Other Experimental Animal Models

Treatments with anti-ICAM-1 and anti-LFA-1, on the one hand, or with anti-a4 integrin, on the other hand, have been successfully applied to other models of organ-specific autoimmune diseases, including experimental autoimmune uveitis (EAU)(Uchioet al., 1994) and experimental autoimmune thyroiditis (EAT), respectively (Metcalfe et al., 1993; McMurray et al., 1996).According to the pathogenesis of organ-specific models, early intervention with antiadhesion therapy is mandatory if one wants to block the shift into chronicity. Therefore, homing to the inflammatory foci is more likely to be affected by anti-a4 integrins than by anti-LFA-1 and/or antiICAM-1 therapy, which in some of the studies exerts suppressive effects mainly via interference with T cell activation. In general, potential adverse effects of antileukocyte adhesion therapy should also be considered. Anti-ICAM-1 therapy, for example, may well affect host defense because homozygous ICAM-l-deficient mice, or antiICAM-l-treated animals, exhibit high mortality rates in the staphylococcal arthritis model despite their resistance to joint inflammation; these findings indicate that ICAM-1 may exert a crucial physiological role in host defense against bacteremia (Verdrengh et al., 1996).Although most of the innnunomodulatoiy therapeutic strategies imply the same risk of weakening the host defense mechanisms, in the case of anti-ICAM-1 this risk has been clearly and appropriately evaluated. For most of the other protocols comparable studies are simply missing. D

CYTOKINE NETWORK 1. Experirnental Encephalomyelitis

MODULATION OF THE

Because Thl-polarized CD4' T cells seem to play a pivotal role in experimental allergic encephalomyelitis (EAE) (Steinman, 1996), there have been several attempts to selectively neutralize the effects of IL-2, IFN-y, 0 1 IL-12 in this model. Anti-IL-2 treatment drastically suppresses

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EAE induced by adoptive transfer of MBP-specific T cells; however, it yields only marginal effects in actively induced EAE (Duong et al., 1992). Indeed, if administered simultaneously to the active immunization or the passive transfer, anti-IFN-y antibodies even lead to significant exacerbation of both actively induced and adoptively transferred disease (Duong et al., 1992). Adverse effects of anti-IFN-y therapy during the afferent limb of the immune response to MBP have been confirmed since 1993 (Lublin et al., 1993), suggesting that blocking IFN-y-mediated pathways may interfere with the selection of regulatory suppressor mechanisms. Also, mice with a disrupted IFN-y gene remain susceptible to EAE, showing quite typical infiltrations of the CNS by macrophages, granulocytes, and lymphocytes (Ferber et al., 1996). Furthermore, in BALBlc disruption of the IFN-y gene confers susceptibility to EAE to a mouse strain otherwise resistant to the disease (Krakowski and Owens, 1996). Thus, although Thl mechanisms may be crucial to the development of EAE, IFN--y may not represent the main mediator of this pathway. IL-12, a regulator of IFN known to promote Thl development, enhances disease severity in a EAE model of adoptive transfer with PLP-sensitized T cells (Leonard et al., 1995). Preventive treatment with antibodies that neutralize IL-12 reduces the incidence, severity, and number of relapses of EAE (Leonard et al., 1995), supporting a clear role for this cytokine in the pathogenesis of the disease. This notion holds also true for interphoto receptor-binding protein (1RBP)-induced EAU, which has very recently been successfully treated with anti-IL-12 (Yokoi et al., 1997). Of the Th2 cytokines supposed to exert regulatory functions, IL-10 does not seem to have a univocal role in EAE. Initially, in fact, it was reported that treatment with IL-10 protects Lewis rats from developing EAE, an effect associated with marked reduction of lymphocyte infiltration of the CNS, sustained T cell proliferation in response to MBP, and elevation of anti-MBP antibodies; recent studies, however, report no therapeutic benefits in murine EAE induced by adoptive transfer of encephalitogenic T cells (Cannella et al., 1996).In this study, IL-10 alone proves insufficient to reverse the encephalitogenic effector response of T cells; however, neutralization of IL-10 clearly aggravates EAE when applied immediately before the onset of the clinical signs (Cannella et al., 1996). Treatment with the Th2 cytokine IL-4 also exerts beneficial effects in adoptively transferred EAE (Racke et al., 1994). Thus, priming of MBPspecific T cells in the presence of IL-4 in vitro, prior to the transfer to SJL mice, results in complete loss of the encephalitogenic potential. Administration of IL-4 after T cell transfer also leads to clinical amelioration; this is accompanied by induction of MBP-specific Th2 cells and, in the tissue, by reduction of demyelination foci; TNF-a and IL-2 expression

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in the CNS is also reduced, most likely due to the IL-4-induced generation of Th2 populations. Although a delayed initiation of therapy (Days 6-11) is less effective than a very early protocol (Days 0-ll), the study nonetheless demonstrates that induction of Th2 populations can ameliorate disease even when pathogenic T h l cells are in full activity. Because during the chronic course of autoimmune diseases T cells exported from the thymus are not yet committed to a T h l or Th2 path, immune deviation determined by the manipulation of the cytokine environment might represent a most powerful therapeutic potential (Rocken et al., 1996). Accordingly, enhanced IL-4 expression induced by the administration of all-truns-retinoic acid clearly improves adoptively transferred EAE (Racke et al., 1995a). A recent study in actively induced EAE has taken the approach of directly comparing the modulatory effects of a broad spectrum of cytokines, both of the Thl-like and the Tli2-like groups, using antibodies to IL-6, IFN-y, and TNF-a, as well as recombinant vaccinia viruses expressing exogenous cytokines (IL-lP, IL-2, IL-4, IL-6, IL-10, TNF-a, and IFN-y) (Willenborg et ul., 1995). Although anti-IFN-y treatment exacerbates the disease, anti-IL-6 has no significant effect; anti-TNF-a therapy, in turn, can suppress, not influence at all, or even aggravate disease, depending on the nature of the antibody administered. Recombinant vaccinia viruses expressing IL-lP, IL-2, and IL-10 show therapeutic efficacy, whereas the IL-4-expressing virus can be ineffective or lead to clinical exacerbations. These results, therefore, seem to question the results of several of the investigations reported previously, as well as some general knowledge about cytokine pathways. The vaccinia virus delivery system may at least partly explain such discrepancies because the virally induced cytokine production may be largely influenced by the host microenvironment at preferential sites of viral growth. In addition, the level of cytokine expression cannot be controUled in the host, likely further influencing the replication of the virus. Thus, although the delivery of recombinant cytokine genes via viral carriers may provide a powerful means to modulate the cytokine network in uiuo, the accurate control of the system becomes an important prerequisite to its application. Antibodies that neutralize TNF-a and TNF-P significantly and for a long duration reduce the severity of adoptively transferred EAE (Ruddle et al., 1990). This is consistent with the fact that TNF cytokines exert a direct toxic effect on oligodendrocytes and that MBP-specific T cell clones require TNF-a and/or TNF-fl to express their encephalitogenic properties (Powell et al., 1990). The therapeutic potential of TNF-a blockade is further supported by the successful prevention of actively induced EAE in Lewis rats using rolipram, a iype IV phosphodesterase inhibitor that

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efficiently reduces the TNF-a production by MBP-specific T cell lines (Sommer et al., 1995), although it must be considered that this drug does not ameliorate established EAE (Jung et al., 1996). A recent study with a TNF-a receptor construct, consisting of the extracellular domain of the TNFRp55 fused to a human IgGl heavy gene fragment, demonstrates protection in three different types of EAE in the Lewis rat (Klinkert et al., 1997). Interestingly, therapeutic efficacy is achieved despite the fact that neither the degree nor the cellular proportions of the inflammatory infiltrates are changed in the CNS, indicating a blocking effect of the therapy on terminal effector events in tissue destruction (Fig. 2C). IL-1P, the other cytokine believed to be directly involved in tissue destruction, has been targeted by the administration of recombinant human IL-l@-raduring the preclinical phase of activelyinduced EAE in Lewis rats, resulting in marked amelioration of the disease (Martin and Near, 1995). The immunosuppressive potential of TGF-P, physiologicallyinvolved in tissue repair mechanisms, is also demonstrated by marked inhibition of MBP-specific lymph node cells in vitro and by efficient suppression of adoptively transferred EAE, even if therapy is initiated after the onset of clinical signs (Racke et al., 1991).

2. IDDM In the complex IDDM model, the feasibility of anti-TNF-a therapy remains controversial because timing and/or duration of antibody administration strongly influence the treatment efficacy. Although TNF-a and IFN-y exert direct cytotoxicity on pancreatic P cells in vitro (Campbell et al., 1988), systemic administration of TNF-a paradoxically leads to amelioration of insulitis in NOD mice (Jacob et al., 1990), a therapeutic effect that is even potentiated if TNF-a is administered in combination with IFN-y (Campbell et al., 1991). An 8-week-long therapeutic schedule with anti-TNF-a antibodies increases the incidence of IDDM in NOD mice and fails to affect the adoptive transfer model of IDDM in irradiated NOD mice (Jacob et al., 1992). In contrast, systemic administration of TNF-a as late as 6 days posttransfer, or protracted for 4 weeks during the spontaneously developing disease, reduces significantly the incidence of IDDM. Thus, the potentially deleterious role of TNF-a in IDDM appears essentially local in nature; this is obviously a difficult feature to exploit because TNF-a-neutralizing antibodies are administered systemically.This route of administration may in fact interfere with a number of less characterized functions of TNF-a that counteract the local effects. A very recent publication supports the view that TNF-a plays a central role in insulitis because the transgenic overexpression of a TNF-

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Rp55-human FcIgG3 fusion molecule in NOD mice protects not only from the spontaneous disease but also from an accelerated form of the disease prolvoked by administration of cyclophosphamide; the transgenic condition also protects from adoptive transfer of IDDM by NOD donor spleen cells (Hunger et al., 1997).The protection is associated with reduced incidence and severity of insulitis and with decreased expression of the adhesion molecules ICAM-1 and MAdCAM-1on the venules of the pancreatic islets. Administration of IL-4 (Rapoport et al., 1993)or IL-10 (Pennline et al., 1994),both believed to promote Th2-like development, does protect NOD mice from developing IDDM. However, the role of these cytokines in IDDM may be insufficientlydescribed by a simple Thl/Th2 antagonism (see Fig. 6) because transgenic BALB/c mice overexpressing IL-10 in the pancreas all exhibit periinsulitis. Backcrossing of these transgenic mice with NOD mice results in severe insulitis and acceleration of the onset of diabetes, indicating that IL-10 is not immunoinhibitory in this circumstance, but rather immunostimulatory and autoaggression promoting (Wogensen et al., 1994).

3. Collagen II-lnduced Arthritis Because the T h l polarization is regarded as playing a crucial role in the pathogenesis of arthritis, modulation of the Thl/Th2 balance has attracted much attention recently. According to the notion that IL-12 is a strong T h l inducer, and thereby a disease-promoting cytokine, this cytokine can in fact replace the mycobacteria contained in complete Freund's adjuvant, necessary for the induction of CIA in DBN1 mice, resulting in high incidence and severity of the disease (Germann et al., 1995a). In this very early system, IL-12 acts by promoting the development of IFN-yproducing CD4' T cells and by upregulating (10- to 100-fold) the production of complement-fixing anti-collagen I1 antibodies of the IgG2a and IgG2b isotypes (Germann et al., 1995b), which are in turn indicative of a Thl-like polarization (Fig. 4). IL-12 administration in CIA-resistant C57BU6 mice, or in BALB/c mice immunized with collagen I1 dispersed in oil, also promotes the selection of IFN-y-producing T cells, although it fails to enhance antibody production and to induce arthritis at all; these findings indicate that the strong IFN-.)I T cell response to collagen I1 elicited by IL-12 is insufficient to trigger arthritis (Szeliga et al., 1996). Thus, the arthritogenic potential of IL-12 is rather linked to its ability to induce collagen II-specific B cell response, a function that is widely ascribed to a Th2 fiunction. On the other hand, if IL-12 is neutralized in the course of conventional immunization with collagen in mycobacteria-including adjuvant, there is no significmt protection from CIA (Hess et al., 1996). Also, high doses of

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mouse recombinant IL-12 (1mg/mouse/day) applied for 2 or 3 weeks even reduce the incidence and severity of CIA (Hess et al., 1996).These partially conflicting results clearly underline that the effects of manipulating cytokines in vivo strongly depend on dose, timing, and context of treatment. Indeed, IL-12 seems very well capable of inducing T cells to high production of the Th2 cytokine IL-10 (Trinchieri et al., 1996); the dual mode of action on Thl and Th2 pathways may therefore explain the paradoxical effects in CIA. These findings stress once more that explanatory schemes of autoimmunity based on an oversimplified ThlRh2 paradigm may be insufficient or at least limited to selected models. The effects of IL-10 in CIA, unlike in EAE, seem to fulfill its anticipated immunosuppressive role because treatment with a neutralizing antibody accelerates the onset and increases the severity of arthritis (Kasama et al., 1995). Also, daily treatment with recombinant IL-10 counteracts the progression of established CIA (Walmsley et al., 1996). Remarkably, the suppressive effect of IL-10 can be potentiated if IL-4 is given in combination, whereas IL-4 alone seems insufficient to ameliorate the disease. IL10 and IL-4 in combination not only prevent CIA but also suppress the established disease; the most notable effect in this case is the reduction of cartilage erosions, clearly a much desired effect when devising antirheumatic therapies (Joosten et al., 1997). The cooperative cytokine effects reduce the cellular infiltrations in the synovium and protect against cartilage destruction, an effect apparently due to suppression of IL-lP and TNFa, on the one hand, and to unchanged mRNA levels of the IL-1 receptor antagonist on the other hand (Joosten et al., 1997). These data stress that combinations of regulatory cytokines may affect the cascade of tissuedestructive cytokines in a much more balanced fashion than single treatments, resulting in objective histopathological amelioration of disease. Therapy based on gene transfer of IL-13, another cytokine classified in the Th2-like scheme, also results in amelioration of CIA; this coincides with decreased TNF-a expression, an effect that may at least partially account for the therapeutic benefit (Bessis et al., 1996). In general, treatments aimed at neutralizing TNF-a and IL-p are effective in CIA (Williams et al., 1992; Joosten et al., 1996),in accordance with their apparent role in the effector phase of joint destruction. Because a cysteine proteinase cleaves the 17-kDa active form of IL-1p from its 31-kDa precursor, inhibition of the IL-1p converting enzyme has been exploited for treatment of CIA, resulting in suppression of the established disease (Ku et al., 1996). Early clinical phases of CIA can also be suppressed using a human TNFRp55-Ig Fc fusion protein, resulting in reduction of cartilage erosions (Williams'et al., 1995). Notably, the antierosive properties of anti-TNF-a treatment can be enhanced considerably by the coadminis-

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tration of anti-CD4 mAbs; this synergistic effect seems at least partially ascribable to prevention of the natural neutralizing antibody response to the heterologous TNFRp55 construct (Williams et al., 1995). The comparative effects of TNF-a or IL-1P on established CIA have been recently studied using a neutralizing antibody to these cytokines or recombinant IL-lra, which binds both IL-1P and IL-la. Interestingly, the beneficial effects of anti-TNF-a therapy remain confined to a short time frame after disease onset, whereas there is little or no effect on the fully established disease. In contrast, IL-lra administration ameliorates both early and later stages of CIA, not only reducing the degree of cartilage destruction but also restoring the proteoglycan synthesis of chondrocytes, which are in turn typically suppressed in arthritic cartilage. TGF-01 also counteracts many effects of TNF-a and IL-10 in tissue destruction and is a potent immunosuppressant itself, qualifying as potentially useful in combatting arthritis. Indeed, treatment of DBA/l mice is effective in preventing CIA (Kuruvilla et al., 1991). The role of IFN-y in arthritis seems to be at least as complex as in EAE. Indeed, a recent study demonstrates that IFN-y can enhance or suppress CIA depending on the timing of administration (Boissier et al., 1995). Amelioration ensues upon early application of IFN-y, whereas later schedules do not change the disease or even aggravate it. Thus, IFN-y may be a key proinflammatory mediator during the afferent limb of the immune response (Fig. l),but a regulatory one in established disease. This bimodal function renders IFN-y-based immunomodulation in arthritis a difficult and perhaps risky task.

4 . Experimental Animal Models for SLE Quite in contrast to arthritis, neutralization of IFN-y by mAbs (Jacob et al., 1987') or soluble recombinant IFN-y receptor (Ozmen et al., 1995) is very effective in suppressing glomerulonephritis and/or prolonging the survival of ?JZB/NZW F1 mice. In the early pathogenesis of lupus nephritis, the lymph nodes express high quantities of IFN-y, which may account for the enhanced MHC-I and MHC-I1 expression in diseased kidneys (Halloran et al., 1988; Manolios et al., 1989). TNF-a and IL-1P also increase in the affected kidneys, as they do in expanded T cell subsets isolated from several strains of lupus-prone mice undergoing glomerulonephritis (Murray and Martens, 1989; Brennan et aE., 1989), with the exception of NZB/ NZW F1 mice. In the latter strain of mice the production of TNF-a is rather decreased most likely due to mutations in the 3' untranslated regions of the TNF-a gene in these mice (Jacob et al., 1996).In this case, replacement ther.apy by systemic administration of high doses of TNF-a is in fact beneficial ( Jacob and McDevitt, 1988). Anti-IL-10 aIso suppresses

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nephritis and prolongs the survival of the animals, also apparently in connection with the upregulation of endogenous TNF-a (Ishida et al., 1994). The example of NZBNZW F1 mice illustrates very well the complexity of the cytokine regulation and, therefore, that of cytokine-based therapy. In addition to the aspects previously mentioned, one must also consider that genetic factors unrelated to the MHC-I1 constellation have a considerable impact on disease susceptibility and clinical phenotype in different inbred strains of autoimmunity-prone rodents; this genetic makeup may critically affect, among other factors, the cytokine regulation. Recent genetic analysis on a whole genome scale has enabled the chromsome localization of almost 40 gene loci that predispose to autoimmunity in animal models (Vyse and Todd, 1996). Nine identified loci (in addition to the ZprlFas and gZdlFas ligand loci; Nagata and Suda, 1995)are associated with murine SLE; some of them map to chromosomal regions that harbor candidate cytokine genes. In the susceptibility loci SLE 1-3 the candidate cytokine genes IL-10, TGF-Pl, IFN-a, IFN-P, and TNF receptor 2 are localized, whereas the Lbw4 locus harbors the genes for the IL-5 receptor and the TNF receptor 1; TNF-a is contained in the Lbwl, and the genes for IFN regulatory factor 1, IL-4, and IL-5 map to the Lbw8 locus (Theofilopoulos, 1995). Nonetheless, each of these loci still spans over a considerable distance on the respective chromosome. Ongoing mapping in murine SLE and other models will unravel in the near future whether candidate polymorphic cytokine genes contribute to disease susceptibility and add to our understanding of the complex regulation of autoimmune diseases by cytokines.

5. Suminuy It is difficult to draw general conclusions from the various cytokinebased treatments in the different models of autoimmunity. However, it appears that TNF-a and IL-1 are promising targets of therapeutic intervention due to their direct role in tissue destruction, although their role in host defense and tumor surveillance must be carefully considered in longterm treatments. It is also clear that several treatments can prevent experimental autoimmune diseases but not suppress established syndromes. Alternatively, neutralization of chemokines, such as MIP-la or MIP-2, which block the entry of mononuclear cells and neutrophils responsible for a considerable part of tissue damage in several experimental autoimmune disorders, may be very effective, as in the case of CIA (Kasama et d., 1995) and EAE (Karpus et a1.,1995). The immunosuppressive cytokine TGF-P, naturally upregulated in healing processes, also appears to be very useful in antagonizing tissue destruction in several models. With regard

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to treatments aimed at modifying the polarization of Thl/ThZ-like subsets, the only possible conclusion at this time is that protocols favoring a Thl > Th2 shift tend to be beneficial. WITH E. INTERFERENCE

TIIE

IL-2/IL-2 RECEPTORSYSTEM

IL-2-dependent expansion of activated T cells can be specifically influenced by antibodies or irninunotoxins that interfere with the IL-YIL2 receptor (IL-2R) system. Thus, an anti-IL-2R antibody prevents the development of CIA in DBN1 mice (Banerjee et al., 1988)and administration of the diphtheria toxin-conjugated IL-2 (DAB486IL-2), which selectively kills cells bearing the high-affinity form of IL-2R delays the onset arid reduce:; the severity of adjuvant arthritis in the rat (Bacha et al., 1992). In combination with subtherapeutic doses of CSA, an anti-IL-2R antibody protects against the development of IDDM in diabetes-prone BB/OK rats (Hahn et al., 1988) and it prevents EAE induced by adoptive transfer of eiicephalitogenic T cells (Engelhardt et d . , 1989). Interestingly, this treatment only slightly influences the actively induced EAE (Engelhardt et al., 1989),probably due to different levels of expression of IL-2R on in vitro- and in uivo-activated MBP-specific T cells. From these few examples it appears that biological targeting of the IL-2/IL-2R complex does not represent it real advantage over the use of xenobiotic drugs like CSA. Given comparably potent or even superior immunosuppressive effects, the administration of xenobiotics is simpler and bound to have fewer adverse effects than antibodies or irnmunotoxins,especially in view of long-term applications.

F. SUPPF;ESSION OF CD4' T CELLS The large number of studies on therapeutic interference with the CD4 coreceptor shows that anti-CD4 rnAbs exert different degrees of cytotoxicity on their target cells; accordingly, they can be classified as depleting or nondepletimg. The nondepleting ni Abs are very attractive because they offer a clear therapeutic potential while circumventing the danger of general immunosuppression. Their functional effects include blockade of the interaction with TCR or MHC, separation of signal transducing kinase ~ 5 6 'froin " ~ the TCWMHC complex (Haughn et al., 1992),and transduction of a negative signal through inappropriate activation of the ~ 5 6 'molecule '~ or p56'"k-independent mechanisms (Zerbib et al., 1994). The different modes of molecular interaction may at least partially account for different therapeutic effects shown by different anti-CD4 mAbs because the fine epitope specificity of the inAbs may crucially influence not only the quantity of CD4 modulation but also its quality.

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A nondepleting mouse anti-rat CD4 mAb, shown to prevent or halt EAE, appears able to induce a Th2-type of cytokine response in the mixed lymphocyte reaction (MLC) in vitro. When CD4' T cells are activated by allogeneic stimulator cells in the presence of this anti-CD4 mAb, the production of IFN-y (but not that of IL-2) is completely inhibited in the primary reaction; in the secondary challenge, the absence of the mAb elicits a dramatic synthesis of IL-4 and IL-13 mRNA (Stumbles and Mason, 1995). Interestingly, the inhibition of IFN-y in the primary MLC is reversed in the secondary MLC; this reversed suppression may provide a reasonable explanation for some effects of anti-CD4 treatment of EAE; that is, splenocytes from protected mice are still capable of transferring EAE upon in vitro stimulation with MBP (Sedgwick and Mason, 1986). The interesting question of the molecular mechanism underlying the shift of the cytokine response remains open, however. Recently, a nondepleting anti-CD4 mAb has proven effective in CIA, with a reduction of IgG2a (but not IgG1) anti-collagen I1 levels (Chu and Londei, 1996). In this study, lymph node T cells isolated from treated mice show a Th2-like cytokine pattern in response to challenge with collagen 11, i.e., a reduction of IFN-y and increase of IL-4. An interesting strategy to target the CD4 coreceptor is to use peptidic analogs of the CD4 molecule itself (Jameson et al., 1994). In the PLP 139-151-induced model of EAE, for example, a CD4 analog exerts its effect by antigen-specific suppression of the lymph node T cell responses, whereas recognition of alloantigen or other nominal antigen remains unaffected (Marini et al., 1996). Futhermore, a single administration of the peptide during the acute phase of EAE induces long-term resistance to a second antigen challenge; analysis of the cytokine profile during late stages of EAE shows significant reduction of both Thl- and Th2-like cytokines. Notably, the peptide approach can suppress T cell responses if the animals are rechallenged with the antigen during the natural recovery phase, indicating that the treatment targets antigen-specific memory T cells; this is of particular advantage in comparison with different anti-CD4 mAbs, which preferentially eliminate resting, naive CD4' T cells rather than memory effector T cells (Chace et al., 1994). OF CD8+ T CELLS G. SUPPRESSION Several effector pathways involving CD4' and CD8+T cells may mediate destruction of p-cells during the development of IDDM in NOD mice, although the linkage of disease susceptibility to MHC-I1 genes seems to privilege a role for CD4' T cells, which is further supported by the effectiveness of anti-CD4 treatment (Shizuru et al., 1988). CD8+ T cells may nonetheless significantly contribute to the pathogenesis of IDDM, as

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suggested Iiy several findings: the adoptive transfer of rapid-onset diabetes via the use of CD8t CTL clones derived from intraislet lymphocytes of NOD mice (Wong et al., 1996); the absence of islet pathology in p2 niicroglobulin-deficient mice (NOD p2m null) (Katz et al., 1993); and the return of irisulitis (but not diabetes) in islet-specific reconstitution of INFy-inducible MHC-I expression in NOD p2m null mice (Kay et al., 1996). Accordingly, anti-CD8 treatment protects against the development of both irisulitis and IDDM. However, this effect is only observed within a discrete age period-between 2 and 5 weeks (Wang et al., 1996). Anti-CD8induced depletion of CD8' T cells is most likely due to an inhibitory influence on the priming and expansion of autoreactive, islet-specific CD4+ T cells because anti-CD8 treatment remains ineffective in a T cell receptor transgenic mouse model of insulitis in which the CD4+ T cell repertoire is highly skewed for antiislet cell reactivity (Wang et al., 1996). Although anti-CD8 treatment does not influence the autoimmune syndrome in MRL Zprllpr mice, which seems instead exquisitely sensitive to anti-CD4 therapy (Merino et al., 1995), experimental arthritides, (e.g., proteoglycan (aggrecan)-induced arthritis in BALB/c mice or antigeninduced arthritis) react to anti-CD8 administration with a clear aggravation of disease severity (Banerjee et al., 1992). This indicates that, at least in arthritis, the function of CD8' T cells may be more univocally oriented to immunoregulation. H. INTERFERENCE WITH T CELLRECEPTORCOSTIMULATION Costimulatory signals are crucial in determining whether antigen recognition leads T cells to activation or anergy. Sole ligation of the TCR does not stimulate naive T cells to proliferate and differentiate into armed effector T cells because clonal expansion, cytokine production, or cytolysis require a second costimulatory signal. In T cell activation, signal transmission via the TCR complex and its coreceptor CD4 is accompanied by a variety of receptor interactions at the surface of the antigen presenting cell involving ligation of the followingpairs: integrin LFA-1 (CDlldCD18) and its countereceptors (ICAM-1, I-CAM-2, and ICAM-3); CD2 and CD58 (LFA-3 in humans) or CD48 in rodents; CD40L-CD40 pair; CD44H and its as yet unidentified countereceptor; and the B7-CD28/CTLA-4 pathway. Upon fulfillment of the geometrical requirements for TCR ligation and costimulation, a complex cascade of receptor-ligand interactions can finally initiate. The CD40-CD40L interaction rapidly upregulates CD44H and ICAM-1 on T cells, which in turn mediates CD28-independent costimulation (Shinde et al., 1996). Of note, CD40L must strongly contribute to TCWCD3 costimulation and T cell activation because CD40L-deficient

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mice, carrying a transgenic TCR specific for MBP, fail to develop EAE (Grewal et al., 1996). The essential role of costimulation in maintanance of tolerance, as well as the importance of CTLA-4 in regulation of autoreactive T cells, offers two avenues for therapeutic intervention in autoimmune diseases: the blockade of T cell activation through B7-CD28 or the triggering of B7CTLA4 interactions to downregulate activated, autoreactive T cells. These possibilities are currently being tested in different models of autoimmune diseases. Active or adoptively transferred EAE, for example, can be suppressed by chimeric recombinant constructs of CTLA-4 fused to constant Ig domains (CTLA-4-Ig), which exhibit high avidity for B7-1 and B7-2 (Cross et al., 1995; Perrin et nZ., 1995; Khoury et al., 1995; Arima et al., 1996); notably, prevention in this case is almost absolute, lasting well after cessation of therapy and causing a significant reduction of demyelinization foci (Cross et nl., 1995).These results, however, do not indicate that CTLA4-Ig acts by inducing tolerance because T cells maintain their in vitro responsiveness to the immunogen, and splenocytes isolated from treated mice are still capable of transferring disease to naive recipients (Cross et al., 1995). Protection against adoptively transferred EAE is also achieved if encephalitogenic T cells are exposed to CTLA-4-Ig in vitro or if CTLA4-Ig is coadministered with MBP-activated T cells upon transferral in recipient mice. In contrast, there is no influence of CTLA-4-Ig therapy after T cell transfer, indicating either that alternative costimulatory pathways may be involved in established EAE or that systemic routes of administration do not yield sufficient levels of the Ig construct in the brain (Perrin et al., 1995). Treatment of established phases of experimental autoimmune disorders remains a moot point for most immunotherapeutic approaches, and therapy with CTLA-PIg represents no clear exception to this rule. Accordingly, there are reports of marked suppression of EAE (Khoury et al., 1995) or no effects at all (Arima et al., 1996). The efficacy highly depends on the dosage because a single administration at Day 2 postimmunization prevents disease, whereas multiple dosing of CTLA-4-Ig (from Day 1 to 17) enhances the disease (Racke et d., 1995b). When therapeutically successful, treatment with CTLA-4-Ig seems nonetheless associated with immunohistological signs of a Thl > Th2-like shift in the CNS (Khouryet al., 1995),whereas exacerbation is accompanied by enhanced production of TNF-a, IFN-y, and IL-2, hence supporting a linking role of CTLA-4 in cytokine regulation and disease attenuation (Perrin et ul., 1996). The in vivo administration of anti-B7-1 and B7-2 mAbs suggests distinct roles of these receptor ligands in experimental autoimmunity. In actively

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induced E 4E, a single anti-B7- 1treatment significantly suppresses disease, whereas anti-B7-2 treatment leads to EAE exacerbation (Racke et al., 1995b).Also, in uitro exposure of MBP-specific T cells to a combination of both mAbs blocks their capacity of adoptively transferring disease. Delayed onset and reduced clinical severity result from transfer of in uitro-treated encephalitogenic T cells (Hacke et al., 1995b). Also, anti-B7-l effectively protects against the development of EAE induced by PLP 139-151 in SJL mice, whereas anti-B7-2 increases the severity of the same model (Kuchroo et al., 1995).The beneficial effects of anti-B7-l are associated with induction of Th2 clones; once transferred, these clones prevent the induction of EAE or abrogate established disease. Thus, manipulations of the B7-CD28KTLA-4 pathway seem to divert the commitment of precusor T cells into ii Thl or Th2 lineage (Kuchroo et al., 1995). Most significantly, the application of a non-cross-linking Fab fragment of anti-B7-l mAbs well into the established chronic phase of relapsing EAE induced by PLP 139-151 blocks the recurrence of the relapses, limiting at the same time the epitope spreading characteristic of chronic phases of autoimmune diseases (Miller et al., 1995). This finding represents a clear breach of the frustrating experience that most experimental immunotherapies have a limited therapeutic window, much too early to be of unquestionable significance for future application in human chronic autoimmune disorders. This remarkable report also contributes the observation that the use of a complete mAb instead of a Fab fragment enhances disease (Miller et al., 19951, suggesting important mechanisms of signaling upon receptor crosslinking (Hirokawa et al., 1995). Modulation of the B7-CD28KTLA-4 pathway is also effective in IDDM, albeit with some remarkable differences from EAE (Lenschow et nl., 1995). CTLA-4-Ig treatment at the onset of insulitis greatly reduces the incidence of diabetes; however, there is no reversal of the histopathological signs of disease. A protective effect, although less potent, is also seen upon administration of anti-B7-2. A later therapeutic schedule is in turn not beneficial, indicating that blockade of costimulatory signals by antiB7-2 and CTLA-4-Ig acts within a limited stage of disease developmentafter the beginning of insulitis but before onset of overt diabetes. Interestingly, antibody treatment with an anti-B7-l mAb accelerates the development of disease in female NOD mice and induces disease in otherwise resistant males (Lenschow et al., 1995). Combined anti-B7-1/B7-2 treatment can also accelerate the development of IDDM, suggesting that the disease-promoting effect of anti-B7-l administration is dominant in the combination with the protective anti-137-2 (Lenschow et al., 1995). As pointed out previously, therapeutic trials aimed at treating established phases of experimental inodels of autoimmunity are of special interest

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because a curative approach is more relevant to human diseases than protective protocols. It is therefore remarkable that CTLA-4-Ig treatment of NZB/NZW Fl mice at a stage of lupus characterized by 40% lethality suppresses the systemic disorder and prolongs the survival of the affected mice (Finck et al., 1994). Complete suppression of established disease is also achieved in the case of CIA, in which the CTLA-4-Ig construct, or the combined treatment with anti-B7-1 and anti-B7-2 mAbs, suppresses the production of anti-collagen I1 IgGl and IgG2a. In this case, lymph node T cells of treated animals respond with diminished production of IFN-y upon in vitro stimulation, suggesting a diminished T h l response (Webb et al., 1996). The possible involvement of the CD40/CD40L system has also attracted interest because CD40L-deficient mice carrying a transgenic TCR specific for MBP show resistance to the development of EAE (Grewal et al., 1996). Consistent with these findings, treatment with anti-CD40L mAbs prevents PLP-induced EAE. More important, this treatment dramatically ameliorates established EAE, even if the mAbs are administered almost at the clinical peak of disease (Gerritse et al., 1996). In CIA, however, the results of this treatment are more modest in that only preventive trials are effective (Dune et al., 1993). The functional importance also renders the interaction between CD2 and CD58 (or CD48 in rodents) a promising target for immunosuppressive strategies. Indeed, anti-CD2 treatment in diabetes-prone BBNor rats prevents IDDM (Barlow and Like, 1992), although it is unclear whether treatment blocks T cell activation or rather depletes T cells (Barlow and Like, 1992). EAE can also be successfully prevented or treated by the application of different anti-CD2 antibodies (Jung et al., 1995). The antiCD2 mAb 0x34, for example, which persists on lymphocytes for at least 11days, completely protects against adoptively transferred EAE if administered simultaneously to the cell transfer and 4 days later, resulting in inhibition of T cell infiltration in the CNS (Jung et al., 1995). A single injection of this mAb, however, although transiently depleting T cells and modulating the expression of CD2 antigen, does not inhibit T cell activation nor diminish the degree of T cell responsiveness. In general, the therapeutic feasibility of modulating TCR costimulation seems to be supported by preclinical studies in several experimental models of autoimmune diseases. The most attractive feature of this approach is the selective targeting of T cells undergoing activation, which allows dangerous effects on the globality of the immune responses to be avoided. This represents a clear advantage over treatments that delete whole T cell subsets or target central cytokines. Also, in contrast to approaches that interfere most directly with TCR functions, the manipulation of costimu-

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latory signals does not require the precise identification of antigenic structures or that of individual polymorphic MHC or TCRs. Different studies point out that the costimulatory requirements of the pathogenic T cells do not necessarily remain constant during the course of autoimmune diseases. In most of the studies, the effectiveness of given agents remains confined to the induction phase or the afferent limb of disease manifestations. There are, however, two remarkable exceptions, i.e., the efficacy of anti-B7-l Fab in relapsing forms of EAE and that of CTLA-4-Ig in late stages of murine lupus. Both studies, therefore, single out the B7-CD28KTLA-4 costimulation pathway as the most promising target of immunotherapy in terms of blockade of ongoing pathological processes. I. MHC AS

A

TARGET OF IMMUNOMODULATION

Prior to the application of rationally designed peptides, other therapeutic approaches showed the feasibility of interfering with antigen presentation. Thus, administration of mAbs directed to the NODI-A antigen (the IDDMpredisposing MHC-I1 analog in NOD mice) prevents the development of IDDM in this mouse strain, although the effect involves the induction of CD4' suppressor cells rather than passive blockade of MHC-I1 molecules (Boitard et al., 1988). EAE can also be prevented, for example, through an antibody directed to an MHC-IVautoantigen complex that interferes with T cell activation in an epitope-specific manner in vitru (Aharoni et al., 1991) or by active immunization with synthetic peptides derived from the I-A P chain. The latter leads to allele-specific (I-As) protection against actively induced EAE in [ SJL( I-As) X BALB/c(I-Ad)] F1 mice; the peptide from the allele that is not linked to EAE (I-Ad) remains ineffective. The mechanism of action of I-As-derived peptides seems due, at least in part, to induction of anti-MHC-I1 autoantibodies that inhibit T cell proliferation in response to MBP (Bright et al., 1996). Peptides binding to the same MHC-I1 molecule can compete with one another for presentation to T cells. Thus, selective immunosuppression can be achieved by blocking the binding sites of MHC-I1 molecules genetically associated with autoimmune diseases, thereby preventing presentation of autoantigens to pathogenic T cells. The application of this theoretically appealing concept is in fact successful in preventing EAE and IDDM. A peptide with high affinity to IAs prevents EAE in SJLJJ mice if coadministered with the encephalitogenic peptide during or immediately before immunization (Lamont et al., 1990). Similar protection is obtained with a nonimmunogenic non-self-peptide in PLJJ mice (Gautam et al., 1992). Experimental autoimmune myasthenia gravis (EAMG) can also be prevented through the use of an ovalbumin peptide binding to the rat class-

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I1 molecule RTl.B(L) coadministered with the acethylcholine receptor (AChR) upon immunization. This treatment drastically reduces the formation of autoantibodies against the AChR (Wauben et al., 1996), which, as already mentioned, is the main pathogenetic mechanism of this disorder. Likewise, a synthetic peptide specific for the NOD MHC-I1 Aag7a bg7(Ag7) prevents IDDM in NOD mice (Hurtenbach et al., 1993). This study raises an important technical point, namely, the necessity for sustained plasma levels of the antagonistic peptide, and proposes to prolong the biological half-life of the peptide using D amino acids in the synthesis of the amino- and carboxy-terminal ends (Hurtenbach et al., 1993). Despite its good promises, the effects of MHC blocking may be complicated by the imniunogenicity of antagonistic peptides. In a IDDM model induced by transfer of splenocytes into irradiated recipients, for example, only immunogenic, but not tolerated peptides exhibit disease-blocking effects (Vaysburd et al., 1995). Another problem, especially in view of long-term treatment of chronic autoimmune diseases, is the failure to demonstrate long-lived MHC saturation both in vivo and in vitro (Ishioka et al., 1994). J. T CELLRECEPTORANTAGONISTS A N D ALTEREDPEPTIDE LIGANDS The concept of developing antagonistic peptides can be applied in a mirror-like fashion to target the TCR. Substitution of single amino acids in the immunogenic peptides, for example, stimulates T cells to perform certain Thl or Th2 effector functions without inducing cell proliferation (Sloan-Lancaster and Allen, 1996). Upon immunization with PLP 139-151, a T cell antagonistic peptide can protect against the development of EAE and ameliorate disease if administered at the onset of the clinical signs (Kuchroo et al., 1994). The rationale for designing the antagonistic peptide in this particular study is very instructive. Following a thorough structural analysis of the in vitro recognition of the iinmunogen, using six different T cell clones that use a diverse set of genes for the a and p chains of the TCR, amino acids have been substituted within the two contact sites of the peptide with the TCR. The sole W to E exchange in position 144 of the primary TCR contact site of the PLP 139-151 peptide (PLP 139-154 W > E 144) results in prevention of EAE upon immunization with the native PLP 139-151 due to the induction of T cells that are cross-reactive with the native hgand and produce Th2-like cytokines (IL-4 and IL-10) and ThO-like cytokines (IFN-y and IL-10) (Kuchroo et al., 1994). Of note, the adoptive transfer of T cell lines that are generated upon exposure to the altered peptide ligand confers protection from EAE (Nicholson et al., 1995).

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These results suggest that immune deviation is one important mechanism by which altered peptide ligands of the TCR inhibit autoimmune diseases. A major effect on the niodulation of cytokine responses is supported by a study of h4BP 87-99-induced EAE; of three altered peptide ligands, only the MBP 87-99 (91 K > A) substitution results in protection and reversal of EAE. This is accompanied by suppression of IFN-y and TNF-a responses in cells isolated from draining lymph nodes upon in uitro challenge with the native MBP 87-99 peptide (Karin et al., 1994). A MBP 87-99 (96 P > A) peptide also exerts dramatic therapeutic effects on established EAE induced by adoptive transferral of MBP 87-99-specific T cells. The therapeutic benefit is not only clinical but also histopathological, with regression of inflammatory infiltrates and disappearance of heterogeneous T cell infiltrates; only T cells with characteristics similar to those used to induce E.4E in the first place remain present in the lesions of the CNS (Brocke ot al., 1996). In this study, neutralization of IL-4 reverses the tolerant state induced by the MBP 87-99 (96 P > A) peptide. The altered peptide ligation thus seems to provide selective signals that result in an efflux of T cells recruited in the initial lesions; this wash out may be the result of intralesional downregulation of TNF-a and concomitant rise of IL-4. TCR antagonism or MHC competition, on the other hand, are unlikely to account for the therapeutic effects because MBP 87-99 (96 P > A) is a partial agonist for the encephalitogenic T cells and fails to inhibit their in vitro proliferation. The most likely explanation for the therapeutic effect of the altered peptide ligand approach remains immune deviation (Fig. 6). Specific inimunotherapy based on altered peptide ligation, however promising, faces the obvious problem that in some experimental models, especially in human autoiminiine diseases, the autoreactive pathogenic T cells and their antigen specificities are still unknown; this clearly hampers the rational design of appropriately modified peptide ligands. In the case of multiple sclerosis, however, this promising approach may find its way into clinical trials because there is already some information available 011 the fine specificity of myelin-specific autoreactive T cells. Another theoretical obstacle is that the therapeutic effectiveness may fade in the case of substantial determinant spreading, which leads by definition to diversity ofthe T cell repertoire. In this respect, encouragng data show that targeting immunoclominant T cell clones in EAE lesions may transactingly suppress encephalitogenic T cells with a diverse repertoire (Brocke et al., 1996).

K. ACTIVATION OF REGULATORY T CELLSBY T CELLAND TCR PEF~TIDE VACCINATION T cell vaccination has been proposed in pioneer studies in EAE, adjuvant arthritis, and EAT (Ben-Nun et al., 1981; Holoshitz et al., 1983; Maron

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et al., 1983), with the attenuation protocols varying from irradiation to pressure or chemical cross-linking. The disease resistance resulting from T cell vaccination is transferrable to naive recipients by regulatory T cells of both CD4+ and CD8' phenotype (Lider et al., 1988). Anticlonotypic T cells are the major regulators that recognize clonotypes of the TCR. Protection by these cells is mediated through cytotoxic effects but may include other suppressive mechanisms. Regulatory T cells recognizing cellular markers other than the TCR clonotype may also be important, in concert with the antiidiotypic response. It is conceivable that T cell vaccination acts through the same regulatory pathways preexisting and naturally operating in the periphery. Thus, clonotypic networks may be preshaped in the physiological situation to accommodate and regulate a limited number of autoreactive T cells within a certain tolerable limit. This hypothesis is supported by recent data showing that inactivation of TCR peptide-specific CD4 regulatory T cells induces chronic EAE in an otherwise self-limiting model induced by MBPAc 1-9 (Kumar et al., 1996). Thus, a failure to generate regulatory T cells that participate in the reestablishment of peripheral tolerance after an acute flare may cause or favor chronic autoimmune conditions. The aim of T cell vaccination is therefore the reestablishment of a physiologic balance in the clonotypic network. Once preclinically tested, the effects of T cell vaccination remain limited to protection against the development of disease rather than reversal of the established signs, with the possible exception of adjuvant arthritis (Lider et al., 1987). Another problem is illustrated by vaccination with attenuated AChR-specific T cell lines in EAMG; this treatment induces AChR-specific suppressor cells in the spleen, but it also enhances AChR antibody responses (Kahn et al., 1990). Thus, T cell vaccination can turn out a seemingly double-edged sword. A novel approach in TCR vaccination is represented by the injection of DNA encoding the Vb 8.2 region of a TCR critical to the pathogenesis of EAE (Waisman et al., 1996). This approach ameliorates MBPAc 1-20induced EAE, inducing the surge of peptide-specific regulatory T cells characterized by a T h l > Th2-like shift of their cytokine production. Of note, the efficacy of this approach extends to EAE induced by the entire MBP molecule, although the latter certainly shapes a more diverse T cell repertoire than the 1-20 peptide. The TCR peptide vaccination also protects DBN1 mice from developing CIA, as shown by the use of a recombinant TCR domain derived from the Val1.l-JA17 gene, a gene used by a T cell hybridoma specific for an immunodominant epitope of collagen I1 (Rosloniec et al., 1995). Similarly, TCR peptide vaccination with VplO peptides blocks the development of

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CIA in BUB/BnJ mice (Haqqi et al., 1996). This approach elicits the formation of antibodies that react with self-TCR; these are likely to account for the downregulation of both T cell and B cell pathogenic responses observed in the study. Antibodies against certain V gene segments of the TCR effectively protect against autoimmune diseases characterized by limited V gene usage. The administration of anti-VP 8.1,2 or anti-VP 5.1,2 mAbs protects DBA mice from developing CIA (Chiocchia et al., 1991);likewise, a combination of anti-Vp 8.2 and anti-Vp 13 protects completely against MBP-induced EAE (Zaller et al., 1990). However, this type of treatment becomes much less effective in models characterized by diverse T cell repertoire, for example, in the case of the relapsing form of EAE induced by PLP 139-151 (Whitham et al., 1996). The attempt to treat autoimmune diseases by activation of regulatory cells is very challenging. However, the therapeutic effects of T cell vaccination, as well as those of TCR peptide vaccination, are mostly confined to prevention of disease models. It remains to be demonstrated how this type of therapy influences established phases of chronic disorders.

L.

ANTIGEN-SPECIFIC TOLERANCE

EAE is the most extensively used model for the study of orally induced tolerance. Initial studies in Lewis rats, with oral administration of high dose of MBP (in four feedings a total of 20 mg in 1 week) before immunization, show delayed onset, reduction of incidence, and clinicalhistopathological improvement of the disease; these effects are accompanied by a decrease of T cell proliferation and a slight reduction of antiMBP antibodies in response to MBP (Bitar and Whitacre, 1988). Notably, high doses (10-20 mg) are necessary to cause the effect, whereas 2 mg remains ineffective; heterologous MBP is also necessary (human or pig), whereas rat MBP is ineffective. Similar therapeutic benefits can also be achieved with a low-dose regimen of oral MBP (<2 mg) with highest efficiency between 25 and 500 p g given in three doses before immunization. A dose of 50 pg seems optimal to block the development of clinical and histopathological signs of EAE (Higgins and Weiner, 1988). A weak effect is also observed if oral feeding is performed in the latency phase between immunization and clinical onset of EAE (see Fig. 4 for overview). Equimolar amounts of MBP peptides are also effective, with nonencephalitogenic peptides more potent than encephalitogenic ones. A T cell population enriched in CD8' cells (85% purity) seems capable of transferring tolerance to naive recipients, with suppression of proliferative responses of MBP-sensitized lymph node cells in vitro (Lider et al., 1989). Thus,

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the dose ranges seem very much to reflect high versus low zone tolerance mechanisms. The study of EAE induced by spinal cord homogenates, which elicit chronic relapsing forms of the disease in Lewis rats and guinea pigs, allows to estimate the potential of oral tolerization to halt further progression of an already established disease (Brod et al., 1991).Initiation of MBP feeding during the first spontaneous remission markedly decreases the frequency and severity of the subsequent relapses, reducing in parallel the inflammatory infiltration and demyelination in the CNS. These results bear obvious implicationswith respect to the potential application in human autoimmune diseases characterized by chronicity, cycling relapses, and remission. The mechanisms contributing to the therapeutic benefits of oral tolerization have been partly elucidated. In vitro, cells isolated from MBP-fed rats suppress proliferation of an OVA-specific T cell line across a transwell (Miller et al., 1991), a phenomenon defined as bystander suppression. The cells from MBP-fed rats suppress OVA cells only upon triggering by the fed antigen. Likewise, T cells from OVA-fed mice suppress MBP-specific T cells across the transwell when they are challenged with OVA. Antigendriven bystander suppression also operates in vivo. Lewis rats fed with OVA, and then subcutaneously immunized with MBP in adjuvant plus OVA, remain resistant to EAE; in contrast, animals are not protected if fed with OVA and subcutaneously immunized with MBP in the absence of OVA. The protective effect can be adoptively transferred by CD8+ T cells. Substitution of OVA with bovine serum albumin attains identical results, indicating that suppressor CD8+T cells are triggered in an antigenspecific fas’hion although their effects are mediated via a nonspecific suppressor factor. This factor appears to be TGF-01 (Miller et at., 1992a). Thus, in order to achieve bystander suppression, autoreactive T cells must reside in the physical vicinity of T cells that have been tolerized by exposure of antigen through the gut. By definition, this requires that the antigen used for tolerization is also expressed in the target tissue of autoimmune attacks. According to this concept, the mucosal administration of only one antigen may be sufficient to suppress the action of autoaggressive cells against any of the autoantigens in the target tissue. In animals orally tolerized to MBP, the marked reduction of inflammatory infiltration in the CNS is accompanied by reduced expression of inflammatory cytokines (IL-1, IL-2, IL-6, IL-8, TNF-a, and IFN-y) and upregulation of TGF-0 (Khoury et al., 1992).LPS, known to augment the effects of oral tolerance, enhances the local expression of IL-4 and PGE2 when applied in combination with oral MBP, presumably due to activation of additional populations of immunoregulatory cells. Thus, orally activated regulatory cells modulate the cytokine milieu in the target organ of autoim-

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munity; the local cytokine expression shifts from a Th l-dominated pattern (typical of established EAE, as mentioned previously) to a Thl-suppressed pattern, a phenotype typical of natural recovery. Furthermore, there is experimental evidence that oral feeding induces a mucosally derived Th2like regulatory population, as shown by the analysis of T cell clones from inesenterial lymph nodes in MBP-fed SJVJ mice. These cells are CD4' and identical to encephalitogenic CD4' clones in TCR V gene usage, M HC restriction, and epitope specificity. However, they produce TGF-P with various amounts of IL-4 and IL-10 and are able to suppress EAE induced by PLP or MBP (Chen et nl., 1994). Similar to the oral route, the intravenous administration of MBP suppresses E,4E in an antigen-specific fashion; however, there is no evidence of active suppression (Miller et nl., 1993).Upon intravenous administration, the nonencephalitogenic peptide MBP 21-40 does not suppress MBP 71-90-induced EAE in Lewis rats, whereas the orally administered MBP 21-40 does. In PLP-induced EAE, intravenously or orally administered PLP suppresses disease; MBP exerts suppression via oral but not intravenoiis route. The intravenous route seems prone to induce clonal anergy. Anergy is a state of lymphocyte unresponsiveness characterized by lack of proliferation, IL-2 production, and diminished IL-2R expression (Schwartz, 1990). Clonal anergy seems to be associated not only with intravenous tolerization but also with oral tolerization with high doses of MBP (Whitacre et al., 1991). Different protocols appear to critically bias the outcome of oral tolerance; bystander suppression is then associated with doses of antigen in the range of 1-5 mg, whereas anergy results from doses in the range of 20 mg (Friedman and Weiner, 1994).Mice transgenic for w OVA-specific TCR are also critically sensitive to antigen dosage (Chen et al., 1995).In this model, lower doses of antigen induce T cells that secrete TGF-P, IL-4, and IL-10; high doses provoke peripheral deletion of antigen-specific Thl and Th2 cells by activation-induced apoptosis. Of note, TGF-&secreting T cells (Th3-like cells) are remarkably resistant to peripheral deletion, perhaps because of the central role of TGF-P in promoting IgA synthesis in the gut (Coffman et al., 1989). The mechanisms underlying oral tolerance seem quite complex and comprise, depending on the dosage of antigen, the activation of regulatory CD8' and CD4' T cell subsets, induction of anergy, or peripheral deletion. Irrespective of different mechanistic effects, oral tolerance demonstrates a remarkable therapeutic potential in different EAE models. In fact, not only the preventive approach but also the treatment of established disease is very effective, as recently demonstrated by the suppression of relapses in an MBP-induced model in B10.PL mice (Meyer et nl., 1996).

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In contrast to more optimistic reports in earlier studies, evidence is growing that oral tolerance may be associated with adverse effects; oral feeding of low doses of MBP (0.4 mg) exacerbates chronic relapsing EAE in B1O.PL mice (Meyer et al., 1996). On the other hand, unpredictable effects may derive from the choice of the species from which the tolerogen is derived as well as from the routes of tolerization because these variables profoundly affect the clinical outcome (Miller et al., 1992b; Javed et al., 1995). Indeed, oral administration of the MBP Ac 1-11 peptide is ineffective over a wide range of doses (Metzler and Wraith, 1993), whereas the same peptide profoundly inhibits EAE if administered via a single nasal administration before immunization. In NOD mice, oral administration of low doses (1mg) of porcine insulin during the first year of life results in delay in the onset of IDDM and reduction of the histopathological severity of insulitis (Zhang et al., 1991), without apparent metabolic side effects of insulin. Adoptive protection in this model can be achieved by transfer of spleen cells but not by T celldepleted spleen cells. Oral tolerance is also effective in a viral model of autoimmune disease (von Herrath et al., 1996).Transgenic mice expressing the viral nucleoprotein of lymphocytic choriomenigitisvirus (LCMV)under the control of the rat insulin promoter develop diabetes upon infection with the LCMV. Oral feeding of insulin, even if initiated 10 days after LCMV inoculation, prevents diabetes in more than 50% of the transgenic animals. In the pancreatic islets the cytokine pattern shifts in this case from a Thl-like predominance to a Th2RGF-/3 profile. The nasal route of administration appears at least as promising as the oral route. In NOD mice, intranasal insulin administered during the subclinical stage of IDDM decreases the incidence of the disease as well as the histopathological signs of insulitis (Harrison et al., 1996). Both oral and nasal administration of AChR protect Lewis rats from developing EAMG, with the nasal protocol requiring only microgram amounts of the antigen compared to 5 mg necessary for oral tolerization. Tolerization is followed by reduction of anti-AChR antibodies and a decrease of AChR-specific B cells in the lymph nodes. Because the affinity of the anti-AChR antibodies is also reduced, mucosal treatments seem to counteract the development of AChR-specific B cells (Wang et al., 1995). T cells in the draining lymph nodes are also clearly influenced, as shown by a decrease of their proliferation rates and production of IFN-y (Ma et al., 1995). The influence of AChR mucosal tolerization on the cytokine patterns, in turn, can be traced as far as the popliteal and inguinal lymph nodes. Lymph nodes from untreated EAMG rats contain elevated numbers of IFN-y, IL-4, and TGF-/3 mRNA expressing cells. Oral or nasal tolerance decrease the numbers of AChR-reactive IFN-y and IL-4 expressing cells

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but upregulate the TGF-P mRNA-positive cells in lymphoid organs (Ma et al., 1996). These results suggest a central role of IFN-y and IL-4 in the development of EAMG and, on the other hand, the importance of TGF-P in the induction of tolerance. Protection of EAMG can be achieved by different protocols with autologous or heterologous AChR or else with different time schedules for the initiation of feeding, i.e., before or after immunization (Drachman et al., 1996),with dose and purity of the antigen representing additional variables. When therapy initiates after immunization, clinical amelioration of EAMG is achieved; however, it is in association with a paradoxical rise of antiAChR autoantibodies. EAU can also be prevented by feeding with retinal S antigen in complete Freunds adjuvant (Nussenblatt et al., 1990). The treatment reduces the histological damage of the eye and suppresses T cell proliferation in response to the antigen. Because anti-CD8 antibodies block this suppression, CD8+ T cells may have a primary role in suppression. Interestingly, only uveitogenic peptides (but not nonuveitogenic ones) are tolerogenic to about the same extent as S antigen itself (Singh et al., 1992). The nasal route of tolerization is also highly effective in this model, allowing a reduction of the tolerogenic dose from mg to pm quantities (Dick et al., 1993). The combination of S-Ag as uveitogen with OVA as oral tolerogen, in analogy to the classical experiments in EAE (Miller et al., 1991),exerts a suppressive effect in OVA-fed animals only if OVA is coinjected with S antigen during the induction of the disease, but not if the activation of the OVA-specific orally sensitized T cells is initiated at a distant body district, for example, by OVA immunization at the contralateral leg. This indicates that bystander suppression in this model might not occur in the target organ but rather peripherally at the site of induction of the autoreactive cells (Wildner and Thurau, 1995). Oral administration of collagen I1 reduces incidence and severity of CIA in rats and mice upon preventive protocols (Thompson and Staines, 1986a; Nagler-Anderson et al., 1986). The therapeutic effect is accompanied by reduction of antibodies of IgG2 and an increase of IgGl antibodies, implying a bias toward aTh2-type response (Thompson et al., 1993).Experiments on adoptive transfer of tolerance indicate that both CD4' and CD8+ populatioiis are important for the suppressor effect. The oral tolerogenicity of collagen I1 is critically influenced by the intactness of its protease-resistant triple-helical conformation; thus, if the molecule is rendered more sensitive to the acidic pH of the stomach and to the intestinal proteases by means of heat denaturation, its tolerogenic potential via the oral route will be diminished. Accordingly, the tolerogenic potential of free a-chains of collagen remains unaffected if heat-denatured

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collagen I1 is given intravenously (Thompson and Staines, 198613).Escaping the intestinal tract has recently proven successful, because heat-denatured collagen I1 via the nasal route prevents rat CIA (Staines et al., 1996). The nasal tolerization, similar to the oral route, is paralleled by a shift of the specific antibodies in favor of the IgGl isotype and by a reduction of T cell reactivity to both the tolerogenic peptide and the entire collagen I1 molecule. The feasibility of using cross-reactive immunodominant T cell epitopes, in turn, has been shown using a bovine-specific collagen I1 peptide (CB11 184-198), which delays the onset and reduces the severity of CIA when given prior to disease induction. The heterologous approach has also proven successful upon administration of a human collagen I1 peptide (CB11 250-270), which markedly reduces the severity of CIA in DBN1 mice and diminishes specific T cell proliferative responses (Khare et al., 1995). Other protocols with heterologous or synthetic peptides have also been successfully applied using, for example, chicken C B l l 133-146 and 181209 (Myers et al., 1989,1995);however, with the goal of protecting against heterologously induced CIA, these studies are less relevant to the question of treating autoimmune diseases caused by autoreactive T cells. It is difficult to draw general conclusions from the numerous studies on antigen-specific tolerization. Already the term antigen-specific is not precise because the occurrence of bystander suppression implies that an antigen-driven step is followed by an unspecific step in the effector phase. The term “tolerance” is also debatable in that active suppressor mechanisms also become involved in the process. In addition, the risk that mucosal tolerization aggravates autoimmune diseases has been considered much more carefully in recent studies. IDDM can also be precipitated by OVA feeding in C57BV6 mice that specifically express OVA under the control of a rat insulin promoter in the pancreatic islets (RIP-OVA mice). Upon OVA feeding, these mice develop diabetes and T cell cytotoxicity against pancreatic P cells (Blanas et al., 1996). Interestingly, however, diabetes develops only in bone marrow chimeras of irradiated RIP-OVA mice, a model designed to reproduce a particular genetic predisposition background characterized by enhanced frequency of precursor CD8+ cells with specificity for pancreatic islet antigens-a background by itself insufficient to cause spontaneous diabetes. The mechanism of antigen-induced bystander suppression is attractive for treating human autoimmune diseases because it is aimed at suppressing T cell responses to autoantigens without requiring the exact knowledge of their specificity and, at the same time, without inducing general immunosuppression. However, convincing immunohistological evidence for by-

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stander suppression in the target organ of autoimmunity has been shown thus far only in the CNS infiltrations of EAE. Oral tolerization also poses other major technical problems, such as the dose and the gastrointestinal breakdown of the antigen. In this sense, the nasal route offers a simpler and more reproducible environment for antigen application. However, despite the technical problems and the as yet undefined risks, “eating (or spraying) the way towards irninunosuppression” ( MacDonald, 1994) remains very attractive.

M

DO WE LEARN FROM APPLYING IMMUNOTIIERAPEUTIC I N EXPERIMENTAL M o i x u OF STRATEGIES A U T O I M M U N E D ISORD EH \ ?

WHAT

During the past decade, the ever-growing knowledge about basic mecliariisins of antigen recognition, T cell activation, cytokine patterns, and tolerance has led to a hitherto unprecedented abundance of means to modulate adverse autoimmune responses. However, despite such abundance, even relatively “simple” experimental inodels of autoimmunity remain difficult to treat. Indeed, only a few agents (and only on certain protocols) are effective in full-blown or chronic phases of established disorders. These few examples are the treatment of CIA with IL-4 + IL-10 or IL-1P-RA; that of lupus gloinerulonephritis with CTLA-4-Ig; the altered peptide ligation in EAE; the oral tolerization; the treatment with anti-B7-l Fab or the administration of a CD4 peptide analog in relapsing models of EAE. It seems encouraging that some inimunotherapies are effective at least in early effector phases of experimental disorders, for example, anti-TNFa treatment in CIA or anti-CD40L in EAE. If ever applicable in human autoiminune diseases, these treatments could be useful in handling recently diagnosed diseases. The majority of the immunotherapeutic agents prove beneficial only in preventive trials. Although we must appreciate that protective protocols enrich our view of possible mechanisms of disease suppression, especially those that prevent relapses of established disease, the main scope of preclinical research remains the identification of strategies that represent an objective advantage in the treatment of established human autoimmune disorders. In this respect, some of the studies reviewed provide evidence that combined therapy (whether additive or synergistic), anti-CD4 mAbs or IL-4 + IL-10, results in for example, anti-TNF-a much clearer effects than the single agents. The variety of substances currently available may turn useful for future investigations on optimal combination approaches. It is clear that combination therapy, similar to individual principles, must respect as much as possible the capacity of the immune system to attend to tumor surveillance and host defense against infectious agents. In this

+

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respect, antigen- and TCR-specific approaches would clearly be preferable because the desired immunosuppression remains selective. The theoretical roundness of specific intervention, in turn, fights the notion that even if the primary autoantigen of each disease should be known, the spreading of autoimmunity to secondary determinants during chronicity limits the applicability, if at all, to newly diagnosed patients. The knowledge that determinant spreading may follow predictable rules of hierarchy (Yu et al., 1996), on the other hand, may greatly simplify this problem. In EAE, for example, the predictability of the spreading cascade, and its invariant relationship to the occurrence of relapses, has been exploited to achieve epitope-specific blockade of the progression of the disease into a chronic phase (Yu et nl., 1996; McRae et al., 1995). Accordingly, the systemic administration of high doses of spreading encephalitogenic peptides (but not that of nonspreading encephalitogenic peptides) arrests the further progression of established EAE (Yu et al., 1996). This possibility, together with the finding that an altered peptide ligand can be effective despite the diversity of the T cell repertoire in the CNS lesions of EAE (Brocke et al., 1996), is therefore most encouraging. The application of peptidic determinants or altered peptide ligands via the nasal route offers additional possibilities for antigen-specific treatment. The broad application of such strategies still requires a more accurate knowledge about the lymphocyte specificities in chronicity, not only in the human autoimmune diseases but also in experimental autoimmunity. It remains unclear whether the genetic variability in human autoimmune disorders will ever allow the definition of predictable epitope hierachies; attempts to modulate the cytokine network or to interfere with costimulatory pathways may have more favorable chances to find their way into clinical application. The results of various treatments of autoimmune diseases bring to surface another general problem; that is, the remarkable plasticity of the immune system, conceivably developed during evolution. The sensitivity of the therapeutic response to contextual influences is becoming common experience in a number of studies; thus, opposite or apparently paradoxical effects can be achieved if the timing of the drug administration is varied or if the cytokine milieu is even slightly altered. Learning more about the contextual requirements of immunomodulation remains a major prerequisite in view of the therapeutic exploitation of immunotherapy. The experimental models of autoimmune diseases are clearly distinct disorders of highly structured autoimmunity; despite the fact that they share some immunopathogenetic pathways, they rely on quite different polygenetic backgrounds. Thus, the beneficial effects of a certain treatment in one model cannot necessarily be extrapolated to another. The increasing

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knowledge about the genetic background, which it is hoped will deepen through gene mapping of disease-susceptibility loci, will probably contribute to the definition of the differences in therapy response and may greatly help in the selection of the appropriate treatment protocols even for human autoimmune diseases.

ACKNOWLEDGMENTS Part of the work presented has been supported by research funds. The excellent secretarial help of Mrs. Martina Seidel is gratefully acknowledged.

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INDEX

A Actinomycin D and AU-binding factor, 31 and c$x 4RE, GM-CSF discrepancies, 21 and c$os mRNA, 28 effect on rnRNA decay, 2-,3, 7 IL-2 mRNA and, 11, 12 Adenosine triphosphate binding cassette, 191, 213-214, 239-240 dependent aminophospholipid translocase, 64 and necrosis, 73, 86 and permeability transition pore, 80-83, 86, 87. Adenosine-iiridiiie-rich elements, 19-22 Adhesion blocking, in autoimmune dispases. 365-371 integrin-dependent. 168 intracellular adhesion molecule-1, 26-27, 336, 367-371, 375 receptors, and CD45, 100 Adriamycin, 10,5, 224 Allergic encephaloinyelitis,see Encep~ialoniyelitis,experimental allergic Allergic reactions, 172-173 Allograft rejection, 179 Alzheimer’s disease, 23 Ainyloid protein precursor, 22-23, 35 Antibodies, w e also Monoclonal antibodies anti-CD3 5, 12, 13, 15 anti-CD23, 13 against 11,-1, 13 Antigens, see d s o Superantigen; specific antigen,Y differenti.ition expressed hy macrophages and monocytes, 227-298 419

and macrophage characterization, 298-303 endoplasmic reticulum processing of, 197-199 generation of peptides by, 199-205 and peptide transporter, 193-197 presentation of, and proteasome activators, 204-205 recognition of, and triggering of interleukin-2, 92 specific tolerance to, 389-395 stabilizing cytokine inRNA in lymphoid cells, 31. tumor-associated, 257, 284 Antigen-specific tolerance, 389-397 Apoptosis in anucleate cells, 70 as autoiinniune disease therapy, 337 calcirnn ions and, 52, 56, 64,65, 71 caspases role in, 57-61 CD45 role in, 98-101 cell structure changes, 54-55, 64 common pathway, 54-88 degradation phase, 54-65 effector phase, 65-88 executioner, 66-88, 120-124 genetic susc:eptihilityto, 68-69 inducers of, 53-54, 74 initiation phase, 89-122 magnesium ions and, 56 necrosis and, 55, 62, 72-73, 85-86 potassium ions and, 65 private pathways, 53, 63, 83, 89-122 Ras role in, 168 recognition of cells in, 56 regnlatory processes of, 51, 54 rescue from, 111-113 signal transduction pathways and, 122 zinc ions and, 55, 56.

420

INDEX

Arthritis, see also Rheumatoid arthritis collagen-induced, 369-371, 375-377, 393-394 non-rheumatoid juvenile, 352 Atopic disorders, 115-116 ATP, see Adenosine triphosphate Autoimmune diseases, see also specijic diseases

animal model studies, 365-397 anti-CD7 monoclonal antibodies and, 358-359 apoptosis and, 51, 100 CD30 and, 115-116 cytokine treatment, 337-347 interleukin-2 targeted approach to, 359-361 major histocompatibility complex and, 385-386 monoclonal antibodies and anti-CD4, 347-353 anti-CD5, 353-358 anti-CD7, 358-359 peptide transporters and, 226-228, 238-239 preventive therapy, 395 T cell receptor costimulation and, 381-385 AUUUA motifs AU-B and AU-C binding requirement, 34 in AU-binding factor, 30-31 in c-fos and c-myc mRNAs, 27-28 in ICAM-1, 27 long term repeats and, 22 in mRNA regulation, 5-8, 11-15, 19 Azathioprin, 356

B Baculovirus, 60, 120, 216-217 Basophils, 173 B cells anti-C5 therapy and, 353, 356 antigen-receptor signaling in, 111 apoptosis in, 96 Bcl-2, and mitochondrial function of, 75 and CD45, 98-99 CD69 and, 14 FcyRII in, 278 CM-CSF production, 8 IL-10 production, 13

immunoglobulin mRNA levels in, 17 lymphoma, and peptide transporter, 236 peptide transport and human, 239 protooncogene levels in, 5 Bcl-2 and antagonist, Bax, 83 and apoptosis, 69-70, 73, 76, 80, 91-92 associated proteins, 83-84 channel-like function, 74 genes for, 74, 91 mitochondrial permeability transition and, 74, 76, 80, 123 and necrosis, 73 and PC12 cells, 75 related proteins, 54, 83-84 and signal transduction modules, 107-111 h-globin, 19, 20, 25, 26 B7 molecules, 280, 382-385 Bone marrow cells CD34 progenitor, 6 deletion of derived, 89 primate stromal, 14 sialoadhesin in, 275 as source of F480 antigen, 272,273 Brain microglia, macrophages in, 272 Brefeldin A, 174, 196 Burn patients, 167

C CaaX substrates CaaX prenyltransferases, 152-157, 175 farnesyltransferase, 152-157, 164-165, 175-178 geranylgeranyltransferase type-I, 152-57, 177-178 type-11, 152, 158-162 Calcium ionophore, 5, 13, 15, 16, 31 Calcium ions in antigen-receptor-induced activation, 93, 95-97 in apoptosis, 64,65, 71 and AU-binding factor regulation, 32 and endonuclease activation, 56, 65, 71 and macrophage adhesion, 274-275 and mitochondrial membrane, 80, 83, 87, 96-97, 122-123 in thymocyte selection, 100-101 Calpains, 61, 62-63

INDEX

Camptothecin, 105 Candida albrcans, 282 Carboxymethylation,162, 172 Carcinomas breast, 23'7-238 cervical, 237 colon adenocarcinoma, 236 colorectal, 237 hepatocellular, 236 KB epidermal cells, 105 lung, non-small cell, 237 murine CMT64.5,237 pancreas, lung and colon, 149 peptide transporters and, 224, 236-238 prostate, and peptide transport, 237 small cell lung, 236 Caspases and anUCledte cells, 70 in apoptoais, 57-61, 70-72, 85, 88, 123-124 autoamplification in, 72 and Bcl-2, 111 inhibitors of, 60 and interferon-y processing, 72 and interleukin-@processing, 72 and mitochondrial membrane, 76 and necrotic cell death, 73 and permeability transition, 83 and phosphatidylserine, 64 subcellular localization of, 60 CD2, 16 CD3, 16, 34, 93-97, 100-101, 266 CD4 and antigen recognition, 93 and apoptosis, 97 in autoimmune diseases, 333-334, 347-352 and encephalomyelitis, 379-380 and Fv receptors, 259-261 and rheumatoid arthritis, 347-353 and T cell differentiation. 17 CD5, 111, :353-357 CD7, 358-,359 CD8, 93, 191, 193-194, 334, 380-381 CD11, 365--367 CD14, 295 CD16, 278--279 CD18, 369-371 CD28, 14, 111, 122, 261, 280 CD30, 114-116 CD31, 280-281

421

CD32, 278-280 CD38, 10, 91-92 CD44, 295-296 CD45, 15, 93, 98-101,296-297 CD51C, 356-358 CD52, 362-365 CD64, 276-278 CD71, 297 CD80, 280 CD86, 280 Cell cycle and apoptosis, 70 GGTase-I inhibitors and, 178 Racl and, 150 and Ras mutation, 151 Cell morphology, 149-50, 174 Cell surface receptors, mRNA regulation of, 14-16, 36 Ceramide, 101-102, 105, 120-122, 168 c-fus actinomycin-D and, 2-3 antigen receptor signal transduction and, 94 AUUUA motifs in mRNA, 19, 27 cis elements and, 18, 21 in fibroblasts, 27 instability detenninants in, 27-28 mRNA decay, 2, 21 protein binding to, 35 truncation of, 31 Chelation, 32, 64,71 Chemotaxis, impaired, 167 Cholesterol, 151, 175, 179 Chromatin condensation, in apoptosis, 54, 56, 63-64 Cisplatinum, 105 c-jun, 94, 105 c-myc and apoptosis, 69-70 coding region instability element, 35 and mRNA regulation, 5, 18, 21-22, 27-29 3' untranslated region of, and AU-A, 34 Collagen 11, 393-394 Collagenous receptors, 286-289 Con A, 11, 13, 15 Crohn's disease, 338, 342 C-terminal peptides anchored at, 220-225 processing events at, 151-164, 171-172

422

INDEX

Cycloheximide and apoptosis induction, 69 and AU-binding factor. 31 and CD45 isoforms, 15 and c-jun mRNA, 5-6 and GM-CSF, 15, 19 interleukins and, 11, 12, 13, 15 and lymphotoxin, 14 perforin mRNA and, 26 Cyclophilins, 55, 80 Cyclosporin A, 11-15, 80, 333 Cytochrome c, 74-78, 85-88 Cytokine receptors, 14-16,293-294 Cytokines, see also specijic cytokine and autoimmune disease therapy, 337-347 AUUUA motifs in, 22 AUUUUA motifs in, 13 deprivation of, 105 in experimental autoimmune diseases, 371-379 genes coding for, 93 irradiation effect on, 9 mRNA regulation and, 8-14, 17, 31, 36 network modulation experiments, 371-378 nucleotide sequence in 3’UTR of, 19 rescuing lymphocytes from apoptosis, 91 tumor necrosis factor effect on, 12. Cytomegalovirus, 205, 235, 240 Cytoplasm, 34, 54 Cytoplasts, 70, 75 Cytosol, 32, 71, 76, 86, 191

mitochondrial, 55 nuclear, 76 oligonucleosomal, 55, 56, 63-64 Doxoruhicin, 105, 224

Encephalomyelitis,experimental allergic adhesion blocking in, 365-367 anti-CD4 monoclonal antibody and, 379-380 antigen-specific tolerance in, 389-391 cytokines and, 371-374 spreading peptide administration, 396 T cell receptor costimulation and, 381-385 Endocytosis, 172-173, 278 Endonucleases, in apoptosis, 52-57, 124 Endoplasmic reticulum antigen processing in, 191, 197-199 export of peptides from, 233-234 peptide loading of class I molecules in, 205-209 peptide transport in, 192, 196, 214-215 Endosomes, regulation of, 174 Epstein-Barr virus, 115, 324 Erythroid precursors, 26 Escherichia coli, 30, 324 Etoposide, 105, 224 Exocytosis, 172-174

F Daunorubicin, 107, 119 Deadenylation, 21, 28 Death-inducing signaling complex, 116-119 Degrannlation, 172- 174 Diabetes mellitus, insulin-dependent and albumin-induced CD8+ T cells, 334 animal studies in, 367-369, 380-381, 383-386, 394 anti-TNF-a! experiment on, 374-375 and ICAM-1, 367-369, 375 interleukin-2 targeting, 359-361 peptide transporter and, 238-239 Differentiation, in thymus, 17-18, 89-91 DNA fragmentation of, 52, 54, 55-56

Farnesyltransferase, 152-157, 164-165, 175-178 Fa as apoptosis inducer, 53, 61, 92 caspases and, 57, 61, 72 role in apoptosis, 114-116 surface receptor, 100 TCWCD3.94 FcyR monoclonal antibodies, 276-279 Fibroblasts and apoptosis, 91 Bcl-2 and mitochondrial function, 75 c-fos mRNA, 5, 27 cycloheximide and, 6, 11 cytomegalovirus, peptide transport and, 235 FTase inhibitors in, 176

423

INDEX

GM-CSF production, 8, 9 phorbol e::ter and, 11 prenylated proteins in, 174 protein production, 9 Rho proteins and, 150 Rs mRNA in 3T3, 27 TNF-a and, 11 transition from Go to S, 27 ultraviolet light on, 27 Fibrosarcoid cells, 87 Fibrosarcoma, murine, 237 Fluorochronles, 77-78 F480 monoclonal antibody, 272-275 FTase, see Famesyltransferase FV receptors, single chain, 258-266

Galectin-1, 99-100 Genes apoptosis-inhibitory, 74 involved in T cell activation, 93 killer, 68-69 LMP2 and LMP7, 203, 210 and MHC class I-bound peptides, 193 posttranscriptional regulation of, 1-49 regulation of peptide transporter, 209-211 and rheumatoid arthritis, 315-332 rheumatoid arthritis susceptibility, 315-317 Saccharornyces cereoisiae, 157 sialodhesin, 276 US6, 235 Geranylgeranyltransferase type-I, 152-57, 177-178 type-11, 1!52, 158-162 Glomerulonephritis, 377-378 Clucocorticoids. 7, 12, 56, 71, 75 Glutathione. 25, 64-65, 70 GM-CF, see Granulocyte-macrophage colony-stimulating factor Granulocyte-macrophage colony-stimulating factor Act-D and, 2, 19 anti-CD2R plus anti-CD3 and, 11 antigen and, 8 AU-binding factor and, 33, 34 AUUUA irepeats in mRNA, 19-20 calcirim flux and, 10 and c-kit, 6

irramation and, 9 mitogenic anti-cell surface antibodies, 8 protein secretion levels, 13 3’ untranslated region of, 34 TNF-a, TPA or cycloheximide and, 14 Granulocytes FcgHII and FcgRIII in, 278 Ras activation in, 167 Granulomatous disease, chronic, 169 Granzyme B, 70, 72 Growth factor, 70, 71, 84, 108-110, 111 Guanine nucleotide exchange factor, 147, 164, 166 Guanine nucleotides, 93, 147, 148, 164, 166, 169-170 Guanosine triphosphatase, 147-148, 164-165, 173-174

H Hamsters, Syrian, and TAP, 211-213, 225-228 Heart, 86, 87, 284 Heat shock, 105, 122, 197, 205; see also Bum patients Hematopoetic progenitor cells, murine, 6 Hepatitis, 165-166, 352 Hepatocytes, 87 Herpes simplex virus, 234-235, 240 HL-60 cells, 62, 75 Hodgkin’s lymphoma, CD30 and, 115, 119 Human immunodeficiency virus, 97, 115, 258 Hybridomas, 17, 97, 261, 266 EIyperoxia, 25 Hypoxia, 14, 53, 71

1 ICAM-1, see Intracellular adhesion molecule-1 IL-1, see Interleukin-1 IL-2, see Interleukin-2 IL-3, see Interleukin-3 IL-4, see Interleukin-4 IL-6, see Interleukin-6 IL-7, see Interleukin-7 IL-10, see Interleukin-10 IL-11, .see Interleukin-11

424 IL-12, see Interleukin-12 IL-13, see Interleukin-13 Immunodeficiency, 51 Immunoglobulin E receptors, 172-173 Immunoglobulin G , 278-279 Immunomodulation, context of, 396-397 Inflammation ICAM-1 and, 27 and IgE receptors on mast cells, 172-173 mediators of, 19, 121, 172-173, 282 and Ras, in granulocytes, 167, 169-170 selectins and, 285 stimuli of, 284 tumor necrosis factor and, 12 Inflammatory skin diseases, 179 Influenza vinis, 194, 197, 199 Insulin, 111 Insulin-like growth factor 11, 21, 25 Integrins, 100, 168, 291-293 Interferon-a, autoimmune effects of, 346 Interferon-p, 10, 342-343 Interferon-y, 346, 375-377 AUUUA repeats in, 19 CD23, IL-4 and, 16 and c-fos mRNA, 5 and c-myc mRNA, 5 and encephalomyelitis,373-376 and GM-CSF mRNA, 8 and ICAM-1 mRNA, 27 IL-7 and, 10 inducing factor, 72 and macrophage B7 levels, 280 and monocyte microbial activity, 300 and peptide transporter, 210 and proteasomes, 202-203 receptors for, 15 and TAPAMP defects in lung carcinoma, 236 T cell regulation and, 8 and TNF-a message, 12-13 Interleukin-1 and collagen 11 arthritis, 377 ligation to IL-1 receptor, 15 PKC-dependent stabilization of, 11 production of, 10-11 and rheumatoid arthritis, 339, 340, 341, 344 in thymic epithelial cells, 17 Interleukin-2 antigen-recognition and triggering of, 92

INDEX

and apoptosis in T-lymphocytes, 91-92 and autoimmune disease therapy, 359-362 and encephalomyelitis animal model, 371, 379 and LAK cells, 265 in MLA-144 and FL5.12, 22 in natural killer cells, 26 receptor, 166, 359-361, 379 recombinant, 13, 261-262 and regulation of mRNAs, 6, 8, 9, 11-12, 13 and serinelthreonine PKB, 111 3' untranslated region of, and AU-A, 34 and transferrin receptors, 16 viral insertions in 3' UTR region of, 31 Interleukin-3, 6, 13, 22, 31, 108-109 Interleukin-4 receptor, 15 and regulation of mRNAs, 6. 10, 12-13, 16 and rheumatoid arthritis, 345-346 Interleukin-6 receptor, 15-16 and regulation of mRNAs, 13-14, 15 and rheumatoid arthritis, 340, 344-345 in thymic epithelial cells, 17 Interleukin-7, 10, 13 Interleukin-10, 338, 345, 373, 376 Interleukin-11, 14 Interleukin-12 blockade of, 10 and collagen 11 arthritis, 375-376 and encephalomyelitis, 372 with natural killer cells, 10 and perforin in NK cells, 26 recombinant, 10 Interleukin-13, 345, 376 Iron, 16, 21-22 Iron response element, 24, 32 Irradiation, 7, 9, 12, 102, 119, 122; see also Radiotherapy Ischemia, 122 Intracehlar adhesion molecule-1, 26-27, 336, 367-371,375

J JNK, see Jun-N-terminal kinase Jun-N-terminal kinase, 93, 150 Jurkat cells, 11, 30, 56, 96, 105

425

INDEX

Kaposi’s sarcoma-associated human herpes virus-8, 60 Kavorrhexis 54 Kidneys, 87, 377-378 Kinases, 61, 101-107, 111-113, 122, 149; see also Protein kinase B; Protein kinase C Kupffer cells, 272, 300

L Lactacystin, 6 - 6 2 , 199

LAK cells. see activated killer ceUs; Lymphcikine Lamins, 61, 63 Langerhans #cells,272, 273, 300 Lectins, 99-100, 281-285 Leukemia, 51, 149, 224, 236 Leukocytes homeostasis mechanism of, 91-93 and methylation of prenylated proteins, 171- 172 prenylation inhibitors affecting, 178-179 Hab proteins and, 172-174 Rho/Rac pathway and, 168-169 skeletal organization of, 168-169 tethering, 285 trafficking, 27 Lipids, 14-15, 65, 150, 171 Lipopolysaccharides and clfos mRNA, 5 and chemokine mRNAs, 14 and macrophages, 10, 12-13, 172, 280 stabilizing cytokine mRNA, 31 Liver, 87, 272, 273, 275; see also Hepatitis Lungs, 105, 120, 284 Lymphadenopathy, 51, 100 Lymph nodes, as source of F4/80 antigen, 273 Lymphoblastoid cells, 207, 225 Lymphocytes; see also B cells; Lymphoid cells; T ceUs apoptosis in peripheral, 78 apoptosis versus necrosis in, 86 AU-binding factor in resting, 31 CDC42 and, 150 homing, 285 hyperplasia, 51

permeability transition pores in, 87 Racl and, 150 RAG-1 and RAG-2 genes in, 17-18 tumor-infiltrating, 258 Lymphocytic choriomeningitis virus, 392 Lymphocytopenia, 351 Lymphoid cells Burkitt, 236-237 cytokine mRNA stabilization in, 31 EL4,and Fv receptors, 259-266 mannose receptor macrophages and, 284 inRNA degradation in, 17 order of apoptosis events in, 78 Lymphokine-activatedkiller cells, 257-258, 262-266 Lymphomas, 28, 51, 236, 259 Lymphotoxin, 14

M Macrophages differentiation antigens and, 271-314 interleukin-4 and murine, 12-13 lipopolysaccharide-activated serum and, 10, 172 prenylated proteins in mnrine, 174-175 in skin, 284 Macrosialin, 273, 289-91 Magnesium ions, 32-33, 56, 80 Major histocompatibility complex chss I, 191-199, 205-209, 225, 239, 281 class 11, 191, 193, 300, 315-332, 385-386 peptide antigens and, 191 and rheumatoid arthritis susceptibility, 315-332 and T cell selection, 89 Mannose receptors, 281-284 Mast cells, 13-14, 172-173, 278-279 Measles virus, 115-116 Membrane binding, 162-164, 170 Membrane molecules, macrophages and, 257-314 Methotrexate, 340, 351, 356 Methylation, 162-163, 1M-65, 171-172 MHC, see Major histocompatibility complex Mitochondria apoptotic membrane changes, 74, 76. 87, 123 BCL-2 in, 110

INDEX

calcium and, 87, 96-97 ceraniide effed on, 122 as executioner, in apoptosis, 73-88, 123-124 in thyniocytes, 91 Mitogen-activated protein kinase, 148-149 Mortoclonal antibodies anti-B7-l and anti-B7-2, 382-384 anti-CD2, 384-385 anti-CD4, 333, 347-353, 379-380 aIlti-CD5, 353-358 aIlti-CD7, 358-359 anti-CD52, 362-365 anti-tumor necrosis fiactor-a. 338-342 and rheumatoid arthritis, 336, 338-342, 344-345, 347-359 against tumor antigens, 258-266 Monocytes ainyloid protein precursor in, 23 CD2 mRNA levels in, 16 CFS- 1 in phorbol-stirnulated, 15 differentiation antigens expressed by, 271-298 F4/80 in peripheral blood, 272 GM-CSF production, 8 interleukins and, 13, 15 preiiylated proteins in human. 174 protein kinase C and, 11 Mouse antigen differentiation in, 271-314 arthritis studies in, 339, 340, 369-371, 375-377, 393 BCL-2 studies in, 107 CD30 experiments in, 116 diabetes studies in, 374-375, 380-381, 383-386, 392, 394 adhesion molecule blocking, 367-369 cytokiiie modulation and, 374-375 oral albunrin and, 334 FTase inhibitors i n tumors in, 176 FV receptor studies in, 259-266 tnacrophage and niyeloid dendritic cells in, 271-314 peptide transport and, 192-195, 211-213, 225-228, 236, 237, 239 protein tyrosine kinase studies in, 97-98 protein tyrosine phosphatase studies in, 99-100 sphingoinyelinase studies in, 120

stress kinase studies on, 106 systemic lupus in, 337, 377-378, 384-385 mHNA ainyloid protein precursor, 22-23, 35 AU-containing, 19-20, 34, 35 chimeric, 20-21, 25 cis elements, 18-29 coding for CD isoforms, 15 cytokine, 8-14, 19, 31 decay rate, 1-3, 6, 21 in developing thymus, 17 differentiation of, 18, 29 posttranscriptional control, 4-5 in T cells, 1-50 trans factors, 29-36 translation measurenient, 3-4 Mucins, 290-291 Multiple sclerosis anti-CD52 therapy and, 364 CD4 monoclonal antibody and, 352 interferon-p treatment of, 342-343 peptides and, 238, 387 Mutagenesis, 23, 24, 26, 27, 31 Mycobacterium tuhercfdosis, 195, 282 Mycoplasma penetrans, 56 Myelin hasic protein, 389-392 Myeloid cells, 278-279, 282 Myeloid mast cell progenitors, 6 Myeloma, 28 Myocardiocytes. 86, 87

N Natural killer cells as apoptosis inducers, 53 CD69 and, 14 FcyRIII in, 278-279 and interleukin-2, 26 peptide transporter and, 194, 236 perforin in, 26 Necrosis, 55, 62, 72-73, 85-86 Neurons, 72, 86, 87 Neutrophils IL-1 and TNF-a effect on, 11 impaired cheinotaxis in, 167, 169 impaired phagocytic function in, 167 Hab proteins and, 173-174 Ras activation in, 167 Nitric oxide, in apoptosis, 60, 80, 123

INDEX

Nonlyinphoid cells, apoptosis in. 78 Nucleolin. 35--36 ~uclerls apoptosis and, 54. 70, 76, 85, 122 endonuc1ea:ies from, in uitro, 56 ribonuclear proteins of. 35

Oncogenes, products of, 54 Organelle nltrastructrlre, apoptosis and. 55 Oxygen, rract;ve species and apopto:;is, 64-65. 70-71, 76, 77 and perinea.bility transition pore, 80, 123

P Peptides; .see t~lsoPeptide transporter altered, in T cell receptors, 386-:389 antigenic, 199-203 arthritogenic shared epitope, 318-326 binding and transport assays, 216-219 effect o n T cell clones, 14 export from endoplnsinic reticulum, 233-234 and perinetbility transition pore. 80 photolahile, 231-233 nibella-derived, 322 selectivity and specificity. 217, 218-228 targeting of, to transporter, 205 Peptide transporter, 191-255: see also Peptides antigen presentation and, 193-197 biochemical characteristics of, 228-23 1 cheinotherzipy resistance and. 224 deficiency, 192, 194, 209, 235-236 diseases, 2214-239 genes for, 209-211, 235 kinetic parameters, 228-229 molecular structure, 214-215 nucleotide usage, 229-231 peptide binding and transport assays, 216-219 as peptide ,supplier for MHC molecules, 192-1!>9 proteins of human, 211-213, 22.5, 227 species differences, 211-213, 219, 225-228 structure and fiinction, 231-2

427

Permeability transition pore. 74, 80-88. 110, 123 Peyer’s patches, macrophages in, 273 p59’”’,93, 95, 96, 97-98 p53, 7, 69-70, 91 Phagocytes, phospliatidylserine receptors on, 64 Phagocytosis, 167, 282 Phosphatidylserine, 64,70. 76. 80 Phospliolipase C-71, 9-3-95 Phosphorylation, :32. 35, 86. 93, 109-110 7, 10, 17, 31. 35 Pli~~ohemaglutinin, Pituitary cells, 173 PKB, see Protein kinase B PKC, sce Protein kinase C Plasma cells, 17; see also Lymphoid cells Plasma ineinbrane, 54-55 64, 65, 164 Platelet-derived growth factor, 8 Platelets, 14, 169, 172 Ynetcnzncystis carinii, 282, 3f;l Polychontiritis. 352 Polysolnes, 4, 21, 33, 35 Potassiiim ions, in apoptosis. 65, 95, 175 Prenylation, of Has GTPase proteins, 145-189 Prenykransferase, 152- 157, 175-178 Procoagulant enzymes, 64 Prostaglandins, 13, 345 I’roteases. 54, 61-64, 199-200; see &J Caspases; Proteasomes Proteasomes, 61-62, 199-205 Protein l)inding, 22-23 Protein kiirase B. 111-113 Protein kinase C, 11, 12. 19, 32, 9.5 Proteins AIF. 85, 87, 88 in apoptosis, 54, 74, 76, 85, 124 Anf-1, 33-34 A U U U A HNA-binding, 30-35 cataluse-specific, HNA-binding, 25 cytokine and protooncogene inHNA-binding, 35 digestion of, by caspases, 60 heat shock, 197, 205 interactions with mHNA, 22-23, 29-30 myelin basic, 389-391 non-AUUUA RNA-binding, 35-36 of permeability transition pore. 80 prenylation of Has GTPase C-terminal methylation of, 157-1.58, 171- 172

428 heterotrimeric G, 164-165 Rab, 148, 158-162, 165-166, 172-174 Rac, 147, 169-170 Rap, 169-170 Ras, 146, 148-149, 163-164, 166-168 Rep, 161-162 Rho, 147, 171, 174, 177-178 Rho/Rac, 148-150, 168-169 Protein synthesis, 15, 16, 19, 36 Protein tyrosine kinases, 93, 97-98 Protein tyrosine phosphatase, 93, 98-101 Proteolysis, 163 Protonophores, 80 Protooncogenes, 5-8, 19, 22, 27-29, 36; see also specific protooncogenes Psoriasis, 238, 357, 361 FTK,see Protein tyrosine kinases Pyknosis, 54

Rab proteins, 148, 158-162, 165-166, 172- 174 Rac proteins, 169-170 Radiotherapy, 53 Rapamycin, 9, 12, 333 Rap proteins, 169-170 Ras-GAP, 94 Ras proteins activation hy GTPase cycle, 147-149 in apoptosis, 168 cancer, and mutation of, 177 C-terminal events and, 163-164 function in immune cells, 146, 164 growth inhibition and, 167 prenylation of, 150- 152 Rho-Rac pathway, link to, 148-149 signaling pathway of, 146, 166-168 Ras superfamily, 145-157 Rauscher virus, 194-195 Reactive oxygen species, in apoptosis. 64-65, 70-71, 77, 123 Redox status, apoptosis and, 54, 64-65, 70-71, 88 Reiter’s syndrome, 238-239 Rep proteins, 161-162 Respiratory burst, 169 Respiratory chain inhibitors, 80 Retina, 161, 164

INDEX

Rhabdomyosarcoma cells, 105 Rheumatoid arthritis animal studies on, 339, 340, 369-371 anti-CD4 therapy, 349-353 anti-CD5 therapy, 356-358 anti-CD7 therapy and, 358-359 anti-CD52 therapy and, 362-364 anti-116 therapy and, 344-345 anti-TNF-a therapy, 338-341 apoptosis induction therapy, 337 CD30 and, 115-116 CD51C trials in, 356-358 cytokines and, 345-347 erosive versus non-erosive, 327 gene therapy, 337 and ICAM-1,369-371 interferon-y therapy, 346 interleukin-1 blockade and, 339, 341, 344 interleukin-2 targeting therapy, 359-360 juvenile, and oral collagen, 335 monoclonal antibody therapy, 336, 347-353 prognostic indicator for, 326-327 role of peptide transporter in, 238-239 susceptibility genotype, 315-332 Rho proteins, 147, 171, 174, 177-178 Rhomac proteins, 148-150, 160-161, 168-169 Ribonucleases, 20, 31 Ribonucleotide reductase, 24-25, 35, 36 Ribosomes, 20, 24

Schizosaccharomyces pomnbe, 56 Selectins, 285 Sendai virus epitopes, 194, 196 Septic shock, 12 Serine proteases, 61, 62-64 Serine-threonine kinases, 6, 93, 101, 121 Serine threonineRKB, 111-113 Serine-threonine protein kinases, 120 Serine-threonine tyrosine kinases, 6, 93, 120-121, 122 Sialoadhesin, 275-276 Signal transduction pathways antigen receptor-mediated, 93-95 in apoptosis and apoptosis-resistance, 122 Bcl-2 and modules of, 107-111

INDEX

Kas role in, 146-147, 166-168 resulting in permeability transition, 83 surface receptor to death effector, 89 Skeletal muscle, macrophages in, 284 Skin, macrophages in, 284 Somatostatin, 95 SOS, see Guanine nucleotide exchange factor Sphingomyelin, 120-122, 168 Spleen, 272, 275-276 Splenocytes, 56, 239, 262-266, 273 Splenomegaly, 51 Staphylococcus atireus, 351 Staurosporine, 69, 70, 71, 75 Stress fiber formation, 150 Stress kinases, 101-107 Substance P; 11-12 Superantigeris, 97 Superoxide production, 25, 85 Systemic lupus erytheinatosus animal studies of, 337, 377-378 CD30 and, 115-116 CD51C studies on, 357 CD4 monoclonal antibody and, 352 and tumor necrosis factor, 338

T T. acidophihtm, 200 TAP, see Peptide transporter Tapasin, 207-208 T cell hybridomas, 78-80, 97 T cell-mediated killing, 27 T cell receprors antagonists, and autoimmune disease, 386-,387 and CD4, 381-385 CD3 and, 16, 34, 93-96, 100-101, 266 and CD2H costirnulation, 12 costimulalion, interference with, 381-385 Ras pathway and, 166-168 and rheumatoid arthritis susceptibility, 322-324 self-recognizing, 89-91 and thymocyte deletion, 68-69 transgenic 11-Y, 97, 99 T cells activation 8-9, 16-17 antigen-receptor signaling in, 111 apoptosis in, 51-144

429

as apoptosis inducers, 53 Bcl-2, and mitochondria1 function of, 75 CD8+and, 191, 193-194 CD 45 and. 98-101 CD4 monoclonal antibody and, 348 cytoskeletal defects in, 169 differentiation of, 17, 89-91 elimination of, and rheumatoid arthritis, 353-358 Fc receptors in, 278-279 FV receptors and, 258-266 GM-CSF production and, 8, 258 HLA class 11 expression in, 16-17 homeostasis of, 91-93 interleukin-lWNK effect on human, 10 and interleukins, 13, 14 major histocompatibility complex molecules and, 89-91 memory, 91-92, 285, 356 mHNA regulation and function of, 1-50 peptides from intruders and, 191, 230 pokeweed mitogen effect on, 5 protooncogene levels, 5 Kas pathway in, 166-168 self-reactive, 93, 321 specific antigen recognition, 89, 93 transferrin receptors in, 16 tumor eradication and, 257-258 tumor necrosis factor and, 14 vaccination with, 387-389 TCWCD3 receptor complex, 93-97, 100-101, 266 Tetracycline, as mRNA promoter, 3 Thalidomide, 13 Themnoplasma acidophilum, 200 Thymocytes antigen-specific activation of, 95-96 apoptosis in, 78-80, 90-91 Bcl-2, and mitochondria1 function of, 75 CD8' and, 97 CD45 and, 99-100 chromatin condensation in rat, 56 differentiation and selection of, 17-18, 89-91, 106-107 etoposide effect on, 62 gfucocorticoid-treated, 52, 56, 57, 61-62, 68-69 immature, and thymic stromal cells, 95 major histocompatibility complex and, 89-91,97

430

INDEX

mitochondria1transmembrane potential, 91 positive and negative selection, 89-91 with self-recognizing T cell receptors, 89-91 sphingomyelinase and, 120 T cell receptors and, 68-69, 95-97 thymic stromal cells and, 95 Thymus, 17, 115, 239; see also Thymocytes Thyroid disease, 346, 371 Tissue destruction, autoimmune, 368, 378 T lymphoma cells, murine, 7, 193, 195 Tolerance, antigen-specific, 389-395 Toxins, 53, 122, 359-361 Transferrin receptors, 15-16, 24, 297 Transforming growth factor ligation of receptor, and apoptosis, 53 transforming growth factor a, 17 transforming growth factor b, 6, 24-25, 36, 377, 378 Tumor necrosis factor and cytokine stimulation, 12 and encephalomyelitis,373-374 sphingomyelinases, ceramide and, 120-122 and TCWCD3-triggered thymocyte apoptosis, 91-92 Tumor necrosis factor-a; see also Tumor necrosis factor; Tumor necrosis factor receptor Actinomycin D and mRNA of, 19 in activated murine T cell clones, 14 apoptotic effect in thymocytes, 114 AU-A protein and, 34 in collagen 11 arthritis, 377 in Crohn’s disease therapy, 342 effect on interleukin-1 mRNA, 11 ligation of, and apoptosis, 53, 72 mRNA regulation in, and T cell activation, 8-9 NF-KB activation and, 119-120 produced by T cells with Fv receptor, 258 and rheumatoid arthritis, 338-342 in systemic lupus, 338 Tumor necrosis factor receptor, and apoptosis in thymocytes, 114-116 Tumors; see also Carcinoma; Lymphoma and adoptive immunity, 257-269 CD30 and, 115 FTase inhibitors and, 177-179 Ioss of TAP expression in human, 236-238 lymphocyte-derived,51 macrophages, binding to, 284

Tyrosine kinases, 6, 17; see also Protein tyrosine kinases Tyrosine phosphorylation, 17, 93

Ubiquitin system, 204 Ultraviolet light, 27, 102, 103, 105 U-rich sequence-binding proteins, 34 Uveitis, experimental autoimmune, 371

v Vaccination, T cell receptor peptide, 387-389 Vasculitis, 352, 364 Vesicular transport, 174 Vinblastine, 110 Vincristine, 110, 224 Viruses, see also specijic virus as apoptosis inducers, 53 caspase inhibitors in, 60 and cytotoxic T cells, 53 peptide transport inhibition by, 234-235 and T cell death, 93 VP-16, 62, 105, 224

Wiskott-Aldrich syndrome, 169

Y Yeast a-mating factor, 151 endoplasmic reticulum protein in, 198 genes for prenykransferases of, 152 methylation in, 157 prenylated proteins in, 175 proteasomes of, 200-201 Ras2 and adenylyl cyclase, 164

z Zinc, 55, 56, 156, 157 Zinc finger proteins, 32

CONTENTS OF RECENT VOLUMES

Volume 64

Volume 65

Proteasomes and Antigen Processing NF-ILG and NF-KB in Cytokine Gene KElJl TANAKA, NORUYUKI T A N ~ H A S I I I , Hegulation CHIZUKO T5UKUMI, KIN-YAY0KO.I.A. A N D SHIZUO AKIKAA N D TADAM~TSU KIStIIMOTO NAOKI SIIIMRAHA Transporter Associated with Antigen Processing TIMELLIOTT

Recent Advmces in Understanding V(D)J Recombination MAKTI bt GELI .E HT

NF-KB as a Frequent Target for Inirnuno~uppressiveand Anti-Inflammatory Molecules PATKICK A. BAEUENLE A N D VIJAY R.

The Role of Ets Transcription Factors in the Developnrent and Function of the Mammalian Immune System A L E X A ~ D Ec. H BASSllK A N D JEFFHEY M. LEIDEh

BAICIIW4L

Mouse Mammary Tumor Virus: Immunological Interplays between Vinis and IIost A. LUTIIEH AND H A MACIIASANJIV OKBEA

Mechanism of Class I Assembly with PMicroglotlin and Loading with Peptide A N D DAVID R. LEE TEDH HANSEN Ilow Do Ljmphocytes Know Where to Go?: Current Concepts and Enigmas of Lymphocyte Homing MAHKOSALMI AND SIRPA JALKANEN

IgA Deficiency PETEHD. BUKKOWS A N D MAXD. COOPER

Plasma Cell Dyscrasias NOHlIii KO NISIIIMOTO, SACIIIKO SUEMATSU, A N D TADAMITSU KISIIIMOTO

Role of Cellular Immunity in Protection against HIV Infection SAKAII ROWLAND-JONES, RUSUNGTAN, A N D ANDREWMCMICHAEL

Anti-Tumor Necrosis Factor-a MAHCFELDUANN, MI(:IIAELJ. ELLIOTT, JAMES V . WOODY, AND RAVINUEK N. MAINI

High Endothelial Venules: Lymphocyte Traffic Control and Controlled Traffic KKAALA N D REINAE. MEBIUS GEOKC

INDEX

INDEX

431

432

CONTENTS OF RECENT VOLUMES

Volume 66 The Role of CD45 in Signal Transduction LOUISB. JUSTEMENT

The Intrinsic Coagulatioflinin-Forming Cascade: Assembly in Plasma and Cell Surfaces in Inflammation ALLENP. KAPLAN,KUSUMAM JOSEPH, Y O J ~ SHIBAYAMA, SESHA REDDIGARI, BERHANE CHEBHEHIWET, AND MICHAEL SILVERBEHC

HLA Class I1 Peptide Binding Specificity and Autoimmunity JUEHGEN HAMMER, TIZIANA STURNIOLO, CDW Cells in Human Immunodeficiency AND FRANCESCO SINIGAGLIA Virus Type I Pathogenesis: Cytolytic and Noncytolytic Inhibition of Viral Replication Role of Cytokines in Sepsis OTTO0. YANGAND BRUCED. WALKER C. ERIKHACK, LUCIEN A. AAHDEN, AND LAMBERTUS G. THIJS INDEX Role of Macrophage Migration Inhibitory Factor in the Regulation of the Immune Response CHRISTINE N. METZ AND RICHARD BUCALA

Volume 67 CUMULATIVE INDEX

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