Lymphotoxin and Nancy Ruddle1 and Carl F. Ware2,* 1
Department of Epidemiology and Public Health Immunology, Yale University School of Medicine, 815 LEPH, New Haven, CT 06520-8034, USA 2 Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA * corresponding author tel: 858-678-4660, fax: 858-558-3595, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.05004.
SUMMARY Lymphotoxin (LT) and LT are members of the TNF superfamily of ligands that function as essential elements in the organization of lymphoid tissue and initiation of innate and acquired immune responses. LT and LT genes reside in the major histocompatibility complex forming a tripartite locus with TNF. LT is secreted as a homotrimer, but also assembles with LT into a membrane-anchored heterotrimeric complex of 1 2 stoichiometry. LT 3, like TNF, binds TNF receptor type I (55±60 kDa and TNFRII (75± 80 kDa), whereas LT 1 2 binds the LT R. The LT R also binds LIGHT, a related cytokine that signals via HVEM (HveA), and HVEM also binds LT . LT produced by activated T, B, and NK cells mediate inflammatory processes important for immune response to certain viral and bacterial pathogens. Mice deficient in LT or lack formation of lymph nodes, Peyer's patches and have disorganized microarchitecture in the spleen leading to poor immunoglobulin production. The differentiation of natural killer (NK) cells and NK-T cells are impaired in LT-deficient mice leaving them unable to control certain tumors. Lymphotoxins have unique roles, but function in some cases with TNF and LIGHT cytokine systems as an integrated network that orchestrates multiple developmental processes and immune responses.
and Granger (Granger and Williams, 1968) in 1967 as a secreted cytotoxic/cytostatic factor produced by activated lymphocytes. Supernatants from cultures of antigen- or mitogen-stimulated lymphocytes demonstrated a cell-independent cytotoxic activity, one of the many discreet molecules with a broad array of activities collectively known as lymphokines. The cytotoxic activity of LT was extremely similar to the macrophage-produced cytotoxic factor defined by Old and colleagues, known as tumor necrosis factor (TNF) (Old, 1985). Molecular cloning in 1984 revealed that LT and TNF were structurally related molecules, which are now recognized as the prototypic members of a large superfamily of cytokines (Gray et al., 1984). Nearly a decade later, a distinct cell surface form of LT was defined by Ware and Browning (Browning et al., 1993) as a heterotrimer comprised of a subunit of LT with a dimer of a 33 kDa protein, designated LT , the third member of the family.
Alternative names
BACKGROUND
The soluble LT is known as LT , previously called TNF . The cell surface form of LT is comprised of two subunits, LT and LT . Two distinct heterotrimers are formed, LT 1 2 (major form) and LT 2 1. For the purposes of cataloging human genes, members of the TNF ligand and receptor superfamilies have been assigned numerical indicators (http://www.gene.ucl.ac.uk/users/hester/tnftop.html). LT alias is TNFSF1; LT is TNFSF3.
Discovery
Structure
Lymphotoxin (LT) was discovered simultaneously by Ruddle and Waksman (Ruddle and Waksman, 1968)
LT is a secreted glycoprotein of 171 amino acids with a secondary structure of antiparallel sandwich
436 Nancy Ruddle and Carl F. Ware structure that assembles into a relatively compact trimer, a feature common to all members of the TNF superfamily. LT is a type II transmembrane glycoprotein (N-terminal cytosolic tail) that lacks a signal cleavage sequence and is retained in the membrane. LT is biologically active as a heterotrimer with LT . Molecular modeling predicts that LT also assumes a secondary structure similar to LT , TNF or CD40 ligand. The LT ligand remains anchored to the cell membrane and as such, acts in a localized fashion that requires cell-to-cell contact. By contrast, LT lacks a retained transmembrane domain and is exclusively secreted as a homotrimer. Species conservation between mouse and human LT and LT is 75%.
Main activities and pathophysiological roles The immediate TNF family consists of four structurally related ligands: lymphotoxin (LT) , LT , TNF, and LIGHT, and their four receptors: TNFRI, TNFRII, LT R, and HVEM (Figure 1) (Smith et al., 1994; Ware et al., 1995, 1998; Wallach et al., 1999). Figure 1 The immediate LT family. Depicted are the individual cytokine systems that utilize the common set of receptors which defines members of the immediate LT family. Arrows indicate high-affinity ligand±receptor interactions, the dashed line indicates the low-affinity binding of LT to HVEM. HSVgD is the envelope glycoprotein gD of herpes simplex virus. DcR3 is the same as OPG2 or TR6 and exists as a soluble product that also binds Fas ligand. Not pictured is LT 2 1 as the functional significance is not known, however it binds TNFRI and TNFRII.
A system is defined as a specific ligand and receptor pair, however several ligands and receptors have more than one cognate. The LT and TNF systems function as critical factors for the organization of lymphoid tissue required for efficient cellular and humoral immune responses (Fu and Chaplin, 1999). The role of the LIGHT/LT /HVEM system is less studied at this point. The receptors show overlapping, but unique ligand-binding patterns. The LT cytokine system is comprised of two gene products, LT and LT , that form three distinct ligands, a secreted homotrimer of LT (LT 3), and two membraneanchored heterotrimers, LT 1 2 and LT 2 1, that have unique receptor specificity. LT 3, like TNF, binds two receptors, TNFRI and TNFR2. By contrast, LT 1 2 signals via LT R, whereas LIGHT interacts with HVEM, an entry factor for herpes simplex virus, as well as the LT R. HVEM is also a third receptor for LT . LT 2 1 binds TNFRI and TNFRII and with low affinity to LT R. This complex pattern of ligand-binding interactions indicates the highly integrated nature of the physiologic activities of these cytokines. Most strikingly, mice genetically deficient in LT and have defects that affect developmental processes (failure to form lymphoid organs) and have differentiative effects on the immune system. The LT complex is required for the formation of peripheral lymphoid tissue (lymph nodes and Peyer's patches), as well as the normal segregation of T and B lymphocytes into distinct compartments in the spleen, the formation of germinal centers. The failure of these tissues to form and organize may be due to the loss of common progenitor cells for NK cells, NK-T cells, and the Peyer's patch inducer cells. In contrast, TNF deficiency leads to defects in germinal center formation and a profound loss of defense against pathogens. Following immunization, LT production by B lymphocytes is required for the formation of germinal centers essential for Ig class switching, most likely by the recruitment and differentiation of antigenpresenting follicular dendritic cells. Surface LT complex expressed by CD4+ TH1 effector cells following antigen stimulation may be responsible for the formation of tertiary lymphoid tissues at sites of chronic inflammation. TNFRI is required for lymphocyte infiltration, whereas the LT R is required for germinal center formation and the ratio of naõÈ ve and memory T cells in tertiary lymphoid tissue. The role of the LIGHT/HVEM system in lymphoid tissue organization remains to be elucidated; however, LIGHT specifically interferes with herpesvirus entry via HVEM, suggesting an antiviral role for this cytokine. Together these results indicate that TNF, LT , and LIGHT form an integrated signaling network
Lymphotoxin and that regulates tissue organization essential for efficient immune responses to viral and other pathogens.
GENE AND GENE REGULATION
Accession numbers Human LT (lta): X01393 Human LT (ltb): L11016 Murine LT (lta): M17015 Murine LT (ltb): U16984
Chromosome location TNF, lta, and ltb form a contiguous cytokine locus that resides on chromosome 6p21.3 (chromosome 17 in mouse) within the major histocompatibility gene complex (MHC) (Figure 2). The LT , LT , and TNF genes show similar intron and exon organization, which is largely conserved for all members of the TNF superfamily. The flanking regions have been entirely sequenced in both human and mouse and did not reveal any additional cytokine genes.
Relevant linkages The presence of the TNF/LT locus in the middle of the MHC has driven an examination of these cytokines for their potential role in MHC-linked autoimmune diseases. LT exhibits a polymorphism (Asn26 to Thr26) initially linked to altered secretion Figure 2 Genetic organization of the human TNF/ LT locus. Upper panel, vertical boxes denote genes in the MHC. Lower panel depicts the exons and arrows indicate direction of transcription. There are no other open reading frames intervening between the LT , TNF, and LT genes. LST1 is leukocyte-specific transcript 1, the homolog of the mouse B144 transcript, with several alternatively spliced isoforms that is predominantly expressed in monocytes and induced by IFN .
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of LT ; however, this substitution does not alter complex formation with LT and does not affect activity in cytotoxic assay. Analysis of the murine TNF/LT locus has not revealed any direct disease linkage with known autoimmune disease phenotypes.
Regulatory sites and corresponding transcription factors Although substantial work has been done on determining the transcriptional regulatory regions in TNF, no significant work has been published on LT or LT regulatory regions. However, context and tissuespecific control elements must exist as the LT and LT mRNAs are expressed independently of TNF, and LT mRNA is found in the absence of LT ; however mRNA levels are coordinately expressed during T cell activation.
Cells and tissues that express the gene LT and mRNAs are detected in spleen and lymph nodes, and peripheral blood. In general, mature T, B, and NK cells can express LT and LT 1 2, as well as certain embryonic progenitors for NK cell and NK-T cell lineages. LT gene expression has not been identified in nonlymphocytic cell lines, unlike TNF, which has broad tissue expression. LT and the LT 1 2 complex are expressed rapidly but transiently following T cell activation. LT 1 2 is expressed by both CD8 and CD4 subsets of activated T cells, but restricted to TH1 effector cells. TH2 cells differentiated in the presence IL-4 or IL-10 do not secrete LT and lack cell surface LT 1 2. B cells that express LT are responsible for maintenance of T and B cell zones, as well as B cell follicles in the spleen. The early progenitor for lymph node development having characteristics of a lymphocyte-like cell identified by CD4+CD3ÿ markers is positive for LT . The progenitor cell that migrates to the intestine responsible for Peyer's patch development is cKit+, IL-7R +, CD4+, 4 7+ and expresses LT after induction by IL-7.
PROTEIN
Accession numbers Human LT : D12614 Human LT : L11015
438 Nancy Ruddle and Carl F. Ware Mouse LT (mustnfba): M16819 Mouse LT : U16985 The crystal structure for human LT ±TNFRI complex is 1TNR (www.rcsb.org/pdb).
Sequence The primary sequence for human and mouse LT (Figure 3a) aligned to show conserved residues indicates a high degree of homology. The conserved sequence that defines this family of ligands lies primarily within the strand scaffold based on the crystal structure of LT . LT is also conserved between mouse and human except that the mouse LT gene omits a splice site resulting in an inframe insertion of 60 residues prior to the trimerization
domain and may extend the trimer from the cell surface (Figure 4).
Description of protein Human LT is encoded by a 1.4 kb mRNA and contains 205 amino acids with a single N-linked glycosylation site that is utilized in the mature protein along with uncharacterized O-linked glycosylation sites (Table 1). LT forms a homotrimer and also heterotrimers with LT . The protein LT trimer is exclusively secreted and stable. LT is encoded by a 0.9 kb mRNA and contains 244 amino acids with a single N-linked glycosylation site that lacks a signal cleavage site, and thus is retained as membrane protein. The surface form of LT is unique in this family (Ware et al., 1995). Surface
Figure 3 Sequence alignment of human and mouse LT . (a) Sequences were aligned using ClustalW program (PAM series) where conserved residues are outlined and shaded. The arrow indicates the position of the signal cleavage site in human LT . The bars denote the positions of the strand scaffold after the nomenclature of Eck et al. (1992). (b) Structural features of LT and TNFR60. Upper panels depict the strands of a single LT subunit. The wide bands are the strands and the smaller rope-like bands represent the connecting loops. The left upper panel shows the positions of D50 and Y108 in the solvent-exposed orientation, where the two other subunits of the LT trimer are not visible. In the upper right panel the subunit has been rotated 180 revealing the interior sheet, where the dark areas represent residues conserved among the superfamily. Lower left panel depicts a receptor-binding site on LT composed of two subunits. The third LT subunit is in the background in outline. D50 is in the A±A00 loop and Y108 in the D±E loop. The dark bands identify the positions of the residues that contact TNFR60. The lower right panel depicts the TNFR60 extracellular domain. The receptor orientation has been rotated counterclockwise 90 (around the vertical axis) exposing the contact residues (dark areas); disulfide bonds are dashed lines; the demarcation of the cysteine-rich motifs (D1±D3) are indicated on the right side. In this orientation the ligand is attached to a T cell membrane at the bottom, and the receptor is anchored to the target cell at the top. The views were generated from Banner et al. (1993) (available as 1TNR PDB, www.pdb.bnl.gov/cgi-bin/ pdbmain) as visualized RasMol (v2.6.1) available at www.umass.edu/microbio/rasmol/ rasnew.htm.
Lymphotoxin and Figure 3 (Continued ) (b)
LT is a heteromeric complex assembled from two subunits, LT and LT , whereas the other TNFrelated ligands are homotrimers. The association between the two LT subunits occurs during biosynthesis and generates two types of heterotrimers, LT 1 2 and LT 2 1. The LT 1 2 complex represents the most abundant form, accounting for > 95% of the complex on the surface of activated T lymphocytes. LT concurrently assembles as a homotrimer and is secreted, whereas expression of LT protein by itself has not been detected.
Discussion of crystal structure The primary structure of TNF and LT contains eight strands (designated A±H) that fold into an antiparallel sheet sandwich (Figure 3). This subunit assembles into a compact trimer, with the end-to-face
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interaction about a 3-fold axis of symmetry. This generates a molecule resembling a cone with a large flat base and narrow top. The internal sheet is rich in hydrophobic residues that create a large surface area where subunits interface in the trimer. The discreet regions of homology that define this family are primarily found within the strand scaffold on the face of the internal sheet. On the solventexposed surface there is also significant conservation of the residues preceding the loops connecting the strands. Two of these loops, A±A00 and D±E, contain residues important for receptor binding. Mutational analyses and X-ray crystallography studies indicate that the TNFRI-binding site on the LT trimer is located in the cleft formed between two interacting subunits (Banner et al., 1993) (Figure 5). Mutational analysis of TNF and LT suggested that two loops, A0 ! A00 and the D ! E, which are on opposite sides of the ligand monomer creating an asymmetric subunit, are critical for receptor binding. Selected mutations within these loops do not alter trimer formation, but decrease receptor interactions. A comparison of the sequences within these loops reveals nonconservative changes among the ligands in this family (Figure 6). Mutation of D50 in LT adjacent to R51, which contacts residues Y108 and P113 between monomers A and C, indicates that residues involved in trimer formation are also important to receptor binding. Domains 2 and 3 of TNFRI bind at the cleft formed between A and C subunits of the LT trimer. Each receptor domain has two distinct ligand-binding regions, a lower and upper region. The lower region consists of loop 1 through the middle of the three central Cys (C73) making contacts with residues in both A and C subunits of LT . The main contacts are in the loop of the C subunit and A ! A00 loop of the A subunit, typically described as the base of the ligand. Residues in loop 1 of domain 2 show primarily hydrophobic interactions with the D ! E loop of LT where Y108 is critical; D50 in the A ! A00 loop is important in binding the CxxCxxC region where the primary contacts are polar. The ligand±receptor complex, as it would exist on the surface of juxtaposing cells, shows the narrow end of the elongated trimeric ligand oriented outward from the cell membrane. This provides the proper orientation for interaction of cell surface ligands and receptors during cell±cell contacts.
Important homologies LT and LT are defined as members of the TNF superfamily of type II transmembrane proteins
Figure 4 Sequence alignment of human and mouse LT . Sequences were aligned by ClustalW program and conserved residues are outlined and shaded. The bars denote the positions of the cytoplasmic domain, dashed line the transmembrane domain and the arrow shows the region encompassing the trimerization domain which contains the residues involved in receptor binding.
Table 1 Selected physical properties of human LT , LT , and TNF Property
LT
LT
TNF
Amino acids (mature form)
171
240 (244)a
233
NA
76c
Signal/cleavage site
34
b
Transmembrane domain
NA
30
21c,d
Cytoplasmic tail
NA
15±18e
30c
Extracellular domain (membrane extension)
NA
36
33
Receptor-binding domain
137
d
a
160
157
Molecular mass (kDa) Sequence
17
25
26 membranec 17 secreted
Observed
25
33
26 membranec 17 secreted
N-glycosylation sites
1
1
0
Secreted form
Yes
No
Yes
a
Human LT has an inframe ATG codon that could act as the translational start site; however, this ATG is very close to the 50 end of the RNA and is probably not utilized very efficiently (Browning et al., 1993). The ATG is followed by a CTG three codons downstream and clones possessing only the CTG start are functional and, moreover, amino acid sequencing of LT indicated that this CTG encoded the N-terminal amino acid of the mature protein. The GC-rich region following this CTG may slow ribosomal scanning sufficiently to allow the CTG to function efficiently. It is possible that both the ATG and CTG start codons are utilized. b Determined by microsequencing of purified mature LT . c
TNF exists in secreted and membrane bound forms. The 76 residue `leader sequence' has been shown to function as a membrane-anchoring domain and is the cleavage site generating the 17 kDa soluble form. d Putative membrane-spanning region based on the hydrophobic character of the amino acid residues.
Lymphotoxin and Figure 5 (a±d) Lymphotoxin structure and theoretical receptor-binding sites in LT complexes. Structural model of LT viewed as a ribbon diagram showing strands for (a) single subunit (side view, solvent exposed surface, Nand C-terminus are located at the bottom) or (b) as a trimer (view from the base) showing the positions of tyrosine 108 (Y108, in red), and aspartic acid 50 (D50, in blue) on opposite sides of the subunit. R denotes the position of the TNFR60-binding sites. The cartoons are based on the crystal structure of the LT ±TNFR60 complex solved by Banner et al. (1993) using Protein Data Bank file 1TNR as visualized with RasMol v2.6. Cartoon showing the theoretical positions of Y108 (red) and D50 (blue) of LT in the heterotrimeric ligands LT 1 2 (c) and LT 2 1 (d) X and Z in LT denote residues in LT equivalent to D50 (A±A00 loop) and Y108 (D±E loop). Reprinted with permission from Williams-Abbott, L., Walter, B. N., Cheung, T. C., Goh, C. R., Porter, A. G., and Ware, C. F. (1997). The lymphotoxin- (LT ) subunit is essential for the assembly, but not receptor specificity, of the membrane-anchored LT 1 2 heterotrimeric ligand. J. Biol. Chem. 272, 19451± 19456. (Full colour figure can be viewed online.)
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CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce See Table 2.
Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Based on cell types that produce LT and LT , physiologic stimuli include antigen recognition via the TCR and the B cell antigen receptor. LT are also produced by innate defenses including NK cells. IL-2 is a potent activator of surface LT expressed by NK and LAK cells. The nature and source of the signals that induce expression of LT during development are unknown, but IL-7 has been implicated as an inducer for the Peyer's patch inducer. Shed forms membrane TNFRI or TNFRII form soluble `decoy' receptors that can inhibit LT activity, however LT R is not shed and no naturally occurring decoys have been identified. Ecto domains of the LT receptors produced as fusion proteins with the Fc region of IgG create high-affinity inhibitors for their respective ligands and are in clinical use (TNFR:Fc aka EnbrelTM).
RECEPTOR UTILIZATION LT binds to TNFRI and TNFRII with high affinity ( 0.1 nM), and to HVEM with low affinity (50 nM); LT 1 2 binds to the LT R ( 1 nM), whereas the LT 2 1 complex binds LT R weakly (25 nM), but TNFRI and TNFRII with high affinity ( 10 nM) (Browning et al., 1996a).
IN VITRO ACTIVITIES that engage the cognate superfamily of TNF receptors.
Posttranslational modifications See Table 1.
In vitro findings Because LT binds to the same TNFR as TNF their in vitro biological responses are very similar. However, based on dose±response curves, LT is typically less potent compared with TNF in inducing most functions except for the death of L929 cells. LT behaves as a partial agonist of TNF in some assays,
442 Nancy Ruddle and Carl F. Ware Figure 6 Alignment of the receptor-binding domains of the immediate LT/TNF family. Sequences start at the A strand to the C-terminus and were aligned using ClustalW program. Identical residues are boxed and shaded.
Table 2 Cell types producing lymphotoxins Cell type
Inducer
Effect
TH1
Ag recognition
Inflammation in tissues
B cells
Ag recognition, Germinal center CD40 activation formation
NK cells/LAK IL-2 cells
Inflammation/antiviral
Peyer's patch progenitor
Differentiation/PP formation
IL-7
receptors made as fusion proteins between the ecto (ligand-binding) domain of the receptor and the Fc region of IgG. The LT R:Fc fusion protein injected into pregnant mice will block further development of lymph nodes or Peyer's patches (Rennert et al., 1996) and can be used to block inflammation in a mouse model of inflammatory bowel disease. Soluble TNFR2:Fc is an approved drug for the treatment of rheumatoid arthritis (EnbrelTM).
Bioassays used such as induction of MHC molecules, ICAM-1 and other types of markers of inflammatory reactions. LT 1 2 displays even weaker potency for induction of proinflammatory markers on various cells, including endothelial cells, prompting its description as a noninflammatory cytokine (Hochman et al., 1996).
Regulatory molecules: Inhibitors and enhancers The most efficient inhibitors of LT and TNF bioactivity are soluble versions of their respective
Human LT is traditionally assayed by L929 cell cytotoxicity assay, but this assay does not differentiate LT and TNF. Mouse LT is less potent than mouse TNF in the L929 in vitro assay. Thus, other modern methods, such as ELISA using noncrossreactive monoclonal antibodies, are recommended to specifically detect LT . LT 1 2 cytotoxic activity is not apparent on L929 cells, but can be detected on the human HT29 adenocarcinoma line (Browning et al., 1996b). Cell death by LT , LT 1 2, and TNF measured on the HT29 cell requires IFN . LT 1 2 will also induce expression of ICAM-1 and VCAM and chemokine secretion.
Lymphotoxin and
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles Lymphotoxins play a role in the initiation of inflammation, which presumably contributes to normal defense against invading pathogens. The molecular form of LT in inflammation appears to be predominantly LT homotrimer, although a role for LT in some inflammatory states has been suggested. LT mRNA has been found in several inflammatory processes in humans in mice, and LT can induce expression of adhesion molecules and chemokines in vitro. LT does not appear to be able to induce adhesion molecules in vitro but can induce chemokines. LT and LT may play normal roles in tumor immunity. LT induces apoptosis (programmed cell death) in several tumor lines in vitro and LT 1 2 induces killing of a more limited array of tumor cells. This effect is due to the action of a distinct types of receptor, LT uses TNFRI, a death domain proapoptotic signaling receptor that directly activates the caspase pathway, whereas LT 1 2 uses the LT R, a TRAF-binding receptor and a potent activator of apoptotic inhibitory pathways, such as NFB transcription factor. The relative lack of readily available recombinant derived murine LT or LT 1 2 (in the absence of contaminating LT or LT 2 1) has limited animal studies involving direct injection of these molecules. However, insight into normal physiologic roles has been gained through the use of a reagent that is specific for LT , namely the LT R:Fc fusion protein. When injected into pregnant mice this molecule
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has profound effects on the development of lymphoid organs in that most lymph nodes (with the exclusion of mesenteric lymph nodes) are inhibited in their development. The conclusion of these studies, that LT 1 2 (which is bound by LT R:Fc) plays a profound role in lymphoid organ development, was also reached in studies in which LT or LT or the LT R were selectively ablated.
Knockout mouse phenotypes The phenotype of the LT knockout mice includes the absence of all lymph nodes and Peyer's patches (Table 3). The spleen is also disrupted with a loss of germinal centers and follicular dendritic cell networks. The absence of germinal centers has some effects on humoral immunity, but these are not allencompassing in that not all isotypes are affected. The most profound effects are on serum and fecal IgAs, which are almost completely absent. The initial studies indicated that LT was crucial for lymphoid organ development, but did not reveal whether this was in the form of LT 3 or LT 1 2. Experiments with LT knockout mice suggest that both forms contribute to these processes, but that LT 1 2 plays the most important role. Thus LT ÿ/ÿ mice are missing most lymph nodes, do not have Peyer's patches, and have disorganized spleens (Table 3). These animals do have mucosal and cervical lymph nodes, however, indicating that a form other than LT 1 2 does participate in the development of these organs, presumably LT 3 or possibly LT 2 1. The disorganization of the spleen in LT ÿ/ÿ mice is less extreme than in LT ÿ/ÿ mice and there is the occasional peanut agglutinin (PNA)-positive area around the central arteriole, suggesting a primitive
Table 3 LT and LT knockout mouse phenotypes Gene
Phenotype
Authors
LT
No lymph nodes, no Peyer's patches, disorganized spleen Reduced IgA No germinal centers Deficient NK and dendritic cells Resistance to experimental allergic encephalomyelitis (EAE)
De Togni et al., 1994; Banks et al., 1995; Koni et al., 1997; Matsumoto et al., 1996
LT
No peripheral lymph nodes (mesenteric and cervical lymph nodes present) No Peyer's patches Disorganized spleen No splenic germinal centers Reduced IgA Somewhat diminished susceptibility to EAE
Koni et al., 1997; Alimzhanov et al., 1997
444 Nancy Ruddle and Carl F. Ware form of germinal center. When all studies carried out with LT ÿ/ÿ, LT ÿ/ÿ, TNFÿ/ÿ, TNFRIÿ/ÿ, and LT Rÿ/ÿ mice are taken into account, one reaches the conclusion that it is likely that there is some contribution of all these molecules to normal lymphoid organ development. There appears to be a cooperation between the various ligands and their receptors and there may even be a role for additional molecules.
foci for continued antigen presentation and T and B cell activation in microbial infections or in autoimmune disease leading to defense in the first instance and pathogenesis and epitope spreading in the second. As yet, there have been no reports of LT transgenic mice. Since LT requires the presence of LT , it is unlikely that any effect would be seen unless such mice were prepared in a situation in which LT was also expressed by the same cells.
Transgenic overexpression
Pharmacological effects
When LT is expressed under the control of the rat insulin promoter (RIPLT mice), the transgene is expressed in the pancreas, as expected from this cell-specific promoter, but also in the kidney (Table 4). Inflammation, consisting of T cells, B cells, macrophages, follicular dendritic cells, and dendritic cells is seen at the sites of transgene expression. The inflammation is mediated by LT 3 and does not require LT . RIPLT mice crossed to LT ÿ/ÿ mice and then backcrossed to LT (RIPLT.LT ÿ/ÿ mice) still exhibit inflammation, though the phenotype of the infiltrating cells is altered with a reduction in the proportion of the L-selectinhi T cells. There is also an increase in expression of adhesion molecules (VCAM, ICAM, MAdCAM, PNAd) and certain chemokines at the sites of transgene expression. The pancreatic and kidney infiltrates exhibit many similarities to lymphoid organs. The cellular composition, segregation into T cell and B cell areas, presence of high endothelial venules and their adhesion molecules, along with the presence of antibodysecreting plasma cells after immunization, all suggest that these infiltrates have the characteristics of `tertiary lympoid organs' seen in many cases of chronic immunologic stimulation including Hashimoto's disease, multiple sclerosis, and rheumatoid arthritis. It has been proposed that the process by which tertiary lymphoid organs develop in the adult be called `lymphoid neogenesis'. In the case of chronic LT expression in transgenic mice, the cytokine appears to recapitulate its role in ontogeny by establishing a new lymphoid organ. Such tertiary lymphoid organs could act as
Detailed pharmacologic actions of LT and LT 1 2 have not been reported.
Interactions with cytokine network IL-2 is a potent inducer of surface LT 1 2 and LT secretion. IL-4 and IL-10 as well as TGF can act to suppress LT production.
Endogenous inhibitors and enhancers Soluble TNF receptors released during inflammation may function to dampen LT biological effects. IFN is a potent enhancer of TNF and LT activities that may contribute to increased effects in vivo.
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects Circulating levels of these cytokines are not detected in normal plasma, but levels can rise during acute inflammation.
Table 4 LT transgenic overexpression Gene
Promoter
Organ expression
Phenotype
References
LT
Rat insulin promoter (RIP)
Pancreas, kidney, skin
Inflammation
Picarella et al., 1992
LT
Rat insulin promoter (RIP)
Pancreas, kidney, skin
Lymphoid neogenesis
Kratz et al., 1996
Lymphotoxin and
Role in experiments of nature and disease states The several instances in which LT mRNAs are elevated in human disease states are summarized in Table 5. These include several instances of chronic inflammation, such as multiple sclerosis, juvenile rheumatoid arthritis, juvenile spondylarthropathy, and Crohn's disease. On the other hand, reports have conflicted regarding whether LT is elevated in adult rheumatoid arthritis. Even in those cases in which LT is elevated in a disease state, one cannot determine whether the elevated cytokine levels contribute to the inflammation or are a side-effect. One interesting observation is the elevated level of LT in the serum of individuals with hypercalcemia of adult T cell leukemia (ATL). ATL is caused by infection with HTLV-I and the tax gene of that virus has been shown to induce LT directly through activation of NFB. LT has been associated with osteoclast activation and could contribute to the pathogenesis of HTLV-I through its effects on osteoclast activation and bone breakdown. LT mRNA is elevated in B cells in chronic lymphocytic leukemia (CLL) and it has been reported that recombinant LT stimulates proliferation of B cells from CLL patients, and thus apparently acts as an autocrine growth factor. In most cases noted in Table 5, LT appears to be acting through its homotrimeric form.
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LT elevation has been reported in a few instances of human disease associated with chronic autoimmune inflammation, namely multiple sclerosis and sarcoidosis. Interestingly, elevated levels of LT mRNA expression have also been seen in two cases of microbial disease: tuberculosis and Pseudomonas aeruginosa infection. (In neither case was LT also analyzed, indicating that even if LT protein is made in these situations, its molecular form and activity are unknown.) Again, it has not been determined whether the presence of LT is a reflection of its pathogenic role or its role as a marker of inflammation. The fact that LT and LT are elevated in certain disease states has thus far not had any diagnostic utility.
IN THERAPY
Preclinical ± How does it affect disease models in animals? It has been difficult to determine the effect of LT administration in disease models in animals because of the above noted scarcity of appropriate recombinant-derived materials. However, administration of recombinant human LT 3 to bio-breeding (BB) rats or nonobese diabetic (NOD) mice prevents the development of insulin-dependent diabetes mellitus that usually occurs spontaneously in these strains.
Table 5 LT and LT pathophysiological roles in normal humans and disease states and diagnostic utility Gene
Disease
Organ
References
LT
Crohn's disease
Increased secretion from mucosa
Noguchi et al., 1998
LT
Multiple sclerosis
Presence in acute and chronic active lesions in brain
Selmaj et al., 1991a
LT
Adrenoleucodystrophy
Elevated levels in microglia in brain
Selmaj et al., 1991b
LT
Hypercalcemia of adult T cell leukemia
Elevation in serum
Ishibashi et al., 1991
LT
Juvenile rheumatoid arthritis
Synovial tissue
Grom et al., 1996
LT
Bulbous pemphigoid
Elevated expression in blisters
Jeffes et al., 1989
LT
Non-Hodgkin's lymphoma
Elevated expression in serum
Warzocha et al., 1998
LT
Pregnancy
Decrease in amniotic fluid and plasma in term labor onset
Laham et al., 1997
LT
Multiple sclerosis
Elevated in astrocytes and oligodendrocytes in brain
Selmaj et al., 1991b
LT
Sarcoidosis
mRNA expression in granulomas
Bergeron et al., 1997
LT
Tuberculosis
mRNA expression in granulomas
Bergeron et al., 1997
LT
Pseudomonas aeruginosa infection
mRNA expression in cornea
Kernacki et al., 1998
446 Nancy Ruddle and Carl F. Ware Table 6 Effect of inhibitors on disease models in animals Reagent
Use
Effects
References
Human LT
Administration to BB rats
Inhibits diabetes
Takahashi et al., 1995
LT ÿ/ÿ
Experimental allergic encephalomyelitis (EAE)
Prevents disease
Suen et al., 1997
LT ÿ/ÿ mouse
EAE
Reduces susceptibility to disease
Suen et al., 1997
Anti-human LT
Rat xenohepatic transplant
Inhibits thymic apoptosis associated with acute rejection
Yamaguchi et al., 1998
LT R:Fc
Pregnant mice
Inhibits secondary lymphoid organ formation in offspring
Rennert et al., 1996
LT R:Fc
Colitis model
Inhibits disease
Mackay et al., 1998
These studies suggest that LT , as is the case with TNF, can play a protective role at certain stages in these autoimmune diseases. There have been several studies in which the roles of TNF receptor antagonists have been evaluated in autoimmune diseases. Because LT 3 binds to these receptors, such studies help to define the role of that cytokine in disease in some instances, particularly those in which CD4 TH1 cells are evaluated. In other instances, antibodies that may crossneutralize murine TNF and LT have been used and the results of these studies are relevant to the role of these cytokines in murine disease models. One example outlined in Table 6 describes the effect of anti-TNF on transfer of experimental allergic encephalomyelitis. There have been numerous other examples, and they are summarized in the chapter on TNF. Because of the ambiguity regarding whether these antibodies neutralize only TNF, this is the only study that will be listed here. The absence of readily available antibody that neutralizes only LT and none of the other forms, leaves these studies somewhat in question. The studies with LT knockout mice suffer from some difficulties because of the impairments in lymphoid organ structure. However, it is clear that LT -deficient mice can mount some immune responses, though they are completely resistant to EAE. There have been no published reports regarding the administration of recombinant LT or the LT complex to evaluate their role in animal models. However, the role of the LT 1 2 complex has been studied in several disease models in animals through the use of the aforementioned LT R:Fc fusion protein. This reagent inhibits the development of colitis in mice.
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