Lymphotoxin β Receptor

Lymphotoxin Receptor 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, LaJolla 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.2000.16003.

SUMMARY The LT receptor (LT R), a member of the TNFR superfamily, functions as an essential element in the organization of lymphoid tissue and initiation of innate and immune defenses. The LT R is a type 1 transmembrane glycoprotein with four cysteine-rich motifs in the ectodomain involved in binding to ligands, LT1 2 and LIGHT. LT R gene maps near TNFRI and CD27 on chromosome 12p13. LT R signaling induces NFB and Jnk/AP-1 transcription factors and activates a slow apoptotic death in certain adenocarcinoma cell lines. The LT R cytoplasmic tail binds TRAF2, TRAF3, and TRAF5, but not TRAF6 to propagate signaling. Mice deficient in LT R lack peripheral lymphoid organs and have disorganized splenic microarchitecture, similar to mice deficient in LT or LT genes. LT R is expressed on stromal tissue and thus receives signals from activated lymphocytes that mediate tissue organization. The LT R±LT1 2 cytokine system has unique roles, but functions in some cases with TNFRI and II, and HVEM cytokine systems as an integrated network that orchestrates multiple developmental processes and immune responses.

BACKGROUND

Discovery The lymphotoxin receptor (LT R) was initially identified as a transcript in somatic cell hybrids

expressing genes on chromosome 12p and recognized as a cysteine-rich TNFR-like domain (Baens et al., 1993). The ligand specificity for LT1 2 was defined by Crowe et al. (1994) and provided key evidence that explained the unusual phenotype of LT-deficient mice and proved that surface lymphotoxin was unique and had a biological role distinct from that of TNF and LT. Recently, Mauri et al. (1998) showed that LT R also recognized another closely related ligand, LIGHT.

Alternative names LT R was previously referred to as TNF receptorrelated protein (TNFRrp). For the purposes of cataloging human genes, the Human Gene Nomenclature Committee, HUGO (http://www.gene.ucl.ac.uk/ users/hester/tnftop.html), has assigned members of the TNF ligand and receptor superfamilies numerical indicators. LT R is TNFRSF3.

Structure The LT R is a type 1 single transmembrane glycoprotein of 435 amino acids that contains a cysteinerich extracellular domain, which defines it as a member of the TNFR superfamily (Figure 1) (Ware et al., 1998). LT R maps to chromosome 12p13 near TNFRI and CD27. The LT R binds three known ligands: LT1 2 and LIGHT with high affinity, and the LT2 1 complex with low affinity. The LT R

1640 Nancy Ruddle and Carl F. Ware Figure 1 Main features of the lymphotoxin receptor.

Figure 2 The immediate LT/TNF family. Depicted are the individual cytokine systems that utilize the common set of receptors which defines members of the immediate LT family. Arrows indicate highaffinity 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 LT2 1 as the functional significance is not known, however it binds TNFRI and TNFRII with high affinity and LT R with low affinity.

signals via members of the TRAF (TNF receptorassociated factors) family of zinc ring finger proteins that mediate activation of NFB, AP-1 transcription factors, and cell death. The mouse LT R is highly homologous to the human version (68% identity).

Main activities and pathophysiological roles The LT R is one of five receptors that interact with a common group of ligands including LT, TNF, LT , and LIGHT, which form individual cytokine systems, and together define the immediate TNF/LT family (Figure 2) (Smith et al., 1994; Ware et al., 1995; Wallach et al., 1999). The LT R is now proven to be the critical signaling molecule for the LT- complex. It is required for the formation of secondary lymphoid tissue, lymph nodes (LN) and Peyer's patches (PP) (Fu and Chaplin, 1999). The LT R contributes to the signaling for organization of T and B cells in the spleen, and germinal center formation during immune responses. LT- and LT -deficient mice also exhibit the loss of progenitor cells required for NK and NK-T cell developmentand maturation. The LT R is expressed on stromal cells, while the ligands are produced by activated lymphocytes. This unidirectional signaling from lymphocyte to stroma is thought to organize the architecture of the tissue to convene efficient immune responses. The physiologic signaling by LT R activated by its other ligand, LIGHT is just beginning to be explored.

GENE

Accession numbers Mouse LT R cDNA: U29173, L38423 (for gene organization see Force et al., 1996) Human LT R cDNA: L04270

Chromosome location and linkages The gene encoding mouse LT R contains 10 exons spanning 6.9 kb and maps to chromosome 6 closely linked to genes for TNFRI (CD120a) and CD27 (Figure 3). Approximately 1.5 cM separates ltbr and tnfr1. This region exhibits conserved synteny with human chromosome 12p13. The human gene encoding LT R spans  9 kb with an intron/exon arrangement very similar to that of TNFRI. The promoter region lacks TATA and CAAT sequences and resembles that of a housekeeping-like gene, and is

Lymphotoxin Receptor 1641

2 Kr as

r, T

b3

Ltb

Gn

f Ra

Rh

o

nfr

1

Figure 3 Genetic organization of the murine LT receptor. (a) Chromosomal map location of Ltbr and Tnfr1 on chromosome 6. The black circle represents the centromere. The relative position of each locus pair is illustrated by vertical hash marks. The recombinational differences between loci and standard errors are shown in parentheses and conserved synteny with the human map locations for the indicated genes are shown below. (b) Exon organization of Ltbr. Each exon is represented as a black rectangle. (c) The mLT R cDNA represented as a compilation of its exons. The number of each exon is given above and the amino acid position located at the intron/exon junction is given below denoted by a vertical hash mark. (d) Schematic of the mLT R protein. L, leader sequence; D1±4, the ligand-binding region including the cysteine-rich repeats; TM, transmembrane domain; CYTO, the cytoplasmic signaling domain. Reproduced with permission from Force et al. (1996).

similar to the promoter found in tnfr1 and consistent with the constitutive expression of this protein. Disease linkage has not been identified for ltbr; however, a spontaneous fever syndrome has been mapped to 12p13, although initial analysis indicates mutations that elevate expression of TNFRI causing an inflammation-based disease.

PROTEIN

Accession numbers Mouse LT R cDNA: U29173, L38423 Human LT R cDNA is L04270

Sequence See Figure 4.

Description of protein LT R is translated from a 2.2 kb mRNA with a theoretical mass of 46.7 kDa. The observed mass of 61 kDa indicates that the two potential N-glycosylation sites are probably utilized. Analysis of the human LT R cDNA sequence encodes a predicted 435 amino acid protein sharing 41% and 46% homology with TNFRI and TNFRII, respectively. LT R has a ligand-binding ectodomain with four cysteine-rich pseudo repeats followed by a short proline-rich membrane proximal region, characteristic of the TNFR superfamily. The ligand binding domain of LT R has characteristics of both TNFRI and TNFRII in the positioning of the cysteine residues and can be readily modeled on the TNFRI structure (Guex and Peitsch, 1997). Similarity with TNFRI is found in the first and second domains. In contrast, the equivalent of loop 1 in domain 3 of LT R is dramatically shortened, having only five residues, and in this regard is highly similar to domain 3 of TNFRII. The fourth domain of LT R resembles TNFRII more closely than TNFRI. An additional similarity between LT R and TNFRII is the proline-rich region proximal to the membrane-spanning sequence, suggested the formation of a `stalk' extending from the cell surface. The cytoplasmic portion of LT R shares limited homology with other members of the family by sequence analysis but is grouped functionally with those other members that bind to TRAF signaling molecules, such as CD40, CD30, and CD27.

Relevant homologies and species differences Mouse and human LT Rs are homologous (68% identity) over the entire length of the sequence, and are similar to many other receptors in this family. The mouse LT R lacks 20 amino acids overall, and 12 of those are at the C-terminal tail.

1642 Nancy Ruddle and Carl F. Ware

Figure 4 Sequence of the LT R. Sequences were aligned with ClustalW program. The cysteine-rich domains (CRD) are denoted by solid bars above the sequence. The transmembrane region is identified by a dashed line and the TRAF-binding region in the cytoplasmic domain by a stippled line.

Lymphotoxin Receptor 1643

Affinity for ligand(s)

SIGNAL TRANSDUCTION

LT R binds membrane forms of LT1 2 and LIGHT with high affinity, and also to their recombinant soluble forms, but only weakly to LT2 1 (Table 1). It cannot be excluded that relatively weak interactions between receptor and ligand in the soluble phase may be highly significant in the context of membrane localization where the avidity may be increased substantially.

Associated or intrinsic kinases

Cell types and tissues expressing the receptor The LT R is expressed on stromal cells in lymphoid tissue such as thymus and spleen and absent on lymphocytes as determined by immunohistochemistry (Murphy et al., 1998). The LT R is expressed on normal dermal fibroblasts, normal bronchial airway epithelial cells, but absent on human peripheral blood derived mononuclear cells including T and B lymphocytes. Most adherent cell lines including FDC-1, follicular dendritic cell line, U937 promyelomonocytic cell line, HT-29 colon adenocarcinoma, HeLa cervical carcinoma line, and the HEK 293 embryonic kidney cells express LT R as measured by cell surface staining (Murphy et al., 1998).

Release of soluble receptors Unlike TNFRI or TNFRII, no evidence indicates that LT R is shed or has alternate spliced forms that create soluble versions.

The binding of the trivalent ligand, which induces an ordered aggregation or `clustering' of receptors, initiates signal transduction by TNFR family members. Antireceptor antibodies mimic receptor clustering, and for many receptors mere overexpression can lead to activation of signal transduction pathways. The LT R, as well as all the other related TNFRs, has no intrinsic kinase or other enzymatic activities encoded by its cytosolic domain. Rather, signal transduction occurs through the recruitment of cytosolic adapters, which directly or indirectly confer enzymatic activity to the receptor. Recent findings indicate that the mutation in the alymphoplasia (aly) mutant mice, which lack lymph nodes and PPs, is due to NFB-inducing kinase (NIK). This result indicates that NIK is likely to be required for LT R-specific signaling (Shinkura et al., 1999).

Cytoplasmic signaling cascades The LT R activates gene transcription and can activate apoptosis, although it is not a death domain receptor, which interact directly with adapters for caspases. The LT R utilizes select members of the TRAF family (Arch et al., 1998) to propagate signals received via LT1 2 and LIGHT (VanArsdale et al., 1997) (Figure 5). TRAF2, 3, and 5 are known to bind directly to the cytosolic domain of the LT R. The

Table 1 Receptor specificity of the LT/TNF cytokine system Ligand

Forma

Binding receptor

Affinity ( Kd=nM)

LT

Soluble

TNFRI and TNFRII

High (0.1)b

LT1 2

Membrane

LT R

High (0.5)

LT2 1

Membrane Soluble

TNFRI and TNFRII LT R

High (0.5) Low (20)

LIGHT

Membrane Soluble

LT R

High (1) High (0.5)

TNF

Soluble

TNFRII and TNFRI

High (0.1)b

a Membrane ligands used to measure binding avidity were expressed on Tn5 insect cells infected with recombinant baculoviruses or by transfection of 293T cells with LT or LT cDNAs. Soluble ligands were purified proteins that were radioiodinated with 125I. With ligands expressed on cells binding specificity was performed using graded amounts of LT R:Fc or TNFRI:Fc fusion proteins and detection of bound fraction by flow cytometry. With soluble ligands, competitive binding assays were performed with receptor:Fc proteins bound to microplate wells and [125I]LT1 2. Soluble LIGHT LT R interactions were determined with Plasmon resonance (Biacore). b

Values derived by Scatchard analysis for ligands bound to mammalian cells.

1644 Nancy Ruddle and Carl F. Ware Figure 5 Signal transduction pathways for the LT R. This cartoon depicts the LT R clustered by its trivalent ligands, LT1 2, or LIGHT, which recruits TRAF adapter molecules. TRAF2 and 5 are involved in gene activation through NFB or AP-1 transcription factors. The link between TRAF3 and the caspase-dependent apoptotic pathway is unknown.

TRAF-binding region is located within a proline-rich sequence between residues 345 and 396 (Force et al., 2000). This is also a region that binds to the hepatitis C virus core protein (Matsumoto et al., 1997). TRAF2 and 5 are potent inducers of NFB and AP-1 transcription factors. TRAF3, however, appears to be involved in signaling cell death as dominant negative mutants of TRAF3 inhibit LT R signaling of cell death, but not NFB activation (Force et al., 1997). Thus the LT R signaling pathways bifurcate and appear to be independently controlled (Figure 5). NIK may mediate some signaling events for the LT R. One likely scenario envisions TRAF2 or 5, both of which interact directly with NIK, acting as the adapters coupling LT R to IB kinases essential for activation of NFB. However, the NIK phenotype in aly mice is not recapitulated by knockout of TRAF2 or 5, suggesting either a redundancy of function or that an alternate mechanism exists for LT R to activate NFB. At present, it is not understood how TRAF3 may be connected to the death pathway. It is possible that TRAF3 acts as an adapter that connects LT R to TRADD, which in turn interacts with FADD thus coupling directly to caspase 8, which activates caspase 3, the executioner caspase.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated The ability of LT R to activate both NFB and AP-1 transcription factors suggests that signaling will elicit inflammation and cellular stress responses. The plethora of genes activated via these transcription factors makes it difficult to predict specific responses. Cellular responses will also be controlled in part by the strength and duration of LT R-generated signals, as well as within the context of the tissue and cell type. LT R can activate expression of adhesion molecules such as ICAM-1 in follicular dendritic cells (FDCs) and is presumably involved in forming FDC clusters, which are absent in LT R knockout mice. The induction of adhesion molecules by the LT R is weak compared to that by TNF, especially in other cells types. In vivo, MAdCAM expression by FDCs is suppressed by treatment with LT R:Fc fusion protein. LT R also induces chemokine expression which attracts leukocytes and other cells. Intriguingly, mice deficient in the chemokine receptor BLR1 (Forster et al., 1996) partially resembles the phenotype of LT Rÿ/ÿ mice.

Genes induced Because NFB and AP-1 transcription factors are activated by LT R, it is expected that a subset of the genes regulated by these factors will be induced (Whitmarsh and Davis, 1996).

Promoter regions involved The NFB and AP-1 transcriptional regulation systems are well studied and found in numerous genes (see reviews by Whitmarsh and Davis, 1996; Ghosh et al., 1998).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY The distinct receptor specificity of LT1 2 complex for the LT R hinted that new functions would be

Lymphotoxin Receptor 1645 associated with this cytokine±receptor system. The production of LT-deficient mice has revealed molecular insight into fundamental processes central to the immune response (De Togni et al., 1994; Banks et al., 1995; Futterer et al., 1998). LT-deficient mice have no detectable popliteal, inguinal, para-aortic, mesenteric, axillary, or cervical lymph nodes, whereas LT deficient mice have mesenteric and cervical lymph nodes (Koni et al., 1997; Alimzhanov et al., 1997). Since mice deficient in TNF have a normal lymph node structure (Pasparakis et al., 1996), LT and TNF are not functionally redundant molecules. Recent evidence has shown that NK and NK-T cells are absent in LT-deficient mice (Iizuka et al., 1999). Interestingly, no easily detectable defect in T lymphocyte number or function could be ascertained by traditional assays. However lack of NK-T cells and no circulating E 7‡ T cells indicates that at least these subsets are abnormal. There was, however, an increase in circulating B cells in the LT-deficient animals. Bone marrow cells obtained from LT-deficient mice were able to home to both spleen and lymph nodes of SCID or lethally irradiated wild-type mice, indicating no intrinsic defect in the hematopoietic compartment. In contrast, normal bone marrow was unable to reconstitute lymph nodes in irradiated LT-deficient mice, indicating that lymph node organogenesis is developmentally fixed. Since mice deficient for the TNF receptors TNFRI and TNFRII, or TNF had no defects in lymph node organogenesis, it was hypothesized that an interaction between membrane-bound LT and LT R located on the stromal elements might play a role in lymph node genesis (Crowe et al., 1994). Three approaches were taken, two using an LT R:Fc fusion protein to neutralize ligands either expressed as a transgene (Ettinger et al., 1996) or injected into pregnant mice for introduction into embryonic circulation (Rennert et al., 1996), and the other by interruption of the LT and LT R locus (Koni et al., 1997; Alimzhanov et al., 1997; Futterer et al., 1998). The LT R:Fc protein does not block soluble, trimeric LT binding with TNFRI, however it does block the activity of LIGHT. LT -deficient mice and mice that were treated in utero with LT R:Fc fusion protein have no histologically detectable inguinal and popliteal lymph nodes nor PPs. As this matches the phenotype of LT and LT mice it is unlikely that LIGHT plays a role here. Temporal development of lymph nodes could be determined by administering the LT R:Fc fusion protein at different days of gestation; the full effect of LT R:Fc must be administered prior to day 12 of gestation. By this staging, PPs were the latest in sequence to develop. By contrast the mice with LT R:Fc transgene had normal lymph node development, but

PPs were reduced or absent, probably due to low neutralizing levels of LT R:Fc until late and/or the mutated Fc region did not cross the placental barrier. LT Rÿ/ÿ mice, and LT- and LT -deficient mice, display disorganized splenic architecture. The other shared phenotypes of the LT Rÿ/ÿ and LTÿ/ÿ mice was the absence of colon-associated lymphoid tissues and all lymph nodes. Other phenotypes observed in the LT Rÿ/ÿ mice included loss of E 7high integrin‡ T cells and loss of marginal zones, T/B cell segregation, and follicular dendritic cell networks in the spleen. Peanut agglutinin‡ cells were aberrantly detectable around central arterioles. In contrast to TNF receptor p55ÿ/ÿ mice, antibody affinity maturation was impaired. Since LT Rÿ/ÿ mice exhibit distinct defects when compared to LTÿ/ÿ and LT ÿ/ÿ mice, for example, affinity maturation of the antihapten Ig response occurs almost unimpaired in TNFRIÿ/ÿ mice and is not markedly impaired in LTÿ/ÿ mice, it implies that other ligands may be able to activate LT R. Here LIGHT becomes a lead candidate (Mauri et al., 1998). Nonetheless, the LT R is essential for generation of lymphoid tissues. In addition to the LT system regulating lymph node genesis, several genes have been identified that also play a role in this process. Mice deficient for the early hematopoietic- and lymphocyte-restricted transcription factor Ikaros, lack lymph nodes and PPs. Mice that developed a spontaneous autosomal recessive mutation, termed aly (alymphoplasia), also lack lymph nodes and this defect is attributable to a mutation in NIK, which links the LT R to activation of NFB (Shinkura et al., 1999). LT R:Fc injected into pregnant mice recapitulated the NK cell defect observed in LTÿ/ÿ mice. The lack of functional NK cells has serious consequences, including the inability to reject some tumors (Iizuka et al., 1999; Ito et al., 1999).

Unique biological effects of activating the receptors Although in vitro the LT R shares significant overlap with TNFRI, in vivo studies clearly indicate these receptors play distinct and nonredundant functions.

Phenotypes of receptor knockouts and receptor overexpression mice See Table 2.

1646 Nancy Ruddle and Carl F. Ware Table 2 Phenotypes of mice deficient in members of the immediate LT/TNF family Lymph nodes Gene:

mes

cer

Spleen axi

ing

pop

PP

Thymus

cec

LT Rÿ/ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

‡

‡

TNFRIÿ/ÿ

‡

‡

‡

‡

‡



ÿ

‡

‡

LTÿ/ÿ



ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

‡

‡

LT ÿ/ÿ

‡

‡

ÿ

ÿ

ÿ

ÿ

‡

‡

‡

TNFÿ/ÿ

‡

‡

‡

‡

‡

‡

‡

‡

‡

Splenic architecture

Germinal centers

Gene:

T&B zones

MZ sinuses

Moma-1‡ M

Sialoadhesion

MAdCAM-1

MZB cells

PNA‡ clusters

FDC network

LT Rÿ/ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

TNFRIÿ/ÿ

‡

‡

‡

‡

ÿ

‡

ÿ

ÿ

LTÿ/ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

ÿ

LT ÿ/ÿ

‡

ÿ

ÿ

ÿ

ÿ

ÿ

‡

ÿ

TNFÿ/ÿ

‡

‡

‡

‡

ÿ

‡

‡

ÿ

Immune response Isotype switching

Affinity maturation

E 7 T cells

Gene:

sRBC

Hapten-Ag

Low Ag

High Ag

LT Rÿ/ÿ

ÿ

‡

ÿ

‡

TNFRIÿ/ÿ

ÿ

‡

‡

‡

‡

LTÿ/ÿ

ÿ

‡

ÿ

‡

ÿ

LT ÿ/ÿ

ÿ

‡

‡

‡

ÿ

TNFÿ/ÿ

ÿ

‡

ÿ

‡

‡

mes, mesenteric; cer, cervical; axi, axillary; ing, inguinal; pop, popliteal; PP, Peyer's patches; cec, cecal; MZ, marginal zone; MAdCAM-1, mesenteric addressin cell adhesion molecule-1; PNA, peanut agglutinin; FDC, follicular dendritic cell; sRBC, sheep red blood cell; E 7, integrin; Ag, antigen.

Human abnormalities None described to date.

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain The LT R:Fc fusion protein is a potent neutralizing agent for LT1 2 and LIGHT. Mackay et al. (1998) investigated the action of this inhibitor in two independent rodent models of colitis. LT R:Fc

attenuated the development of both the clinical and histological manifestations of the disease and was as efficacious as antibody to TNF. This lead to the conclusion that LT pathway plays a role in the development of colitis and represents a potential novel intervention point for the treatment of inflammatory bowel disease.

Effects of inhibitors (antibodies) to receptors Anti-LT R monoclonal antibodies have been shown to induce death of certain adenocarcinomas and also

Lymphotoxin Receptor 1647 show an effect on growth of tumors in vivo (Browning et al., 1996), suggesting the possibility of their use in antitumor therapy.

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