TNF Receptors

TNF Receptors Bharat B. Aggarwal1,*, Ajoy Samanta1 and Marc Feldmann2 1

Cytokine Research Laboratory, Department of Bioimmunotherapy, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, PO Box 143, Houston, TX 77030, USA 2 Kennedy Institute of Rheumatology, 1 Aspenlea Road, Hammersmith, London, W6 8LH, UK * corresponding author tel: 713-792-3503, fax: 713-794-1613, e-mail: [email protected] DOI: 10.1006/rwcy.2000.16001.

SUMMARY The TNF receptors, commonly referred to as type I and type II with a molecular mass of 55±60 kDa and 75±80 kDa, respectively, share a cysteine-rich extracellular domain and a distinct transmembrane domain. Type I receptor is expressed in all cell types, whereas type II is expressed only by cells of the immune system and on endothelial cells. Most TNF signals are mediated through the type I receptor; the precise role of type II receptor is still unclear. The cytoplasmic domains of both receptors lack any enzymatic activity. The death domain present in the cytoplasmic portion of the type I receptor is known to recruit at least 20 different proteins to form a cascade leading to activation of various cellular responses including apoptosis, nuclear transcription factor NFB, and cJun N-terminal kinase. Soluble forms of both types of TNF receptors, consisting of an extracellular domain, have been identified in in vitro cell culture conditioned media and in serum, urine, synovial fluids, and cerebral spinal fluids of patients with various diseases. The soluble form of the type II receptor has been approved for human use in rheumatoid arthritis.

BACKGROUND

Discovery That there are specific high-affinity cell surface receptors for TNF were first discovered in 1985 (Aggarwal et al., 1985). By crosslinking the ligand with the receptor through reversible and irreversible crosslinkers and by immunoaffinity chromatography, the TNF receptor was isolated as a protein with an approximate molecular mass of 70 kDa (Stauber et al.,

1988 and references therein). In 1990, the cDNA for two different TNF receptors with a predicted molecular mass of 55±60 kDa or 75±80 kDa were cloned and thus referred to as p60 (or p55) and p80 (or p75) (Loetscher et al., 1990; Schall et al., 1990; Smith et al., 1990).

Alternative names The smaller TNF receptor is referred to as p60, p55, type I, or CD120a receptor and the larger form as p80, p70, p75, type II receptor, or CD120b.

Structure Both TNF receptors are type II transmembrane proteins consisting of an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain (ICD) (Figure 1). The ECDs of both receptors contain four well-conserved cysteine-rich domains (CRDs). The amino acid sequences of the ICD of the two receptors are quite dissimilar and lack any intrinsic enzymatic activity.

Main activities and pathophysiological roles In general, most TNF proinflammatory activities are mediated through the p60 receptor; the role of the p80 receptor is less clear (Tartaglia and Goeddel, 1992; Gruss and Dower, 1995). The p60 receptor has been shown to activate apoptosis, nuclear transcription factor NFB, and c-Jun N-terminal kinase (JNK) (Chainy et al., 1996 and references therein). In addition, the p60 receptor has been implicated in

1620 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann TNF-mediated inflammation, viral replication, and protection against bacterial and fungal infections. In selective studies, the p80 receptor has been shown to mediate cytotoxicity and primary thymocyte and T cell proliferation (Tartaglia et al., 1993a). The overexpression of p80 receptor can also cause activation of NFB and JNK (Haridas et al., 1998 and references therein).

GENE

Accession numbers p60 receptor: M75866 p80 receptor: S63368

Sequence The p60 and p80 TNF receptors are each encoded by a single gene. The p60 receptor is encoded by at least three exons. The gene structure for the human p80 receptor, elucidated by Santee and Owen-Schaub (1996), spans approximately 43 kbp, consists of 10 exons (ranging in length from 34 bp to 2.5 kbp) and nine introns ranging from 343 bp to 19 kbp. Consensus elements for transcription factors present in the promoter region include T cell factor 1 (TCF-1), Ikaros, AP-1, CK-2, IL-6 receptor E (IL-6RE), ISRE, GAS, NFB, and SP-1 in the 5 0 -flanking region. The unusual (GATA)n and (GAA)(GGA) repeats were found within intron 1.

Chromosome location and linkages The gene for the human p60 receptor is located on chromosome 12p13 and the p80 receptor gene is on chromosome 1p36.

PROTEIN

Accession numbers p60 receptor: M75866 p80 receptor: S63368

Description of protein The p60 receptor has 426 amino acid residues consisting of an ECD of 182 amino acids, a TMD of 21

amino acids, and an ICD of 221 amino acids. From this the predicted molecular mass of this receptor is about 47.5 kDa. Since the apparent molecular mass of the p60 receptor is between 55 and 60 kDa, the difference most probably is due to three potential N-linked glycosylation sites present in the ECDs of the receptors. The ECD of p60 receptor has a net charge opposite to that of the TNF, suggesting electrostatic interaction. The p80 receptor is a 46 kDa protein, and it consists of 439 amino acid residues with an ECD of 235 amino acids, a TMD of 30 amino acid residues, and an ICD of 174 amino acids. This receptor is also glycosylated (Tartaglia and Goeddel, 1992; Gruss and Dower, 1995). The ECDs of both p60 and p80 receptors contain four cysteine-rich domain repeats, each consisting of six cysteine residues. The ICDs of the two receptors, however, are completely distinct, indicating distinct signaling pathways. The most striking feature of the ICD of the p60 receptor is a region of approximately 80 amino acid residues near the C-terminus called the death domain (DD) because of its importance in TNF-mediated cell death (Tartaglia et al., 1993b). This region is homologous to Fas, death receptor (DR)-3, DR4, DR5 and DR6, all the receptors implicated in cell death (Ashkenazi and Dixit, 1998). The p80 receptor, in contrast, lacks a DD but contains a serine-rich region that undergoes phosphorylation in a ligand-independent manner (Pennica et al., 1992; Darnay et al., 1994a; Beyaert et al., 1995). The crystal structure of the p60 receptor bound to TNF has been solved. This structure predicts that one ligand trimer brings three receptor chains together to form a complex. The receptor binds in three grooves in the ligand trimer formed by the subunit interfaces, so each receptor makes contact with two subunits (Banner et al., 1993). Initially it was found that the TNF receptor binds TNF with the same affinity as it binds to lymphotoxin (LT) (Aggarwal et al., 1985). Later it was found that both p60 and p80 forms of the TNF receptors can bind both LT and TNF with comparable affinities (for references see Tartaglia and Goeddel, 1992).

Relevant homologies and species differences The cDNA of mouse TNF receptor has also been cloned (Barrett et al., 1991; Lewis et al., 1991). The sequence homologies show that the human p60 and p80 receptors are 64% and 62%, respectively, identical to the corresponding mouse receptor. The p60 receptor, however, is most conserved in the ECD

TNF Receptors 1621 (70%) whereas the p80 is conserved in the ICD (73%). This may explain why human p60 receptor binds both human and murine TNF whereas human p80 receptor binds only human and not mouse TNF (Lewis et al., 1991). The ECD of murine p60 and p80 are 28% identical to each other. The p60 and p80 form of the TNF receptors are also homologous to several other members of the TNF receptor superfamily characterized by the presence of cysteine-rich domains in their ECDs. These include Fas, DR3, DR4, DR5, DR6, NGF (31%), RANK, CD40 (40%), CD27, CD30, Ox40, and 4-1BB (for references see Gruss and Dower, 1995; Ashkenazi and Dixit, 1998). The major area of homology between these receptors, which is in their ECD, may range from 25 to 30%. In addition, several viral open reading frames (ORF) have been found to encode for soluble TNF receptor-like molecules. This includes SFV-T2 in Shope fibrosarcoma virus and Va53 or SaIF19R in vaccinia virus, MYX-T2, G4R, CrmB, and CrmD.

1985). Since then several agents have been found to regulate TNF receptors, including IFN, IFN , IL-2, IL-4, and phorbol ester (Table 1; for references see Aggarwal and Natarajan, 1996). Interestingly, TNF can also both upregulate and downregulate its own receptors in a cell type-specific manner. Table 1 Agents that can regulate TNF receptors in different cells Protein kinase C activators

Phorbol esters

Protein kinase C inhibitors

Staurosporine

Cytokines

IFN IL-2 IL-4 IL-6 IL-8 TNF Thyroid-stimulating hormone

Affinity for ligand(s) Both types of TNF receptor have a high affinity for TNF, in the range of 0.1±1 nM. Most cells exhibit a receptor density of around 1000 sites/cell but in some it is as high as 5000 sites/cell. A recombinant human TNF has been engineered that binds either the p60 or p80 form of the TNF receptors. TNF mutated at R32W and S86T binds to the p60 and that mutated at D143N and A145R exclusively binds to the p80 forms, thus suggesting that p80 receptor binds at the C-terminal of the cytokine whereas p60 binds more towards the N-terminus (Loetscher et al., 1993; Van Ostade et al., 1994; Haridas et al., 1998).

Cell types and tissues expressing the receptor The p60 form of the TNF receptor is expressed by all cell types examined to date. The p80 receptor, in contrast, appears to be expressed by the cells of the immune system and hematopoietic cells such as macrophages, neutrophils, lymphocytes (B cells and T cells), thymocytes, and mast cells. Endothelial cells, cardiac myocytes, and prostate cells have also been shown to express the p80 receptor.

GM-CSF Microtubule depolymerizing agents

Nocodazole Vincristine Vinblastine Colchicine Podophyllotoxin P-Lumicolchicine

Protein kinase A activators

Dibutyryl cAMP Forskolin

Others

Hydrogen peroxide Retinal Butyrate Glucocorticoids Lipopolysaccharide Taxol fMLP Complement (C5a) Calcium ionophore Platelet-activating factor Leukotriene B4 Lectins Sulfasalazine

Regulation of receptor expression

Okadaic acid

When the TNF receptor was first discovered, it was found to be upregulated by IFN (Aggarwal et al.,

Pervanadate

Iodoacetic acid

1622 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann So far, there is no report of differential regulation by one type of TNF receptor over another. There is also very little known about the regulation of TNF receptor at the transcription, translation, or posttranslation levels.

Release of soluble receptors The TNF receptor was first purified in its soluble form (Seckinger et al., 1989, 1990; Kohno et al., 1990; Nophar et al., 1990; Gatanaga et al., 1990; Lantz et al., 1990). Studies during the last decade have shown that both the p60 and p80 forms of the TNF receptor are released in cell culture-conditioned medium in response to a variety of stimuli. In addition, the soluble forms of both receptors have been detected in vivo, in serum, synovial fluids, cerebral spinal fluids, ovarian ascites and urine (Engelmann et al., 1989; Seckinger et al., 1989). The levels of these receptors appear to be elevated when a person is attacked by a pathological condition (Aderka et al., 1993; Deloron et al., 1994). In general, there are higher levels of soluble p80 receptors (2±4 ng/mL) than the soluble p60 receptors in normal sera. These receptors appear to be able to bind TNF with almost the same affinity as the transmembrane receptor. How these receptors are released is not fully understood. Metalloprotease inhibitors block both TNF and TNFR release with similar IC50 values by blocking. TNF-converting enzyme (TACE), the enzyme that causes the release of TNF, is also involved in the release of the receptor. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and TGF. The phenotype of mice lacking TACE suggests an essential role for soluble TGF in normal development (Peschon et al., 1998).

SIGNAL TRANSDUCTION

Associated or intrinsic kinases The ICD of both the p60 and p80 receptor lacks homology to the catalytic domain of either Tyr or Ser/Thr-specific protein kinases or to nucleotidebinding proteins. The cytoplasmic domains of both the p60 and p80 receptors have been shown to bind to distinct serine/threonine kinases and cause the phosphorylation of the receptor (Darnay et al., 1994a, 1994b, 1995; Beyaert et al., 1995). In the case

of the p60 receptor, the DD recruits a protein called TRADD which binds to TRAF2. TRAF2 has been shown to recruit a serine/threonine kinase called NIK that binds to IKK and IKK (for references see Wallach et al., 1997) (see also The TNF Ligand and TNF/NGF Receptor Families).

Cytoplasmic signaling cascades A series of proteins have been identified that bind to the p60 receptor, leading to various cellular responses (Table 2; for references see Tewari and Dixit, 1996; Darnay and Aggarwal, 1997; Singh and Aggarwal, 1998). Three major activities assigned to the p60 receptor include activation of NFB, JNK, and apoptosis (Figure 1). As indicated above, the DD present in the ICD of p60 receptor binds to TRADD, which binds to TRAF2, which in turn binds to NIK (for references see Wallach et al., 1997) (see also The TNF Ligand and TNF/NGF Receptor Families). NIK then binds to activate IKK and IKK , which cause the phosphorylation of IB-, leading to its degradation through a ubiquitin-dependent pathway, thus releasing in the cytoplasm the p50/p65 and other heterodimers of NFB, which is then translocated to the nucleus to bind to the DNA, resulting finally in NFB-dependent gene activation (Figure 2). The cytoplasmic protein TRADD has also been found to recruit FADD, which in turn binds to FLICE, and the latter activates a family of aspartatespecific cysteine proteases, called caspases, which are responsible for inducing apoptosis or cell death (for references see Tewari and Dixit, 1996; Darnay and Aggarwal, 1997; Salvesen and Dixit, 1997; Singh and Aggarwal, 1998). Although it has been shown that TRAF2 is needed for JNK activation through the p60

Figure 1 Architecture of the p60 and p80 forms of the TNF receptors and their function.

TNF Receptors 1623 Table 2

Proteins known to interact with the cytoplasmic domain of the TNF receptors

Protein

Interacting partner

p60 receptor TNF receptor-associated death domain protein (TRADD)

p60 receptor

Sentrin

p60 receptor

Factor-associated with N-Smase activation (FAN)

p60 receptor

MAP kinase-activated death domain protein (MADD)

p60 receptor

TNF receptor-associated protein (TRAP1)

p60 receptor

TNF receptor-associated protein (TRAP2)

p60 receptor

p60 TNF receptor-associated kinase 60-TRAK

p60 receptor

BRE (brain and reproductive organ expression)

p60 receptor

55.11 protein

p60 receptor

TNF receptor-associated factor (TRAF2)

TRADD

Receptor-interacting protein (RIP)

TRAF2

NFB-inducing kinase (NIK)

TRAF2

Apoptosis signal-regulating kinase 1 (ASK1)

TRAF2

Germinal center kinase (GCK)

TRAF2

TRAF2-interacting protein (I-TRAF/TANK)

TRAF2

Cellular inhibitor of apoptosis (cIAP1)

TRAF2

Cellular inhibitor of apoptosis (cIAP2)

TRAF2

A20 zinc finger protein

TRAF2

Silencer of death domains (SODD)

p60 receptor

Fas-associated death domain protein (FADD/Mort 1)

TRADD

FADD-like ICE (FLICE/MACH)

FADD

FLICE-interacting protein (I-FLICE/CASH/FLIP/MRIT)

FLICE

p80 receptor TNF receptor-associated factor (TRAF2)

TRAF1

TNF receptor-associated factor (TRAF1)

TRAF2

Cellular inhibitor of apoptosis (cIAP1)

TRAF2

Cellular inhibitor of apoptosis (cIAP2)

TRAF2

p80-TRAK

p80 and p60 receptor

receptor, how TRAF2 induces JNK activation is not clear (Figure 2). Recent evidence indicates that apoptosis signal-regulating kinase 1 (ASK1) interacts with TRAF2 and activates JNK (Nishitoh et al., 1998). ASK1 is a MAPKKK that activates SEK1/ JNK and MKK6/p38 signaling cascades. Another study indicates that interaction of TRAF2 with germinal center kinase (GCK) leads to activation of JNK (Yuasa et al., 1998). Yuasa's group also showed that the interaction of TRAF2 with RIP leads to activation of both JNK and p38 MAPK.

Some studies indicate that the p80 receptor can also activate apoptosis, JNK and NFB (Haridas et al., 1998). The p80 receptor lacks the DD. Exactly how the p80 receptor mediates these responses is not fully understood, but its ICD is known to bind to TRAF2 directly, which can activate NFB and JNK through a similar cascade used by the p60 receptor (see chapters on TNF and The TNF Ligand and TNF/ NGF Receptor Families). In addition to these responses, the p60 receptor is known to activate a family of kinases, the mitogen-

1624 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann Figure 2

Signaling cascade leading to activation of NFB, JNK, and apoptosis.

activated protein kinases (MAPK) as outlined in Figure 3. In addition, the p60 receptor has also been shown to activate various other kinases including germinal center kinase (GCK) (Pombo et al., 1995), apoptosis signal-regulating kinase (ASK1) (Nishitoh et al., 1998) and p21-activated kinase (PAK) (Table 3). Besides kinases, the p60 receptor activates acidic and neutral sphingomyelinases to induce the release of ceramide which is involved in downstream signaling (Kim et al., 1991 and references therein). This receptor is also known to activate various phospholipases leading to the release of arachidonic acid and its metabolites, the prostaglandins. The p60 receptor is also a potent inducer of reactive oxygen intermediates. The signals mediated through each type of receptor are listed in Table 4.

DOWNSTREAM GENE ACTIVATION

transcription factor described to date. The activation occurs within 5 minutes and with as little as 1 pM TNF (Chaturvedi et al., 1994). How the p60 receptor activates this transcription factor is shown in Figure 2. The activation of another transcription factor, AP-1, by the p60 receptor is mediated through JNK (Brenner et al., 1989).

Genes induced The TNF receptors mediate the induction of a wide variety of genes that are involved in autoimmunity, inflammation, viral replication, cell proliferation, tumorigenesis, and tumor metastasis. Some of these genes are listed in Table 5. This includes genes for inflammatory mediators, acute phase response proteins, cytokines, receptors, members of the MHC, enzymes, oncogenic proteins, cell adhesion molecules, and growth regulatory molecules (for references see Aggarwal and Natarajan, 1996).

Transcription factors activated

Promoter regions involved

TNF activates various transcription factors (Table 5). Perhaps most important of these is NFB, which is responsible for many of the inflammatory effects of the TNF. TNF is the most potent activator of this

The promoter of the inflammatory genes activated by the TNF receptor contains NFB-binding sites. Other genes also contain AP-1, SP-1, or c-myc-binding sites needed for TNF receptor-mediated activation.

TNF Receptors 1625 Figure 3 Activation of MAPK pathways by TNF receptors.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors As indicated above, most activities of TNF are mediated through the p60 receptor (see Table 4). There are some reports which suggest that proliferation of thymocyte and circulating T lymphocytes and inhibition of early hematopoiesis are mediated through the p80 receptor (Tartaglia et al., 1993a; Jacobsen et al., 1994). It has been proposed that the p80 receptor may concentrate the ligand to be passed on to the p60 receptor. There are also reports suggesting that the p80 receptor is unique in mediating signaling initiated by the transmembrane form of TNF (Grell et al., 1995). In contrast to the effects of TNF and p60 receptor-specific TNF mutein, p80

receptor-specific TNF mutein given to baboons failed to induce inflammation or shock, failed to induce hemodynamic changes, and failed to induce the plasma appearance of IL-6 and IL-8 (Van Zee et al., 1994; Welborn et al., 1996). It also increased baboon thymocyte proliferation in vitro. In addition, local skin necrosis and tissue neutrophil infiltration was seen after subcutaneous administration only of the TNF and TNF-p60 mutein, not with the p80-specific mutein. A similar role of the p80 receptor was discovered by administering receptor antibodies (Sheehan et al., 1995).

Phenotypes of receptor knockouts and receptor overexpression mice The deletion of genes for p60 or p80 receptor have quite distinct effects in mice (Table 6). Mice deficient in p60 receptor are resistant to endotoxin-induced shock but are susceptible to infection by Listeria monocytogenes (Pfeffer et al., 1993). The p60 receptor

1626 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann Table 3 Kinases activated by TNF receptors P60 receptor

p42/44 Mitogen-activated protein kinase (MAPK or Erk)

Table 4 Signals transmitted through each type of the TNF receptors p60 receptor

MAPK kinase (MAPKK or MEK)

Induction of c-fos

MAPKK kinase (MAPKKK or MEKK; MKK4, MKK7)

Stimulation of protein kinase C Stimulation of sphingomyelinase

p38 MAPK

Stimulation of phospholipase A2

c-Jun N-terminal protein kinase (JNK or SAPK)

Production of diacylglycerol Production of ceramide

NFB-inducing kinase (NIK)

Induction of IL-6

IBa kinase (IKK-)

Induction of Mn superoxide dismutase mRNA

IBa kinase (IKK- ) Germinal center kinase (GCK)

Prostaglandin E2 synthesis

Apoptosis signal-regulating kinase 1 (ASK1)

Induction of IL-2 receptor HLA class I and II Ag expression

Receptor interacting protein (RIP)

Antiproliferation/cytotoxicity/ apoptosis

p21-activated protein kinase (PAK) hsp27 and -casein kinase (65 kDa)

Growth stimulation

c-raf-1 kinase

Endothelial cell adhesion molecules

Protein kinase C

Generation of lymphocyte-activated killer (LAK) cells

Casein kinase 2 pp90rsk

Proliferation of natural killer (NK) cells

Cyclin-dependent kinase (CDK) 2 Ceramide-activated protein kinase (CAPK)

p80 receptor

Induction of NFB activation

Antiviral activities p80 receptor

Induction of NFB

HPK/GCK-like (HGK)

Proliferation of thymocytes

TGF -activated kinase (TAK1)

Induction of IL-6

c-Jun N-terminal kinase (JNK)

Generation of NK and LAK cells DNA fragmentation

Both NIK and ASK1 belong to the MAPKKK family. All kinases are Ser/Thr kinases except MEK, which is a dual specificity kinase and phosphorylates at Ser-Thr and Tyr residues. For relationships between these kinases see Figure 2.

was also found to control early graft-versus-host disease (Speiser et al., 1997). These results also suggest that the p60 receptor plays an important role in septic shock and in protection from bacterial infection. Another study indicated that such mice are also resistant to TNF-mediated toxicity (Rothe et al., 1993). Interestingly, however, deletion of the gene for the p80 receptor in mice also decreased the sensitivity to TNF (Erickson et al., 1994). In addition, p80 receptor-deleted mice had normal T cell development though they exhibited depressed Langerhans cell migration and reduced contact hypersensitivity (Wang et al., 1997).

Antiproliferation/cytotoxicity/ apoptosis

There are limited studies on the effects of TNF receptor transgenes in vivo. It was found that the production of the human p80 receptor in transgenic mice results in a severe inflammatory syndrome involving mainly the pancreas, liver, kidney, and lung, and characterized by constitutively increased NFB activity in the peripheral blood mononuclear cell compartment (Douni and Kollias, 1998). Mice have been generated in which the genes for the signaling proteins through which p60 receptor mediates its effects (Table 3) are deleted. The effect of these gene deletions on p60 signaling has been examined (see Table 6). For instance the deletion of TRAF2, FADD, FLICE, caspase 9, and the caspase 3

TNF Receptors 1627 Table 5 Genes and transcription factors activated and induced by TNF Transcription factors

Oncogenes

Table 5 (Continued ) Transcription factors

SP-1 c-fos

Haptoglobin

c-jun/AP-1

C3 complement

NFB

TNF-stimulated gene 14

IRF-1

MHC proteins

MHC class I and II

c-myc

Viruses

HIV

c-abl

Enzymes

Acyl-CoA synthetase Stearoyl-CoA desaturase 1

c-sis Egr-1 p53 Cytokines

Receptors

SP-1

TNF

Other proteins

Plasminogen activator inhibitor I and II MnSOD

IL-1

2 0 ,5 0 -oligoadenylate synthetase

IL-6

Glycoprotein Ib

IL-8

HMG-CoA reductase

IFN

B12

MCP-1

GLUT-1

NCF

B94

MCAF

Phosphodiesterase

G-CSF

M1, M2

GM-CSF

PCNA

M-CSF

Endothelin 1

HB-EGF

A20

Activin A

Cyclin 1

Basic FGF

Secretory component

PDGF

CD4 surface antigen

TGF

Dimethylnitrosamine

NGF

Cystic fibrosis transmembrane

fMLPR IL-1R IL-2R EGFR

Conductance regulator gene VP16

IL-6R (gp130) IFN R PTHR Cell adhesion molecules

VCAM-1 ICAM-1 ELAM-1

Inflammatory mediators

Collagenase Stromelysin Tissue factor 1 acid glycoprotein

gene was lethal in mouse (Yeh et al., 1997, 1998; Woo et al., 1998; Varfolomeev et al., 1998; Hakem et al., 1998), whereas deletion of Apaf-1 was not (Yoshida et al., 1998). Embryonic lethality suggests a critical role of FADD and FLICE in development. This role must be independent of TNF receptor as deletion of either p60 or p80 has no effect on the survival of the animals. Cells derived from FLICE or FADD knockout animals could be activated for NFB and JNK but not apoptosis, suggesting a critical role of FADD and FLICE in TNF-induced apoptosis.

1628 Bharat B. Aggarwal, Ajoy Samanta and Marc Feldmann Table 6 Effect of gene deletion for TNF receptors and receptor-associated proteins on the mouse phenotypes Deletion

Phenotype

p60

Resistant to endotoxin-induced shock Susceptible to infection by Listeria monocytogenes Resistant to TNF-mediated toxicity Controls early graft-versus-host disease Controls induction of autoimmune heart disease Mediates deletion of peripheral cytotoxic T lymphocytes in vivo Expression of adhesion molecules and leukocyte organ infiltration

p80

Decreased the sensitivity to TNF Normal T cell development Depressed Langerhans cell migration Reduced contact hypersensitivity

TRAF2

Appeared normal at birth but became progressively runted and died prematurely Atrophy of the thymus and spleen and depletion of B cell precursors Elevated serum TNF levels Normal NFB activation Severe reduction in TNF-induced JNK activation Increased sensitivity to TNF-induced cell death

FADD/Mort-1

Lethal, mice do not survive beyond day 11.5 of embryogenesis Signs of cardiac failure and abdominal hemorrhage Cells are resistant to p60 receptor-mediated apoptosis

FLICE/caspase 8

Lethal in utero Impaired heart muscle development and congested accumulation of erythrocytes Low recovery of hematopoietic colony-forming cells TNF-activated NFB and JNK but not apoptosis

Caspase 3

Reduced viability of animals Defective neuronal apoptosis and neurological defects Show cell type-specific inihibition of DNA fragmentation

Caspase 9

Embryonically lethal Defective brain development with decreased apoptosis Cells are resistant to apoptosis induction by various agents Normal TNF-induced apoptosis in some cells but not in others

Apaf-1

Animals are viable but exhibit craniofacial abnormalities with hyperproliferation of neuronal cells Cells are resistant to apoptotic stimuli but not to Fas Impaired processing of caspase 2, 3, and 8

Human abnormalities There are several familial disorders characterized by periodic fever. The most common is the recessive

familial mediterranean fever (FMF) which is caused by a polymorphism of pyrin, a protein expressed predominantly in leukocytes homologous to nuclear factors (The International FMF Consortium, 1997).

TNF Receptors 1629 Familial hibernian fever (FHF) is a dominant disease, found in several large Irish/Scottish families, which has longer inflammatory attacks than FMF, and responds less well to colchicine. FHF has been studied by genetic analysis, and the susceptibility mapped to the distal part of chromosome 12p, an area encoding CD4, LAG-3, CD27, complement genes CIr and CIs, and the p55 TNFR (McDermott et al., 1998). As during attacks the patients have low levels of soluble p55 TNFR compared to controls, this latter gene was analysed in seven families. In each family, a single nucleotide substitution was found, most in the first cysteine-rich domain of the extracellular region, some in the second cysteine-rich domain of the extracellular region (McDermott et al., 1999). In the affected individuals, soluble p55 TNFR levels were half those of normals (500 pg against 1 ng/mL), whereas levels of p75 soluble TNFR were normal. The affected individuals have increased levels of membrane-bound p55 TNFR, with a similar binding affinity for TNF. Shedding of p55 TNFR after activation, e.g. with PMA, was reduced, with more membrane TNFR and less in the supernatant. These studies confirm that the p55 TNFR is a major receptor for inflammation, and that its regulation by shedding is critical to the regulation of inflammation. Regulatory Roles of TNF Receptor Cleavage TNF receptors signal after they have been aggregated, usually by the agonist, TNF, or experimentally by antibodies. Thus the density of TNFR is important in facilitating signaling, and TNFR cleavage will reduce TNFR signaling by reducing TNFR density (Chan and Aggarwal, 1994). Moreover, the soluble TNFR can bind TNF and so can act as a competitor to the cell surface receptors (Engelmann et al., 1989; Seckinger et al., 1989; Olsson et al., 1989). The concentration of soluble receptors in the serum in normal healthy individuals is high, about 1 ng/mL of p55 and 2±4 ng/mL of the p75 receptor. In inflammatory diseases these levels are increased significantly and this has been well documented in rheumatoid arthritis, with levels increased in synovial fluid several fold higher than in the serum, where the levels are also augmented (Cope, 1992; Roux-Lombard, 1993). It is likely that a major function of soluble TNFR is to augment the clearance of TNF from the serum. This concept is supported by studies in volunteer patients or primates given TNF i.v., who had transient rises in serum TNF and subsequent rises in serum TNFR with rapid clearance of the serum TNF (Van Zee et al., 1992; Bemelmans et al., 1993). Furthermore, studies in nephrectomized mice have supported this conclusion.

THERAPEUTIC UTILITY See chapter on TNF, where the effects of TNF blockade especially in rheumatoid arthritis and Crohns' disease are discussed. So far, two TNFblocking agents have been licensed for use: EnbrelTM, a p75 dimeric Fc fusion protein (Weinblatt et al., 1999) and RemicadeTM, a chimeric anti-TNF antibody. More are in clinical trials. The second aspect is the use of soluble TNFR to monitor inflammatory disease activity. It has been found that in a wide spectrum of diseases, such as systemic lupus erythematosus (Aderka et al., 1993), rheumatoid arthritis (e.g. Cope et al., 1992; Roux-Lombard et al., 1993) and cancer (e.g. Aderka et al., 1991), the levels of soluble TNFR are augmented as disease activity gets worse, and so it is a useful marker of disease activity.

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LICENSED PRODUCTS Etanercept (EnbrelTM), a dimeric fusion protein of p75 TNFR coupled to IgG1 Fc (Immunex/AHP). TNF p60 receptor antibodies supplied by StressGen Biotechnology Corp. (www.stressgen.com/reagents) TNF p80 receptor antibodies Soluble p60 receptor Soluble p80 receptor Enbrel (soluble p80 ECD fused to Fc) supplied by Immunex Corporation for use in advanced rheumatoid arthritis