The tumour necrosis factor receptorassociated

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1 The tumour necrosis factor receptorassociated periodic syndrome: Silvia Stojanov and Michael F. McDermott The tumour necrosis factor receptor (TNFR)-associated periodic syndrome (TRAPS) is an autosomal dominant, multisystemic, autoinflammatory disorder caused by mutations in the TNFR1 gene (TNFRSF1A). TRAPS seems to be the most common hereditary periodic fever (HPF) syndrome in some Western populations, and the second most prevalent HPF worldwide, behind familial Mediterranean fever (FMF). The proteins involved in susceptibility to TRAPS (TNFRSF1A) and FMF (pyrin) are both members of the death-domain-fold superfamily. Mutations affecting these proteins might cause dysregulation of innate immune responses, with a propensity to autoinflammation. Most TRAPS patients have reduced blood levels of soluble TNFRSF1A between attacks, with an inappropriately small increase during bouts of fever. The pathogenesis of the hyperinflammatory state in TRAPS has been variously ascribed to a shedding defect of TNFRSF1A from the cell surface resulting in increased TNF inflammatory signalling, or impaired TNF apoptotic signalling. Some lowpenetrance TNFRSF1A variants also contribute to the clinical phenotype in individuals carrying other HPF-associated mutations, and have been reported in several disorders such as Behçet s disease and systemic lupus erythematosus. Synthetic anti-tnf agents provide a rational form of therapy for TRAPS, and have been shown to delay or indeed prevent development of systemic amyloidosis (AA type), a life-threatening complication in this condition. Silvia Stojanov (corresponding author) Postdoctoral Fellow, Genetics and Genomics Branch, NIAMS, National Institutes of Health, Building 9, Room 1W111, 9 Memorial Drive, Bethesda, MD , USA. Tel: ; Fax: ; stojanos@mail.nih.gov Michael F. McDermott Professor of Experimental Rheumatology, Academic Unit of Musculoskeletal Disease, University of Leeds, Clinical Science Building, St. James s University Hospital, Beckett Street, Leeds, LS9 7TF, UK. Tel: +44 (0) ; Fax: +44 (0) ; m.mcdermott@leeds.ac.uk Silvia Stojanov s contributions are a work of the United States Government and are not protected by copyright in the United States. 1

2 The tumour necrosis factor receptor (TNFR)- associated periodic syndrome (TRAPS; MIM ) is found worldwide, and is a key representative of the autoinflammatory syndromes (Ref. 1). These syndromes are distinguished from autoimmune diseases by the absence of both high-titre autoantibodies and autoantigen-specific T cells. The majority of these syndromes are inherited, and the clinical presentation is typically associated with recurrent fever episodes and systemic inflammation involving skin and serosal linings; therefore, the name hereditary periodic fever syndromes (HPFs) has been adopted. The autosomal recessive HPFs include familial Mediterranean fever (FMF; MIM ), which is the most prevalent HPF worldwide, and hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS; MIM ). In addition to TRAPS, the autosomal dominant disorders include the recently recognised cryopyrin-associated periodic syndromes (CAPS), comprising familial cold autoinflammatory syndrome (FCAS; MIM ), Muckle Wells syndrome (MWS; MIM ), and chronic infantile neurological cutaneous and articular syndrome or neonatal-onset multisystemic inflammatory disease (CINCA/NOMID; MIM ) (Ref. 2). Identification of the mutated proteins for these syndromes (Table 1) has led to an appreciation that most of the proteins involved, including pyrin (mutated in FMF), cryopyrin (mutated in CAPS) and TNFR (mutated in TRAPS), are members of the death-domain-fold (DDF) superfamily of molecules, which are intimately involved in innate immunity. The altered function of these mutated proteins plays a key role in the hyperinflammatory response found in HPFs. TRAPS is the most thoroughly characterised autosomal dominant HPF, and was comprehensively reviewed three years ago (Ref. 3). Recent findings indicate new pathophysiological and therapeutic aspects that warrant an update of the knowledge base of this disease. TNF and TNFR signalling TNF is a highly pleiotropic cytokine that, in addition to triggering apoptosis of certain tumour cells, is a major mediator of the inflammatory response. Thus, inappropriate TNF production or sustained activation of TNF signalling is implicated in the pathogenesis of a wide spectrum of infectious, malignant, autoimmune and autoinflammatory diseases (Refs 1, 4). Human TNF is primarily produced by activated macrophages. After the initial synthesis of a precursor protein of 233 amino acids, the N- terminal 76 amino acids are cleaved by a specific metalloproteinase, TNF-α-converting enzyme (TACE or ADAM17), to yield the mature trimeric Table 1. Mutated genes identified in hereditary periodic fever syndromes Hereditary periodic Inheritance Gene; chromosome Encoded protein fever syndrome FMF Autosomal recessive MEFV; 16p13 Pyrin/marenostrin HIDS Autosomal recessive MVK; 12q24 Mevalonate kinase TRAPS Autosomal dominant TNFRSF1A; 12p13 TNFRSF1A FCAS Autosomal dominant CIAS1/NALP3; 1q44 Cryopyrin MWS Autosomal dominant CIAS1/NALP3; 1q44 Cryopyrin CINCA/NOMID Autosomal dominant CIAS1/NALP3; 1q44 Cryopyrin or de novo Abbreviations: FMF, familial Mediterranean fever; HIDS, hyperimmunoglobulinaemia D and periodic fever syndrome; FCAS, familial cold autoinflammatory syndrome; MWS, Muckle Wells syndrome; CINCA/ NOMID, chronic infantile neurological cutaneous and articular syndrome or neonatal-onset multisystemic inflammatory disease; TNFRSF1A, tumour necrosis factor receptor superfamily 1A; TRAPS, tumour necrosis factor receptor (TNFR)-associated periodic syndrome. 2

3 form, consisting of three identical 157 amino acid subunits (Ref. 5). TNF signals through two distinct receptors TNFRSF1A (also known as TNFR1, p55tnfr, CD120a) and TNFRSF1B (TNFR2, p75tnfr, CD120b) which exist as membrane-bound or soluble homotrimers. These two receptors belong to the TNFR superfamily (TNFRSF), which comprises at least 20 different cytokine receptors (Refs 6, 7). TNFRSF1A is constitutively expressed in most tissues, whereas expression of TNFRSF1B is typically confined to cells of the immune system. In the vast majority of cells, TNFRSF1A is the key mediator of TNF signalling. The extracellular region of TNFRSF1A consists of four cysteine-rich domains (CRD1 4), each containing six cysteine residues that form three disulphide bonds (Fig. 1). Three receptor molecules bind symmetrically to one TNF trimer (Ref. 8). The membrane-distal CRD1 has been shown to mediate homophilic interaction of receptor molecules in the absence of ligand (Ref. 8), and this so-called pre-ligand assembly domain (PLAD) might serve to keep receptors in a silent, homotrimerised state that prevents spontaneous autoactivation. The CRD2 and CRD3 domains interact with the lateral grooves of the trimeric TNF, which are formed between each pair of its three protomers (Ref. 9). Besides the CRDs, TNFRSF1A contains a transmembrane region and an intracellular death domain (DD) (Fig. 1). TNFRs use diverse sets of signalling molecules to initiate cell death and survival; the pathways that determine whether a cell proliferates or undergoes apoptosis in response to TNF are complex and not fully understood. TNFRSF1A might signal through the DD, which can then initiate apoptosis-related signalling cascades through Fas-associated death domain (FADD) and caspase-8 activation (Fig. 2), thereby generating a death-inducing signalling complex (DISC). Alternatively, TNFRSF1A signalling might induce cell survival by activation of two major transcription factors: nuclear factor kappa B (NF-κB) and c-jun. NF-κB activation occurs through the formation of an intracellular complex containing several adaptor proteins, such as TNFR1-associated death domain (TRADD), TNFR-associated factor (TRAF2) and receptorinteracting proteins (RIPs) (Fig. 2). These two distinct intracellular signalling complexes have been described in TNF-induced NF-κB activation and apoptosis in the HT1080 fibrosarcoma cell line (Ref. 10). The first complex (plasma-membrane-bound complex I) rapidly activates NF-κB; the second complex is located in the cytoplasm and involves the association of TRADD and RIP1 with FADD and caspase-8 (Fig. 2). When NF-kB is activated by complex I, expression of the caspase-8 inhibitor c-flip(l) is subsequently induced, which ensures cell survival by not releasing caspase-8/10 from the DISC (complex II). When the initial signal (through complex I and NF-κB) fails to be activated, then signal transduction switches, through complex II, to the apoptotic pathway. Recent studies provide evidence that TNFmediated NF-κB activation requires recruitment of TNFRSF1A to cholesterol- and sphingolipidenriched membrane microdomains, termed lipid rafts (Ref. 11). Interference with the lipid raft organisation has been shown to switch TNF signalling from NF-κB activation to apoptosis. The proper execution of apoptosis, however, is critically dependent on another step: the endocytosis of TNFRSF1A, which results in endocytic vesicles containing activated TNFR1 complexes. Endocytosis is mediated by the TNFR1 internalisation domain (TRID), which is located distal to the transmembrane region within the cytoplasmic receptor sequence, and allows subsequent DISC formation (Ref. 12). Activation of neutral sphingolipidase, which promotes the generation of ceramide that forms the basic structural unit of the sphingolipids, is a further necessary step for the complete development of the cytotoxic program induced by TNF (Ref. 13) and plays a critical role in activating cell death signals initiated by cytokines such as TNF. Thus, the fate of individual cells receiving a TNF signal depends upon a variety of modulators and might also vary according to the presence of particular mutated molecules and the signalling status of the cell. TNF-induced activation of NF-κB NF-κB coordinates the activation of numerous genes in response to pathogens and proinflammatory cytokines, and is therefore a vital component in the development of acute and chronic inflammatory diseases. NF-κB is retained in the cytoplasm in association with inhibitory proteins, called inhibitors of NF-κB (IκBs), which include IκBα, IκBβ and IκBε. A multisubunit IκB kinase, termed the IKK complex, also located in the cytosol, contains two subunits with protein kinase activity: the inhibitory κb kinases 1 and 2 3

4 a Domain organisation of TNFRSF1A Extracellular cysteine-rich domains Transmembrane region Intracellular death domain b 3-D CRDs showing TRAPS mutations CRD1 CRD2 CRD3 CRD4 I170N Cell membrane N H69fs L67P C15 C55R/S C98Y F112I C70S/ N65I C29F R/Y/G C114 H22Q/Y T61I R104Q C30R/S/Y Y20D/H P46L deld42 F60L S86P C117 C129 C73/R/W CH 3 C43R/Y 3 C88/R/Y CH 3 O OCH O T50K/M R92Q/P C33/G/Y HO HO C120 HO C76 C52F/R C96Y C137 APFT L39F insertion C Y38C G36E T37I CRD1 CRD2 CRD3 Schematic of the TNF receptor TNFRSF1A, showing TRAPS mutations in the extracellular cysteine-rich domains and transmembrane region Expert Reviews in Molecular Medicine C 2005 Cambridge University Press Figure 1. Schematic of the TNF receptor TNFRSF1A, showing TRAPS mutations in the extracellular cysteine-rich domains and transmembrane region. (a) Domain organisation of TNFRSF1A. TNFRSF1A contains four extracellular cysteine-rich domains (CRD1 4), each containing six cysteine residues involved in the formation of three disulphide bonds. Besides a transmembrane region, TNFRSF1A also has a distinct intracellular region containing a death domain (DD), which can initiate apoptosis-related signalling cascades or induce cell survival (Fig. 2). I170N is the only TNFRSF1A mutation described so far in the transmembrane region of the receptor. (b) Three-dimensional structure of CRDs 1, 2 and 3 showing mutations associated with tumour necrosis factor receptor (TNFR)-associated periodic syndrome (TRAPS) in bold. Mutations involving cysteine residues are marked in red and disrupted intrachain disulphide bonds are shown as thick red bars. Some mutations marked in turquoise prevent (e.g. Y20H, Y20D, D42del) or cause (e.g. I170N) the formation of a hydrogen bond, with possible consequences for the spatial structure of the receptor or cleavage site. Structurally conserved regions of the CRDs are represented by thicker blue lines. The images of CRD1 and 2 are adapted from Ref. 23 ( 2001 by The American Society of Human Genetics), with permission from Dr I. Aksentijevich, and The University of Chicago Press. 4

5 TNF TNFRSF1A U U U TRAF2 TRADD RIP P IκBα p65 p50 p65 TRADD TRAF2 RIP IKK activation IκBα phosphorylation and polyubiquitination p50 Active NF-κB Survival pathway Dissociation of TRADD, TRAF2 and RIP from TNFRSF1A c-flip(l) TNF-induced cell survival and cell death pathways FADD Expert Reviews in Molecular Medicine C 2005 Cambridge University Press TRAF2 TRADD TRADD TRAF2 Apoptotic pathway Caspase-8/10 Figure 2. TNF-induced cell survival and cell death pathways. (a) Cell survival. Signalling through the tumour necrosis factor (TNF) receptor TNFRSF1A induces cell survival by activation of two major transcription factors: NF-κB (illustrated here) and c-jun (not shown). NF-κB activation occurs through the formation of an intracellular complex ( complex I ) containing several adaptor proteins, such as the death-domain-containing proteins TRADD and RIP (which interact with the death domains of TNFRSF1A) and TRAF2. After TNFRSF1A activation, primarily RIP interacts with and activates the inhibitory κb kinase (IKK) signalosome (comprising IKK1, IKK2 and IKKγ/NEMO). This in turn results in phosphorylation, polyubiquitination and degradation of the NF-κB inhibitor IκBα. This process allows translocation of the NF-κB p50 p65 heterodimer to the nucleus to bind DNA and induce gene expression. The cell survival pathway also activates the caspase-8 inhibitor c- FLIP(L), which results in suppression of the alternative, cell death pathway. (b) Cell death. When NF-κB is not activated upon TNFRSF1A-mediated signalling, the lack of inhibition by c-flip(l) results in activation of the apoptotic pathway via caspase-8/10. Following dissociation of TRADD, RIP and TRAF2 from TNFRSF1A, the death domains of TRADD (and/or RIP) associate with Fas-associated death domain (FADD), leading to the recruitment of caspase-8/10 to the complex ( complex II ) and caspase-8/10 activation by induced proximity. Abbreviations: c-flip, cellular FLICE (Fas-associated-death-domain-like IL-1β-converting enzyme)-inhibitory protein; FADD, Fas-associated death domain; NEMO, NF-κB essential modifier; NF-κB, nuclear factor κb; P, phosphorylation; RIP, receptor-interacting protein; TRADD, TNF-receptor-associated death domain; TRAF2, TNFreceptor-associated factor 2; U, ubiquitin. RIP RIP FADD Caspase-8/10 activation 5

6 (IKK1α and IKK2β). The third component of this IKK complex is NF-κB essential modulator (NEMO), a protein that interacts directly with the kinase subunits and is required for activation of the complex in response to extracellular or intracellular stimuli. The signalosome complex of IκB kinases binds to RIP, drawing the signalosome into direct contact with the TNFR complex (Ref. 14) (Fig. 2). The NF-κB family of transcription factors comprises five different gene products [p50, p52, p65 (RelA), c-rel, RelB] that are capable of homoand heterodimerisation to produce generegulatory complexes with different properties. The classical pathway of NF-κB activation is typified by an inflammatory response operating through the p50 p65 heterodimer, which is retained in the cytoplasm by the inhibitory protein IκBa. Pro-inflammatory cytokines, such as TNF and interleukin 1β (IL-1β), cause activation of the IKK complex, resulting in phosphorylation, polyubiquitination and degradation of IκBa. This process allows translocation of the p50 p65 heterodimer to the nucleus to bind DNA and induce gene expression (Fig. 2). The complexity of the TNF NF-κB signal transduction pathway was recently addressed by an integrated approach employing proteomic pathway mapping based on tandem affinity purification, liquidchromatography tandem mass spectrometry, network analysis and directed functional analysis with RNA interference (Ref. 15). This approach should allow a more holistic understanding of disease pathways as well as the identification of novel therapeutic targets. TNFR shedding The extracellular domain of TNFRSF1A can be proteolytically cleaved from the cell surface, thereby releasing receptor fragments, referred to as soluble TNFRSF1A (stnfrsf1a) (Ref. 16), into the plasma. This shedding mechanism is mediated by the actions of TACE (Ref. 17), in addition to further zinc metalloproteinases, other uncharacterised proteases (also known as sheddases ) and the proteasome (Ref. 18) (Fig. 3a). Recent data have shown that a multifunctional ectoprotein, aminopeptidase regulator of TNFR shedding (ARTS-1), can also bind to the TNFRSF1A extracellular domain and promote receptor shedding (Ref. 19). A second mechanism, independent of ectodomain cleavage, by which soluble cytokine receptors might be released into the extracellular compartment has been proposed by Hawari et al. (Ref. 20), who described the release of full-length TNFRSF1A via exosome-like vesicles (Fig. 3b). Exosomes are small membrane-enclosed vesicles corresponding to the internal vesicles of endolysosome-related multivesicular bodies that are released from the cell via exocytic fusion with the plasma membrane. Consistent with this hypothesis, supernatants from human vascular Cell membrane Early endosome Multivesicular endosome Metalloproteinase Full-length stnfrsf1a Cleaved stnfrsf1a Figure 3. Proposed mechanisms for generation of cleaved or full-length soluble TNFRSF1A. (a) Metalloproteinase-induced cleavage of cell-surfacebound tumour necrosis factor receptor 1 (TNFRSF1A) upon TNF stimulation. (b) Generation of full-length TNFRSF1A through an exosome-associated release. Cell-surface-associated TNFRSF1A is internalised to early endosomes, which transform to endosomes containing internal vesicles (multivesicular endosomes), and is localised mostly at the surface of the internal vesicles. Subsequently, fusion of some of these multivesicular compartments with the plasma membrane leads to the release of full-length TNFRSF1A into the extracellular environment via exosomes. a Exosomes Proposed mechanisms for generation of cleaved or full-length soluble TNFRSF1A Expert Reviews in Molecular Medicine C 2005 Cambridge University Press b 6

7 endothelial cells were shown to contain only fulllength TNFRSF1A that can be sedimented by high-speed centrifugation, float on sucrose gradients at a density of 1.1 g/ml, and are associated with vesicles of diameter nm, which are all properties typical of exosomes (Ref. 20). As full-length protein is the predominant form of TNFRSF1A in human plasma, the exosomerelease mechanism may in fact be the main source of extracellular receptor. Genetics of TRAPS Familial Hibernian fever, which was first described in an Irish Scottish family (Ref. 21) in 1982, has since been renamed TRAPS following the discovery of TNFRSF1A as the susceptibility gene. TRAPS is found in most populations, as reflected by reports of patients of very varied ethnic background, including: European (several); African-, Native- and South-American; Australian; New Zealand; Asian; and Arabic (Refs 3, 22, 23, 24, 25, 26, 27). The TNFRSF1A gene, comprising 10 exons, is located on chromosome 12p13. To date, at least 46 different pathogenic TNFRSF1A mutations have been identified in a total of 195 symptomatic patients, as well as at least 24 asymptomatic carriers. These mutations are listed in the INFEVERS database ( Refs 28, 29). (Unfortunately two different numbering systems are used for TNFRSF1A mutations. Throughout this article, as in most published reports, we begin the numbers with the leucine residue that follows the signal peptide cleavage site. A second system, which includes the signal peptide, results in numbers that are 29 greater; for example, the mutation denoted T50M here would be referred to as T79M in the alternative system.) The majority [43 (93.5%)] of TNFRSF1A mutations are single-nucleotide missense mutations within exons 2, 3, 4 and 6; one mutation (2.2%) involves a splice site in intron 3, and, more recently, two deletions (4.3%) D42Del (Ref. 25) and H69fs (INFEVERS database) have also been reported. The H69fs mutation arises from an outof-frame 3 bp deletion resulting in a truncated protein owing to a stop codon at residue 338 (His98ArgfsX338); the splice site variant in intron 3 results in a four amino acid insertion. All mutations except one (98%) are located in the extracellular domains (Table 2; Fig. 1); the single exception is I170N, located very close to the receptor cleavage site, between Asn172 and Val173, which is adjacent to the transmembrane domain (Ref. 30). Mutations have been reported for 10 of the total 12 cysteines found in the first two cysteinerich N-terminal extracellular domains (Fig. 1); C15 in the PLAD region and C76 in CRD2 are the exceptions. As the majority of asymptomatic carriers [19/24 (79%)] have noncysteine mutations, the penetrance of cysteine mutations appears to be higher, and these mutations also tend to be associated with a more severe phenotype (Refs 23, 24). Overall, at least eight cysteine residues (C30, 33, 43, 52, 55, 70, 73, and 88) are mutation hot spots with more than one documented mutation; the Y20, H22, T50 and R92 residues are also mutational hot spots. A positive family history has been found for 33/40 (82%) mutations; however, numerous de novo TNFRSF1A mutations have also been reported in patients with neither parent possessing the mutation in question [T50K and G36E (Ref. 31), Y20D (Ref. 27) and C70 (Ref. 32)], therefore necessitating consideration of the disease even in cases without a positive family history. Genetic heterogeneity Not all patients presenting with a TRAPS-like illness have mutations in the coding region of TNFRSF1A (Refs 23, 24, 25). Genetic screening of 18 families with multiple affected members, and 176 sporadic (nonfamilial) patients with a clinical picture compatible with TRAPS, detected TNFRSF1A mutations in 50% of familial cases and only 2% of sporadic cases (Ref. 25). Furthermore, 3 of the 12 affected members in the originally described TRAPS family did not harbour the C33Y mutation, despite presenting with similar clinical symptoms. A moderate-to-severe TNFRSF1A shedding defect (see below) was found in some affected members of mutation-negative families, with reduced plasma stnfrsf1a levels in 50% of these patients (Ref. 25). These data suggest that other loci play a role in the pathogenesis of a TRAPS-like phenotype, with unknown genes and modifying factors presumably being responsible. Genotype phenotype analysis A proven serious long-term complication of TRAPS is the development of systemic amyloidosis, due to the accumulation of misfolded fragments of serum amyloid A (AA 7

8 Table 2. Summary of mutations in the tumour necrosis factor receptor 1 gene (TNFRSF1A) a Nucleotide Amino acid Exon location CRD 145 T_C Y20H Exon T_G Y20D Exon C_T H22Y Exon C_G H22Q Exon G_T C29F Exon T_C C30R Exon G_C C30S Exon G_A C30Y Exon T_G C33G Exon G_A C33Y Exon 3 1 C G_A Splice junction Intron G_A G36E Exon T_C T37I Exon A_G Y38C Exon G_C L39F Exon 3 1 c211_213del DD42del Exon T_C C43R Exon G_A C43Y Exon C_T P46L Exon C_A T50K Exon C_T T50M Exon T_C C52R Exon G_T C52F Exon T_C C55R Exon G_C C55S Exon C_G F60L Exon C_T T61I Exon A_T N65I Exon T_C L67P Exon 3 2 c293_295del H69fs Exon T_C C70R Exon T_A C70S Exon T_G C70G Exon G_A C70Y Exon T_C C73R Exon C_G C73W Exon T_C S86P Exon T_C C88R Exon G_A C88Y Exon G_C R92P Exon G_A R92Q Exon G_A C96Y Exon G_A C98Y Exon G_A R104Q Exon T_A F112I Exon T_A I170N* Exon 6 a At least 46TNFRSF1A mutations have been described so far. All sequence variants shown in this table are included in the INFEVERS database ( Of the mutations involving cysteine residues, all except one (98%) are located in the extracellular domains; the single exception is the I170N mutation, located very close to the receptor cleavage site, between Asn172 and Val173, which is adjacent to the transmembrane domain. (This mutation was reported as I199N to include the 29 amino acid signal peptide that is not present in the mature peptide.) type), an acute-phase reactant which is produced by the liver in response to systemic inflammation. The current data indicate an amyloidosis rate of 14% in TRAPS: 28 patients out of 199 mutation- 8

9 positive patients and asymptomatic carriers developed systemic amyloidosis. However, the percentage of amyloidosis patients with cysteine mutations has dropped from an initial estimation of 93% (Ref. 3) to 64%, based on reports in the current literature. A total of nine patients have presented with multiorgan amyloidosis, of whom six have cysteine substitutions and three have the T50M mutation (Ref. 33). These findings confirm the severity of cysteine mutations in TNFRSF1A, but also indicate that, over time, many, if not most, noncysteine mutations may also lead to amyloidosis. Although low-penetrance variants of TNFRSF1A and MEFV (the causative gene for FMF) are not major susceptibility factors for the development of amyloidosis, they might have significant pro-inflammatory effects in the chronic inflammatory disease state (Ref. 34). Apart from cysteine mutations, there is no clear-cut correlation between genotype and phenntype, although the clinical presentation associated with the R92Q variant seems to be milder, and some mutations at residues T50 and T61 may be associated with more severe inflammation with possible central nervous system (CNS) involvement (discussed later) and systemic lupus erythematosus (SLE), respectively (Refs 1, 35, 36, 37, 38). However, the vast majority of T50M mutations do not have CNS disease. Pathogenesis of TRAPS Defective TNFRSF1A shedding in TRAPS Soluble (s)tnfrs are constitutively released into the circulation (Ref. 39), and their level may increase during the course of various disease states, such as inflammation (Ref. 40) and after TNF stimulation (Refs 41, 42). Receptor shedding may desensitise cells to TNF action by reducing surface expression of TNFRSF1A. In addition, stnfrs may serve as physiological TNF-neutralising agents because they maintain their ability to bind ligand, thus preventing its interaction with cell-surface receptors. The importance of TNFRSF1A cleavage in the regulation of cellular TNF responsiveness has been underlined by the demonstration of cleavage-resistant TNFRSF1A mutations in TRAPS patients (Refs 1, 23, 25). These patients have reduced blood levels of stnfrsf1a between attacks, which increase to a much lesser extent during attacks than in other inflammatory conditions (e.g. rheumatoid arthritis), where stnfrsf1a can reach times normal levels (Ref. 1). Consistent with these findings, peripheral blood leukocytes from C52F patients exhibited increased cell-surface TNFRSF1A levels, and decreased shedding of TNFRSF1A following stimulation with phorbol myristate acetate (PMA). However, a generalised metalloproteinase defect was excluded in TRAPS as these abnormalities were not found for TNFRSF1B, thus pointing to a specific TNFRSF1A shedding defect. The resulting lack of stnfrsf1a to antagonise TNF through negative feedback and the sustained TNF signalling through nonshed TNFRSF1A receptors has been proposed as a cause of the persistent inflammation in TRAPS. A similar condition has been induced in mice when TNFRSF1A shedding was prevented by mutating the receptor cleavage site, thus producing a phenotype with similarities to TRAPS (Ref. 43). However, more-recent experiments indicate that shedding defects are influenced not only by specific mutations but also by different cell types (Refs 44, 45). In fact, 20 out of the 23 TNFRSF1A mutations (87%) for which data are currently available are associated with reduced stnfrsf1a levels, whereas only about 50% (8/15) of these (C30S, P46L, T50M, T50K, C52F, T61I, F112I, I170N) are associated with defective TNFRSF1A shedding by leukocytes. Analysis of leukocytes and dermal fibroblasts from C33Y patients demonstrated a marked reduction in shedding of TNFRSF1A only in the skin cells (Ref. 44). In view of these data, in addition to the findings of TNFRSF1A shedding defects and low stnfrsf1a levels in autosomal dominant TRAPS-like patients who are negative for the TNFRSF1A mutation (Ref. 25), we can postulate that other pathophysiological mechanisms may be involved in the disease phenotype. In particular, the contribution of the TNFR-exocytosing mechanism to stnfrsf1a levels, and to the pathogenesis of TRAPS, remains to be elucidated. Impaired intracellular TNFRSF1A trafficking and TNF binding in TRAPS Several studies have reported on the intracellular consequences of TNFRSF1A mutations in TRAPS patients. In a transfection system using a tetracycline-regulated expression system in which the human embryonic kidney (HEK)-293 cell line expressed wild-type (WT) TNFRSF1A or various TNFRSF1A mutants (R92Q, T50M, C33Y, C52F), both the WT and mutant forms of full-length TNFRSF1A spontaneously induced apoptosis and 9

10 IL-8 production, whereas all cell clones lacking the cytoplasmic signalling domain ( sig) did not (Ref. 46). A substantial degree of TNFRSF1A shedding was induced upon treatment with PMA in all sig cell clones (Ref. 44). Similar results were obtained in dual-transfectant cell lines (cotransfected with mutant and WT sig TNFRSF1A). Thus, the intracellular region of TNFRSF1A seems not to be essential for receptor shedding, but is necessary for the functional activity of the WT and mutant receptors. Todd et al. also showed that full-length WT TNFRSF1A was expressed in both the cytoplasm and on the cell surface of transfected HEK-293 cells, whereas the mutant full-length receptors showed strong cytoplasmic but reduced cellsurface expression (Ref. 46). The WT and mutant sig TNFRSF1A receptors were all expressed at the cell surface, but some of the mutant receptors were retained in the cytoplasm. Furthermore, mutants with surface-expressed sig TNFRSF1A were defective in their ability to bind TNF. The cytoplasmic region of the receptor is required for its accumulation in the trans-golgi apparatus, which is thought to serve as a reservoir for receptor translocation to the cell surface (Ref. 47). Thus, in this transfection system, TRAPS-associated mutations within the CRD1 of TNFRSF1A inhibit intracellular trafficking and TNF binding, whereas the signalling properties of the cytoplasmic DD are preserved. By extrapolating from the Dsig TNFRSF1A data, it is possible that some of the TRAPS-related mutations may have significant structural effects on TNFRSF1A conformation that affect the subcellular distribution and/or functional properties of the receptor: a total of 22 of the 46 TNFRSF1A mutations (48%) involve cysteine residues and, of the other amino acid substitutions, four mutations are thought to prevent (Y20H, Y20D, D42del) (Refs 24, 25, 27), or cause (I170N) (Ref. 30) the formation of a hydrogen bond. TNF-induced apoptosis defect in TRAPS Siebert et al. showed that TNF-induced activation of NF-κB was decreased in primary dermal fibroblasts from a TRAPS patient with the C43S TNFRSF1A mutation and TNF-induced apoptosis was also significantly decreased in these cells (Ref. 45). Nevertheless, these fibroblasts were capable of producing IL-6 and IL-8 in response to TNF. This study suggests that, in some TRAPS patients, there may be defective TNFinduced nuclear signalling and apoptosis. It was proposed that cells from these patients might have a longer survival time because of impaired apoptosis, and thus remain capable of producing pro-inflammatory cytokines and resultant inflammatory pathology. TNF-independent NF-κB activation in TRAPS A recent study using a new tightly regulated doxycycline-dependent expression system revealed an interaction of WT and mutant (T50K and P46L) TNFRSF1A in the absence of TNF (Ref. 48). Upregulation of the WT or mutant receptor caused downregulation of the other receptor or vice versa. Furthermore increased expression of mutant TNFRSF1A (T50K) was associated with an increase of NF-κB p65 subunit activation, which did not occur after WT receptor expression. Thus, part of the inflammatory process in TRAPS may be induced by a TNF-independent upregulation of TNFRSF1A with subsequent NF-κB activation. Low-penetrance TNFRSF1A mutations associated with other inflammatory diseases Of all currently identified TNFRSF1A mutations, the P46L and R92Q variants have each been found in 1 2.5% of normal control chromosomes (Refs 23, 24, 35) and the more recently identified T61I variant has been reported in 1 4.2% of healthy Japanese control populations (Refs 37, 38). These three variants are generally considered to be low-penetrance TNFRSF1A mutations, as they are present in symptomatic patients with TRAPS as well as unaffected individuals. Furthermore, the T61I and P46L variants are both associated with a TNFRSF1A shedding defect in monocytes (Refs 37, 38, 49) and R92Q carriers may show reduced stnfrsf1a levels in plasma. An unexpectedly high allelic frequency of 9.8% has been reported for P46L in two distinct sub- Saharan West African populations, including a cohort of 145 patients with sickle cell anaemia and 349 healthy controls (Ref. 22). By contrast, P46L is present in just 1.9% of African-American control chromosomes (P < 0.005). Given the known involvement of TNFR in infectious diseases, this polymorphism may confer some biological advantage to carriers, in terms of an ongoing biochemical inflammation even between febrile 10

11 episodes that might protect carriers against infectious agents. The possibility that low-penetrance TNFRSF1A variants might contribute to the degree of nonspecific inflammatory responses, with beneficial effects in case of Native-African populations or more deleterious effects in other populations, is underlined by the following findings. The P46L and R92Q variants are sometimes associated with atypical TRAPS features, such as pericarditis (Ref. 24), myocarditis and sacroiliitis (P46L) (Ref. 50), panniculitis (R92Q) (Ref. 51) or early arthritis (Ref. 23). Furthermore, R92Q has been reported in a patient with periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) as a child (Ref. 52) and has been associated with an increased risk of extracranial venous thrombosis in patients with Behçet s disease (Ref. 53). The T61I and R104Q TNFRSF1A mutations have been recently identified in 2% and 8.3%, respectively, of Japanese SLE patients, although there was no statistically significant difference compared with the control population (Refs 37, 38). These findings raise the question of the role of the TNFRSF1A mutations in the pathogenesis of autoimmune disease such as rheumatoid arthritis, SLE and Behçet s disease, and point to further unidentified genetic and/or environmental modifying factors that influence the phenotypic expression of these mutations. Recently, at least six patients with periodic fever syndromes have been described in whom genetic screening revealed a combination of the P46L, R92Q or Y20D mutation with one or two MVK mutations (causative for HIDS) or MEFV mutations (Refs 27, 35, 49, 54, 55). The disease phenotype of these patients was varied, but for the most part showed a mixture of two diseases (TRAPS and HIDS, or TRAPS and FMF) with a tendency towards atypical clinical presentations. These findings underline the fact that lowpenetrance mutations might contribute to the clinical phenotype in individuals carrying other HPF mutations, whereas more-severe mutations, such as Y20D (TRAPS) or V726A (FMF), segregate with specific monogenic disorders (Ref. 56). Clinical features of TRAPS Although the phenotype and clinical severity of TRAPS may vary considerably, characteristic features include recurrent prolonged fevers, abdominal pain, migratory myalgia and cutaneous inflammation, as well as ocular symptoms (Ref. 3). Based on data from 153 TRAPS patients described so far in the literature, the median age of onset is at 10 years with a wide range of 1 to 63 years. The fever episodes typically last longer than those of the other HPFs, with a median duration of 14 days, which can vary between 2 and 56 days. The most frequent accompanying symptom is abdominal pain [in 77% of cases (118/153)], which has led to a surgical event (appendectomy, laparotomy or even gynaecological intervention) in 33% (40/120) of TRAPS patients. The second most frequent and TRAPS-specific symptom is myalgia, which has been reported in 63.5% (80/ 126) of patients. In the majority of cases, only one area of the body is affected and is associated with tenderness and an exanthema, both typically migrating centrifugally during the attack. Magnetic resonance imaging (MRI) and biopsies revealed monocytic fasciitis or lymphocytic vasculitis rather than myositis as the underlying cause (reviewed in Ref. 3). The accompanying exanthema was reported in 55.2% (80/145) of patients and was characteristically described as erythematous/erysipelas-like, and occasionally as urticarial. A further distinctive feature characteristic of TRAPS is ocular involvement, which occurs in 48.8% (61/125) of cases, with the majority presenting as unilateral periorbital oedema, conjunctivitis and/or uveitis, and more rarely episcleritis or orbital cellulitis. Further symptoms include arthralgia/arthritis in 51% (78/153) and pleuritis in 32% (49/153) of patients. Pericarditis has been described in two patients, each carrying one of the low-penetrance P46L and R92Q mutations (Ref. 24). Sporadic pharyngitis, tonsillitis, cervical lymphadenopathy, diarrhoea and inguinal herniae have been reported in patients with various TNFRSF1A mutations. The report of a few CNS events in TRAPS patients has raised the question of CNS involvement in TRAPS, although the causal relationship remains unclear. Clinical presentations of these cases varied, and included severe neurological illness (T50M and T50K mutations; Refs 1, 36, 57), optic neuritis/papillitis (T50K, C30R; Refs 36, 57, 58), and behavioural changes such as depressive symptoms (R92Q, T50K; Refs 24, 36). T2-weighted magnetic resonance imaging (MRI) of the brain has revealed disease of the white matter with multiple small hyperintense lesions located in the supratentorial 11

12 region in one of three T50K-mutation-positive members of a German family with neurological disease (Refs 36, 57). Although this distribution is not typical of multiple sclerosis (MS) lesions, there is evidence form studies with experimental autoimmune encephalomyelitis (EAE), an animal model for MS, that the entry of autoreactive myelin-specific T cells, the initiators of this inflammatory and demyelinating process, into the CNS parenchyma is dependent on TNFRSF1A signalling (Ref. 59). TNFRSF1A was shown to upregulate the expression of vascular cell adhesion molecule 1 (VCAM-1), an adhesion molecule that is important to leukocyte movement across the blood brain barrier, by astrocytes. Given the fact that the inflammatory and demyelinating process is mediated at least in part by TNF, the hyperinflammatory state in TRAPS patients could predispose to a demyelinating event. By contrast, the above-mentioned patient had received anti-tnf treatment, during an exacerbation of her neurological symptoms, thus pointing to the possibility of a TNF-antagonistinduced side effect (Ref. 60). Careful examination of potential disease- or treatment-related events is therefore crucial for our further understanding and management of disease in these patients. TRAPS patients manifest a nonspecific increase of acute-phase reactants such as C- reactive protein (CRP), erythrocyte sedimentation rate (ESR), serum AA and fibrinogen during attacks, as is the case with other autoinflammatory fever syndromes. However, even during symptom-free intervals, these patients can be shown to have an intermediate degree of biological inflammation, which increases the risk of amyloidosis. The number, intensity and duration of the attacks vary considerably between TRAPS patients. Factors known to trigger attacks include physical exertion, viral infections (which may be minor), trauma and ethanol consumption. The intervals between attacks may last from several months to several years, during which the patient is likely to be in a hyperinflammatory state, with associated raised acute-phase proteins, even in the absence of fever (Ref. 25). Clinical implications/applications The discovery of the underlying genetic defect in TRAPS allows comprehensive screening of the TNFRF1A gene in suspected cases; before the availability of DNA-based tests many people with HPFs went undiagnosed and were exposed to unnecessary surgical interventions. Furthermore, the insights gained into the pathophysiology of TRAPS have resulted in synthetic anti-tnf agents being used as a rational form of therapy; these are especially advantageous given the fact that although corticosteroids can be effective in more severe episodes, they often require escalating doses, with subsequent side effects. As the affinities of soluble receptors are generally low compared with their membraneintegrated forms, TNF-neutralising agents designed for clinical use have been engineered as dimeric IgG fusion proteins. One such agent is etanercept (Enbrel), which is a human TNFR (p75) Fc fusion protein comprising two TNFRSF1B receptors linked by an IgG1 Fc fragment (Ref. 61). Etanercept is administered subcutaneously at a dose of 25 mg (paediatric dose 0.4 mg/kg) twice weekly and, to date, more than 30 TRAPS patients harbouring a total of at least ten different TNFRSF1A mutations have been treated with the drug (Refs 31, 33, 36, 49, 51, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71). In an open-label clinical trial that comprised seven TRAPS patients who received etanercept twice weekly over 6 months, a significant decrease of the disease activity scores for well-being and pain/stiffness was shown for five and six patients, respectively (Ref. 68). Similar effects were obtained for a reduction of acute-phase reactants, and five corticosteroid-responsive patients required significantly less corticosteroids. During the 6- month treatment period, the drug was found to be safe and effective. However, long-term studies are required in these patients given the recently described possible association of anti-tnf treatment with an increased risk of neurological complications (Ref. 36). Furthermore, although not described in TRAPS patients treated with etanercept, the drug can promote the reactivation of tuberculosis (Refs 72, 73); thus, purified protein derivative (PPD) tests are mandatory to exclude quiescent tuberculosis before anti-tnf therapy initiation. The initial response to etanercept may wane with ongoing therapy (Ref. 49), leading to the need for alternative therapies. Among the anti-tnf agents, infliximab, a mouse human chimeric IgG1 monoclonal antibody to TNF, has not been effective in one patient with TRAPS (Ref. 68). Similar disappointing results were shown for the recombinant humanised fusion protein of two TNFRSF1A receptors and IgG1 (p55tnfr Ig), 12

13 which has been used to treat another TRAPS patient (Ref. 68). This surprising result might be explained by a high incidence of neutralising, antip55tnfr Ig antibodies, which were found to be induced by a nonhuman amino acid epitope within the molecule, therefore reducing efficacy (Ref. 74). Colchicine, an alkaloid neutral, is the drug of choice for prophylaxis against FMF attacks, as it inhibits assembly of microtubles and mitotic spindle formation, thereby preventing activation of neutrophils (Ref. 75). Only limited efficacy has been reported for colchicine therapy in TRAPS as it was found to be effective in only 3/14 TRAPS mutations (21.4%) (G36E, R92Q, F112I; Refs 31, 76, 77). Treatment of a TRAPS patient with sirolimus, an inhibitor of T-cell activation, was reported to be beneficial in one patient (Refs 24, 78). Anakinra, a recombinant form of IL-1 receptor antagonist (IL-1ra), has produced a beneficial effect in another patient, who was unresponsive to etanercept (Ref. 71). These anecdotal reports suggest that the IL-1 cytokine may be an important component in the pathogenesis of TRAPS, in addition to TNF and its receptor. Despite etanercept having been clinically effective in TRAPS patients harbouring the C43Y (Ref. 71), G36E (Ref. 31) or C52F (Ref. 66) mutations, the subclinical acute-phase response was not controlled during asymptomatic intervals. This highlights the importance of monitoring acutephase reactants during symptom-free intervals in patients in order to have a risk estimate for the development of amyloidosis, which is also influenced by the underlying TNFRSF1A mutation. Anti-TNF therapy is effective in the prevention of amyloidosis, with even the possibility of amyloidosis regression (Ref. 68), but control of the acute-phase response is necessary. Furthermore, etanercept combination therapy, which could include prednisone, colchicine or anakinra, may be necessary to achieve sufficient regulation of the cytokine-modulated metalloproteinase enzymes (Refs 79, 80) that participate in amyloidogenesis by serum AA proteolysis (Ref. 81). Thus, serum AA production and remodelling of the extracellular matrix after AA deposition might be influenced by a combination of therapies. Research in progress and outstanding research questions A major effort is currently under way to discover novel HPF genes, as it is apparent that other disease loci are involved in producing a TRAPSlike phenotype. One of the most significant problems encountered is a lack of informative families for linkage studies and thus it has been necessary to use alternative approaches to identify novel disease genes. It is hoped that advances in proteomics technology, including protein chip and mass spectrometry, will help to delineate interactive pathways involving the mutated TNFRSF1A molecule. DNA microarrays have been developed for RNA expression profiling of cells from these patients. Studies on RNA and protein expression in cells from mutationnegative patients between and during attacks, and pre- and post-therapy, if possible, should provide insights into key pathways and protein interactions involved. In addition, a re-sequencing chip that will allow up to 100 candidate genes to be interrogated simultaneously on a single chip (300 kb of sequence) is currently under development. Other outstanding research questions include the reason for functional differences between cell types in TRAPS patients, as well as the varied phenotypes associated with particular mutations. A knock-in mouse model of TRAPS is being developed, in addition to various in vitro transfected cell systems, in order to investigate pathological mechanisms. A clinical trial evaluating the efficacy of etanercept in decreasing the frequency and severity of symptoms in TRAPS has recently been conducted, with promising preliminary results (Ref. 67). Nevertheless, long-term surveillance of patients and treatment monitoring will be necessary to evaluate the occurrence of rare adverse events. In the future, it will also be necessary to develop alternative treatment options to avoid potential side effects of anti-tnf therapy and to control the various pathways involved in disease pathogenesis. If further data confirm the defective step(s) in the apoptotic pathway in TRAPS patients, it may eventually be possible to intervene intracellularly with targeted therapies (e.g. small molecules taken orally), to correct the imbalance between NF-kB activation and apoptosis (Ref. 4). Concluding remarks Since the discovery of the genetic basis for TRAPS, tremendous advances have been achieved in terms of our understanding of the genetics and pathophysiology of this autoinflammatory 13

14 disorder as an example of an innate immune deficiency. The hyperinflammatory state of patients seems to be of an ongoing nature dependent on an unstable equilibrium between a growing number of triggers and mediators. The clinical and molecular complexity of TRAPS seems to be reflected by the activation of inflammatory and apoptotic pathways. Given the promising preliminary results for biological therapies in TRAPS, the first steps have already been taken to improve the patient s quality of life and overall prognosis, especially with regard to the prevention of amyloidosis. Acknowledgements and funding We thank Daniel Kastner, MD, PhD and Ivona Aksentijevich, MD for helpful advice. This work was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health, and the Charitable Foundation of the Leeds Teaching Hospitals. We also thank the anonymous peer reviewers of this article for their constructive comments. References 1 McDermott, M.F. et al. (1999) Germline mutations in the extracellular domains of the 55 kda TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 97, , PubMed: McDermott, M.F. and Aksentijevich, I. (2002) The autoinflammatory syndromes. Curr Opin Allergy Clin Immunol 2, , PubMed: Hull, K.M. et al. (2002) The TNF receptorassociated periodic syndrome (TRAPS): emerging concepts of an autoinflammatory disorder. Medicine (Baltimore) 81, , PubMed: Palladino, M.A. et al. (2003) Anti-TNF-alpha therapies: the next generation. Nat Rev Drug Discov 2, , PubMed: Black, R.A. et al. (1997) A metalloproteinase disintegrin that releases tumour-necrosis factoralpha from cells. Nature 385, , PubMed: Locksley, R.M., Killeen, N. and Lenardo, M.J. (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104, , PubMed: McDermott, M.F. (2001) TNF and TNFR biology in health and disease. Cell Mol Biol (Noisy-legrand) 47, , PubMed: Chan, F.K. et al. (2000) A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science 288, , PubMed: Banner, D.W. et al. (1993) Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell 73, , PubMed: Micheau, O. and Tschopp, J. (2003) Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, , PubMed: Legler, D.F. et al. (2003) Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalphamediated NF-kappaB activation. Immunity 18, , PubMed: Schneider-Brachert, W. et al. (2004) Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity 21, , PubMed: Luberto, C. et al. (2002) Inhibition of tumor necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase. J Biol Chem 277, , PubMed: Zhang, S.Q. et al. (2000) Recruitment of the IKK signalosome to the p55 TNF receptor: RIP and A20 bind to NEMO (IKKgamma) upon receptor stimulation. Immunity 12, , PubMed: Bouwmeester, T. et al. (2004) A physical and functional map of the human TNF-alpha/NFkappa B signal transduction pathway. Nat Cell Biol 6, , PubMed: Wallach, D. et al. (1991) Soluble and cell surface receptors for tumor necrosis factor. Agents Actions Suppl 35, 51-57, PubMed: Reddy, P. et al. (2000) Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme. J Biol Chem 275, , PubMed: Levine, S.J. et al. (2005) Proteasome Inhibition Induces TNFR1 Shedding from Human Airway Epithelial (NCI-H292) Cells. Am J Physiol Lung Cell Mol Physiol PubMed: Cui, X. et al. (2002) Identification of ARTS-1 as a novel TNFR1-binding protein that promotes TNFR1 ectodomain shedding. J Clin Invest 110, , PubMed: Hawari, F.I. et al. (2004) Release of full-length 55- kda TNF receptor 1 in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors. Proc Natl Acad Sci U S A 101,