Advances in the understanding of familial Mediterranean fever and possibilities for targeted therapy

Summary

Familial Mediterranean fever (FMF) is a systemic autoinflammatory disorder characterized by seemingly unprovoked recurrent episodes of fever and serosal, synovial, or cutaneous inflammation. FMF is caused by recessively inherited mutations in MEFV, which encodes pyrin, and most of the mutations are present in the C-terminal end of the protein encoding B30.2 domain. The FMF carrier frequencies are extremely high in several eastern Mediterranean populations. Pyrin is expressed in granulocytes, monocytes, dendritic cells, and synovial fibroblasts. Pyrin regulates caspase-1 activation and consequently interleukin-1β production through the interactions of its N-terminal PYRIN domain and C-terminal B30.2 domain with an adaptor protein, apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) and caspase-1 respectively. Pyrin is cleaved by caspase-1 and the cleaved N-terminal fragment translocates to nucleus and enhances ASC-independent nuclear factor (NF)-κB activation through interactions with p65 NF-κB and IκB-α. In addition to the regulatory role of pyrin for caspase-1, the cleavage of pyrin provides an important clue not only in understanding the molecular pathogenesis of FMF but also in developing new therapeutic targets for FMF.

The innate immune system plays a pivotal role in the initial host response to a broad spectrum of microbial organisms. Genetic disorders in the regulation of innate immunity lead to a family of illnesses, called systemic autoinflammatory diseases, that are characterized by seemingly unprovoked episodes of fever and inflammation without high-titre autoantibodies or antigen-specific T cells. Familial Mediterranean fever (FMF, Mendelian inheritance in man (MIM) 249100) is the prototypic recessively inherited autoinflammatory disease, prevalent among multiple populations from the eastern Mediterranean basin, particularly Jews, Armenians, Turks, and Arabs. FMF is characterized by short recurrent bouts of fever and localized inflammation usually involving the peritoneum, pleura, joints, or skin. FMF inflammation is mediated by a massive influx of polymorphonuclear leucocytes into the affected tissues, neutrophilia, and a rapid acute-phase response (Galon et al, 2000; Stojanov & Kastner, 2005). In some patients, progressive systemic AA amyloidosis can lead to kidney failure and death. In most patients, the onset of disease begins before the age of 20 years (Ben-Chetrit & Levy, 1998). A typical attack generally lasts from 12 to 72 h and occurs every few weeks to months. Patients are usually asymptomatic between attacks, but sub-clinical inflammation may persist.

In 1997, the gene responsible for FMF (designated MEFV for Mediterranean fever) was identified on chromosome 16p13·3 by positional cloning. MEFV is comprised of ten exons encoding 781 amino acids, and the protein product has been named pyrin (or marenostrin). So far, more than 60 FMF-associated mutations have been detected (http://fmf.igh.cnrs.fr/ISSAID/infevers/). In the populations of the eastern Mediterranean basin, the carrier frequencies of mutant alleles are extremely high (1:3–1:5 in some populations), and four exon 10 mutations, M680I, M694V, M694I, and V726A account for a large percentage of FMF chromosomes (Touitou, 2003). Such high carrier frequencies could be explained by heterozygote selection that may confer increased resistance to an as yet unidentified infectious agent (International FMF Consortium, 1997). This finding is consistent with the hypothesis that several FMF mutations are found as the wild type (WT) amino acid in primates (Schaner et al, 2001). Recent reports demonstrated that FMF mutations are also found in Spanish, Italian, Greek, Portuguese, Kurdish, non-Jewish Caucasian, Indian, and Chinese (Dode et al, 2000; Domingo et al, 2000; Booth et al, 2001) populations. Moreover, there have been several reports suggesting that FMF mutations may be involved in non-FMF conditions, such as Behçet disease (Touitou et al, 2000; Livneh et al, 2001) and inflammatory bowel disease (Cattan et al, 2000).

Most FMF-associated mutations are single amino acid substitutions (missense mutations), and a few single amino acid duplication/deletion mutations have also been reported. Only two mutations, a frame-shift in exon 2 and a nonsense mutation in exon 10, have been reported to produce truncated proteins. Up to 30% of FMF patients, depending on the population, with clinical symptoms of FMF have only a single demonstrable MEFV mutation within the coding sequences, while the non-coding regions, such as introns, 5′-, and 3′-untranslated region, still remain to be thoroughly analyzed for possible disease-associated variants. Several cases of apparently dominantly inherited FMF have been reported (Booth et al, 2000; Aldea et al, 2004). Although segregation analysis initially indicated a recessive mode of inheritance for FMF, and this was in fact the basis for the positional cloning of MEFV, the frequent identification of seemingly single-mutation cases may be due to the recognition of a broader FMF phenotype as clinicians have had a greater awareness of the disease. At a molecular level, these findings suggest that FMF-associated pyrin mutations might lead to a gain of function, with gene-dosage effect, rather than a loss of function, as would be expected for more straight forward recessive diseases.

Expression of the FMF gene, MEFV

Initially, the expression of MEFV was observed in peripheral blood leucocytes (PBL) and a colorectal adenocarcinoma cell line, SW480 (International FMF Consortium, 1997). The subsequent expression analysis of MEFV in fractionated leucocytes from bone marrow and PBL showed that MEFV is expressed predominantly in neutrophils, the major cell type found in FMF inflammatory exudates, and in eosinophils and monocytes but not in lymphocytes (Centola et al, 2000). MEFV is also expressed in dendritic cells and synovial fibroblasts (Diaz et al, 2004). In monocytes, the expression levels are variable and the expression is up-regulated by proinflammatory agents, such as interferon (IFN) γ, tumour necrosis factor (TNF) α, and lipopolysaccharide (LPS). The monocytic cell lines U937 and THP-1 also express MEFV, but the promyelocytic cell line HL60 expresses MEFV only after granulocytic and monocytic differentiation. In CD34⁺ hematopoietic stem-cells, the expression of MEFV is observed at the myelocyte stage during granulocyte differentiation. MEFV expression is also observed in fibroblasts from synovium, peritoneum, and skin at a lower level compared to neutrophils, and the expression is increased by interleukin (IL)-1β or phorbol myristate acetate (PMA) (Matzner et al, 2000). These observations could account for the predisposition of FMF for serosal, synovial, and skin inflammation.

Pyrin structure and subcellular localization

Clarifying the normal function of pyrin may suggest possible mechanisms of inflammation in FMF. Important clues concerning the function of wild type pyrin came from predictions of domains/motifs within pyrin that have been deduced from computational searches for amino acid sequence similarities between pyrin and other proteins (International FMF Consortium, 1997; Bertin & DiStefano, 2000). Five different domains have been identified within pyrin: (i) a PYRIN domain; (ii) a bZIP transcription factor basic domain; (iii) a B-box zinc finger domain; (iv) an α-helical (coiled-coil) domain; (v) a B30.2 (PRYSPRY) domain. As summarized in Fig 1, each domain has a distinct role in the protein-protein interactions with proteins that are connected to inflammation through regulation of cell death, cytokine secretion, transcriptional regulation, and cytoskeletal signalling. The identification of multiple binding partners suggests that pyrin participates in several molecular pathways. However, taken together with the restricted expression of pyrin in innate immune cells, the major role of pyrin appears to be in the regulation of inflammation.

Fig 1.

The structure of pyrin and its interacting proteins. Pyrin is composed of five distinct domains, the PYRIN domain (residues 1–95), bZIP transcription factor basic domain (residues 266–280), B-box zinc finger domain (residues 375–407), α-helical (coiled-coil) domain (residues 408– 594), and B30.2 domain (residues 598–774). Each domain is responsible for various protein-protein interactions of pyrin: the PYRIN domain with ASC (Richards et al, 2001), the bZIP basic domain and adjacent sequences with p65 and IκB-α respectively (Chae et al, 2008), the B-box and α-helical (coiled-coil) domain with the PAPA protein (PSTPIP1, also known as CD2BP1) (Shoham et al, 2003), and the B30.2 domain with caspase-1 (Chae et al, 2006; Papin et al, 2007) and Siva (Balci-Peynircioglu et al, 2008). Pyrin binds to microtubules through the whole N-terminal half of pyrin (Mansfield et al, 2001), while three residues, serines 208, 209, and 242, which are located between the PYRIN domain and the bZIP domain, are critical for the interaction of pyrin with 14.3.3 (Jeru et al, 2005). The caspases-1 mediated cleavage site of pyrin at Asp330 is indicated between the bZIP basic domain and the B-box zinc finger domain.

The presence of a bZIP transcription factor basic domain and two nuclear localization signals, a basic residue cluster, PLSKREE, beginning at amino acid residue 157, and a bipartite NLS motif at residues 420–437 has suggested that pyrin may work as a nuclear factor (International FMF Consortium, 1997; Centola et al, 1998). However in transfected cells, full-length pyrin exclusively localizes to the cytoplasm, while a rare isoform lacking exon 2 can enter the nucleus (Papin et al, 2000; Mansfield et al, 2001). Nevertheless, immunostaining of various pyrin expressing cells showed that endogenous pyrin is predominantly localized to the nucleus in synovial fibroblasts, dendritic cells, and polymorphonuclear cells, but to the cytoplasm in monocytes (Diaz et al, 2004). Moreover, we have demonstrated that pyrin is cleaved by caspase-1 at Asp330, and the N-terminal cleaved fragment localizes to the nucleus and potentiates nuclear factor (NF)-κB activation (Chae et al, 2008). As pyrin is cleaved, it is yet unclear whether pyrin in the nuclei of the synovial fibroblasts, dendritic cells, and polymorphonuclear cells is full-length or the N-terminal cleaved fragment, which cannot be distinguished by immunostaining with Ab specific to N-terminus.

The N-terminal PYRIN domain and the inflammasome

A critical insight into the normal function of pyrin and the possible pathogenesis of FMF came from the recognition of the N-terminal ~90 amino acid PYRIN domain (Bertin & DiStefano, 2000), which has also been denoted PYD (Martinon et al, 2001), PAAD (Pawlowski et al, 2001), or DAPIN (Staub et al, 2001). The PYRIN domain has been found in more than 20 proteins with putative functions in regulating apoptosis and inflammation. Despite a relatively modest level of amino acid sequence similarity, the structure prediction placed the PYRIN domain as the fourth member of the death domain fold superfamily (DD superfamily) comprised of the death domain (DD), death effector domain (DED), and caspase-recruitment domain (CARD) (Liepinsh et al, 2003). The three-dimensional structure of the DD superfamily is composed of six alpha helices that permit homotypic protein-protein interactions. Through the cognate PYRIN-PYRIN interaction, pyrin can interact with other PYRIN domain-containing proteins, such as the adaptor protein, apoptosis-associated speck-like protein with a CARD (ASC) (Masumoto et al, 1999; Richards et al, 2001).

ASC is composed of an N-terminal PYRIN domain and a C-terminal CARD, and has been shown to oligomerize and mediate the proteolytic activation of caspase-1 in cytoplasmic multiprotein complexes denoted inflammasomes which are essential for the maturation of proinflammatory cytokines IL-1β, IL-18, and IL-33 (Martinon et al, 2002; Agostini et al, 2004), and for the unconventional secretion of leaderless proteins (Keller et al, 2008). Several inflammasomes have been proposed, based on their constituent stress-sensing components, specifically the nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs), such as NACHT, LRR, and pyrin domain–containing protein (NLRP or NALP) 1, NLRP2, NLRP3, and ICE protease–activating factor (IPAF). Recently, an interferon-inducible HIN-200 family member, AIM2 (absent in melanoma 2) has been demonstrated as a component of a new inflammasome that is activated by cytoplasmic DNA (Burckstummer et al, 2009; Fernandes- Alnemri et al, 2009; Hornung et al, 2009). In NLRPs and AIM2 inflammasomes, ASC works as an adaptor molecule that connects the stress-sensing component and pro-caspase-1 through its N-terminal PYRIN domain and C-terminal CARD domain respectively. ASC is not involved in IPAF inflammasome because IPAF has no PYRIN domain, and recently it has also been demonstrated that ASC is not required in the NLRP1 inflammasome but enhances caspase-1 activation by NLRP1 (Faustin et al, 2007).

As denoted by the name, NLRPs are composed of a tripartite domain structure: (i) an N-terminal effector-binding domain, the PYRIN domain; (ii) a nucleotide binding site (NBS) domain of the NACHT subfamily [domain present in the neuronal apoptosis inhibitor protein (NAIP), the major histocompatibility complex (MHC) class II transactivator (CIITA), HET-E (incompatibility locus protein from Podospora anserine), and IP1 (mammalian telomerase-associated proteins)]; (iii) a C-terminal leucine-rich repeat (LRR) domain (Koonin & Aravind, 2000; Inohara & Nunez, 2003; Tschopp et al, 2003). NLRP3, also known as NALP3, cryopyrin, PYPAF1, or CATERPILLER 1.1, is the most interesting because mutations in the gene encoding this protein (denoted NLRP3, previously known as CIAS1), cause three different dominantly-inherited or de novo autoinflammatory diseases (now specifically called cryopyrinopathies or cryopyrin-associated periodic syndromes): Muckle–Wells (MWS) syndrome, familial cold autoinflammatory syndrome (FCAS), and neonatal- onset multisystem inflammatory disease (NOMID), also known as chronic infantile neurological cutaneous and articular (CINCA) syndrome (Hoffman et al, 2001; Aksentijevich et al, 2002). The cryopyrinopathies are characterized by recurrent episodes of fevers, urticarial skin rash, varying degrees of arthralgia/arthritis, neutrophil-mediated inflammation, and an intense acute-phase response. Patients with NOMID/CINCA, the most severe of these syndromes, manifest chronic inflammation of the central nervous system, which can lead to blindness, deafness, or mental impairment. These inflammatory symptoms are caused by constitutively activated NALP3 inflammasome due to the gain-of-function mutations and subsequent increased production of IL-1 β. The blockade of IL-1β signalling with a recombinant IL-1 receptor antagonist IL-1Ra (anakinra) or rilonacept (IL-1 Trap), a fusion protein of the IL-1 receptor, markedly decrease symptoms (Hawkins et al, 2004; Hoffman et al, 2004; Goldbach-Mansky et al, 2006, 2008).

In the NLRP3-inflammasome, ASC binds to NALP3 through its N-terminal PYRIN–PYRIN interactions, and to procaspase-1 through its C-terminal CARD–CARD interactions. CARDINAL recruits a second pro-caspase-1 molecule to the complex. This brings two molecules of procaspase-1 into close proximity, leading to proteolytic activation and the subsequent release of the active catalytic domains, p20 and p10 (Fig 2) (Agostini et al, 2004). Active caspase-1, in turn, cleaves the 31-kDa precursor form of IL-1β into its biologically active 17-kDa fragment. Given that ASC also interacts with pyrin through PYRIN domain, it is possible that pyrin is somehow involved in the NLRP2/3 or AIM2 inflammasome as a modulator, or pyrin itself is a component of an inflammasome. Thus, most studies for the function of pyrin in the innate immune system have been focused on the regulation of the caspase-1 activation and subsequent IL-1β secretion.

Fig 2.

Proposed role of pyrin and pathogenesis of FMF. As a representative inflammasome, structural organization of the NLRP3 inflammasome is shown. In the inflammasome, p20 and p10, active caspase-1 subunits are produced by induced proximity-mediated autocatalysis. The wild-type B30.2 domain of pyrin interacts with the p20 and p10 subunits, and consequently preventing their assembly into active p20/p10 heterodimer. FMF-associated pyrin B30.2 mutants interacts with p20 and p10 less than WT pB30.2 domain, thereby permitting p20/p10 heterodimer assembly, IL-1β activation, and induction of inflammation. The active p20/p10 heterodimer cleaves pyrin at Asp330 which is located between the bZIP basic domain and the B-box zinc finger domain. The N-terminal cleaved fragment interacts with p65 and IκB-α through the bZIP basic domain and adjacent sequences respectively, by which NF-κB is activated and the expression of inflammatory genes are induced.

Regulation of IL-1β secretion through the N-terminal PYRIN domain of pyrin

In various experimental systems pyrin has been shown either to potentiate or to inhibit inflammasome activation. An inhibitory role of pyrin in caspase-1 activation has been proposed in our study with a mouse model expressing a hypomorphic truncated form of pyrin. The pyrin-truncation mice are hypersensitive to endotoxin, and their peritoneal macrophages exhibit heightened caspase-1 activation and IL-1β production in response to LPS, relative to WT littermates (Chae et al, 2003). In accordance with in vivo and ex vivo data from the pyrin-truncation mice, retroviral transduction of full-length mouse pyrin into the mouse macrophage cell line, RAW 264·7 cells (not expressing endogenous pyrin), resulted in suppression of IL-1β secretion. On the other hand, when pyrin is transfected with ASC, caspase-1, and IL-1β into 293T human embryonic kidney cells, pyrin may actually activate caspase-1 and thus increase the IL-1β secretion (Yu et al, 2006). Moreover, depending on the experimental system, ectopic silencing of pyrin in THP-1 human monocytic cells resulted in augmentation (Chae et al, 2006; Papin et al, 2007) or suppression (Seshadri et al, 2007; Yu et al, 2007) of IL-1β secretion.

The importance of pyrin’s role in the regulation of IL-1β secretion is also supported by molecular studies of a dominantly inherited autoinflammatory disorder, the syndrome of pyogenic arthritis with pyoderma gangrenosum, and acne (PAPA) (MIM 604416), which, like FMF, can present with recurrent episodes of sterile pyogenic arthritis. Patients with PAPA syndrome also manifest cystic acne, sterile abscess formation at the sites of injections, and severe cutaneous ulcers known as pyoderma gangrenosum. PAPA syndrome is caused by missense mutations in the gene encoding CD2-binding protein 1 (now designated proline serine threonine phosphatase interacting protein; PSTPIP1) (Wise et al, 2002). Pyrin directly interacts with PSTPIP1 through the interaction between B-box/coiled-coil domains of pyrin and SH3/coiled-coil domains of PSTPIP1 (Shoham et al, 2003). PSTPIP1 is phosphorylated at Y344 by a tyrosine kinase, cAbl, and the phosphorylation of PSTPIP1 increases its interaction with pyrin. PAPA-associated mutants show increased levels of tyrosine phosphorylation perhaps due to decreased interaction with PEST-type protein tyrosine phosphatases (Wise et al, 2002), and, consequently, increased interaction with pyrin and increased IL-1β production. Indeed, peripheral blood leucocytes from a clinically active PAPA patient showed markedly increased IL-1β production. However it is still ambiguous whether the increased IL-1β production through the increased interaction of mutant PSTPIP1 with pyrin is a dominant negative effect on the suppressive function of pyrin on caspase-1 activation (Shoham et al, 2003) or is result of potentiating function of pyrin in caspase-1 activation (Yu et al, 2007).

Regulation of IL-1β secretion through C-terminal B30.2 domain of pyrin

Although the normal role of pyrin in the regulation of IL-1β secretion is still ambiguous, another important segment of pyrin for the regulation of caspase-1 activation resides in the C-terminal B30.2 domain where most of the major FMF-associated mutations are present. The B30.2 domains, comprising the combination of C-terminal SPRY (the dual-purpose splA kinase and the ryanodine receptor) domain with a N-terminal PRY subdomain, are found in a variety of cellular proteins with various functions, and may be protein-interacting modules, which recognize a specific individual partner protein rather than a consensus sequence motif (Henry et al, 1998; Grutter et al, 2006). Through direct interaction between the B30.2 domain of pyrin and the catalytic domains of caspase-1, pyrin binds to procaspase-1 as well as the processed active p10 and p20 catalytic subunits (Chae et al, 2006; Papin et al, 2007). Furthermore, the B30.2 domain also mediates the interactions of pyrin with the major components of inflammasomes, such as NLRP1, NLRP2, NLRP3, and caspase-5 (Papin et al, 2007). These interactions resulted in the inhibition of the caspase-1 activation and subsequent IL-1β secretion. Since the B30.2 domain binds to p10 and p20 subunits of caspse-1, it is suggested that pyrin inhibits not only the proteolytic activation of procaspase-1 but also the activity of p10p20 heterodimer, the active form of caspase-1. Moreover, the B30.2 domain of pyrin also interacts with proIL-1β, and this interaction also contributes additional blockade of IL-1β secretion (Papin et al, 2007).

Because most of FMF-associated mutations are clustered in the C-terminal B30.2 domain of pyrin, the inhibition of caspase-1 through the B30.2 domain of pyrin appears to be a key point for the adequate molecular explanation of the pathogenesis of FMF. In our transfection study, we have shown that pyrin proteins with one of the three major FMF-associated mutations (M680I, M694V, and V726A) still bind caspase-1 but the interactions are substantially decreased compared to WT pyrin (Chae et al, 2006). Moreover, in a model for the interaction between the deduced structure of pyrin and the crystal structure of caspase-1, M680I and M694V pyrin mutations are located in the putative binding interface. Consequently, the diminished interaction of mutant pyrin with caspase-1 attenuates the inhibitory effect of pyrin on IL-1β secretion. This finding provides another line of evidence that pyrin has a role in the inhibition of IL-1β production, and FMF is caused by uncontrolled activation of caspase-1 and subsequent IL-1β secretion that also provide an important therapeutic target for FMF. However in a different transfection study, a difference was not observed in the interaction between M694V mutant and WT pyrin with caspase-1 (Papin et al, 2007), and this contradiction may be due to different transfection studies. Thus, further endogenous studies are required using cells from FMF patients or a mouse model with mutant pyrin proteins, to address whether the FMF-associated mutations affect the interaction of pyrin with caspase-1 or not.

Cleavage of pyrin by caspase-1

Although many caspases are involved in apoptosis, the major role of caspase-1 is proteolytic processing of inflammatory IL-1 family cytokines. Recently, we have demonstrated that even though there is no similarity of pyrin with IL-1 family cytokines, caspase-1 cleaves pyrin at Asp330 (Fig 1), producing a 330-residue N-terminal fragment that enhances ASC-independent NF-κB activation and a 451-residue C-terminal fragment (Chae et al, 2008). Moreover, the mutant pyrin proteins harbouring FMF-associated B30.2 mutations are cleaved more efficiently than WT pyrin by caspase-1. Consequently, in the peripheral blood mononuclear cells (PBMCs) from FMF patients who have mutations on B30.2 domain of pyrin, more than 70% of pyrin proteins are present as a cleaved form, while full-length pyrin is predominant in the PBMCs from healthy individuals, despite individual variations in the amounts of cleaved pyrin. The increased sensitivity of pyrin with B30.2 domain mutations to caspase-1 cleavage can be explained by the differential binding and inhibitory effect of B30.2 mutant pyrin for caspase-1 as mentioned in the previous section (Chae et al, 2006), or by a possible conformational change of mutant pyrin that render them more susceptible to caspase-1 cleavage. The pyrin cleavage introduces a new level of complexity in the subcellular localization of the protein. As mentioned above, when the gene encoding pyrin, MEFV, was cloned, pyrin was predicted to be a nuclear factor based on the analysis of primary structure (International FMF Consortium, 1997). The nuclear localization of N-terminal cleaved pyrin re-opened the possibility that pyrin may be a nuclear factor. Similar to pyrin, it has also been shown that MyD88 adapter-like (Mal), an adaptor protein involved in Toll-like receptor (TLR)2 and TLR4 signalling, interacts with caspase-1, is cleaved by caspase-1, and subsequently activates NF-κB (Miggin et al, 2007).

Activation of NF-κB through N-terminal cleaved pyrin

Pyrin also has been demonstrated to have a role in the regulation of NF-κB activation in conjunction with ASC as do other PYRIN domain-containing proteins, such as NLRP3 (Manji et al, 2002), PAN1/NALP2/PYPAF2 (Bruey et al, 2004), NLRP6/PYPAF5 (Grenier et al, 2002), NLRP12/PYPAF7/Monarch1 (Wang et al, 2002; Williams et al, 2005), NLRP10/PYNOD (Wang et al, 2004), POP1 (Stehlik et al, 2003), and POP2 (Bedoya et al, 2007). However, the true effect of pyrin on NF-κB activation is not obvious because in different transfection studies, co-expression of pyrin with ASC has been shown to have positive, negative, or no regulatory effects on ASC-dependent NF-κB activation (Stehlik et al, 2002; Dowds et al, 2003; Masumoto et al, 2003; Yu et al, 2006). Moreover, the role of ASC on NF-κB activation is also ambiguous, showing a dual role in either inhibition or activation of NF-κB, depending on cellular context (Stehlik et al, 2002), or no critical role in NF-κB signalling in the studies using ASC knock-out mice (Mariathasan et al, 2004). Regardless of the discrepancies in the effect of full-length pyrin on NF-κB activation, the N-terminal cleaved fragment of pyrin appears to act as a potent enhancer for the activation of NF-κB. Pyrin may enhance NF-κB activity in two possible ways, facilitating the entrance of p65 NF-κB into the nucleus and enhancing the degradation of IκB-α, and both were mediated through the interactions of N-terminal cleaved pyrin with both components (Chae et al, 2008). As summarized in Fig 2, the differential sensitivity of mutant and WT pyrin to caspase-1 cleavage and subsequent activation of NF-κB through the N-terminal cleavage fragment of pyrin suggest an additional mechanism for the pathogenesis of FMF by which pyrin mutations lead to heightened potential for inflammation through NF-κB.

The interaction with p65 takes place through a possible bZIP transcription factor basic domain of pyrin (residues 266–280) (International FMF Consortium, 1997), and the interaction of IκB-α to the region adjacent to the bZIP domain where no known motifs have been identified. The bZIP domain is found in a large number of eukaryotic transcription factors as a bipartite DNA binding structure that consists of a region enriched in basic amino acids (14–20 a.a.) followed by a leucine zipper that is characterized by several leucine residues regularly spaced at seven-amino acid intervals. Whereas the basic region directly contacts the DNA, the leucine zipper mediates homodimerization and heterodimerization of protein monomers, which provide the specificity of the binding on the target of DNA (Shuman et al, 1990). A group of well known bZIP domain-binding proteins are the NF-κB/Rel family of transcription factors. The binding of NF-κB to the bZIP domain is mediated by the 300 residue N-terminal Rel homology domain, which also has a role in the DNA binding, dimerization and nuclear targeting functions, as well as interaction with IκB. Through the Rel domain-bZIP interaction, p65 has been demonstrated to interact with various transcription factors such as CCAAT enhancer-binding protein (C/EBP) family members, BZLF1 (bZIP transactivator of Epstein-Barr virus) and AP-1 transcription factor (Fos/Jun) (Stein et al, 1993; Gutsch et al, 1994; Hu et al, 2002). Therefore it is not surprising that N-terminal cleaved pyrin can interact with p65. However the bZIP domain of pyrin is partial and does not seem to have the leucine-rich part that is important for interaction with p65. Nevertheless, the interaction of N-terminal cleaved pyrin with p65, which is mediated through the potential basic DNA-binding part at residues 266–280 of pyrin, suggests not only a new mechanism for Rel homology-bZIP interaction but also that pyrin may act as a transcription factor that may modulate p65 in augmentation of gene expression. Moreover, the regulatory role of N-terminal cleaved pyrin on NF-κB seems to be specific to p65 because N-terminal cleaved pyrin cannot interact with p50, although it has a Rel homology domain. Similarly, it has been demonstrated that the interaction of Fos and Jun with NF-κBs is also exclusive to p65 (Stein et al, 1993). In the other hand, C/EBP family members have been shown to interact both with p65 and p50 (LeClair et al, 1992; Stein et al, 1993). Thus it can be speculated that small differences in the Rel homology domains or the transactivation region among the NF-κB family members might account for the specificities of interactions.

The interaction of N-terminal cleaved pyrin with IκB-α induces the calpain-mediated degradation of IκB-α, which is also different from the classical NF-κB activation pathway. Although IκB-α degradation is attributed primarily to the ubiquitin-proteosome pathway, a number of calpain-mediated proteolytic mechanisms have been described for IκB-α degradation (Miyamoto et al, 1998; Baghdiguian et al, 1999; Han et al, 1999; Schaecher et al, 2004). Calpains are a family of calcium-dependent, non-lysosomal cysteine proteases that are comprised of two subunits, a large 80-kDa subunit and a smaller 30-kDa subunit. The large subunit mediates the catalytic function and contains a calmodulin-like domain (CaMLD) that can bind to the PEST domain of IκB-α for proteolytic cleavage (Shumway et al, 1999). The cleavage by calpain takes place at the N-terminus of IκB-α and produces ~30-kDa intermediate fragment that cannot be detected by the classical ubiquitin-proteosome mediated degradation (Han et al, 1999; Chen et al, 2000; Schaecher et al, 2004). Thus, one of the lines of evidence for the calpain-mediated IκB-α degradation is the presence of a 30-kDa intermediate form of IκB-α that can be detected not only from the co-transfected cells with N-terminal cleaved pyrin and IκB-α but also from PBMCs of FMF patients who produced substantially increased amount of cleaved pyrin. Taken together with the N-terminal cleaved pyrin-dependent production of 30-kDa IκB-α intermediate and the increase of calpain activity in monocytic cells expressing N-terminal cleaved pyrin, it is possible that the N-terminal cleaved pyrin enhances the activity of calpain. However it is unclear whether the N-terminal cleaved pyrin has a direct role on calpain activation or not, even though the direct interaction of N-terminal cleaved pyrin with IκB-α is critical for the calpain-mediated IκB-α degradation.

Colchicine treatment

The most effective therapy for FMF patients is daily oral treatment with colchicine which prevents both acute attacks and serum amyloid A (SAA) amyloidosis (Goldfinger, 1972; Dinarello et al, 1974; Zemer et al, 1974), although the mechanism underlying these effects is not completely understood. It is thought that one of the major effects of colchicine on FMF inflammation is in modulating cytoskeletal structures, such as microtubules in neutrophils, because colchicine is known to depolymerize microtubules by interacting with microtubules and accumulates in granulocyte after oral administration, and pyrin is expressed predominantly in granulocytes and also associates with microtubules (Chappey et al, 1993; Centola et al, 2000; Mansfield et al, 2001). It has also been demonstrated that colchicine prevents inflammation by inhibiting neutrophil chemotaxis (Bar-Eli et al, 1981) and reducing serum inflammatory cytokine levels, such as IL-6, IL-8, and TNF-α in FMF patients (Kiraz et al, 1998). The reduction of inflammatory cytokine levels by colchicine can be explained by the finding that colchicine suppresses NF-κB activation by attenuating the calpain-mediated IκB-α degradation that is enhanced by N-terminal cleaved pyrin (Chae et al, 2008). Nevertheless, colchicine is not effective in all FMF patients and it has some toxicity. Thus the finding that colchicine suppresses the NF-κB activation is expected to shed light not only on understanding the mechanism of colchicines action, but also in developing new therapeutic targets for FMF, such as calcium channels or calpain because it is directly involved in the NF-κB activation in response to the cleavage of pyrin, and the FMF-associated mutant pyrin proteins are cleaved more efficiently than WT pyrin.

Cytokine treatment and cytokine blocking

Although there are discrepancies in the underlying mechanism, a major role of pyrin is the regulation of IL-1β secretion. Whether the normal function of pyrin is to inhibit or activate IL-1β secretion, the inflammatory symptoms of FMF are thought to be triggered by IL-1β which may be abnormally induced by mutations in the C-terminal B30.2 domain of pyrin. Thus the blockade of IL-1β may prove to be a fascinating adjunctive therapy for FMF. Indeed, in several case reports, colchicine-resistant FMF patients have shown immediate and sustained resolution of symptoms when treated with the IL-1 receptor antagonist, anakinra (Chae et al, 2006; Belkhir et al, 2007; Kuijk et al, 2007; Calligaris et al, 2008; Roldan et al, 2008). The dramatic therapeutic effect of anakinra is better known in another group of autoinflammatory diseases, the cryopyrin-associated periodic syndromes (Hawkins et al, 2003; Hoffman et al, 2004; Goldbach-Mansky et al, 2006). One issue with the treatment of anakinra is that it should be administered daily by subcutaneous injection. Recently, it has been reported that the weekly administration of the long-acting IL-1 inhibitor Rilonacept (IL-1 Trap) leads to immediate resolution of the inflammatory symptoms in patients with FCAS (Goldbach-Mansky et al, 2008; Hoffman et al, 2008). Thus, IL-1 Trap may also be expected to ameliorate the symptoms in FMF.

IFN-α may be another adjunctive therapy for FMF since early administration of IFN-α injections at the onset of attack has shown reduction of attack length and/or severity in some cases of FMF (Tankurt et al, 1996; Tunca et al, 1997, 2004; Tweezer-Zaks et al, 2008). Moreover it is intriguing that colchicine has a role on macrophages in down-regulating the LPS-induced production of GM-CSF which is central to the proliferation, maturation, and functional competence of granulocytes and monocytes (Rao et al, 1997). Therefore, the blockade of GM-CSF by neutralizing antibody can be considered as a possible, but untested, alternative therapeutic option for FMF patients.

Reactive oxygen species (ROS) as a possible target

The underlying mechanism for a massive influx of polymorphonuclear neutrophils (PMN) into the affected tissues during the FMF attack could possibly be explained by ROS, since the neutrophils from FMF patients produce high levels of ROS, O²⁻ without any stimulation (Sarkisian et al, 1997) and the activation of phagocytic NADPH oxidase can be induced by IL-1β (Bonizzi et al, 2000). ROS induce chemokines responsible for the recruitment of granulocytes and monocytes into sites of injury or infection, and also have a role in the regulation of IL-1β production through the activation of the inflammasome (Dostert et al, 2008; Meissner et al, 2008). It is noteworthy that ROS-induced chemokine production is controlled by the plasma membrane Ca²⁺-permeable channel TRPM2 (Yamamoto et al, 2008) that is abundantly expressed in inflammatory cells including monocytes, neutrophils and T lymphocytes (Perraud et al, 2001; Hara et al, 2002; Massullo et al, 2006). Moreover, Trpm2 knock-out mice demonstrate attenuation of chemokine production, neutrophil infiltration and ulceration in a ROS-associated inflammation model (Yamamoto et al, 2008). Thus, the blocking of ROS by ROS-scavenger such as N-acetylcysteine or functional inhibition of TRPM2 can be considered as possible new therapeutic strategies for FMF.

Conclusion

Given that pyrin is composed of multiple domains (Fig 1), this protein may have various roles in regulation of the innate immune response. Nevertheless, it is thought that the major role of pyrin is the regulation (up or down) of caspase-1 activation, and the inflammatory phenotypes of FMF are induced by IL-1β and NF-κB, which are abnormally activated by FMF-associated mutations in the C-terminal B30.2 domain of pyrin. Therefore the blockade of IL-1β signalling or NF-κB activation may be possible targets for the treatment of FMF.