Neutrophil Phenotyping in Periodic and Chronic Arthritis

Disease-causing alleles of MEFV occur exclusively within populations derived ancestrally from the Mediterranean region including Turkish, Armenian, Jewish and Arab populations. Some FMF mutations predate the divergence of these populations into distinct ethnic groups and are estimated to have arisen more than 15,000 years ago (1). The carrier frequency of FMF disease-causing alleles is very high – approximately 1 in 5 individuals are carriers in the affected populations within the Mediterranean region (5-8). FMF therefore has among the highest carrier frequencies of any known Mendelian disease that exhibits geographical or ethnic specificity.  This frequency exceeds that of cystic fibrosis or sickle cell anemia within Caucasian and African populations, respectively. The fact that FMF disease-causing alleles are common, that they arose in a limited geographical area and have been maintained for millennia within this region, and that multiple mutations occur in distinct populations within that region suggests that a selective advantage for heterozygotes exists.

Pyrin structure.
The MEFV cDNA is 3.7 kb in length and codes for a 781 amino acid polypeptide, denoted pyrin (1, 2) (Fig. 2A). At the time of its identification no proteins with sequence homology to the N-terminal regions of pyrin were reported. Recently, 4 novel regulators of apoptosis with strong sequence homology to the N-terminal 85 amino acids of pyrin have been cloned and functionally characterized. The region of sequence similarity was denoted a Pyrin-like domain (PYD) (9, 10). The PYD mediates homo- and heterotypic protein-protein interactions among proteins carrying this motif, suggesting it mediates the formation of apoptotic-regulatory multi-protein complexes (11, 12). Our data and that of our collaborator Dr. Junji Sagara, suggests that the PYD of pyrin mediates pyrin binding to a known myeloid-specific apoptotic regulator ASC (apoptosis spec-like protein with a CARD domain), suggesting that like ASC pyrin may regulate apoptosis in myeloid cells.

The carboxyl terminal region of pyrin (~411 amino acids) bears sequence-similarity to the RoRet/B30.2-like family of proteins (4, 13). This family consists principally of nuclear effector molecules that regulate inflammation, hematopoiesis, oncogenesis, and embryonic development (4, 13-15). The majority of family members are transcriptional regulators, including RPT-1, a murine T-cell specific gene that attenuates the IL-2 receptor and the HIV LTR promoters; PML, a growth suppressor and transcription factor involved in myelomonocytic differentiation, cell cycle regulation, apoptosis, tumorgenesis; and Ro52, a target of autoantibodies in systemic lupus erythematosus and Sjögren's syndrome. Biochemical analyses of Ro52 demonstrating sequence-specific DNA-binding and transcription-factor-like activity were done by Dr. Bart Frank of our group (16).  

The likely function of the conserved protein motifs of pyrin within this C-terminal region are consistent with a nuclear effector function.  These include two nuclear localization signals (NLS) and a basic putative DNA-binding domain usually associated with a leucine zipper, denoted a b-ZIP motif.

Pyrin and control of TNF-a   signaling.
TNF-a is of principal importance in promoting inflammation in human autoimmune and autoinflammatory diseases, demonstrated by the ability of TNF- a antagonist to block disease progression in several disorders including rheumatoid arthritis, psoriatic arthritis, and Crohn’s disease (17-19). In neutrophils, TNF-a induces or enhances CD11b/CD18 expression and endothelial adhesion, reactive oxygen intermediate (ROI) release, degranulation, phagocytosis, and antibody-dependent cell-mediated cytotoxicity (20-22). TNF-a also potently modulates the inflammatory response by regulating apoptosis. We have observed that pyrin over-expression biases TNF- a -activity towards cell survival in vitro, suggesting pyrin influences TNF-a signaling.

The fact that pyrin mediates TNF-a signaling suggests the protein plays a general role in inflammation control and that the effects of the protein may not be limited to FMF patients Interestingly, mutations in MEFV have recently been implicated in the etiology of Behcet’s disease and inflammatory bowel disease suggesting that modulation of TNF-a signaling by pyrin may be of general consequence in human inflammatory disorders (23-26). Delineation of the normal function of pyrin in the context of TNF-a -signaling will likely have therapeutic consequences for FMF patients, in that pyrin-pathway agonists should be of utility in treating these patients. Moreover, once defined, the pyrin regulatory pathway may provide an entirely new set of therapeutic targets of broad relevance in the context of TNF-a and neutrophil-mediated pathology. The relevance of this potential is enhanced by the fact that specific targeting of neutrophil function through rational drug design has not been used in the past for developing anti-inflammatory agents.