Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, is involved in the breakage of self-tolerance in anti-CCP-positive rheumatoid arthritis. This reaction is catalyzed by peptidyl arginine deiminases (PADs), of which PAD2 and PAD4 are thought to play key pathogenic roles. Small-molecule PAD inhibitors such as the pan-PAD inhibitor BB-Cl-amidine, the PAD2-specific inhibitor AFM-30a, and the PAD4-specific inhibitor GSK199 hold therapeutic potential and are useful tools in studies of citrullination. Using an ELISA based on the citrullination of fibrinogen, we found that AFM-30a inhibited the catalytic activity of PADs derived from live PMNs or lysed PBMCs and PMNs and of PADs in cell-free synovial fluid samples from RA patients, while GSK199 had minor effects. In combination, AFM-30a and GSK199 inhibited total intracellular citrullination and citrullination of histone H3 in PBMCs, as determined by Western blotting. They were essentially nontoxic to CD4+ T cells, CD8+ T cells, B cells, NK cells, and monocytes at concentrations ranging from 1 to 20 μM, while BB-Cl-amidine was cytotoxic at concentrations above 1 μM, as assessed by flow cytometric viability staining and by measurement of lactate dehydrogenase released from dying cells. In conclusion, AFM-30a is an efficient inhibitor of PAD2 derived from PBMCs, PMNs, or synovial fluid. AFM-30a and GSK199 can be used in combination for inhibition of PAD activity associated with PBMCs but without the cytotoxic effect of BB-Cl-amidine. This suggests that AFM-30a and GSK199 may have fewer off-target effects than BB-Cl-amidine and therefore hold greater therapeutic potential.

• Klíčová slova
• cell viability, citrullination, peptidyl arginine deiminase (PAD), rheumatoid arthritis, small-molecule PAD inhibitors,
• MeSH
• aktivace enzymů MeSH
• histony metabolismus   MeSH
• inhibiční koncentrace 50 MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• náchylnost k nemoci MeSH
• peptidylarginindeiminasa typu 2 antagonisté a inhibitory   metabolismus   MeSH
• peptidylarginindeiminasa typu 4 antagonisté a inhibitory   metabolismus   MeSH
• revmatoidní artritida etiologie   metabolismus   patologie   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histony MeSH
• inhibitory enzymů MeSH
• PADI2 protein, human MeSH Prohlížeč
• PADI4 protein, human MeSH Prohlížeč
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH

OBJECTIVE: Peptidylarginine deiminase 2 (PAD2) and PAD4 are expressed in the synovium of rheumatoid arthritis (RA) patients and catalyze citrullination of arginine residues in proteins targeted by anti-citrullinated protein antibodies (ACPAs). Little is known about the relative importance of PAD2 and PAD4 in generating citrullinated self-antigens. Here we investigate the ability of PAD2 and PAD4 to generate citrullinated targets for ACPAs in four human proteins. METHODS: Synovial fluid (SF) and plasma were collected from 42 RA patients. Human fibrinogen, human alpha-enolase (ENO1), human histone H3, and human serum albumin (HSA) were citrullinated in vitro by PAD2 or PAD4. The total degree of citrullination was determined using the anti-modified citrulline approach. Antibody binding to native and citrullinated proteins was measured by ELISA. RESULTS: ACPAs within pooled SF from multiple RA patients reacted equally well with, and cross-reacted with, PAD2- and PAD4-citrullinated fibrinogen. ACPAs from most individual patient SF and plasma samples bound equally well to PAD2- and PAD4-citrullinated fibrinogen or ENO1. When histone H3 was used as target, PAD4 was generally superior in generating epitopes recognized by ACPAs. No binding to citrullinated HSA was observed. CONCLUSION: In most patients, PAD2 and PAD4 are equally efficient in generating citrullinated target sites for ACPAs in fibrinogen and ENO1. The binding of autoantibodies to histone H3 was generally higher after citrullination with PAD4 than with PAD2. Citrullinated HSA is not a target for ACPAs.

• MeSH
• citrulinace MeSH
• fibrinogen imunologie   MeSH
• fosfopyruváthydratasa imunologie   MeSH
• histony imunologie   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH
• peptidylarginindeiminasy metabolismus   MeSH
• protilátky proti citrulinovaným peptidům metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• revmatoidní artritida enzymologie   imunologie   MeSH
• sérový albumin imunologie   MeSH
• synoviální tekutina MeSH
• techniky in vitro MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fibrinogen MeSH
• fosfopyruváthydratasa MeSH
• histony MeSH
• PADI2 protein, human MeSH Prohlížeč
• PADI4 protein, human MeSH Prohlížeč
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH
• peptidylarginindeiminasy MeSH
• protilátky proti citrulinovaným peptidům MeSH
• rekombinantní proteiny MeSH
• sérový albumin MeSH
• vápník MeSH

BACKGROUND: Calgizzarin (S100A11) is a member of the S100 protein family that acts in different tumors by regulating a number of biologic functions. Recent data suggest its association with low-grade inflammation in osteoarthritis (OA). The aim of our study is to compare S100A11 expression in the synovial tissues, synovial fluid and serum of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to characterize the potential association between S100A11 and disease activity. METHODS: S100A11 protein expression was detected in synovial tissue from patients with RA (n = 6) and patients with OA (n = 6) by immunohistochemistry and immunofluorescence. Serum and synovial fluid S100A11 levels were measured by ELISA in patients with RA (n = 40) and patients with OA (n = 34). Disease activity scores in 28 joints based on C-reactive protein (DAS28-CRP) were used to assess disease activity. Cytokine content in peripheral blood mononuclear cells (PBMCs), synovial fibroblasts (SFs) and synovial fluid was analysed by ELISA, western blotting or cytometric bead array. RESULTS: S100A11 expression was significantly up-regulated in the synovial lining and sublining layers (p < 0.01) and vessels (p < 0.05) of patients with RA compared to patients with OA, and was associated with fibroblasts and T cells. S100A11 was significantly increased in synovial fluid (p < 0.0001) but not in serum (p = 0.158) from patients with RA compared to patients with OA when adjusted for age and sex. Synovial fluid S100A11 correlated with DAS28 (r = 0.350, p = 0.027), serum CRP (r = 0.463, p = 0.003), synovial fluid leukocyte count (r = 0.677, p < 0.001), anti-cyclic citrullinated peptide antibodies (anti-CCP) (r = 0.424, p = 0.006) and IL-6 (r = 0.578, p = 0.002) and IL-8 (r = 0.740, p < 0.001) in synovial fluid from patients with RA. PBMCs and SFs isolated from patients with RA synthesized and spontaneously secreted higher levels of S100A11 in comparison with PBMCs and SFs from patients with OA (p = 0.011 and 0.03, respectively). S100A11 stimulated the production of the pro-inflammatory cytokine IL-6 by PBMCs (p < 0.05) and SFs (p < 0.01). CONCLUSIONS: Our data provide the first evidence of S100A11 up-regulation and its association with inflammation and disease activity in patients with RA.

• Klíčová slova
• Calgizzarin, Disease activity, Inflammation, Rheumatoid arthritis, S100 proteins,
• MeSH
• biologické markery MeSH
• dospělí MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• mediátory zánětu metabolismus   MeSH
• progrese nemoci * MeSH
• proteiny S100 metabolismus   MeSH
• průřezové studie MeSH
• revmatoidní artritida diagnóza   metabolismus   MeSH
• senioři MeSH
• synoviální tekutina metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• mediátory zánětu MeSH
• proteiny S100 MeSH
• S100A11 protein, human MeSH Prohlížeč