The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33-induced cytokine secretion revealed high IFN-γ and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 phenotype and secreted higher amounts of IFN-γ than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA.

Objectives: To test whether the active metabolite of leflunomide (LEF-M), in addition to blocking the proliferation of activated lymphocytes by inhibiting dihydro-orotate dehydrogenase (DHODH), influences the transendothelial migration (TEM) of peripheral blood mononuclear cells (PBMC). Methods:In an in vitro model of PBMC transmigration through an endothelial cell (EC) barrier, PBMC were re-collected in three groups: cells not adherent to the EC, cells bound to, and cells which had migrated through, the EC layer. Experiments in which cells were pretreated with LEF-M (in the absence or in the presence of uridine) were compared with parallel experiments in the presence of medium alone. Results: Preincubation of EC with LEF-M led to a 36 (SEM 16)% reduction in PBMC TEM (p,0.05). Likewise, preincubation of PBMC induced a reduction in their TEM of 39 (9)% (p,0.005). Incubation of both PBMC and EC with LEF-M had an additive effect (mean reduction of 48 (6)%, p,0.005). Incubation of PBMC with LEF-M also decreased monocytic CD44 expression (p,0.005) and PBMC-hyaluronan binding (p,0.05). Incubation of cells with LEF-M and uridine in addition to LEF-M reversed the inhibition of migration, suggesting that the observed effects were due to DHODH inhibition. Fluorocytometric analysis of PBMC subsets within the migrated population showed a decrease of monocytes, but not of B or T cells, after LEF-M treatment. Conclusions: LEF-M reduces monocytic adhesion molecule expression and TEM and may thus interfere with monocyte and EC activities in RA. Thus, the clinical effects of leflunomide may, at least in part, be due to blocking cell traffic into the inflamed synovia.

We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). An indirect immunofluorescence test and rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients´ sera. Overall 33/60 patients with JIA had sera positive for AKA (55 %, P = 0,0001) ranging from 1:10 to 1:160 dilutions. Following idiopathic arthritis of childhood classification criteria AKA occurred in 2/7 patients with systemic disease (28,6 %), in 13/30 patients with RF negative polyarthritis (43,3 %, P = 0,008) and in 15/18 RF positive polyarthritis (83,3 %, P = 0,000002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence of AKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 18/29 patients (62%) with severe JIA and in 12/26 patients (46,2 %) with non-severe disease, however this did not reach statistical significance (P = 0,18). We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 55,6 % patients with active JIA and in 48,6 % patients in the complete or near remission. Acknowledgement: This research was supported by a European Commission (Acronym: EUROBANK, contract no: QOL-2000-14.1), web site http://www.ncl.ac.uk and by grant of 2nd Medical Faculty, Charles University in Prague, VZ no. 111300003. P2The significance of antibodies to cyclic citrullinated peptide, antikeratin antibodies, antiperinuclear factor, rheumatoid factor isotypes and HLA shared epitope in prediction of erosive disease in early rheumatoid arthritis patients J Vencovsky, L Sedova, S Machacek, J Gatterova, V Pesakova, J Kafkova and O Krystufkova Institute of Rheumatology, Prague, Czech RepublicObjectives: To evaluate a predictive value of autoantibody examinations in development of erosive disease in early rheumatoid arthritis (RA). Patients and methods: One hundred and fourteen patients with disease duration less than 2 years after the onset of symptoms were investigated. Only patients who fulfilled the diagnostic criteria for RA either at the beginning of the disease or during the follow-up period were included. The antibodies to cyclic citrullinated peptide (anti-CCP) (Immunoscan RA, Euro-diagnostica, The Netherlands), IgM, IgA and IgG rheumatoid factors (RF) were measured by ELISA, antikeratin antibodies (AKA) and antiperinuclear factor (APF) were detected by indirect immunofluorescence, and the presence of HLA shared epitope (HLA SE) was detected by PCR with sequence specific primers. Patients were divided into two groups, either with erosive or non-erosive changes present on the hand or/and feet radiographs at the end of 24 months follow-up. Results: Seventy-six (66.7%) patients developed bony erosion, whereas 38 (33.3%) remained without destructive changes. The initial anti-CCP, AKA, APF, IgM RF, IgA RF, IgG RF ...

We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). An indirect immunofluorescence test and rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients´ sera. Overall 33/60 patients with JIA had sera positive for AKA (55 %, P = 0,0001) ranging from 1:10 to 1:160 dilutions. Following idiopathic arthritis of childhood classification criteria AKA occurred in 2/7 patients with systemic disease (28,6 %), in 13/30 patients with RF negative polyarthritis (43,3 %, P = 0,008) and in 15/18 RF positive polyarthritis (83,3 %, P = 0,000002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence of AKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 18/29 patients (62%) with severe JIA and in 12/26 patients (46,2 %) with non-severe disease, however this did not reach statistical significance (P = 0,18). We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 55,6 % patients with active JIA and in 48,6 % patients in the complete or near remission. Acknowledgement: This research was supported by a European Commission (Acronym: EUROBANK, contract no: QOL-2000-14.1), web site http://www.ncl.ac.uk and by grant of 2nd Medical Faculty, Charles University in Prague, VZ no. 111300003. P2The significance of antibodies to cyclic citrullinated peptide, antikeratin antibodies, antiperinuclear factor, rheumatoid factor isotypes and HLA shared epitope in prediction of erosive disease in early rheumatoid arthritis patients J Vencovsky, L Sedova, S Machacek, J Gatterova, V Pesakova, J Kafkova and O Krystufkova Institute of Rheumatology, Prague, Czech RepublicObjectives: To evaluate a predictive value of autoantibody examinations in development of erosive disease in early rheumatoid arthritis (RA). Patients and methods: One hundred and fourteen patients with disease duration less than 2 years after the onset of symptoms were investigated. Only patients who fulfilled the diagnostic criteria for RA either at the beginning of the disease or during the follow-up period were included. The antibodies to cyclic citrullinated peptide (anti-CCP) (Immunoscan RA, Euro-diagnostica, The Netherlands), IgM, IgA and IgG rheumatoid factors (RF) were measured by ELISA, antikeratin antibodies (AKA) and antiperinuclear factor (APF) were detected by indirect immunofluorescence, and the presence of HLA shared epitope (HLA SE) was detected by PCR with sequence specific primers. Patients were divided into two groups, either with erosive or non-erosive changes present on the hand or/and feet radiographs at the end of 24 months follow-up. Results: Seventy-six (66.7%) patients developed bony erosion, whereas 38 (33.3%) remained without destructive changes. The initial anti-CCP, AKA, APF, IgM RF, IgA RF, IgG RF ...

Objectives In the present study we followed activation of STAT1 and 3 during zymosan-induced arthritis (ZIA) in wildtype (wt) and IL-6-/-mice. We also studied expression of the STAT inhibitors Suppressor Of Cytokine Signalling (SOCS) 1 and 3 during the arthritis. Methods Zymosan induced Arthritis was elicited by intra-articular injection of zymosan as described in (2). Protein lysates of inflamed and control synovia were subjected to Western blotting for both total and phosphorylated STAT1 and STAT3. RT-PCR was performed for STAT3, SOCS1 and SOCS3. Results The primary inflammation lasted for a week in both strains. In wt mice this inflammation became chronic until the end of the experiments at week 4, while it decreased rapidly in IL-6-/-mice. In wt mice STAT3 became activated from day 1 until the end of the experiments. Also the level of STAT3 mRNA and protein increased. STAT1 in contrast only became activated during the chronic phase after day7. During the arthritis there was an increase of SOCS1 and SOCS3 mRNA expression. In IL-6-/-mice STAT3 only became activated at day 1. No activation of STAT1 occurred in IL-6-/-mice. Conclusion STAT3 activation occurred during both the primary and chronic inflammation in wildtype mice. During the first week IL-6-/-mice also developed a primary inflammation. IL-6 is not the only cytokine that activates STAT3, explaining STAT3 activation at day 1 in IL-6-/-mice. Lack of further STAT3 activation during the primary inflammation suggests a major role for IL-6 in STAT3 activation. STAT1 only became activated in the chronic phase. Arthritis increased expression of SOCS1 and 3, but this seemed not enough to inhibit STAT activation. Future research will have to address the importance of activated STAT1 and 3 for chronic synovitis and the possibility of inhibiting these signalling molecules in the inflamed synovium. REFERENCES1 de Hooge ASK, van de Loo FAJ, Arntz OJ, van den Berg WB. Involvement of IL-6, apart from its role in immunity, in mediating a chronic response during experimental arthritis.