| Objective:Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitis, symmetrical and destructive joint disease. It’s pathogenesis is not well known. Studies have reported that calreticulin (CRT) may be related to the pathogenesis of RA. In this study, a variety of immunological techniques were used to analyze the expression of CRT in serum, synovial fluid and synovium in patients with RA. Correlations of CRT serum levels with disease activity [Disease Activity Score for28joints (DAS28)], erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were assessed. Serum CRT levels were also detected in RA patients whose RF, anti-CCP and anti-MCV antibodies were positive and negative.And to build TRAIL-induced Jurkat T cells apoptosis model to explore the effects of CRT in the resistance of T-cell apoptosis in RA. The purpose of this study is to explore the role of CRT in the pathogenesis of RA, especially in the inflammatory response, and provide the experimental basis for the diagnosis and treatment of RA.Methods:1. Serum CRT levels were measured by ELISA in70patients with established RA,30systemic lupus erythematosus (SLE),25other autoimmune disease,20osteoarthritis (OA), and35of healthy controls (HC). CRT levels in synovial fluid were measured by ELISA in10patients with RA and16patients with OA. Sera CRT content was determined by Western blot.2. The expression of CRT in RA and OA synovium was detected by immunohistochemistry.3. Correlations of CRT serum levels with disease activity [Disease Activity Score for28joints (DAS28)], erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were assessed. Serum CRT levels were also detected in RA patients whose RF, anti-CCP and anti-MCV antibodies were positive and negative.4. The ability of TRAIL binding to CRT was detected by ELISA. 5. The viability of Jurkat T cell was detected by reagents (Muse), and the effect of TRAIL on the proliferation of Jurkat T cells was detected by MTT.6. The ability of calreticulin to inhibit TRAIL-mediated apoptosis was assessed by flow cytometry.Results:1. Serum CRT levels in RA patients (4.817±2.425ng/ml) was significantly higher (P<0.05) compared with those in the serum of OA (3.574±0.942ng/ml), SLE (4.013±1.536ng/ml), other autoimmune disease (3.882±0.837ng/ml) and HC (3.726±0.627ng/ml). Though there was increased CRT in synovial fluid of RA, no significance diffience between RA and OA group was found (mean3.700±0.220ng/ml and3.563±0.153ng/ml; p=0.601). Western blot was used to detect the serum CRT in RA, SLE, OA and HC simultaneously when CRT levels were detected using ELISA. CRT levels were increased in patients with RA compared with SLE, OA and HC.2. Pathology showed that CRT was observed in endothelial cells, synovial fibroblasts, macrophages and perivascular areas in RA synovium. In OA, CRT was observed in perivascular areas and synovial fibroblasts.3. A significant correlation was observed between CRT levels in sera and DAS28in RA patients (R2=0.445, P<0.001). It was found that CRT levels positively correlated with ESR ((R2=0.567, p<0.0001) and CRP ((R2=0.255, p<0.0001). Patients with RA who were positive for RF had increased levels of CRT compared to seronegative patients but with no significance (mean5.152±o.467ng/ml and4.157±o.603ng/ml; p=0.192). Patients with RA who were positive for anti-CCP and anti-MCV had significantly increased levels of CRT compared to seronegative patients (mean5.387±o.468ng/ml and2.928±o.143ng/ml; p=0.003;5.568±0.510ng/ml and3.091±o.144ng/ml; p=0.001).4. The ability of TRAIL binding to CRT was detected by ELISA. CRT binding to TRAIL has been observed previously, and this was confirmed by our results on ELISA. The normalized results showed that CRT bound to TRAIL in a dose-dependent manner.5. We evaluated TRAIL inhibiting the proliferation of Jurkat T cells at12and24hours. Jurkat T cells treated with50ng/ml and100ng/mlTRAIL were assessed at12and24hours, at12hours,100ng/ml TRAIL treated cell showed a significant difference compared with the control group (P<0.05). At24hours,50ng/ml and100ng/ml TRAIL showed a significant difference compared with the control group (P <0.05).6. The addition of CRT alone (4,8,16ng/ml) had no significant effect on Jurkat cell death. Furthermore, the addition of CRT (4,8, or16ng/ml) with100ng/ml TRAILsignificantly decreased the number of early apoptotic cells by between2%and10%as compared with TRAIL treatment alone.Conclusions:1. In RA, serum CRT levels were increased in patients with RA compared with those controls. Our results suggest that CRT may play a role in inflammation reaction and joint destruction of RA.2. Moreover, a significant correlation was observed between serum CRT levels and disease activity in RA. It might be used as a potential biomarker for clinical diagnosis and provide additional information regarding disease activity along with the traditional indices such as ESR and CRP.3. Calreticulin had the capacity to bind directly to TRAIL and to inhibit TRAIL-mediated apoptosis of Jurkat T cells, and thus might play a role in inhibiting apoptosis of inflammatory T cells in RA. |
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