Objective
To investigate the anti-apotoptic effects of calreticulin (CRT) on tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated T cells apoptosis in rheumatoid arthritis (RA).
Methods
Levels of CRT in the synovial fluid from patients with RA (23 cases) and osteoarthritis (OA) (36 cases) were detected with enzyme-linked immunosorbent assay (ELISA); the expression and localization of CRT in RA and OA synovial tissue were detected by immunohistochemistry. The capacity of CRT binding to TRAIL was measured by ELISA; the effect of TRAIL on Jurkat T cell proliferation was determined by the methylthiazolyl tetrazolium (MTT) assay, CRT on TRAIL-mediated apoptosis by flow cytometry analysis. The t-test and SNK-q test were used for statistical analysis.
Results
① CRT were significantly up-regulated both in synovial fluid and tissue from RA than those from OA [ (5.6±2.5) ng/ml and (3.2±1.3) ng/ml, t=6.233, P<0.05; 32 787±18 294 and 7 945±2 765, t=15.662, P<0.01]. ② Binding rate of CRT-TRAIL increased to (117±9)% and(155±14)% with TRAIL stimulation (1 μg/ml and 2 μg/ml, respectively) compared to the controls (q=3.858, 8.647; P<0.01). ③ The proliferation of Jurkat T cell was inhibited after co-cultured with TRAIL, at 12 hours, 100ng/ml TRAIL treated cell showed a significant decreased survival rate and at 24 hours(q=7.532, P<0.05), and the similar results were shown both with 50 ng/ml and 100 ng/ml TRAIL (q=9.211, 12.879; P<0.01). ④ T cell early apoptosis was suppressed by the combination of CRT and TRAIL: with CRT (4, 8 and 16 ng/ml), the corresponding apoptotic rates were (30.7±1.1)%, (24.5±1.1)% and (22.3±1.5)% respectively, which showed significant differences compared with the control group (32.5±1.4)% (q=3.82, 12.17, 15.39; P<0.05).
Conclusion
Increased levels of CRT are detected in the synovial fluid and tissue in RA; and CRT is expressed in inflammatory cells in synovial tissue from RA patients. In addition, CRT can partly inhibit TRAIL-mediated apoptosis of Jurkat T cells via binding to TRAIL, which may suggest its potential role in the pathogenesis especially in inflammation of RA.
Key words:
Arthritis, rheumatoid; Calreticulin; Tumor necrosis factor-related apoptosis-inducing ligand
Objective
To investigate the neutrophil extracellular traps (NETs) formation and their molecular mechanisms induced by serum amyloid A (SAA) in rheumatoid arthritis (RA).
Methods
Neutrophils were isolated from peripheral blood of RA and healthy volunteers. ① Neutrophils were cultured in vitro, the formation of NETs was observed and their percentage was calculated. ② Neutrophils were cultured in vitro, divided into six groups: control, SAA, [SAA+anti-Toll like receptro4 (TLR4)-Ab], LPS, (LPS+anti-TLR4-Ab) and anti-TLR4-Ab. Appropriate stimulation was conducted for each group. NETs formation and their percentages were investigated. The concentration of DNA in supernatant was detected by fluorescent staining. F test and t test were used for statistical analysis.
Results
① The purification of isolated neutrophils was higher than 95%. The network which was collocated with the spreading neutrophils nucleus and neutrophil elastase under the microscope, was NETs. In the RA group, the formation of NETs induced by SAA was significantly more than control [(19.1±0.8) vs (7.4±0.5), t=12.30, P<0.05]. ② However, after pretreated with anti-TLR4 antibody, NETs formation was significantly less than the SAA group [(5.7±0.4) vs (14.7±1.1), t=7.825, P<0.05]. Moreover, the fluorescence intensity of DNA in supernatant was significantly higher in SAA group than that of anti-TLR4-Ab pretreatment group[(18.7±0.7) vs (12.9±0.8), t=5.552, P<0.05]. The concentration of DNA in supernatant of SAA group was higher than that of anti-TLR4-Ab pretreatment group[(36.9±1.3) μg/ml and(16.3±0.6) μg/ml, t=14.41, P<0.05].
Conclusion
SAA can induce the formation of NETs from neutrophils by binding to TLR4 in RA.
Key words:
Arthritis, rheumatoid; Serum amyloid A protein; Toll-like receptor 4; Neutrophil extra-cellular traps
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)