Osteoclast Precursors In Peripheral Blood Of Patients With Rheumatoid Arthritis

PartⅠ: Osteoclast Precursors in Peripheral Blood of Patients with Rheumatoid ArthritisObject:To investigate osteoclast precursor (OCP) and tumor necrosis factor-α(TNF-α) in peripheral blood of patients with rheumatoid arthritis (RA) .Method: The peripheral blood mononuclear cells from 7 cases of RA patients and 9 healthy controls were cultured in the medium containing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factorκB-ligand (RANKL). After being cultured for 14 days, cytochemistry was applied to detect tartrate-resistant acid phosphatase (TRAP) expression and the cells with positive TRAP and≥3 nuclei were counted and defined as OC. Bone resorption assay was used to evaluate OC function. ELISA was used to measure serum TNF-αlevel in 15 cases of RA and 9 healthy controls. The relationship was analyzed in RA patients between OC precursor number and serum TNF-αlevel as well as the disease activity indicators including erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP).Results:1. Significantly higher OC production was observed in peripheral blood of RA patients than that of healthy controls. The OC number per ten fields were 27.8±10.8 (±SD) and 14.4±6.1 respectively, (p<0.05) ; 2. Serum TNF-α. level was significantly higher in RA patients (18.8±5.2 pg/ml) than in healthy controls (9.5±3.0 pg/ml) (P<0.05). 3. Significant positive correlation was observed between the OC number and ESR in RA patients (r=0.912, p=0.000) ; 4. In RA patients, serum TNF-αlevel was found to have significant positive correlation with ESR (r=0.936, p=0.000) ; 5. Significant positive correlation was observed between the OC number and the serum TNF-αlevel (r=0.913, p=0.000). Conclusion: In RA, there are more OC precursors in peripheral blood and higher serum level of TNF-α. Both might be responsible for increased OC activity in inflammatory site, leading to joints destruption.PartⅡ: Effect of methotrexate on Osteoclast Precursors in Peripheral Blood of Patients with rheumatoid arthritisObject:To investigate the effect of methotrexate (MTX) on OC production and TNF-αsecretion of peripheral blood mononuclear cells (PBMCs) from rheumatoid arthritis (RA) patient.Method: PBMCs from 3 healthy controls were cultured in the medium containing different concentration of MTX (0, 1, 10, 102, 103 and 104 nM). CCK8 was used to evaluate the toxicity of MTX on PBMCs. PBMCs from 8 cases of RA patients were cultured in the medium containing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factorκB-ligand (RANKL), together with different concentration of MTX (0, 1, 10 and 102 nM), After being cultured for 14 days, cytochemistry was applied to detect TRAP expression and the cells with positive TRAP and≥3 nuclei were counted and defined as OC. Culture supernatant was collected at day 2 of culture and TNF-αlevel was measured with ELISA.Results:1. The viability of PBMCs was significantly decreased when medium MTX concentration was≥103 nM. When MTX concentration was 1 nM, 10 nM and 102 nM, the decrease of viability was not statistically significant. 2. The OC production of PBMCs was significantly decreased when cultured with 10 nM MTX and 102 nM MTX, comparing with no MTX (0 nM). The OC number per ten fields were 26.3±7.6 (±SD), 13.6±6.0 and 35.8±6.6 respectively, (p<0.05), TNF-αlevel in culture supernatant was significantly lower when cultured with 10 nM MTX (45.3±4.7 pg/ml) and 102 nM MTX (39.9±3.0 pg/ml), comparing with no MTX (101.3±1.5 pg/ml). (P<0.05). Conclusion: MTX inhibits the capability of PBMCs differentiating into OC and secreting TNF-α, which might be part of mechanism through which MTX treats RA.