Fig. 7

B cells from RA patients inhibit OB differentiation. a–c Peripheral blood (PB) B cells were purified, as in Fig. 2A and stimulated (S) with CpG2006+anti-Ig (A+G+M) Ab or vehicle (U) for 4 h. a CCL3 and TNF mRNA expression was detected by qPCR. *p < 0.05. b CCL3 and TNF protein expression levels were assessed in the culture media by ELISA. *p < 0.05. c Conditioned medium (40% by volume) from B cell culture were co-cultured with human MSCs ± anti-CCL3 neutralizing Ab ± anti-TNF neutralizing Ab in OB-inducing medium for 3 days. ALP+ area was measured. *p < 0.05 vs. U, #p < 0.05 vs. S-IgG. d PB B cells from RA patients and healthy controls (HC) were co-cultured with human MSCs in OB-inducing medium for 3 days. ALP staining was performed. *p < 0.05 vs. HC. e The expression levels of ALP and Runx2 in hMSCs in d were measured by qPCR. Fold-changes were calculated by dividing patient values by the value from HC cells. Values represent individual HC/RA. *p < 0.05 vs. HC. f RA synovium was stained with Abs to CD20 (B cells), CCL3 and TNF. White arrows indicate the cells with dual staining. Bar = 25 μm. 3 or 6 patients and their controls were included in each experiment. All of these experiments were repeated at least once with representative images and quantifications shown. All error bars represent s.e.m. One way ANOVA followed by Dunnett’s post-hoc multiple comparisons was performed for c. Two-tailed unpaired Student’s t-test was performed for all the others