Fig. 2

B-cell receptor (BCR)/CD40-induced receptor activator of nuclear factor kappa-B ligand (RANKL) expression in switched-memory B cells is augmented by interferon gamma (IFN-γ) but is suppressed by interleukin-21 (IL-21). a Naïve (IgD⁺CD27^–), IgD⁺-memory (IgD ⁺ -m, IgD⁺CD27⁺) and switched-memory (Sw-m, IgD^–CD27⁺) B cell subsets from HC were purified by flow cytometry and stimulated with or without BCR/CD40. RANKL expression at mRNA and protein levels was detected by quantitative PCR and flow cytometry, respectively. The values are the mean ± SEM of three independent experiments (n = 5, **P < 0.005, with reference to BCR/CD40-stimulated sw-m B cells). b Purified switched-memory B cells from HC were incubated with IFN-γ, tumor necrosis factor alpha (TNF-α), IL-2, IL-4, IL-6 + IL-6 receptor, IL-17, or IL-21 in addition to BCR/CD40 simulation. RANKL mRNA expression was analyzed by quantitative PCR (n = 4, *P < 0.05, with reference to BCR/CD40-stimulated sw-m B cells). Expression of RANKL and CD80 was detected by flow cytometry (n = 4). c Purified switched-memory B cells from HC were stimulated via BCR/CD40, IFN-γ or IL-21, and mRNA expression of indicated molecules was analyzed by quantitative PCR. The values are the mean ± SEM of three independent experiments (n = 4, *P < 0.05, **P < 0.005, with reference to BCR/CD40-stimulated sw-m B cells). d Purified switched-memory B cells from HC were cultured with BCR/CD40 stimulation and/or IFN-γ, and CXCR3 mRNA expression was analyzed (n = 3, *P < 0.05, with reference to IFN-γ-stimulated sw-m B cells). Expression of RANKL and CXCR3 was detected by flow cytometry (n = 7). e Purified switched-memory B cells from HC were stimulated with the indicated dose of BCR/CD40 in the presence or absence of CXCL10 (100 ng/ml) and RANKL mRNA expression was analyzed. BCR/CD40 stimulation was carried out at suboptimal or optimal dose (n = 3, *P < 0.05). a–e mRNA expression of each molecule was detected by quantitative PCR after 24 hours culture. Expression of RANKL, CD80 and CXCR3 was detected after 48 hours culture. OPG osteoprotegerin, N.D. not determined, No stim no stimulation, NS not significant