Fig. 2

CXCL10 stimulates production of osteoclastogenic cytokines in CD4⁺ T cells through TLR4 and CXCR3. a, b CD4⁺ T cells were isolated from WT, Tlr4 ^–/–, and Cxcr3 ^–/– mice, and serum-starved CD4⁺ T cells were cultured with or without CXCL10 (100 ng/ml) for 24 hours. RANKL, TNFα, IL-6, CXCR3, and TLR4 mRNA levels were analyzed by real-time PCR (a). RANKL, TNFα, and IL-6 protein levels in culture media were quantified by ELISA (b). c CD4⁺ T cells from WT, Tlr4 ^–/–, and Cxcr3 ^–/– mice were serum starved and treated with or without 500 ng/ml ionomycin and 20 ng/ml PMA (P/I) for 6 hours. RANKL mRNA levels and RANKL protein levels in culture media were measured by real-time PCR (left panel) and ELISA (right panel), respectively. d CD4⁺ T cells were serum starved and treated with or without CXCL10 (100 ng/ml) for 24 hours. Total cell lysates were immunoblotted with the indicated antibodies. e, f Serum-starved WT CD4⁺ T cells were preincubated with DMSO or 10 nM CsA and then stimulated with or without CXCL10 (100 ng/ml) for 24 hours. RANKL mRNA levels were analyzed by real-time PCR (e). NFATc1 protein levels were measured by western blotting (f). g, h CD4⁺ T cells from the indicated mice were transduced with the retroviral vectors pMX-IRES-EGFP (pMX) or pMX-CA-NFATc1-IRES-EGFP (pMX-CA-NFATc1). The cells were serum starved and cultured with or without CXCL10 (100 ng/ml) for 24 hours. Total cell lysates were immunoblotted with the indicated antibodies (g). RANKL mRNA levels (h, left panel) and RANKL protein levels in culture media (h, right panel) were measured. Results shown are representative of three independent experiments (n = 3), and values are expressed as mean ± SD. *P < 0.001 by one-way ANOVA followed by Dunnett’s test. CXCL10 C-X-C motif chemokine 10, RANKL receptor activator of nuclear factor kappa-B ligand, TNFα tumor necrosis factor alpha, IL-6 interleukin 6, Tlr4 toll-like receptor 4, Cxcr3 CXC chemokine receptor 3, WT wild-type, NFATc1 nuclear factor of activated T cells, cytoplasmic 1, DMSO dimethyl sulfoxide, CsA cyclosporin A, ND not detected, n.s. nonsignificant