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GM-CSF expressed by in vivo and in vitro activated T-lymphocytes suppresses osteoclast differentiation of murine bone marrow cells stimulated by RANKL and M-CSF

We investigated the role of different modes of T lymphocyte (lyT) activation in bone-marrow (BM) osteoclast differentiation – ConA-pulse, anti- CD3/CD28 antibody-treatment, and in vivo- and in vitro-alloactivation. Osteoclast-like cells (OCL) were generated by incubating murine BM (C57B6/J6) overnight with 5 ng/mL M-CSF, followed by harvesting of non-adherent immature hematopoietic monocyte/macrophage-progenitors, cultured then for 5 days with receptor activator of NF-kappaB ligand (RANKL) and M-CSF. Addition of activated lyT or conditioned medium (CM) to early stages of BM cultures suppressed OCL formation. All modes of in vivo and in vitro lyT activation produced similar inhibitory effects on osteoclastogenesis. Both CD4- and CD8-subpopulations of lyT were equally effective in the suppression of osteoclastogenesis. OCL inhibitory effect of activated lyT was specifically associated with the increased GM-CSF expression, since osteoclastogenesis could be partially restored after addition of anti-GM-CSF antibodies to BM cultures treated with activated lyT-CM. Addition of activated lyT or CM to BM cultures supplemented with RANKL and M-CSF caused the downregulation in the gene expression for RANK, c-Fms, c-Fos and calcitonin receptor by quantitative PCR. The suppression of RANK and c-Fms as OCL markers, together with the enhancement of CD11c as dendritic-cell marker, were confirmed by flow- cytometry. Finally, blocking of lyT activation with cyclosporine A abrogated inhibitory effects of lyT on osteoclast differentiation. In conclusion the inhibitory role of activated lyT on osteoclastogenesis is mediated through GM-CSF- release, which may be a significant regulatory- loop and possible therapeutic target to counteract activated bone resorption mediated by lyT-derived cytokines in inflammatory and immune disorders.

T cells; Cytokines; Cytokine Receptors; Cell Activation; Cell Differentiation

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