A potent interleukin-6 (IL-6) antagonist (Sant 5), which binds tightly to the 6α receptor but has impaired gp130 heterodimerization, has been developed recently by site-directed mutagenesis of human IL-6. We report here that Sant 5 inhibitS IL-6-stimulated osteoclast-like multinucleated cell (MNC) formation in human marrow cultures but also inhibits the stimulatory effects of IL-1 or tumor necrosis factor alpha (TNF-α in these cultures. We further show that a neutralizing antibody to IL-6 also inhibits the stimulatory effects of IL-1 or TNF-α in these cultures. In contrast, Sant 5 had no effect on parathyroid hormone related protein (PTHrP) or 1,25- dihydroxyvitamin D3 stimulated MNC formation in human marrow cultures. Transfection of a human marrow stromal cell line, which normally induces osteoclast formation through production of IL-6, with the Sant 5 cDNA driven by a cytomegalovirus (CMV) promoter blocked the capacity of these cells to stimulate osteoclast-like cell formation. These Sant 5 transfected cells and conditioned media from these cells also inhibited the stimulatory effects of the parent cell line on MNC formation. These data suggest that IL-6 mediates the effects of IL-1 and TNF on human osteoclast formation, but in contrast to murine systems, does not mediate the effects of PTHrP. These data further demonstrate that stromal cells transfected with the Sant 5 cDNA can constitutively produce high levels of the IL-6 antagonist and inhibit osteoclast formation in vitro.
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