Stavros C. Manolagas

Endogenous production of tumor necrosis factor by primary cultures of murine calvarial cells: influence on IL-6 production and osteoclast development

We have previously shown that tumor necrosis factor (TNF) and interleukin-1 (IL-1) acted synergistically to stimulate the production of IL-6 by bone marrow stromal and osteoblastic cells; and that an antibody to IL-6 inhibited TNF-induced osteoclast development in murine calvarial cell cultures. Prompted by this evidence, we have now examined whether TNF and/or IL-1 are produced by murine calvarial cells, and whether these cytokines are involved in IL-6 production and osteoclast formation. When cultured under basal conditions, calvarial cells produced TNF and IL-6, and were able to form bone resorbing osteoclasts. A neutralizing antibody against TNF suppressed both basal IL-6 production and the formation of bone resorbing osteoclasts. The anti-TNF antibody also inhibited IL-6 production in response to exogenous IL-1 or parathyroid hormone (PTH). In contrast, a neutralizing anti-IL-1 receptor antibody had no effect on basal, TNF- or PTH-stimulated IL-6 production. These findings suggest that TNF, but not IL-1, is produced by murine bone cells and that endogenous TNF induces the IL-6 production, osteoclast formation, and bone resorption exhibited by these cultures under basal conditions. Furthermore, bone cell-derived TNF amplifies the stimulatory effect of exogenous IL-1 or PTH on IL-6 production by calvarial cells.

The effects of γ-interferon on human peripheral blood monocyte/macrophage-mediated bone particle degradation

gamma-Interferon (IFN-gamma) has recently been demonstrated to inhibit the ability of mononuclear phagocytes to degrade bone particles. We have further addressed the specificity, potency and mechanism of this activity using human recombinant IFN-gamma. Adherent peripheral blood mononuclear leukocytes from normal human volunteers were cultured with washed, sieved (less than or equal to 75 microns) 45Ca-labelled rat bone particles for 3 days, after which bone particle degradation (7.1 +/- 1.6%, n = 11) was calculated from the fraction of 45Ca released into the medium. As little as 5 U/ml IFN-gamma significantly suppressed bone particle degradation and 50 U/ml was associated with consistent marked suppression (74.0 +/- 3.5% inhibition, P less than 0.001, n = 11). IFN-gamma was not suppressive if added to cells 24 h or more after exposure to bone particles. Addition of indomethacin (10 microM) did not reverse the effect of IFN-gamma, suggesting that it was not prostaglandin-mediated. In addition, 1,25(OH)2D3 (10 nM) did not remove the inhibitory effect of IFN-gamma. Contact of mononuclear phagocytes with bone particles and secretion of soluble factors from these cells have both been demonstrated to play a role in their ability to degrade bone particles. IFN-gamma (50 U/ml) inhibited monocyte/macrophage interaction with another unopsonized surface, i.e., one micron fluorescent latex particles, decreasing the number of internalized particles from 12.6 +/- 2.9 per cell to 5.9 +/- 1.4 per cell (P less than 0.01, n = 15), as measured using flow cytometry. However, binding of bone particles by the cells was not diminished by IFN-gamma. Exogenous alpha-interferon and human recombinant IL-1 beta, TNF-alpha, and lymphotoxin did not alter bone particle degradation. In addition, endogenous IL-1 beta release from human monocyte/macrophages exposed to bone particles was negligible and unaffected by IFN-gamma. We conclude that IFN-gamma is a potent and specific inhibitor of monocyte/macrophage-mediated bone particle degradation, and that this activity does not appear to be due to effects on the ability of monocytes to bind bone particles or to release IL-1 in response to the particles.