Up-regulation of TNF-producing T cells in the bone marrow: a key mechanism by which estrogen deficiency induces bone loss in vivo

Abstract

In vivo studies have shown T cells to be central to the mechanism by which estrogen deficiency induces bone loss, but the mechanism involved remains, in part, undefined. In vitro, T cells from ovariectomized mice produce increased amounts of tumor necrosis factor (TNF), which augments receptor activator of NF-kappa B ligand (RANKL)-induced osteoclastogenesis. However, both the mechanism and the relevance of this phenomenon in vivo remain to be established. In this study, we found that ovariectomy increased the number of bone marrow T cell-producing TNF without altering production of TNF per T cell. Attesting to the essential contribution of TNF, ovariectomy induced rapid bone loss in wild type (wt) mice but failed to do so in TNF-deficient (TNF(-/-)) mice. Furthermore, ovariectomy induced bone loss, which was absent in T cell-deficient nude mice, was restored by adoptive transfer of wt T cells, but not by reconstitution with T cells from TNF(-/-) mice. These findings demonstrate the key causal role of T cell-produced TNF in the bone loss after estrogen withdrawal. Finally, ovariectomy caused bone loss in wt mice and in mice lacking p75 TNF receptor but failed to do so in mice lacking the p55 TNF receptor. These findings demonstrate that enhanced T cell production of TNF resulting from increased bone marrow T cell number is a key mechanism by which estrogen deficiency induces bone loss in vivo. The data also demonstrate that the bone-wasting effect of TNF in vivo is mediated by the p55 TNF receptor.

Figure 1

Flow cytometry profiles of anti-TNF Ab staining of CD90⁺ cells from ovx and sham-operated mice. Data are plotted as fluorescence intensity vs. cell number. Histograms are representative of results obtained in triplicate experiments. (a) Isotype control. (b and c) Sham and ovx cells. Data are representative of triplicate experiments. Variability among experiments was <10%.

Figure 2

TNF^−/− mice are protected against ovx-induced bone loss. Trabecular BMD (mean ± SEM) of excised tibiae was measured by pQCT both in intact wt and TNF^−/− mice of 10 weeks of age and in additional groups of 10-week-old wt and TNF^−/− mice, 4 weeks after ovx or sham operation. Data (n = 6 per group) are expressed as percent difference from intact mice. *, P < 0.05 compared with all other groups.

Figure 3

Ovariectomy induces bone loss in nude mice injected with T cells from wt (A) but not TNF^−/− (B) mice. Trabecular BMD (mean ± SEM) of excised tibiae was measured by pQCT both in intact mice at 10 weeks of age and in additional groups of mice of the same age, 4 weeks after ovx or sham operation. Data (n = 6 per group) are expressed as percent difference from intact mice. *, P < 0.05 compared with all other groups.

Figure 4

Mice lacking the p55 TNF receptor are completely protected against ovx-induced bone loss. Trabecular BMD (mean ± SEM) of excised tibiae was measured by pQCT both in intact wt, p75^−/−, and p55^−/− mice at 10 weeks of age and in additional groups of mice of the same age, 4 weeks after ovx or sham operation. Data (n = 6 per group) are expressed as percent difference from intact mice. *, P < 0.05 compared with all other groups except p75^−/− ovx mice.