Interleukin-1 and tumor necrosis factor activities partially account for calvarial bone resorption induced by local injection of lipopolysaccharide

Abstract

The present study was undertaken to test the hypothesis that tumor necrosis factor (TNF) and/or interleukin-1 (IL-1) activity mediates lipopolysaccharide (LPS)-induced bone resorption in vivo. To test this hypothesis, Escherichia coli LPS or Porphyromonas gingivalis LPS was injected into the subcutaneous tissues overlying mouse calvariae. Histological sections, prepared from the center of the lesion, were stained for tartrate-resistant acid phosphatase, and histomorphometric analysis was performed to quantify the osteoclast number and the area of bone resorption. In time course experiments using normal mice, a peak of bone resorption occurred 5 days after endotoxin stimulation. In dose-response experiments, IL-1 receptor type 1 deletion (IL-1R(-/-)), TNF double-receptor p55/p75 deletion (TNF p55(-/-)/p75(-/-)), combined TNF p55 and IL-1 receptor type 1 deletion (TNF p55(-/-)/IL-1R(-/-)), and IL-1beta-converting enzyme-deficient (ICE(-/-)) mice and the respective wild-type mice were injected with 500, 100, or 20 micrograms of P. gingivalis LPS and sacrificed 5 days after LPS injection. At the highest dose (500 micrograms), significant decreases in osteoclast number occurred in mutant mice compared to wild-type mice: (i) a 64% reduction for the TNF p55(-/-)/IL-1R(-/-) mice, (ii) a 57% reduction for the IL-1R(-/-) mice, (iii) a 41% reduction for the TNF p55(-/-)/p75(-/-) mice, and (iv) a 38% reduction for the ICE(-/-) mice. At the two lower doses, bone resorption was apparent but no significant differences between mutant and wild-type animals were observed. The present data indicate that at higher doses, LPS-induced bone resorption is substantially mediated by IL-1 and TNF receptor signaling. Furthermore, IL-1 receptor signaling appears to be slightly more important than TNF receptor signaling. At lower LPS doses, other pathways leading to osteoclast activity that are independent of TNF and IL-1 are involved.

FIG. 1

Light micrographs of mouse calveriae injected with 500 μg of LPS. Histological sections of the calvarial bone were stained for TRAP activity. Red-staining cells are osteoclasts in Howship’s lacunae. Magnification, ×200.

FIG. 2

Time course study of bone resorption induced by a local injection of 500 μg of E. coli LPS (n = 5). Bars represent means and standard errors. Mice were sacrificed after 2, 5, 9, and 14 days, and their calvarial bones were processed for histomorphometry. (A) Osteoclast index; (B) osteoclast-covered surface. The highest peak of bone resorption (P < 0.01) occurred on day 5. ∗, statistical significance at P ≤ 0.01.

FIG. 3

Light micrographs of calveriae of wild-type (A and B) and mutant (C to F) mice injected with 500 μg of LPS (B to F) or saline (A). Histological sections of the calvarial bone were stained for TRAP activity. Red-staining cells are multinucleated cells in Howship’s lacunae. Results similar to those in panel B were obtained for the wild-type controls corresponding to ICE^−/− mice (data not shown). Magnification, ×200.

FIG. 4

Dose-response study. Three doses (20, 100, and 500 μg) of P. gingivalis LPS were applied to the mouse calveriae. Bars represent means and standard errors. Fisher’s analysis of variance (P < 0.01) was performed on histomorphometric data for the calvariae of wild-type, TNF p55^−/−/p75^−/−, IL-1R^−/−, and TNF p55^−/−/IL-1R^−/− mice (n = 6 in each group) injected with either P. gingivalis LPS or saline. (A) Osteoclast index. *, significant difference for the wild-type mice injected with 500 μg compared with the mutant mice (P ≤ 0.01); #, significant difference for the TNF p55^−/−/p75^−/− mice compared with the IL-1R^−/− and TNF p55^−/−/IL-1R^−/− mice (P ≤ 0.01). (B) Osteoclast-covered surface. Symbols are as in panel A. A slight increase was seen in the TNF p55^−/−/IL-1R^−/− mice injected with 100 and 20 μg of LPS compared with the mice injected with 500 μg of LPS. For the osteoclast activity (micrometers per osteoclast), a statistically significant difference was observed only between the mice injected with 500 μg of LPS and the low-dose groups (data not shown).