We studied the in vitro effect of cyclosporin-A (CyA) on bone resorption using a fetal rat long bone-resorbing assay. CyA inhibited both PTH-stimulated and unstimulated bone resorption. The inhibitory effect of CyA on basal resorption was dose dependent, and it was more pronounced during the second period (less than or equal to 0.1 microgram/ml) of culture (days 5-7) than during the first period (days 2-4). A cytotoxic effect was ruled out by the absence of decrease in [3H]thymidine incorporation into bones up to a concentration of 5 micrograms/ml CyA. Histomorphometry performed after 4 and 7 days of culture showed that CyA (1 microgram/ml) decreased the number of osteoclasts per bone section after 7 days of culture (23.5 +/- 4.0 vs. 41.7 +/- 2.9 osteoclasts/bone section; P less than 0.05), but not after 4 days (25.6 +/- 3.3 vs. 23.0 +/- 2.5). These data suggested an effect of CyA on osteoclastic differentiation rather than on the function of mature osteoclasts. We further assessed the mechanisms of the inhibitory effect of CyA on osteoclastic differentiation in order to determine 1) the level of this action (proliferation and/or fusion of osteoclast precursors), and 2) if this action is direct or indirect. Autoradiographic studies were performed on bone sections after incubation of bones with [3H]thymidine for the last 48 h of culture. CyA decreased slightly but significantly the percentage of labeled nuclei per osteoclast and the number of osteoclasts containing at least one labeled nucleus (20.2 +/- 0.7 vs. 33.2 +/- 3.5; P less than 0.02). Moreover the number of nuclei per osteoclast was decreased after 7 days in CyA-treated bones (2.4 +/- 0.05 vs. 3.0 +/- 0.1; P less than 0.02). Taken together these results demonstrate that CyA slightly decreased the proliferation of osteoclast precursors, but markedly decreased their fusion. Similar effects were observed in cultures of rat marrow macrophages. CyA (1 microgram/ml) inhibited the fusion of macrophages into multinucleated cells elicited by 1 nM 1,25-dihydroxyvitamin D3, but had only a slight effect on the proliferation of these cells, as assessed by autoradiography. CyA also inhibited the formation of multinucleated cells and the fusion index in long term cultures of human cord blood monocytes, a cellular model for osteoclastic differentiation. By contrast, CyA had no effect on the formation of myotubes by fusion of cultured mononucleated rat myoblasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased bone resorption is a mechanism contributing to bone loss in the postmenopausal period. Cytokines are involved in osteoclastic differentiation and, therefore, may play a role in the regulation of bone resorption. Several previous works showed the implication of interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF alpha) in the modulation of bone remodeling. This study determines the concomitant production of the three cytokines and tests the bone-resorbing activity of peripheral monocyte supernatants. Four groups of women were studied: premenopausal women (n = 13; mean age, 47 +/- 0.9 yr), untreated postmenopausal women (n = 21; mean age, 52 +/- 0.6 yr), postmenopausal women treated with estrogens (n = 14; mean age, 54.2 +/- 1.1 yr), or postmenopausal women treated with ethanehydroxydiphosphonate (n = 12; mean age, 53.2 +/- 2 yr). Assignment to clinical groups was verified by plasma FSH and estradiol determinations. Lumbar spine bone mineral density was significantly higher in the premenopausal women group than in the three postmenopausal groups. Peripheral blood monocytes were cultured for 48 h with 20% autologous plasma, and after stimulation with lipopolysaccharides. IL-1, IL-6, and TNF alpha levels were measured by RIA in the monocyte surpernatants. The three cytokines were highly correlated to each other, IL-1 with IL-6 (r = 0.76; P < 0.001), IL-1 with TNF alpha (r = 0.89; P < 0.001), and IL-6 with TNF alpha (r = 0.89; P < 0.001). The mean levels of the three cytokines could not be compared because of the variations in the values. However, a trend toward lower levels in the three cytokines was noted in estrogen-treated women compared to the untreated postmenopausals. The bone-resorbing activity of monocyte supernatants, assessed by fetal long bone-resorbing assay, increased in untreated postmenopausal compared to that in premenopausal women (1.22 +/- 0.08 vs. 0.87 +/- 0.11; P < 0.05). In estrogen-treated patients, this activity decreased to premenopausal levels (0.89 +/- 0.04 vs. 0.87 +/- 0.11; P = NS). The resorbing activity was correlated to IL-1 (r = 0.28; P = 0.03), IL-6 (r = 0.52; P < 0.01), and TNF alpha (r = 0.48; P < 0.01). The addition of cytokine inhibitors and IL-1 receptor antagonist and TNF alpha antibodies to the supernatant bone culture medium induced a significant decrease in the calcium release. Those data show the involvement of several cytokines in the bone resorption process after estrogen deficiency.

We recently showed that indapamide (IDP), a thiazide-related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10