Chronic inflammatory diseases such as rheumatoid arthritis (RA), ankylosing spondylitis, and inflammatory bowel disease are associated with generalized osteoporosis and an increased risk of fractures (1). The pathogenesis of osteoporosis in these conditions is unclear, but there is evidence to suggest that inhibition of bone formation plays a role in association with systemic overproduction of bone-resorbing cytokines, nitric oxide (NO), and prostaglandins (2).
The evidence implicating NO as a mediator of inflammation-induced bone loss stems from the fact that NO production is increased in inflammatory disorders (3). This appears to be due to activation of the inducible NO synthase (iNOS) pathway by cytokines, such as interleukin-1 and tumor necrosis factor α (4), which are expressed at sites of inflammation. Cytokine-induced iNOS activation has also been shown to inhibit osteoblast function in vitro and to stimulate osteoblast apoptosis (5). While the role of iNOS-induced osteoblast apoptosis in the pathogenesis of inflammation-induced bone loss remains unclear, we previously reported that the NOS inhibitor NG-monomethyl-L-arginine inhibited bone loss in a rat model of inflammation-induced osteoporosis (IMO) (6). In those studies, however, we were unable to clearly define the mechanisms involved, because of the poor specificity of the pharmacologic inhibitors then available to probe NOS function.
In the present study, in order to resolve these issues and clarify the role of the iNOS pathway in inflammation-induced bone loss in vivo, we have examined the effects of systemic inflammation on bone in mice lacking a functional iNOS gene.
- Top of page
- MATERIALS AND METHODS
BMD. Total BMD was significantly reduced in animals that had been injected with MgSiO4 compared with controls, due to a fall in both trabecular and cortical components of BMD (Figures 1A–C). The reduction in BMD was significantly attenuated in the iNOS KO mice compared with the WT mice, and this was statistically significant for total, trabecular, and cortical BMD.
Figure 1. Bone mineral density (BMD), osteoblast apoptosis, and nitric oxide (NO) production in wild-type (WT) mice and mice with targeted inactivation of the gene for inducible NO synthase (iNOS knockout [iNOS KO] mice). A–C, In vivo measurements of volumetric BMD. Values are expressed as percentage changes in relation to the values observed in WT sham-operated animals, to correct for the effects of skeletal growth. D, Osteoblast apoptosis on day 21 following inflammation-mediated osteoporosis (IMO) or saline (Sal) treatment. E, NO production (as assessed by urinary nitrate excretion) on day 21 following IMO or saline treatment. Values are the mean and SEM of 7 samples per group. P values indicate differences between iNOS KO and WT mice. ∗∗ = P < 0.005 for within-genotype differences between IMO and control animals.
Download figure to PowerPoint
Bone histomorphometry, body weight, and spleen weight. Consistent with the BMD results, trabecular bone volume, trabecular thickness, and cortical thickness were significantly reduced in mice with IMO compared with controls (Table 1). The reductions in trabecular bone volume and thickness which resulted from IMO were smaller in the iNOS KO mice than in the WT mice. The difference in the skeletal responses to inflammation was obvious on examination of low-power photomicrographs of the long bones (Figures 2A–D). At a cellular level, IMO was associated with a significant decrease in several parameters of bone formation, including osteoblast surface, osteoblast number, and mineral apposition rate. These changes were significantly attenuated in iNOS KO mice compared with WT mice. In contrast, IMO had no significant effect on parameters of bone resorption, which were unchanged from baseline in both iNOS KO and WT mice (Table 1).
Table 1. Changes in proximal tibial bone histomorphometric parameters and in body and spleen weights in response to inflammation-mediated osteoporosis (IMO) in wild-type (WT) mice and mice with targeted inactivation (knockout) of the gene for inducible nitric oxide synthase (iNOS KO mice)*
|Variable||WT mice||iNOS KO mice|
| BV/TV, %||41.86 ± 1.71||16.17 ± 1.51†||32.79 ± 5.03||23.43 ± 2.60‡|
| TrabTh, μM||0.035 ± 0.004||0.018 ± 0.007†||0.024 ± 0.002§||0.021 ± 0.001|
| CtTh, mm||0.135 ± 0.011||0.095 ± 0.004§||0.142 ± 0.014||0.104 ± 0.013¶|
| OcS, %||7.14 ± 1.20||8.76 ± 0.9||7.13 ± 1.17||7.40 ± 0.76|
| ES, %||9.87 ± 1.70||12.96 ± 1.60||8.97 ± 1.93||9.80 ± 0.93|
| NOc/mm2||14.1 ± 1.9||15.1 ± 1.4||15.4 ± 2.3||14.8 ± 0.7|
| ObS, %||8.68 ± 1.44||2.67 ± 0.39†||8.97 ± 1.82||4.72 ± 0.85‡|
| NOb/mm2||46.7 ± 5.7||15.3 ± 2.0†||55.5 ± 8.4||27.7 ± 5.0‡¶|
|Mineral apposition rate, μm/day||0.97 ± 0.09||0.29 ± 0.07†||0.93 ± 0.11||0.63 ± 0.08‡¶|
|Body, % of that on day 0||101.3 ± 5.5||114.3 ± 4.5§||99.2 ± 6.8||117.2 ± 2.6#|
| Spleen, mg||109.1 ± 7.2||390.7 ± 44.7§||99.3 ± 2.4||475.6 ± 22.3¶|
Figure 2. Skeletal responses to inflammation. A–D, Histologic sections of tibiae from WT and iNOS KO mice treated with saline or talc (MgSiO4). A and B, WT and iNOS KO saline-treated control mice, respectively. C and D, Talc-treated WT and iNOS KO mice, respectively, showing noticeably less bone loss in iNOS KO mice. E and F, Fluorescence micrographs of a representative tibial section from a talc-treated WT mouse. E, Nuclei of osteoblasts on the bone surface (arrowheads) and bone marrow cells in the marrow cavity (asterisks) are clearly stained with 4′,6-diamidino-2-phenylindole. F, Fluorescein isothiocyanate staining of apoptotic osteoblasts (arrowheads) in the same section. See Figure 1 for definitions. (Original magnification × 50 in A–D; × 400 in E and F.)
Download figure to PowerPoint
Spleen weight, which was used as an indicator of generalized inflammation, was significantly greater in the IMO groups than in the saline control groups, but did not differ between genotypes (Table 1). Mean (±SEM) initial body weights were 30.5 ± 1.2 gm in the WT mice and 27.8 ± 0.8 gm in the iNOS KO mice. Body weights increased significantly in both the WT and iNOS KO IMO groups compared with controls (Table 1), although this was largely attributable to the weight of the injected MgSiO4 and to localized edema surrounding the sites of MgSiO4 injection. Tibial bone length was unaffected by IMO.
Apoptosis. Apoptosis was detected in 14.0 ± 1.4% and 11.5 ± 1.3% of osteoblasts in WT and iNOS KO mice, respectively (see Figures 1D, 2E, and 2F). In the WT IMO group, the level of osteoblast apoptosis was increased significantly, to 44.8 ± 3.4%, while only a modest increase, to 14.2 ± 3.0%, was noted in the iNOS KO IMO group. The difference between the two IMO groups was highly significant (P < 0.001). Osteoclast apoptosis was not observed in any of the sections. While DAPI staining clearly revealed the presence of osteocytes within bone lacunae, no osteocyte apoptosis was observed.
NO production. Baseline urinary nitrate levels were significantly lower in the iNOS KO mice than in the corresponding WT animals. NO production increased significantly in WT mice injected with MgSiO4, but did not change significantly in iNOS KO mice (Figure 1E). Urinary nitrate levels at day 21 were unchanged from the baseline values on day 0 in the saline-treated mice.
- Top of page
- MATERIALS AND METHODS
This report is the first to describe the use of mice as a model of IMO caused by the subcutaneous injection of MgSiO4. We found that subcutaneous injections of MgSiO4 caused a generalized inflammatory process associated with splenomegaly and significant bone loss. In WT mice, IMO was accompanied by a marked increase in systemic production of NO, as reflected by urinary nitrate/nitrite excretion, while nitrate/nitrite excretion did not change in iNOS KO mice. This confirms that the previously noted increase in NO production in IMO (6) is due to iNOS activation rather than activation of other NOS isoforms.
The inflammatory process in animals injected with MgSiO4 was associated with significant bone loss, as detected by pQCT, which affected both trabecular and cortical compartments of the bone. In accordance with the pQCT data, histomorphometric analysis showed reductions of trabecular bone volume, trabecular thickness, and cortical thickness in animals with IMO. Inhibition of several parameters of bone formation, including osteoblast number, osteoblast surface, and mineral apposition rate, was also observed. In contrast, indices of bone resorption, including osteoclast numbers and resorption surfaces, were not significantly changed from baseline values. These data suggest that the systemic osteoporosis which develops in response to this inflammation model results primarily from decreased bone formation rather than increased osteoclastic bone resorption, which is consistent with the observations in rats with IMO (8) and in humans with chronic inflammatory disease (11).
The abnormalities of bone turnover in IMO, which are observed at sites distant from inflammatory lesions, contrast with the periarticular increases in osteoclast activity that are characteristic of human RA and adjuvant-induced arthritis in experimental animals (12). Periarticular bone loss in adjuvant arthritis has recently been shown to be due mainly to osteoclast activation mediated by up-regulation of receptor activator of nuclear factor κB ligand (RANKL) expression on activated T cells which infiltrate the inflamed joint (12). The lack of an increase in systemic osteoclastic bone resorption in humans with RA (11) and in the IMO model studied here (8) may result from the fact that activated T cells, which promote osteoclastic bone resorption via RANKL expression, tend to accumulate within inflammatory lesions. An additional explanation could be that the stimulatory effects of cytokines and prostaglandins on systemic bone resorption were counterbalanced by suppressive effects of high NO levels on osteoclast activity within the bone microenvironment (13).
A novel observation to emerge from this study was that the reduced bone formation in WT mice with IMO was associated with a significant increase in osteoblast apoptosis. The increase in osteoblast apoptosis was not observed in iNOS KO mice, which is consistent with the fact that the reduction in BMD that resulted from IMO was significantly less in iNOS KO mice than in WT mice, even though the severity of inflammation—as reflected by spleen weight—was similar in the two genotype groups. It is important to emphasize that iNOS KO mice still exhibited a significant reduction in BMD compared with control animals, indicating that factors other than iNOS activation and osteoblast apoptosis contribute to reduced bone formation and bone loss in this model. Further work will be required to investigate the mechanisms involved, but possibilities include direct inhibitory effects of cytokines on osteoblast growth and differentiation and suppressive effects of increased corticosteroid levels on bone formation (14).
Osteoblast and osteocyte apoptosis have previously been shown to contribute to bone loss in corticosteroid-induced osteoporosis (14), and this study demonstrates that a similar mechanism may be at work in inflammation-induced bone loss. Unlike glucocorticoid-induced osteoporosis, we observed no evidence of an increase in osteocyte apoptosis in this study, nor did we observe an increase in osteoclast apoptosis. This suggests that the deleterious effects of iNOS activation and inflammation on bone may be relatively specific for mature osteoblasts.
These observations are consistent with the results of in vitro studies which have shown clearly that high levels of cytokine-induced NO inhibit osteoblast growth and differentiation as well as stimulating osteoblast apoptosis (5, 13). The mechanisms of NO-dependent apoptosis are becoming increasingly well understood, and apoptosis of osteoblasts is known to involve death receptor– and mitochondria-activated pathways, with caspases as the downstream effectors (15, 16). Further studies will be required to define the precise mechanism of osteoblast apoptosis during chronic inflammation.
In conclusion, we have shown that the osteoporosis resulting from MgSiO4-induced inflammation is primarily mediated by reduced bone formation and that this is accompanied by a dramatic increase in osteoblast apoptosis, mediated by activation of the iNOS pathway. When combined with the results of our previous investigations (6), these data demonstrate the importance of the iNOS pathway as a regulator of bone turnover in vivo and suggest that iNOS inhibitors could be of value in the treatment of bone loss in inflammatory conditions such as RA.