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Increased Bone Resorbing Activity of Peripheral Monocyte Culture Supernatants in Elderly Women

INSERM U 349 (M.E.C-S., M.A.D., M.C.deV.), Centre Viggo Petersen, Laboratoire de Biologie Endocrinienne (O.B-P., A.M.G.), Hopital Lariboisière, 75010 Paris; and Department of Hormonology (F.B., M.B.), Hopital Sud, Amiens, France

Address all correspondence and requests for reprints to: Dr. M. Cohen-Solal, INSERM U349, Centre Viggo Petersen, Hopital Lariboisière, 6 Rue Guy Patin, 75010 Paris, France.

	Abstract

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Accelerated bone loss occurs in the years after menopause, and is an ongoing phenomenon in elderly women. The role of cytokines in bone loss after estrogen deficiency has been shown in ovariectomized rat and mice models. In humans, the involvement of bone resorbing cytokines is now well established. In the early years after menopause, monocyte activation leads to increased cytokine production. We have previously shown that the bone resorbing activity (BRA) of peripheral blood monocyte culture supernatants from postmenopausal women is higher than in premenopausal (Pre-M) women. This increased activity was related to interleukin (IL)-1, IL-6, and tumor necrosis factor- levels. We here investigate whether monocyte activation still occurs in older women and whether this relates to bone resorption. We studied 19 healthy Pre-M, and 24 early (E-Post-M, menopause <10 yr) and 24 late (L-Post-M, menopause >10 yr) postmenopausal women. Peripheral blood monocytes were cultured for 48 h with 20% autologous plasma. BRA of monocyte supernatants (expressed as the ratio of monocyte supernatant over control bones supernatant) was assessed using fetal long-bone resorbing assays. Bone resorption was determined by urinary total pyridinoline excretion. BRA was significantly increased in E-Post-M and L-Post-M, compared with Pre-M subjects (1.20 ± 0.10 and 1.15 ± 0.20 vs. 0.73 ± 0.10, respectively, both P < 0.05). Moreover, BRA of bones cultured with the supernatant of Pre-M was lower than BRA of control bones. BRA was significantly correlated with levels of IL-1, IL-6, and tumor necrosis factor- in supernatant. Supernatant IL-1 levels were increased in E-Post-M, compared with Pre-M women (506 ± 180 vs. 122 ± 30, P < 0.05). Similarly, pyridinoline levels were increased in E-Post-M and L-Post-M, compared with Pre-M subjects (8.8 ± 1 and 10.5 ± 0.9 vs. 5.8 ± 0.5, respectively, both P < 0.05). BRA was significantly correlated to pyridinoline levels. These data indicate the presence of monocyte activation in L-Post-M, which may be responsible for the increased bone resorption and bone loss observed in this elderly population.

	Introduction

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INCREASED bone resorption is a major mechanism contributing to bone loss in postmenopausal women. Although bone loss accelerates in the years immediately after menopause, biochemical markers of bone resorption suggest that bone resorption continues many years after menopause (1, 2, 3). Several cytokines are involved in osteoclast recruitment and differentiation and play a role in the regulation of bone remodeling. Estrogen-deficient bone loss may be related to modulation of local bone resorbing factors in the bone microenvironment, such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)- (4, 5). In animal models, injection of IL-6 antibodies (6) or IL-1 receptor antagonist and TNF- inhibitors prevented ovariectomy-induced bone loss in normal mice and rats (7). Furthermore, ovariectomy failed to induce bone loss in IL-6 knock-out mice (8) and transgenic mice overexpressing TNF- antibodies (9). In humans, peripheral monocyte IL-1 release is increased in osteoporotic women with high bone turnover (10, 11). Monocyte-release of TNF- is enhanced in postmenopausal women (12).

We have previously shown that bone resorbing activity (BRA) of the peripheral blood mononuclear cell culture supernatants is increased in 5-yr postmenopausal women, compared with Pre-M women (13). This enhanced activity was related to increased levels of IL-1, IL-6, and TNF- levels. In the present study, we examined this relationship in elderly women (>10 yr post menopause) and further assessed the putative relationship between mononuclear cell activation and bone resorption in both early (E-Post-M) and L-Post-M.

	Subjects and Methods

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Patients

Sixty-seven healthy female volunteers (mainly hospital employees or their relatives) agreed to participate in the study and were classified into three groups (Table 1): 1) premenopausal (Pre-M) women (n = 19, mean age 38 ± 2 yr); 2) early postmenopausal (E-Post-M) women (n = 24, mean age 56 ± 1 yr, menopause < 10 yr); and 3) late postmenopausal (L-Post-M) women (n = 24, mean age 69 ± 1 yr, menopause > 10 yr).

Bone mineral density (BMD, g/cm²) was measured at the femoral neck using the LUNAR DPX-L densitometer (Madison, WI).

Methods

Monocyte culture. Peripheral mononuclear cells were cultured as previously described (13). Thirty milliliters heparinized blood was drawn after an overnight fast. Peripheral mononuclear cells were isolated using a Ficoll/Hypaque gradient and washed twice with phenol-free DMEM (Gibco, France). Cells were resuspended in DMEM at a density of 10⁶ cells/mL and were allowed to adhere in a 24-well tissue culture plate for 1 h at 37 C in a humidified atmosphere. After incubation, the wells were gently washed to remove the nonadherent cells, and the remainders were cultured for 48 h with 1 mL complete medium (DMEM supplemented with 20% autologous plasma). After the culture, supernatants were collected and stored at -80 C until assay for BRA and levels of IL-1, IL-6, and TNF-.

Bone resorbing assays. To assess the biological activity of the monocyte culture supernatants, bone resorbing assays were performed as previously described (13). Twenty-four hours after injection of 100 mCi ⁴⁵Ca into pregnant Sprague-Dawley rats, 19 1-day-old fetuses were removed, and the forearm shafts were dissected free of soft tissues. The fetal long bones were cultured for 24 h in BGJb medium to release unbound calcium, and then for 2 periods of 3 days in the presence of 500 µL testing media. These media were prepared with 70% BGJb and 30% monocyte supernatant for each patient. Control bones were incubated with the same medium (70% BGJb and 30% DMEM containing 20% plasma to replace the supernatant). Moreover, all wells (patients and controls) were supplemented with 30 µL plasma. Supernatants of the 61 patients and control bones were assessed in triplicate wells. Results are expressed as the patient/control ratio of released calcium during the second culture period.

Biochemical markers of bone resorption. Urinary total deoxypyridinoline excretion was measured in fast urine spot sample using high-performance liquid chromatography (14). Data are expressed as the deoxypyridinoline/creatinine ratio (nmol/mmol).

Cytokine assays. Levels of IL-1, IL-6, and TNF- were measured in mononuclear cell culture-conditioned medium. Concentrations of IL-6 and TNF- were measured using commercial RIA kits (Medgenix, Belgium). These specific and sensitive assays are based on coated-tube separation and an oligoclonal system, in which several specific monoclonal antibodies are directed against distinct epitotes of IL-6 and TNF-. IL-6 does not cross-react with the TNF- antibody, and TNF- does not cross-react with the IL-6 antibody. The sensitivity of both assays is 5–6 pg/mL. Respective intra- and interassay coefficients of variation are as follows: IL-6, 6.7% and 8.3%; TNF-, 6% and 7%. Concentrations of IL1-b were measured in the medium by enzyme-linked immunosorbent assay (Medgenix). The sensitivity of this assay is 4 pg/mL, with intra- and interassay coefficients of variation of 4 and 6%, respectively.

Statistical analysis. Data are expressed as means ± SEM. Statistical analyses were performed by ANOVA and Fisher PLSD test. Correlations were tested by single regression and step-wise multiple-regression analysis (Statview II Software, Abascus). Regression models were linearized by log transformation.

	Results

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Clinical data

BMD was measured at the femoral neck. BMD was significantly decreased in E-Post-M (compared with Pre-M) women (0.906 ± 0.030 vs. 0.814 ± 0.020 g/cm², P < 0.01) and in L-Post-M (0.906 ± 0.030 vs. 0.785 ± 0.020 g/cm², P < 0.01).

Urinary deoxypyridinoline excretion, measured as a marker of bone resorption, was significantly increased in both E-Post-M and L-Post-M, compared with Pre-M women (8.8 ± 1.0 and 10.5 ± 0.9 vs. 5.8 ± 0.5 mmol/nmol, respectively; P < 0.01; Fig. 1). This increased bone resorption was not caused by hyperparathyroidism, because there were no differences in intact serum PTH levels among the three groups.

View larger version (30K): [in this window] [in a new window]   Figure 1. BRA and pyridinoline excretion in Pre-M, E-Post-M, and L-Post-M women. BRA of mononuclear cell supernatant was assessed using long-bone resorbing assays. Results are expressed as the patient/control ratio of calcium release during bone culture. Pyridinoline excretion was measured in spot urine sample using high-performance liquid chromatography. *, Different from Pre-M, P < 0.05.

The levels of three cytokines were highly correlated to each others (P < 0.01). Table 1 shows cytokine levels measured in culture supernatants after 48 h. IL-1 levels were significantly increased in E-Post-M (compared with the Pre-M group), whereas levels were reduced in L-Post-M women. In contrast, there was no difference in IL-6 release in any of the three groups, although there was an insignificant trend towards increased levels of TNF- in the L-Post-M women. Moreover, no relationship was found between age and the three cytokines levels in the supernatants, and there was no relationship between the cytokines levels in the supernatants and pyridinoline excretion.

BRA of monocyte supernatants was significantly higher in E-Post-M (compared with Pre-M) women (1.20 ± 0.10 vs. 0.73 ± 0.10, P < 0.05). The same increase was observed in L-Post-M women (1.15 ± 0.20 vs. 0.73 ± 0.10, P < 0.05). However, no difference was observed between the E-Post-M and L-Post-M women (Fig. 1). The BRA of the supernatant of Pre-M women was significantly lower than the BRA from the control bones assessed in the same assay (P < 0.01). BRA of E-Post-M and L-Post-M subjects was insignificantly different from that of control bones.

Relationship between cytokine levels and BRA

BRA of mononuclear cell supernatant was positively correlated to pyridinoline excretion (r = 0.32, P < 0.01). The same relationship was observed with levels of IL-1, IL-6, and TNF- (r = 0.37, r = 0.39, and r = 0.50, respectively, all P < 0.01; Fig. 2). These relationships were independent of age.

View larger version (18K): [in this window] [in a new window]   Figure 2. BRA and cytokine production of mononuclear cell culture supernatants. Mononuclear cells were cultured for 48 h in the presence of 20% autologous plasma. BRA of mononuclear cell supernatant was assessed using long-bone resorbing assays. Cytokine levels were measured as described in Subjects and Methods. Data were linearized by log transformation. Positive correlations were observed between BRA and the levels of the three cytokines. •, Pre-M women; , E-Post-M; , L-Post-M.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Many studies in rat and mice models have provided evidence for the role of cytokines in ovariectomy-induced bone loss. Some reports have also shown involvement of bone-resorbing cytokines in humans. Production of IL-1 and TNF- from peripheral monocyte cultures is increased in postmenopausal and osteoporotic women, compared with Pre-M women (10, 11). IL-1 production increases in the years immediately after menopause and is correlated to the rate of bone turn-over, as assessed by histomorphometry. In the present study, we found that IL-1 secretion was significantly higher in the monocyte culture supernatant isolated from E-Post-M, and it decreased in L-Post-M, compared with the Pre-M group. This may indicate a particular higher monocyte secretion, compared with monocyte in the early years after menopause. As suggested earlier, IL-1 is one of the underlying factors promoting bone resorption in situations of early estrogen deficiency (4). We observed no statistically significant change in IL-6 and TNF- secretion in any study group, although there was a trend towards increased TNF- levels in the elderly subjects. However, we found a positive relationship between BRA and supernatant levels of the three cytokines. Indeed, because they were closely correlated to each other, these cytokines (and some untested others) might have synergistic actions that contribute to bone resorption (15). Therefore, we used bone resorbing assays to reflect the biological activity of the mononuclear cell culture media. Using this method, we have previously shown that BRA of mononuclear cell supernatants is increased in E-Post-M (compared with Pre-M) women. This activity was reduced when women were receiving hormone replacement therapy (13). In the present study, we confirm these findings and demonstrate that this activity remains increased more than 10 yr after menopause. These data suggest that a persistent monocyte activation, which may be responsible for the bone resorption, and may be for bone loss. BRA was correlated to the levels of three cytokines. These results confirm our previous published findings (13), where, using specific antibodies, we showed that IL-1 and TNF- were involved in bone resorption. Although multiple regression analysis of the present study indicated that this activity was mainly related to TNF-, this finding does not exclude effects of the other cytokines. Moreover, it is noteworthy that BRA of supernatants from Pre-M was low (mean ratio < 1) and was significantly lower than that of control bones, suggesting the presence of inhibitory factors in the supernatant. It may be hypothesized that these cells produce inhibitors of bone resorption in estrogen-dependent women, which is abolished in estrogen-deficiency. Furthermore, imbalance between increased production of resorbing cytokines and decreased production of inhibitors could contribute to supernatant BRA.

Bone loss is accelerated at the time of menopause. Several studies suggest that bone loss stops, and that bone mass even increases, 10 yr after menopause (16, 17). These studies may have been biased by their cross-sectional design and the increased prevalence of osteophytosis with age. Moreover, most studies excluded osteoporotic patients, who represent a significant percentage of the elderly population; therefore, the rate of bone loss may have been underestimated. More recent studies show that lumbar spine and femoral neck bone mass decreases with age (17, 18, 19). Moreover, biochemical markers of bone turn-over remain elevated long after menopause (20, 21). Thus, urinary pyridinoline levels are increased in normal elderly women, suggesting persistently high bone resorption (2, 3). Our data are in agreement, because deoxypyridinoline excretion rapidly increased in the E-Post-M subjects and remained at the same level more than 10 yr later. Moreover, the deoxypyridinoline excretion assessed at tissue level paralleled the BRA of mononuclear cell supernatants, indicating that high bone resorption in the elderly may relate to mononuclear cell activation.

Increased levels of PTH may be one of the factors contributing to bone resorption in elderly women. Given that IL-6 could mediate the effects of PTH (22), we determined whether increased PTH secretion could explained the BRA. In fact, the levels of PTH are comparable in Pre-M (as well as E-Post-M and L-Post-M) women. Thus, bone loss in this elderly population is not associated with increased PTH levels. The contribution of IL-6 to bone resorption in estrogen deficiency may be independent of changes in PTH levels.

There is now much evidence that cytokines produced by stromal cells play a major role in the regulation of local osteoclastogenesis and bone loss (5). However, in a previous study, we have shown that the level of cytokine released by peripheral monocytes is highly correlated to the level released by bone marrow monocytes (23). These previous results indicate that the production of peripheral monocytes reflects, in some way, the production of local monocytes. The mechanism of estrogen is still poorly understood, and it probably involves transcriptional factors such as NF-KB. In contrast, the mechanism of monocyte activation after estrogen withdrawal remains unclear. The action of estrogen is indirect, because no response element has been shown in any promoter of those three cytokines. The effect, however, may also involve the regulation of transcription factors (24). Whatever the mechanism of the bone-sparing effect of these hormones, monocyte activation still occurs long after estrogen withdrawal. These data indicate common mechanisms implicated in the pathogenesis of bone loss in both early and late postmenopausal periods. Therefore, treatments that have been shown to be effective in the prevention of postmenopausal bone loss could be applied also in the elderly.

Received October 20, 1997.

Accepted January 29, 1998.

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