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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 6 2892-2898
Copyright © 2002 by The Endocrine Society

Other Original Articles

Evidence that the IL-6/IL-6 Soluble Receptor Cytokine System Plays a Role in the Increased Skeletal Sensitivity to PTH in Estrogen-Deficient Women

Urszula S. Masiukiewicz, MaryAnn Mitnick, Barbara I. Gulanski and Karl L. Insogna

Section of Endocrinology, Yale University School of Medicine, New Haven, Connecticut 06520-8020

Address all correspondence and requests for reprints to: Dr. Urszula Masiukiewicz, Yale University School of Medicine, P.O. Box 208020, 333 Cedar Street FMP 109, New Haven, Connecticut 06520-8020. E-mail: .

Abstract

Estrogen-deficient women show increased skeletal sensitivity to the resorbing actions of PTH. The basis for this effect is not known. To examine the influence of estrogen deficiency on PTH-induced proresorptive cytokine production in humans, the response of five young women to a 36-h infusion of (1–34)human PTH (hPTH) was studied. PTH induced significant increases in circulating levels of IL-6 (mean values, T0->T36 h; 2.2->19.2 pg/ml), IL-6 soluble receptor (IL-6sR; 29.8->67.2 ng/ml), urine N-telopeptide of type I collagen (NTX) (38.6->148 nM bone collagen equivalent/mM creatinine) and serum calcium (2.12->2.62 mmol/liter). To examine the impact of hormonal status on this response, PTH infusions were next undertaken in seven estrogen-deficient and seven estrogen-treated postmenopausal women. When compared with estrogen-treated women, and correcting for differences in baseline values, estrogen-deficient women demonstrated an exaggerated increase in circulating levels of IL-6 (5.0->31.7 vs. 3.2->14.4 pg/ml; P = 0.0001) and IL-6sR (49.2->102.1 vs. 37.7->66.7; P = 0.0001). This was accompanied by greater increases in NTX excretion in the estrogen-deficient women (61.2->201.6 vs. 44.8->114.8, E- vs. E+, P = 0.0001). Estrogen deficiency was not associated with augmented PTH-induced increases in colony-stimulating factor-1, IL-1ß, IL-11, or TNF-{alpha}. In a multiple regression model controlling for group, age, years since menopause both IL-6 and IL-6sR were strong predictors of NTX. These data, along with previous animal studies, support the conclusion that the IL-6/IL-6SR cytokine system plays a role in the increased skeletal sensitivity to PTH seen in estrogen-deficient women.

NORMAL BONE REMODELING requires the action of proresorptive factors released either systemically or in the bone microenvironment, that promote osteoclast recruitment, maturation and function. This resorptive phase is coupled to a matched rebuilding of new bone. Under physiologic conditions, PTH is one of the principal hormones regulating bone remodeling and calcium homeostasis. It is currently believed that PTH regulates bone resorption by inducing osteoblasts and/or stromal cells to produce soluble and cell-surface factors that act on mature osteoclasts to increase their resorptive activity and on osteoclast progenitor cells to increase proliferation. Possible mediators of this PTH effect include RANKL, colony-stimulating factor (CSF)-1, IL-11, and IL-6 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

Increasing evidence suggests that IL-6 may be one of the key cytokines mediating the proresorptive effects of PTH. In vitro, PTH induces stromal/osteoblastic cells to produce IL-6 (3, 4, 5, 6, 7, 8, 9) and PTH-induced bone resorption can be attenuated in a rat osteoblast/osteoclast coculture system by using a neutralizing antibody to the IL-6 receptor (11). Neutralizing IL-6 in vivo, in mice, significantly diminishes the resorptive response to PTH. Consistent with this latter finding, mice with targeted deletion of the IL-6 gene have a markedly attenuated increase in PTH-induced indices of bone resorption during a 5-d infusion of the hormone (12). In addition, IL-6 knockout mice have secondary hyperparathyroidism despite reduced biochemical markers of resorption (13). In humans, circulating levels of IL-6 and its receptor are elevated in states of PTH excess, correlate strongly with markers of bone resorption and revert to normal following the correction of hyperparathyroidism (14, 15).

The ability of exogenously administered estrogen to prevent the accelerated skeletal turnover and bone loss that occurs at menopause is well established. The mechanism(s) by which estrogen exerts this effect has been the focus of continued investigative interest. It has been reported that IL-6 may be important in mediating bone loss induced by estrogen deficiency in so far as neutralizing IL-6 in vivo prevents the increase in osteoclastogenesis seen in estrogen-deficient mice and IL-6 knockout mice do not lose trabecular bone following ovariectomy (16, 17).

We have recently reported that PTH-induced IL-6 production is augmented following estrogen withdrawal in vitro and in vivo in mice (18). In humans, it has been shown that estrogen-deficient women demonstrate increased skeletal sensitivity to the resorbing action of PTH (19). We therefore explored the possibility that augmented PTH-induced production of IL-6 and/or IL-6sR mediates the enhanced resorptive response to PTH seen in estrogen-deficient women.

Materials and Methods

Study subjects

Five young, healthy premenopausal and 14 postmenopausal women (7 estrogen deficient and 7 estrogen treated) were recruited for study from the Yale-New Haven community. Exclusion criteria were smoking, use of medications known to affect bone metabolism, presence of inflammatory disease, or any disease known to affect bone metabolism. Premenopausal women were excluded if they were taking oral contraceptives. All premenopausal women were studied in the early follicular phase of their menstrual cycle and had a negative urine pregnancy test before being studied. None of the postmenopausal women were taking antiresorptive medications other then estrogen before study. All postmenopausal women taking estrogen had been on a stable HRT regimen for at least 6 months before study. Among postmenopausal women taking estrogen, six were taking 0.625 mg/d of conjugated equine estrogen orally, and one was taking 0.05 mg/d of transdermal estrogen. Six patients were taking cyclical progesterone, and one was not on progesterone because she had had a hysterectomy. There was no significant difference in the response to PTH in the one woman who was not taking progesterone compared with the remaining women. Before study, each patient underwent a medical history, physical examination, electrocardiogram, and screening blood tests that included serum calcium, PTH, and a hemogram. The study was approved by the Yale University Human Investigation Committee, and all participants gave written informed consent.

Study protocol

Human (1–34)PTH was synthesized in the William Keck Peptide Synthesis Facility at Yale University, packaged by the Yale New Haven Hospital Investigational Pharmacy and subjected to sterility and pyrogenicity testing before use. It was delivered using an iv pump at a constant rate of 12 pmol/kg·h for 36 h. The dose of PTH was based in part on doses used in prior PTH infusion studies (19) examining the effect of PTH infusion on bone resorption markers in estrogen-treated and estrogen-deficient women and in part on pilot studies we conducted during which PTH was infused for 36, 48, and 72 h. The shortest duration of infusion that resulted in mild hypercalcemia and at least 2-fold increase in bone resorption markers was chosen for the current study. This resulted in a dose of hormone slightly higher than that used in prior studies (19).

Study subjects were admitted to the General Clinical Research Center at 0700 h. Upon admission they had a focused history and physical examination and had two iv lines placed, one for infusion of PTH and one for blood drawing. Study subjects consumed a diet designed to contain 800 mg/d calcium, 2.3 g/d sodium, and 1 g/kg body weight protein/d during their admission. Blood and urine collections were obtained at baseline, at 1 h after initiation of infusion, every 8 h thereafter as well as at the end and 1 h after completion of infusion. Blood was analyzed for serum calcium, phosphate, creatinine, PTH (mid-molecule and 1–34), plasma cAMP, 1,25-(OH)2vitamin D, serum type I collagen carboxyterminal telopeptide (ICTP) and cytokines (IL-6, IL-6sR, IL-1ß, IL-11, TNF{alpha}, CSF-1). Urinary measurements included cAMP, DPD (deoxypyridinoline), and NTX (N-telopeptide of type I collagen).

Measurements

Serum biochemistries and calcitropic hormones. Serum calcium was measured using a Model 2380 atomic absorptiometer (Perkin-Elmer Corp., Norwalk, CT). Mid-molecule PTH was measured as previously described (20). Circulating levels of 1–34 PTH were measured by double antibody RIA using a commercially available kit (Peninsula Laboratories, Inc., Belmont, CA). The lower limit of detection in this assay is 0.30 pg/100 µl. The intraassay and interassay CVs are 4.8% and 6.9% respectively. Nephrogenous cAMP (NcAMP) was calculated as previously described (21). Plasma 1,25(OH)2D was measured using a competitive protein binding assay as previously described (22).

Cytokines. Human IL-6, IL-6sR, IL-1ß, IL-11, TNF-{alpha} and CSF-1 were measured using highly sensitive solid-phase ELISA kits (R&D Systems, Minneapolis, MN). The lower limits of detection for these assays in our laboratory are 0.1 pg/ml for IL-6, 0.14 ng/ml for IL-6sR, 0.1 pg/ml for IL-1ß, 1.9 pg/ml for IL-11, 0.18 pg/ml for TNF-{alpha} and 7.2 pg/ml for CSF-1. The respective intraassay and interassay CVs are; 3.3% and 3.6% for IL-6, 2.3% and 4.7% for IL-6sR, 6.4% and 7.1% for IL-1ß, 3.9% and 5.1% for IL-11, 5.6% and 7.5% for TNF-{alpha}, 3.1% and 4.3% for CSF-1.

Markers of bone resorption. Urine DPD and serum ICTP were measured as previously described (15). Urine NTX was measured by ELISA using a commercially available kit (Ostex International, Inc., Seattle, WA). The sensitivity of the NTX assay is 10 nM BCE (bone collagen equivalents) and the intraassay and interassay CVs are 5.4% and 6.6%, respectively.

Statistical analyses

Baseline characteristics including demographics, measures of calcium metabolism, serum cytokines levels and urinary markers of bone resorption were compared between estrogen-treated and estrogen-deficient groups using t tests. Changes in serum IL-6 and IL-6sR and urinary markers of bone resorption during PTH treatment in premenopausal women (Figs. 1Go and 2Go) were analyzed by repeated measures one-way ANOVA. Paired t test was used to determine when the fold increase for each parameter was first statistically significant from the basal value (Fig. 3Go). Changes in cytokines and urinary markers of bone resorption over time in postmenopausal women in response to PTH infusion were compared between groups using a mixed model analysis of repeated measures with log-transformed data. Pearson correlation coefficients were used to characterize associations between urinary markers of bone resorption, serum cytokine levels, age, and body mass index. Multiple linear regression using backward selection for the dependent variable, NTX, was performed adjusting for potential confounders. For multivariate analyses, change from baseline values were used for urinary markers of bone resorption and cytokine measurements. All analyses were conducted using SAS version 6.12 (SAS Institute, Inc., Cary, NC). All data are presented as the mean ± SEM.



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  		Figure 1. Increases in circulating levels of IL-6 (A) and IL-6sR (B) in young healthy women during PTH infusions. Five young healthy women were continuously infused with (1–34)hPTH at 12 pmol/kg·h. IL-6 and IL-6sR were measured at time 0, 1, 9, 15, 25, 33, and 36 h. P < 0.0001 for effect of PTH treatment on IL-6 and IL-6sR by repeated measures one-way ANOVA.

 


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  		Figure 2. Changes in NTX excretion during PTH infusions in young healthy women. Five young healthy women were continuously infused with (1–34)hPTH at 12 pmol/kg·h. NTX was measured at time 0, 1, 9, 15, 25, 33, and 36 h. P < 0.0001 for effect of PTH treatment on NTX by repeated measures one-way ANOVA.

 


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  		Figure 3. Fold increase (compared with baseline values) in circulating levels of IL-6 (stippled bar), serum IL-6sR (gray bar) and urine NTX (open bar) during PTH infusions in young healthy women. P values for IL-6 vs. NTX were 0.1 at 1 h and < 0.01 for 9, 15, 25, 33, and 36 h. P values for IL-6sR vs. NTX were 0.3 at 1 h, 0.4 at 9 h and < 0.01 at 15, 25, 33, and 36 h.

 
Results

Effect of PTH infusion on circulating levels of IL-6 and IL-6sR and markers of bone resorption in premenopausal women

Because there are no data in the literature describing the effect of PTH infusion on circulating levels of IL-6 and IL-6sR in normal subjects, the response to PTH was initially studied in 5 healthy, premenopausal women (mean age 27.0 ± 9.2, mean body mass index 26.1 ± 1.3). In these studies, (1–34)hPTH was infused for 36 h as described above.

As anticipated, PTH infusion induced a significant increase in NcAMP excretion and in circulating levels of 1,25-(OH)2 D with the mean value for NcAMP rising from 1.4 ± 0.1 at baseline to 5.3 ± 0.2 nmol/100 ml glomerular filtration rate (GFR) at the end of the infusion (a 279 ± 12% increase) and the mean value for 1,25-(OH)2 D rising from 89.5 ± 8.4 to 212.1 ± 17.3 pmol/liter (a 136 ± 8% increase). PTH infusion was also accompanied by a progressive increase in serum calcium levels such that at the end of infusion the mean value for serum calcium was in the mildly hypercalcemic range (2.12 ± 0.01 -> 2.62 ± 0.05 mmol/liter).

As shown in Fig. 1AGo, PTH infusion resulted in a rapid and significant increase in circulating levels of IL-6 with means values rising from 2.2 ± 0.3 pg/ml at baseline to 19.1 ± 1.4 pg/ml at the end of infusion (P < 0.001; a 765 ± 87% increase). Similarly, circulating levels of IL-6sR rose significantly from 29.8 ± 1.9 ng/ml to 67.2 ± 1.9 ng/ml (P < 0.001; Fig. 1BGo; a 128 ± 12% increase). As was previously reported (19), PTH-infusion was associated with a rise in bone resorption markers, with a mean increase in urinary NTX from 38.6 ± 1.8 to 148 ± 19.0 nM BCE/mM creatinine (P < 0.001, Fig. 2Go; a 284 ± 48% increase). The rise in bone resorption markers correlated strongly with the rise in circulating levels of IL-6 (r = 0.99) and IL-6sR (r = 0.99). Similar responses were seen in two other bone resorption markers, urinary DPD and serum ICTP (data not shown).

When expressed as fold elevation and compared with baseline values, IL-6, IL-6sR, and NTX rose significantly by h 1 of the infusion and remained significantly elevated above baseline value throughout the study. However, at every time point the fold increase in IL-6 was greater than that for NTX and, beginning with the 9-h time point, this difference became statistically significant (2.9 for IL-6 vs. 1.2 for NTX, P = 0.0007) and remained so for the duration of the infusion (Fig. 3Go).

Effect of PTH infusion on circulating cytokine levels in postmenopausal women

Having established that PTH infusion induces an acute rise in circulating levels of IL-6 and IL-6sR, postmenopausal women were next studied to determine whether estrogen modulates this effect in humans in vivo. Fourteen women, 7 who were estrogen-deficient (E-) and 7 taking hormone replacement therapy (E+) were infused with PTH as described above. The baseline characteristics of the two postmenopausal study groups are summarized in Table 1Go. Estrogen-deficient women were older (mean age: 63 ± 6, E- vs. 55 ± 4, E+), had higher mean baseline levels of IL-6 (5.0 ± 1.9 pg/ml, E- vs. 3.2 ± 0.5 pg/ml, E+) and higher basal rates of bone resorption as assessed by NTX (61.2 ± 1.7, E- vs. 44.8 ± 2.1, E+ nM BCE/mM creatinine).


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  		Table 1. Baseline parameters for the two groups of postmenopausal women

 
There was no effect of estrogen on the renal responsiveness to infused PTH as evidenced by a comparable percent increase in NcAMP and 1,25-(OH)2 D in the two study groups. Thus, the mean increase in NcAMP excretion in the E+ women was 164 ± 9% (1.8 ± 0.02-> 4.8 ± 0.1 nmol/100 ml GFR) vs. 171 ± 13% (1.6 ± 0.1-> 4.4 ± 0.1 nmol/100 ml GFR) in E+ women (P = 0.27) and the mean increase in 1,25-(OH)2 D was 106 ± 9% (114 ± 5.7-> 234.8 ± 9.9 pmol/liter) vs. 90 ± 10% (114 ± 3.9-> 214 ± 10.3 pmol/liter) respectively (P = 0.28). Consistent with the equipotent response to PTH, changes in circulating levels of immunoreactive (1–34)hPTH levels were nearly identical in both groups throughout the infusion with (1–34)hPTH levels rising in E- women from 13.0 ± 1.0 at baseline to 89.3 ± 5.3 pg/100 µl at the end of the infusion and in E+ women from 13.4 ± 0.9 to 81.4 ± 2.2 (P = 0.1). Despite the identical administered dose of PTH and comparable baseline values of serum calcium (2.3 ± 0.02 E- vs. 2.2 ± 0.05 E+ mmol/liter P = 0.25) the increase in serum calcium was greater in the estrogen-deficient women such that at the end of infusion there was a nearly significant difference in mean serum calcium levels between the groups (2.7 ± 0.1 E- vs. 2.4 ± 0.1 mmol/liter in E+, P = 0.06).

As in the premenopausal group, PTH infusion in the postmenopausal women resulted in a rapid and significant rise in circulating levels of IL-6 and IL-6sR (Fig. 4Go). The increases in circulating levels of both IL-6 and IL-6sR were, however, significantly greater in estrogen-deficient women compared with estrogen-treated women. The mean increment in IL-6 was 27.1 ± 2.3 pg/ml in the E- group (5.0 ± 1.9->31.7 ± 5.1) vs. 11.2 ± 1.0 pg/ml in the E+ group (3.2 ± 0.5->14.4 ± 3.1) (P = 0.0001), a 532 ± 28% vs. 349 ± 14% increase respectively, P = 0.0005. The corresponding increments in circulating levels of IL-6sR were 52.9 ± 3.3 ng/ml (49.2 ± 1.4->102.1 ± 2.6) vs. 29.0 ± 1.6 ng/ml (37.7 ± 1.6->66.7 ± 2.3) (P = 0.0001) in E- and E+ women, a 107 ± 8% vs. 77 ± 4% increase, respectively, P = 0.0005.



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  		Figure 4. PTH-induced increases in circulating levels of IL-6 (A) and IL-6sR (B) in postmenopausal women. Fourteen postmenopausal women (7 estrogen-treated and 7 estrogen-deficient) were continuously infused with (1–34)hPTH at 12 pmol/kg·h. IL-6 and IL-6sR were measured at time 0, 1, 9, 15, 25, 33, and 36 h. P = 0.0001 for group differences over time for both IL-6 and IL-6sR.

 
Adjusting for baseline differences in IL-6 and age the increase in circulating levels of IL-6 over time was significantly greater in estrogen-deficient women (P = 0.0001), indicating that PTH-induced IL-6 production is augmented in the estrogen-deficient state in women. Using the same model and again correcting for baseline differences in age and IL-6sR a similar effect was observed for IL-6sR (P = 0.0001). Because the PTH-induced changes in IL-6 and IL-6sR at the 36 h time-point diverge sharply in the two groups, these analyses were repeated with this final time point eliminated. Even with this last time point eliminated the difference in PTH induced IL-6 and IL-6sR in the two groups remained significant (P = 0.0004 for IL-6 and P = 0.0035 for IL-6sR).

By contrast, there was no difference between groups in the PTH-induced change in circulating levels of TNF-{alpha}, and IL-1ß, with curves for estrogen-deficient and estrogen-treated women almost superimposable (Fig. 5AGo). In response to PTH, there was a significant decline over time in circulating levels of IL-11 in both groups. The observed decline was, however, significantly more pronounced in estrogen-deficient women (P = 0.001; Fig. 5AGo), which is in agreement with our recently reported observation that IL-6 negatively regulates IL-11 production (23). That is, in estrogen-deficient women PTH induced a greater increase in IL-6 resulting in the significant decline in IL-11 in these women. PTH infusion resulted in an increase in circulating levels of CSF-1 in both groups. Contrary to what might be predicted, however, the PTH-induced increase was slightly greater in estrogen-treated women (E+ vs. E-, 566 ± 48-> 902 ± 140 vs. 561 ± 64->693 ± 57, P = 0.06) (Fig. 5BGo).



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  		Figure 5. PTH-induced changes in circulating levels of IL-11, TNF-{alpha}, IL-1ß (A), and CSF-1 (B) in postmenopausal women. Fourteen postmenopausal women (7 estrogen treated and 7 estrogen deficient) were continuously infused with (1–34)hPTH at 12 pmol/kg·h. Cytokines were measured at time 0, 1, 9, 15, 25, 33, and 36 h. NS indicates no statistically significant differences between groups (E + vs. E-) over time for IL-1ß and TNF{alpha}. *, P = 0.001 for IL-11; P = 0.06 for CSF-1.

 
The differential IL-6 and IL-6sR response to PTH correlates with the increased resorptive response to PTH in postmenopausal women

PTH infusion resulted in a rise in bone resorption markers in both groups. The estrogen-deficient women demonstrated a significantly greater rise in markers of bone resorption with a mean increase in urinary NTX of 140.4 nM BCE/mM creatinine (61.2 ± 1.7->201.6 ± 12.1) vs. 70.0 nM BCE/mM creatinine in E+ women (44.8 ± 2.1->114.8 ± 5.0), a 233 ± 9% vs. 156 ± 19% increase respectively (P = 0.01, Fig. 6Go). This difference remained significant after correction for the higher baseline NTX excretion in the E- group.



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  		Figure 6. PTH-induced increases in urinary NTX in the two study groups of postmenopausal women. Fourteen postmenopausal women (7 estrogen treated and 7 estrogen deficient) were continuously infused with (1–34)hPTH at 12 pmol/kg·h. NTX was measured at time 0, 1, 9, 15, 25, 33, and 36 h. P = 0.0001 for group differences over time.

 
In an effort to determine which factor was the most significant predictor of NTX, a multiple regression model was used controlling for group status (E+ vs. E-), age, years since menopause and IL-6 with urinary NTX as the dependent variable. CSF-1 and IL-11 were not included in the analysis because they were regulated in direction opposite to that of IL-6/IL-6sR axis. Serum IL-1 and TNF were not included in the model because the response of these two cytokines to PTH was not influenced by estrogen status. Because of colinearity with IL-6, IL-6sR was included in a separate model in which IL-6sR was substituted for IL-6. In the separate models serum IL-6 and IL-6sR were strong predictors of NTX (P = 0.03 for IL-6, P = 0.022 for IL-6sR).

Discussion

The principal finding of this study is that increased skeletal sensitivity to PTH in the estrogen-deficient women is accompanied by significantly greater production of IL-6 and IL-6 soluble receptor but not of the other cytokines measured. This is the first study to demonstrate the effects of acute PTH exposure on circulating levels of cytokines in humans. Our prior studies have demonstrated that production of IL-6 and its receptor are increased in states of PTH excess and that this increase is paralleled by an increase in bone resorption both in animals and in humans (12, 14, 15). Studies in animals support the conclusion that IL-6 is a proximate mediator of this resorptive response.

In the current study, we demonstrate that circulating levels of IL-6 and IL-6sR rise significantly within 9 h of beginning an infusion of PTH in women. The circulating levels of PTH at the end of the infusion were comparable to those seen in patients with moderate primary hyperparathyroidism. The observed acute rise in circulating levels of IL-6 is in agreement with previously published data showing that IL-6 is an immediate early gene and that its message is up-regulated within 30 min of stimulation with PTH (6). The rise in IL-6 and its receptor in our study correlated with the increase in bone resorption markers in both premenopausal and postmenopausal women.

Levels of IL-6, IL-6sR, and NTX all were increased after the first hour of the infusion although the fold increase was greatest in IL-6. In animals neutralizing IL-6 prevents the PTH-induced rise in bone resorption. We cannot be certain if the same causal relationship obtains in humans but our findings are consistent with the hypothesis that the IL-6/IL-6sR system drives the increase in bone resorption that in turn eventually contributes to the increase in serum calcium. We cannot exclude an absorptive component to the rise in serum calcium due to increased intestinal calcium absorption mediated by the PTH-induced rise in 1,25(OH)2D. However, the higher levels of serum calcium seen at the end of the infusion in the postmenopausal estrogen-deficient women despite an equivalent increase in 1,25(OH)2D in both groups, suggests that the rise in serum calcium is, at least in part, related to activation of bone resorption.

The IL-6/IL-6sR cytokine system remains a strong candidate mediator for the increased skeletal turnover associated with estrogen deficiency. It is well established that, in vitro, estrogen negatively regulates IL-6 gene transcription via a nongenomic mechanism (24, 25, 26). Studies in vivo, in mice, have demonstrated that neutralization of IL-6 prevents the increase in osteoclastogenesis seen in estrogen-deficient mice and IL-6 knockout mice do not lose trabecular bone following ovariectomy (16, 17). The IL-6sR is a critical component of this effector system because it has been repeatedly shown in vitro that IL-6sR augments the effects of IL-6 and, in the case of in vitro osteoclastogenesis assays, appears to be required for the osteoglastogenic effects of IL-6 (27).

Relatively few studies have examined circulating levels of IL-6 in humans (28, 29, 30, 31, 32) and the data examining the effect of estrogen deficiency on circulating levels of IL-6 in euparathyroid women are inconsistent. McKane et al. (30) in a study of 80 nonosteoporotic women, ages 24–87, found no correlation between serum IL-6 levels and menopausal status, serum estrogen concentration, or markers of bone resorption. By contrast, a recent study of 302 postmenopausal women, found that circulating levels of IL-6 were significantly higher in estrogen-deficient women compared with estrogen-treated women (P = 0.017) (29). In addition, findings in a recent prospective observational study by Scheidt-Nave et al. (32) are consistent with the conclusion that IL-6 plays an important role in bone loss in the immediate postmenopausal period. In this prospective study of 89 women, serum IL-6 was by far the strongest determinant of femoral bone loss among women within the first decade after menopause and explained 34% of the variability in absolute bone loss during this time period. Other investigators (31) have found no differences in circulating levels of IL-6 in osteoporotic vs. nonosteoporotic estrogen-deficient women when studied a mean of 16 yr after menopause, suggesting that IL-6 dysregulation plays a role predominately in the early postmenopausal period.

Both in hyperparathyroid and euparathyroid women, accumulating evidence suggests a possible relationship between PTH and accelerated bone loss following estrogen withdrawal. Gray et al. (33, 34) reported that estrogen-deficient women with primary hyperparathyroidism had accelerated rates of bone loss compared with euparathyroid controls and that this bone loss was attenuated by hormone replacement therapy. Khosla et al. (35) found that postmenopausal estrogen-deficient women demonstrate an age- dependent rise in PTH that is accompanied by increases in markers of bone resorption. Estrogen-treated women do not demonstrate either of these changes, suggesting that one mechanism by which estrogen prevents the postmenopausal increase in bone resorption, is by preventing the age-dependent rise in PTH. Finally, as noted, studies have shown that estrogen-deficient women show an increased sensitivity to the resorptive effects of infused PTH. This increased sensitivity can be corrected by the administration of estrogen (19).

Our recent findings (18), together with the data reported here, suggest that increased release of the proresroptive cytokines IL-6/IL-6sR may be one mechanism contributing to increased skeletal sensitivity to PTH in the estrogen-deficient state. We have observed that, in vitro and in vivo, PTH-induced IL-6 production is augmented in the estrogen-deficient state. In vivo, this correlates with an enhanced resorptive response to PTH that can be abrogated by treatment with estrogen. In estrogen-deficient postmenopausal women this augmented release of IL-6/IL-6sR highly correlates with the difference in the resorptive response to PTH when compared with the findings in estrogen-treated women.

By contrast to the pronounced effect of estrogen on PTH-induced changes in circulating IL-6 and IL-6sR, estrogen status did not modulate the effect of PTH on serum levels of IL-1ß, or TNF-{alpha}, whereas estrogen deficiency attenuated the effect of PTH on levels of CSF-1 and was associated with significantly greater suppression of IL-11.

The tissue source(s) of circulating IL-6 and its receptor produced in response to PTH remain unknown. Our in vitro data suggest that bone may be one source (18). However, we have recently reported that PTH also increases IL-6 and IL-6sR production by isolated perfused rat livers (36) and that estrogen modulates this process (37). This suggests that estrogen withdrawal also sensitizes the liver to PTH which may contribute to the greater increase in circulating levels of IL-6 and IL-6sR following PTH exposure in estrogen deficiency.

In summary, the current study, together with earlier experiments in mice and patients with disordered parathyroid function (15), support the conclusion that IL-6 and its soluble receptor are important mediators of PTH-induced bone resorption in vivo and that the increased skeletal sensitivity to PTH in the estrogen-deficient state may be, in part, explained by a greater production of IL-6 and its soluble receptor. Our findings that estrogen treatment restrains the PTH-induced increase in IL-6 and IL-6sR and bone resorption but does not change the response to PTH for other proresorptive cytokines (or changes them in the opposite direction e.g. IL-11 and CSF-1), further bolsters the conclusion that IL-6/IL-6sR cytokine system plays a key role in this process. Because abnormalities in PTH function are common in women with osteoporosis, these findings may be relevant to the pathogenesis of that disease.

Acknowledgments

We gratefully acknowledge the support of Yale’s GCRC (NCRR Grant RR00125) and of the Yale-New Haven Hospital Investigational Pharmacy staff. We thank Drs. Gene Fish (Informatics Core GCRC) and Heather Allore (Yale Pepper Center) for providing helpful statistical consultation.

Footnotes

This work was supported by the NIH (KO8DK02596, to U.S.M.; AG15345, to K.L.I.), the Endocrine Fellows Foundation (to U.S.M.) the Yale Claude D. Pepper Older Americans Independence Center (to K.L.I.) and the Yale-Hartford Foundation Center for Excellence in Geriatrics (to U.S.M.).

Abbreviations: BCE, Bone collagen equivalents; CSF, colony-stimulating factor; DPD, deoxypyridinoline; GFR, glomerular filtration rate; hPTH, human PTH; ICTP, serum type I collagen carboxyterminal telopeptide; NcAMP, nephrogenous cAMP; NTX, N-telopeptide of type I collagen.

Received July 17, 2001.

Accepted March 5, 2002.

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