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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 1 32-38
Copyright © 1997 by The Endocrine Society

Clinical Studies

Low Bone Mineral Density and Peripheral Blood Monocyte Activation Profile in Calcium Stone Formers with Idiopathic Hypercalciuria1

A. Ghazali, V. Fuentès, C. Desaint, P. Bataille, A. Westeel, M. Brazier, L. Prin and A. Fournier

Department of Nephrology-Internal Medicine (A.G., P.B., A.F.), Laboratory of Immunology (V.F., C.D., P.L.), Radiology Department (A.W.), Faculty of Pharmacy (M.B.), Centre Hospitalier Universitaire Amiens-Hôpital Sud, 80054 Amiens Cedex 1, France

Address all correspondence and requests for reprints to: Professor A. Fournier, Department of Nephrology-Internal Medicine, Centre Hospitalier Universitaire Amiens-Hôpital Sud, Avenue Laennec-Salouel, 80054 Amiens Cedex 1, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Calcium stone formers (CaSF) with idiopathic hypercalciuria (IH) have been shown to have decreased bone mineral density (BMD). The mechanism of their bone loss remains obscure. Monokines like interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-{alpha} (TNF-{alpha}), and granulocyte macrophage stimulating factor (GM-CSF) are involved in bone remodeling, but only IL-1 excess has been incriminated in the bone loss of CaSF with IH. Therefore, to more precisely delineate the role of monocyte activation in the pathogenesis of bone loss in these patients, we studied the production of IL-1ß, IL-6, TNF-{alpha}, and GM-CSF by unstimulated or lipopolysaccharide (LPS)-stimulated cultured peripheral blood monocytes in 15 CaSF with IH, in 10 CaSF with dietary calcium-dependent hypercalciuria (DH), and in 10 healthy controls (C). Cytokines were measured in the culture medium by sensitive enzyme-linked immunosorbent assay and vertebral BMD by single energy computed tomography. The decrease of vertebral BMD in IH compared with DH, was confirmed (Z score: -1.2 ± 0.2 vs. -0.5 ± 0.2; P = 0.04; Mann-Whitney). In the supernatant of unstimulated peripheral blood monocytes, IL-1ß and TNF-{alpha} levels were higher in IH than in C (respectively, 40 ± 21 vs. 7 ± 1 pg/mL, P = 0.008 and 236 ± 136 vs. 39 ± 23 pg/mL, P = 0.03); those of GM-CSF were greater in IH than in DH and C (respectively, 52 ± 27 vs. 6 ± 2, P = 0.04 and 6 ± 2 pg/mL, P = 0.01) and those of IL-6 were not significantly different among the groups. After in vitro stimulation by LPS (10 µg/mL), the levels of the various monokines were not significantly different. In IH patients, the post-LPS levels of IL-6 were negatively correlated to vertebral BMD (n = 15, Z = -1.97, P = 0.04; Spearman), whereas those of GM-CSF were positively related to vertebral BMD (n = 15, Z = 2.01, P = 0.04).

In this study, calcium stone formers with IH have bone mineral decrease and a particular profile of peripheral blood monocytes activation. This latter is characterized by a spontaneously increased synthesis of IL-1ß, TNF-{alpha}, and GM-CSF. Furthermore, post-LPS levels of IL-6 and GM-CSF are correlated with vertebral BMD. These results suggest that monocyte activation may be involved in the bone loss of calcium stone formers with IH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN IDIOPATHIC calcium stone formers (CaSF), hypercalciuria is the most frequent risk factor for kidney stone disease (1). Dietary calcium-dependent hypercalciuria (DH) is caused by an excessive intake of calcium, whereas idiopathic hypercalciuria (IH) is defined by the persistence of a hypercalciuria in spite of normal or restricted calcium intake. In this case, there is evidence for bone loss because bone mineral density (BMD) has been found decreased (2). Bone disease in IH patients also has been documented by static and dynamic bone histomorphometry (3). The osteopenia in CaSF may be the consequence of increased bone resorption and/or decreased bone formation. Hydroxyprolinuria, a known marker of bone resorption, is higher in IH than in DH and is correlated with fasting calciuria, suggesting that hypercalciuria in these patients is linked to bone resorption (2). IH patients have mainly normal or low values of plasma parathormone (PTH) (4), suggesting that a PTH-independent mechanism accounts for bone demineralization in these patients. Increased levels of serum calcitriol observed in IH patients (2, 5) are not responsible for bone loss in IH patients because there was a positive correlation between plasma 1.25 (OH)2 vitamin D3 levels and BMD (2). Pacifici et al. (6) have demonstrated an increased production of interleukin-1 (IL-1) by cultured peripheral blood monocytes (PBM) associated with decreased vertebral BMD in patients with fasting hypercalciuria. IL-1 can induce bone resorption by a direct effect through activation of the osteoclasts (7) and through a PG-dependent mechanism (8). In addition, PGs stimulate calcitriol synthesis (9). Thus, in IH the increased bone resorption and/or the decreased bone formation leading to reduction of bone density and the high plasma calcitriol level could result from activation of monocytes and the synthesis of IL-1 (10). Considering that other monokines like tumor necrosis factor-{alpha} (TNF-{alpha}) (11, 12, 13) and IL-6 (14) may have a bone catabolic effect, that granulocyte macrophage stimulating factor (GM-CSF) synthesis is induced by IL-1 and TNF-{alpha} (15, 16), and that this factor modulates the differentiation of osteoclasts (17, 18) and osteoblasts (19), we have measured the synthesis of these cytokines by cultured PBM obtained from patients with IH, from DH patients, and from healthy controls to test whether an abnormal monokine secretion profile could account for the decreased BMD in patients with IH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

Inclusion-exclusion criteria. All calcium stone formers evaluated in our outpatient clinic were considered as potential candidates for the study when they had a history of recurrent calcium renal stones and documentation of a hypercalciuria, i.e. calciuria greater than 0.1 mmol/kg·day while on a calcium-free diet (20). Patients were then excluded when they had known diseases that could affect calcium excretion, bone remodeling, and/or monocyte function (hyperparathyroidism, hyperthyroidism, hypercorticism, granulomatosis, renal tubular acidosis, neoplasm, malignant hemopathy... ), or when they had received a treatment with estrogen, progesterone, corticosteroids, anticonvulsants, sodium fluoride, bisphosphonate, calcitonin, vitamin D and analogs, PG analogs, antiacids, and heparins. Patients treated by thiazides diuretics were included, however, after at least 6 weeks of treatment discontinuation.

According to these criteria and after their informed consent, 25 patients were included in the study.

Protocol. Two 24-h urine collections were obtained from all patients and controls while on a calcium-unrestricted diet but without gelatin. Then, for 7 days, the patients were put on a calcium-restricted diet of about 400 mg by exclusion of all dairy products, as well as on a moderate sodium intake (no salt on the table). They were asked to collect their 24-h urine on day 5–6 of this diet while gelatin rich food were excluded. Fasting 2-h urine samples (0700–0900 h) also were collected on the morning of day 7 after a 12-h fasting. At 0900 h of this day, while still fasting, their blood was drawn and they were given an oral load of 1 g of elemental calcium (as gluconolactate and carbonate of calcium). Their urine was collected during the following 4 h, and their blood was drawn again at 1300 h.

Classification of the patients according to their calciuria. Secondary hypercalciuria being excluded, a consensus exists to consider 4 mg/kg·day (or 0.1 mmol/kg·day) of calciuria on a free diet as the upper limit of normal (1, 20), whereas on a calcium-restricted diet (400 mg/day by excluding dairy products), this upper limit of normal is 0.07 mmol/kg·day (2, 21).

According to these criteria, DH linked to an exaggerated dietary calcium intake was diagnosed in 10 of the patients included in the study because their hypercalciuria was present on free diet and had disappeared on restricted calcium intake. The other 15 patients had persistent hypercalciuria in spite of calcium-restricted diet and were classified as having IH. The justification for this classification was previously discussed (2, 3).

Controls

Ten age- and sex-matched healthy controls also entered the study after giving their informed consent and were explored according to the same protocol as the calcium stone formers.

Methods

Biochemical assay. Plasma concentrations of creatinine, calcium, phosphate, protein, alkaline phosphatase (autoanalyzer DAX 48, Bayer Diagnostics, Puteaux, France), osteocalcin [RIA (22)], calcidiol [radio-competition assay (23)], calcitriol [RIA (24)], and intact PTH [RIA (25)] were measured. In the 24-h urine collected on free and calcium-restricted diet, creatinine, calcium, phosphate, total hydroxyproline (26), lysyl and hydroxylysylpyridinoline (fluorescence detection after HPLC and acid hydrolysis urine extraction (27)) were measured. On the fasting urines, the same determinations were performed. On the 4-h urines after the calcium load, only calcium, phosphate, and creatinine were measured again. All the urinary parameters were expressed by mmol of urinary creatinine to eliminate the urine sampling error.

Evaluation of BMD. BMD was measured using single-energy (80 kVp) quantitative computed tomography (GE CT/T9800, General Electric Europe, Buc, France). This method can select a volume of pure trabecular bone in the center of a vertebrae. It is a sensitive method for detecting changes in bone mass, because it measures density of bone with high remodeling (28). The coefficient of variation of repeated scans is less than 6% if precise localization and calibration tests are performed (29). The accuracy of this technique is improved also by measurements of several vertebrae and the use of the mean of these measurements (30). In this study, BMD measurements were performed at the lumbar spine (L1-L4). They were expressed for each patient as Z-score, which estimates the number of SD below or above the mean value of age- and sex-matched healthy controls.

Mononuclear cell culture procedure. PBM cells were separated from 50 mL heparinized venous blood from all patients and controls on calcium-restricted diet, by centrifugation on a gradient of Ficoll Paque (TechGen International, Les Ulis, France) as previously described (6). PBM were washed twice and resuspended in RPMI 1640 culture medium plus 3% heat-inactivated FCS, 20 mmol/L of HEPES, and 50 µg/mL of gentamycin at a cell concentration of 3 x 106/mL. Endotoxin contamination of medium was evaluated by limulus amoebocyte test (E-TOXATE, Sigma, St. Quentin Fallavier, France) and was below 0.05 U/mL. This quantity was insufficient to increase TNF-{alpha} secretion by PBM (data not shown). Cells were allowed to adhere to plastic in 24-well tissue culture plates for 2 h at 37 C in humidified air with 5% CO2. The nonadherent cells were removed by five washings with RPMI. Adherent cells contained 95 ± 2% monocytes as defined by specific enzyme {alpha}-naphtyl acetate esterase (reference 91-A, Sigma). Adherent cells were cultured for 16 h with 1 mL of medium (RPMI, HEPES, FCS, gentamycin). Half of the cell suspension was then stimulated with 10 µg/mL Escherichia coli lipopolysaccharide (LPS) serotype 0111: B4 (Sigma). After 6 h incubation, all culture supernatants (with or without LPS) were harvested and filtered through 0.2 µm filter (Gelman Science, Ann Arbor, MI) and stored at -80 C until enzyme-linked immunosorbent assay (ELISA) analysis.

Cytokine assays. Cytokine concentrations (IL-1ß, IL-6, TNF-{alpha}, and GM-CSF) were evaluated in the mononuclear cell culture-conditioned medium using sandwich enzyme immunoassay (EASIA, Medgenix Diagnostics, Fleurus, Belgium). The range measures for each assay were 2–1500 pg/mL for IL-1ß, 3–2000 pg/mL for IL-6, 3–1500 pg/mL for TNF-{alpha}, and 3–2000 pg/mL for GM-CSF. The intra- and interassay coefficients of variation are, respectively, 3.4% and 4.6% for IL-1ß, 5.6 and 7.5% for IL-6, 5.2% and 8% for TNF-{alpha}, and 5.6% and 9.6% for GM-CSF. The cross-reaction among all these cytokine measurements is insignificant. All measurements were performed in duplicate. The results were expressed in pg/mL of culture medium.

Statistical analysis. Results were expressed as mean ± SEM. Comparison analysis was done by the Mann-Whitney U test. Correlation studies among cytokine synthesis, bone biochemical markers, and BMD were assessed also by a nonparametric test (Spearman rank correlation test). A P value less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical data and bone remodeling markers

Twenty five CaSF with hypercalciuria on a free calcium diet were included in the study: 10 had DH (9 men, 1 woman; mean age ± SD: 41 ± 6 yr) and 15 had IH (14 men, 1 woman; age: 43 ± 10 yr). They were compared with 10 age- and sex-matched healthy controls (8 men, 2 women; age: 37 ± 5 yr).

Plasma parameters after 12 h of fasting and after calcium oral load are reported in Table 1Go. Fasting plasma levels of phosphate are lower in IH (P = 0.01) and in DH (P = 0.02) patients than in controls, whereas fasting levels of intact PTH are lower only in IH patients (P = 0.02). Plasma levels of calcitriol are higher both in IH and DH patients than in controls (P = 0.04).


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  		Table 1. Plasma parameters (mean ± SEM) before and after the oral calcium load in controls and in patients with DH or IH

 
Urinary parameters after 7 days of calcium-restricted diet, after 12 h of fasting, and after oral calcium load are given in Table 2Go. Calciuria is significantly higher in IH patients than in controls during calcium-restricted diet (P = 0.0003) or in fasting state (P = 0.04). After oral calcium load, calciuria is higher, and its increase is greater in both IH (P = 0.0002) and DH (P = 0.001) patients than in controls. Total hydroxyprolinuria is significantly higher in IH than in DH patients (P = 0.004).


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  		Table 2. Urinary parameters (mean ± SEM) in controls and in patients with DH or IH on calcium restricted diet (Ca R diet), before (fasting state), and after (after Ca load) oral calcium load

 
Figure 1Go shows the respective vertebral BMD (expressed as Z-score) measured in DH (-0.5 ± 0.24) and in IH patients (-1.17 ± 0.21). The Z-score is significantly lower in IH patients than DH patients (P = 0.04) and than in sex- and age-matched controls (P = 0.001).



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  		Figure 1. BMD (Z-score, mean ± SEM) as measured by axial quantitative computed tomography at lumbar spine (L1-L4) in controls (C) and in patients with DH or IH. Significance of the comparison (Mann-Whitney): IH vs. controls: a, P = 0.001; IH vs. DH: b, P = 0.04.

 
Monokine production

As shown in Table 3Go, unstimulated PBM obtained from IH patients secrete higher levels of IL-1ß (P = 0.008) and TNF-{alpha} (P = 0.03) than those from controls. In addition, in the same culture conditions, the levels of GM-CSF are significantly higher in IH than in DH patients (P = 0.02) and in healthy controls (P = 0.01). The basal synthesis of IL-6 is not significantly different between the groups (Fig. 2Go).


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  		Table 3. Levels (mean ± SEM) of GM-CSF, TNF-{alpha}, IL-6, and IL-1ß in the supernatant of cultured PBM without or with in vitro LPS-stimulation in controls and in patients with DH or IH

 


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  		Figure 2. Spontaneous production (pg/mL of culture medium; mean ± SEM) of IL-1ß (A), GM-CSF (B), and TNF-{alpha} by cultured PBM in controls (C) and in patients with DH or IH. Significance of the comparison (Mann-Whitney): IH vs. controls: a1, P = 0.03; a2, P = 0.01; a3, P = 0.008; IH vs. DH: b, P = 0.02.

 
After LPS stimulation, there is no significant difference between the groups in regard to the levels of the various monokines.

To examine the magnitude of PBM activation in IH patients, comparisons were done between the synthesis of cytokines without any in vitro stimulation of PBM in IH patients and those reached by in vitro LPS-stimulation of PBM obtained from healthy controls and DH patients (Table 3Go). It is worth noting that the basal synthesis of IL-1ß, GM-CSF, and IL-6 in IH patients is comparable with the LPS-stimulated synthesis of these cytokines in DH patients and in controls. In contrast, although basal synthesis of TNF-{alpha} is significantly higher in IH patients, its levels remain lower than those reached after LPS stimulation in DH patients (P = 0.002) and in controls (P = 0.003).

To examine the relationships between bone loss and monocyte activation in IH patients, we have performed correlation studies between BMD as assessed by QCT and monokines production by cultured PBM. A significant negative correlation is found between IL-6 levels after LPS stimulation and BMD (n = 15; Z = -1.97; P = 0.04, Spearman) (Fig. 3Go). In contrast, in similar culture conditions, GM-CSF levels are positively correlated to the BMD (n = 15; Z = 2.01; P = 0.04) (Fig. 4Go). Whatever the PBM culture conditions, no correlation is found between IL-1ß and TNF-{alpha} levels and BMD. In addition, no relationship is found between the cytokine synthesis and any urinary or plasmatic parameter of bone remodeling.



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  		Figure 3. Negative correlation between BMD, as assessed by axial quantitative computed tomography at lumbar spine (L1-L4), and IL-6 production by cultured PBM after LPS (10 µg/mL) stimulation in patients with IH. n = 15; Z = -1.97; P = 0.04, Spearman test.

 


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  		Figure 4. Positive correlation between BMD, as assessed by axial quantitative computed tomography at lumbar spine (L1-L4), and GM-CSF production by cultured PBM after LPS (10 µg/mL) stimulation in patients with IH. n = 15; Z = 2.01; P = 0.04, Spearman test.

 
In IH patients, positive correlations are found between levels of GM-CSF and IL-6 (n = 15; Z = 2.01; P = 0.04), between levels of GM-CSF and IL-1ß (n = 15; Z = 2.41; P = 0.01), and between levels of IL-1ß and IL-6 (n = 15; Z = 1.94; P = 0.04) in the culture medium of unstimulated PBM.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that BMD is decreased in CaSF with IH but not in CaSF with DH. This finding confirms our previous reports (2, 31) and is in agreement with other studies demonstrating an osteopenia in IH but not in DH patients (3).

Osteopenia is caused by a disequilibrium between bone resorption and bone formation; in our study, some data suggest an increased bone resorption rather than a decreased bone formation. Urinary calcium excretion always is increased in these patients, but without increased pyridinolinuria, as reported by others (32, 33). However, total hydroxyprolinuria, another urinary marker of bone resorption, is increased in our IH patients, at least when the patients are on calcium-restricted diet, in accordance with our previous study (2). There also is no biochemical evidence for decreased bone formation because plasma values of osteocalcin and total alkaline phosphatase are normal in our IH patients.

The pathophysiological mechanism involved in bone loss of IH patients is likely multifactorial, implicating nutritional, hormonal, paracrine, or autocrine factors (3, 34). PTH-induced bone loss can not be incriminated in this study because all IH patients have normal or low plasma intact PTH, probably because of their high plasma levels of calcitriol. The latter is increased both in IH patients and in DH patients who have normal vertebral BMD. In this study, our interest was focused on cellular factors and, particularly, on cytokines produced by monocytes. Our data demonstrate that PBM obtained from IH patients have spontaneously increased capacity to release IL-1ß comparatively with controls. This result confirms that reported by Pacifici et al. (6) in CaSF with fasting hypercalciuria using bioassay determination of IL-1, which does not differentiate between IL-{alpha} and ß. Furthermore, our study shows that the activated monocytes in CaSF with IH also produce an excess of TNF-{alpha} and GM-CSF. The magnitude of the enhanced spontaneous production of IL-1ß and GM-CSF by cultured PBM isolated from IH patients is thus identical to that obtained from DH patients and healthy controls after LPS in vitro stimulation. On the other hand, although basal TNF-{alpha} production in IH patient is higher than in DH patients and controls, it remains lower than its production after LPS stimulation in DH patients and controls. This particular profile of monokines secretion in IH patients may be explained by a selective activation of some PBM subpopulations specifically secreting certain monokines. Our recent data (submitted to the Congress of the American Society of Nephrology), obtained by flow cytometric evaluation, support an ex vivo activation of some PBM subpopulations in IH patients. Furthermore, the positive correlations between GM-CSF and IL-6, between GM-CSF and IL-1ß, and between IL-1ß and IL-6 synthesis illustrate the general phenomenon of cytokine autocrine and/or paracrine cross-regulation.

The relationship between PBM activation (as assessed by in vitro overproduction of monokines) and the abnormalities that lead to bone loss in CaSF with IH remains to be more investigated. In all the phases of bone remodeling, cytokines and growth factors play a key role in the regulation of bone cell differentiation and functions. Involvement of mononuclear cells in these phases is well known. These cells provide signals for recruiting osteoblast and/or osteoclast precursors at the site of remodeling and for promoting their activation (34). In metabolic bone diseases, previous works demonstrate that PBM could reflect abnormalities of bone marrow mononuclear cells (35, 36, 37). Our study investigates some of the cytokines (IL-1ß, TNF-{alpha}, IL-6, and GM-CSF) with a potential regulatory role in bone metabolism (13, 14, 17, 38).

In humans, involvement of monocyte production of IL-1 has been demonstrated in postmenopausal osteoporotic women (39), but these data were not confirmed by other studies (40, 41). IL-1 has been implicated also in bone loss in myeloma (42) and in Paget’s disease (43). In IH patients, increased levels of IL-1 may be an important factor that mediates bone loss because this monokine resorbs bone in vitro (44), may induce hypercalcemia in vivo (45), and is correlated positively to hydroxyprolinuria and negatively to vertebral BMD in CaSF with fasting hypercalciuria (6). Our results are in contradiction with those reported recently by Weisinger et al. (46), who showed an increased synthesis (ELISA) of IL-1{alpha}, but not of IL-1ß, in hypercalciuric CaSF. This discrepancy between the finding of Weisinger and our data may be explained by the difference in experimental procedures. In fact, to prevent in vitro cells activation, we have cultured PBM for less time (22 h) than in Weisinger’s study (48 h). IL-1{alpha} remains membrane-associated during the first 20 h, whereas about 70% of IL-1ß are secreted during the first 24 h (47). Indeed, we also have tested IL-1{alpha} in the culture medium by ELISA and found a weak level of secreted protein in only 3 patients with IH (data not shown).

TNF-{alpha} is involved in bone resorption (12, 13) but also can inhibit bone formation (12, 48). This cytokine seems to be implicated in bone loss induced by estrogen deficiency (34, 35, 36, 37, 49, 50). Our data are consistent with a possible role of TNF-{alpha} in bone loss of IH patients because its synthesis by cultured PBM is increased spontaneously. Its exact mechanism of action (increase of bone resorption or inhibition of bone formation) remains to be defined precisely.

GM-CSF is implicated also in bone loss of postmenopausal women (36, 40, 51). Its role in osteoclastogenesis and bone resorption is well known (17, 18). However, GM-CSF can act also as an autocrine proliferative factor on human osteoblastic cells (19, 52). GM-CSF secretion by osteoblasts is induced by mononuclear cell release of IL-1 and TNF-{alpha} (53). The higher spontaneous synthesis of GM-CSF by PBM in our IH patients and the positive correlation found between its LPS stimulated synthesis and vertebral BMD suggest that GM-CSF may have an overall protective role in IH patients. These data can possibly reflect the action of this colony-stimulating factor on osteoblast proliferation after their recruitment, to refill the resorptive lacunae. This effect possibly can explain, at least in part, why these patients have a mild bone loss with exceptional report of clinical symptoms (54) in spite of high IL-1 and TNF-{alpha} synthesis.

IL-6 is a potential resorptive factor in vitro and in vivo (14, 55, 56). In humans, IL-6 seems to be implicated in postmenopausal osteoporosis (37), in Paget’s disease (57), and in myeloma (58). In our study, we have found that BMD of IH patients is negatively correlated to IL-6 synthesis by LPS-stimulated PBM, suggesting that this cytokine may participate in abnormal bone remodeling of these patients. In conclusion, BMD decrease in calcium stone formers with IH is associated with spontaneous activation of PBM as assessed by in vitro cytokine synthesis. Our results suggest that IL-1ß, TNF-{alpha}, GM-CSF, and IL-6 may play an important role in the bone loss of IH patients, although direct evidence at the bone level for a causal relationship is still lacking. In these patients, GM-CSF seems to be a protective factor, whereas IL-1, TNF-{alpha}, and IL-6 probably have deleterious effects on bone density. These results reveal a particular profile of induced or inducible PBM activation in IH patients. However, other local factors released from bone or from hematopoietic cells can be implicated in the regulation of bone remodeling. Further studies are required to define the exact mechanism of in vivo monocyte activation and of its link with bone loss.


   Acknowledgments
 
The authors thank M. Froissart, N. Celisse, C. Bilhaut, S. Darret, and J. C. Capiod for their precious collaboration.


   Footnotes
 
1 This study was partially supported by a grant from INSERM Paris, France (Contrat d’Etude Pilote) and by a grant from the ministry of health (PHRC 94, France). Back

Received April 22, 1996.

Revised August 16, 1996.

Accepted August 19, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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