Effect of oestradiol on cytokine production in immortalized human marrow stromal cell lines.

Oestrogen deficiency enhances bone osteoclastogenesis and bone resorption. Evidence of cooperation between stromal cells and osteoclast precursors in mice suggests that oestradiol acts by regulating cytokine release from stromal cells. Bone marrow stroma contains multipotent progenitors that give rise to many mesenchymal lineages, including osteoblasts that may regulate osteoclast differentiation. We immortalized and characterized six human bone marrow stromal cell lines (presence of Stro1, secretion of alkaline phosphatase, osteocalcin, formation of lipid droplets, and presence of alpha and beta oestrogen receptors). The response of cytokines to oestradiol was then evaluated in vitro, as were the phorbol myristate acetate (PMA)-stimulated cytokine levels. Cells had the characteristics of undifferentiated stromal cells (Stro1+, RANK-L+), and expressed alpha-oestrogen receptors. The osteoblast phenotype (amounts of alkaline phosphatase and osteocalcin) was weak and there was a poor capacity to differentiate into adipocytes. These cell lines did not respond to oestradiol by producing interleukin 6 (IL-6), IL-1 or tumour necrosis factor alpha (TNF-alpha) either constitutively or after stimulation with PMA. Moreover, RANK-L and osteoprotegerin expressions were not regulated by oestradiol in vitro. Thus, modulation of these cytokines by stromal cells do not appear to be the mechanism by which oestradiol regulates bone resorption in humans.

Parathyroid hormone-related peptide stimulates proliferation of highly tumorigenic human SV40-immortalized breast epithelial cells.

Parathyroid hormone-related protein (PTHrP) is the main mediator of humoral hypercalcemia of malignancy (HHM) and it is produced by many tumors, including breast cancers. Breast epithelial cells as well as breast cancer tumors and cell lines have been reported as expressing PTHrP and the PTH/PTHrP receptor, suggesting that PTHrP may act as an autocrine factor influencing proliferation or differentiation of these cell types. We investigated PTHrP gene expression, PTH/PTHrP receptor signaling, and PTHrP-induced mitogenesis in three immortalized human mammary epithelial cell lines that exhibit differential tumorigenicity. The most tumorigenic cells expressed the highest levels of PTHrP messenger RNA (mRNA) and protein. We used reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting to detect the PTH/PTHrP receptor transcripts and proteins in all of the three cell lines. Treatment with human PTHrP(1-34) [hPTHrP(1-34)] and hPTH(1-34) increased intracellular cyclic adenosine monophosphate (cAMP) but not free Ca2+ in the nontumorigenic line. These agonists increased both cAMP and free Ca2+ levels in the moderately tumorigenic line, but only increased free Ca2+ in the highly tumorigenic line. Application of the PTH/PTHrP receptor antagonist [Asn10,Leu11,D Trp12]PTHrP(7-34) or PTHrP antibodies reduced [3H]thymidine incorporation in a dose-dependent fashion in the highly tumorigenic cell line but did not affect the other lines. Thus, treatment with a PTH/PTHrP receptor antagonist reduced cell proliferation, suggesting that PTHrP signaling mediated by the phospholipase C (PLC) pathway stimulates proliferation of a highly tumorigenic immortalized breast epithelial cell line.

Calcitonin receptor mRNA in mononuclear leucocytes from postmenopausal women: decrease during osteoporosis and link to bone markers with specific isoform involvement.

Calcitonin inhibits bone resorption via its receptor (CTR) on osteoclasts. Two hCTR isoforms, hCTR1 and hCTR2, give proteins that differ in their structure and signaling pathways. We investigated whether specific isoforms or quantitative changes in total hCTR mRNA were associated with high bone resorption and turnover in menopause or osteoporosis. The hCTR mRNA in mononuclear blood cells of premenopausal (PreM), healthy (PostM), and osteoporotic (OsteoP) postmenopausal women was assessed using reverse-transcriptase polymerase chain reaction. hCTR1 and hCTR2 were investigated for 59 total RNA samples, and semiquantitative analysis of total hCTR mRNA was performed for 71. Serum calcitonin, free urinary deoxypyridinoline (D-Pyr), serum bone alkaline phosphatase (SBAP), and osteocalcin (SOC) were also evaluated. Serum calcitonin levels did not differ in PostM and OsteoP. The prevalence of each isoform was similar in the three groups. Healthy postmenopausal women and OsteoP with hCTR2 had lower bone turnover (D-Pyr: 6.79 +/- 0.54, n = 25; SBAP: 11.63 +/- 1.47, n = 26; SOC: 8.31 +/- 0.58, n = 26) than those without hCTR2 (D-Pyr: 9.90 +/- 1.95, n = 5; SBAP: 21 +/- 5.19, n = 5; SOC: 11.9 +/- 2.10, n = 5; p < 0.05). Total hCTR mRNA levels were not different in PreM and PostM. By contrast, values were strikingly lower in OsteoP (0.57 +/- 0.17, n = 28) than in PostM (2. 25 +/- 0.61, n = 19, p < 0.05) and negatively correlated with bone markers values in both. We suggest that a specific isoform and amounts of total hCTR mRNA are linked to increased bone resorption in postmenopausal osteoporosis.

Inflammatory cytokine response in patients with septic shock secondary to generalized peritonitis.

OBJECTIVES: The aims of this study were the following: a) to assess the proinflammatory cytokine (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1, and IL-6) response in patients with septic shock secondary to generalized peritonitis; and b) to evaluate the influence of bacteremic status, type of peritonitis (acute perforation or postoperative), and peritoneal microbial status (mono- or polymicrobial) on cytokine expression and mortality. DESIGN: Prospective study. SETTING: Surgical intensive care unit of a university hospital. PATIENTS: Fifty-two consecutive patients with septic shock caused by generalized peritonitis. INTERVENTIONS: Routine blood tests, blood cultures, and cytokine assays were performed during the first 3 days after onset of shock. MEASUREMENTS AND MAIN RESULTS: Serum TNF-alpha and IL-6 concentrations were measured by using a radioimmunoassay, and IL-1 concentrations were measured by using ELISA. Median serum concentrations on day 1 were: TNF-alpha, 90 pg/mL; IL-1, 7 pg/mL; and IL-6, 5000 pg/mL. TNF-alpha and IL-6 concentrations decreased significantly between the first and third days of septic shock (p = .0001), whereas IL-1 concentrations remained low. The decrease in IL-6 tended to be more pronounced in the survivors group (p = .057). Median TNF-alpha serum concentrations were higher in bacteremic compared with nonbacteremic patients (151 vs. 73 pg/mL, p = .003). TNF-alpha, IL-1, and IL-6 serum concentrations and mortality were not different between acute perforation vs. postoperative peritonitis and mono- versus polymicrobial peritonitis. CONCLUSIONS: The systemic release of TNF-alpha and IL-6 during septic shock caused by generalized peritonitis was maximal on day 1 and decreased rapidly during the next days. No systemic release of IL-1 was observed. IL-6 serum concentrations remained higher in patients who subsequently died. Among the different features of peritonitis studied, only bacteremia influenced the systemic cytokine response (higher TNF-alpha).

Effects of bone morphogenetic protein-2 on human neonatal calvaria cell differentiation.

Bone morphogenetic proteins (BMPs) are factors that promote osteoblastic cell differentiation and osteogenesis. It is unknown whether BMPs may act on human osteoblastic cells by increasing immature cell growth and/or differentiation. We investigated the short- and long-term effects of recombinant human (rh)BMP-2 on cell growth and osteoblast phenotype in a new model of human neonatal pre-osteoblastic calvaria cells (HNC). In short-term culture, rhBMP-2 (20-100 ng/ml) inhibited DNA synthesis and increased alkaline phosphatase (ALP) activity without affecting osteocalcin (OC) production. When cultured for 3 weeks in the presence of ascorbic acid and inorganic phosphate to induce cell differentiation, HNC cells initially proliferated, type 1 collagen mRNA and protein levels rose, and then decreased, whereas OC mRNA and protein levels, and calcium accumulation into the extracellular matrix increased at 2 to 3 weeks. A transient treatment with rhBMP-2 (50 ng/ml) for 1 to 7 days which affected immature HNC cells, decreased cell growth, increased ALP activity and mRNA, and induced cells to express ALP, osteopontin, and OC at 7 days, as shown by immunocytochemistry. At 2 to 3 weeks, matrix mineralization was markedly increased despite cessation of treatment, and although OC and Col 1 mRNA and protein levels were not changed. A continuous treatment with rhBMP-2 for 3 weeks which affected immature and mature cells reduced cell growth, increased ALP activity and mRNA at 1 week and increased OC mRNA and protein levels and calcium content in the matrix at 3 weeks, indicating complete osteoblast differentiation. These results indicate that the differentiating effects of BMP-2 on human neonatal calvaria are dependent on duration of exposure. Although long-term exposure led to complete differentiation of OC-synthesizing osteoblasts, the primary effect of rhBMP-2 was to promote osteoblast marker expression in immature cells, which was sufficient to induce optimal matrix mineralization independently of cell growth and type 1 collagen expression.

The diuretic indapamide increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium.

Clinical and epidemiological studies suggest that thiazide diuretics can prevent bone loss and decrease the incidence of hip fractures. However, the mechanism of the effect of diuretics on bone is not clearly established. Indapamide (IDP), a sulfonamide diuretic related to thiazides, is used to treat hypertension. Sixty spontaneously hypertensive rats (SHRs) were divided into four groups and treated with or without IDP (1.5 mg/kg/day) during 8 weeks in the presence or absence of a high sodium load (8% NaCl supplementation in the diet). Sodium and calcium excretions were increased in the rats receiving the high sodium load (SHR + 8% NaCl) comparatively with control rats (SHR). IDP decreased and increased, respectively, calcium and sodium excretions. Serum parathyroid hormone (PTH) was unchanged in any group. Bone density was measured at the femur, tibia, and vertebrae, and bone morphometry was performed at the metaphysis of the femur to evaluate bone architecture. Rats fed a high sodium diet had an average 5.5% decreased bone density at every site except the femoral diaphysis. The trabecular bone volume was also decreased (SHR + 8% NaCl vs. SHR, 11.99+/-0.78 vs. 17.51+/-1.5%, p < 0.05). An increase in trabecular separation suggested that these changes were due to increased bone resorption. In the SHR + 8% NaCl + IDP group, IDP increased bone density and trabecular bone volume (SHR + 8% NaCl + IDP vs. SHR + 8% NaCl, 16.52+/-1.04 vs. 11.99+/-0.78%, p < 0.05). Trabecular separation and pyridinoline/creatinine excretion (SHR + 8% NaCl + IDP vs. SHR + 8% NaCl, 136.39+/-9.62 vs. 195.18+/-22.34 nmol/mmol, p < 0.05) were also decreased by IDP. These results show that in rats receiving a high sodium diet, IDP can reverse sodium-induced bone loss and increased bone resorption independently of changes in serum PTH.

Biochemical effects of calcium supplementation in postmenopausal women: influence of dietary calcium intake.

We studied the biochemical effects of calcium supplementation during a 2-mo course in postmenopausal women (x +/- SD: 64 +/- 5 y of age and 14.5 +/- 6.7 y since menopause). The effects on calcium homeostasis and bone remodeling were assessed after 1 and 2 mo of daily administration of either calcium carbonate (1200 mg elemental Ca/d, n = 60) or a placebo (n = 56). The daily dietary calcium intake assessed before the beginning of calcium supplementation was 786 mg/d. We found a significant inverse relation between baseline intact parathyroid hormone (iPTH) and dietary calcium intake before supplementation (r = -0.48, P = 0.0002). A significant increase in urinary excretion of pyridinoline was observed when the dietary calcium intake was lower than the median value. Calcium supplementation resulted in a significant increase in 24-h urinary calcium (39%, P < 0.02) and a significant reduction of bone alkaline phosphatase at 2 mo and of all bone-resorption markers (hydroxyproline, pyridinoline, and deoxypyridinoline) at I and 2 mo without significant changes in 44-68 PTH fragments or iPTH concentrations. When the dietary calcium intake was low (mean +/- SD: 576 +/- 142 mg/d), calcium supplementation was responsible for a greater increase in urinary calcium excretion and a greater decrease in markers of bone turnover. The greatest variations were observed for deoxypyridinoline at 1 and 2 mo (-18.5%, P < 0.05) and for pyridinoline at 1 mo (-16.3%, P < 0.01). Two months of calcium supplementation in postmenopausal women was efficient in reducing markers of bone turnover, with a greater effect in women with a low dietary calcium intake.

Estrogen stimulates PTHrP but not PTH/PTHrP receptor gene expression in the kidney of ovariectomized rat.

The aim of the present study was to test the hypothesis that the decreased renal tubular reabsorption of calcium observed in estrogen deficiency is associated with a local regulation of either PTHrP or PTH/PTHrP receptor genes in the kidney. Rats were randomly sham-operated (S) or ovariectomized receiving either vehicle (OVX) or 4 microg E2/kg/day (OVX+E4) or 40 microg E2/kg/d (OVX+E40) during 14 days using alzet minipumps. Plasma PTH and calcium levels were lower in untreated OVX animals than in all other groups (P < 0.01). Plasma PTH was higher in OVX+E40 than in OVX+E4 (P < 0.05). PTHrP mRNA expression in the kidney was unaffected by ovariectomy but was increased in OVX+E40 (0.984 +/- 0.452 for PTHrP/GAPDH mRNAs expression vs. 0.213 +/- 0.078 in sham, P < 0.01). PTH/PTHrP receptor mRNA expression and the cAMP response of renal membranes to PTH were unaffected by ovariectomy and estrogen substitution. In conclusion, renal PTHrP and PTH/PTHrP receptor mRNAs are not modified by ovariectomy. However, 17beta-estradiol increases renal expression of PTHrP mRNA without evident changes in its receptor expression and function. This may help to explain the pharmacological action of estrogen in the kidney, especially how it prevents the renal leak of calcium in postmenopausal women.

Increased bone resorbing activity of peripheral monocyte culture supernatants in elderly women.

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-alpha 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-alpha 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.

The effects of fibroblast growth factor-2 on human neonatal calvaria osteoblastic cells are differentiation stage specific.

Fibroblast growth factors (FGFs) appear to play an important role in human cranial osteogenesis. We therefore investigated the effects of recombinant human FGF-2 (rhFGF-2) on human calvaria (HC) osteoblastic cells. Immunocytochemical analysis showed that confluent HC cells express both FGF receptors -1 and -2. In short-term culture, rhFGF-2 (0.1-100 ng/ml, 2-5 days) increased HC cell growth and decreased alkaline phosphatase (ALP) activity and type I collagen (ColI) synthesis, as evaluated by P1CP levels. When HC cells were induced to differentiate in long-term culture in the presence of 50 microg/ml ascorbic acid and 3 mM phosphate, HC cells initially proliferated, then ALP activity and ColI synthesis decreased and calcium content in the extracellular matrix increased. Continuous treatment with rhFGF-2 (50 ng/ml) for 1-28 days, or a transient rhFGF-2 treatment for 1-7 days, slightly increased DNA synthesis at 7 days, whereas a late treatment for 8-28 days had no effect on cell growth. The continuous and transient treatments with rhFGF-2 decreased ALP activity, ColI synthesis, and matrix mineralization. This was associated with a transient fall in osteocalcin (OC) production at 7 days. In contrast, the late rhFGF-2 treatment for 8-28 days only slightly inhibited ALP activity and increased matrix mineralization. In addition, both continuous and late treatments with rhFGF-2 increased OC production in more mature cells at 3-4 weeks of culture. We also found that the early and late treatments with rhFGF-2 had opposite effects on transforming growth factor beta2 production in proliferating cells and more mature cells. The results show that rhFGF-2 slightly stimulates cell growth and reduces the expression of osteoblast markers in less mature cells, whereas it induces OC production and matrix mineralization in more mature cells, indicating that the effects of FGF-2 are differentiation stage specific and that FGF-2 may modulate HC osteogenesis by acting at distinct stages of cell maturation.

Cytokine release from marrow mononuclear cells is negatively correlated to cortical elasticity in non-osteoporotic postmenopausal women.

Activation of bone remodeling is likely to be under the control of mechanical factors acting, in part, through soluble local factors. We therefore investigated a relationship between cytokine production by marrow cells and bone elasticity. We studied 36 non-osteoporotic postmenopausal women undergoing hip arthroplasty for hip arthrosis (mean age: 68 +/- 8 years; lumbar BMD Z-score: +0.54 +/- 0.33 SD). Adherent marrow mononuclear cells were cultured for 48 hours with autologous plasma, and supernatants were harvested for PGE2, IL-1, TNF-alpha, and IL-6 measurements. Femoral neck cortical bones were removed during surgery for cortical histomorphometric evaluation and determination of elasticity indices (C33) using ultrasonic transmission method. In this nonosteoporotic population, femoral neck longitudinal elasticity indices were inversely correlated to both cortical thickness (r = -0.58, P < 0.01) and cortical porosity (r = -0.33, P < 0.01). The longitudinal elasticity indices were also negatively correlated to basal IL-1 and TNF-alpha release by adherent mononuclear marrow cells (r = -0.59, P < 0.01; r = -0.60, P < 0.01, respectively). However, no relationship was found between the three cytokines tested and either cortical thickness or porosity. These data show a link between cortical biomechanical properties and local factors involved in bone remodeling. We suggest that increased bone elasticity decreases transmission of strain, which in turn decreases cytokine release from marrow cells. However, whether cytokines influence bone elasticity or vice versa remains to be demonstrated.

High tumor necrosis factor serum level is associated with increased survival in patients with abdominal septic shock: a prospective study in 59 patients.

BACKGROUND: In several studies including patients with septic shock of various origins, high serum cytokine levels have been reported to correlate with poor outcome. The aim of this prospective study was to assess the prognostic value of cytokine serum levels in a group of patients with perioperative septic shock of digestive origin. METHODS: From January 1992 to December 1994, 59 patients were evaluated (mean age, 68 +/- 15 years). From the first day of septic shock to day 7, blood was drawn every day to measure the conventional biologic parameters (white blood cell count, platelet count, hematocrit, blood urea nitrogen level, serum electrolytes level, pH, blood gases, serum lactate level, coagulation parameters, liver function tests) and tumor necrosis factor (TNF), interleukin-1, and interleukin-6. RESULTS: No difference was observed between the 26 survivors and the 33 nonsurvivors with regard to age, gender, and cause of sepsis. On admission, mean platelet count was significantly higher in the survivors than in the nonsurvivors (260 +/- 142 versus 177 +/- 122 10(9)/L; p = 0.01). Mean blood urea nitrogen level was significantly lower in the survivors than in the nonsurvivors (9.6 +/- 9 versus 12 +/- 7 mmol/L; p = 0.04). No difference was observed between survivors and nonsurvivors for the other conventional biologic parameters and for serum interleukin-1 and interleukin-6 levels. Mean serum TNF level tended to be higher in survivors than in nonsurvivors (565 +/- 1325 versus 94 +/- 69 pg/ml; not significant). In the group survivor 9 (35%) of 26 patients had a serum TNF level greater than 200 pg/ml versus 2 (6%) of 33 patients in the nonsurvivor group (p < 0.02). Survival was noted in 6 (100%) of 6 patients who had both a serum TNF level greater than 200 pg/ml and a platelet count greater than 100.10(9)/L versus 1 (11%) of 9 in patients with neither of these criteria (p < 0.01). CONCLUSIONS: In our patients with abdominal septic shock, high serum TNF levels were associated with increased survival. The high serum level of TNF may reflect the efficacy of peritoneal inflammatory response against abdominal sepsis. Although this possibility must be further explored, a score combining the serum TNF level and platelet count could be helpful for the prognostic assessment of patients with abdominal septic shock.

1,25 dihydroxyvitamin D and dexamethasone decrease in vivo Walker carcinoma growth, but not parathyroid hormone related protein secretion.

Parathyroid hormone related protein (PTHrP) is produced by several breast cancers. 1,25 dihydroxyvitamin D (1,25[OH]2D) and Dexamethasone (DEX) have been shown to decrease PTHrP mRNA expression in several cell lines. We therefore tested the in vivo effect of both steroids on PTHrP secretion and tumor development of the Walker carcinoma (WC). WC cells were injected subcutaneously in Fisher rats which were simultaneously treated with either vehicle, or 1,25(OH)2D (0.5 micrograms/kg/d) or DEX (2 mg/kg/d). After 7 days, tumor weight was significantly decreased in the 2 treated-groups as compared to the control group. Vehicle treated-rats developed hypercalcemia, which was also observed in rats treated with 1,25(OH)2D; by contrast, the plasma calcium was significantly decreased in the DEX-treated group compared to vehicle-treated rats. In a dose-effect experiment, this dose of 1,25(OH)2D induced marked hypercalcemia in rats not implanted with WC, but was required to decrease the tumor weight in implanted rats. In both 1,25(OH)2D and DEX-treated groups, plasma PTHrP levels were significantly decreased, but there was a similar correlation between PTHrP plasma level and tumor weight in the three groups. Indeed, the cytosolic PTHrP content/mg tumor was identical in the 3 groups. By contrast, the PTHrP/Actin mRNA in the tumor was significantly decreased in the 1,25(OH)2D group, comparatively to the vehicle and DEX groups. Our results show that Dexamethasone and 1,25(OH)2D decrease WC tumor development in vivo, but do not change the PTHrP secretion by the remaining tumor although steady state PTHrP mRNA content level is decreased by 1,25(OH)2D.

Cytokine-mediated bone resorption in patients with the hyperimmunoglobulin E syndrome.

Hyperimmunoglobulin E syndrome (HIES) is a rare immunodeficiency disorder characterized by increased serum immunoglobulin E levels. Bone fragility is part of this syndrome, which has recently been reported to be also associated with an imbalance in cytokine-secreting lymphocyte subpopulation. It has recently been shown that some cytokines can play a role in the bone fragility following menopause. We therefore investigated six patients (mean age 16.5 +/- 8.5 years) affected by this rare syndrome in order to study their bone remodeling and the possible involvement of cytokines in causing the bone fragility associated with this disease. Three of six patients had suffered long bone fractures; in four of six patients the cortical bone mass measured at the distal radius was two standard deviations below that of the aged-matched controls. Urinary pyridinoline excretion, a marker of bone resorption, was markedly increased in the two youngest patients. Adherent mononuclear cells derived from these patients were cultured in vitro and the bone resorbing activity (BRA) of the culture supernatant was measured by means of a fetal rat long bone assay. The BRA was up to 28% above the basal value. We compared the BRA and the cytokine production by the mononuclear cells of these patients to that of postmenopausal women. The BRA, and the IL1 beta, IL6, and TNF alpha levels in the mononuclear cell culture supernatants were identical for both HIES and postmenopausal women. However, the levels of PGE2 were higher and the levels of interferon-gamma were lower in the HIES patients. In conclusion, increased bone resorption in young patients with the HIES is responsible for the cortical bone loss that leads to a higher incidence of fractures. The high BRA secreted by the mononuclear cells of these patients is similar to that found in mononuclear cells from postmenopausal women. These data provide evidence of potent mononuclear cell activation leading to bone loss in HIES, which could be related to IgE-dependent mechanisms.

Bone resorption at the femoral neck is dependent on local factors in nonosteoporotic late postmenopausal women: an in vitro-in vivo study.

Local mediators of bone resorption may be involved in bone loss in recently postmenopausal women and in osteoporosis. In the present study, we investigated the production of cytokines and the formation of osteoclast-like cells in marrow cultures from 16 late postmenopausal nonosteoporotic women (mean age: 66 +/- 8 years; time after menopause: 15 +/- 8 years) undergoing hip replacement for arthrosis. Marrow adherent mononuclear cells (MMNC) isolated from femoral diaphysis marrow were cultured for 10 days in the absence or in the presence of 1,25(OH)2D3. In vivo bone resorption was concomitantly assessed by histomorphometry on femoral neck bone sections. The number of TRAP+ multinucleated cells obtained after 10 days in MMNC cultured in the presence of 1,25(OH)2D3 correlated with the number of osteoclasts measured on the bone femoral neck biopsies (r = 0.65, p < 0.01), suggesting that the formation of multinucleated cells in vitro could reflect the osteoclast differentiation in vivo. Furthermore, the number of osteoclasts was related to the eroded volume and the trabecular separation of the femoral neck bone biopsies. Finally, the release of interleukin-1 (IL-1), IL-6, and TNF-alpha by cultures of peripheral blood mononuclear cells (PBMC) and MMNC was measured by radioimmunoassay. The cytokine levels of basal and 1,25(OH)2D3-treated MMNC decreased from days 2 to 5 and then reached a plateau to day 10. The number of TRAP+ multinucleated cells obtained after 10 days in MMNC cultures correlated with the basal IL-6 release in the same cultures determined at day 2 (r = 0.55, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral monocyte culture supernatants of menopausal women can induce bone resorption: involvement of cytokines.

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.

In vitro production of cytokines by bone surface-derived osteoblastic cells in normal and osteoporotic postmenopausal women: relationship with cell proliferation.

To evaluate the role of cytokines produced by osteoblasts in the pathophysiology of bone lesions in postmenopausal osteoporosis (PMOP), we have determined by RIA and immunoradiometric assays the levels of prostaglandin E2 (PGE2), interleukin-1 beta (IL-1), tumor necrosis factor-alpha (TNF alpha), and IL-6 released by cultured bone surface-derived osteoblastic (OB) cells isolated from 24 untreated PMOP women with high, low, or normal bone turnover on bone biopsy. OB cells isolated from patients with high bone formation had a 2-fold increased proliferation rate in vitro compared to OB cells from patients with normal or low bone formation or OB cells from age-matched controls. The spontaneous in vitro production per cell protein of PGE2, IL-1, and TNF alpha, but not of IL-6, was 2- to 3-fold lower in rapidly proliferating OB cells isolated from PMOP patients with high bone formation compared to OB cells from patients with normal or low proliferation or control cells. Treatment with 10 nmol/L 1,25-dihydroxyvitamin D (48 h) increased PGE2 levels to normal values in OB cells with a high proliferation rate, but decreased PGE2 production in cells with low proliferation and in control cells, suggesting that the release of PGE2 was dependent on the stage of maturation of OB cells. Significant correlations were found between IL-1 and TNF alpha (r = 0.87; P < 0.001), IL-1 and PGE2 (r = 0.46; P < 0.05), IL-6 and IL-1 (r = 0.39; P < 0.05), and IL-6 and TNF alpha (r = 0.49; P < 0.05), suggesting that the production of these cytokines was under reciprocal control. The results indicate that the in vitro production of PGE2, IL-1, and TNF alpha, but not IL-6, by OB cells isolated from patients with PMOP is related to the proliferation rate of these cells and the rate of bone formation. The variable production of cytokines by OB cells may contribute to the histological heterogeneity of bone formation in PMOP.

The resistance to parathyroid hormone of fibroblasts from some patients with type Ib pseudohypoparathyroidism is reversible with dexamethasone.

Skin fibroblasts from some patients with pseudohypoparathyroidism type Ib (PHP-Ib) are resistant to PTH. To characterize the defect producing PTH resistance in these cells and determine whether the abnormality is potentially reversible, we evaluated the ability of fibroblasts from patients with PHP-Ib to produce cAMP in response to PTH both before and after exposure to dexamethasone, an agent known to increase PTH responsiveness in other cell types. Before dexamethasone treatment, fibroblasts from five of eight patients with PTH-Ib produced reduced amounts of cAMP in response to PTH (but not prostaglandin E1 and forskolin) compared to that of control cells (6.3 +/- 1.0 and 37.3 +/- 6.3 pmol cAMP/100 micrograms protein, respectively). Whereas dexamethasone pretreatment had no effect on cAMP production by control or PTH-responsive PHP-Ib fibroblasts, it resulted in a dose-dependent increase in PTH-induced cAMP production by PTH-resistant fibroblasts to normal levels in four of five cases. Studies evaluating the binding of radiolabeled PTH did not permit quantification of the small number of high affinity PTH receptors present on these cells. We conclude that PTH resistance in PHP-Ib patients with PTH-resistant fibroblasts results from an abnormality in the expression of or coupling to cyclase of high affinity PTH-receptor complexes. Because it is expressed in only some tissues and is reversible, the defect could be regulatory in nature.

Optimal correction of acidosis changes progression of dialysis osteodystrophy.

To investigate an eventual role of acidosis on hemodialysis osteodystrophy we prospectively studied 21 patients who were dialyzed with different amounts of bicarbonate in the dialysate for 18 months. According to the level of bone formation rate (BFR) on a prestudy bone biopsy, patients were split in two subgroups. Inside these two subgroups patients were randomly allocated to two therapeutics groups: 10 patients (group A) were dialyzed with the conventional amount of bicarbonate (33 +/- 2 mmol/liter) in the dialysate; the rest of the patients (group B, N = 11) had 7 to 15 mmol/liter sodium bicarbonate added to the dialysate to obtain 24 mEq predialysis bicarbonate plasma levels. An effective correction of acidosis was shown in group B by a higher predialysis plasma bicarbonate level (15.6 +/- 1 group A vs. 24.0 +/- 0.6 mEq/liter group B, P less than 0.005), which was reached three months after start of the study. Compared to the prestudy bone biopsy, osteoid and osteoblastic surfaces increased in group A but not in group B on the bone biopsies performed at the end of the study. Parathormone plasma level (iPTH), measured with an antiserum which cross reacts with the 44-68 region of PTH molecule, increased during the study in group A but not in group B. This finding suggested progression of secondary hyperparathyroidism (HPT) only in group A patients. Osteocalcin plasma values increased in both groups during the 18 months of the study. Consequently the two subgroups of patients formed on the basis of BFR level were evaluated separately.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationships between histomorphometric features of bone formation and bone cell characteristics in vitro in renal osteodystrophy.

To determine whether abnormal bone cell recruitment or differentiation may be involved in the development of aplastic bone lesion in renal osteodystrophy we have compared histomorphometric parameters of bone formation and in vitro behavior of osteoblastic cells isolated from the trabecular bone surfaces in 37 dialysis patients with osteitis fibrosa, normal bone formation rate, or aplastic bone lesion. The bone cell responses to human PTH-(1-34) (20 nmol/L), as evaluated by intracellular cAMP production, and to 1,25-dihydroxyvitamin D (10 nmol/L), as assessed by osteocalcin synthesis, were not different from normal in patients with low, normal, or high bone formation rates. Osteoblastic cells isolated from patients with a high bone formation rate and markedly elevated serum iPTH and osteocalcin values had a higher than normal DNA replication in primary culture. The peak of [3H]thymidine incorporation, the maximal DNA synthesis, and the area under the growth curve were 4.4- to 6.3-fold increased in osteitis fibrosa compared to those in normal bone cells obtained from age-matched individuals. By contrast, [3H]thymidine incorporation in bone cells from aplastic patients was about 25% of normal and only 5% of the value in osteitis fibrosa. The decreased DNA replication of cultured bone cells in aplastic patients was unrelated to trabecular bone aluminum staining, but was associated with low serum immunoreactive PTH values compared to those in other groups of patients. These results show that high bone formation in uremic osteoitis fibrosa is associated with higher than normal [3H]thymidine incorporation in bone cells in vitro, whereas low bone formation in aplastic patients results from lower than normal DNA replication and suggest that the defective osteoblastic recruitment in aplastic patients may be related to factors other than aluminum, including inappropriate PTH secretion.