Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression.

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.

T cell activation induces human osteoclast formation via receptor activator of nuclear factor kappaB ligand-dependent and -independent mechanisms.

In unstimulated conditions, osteoclast (OC) formation is regulated by stromal cell production of the key osteoclastogenic factors receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the mechanisms of accelerated osteoclastogenesis and bone loss characteristic of inflammatory conditions are poorly understood but appear to involve T cells. In addition, the mechanism by which OCs arise spontaneously in cultures of peripheral blood mononuclear cells in the absence of stromal cells or added cytokines remains unclear. Using a stromal cell free human osteoclast generating system, we investigated the ability of activated T cells to support osteoclastogenesis. We show that when activated by phytohemagglutinin-P (PHA), T cells (both CD4+ and CD8+) stimulate human OC formation in vitro. Although both soluble M-CSF and RANKL were detected in activated T cell supernatants, the presence of M-CSF was not essential for macrophage survival or RANKL-dependent osteoclast formation, suggesting that other soluble T cell-derived factors were capable of substituting for this cytokine. We also found that saturating concentrations of osteoprotegerin (OPG) failed to neutralize 30% of the observed OC formation and that T cell conditioned medium (CM) could superinduce osteoclastogenesis in cultures of purified monocytes maximally stimulated by RANKL and M-CSF. Together, these data suggest that activated T cells support osteoclastogenesis via RANKL-dependent and -independent mechanisms. Although not relevant for T cell-induced osteoclastogenesis, secretion of soluble M-CSF is a previously undescribed property of activated T cells.

Interleukin-7 stimulates osteoclast formation by up-regulating the T-cell production of soluble osteoclastogenic cytokines.

In unstimulated conditions osteoclast renewal occurs as a result of the stromal cell production of the key osteoclastogenic factors, receptor activator of NFkB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Inflammation is known to cause increased osteoclastogenesis; however, the mechanisms responsible for this phenomenon are poorly understood. We now show that interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha), cytokines typically produced in inflammatory conditions, increase the stromal cell production of IL-7. This factor, in turn, up-regulates production of osteoclastogenic cytokines by T cells leading to stimulation of osteoclast (OC) formation. Although T cells were found to produce soluble forms of both RANKL and M-CSF, saturating concentrations of osteoprotegerin failed to inhibit approximately 40% of the OC formation, suggesting that IL-7 acts via both RANKL-dependent and RANKL-independent pathways. Despite the identification of T-cell-secreted M-CSF, this cytokine was not essential for either RANKL-dependent or -independent OC formation, suggesting that T cells secrete other cytokines capable of substituting for M-CSF action. On the basis of our data, we propose a novel mechanism for inflammatory bone loss in which induction of IL-7 from stromal cells by IL-1 and TNFalpha leads to the production of soluble osteoclastogenic cytokines by T cells. Thus, the mechanism by which IL-7 causes bone resorption involves the activation of T cells and the T-cell-dependent augmentation of osteoclastogenesis. (Blood. 2000;96:1873-1878)

A novel T cell cytokine stimulates interleukin-6 in human osteoblastic cells.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a heavy lymphocytic infiltration into the synovial cavity, resulting in the secretion of a variety of cytokines which ultimately leads to destruction of joint tissue. Among the infiltrating cells are activated T cells which produce specific cytokines capable of osteoclast progenitor cell expansion, fusion, and activation. Cultures of activated human T cells and human osteoblasts (hOBs) were used to study the possibility that lymphokines may act on osteoblasts to produce the osteoclastogenic factor interleukin-6 (IL-6). Purified T cells were activated with a combination of anti-CD3 and anti-CD28 antibodies, cocultured with hOBs in direct physical contact or separated by a transwell system, and conditioned media (CM) were assayed for IL-6 production. After a 72 h incubation period, activated T cell-hOB interaction resulted in a 100-fold increase of IL-6 production over basal levels. The immunosuppressant cyclosporine A (CsA) inhibited T cell tumor necrosis factor alpha and IL-6 production but did not inhibit the T cell induction of IL-6 from hOB. Assay of activated T-cell CM on hOB revealed that a soluble factor, not cell-cell contact, was the major inducer of IL-6. The induction of IL-6 mRNA by both activated T cell CM and CsA-treated activated T cell CM was confirmed by Northern blot analysis. Neutralizing antibodies to IL-13 and IL-17 did not affect IL-6 production. These findings suggest that activated T cells produce a novel, potent, IL-6 inducing factor that may be responsible for the bone loss observed in RA patients.

Skeletal casein kinase activity defect in the HYP mouse.

The Hyp mouse, a model for human X-linked hypophosphatemia (XLH), is characterized by phosphate wasting and defective mineralization. Since osteopontin (OPN) is considered pivotal for biological mineralization, we examined the biosynthesis of OPN in osteoblasts of +/Y and Hyp/Y mice. Immunoprecipitation analyses using a specific antibody to OPN revealed that Hyp/Y and +/Y osteoblasts secrete similar levels of OPN as determined by [35S]-methionine biosynthetic labeling, but a reduced phosphorylation was noted after 32P-PO4 biosynthetic labeling. Northern blot hybridization analysis of +/Y and Hyp/Y mice osteoblast mRNAs, using a cDNA probe for mouse OPN, revealed no difference in the steady state levels of osteopontin mRNA. Analysis of casein kinase II activity in +/Y and Hyp/Y mice osteoblast, kidney, heart and liver membrane fractions revealed that casein kinase II activity in the Hyp/Y mice osteoblasts and kidney is only 35%-50%, respectively, of that of the +/Y mice tissues. The accumulated data are consistent with a post-translation defect in the Hyp/Y mouse osteoblast which results in the under-phosphorylation of osteopontin and subsequent under-mineralization of bone matrix.

Gestational exposure to ethanol suppresses msx2 expression in developing mouse embryos.

Ethanol acts as a teratogen in developing fetuses causing abnormalities of the brain, heart, craniofacial bones, and limb skeletal elements. To assess whether some teratogenic actions of ethanol might occur via dysregulation of msx2 expression, we examined msx2 expression in developing mouse embryos exposed to ethanol on embryonic day (E) 8 of gestation and subjected to whole mount in situ hybridization on E11-11.5 using a riboprobe for mouse msx2. Control mice exhibited expression of msx2 in developing brain, the developing limb buds and apical ectodermal ridge, the lateral and nasal processes, olfactory pit, palatal shelf of the maxilla, the eye, the lens of the eye, otic vesicle, prevertebral bodies (notochord), and endocardial cushion. Embryos exposed to ethanol in utero were significantly smaller than their normal counterparts and did not exhibit expression of msx2 in any structures. Similarly, msx2 expression, as determined by reverse transcription-PCR and Northern blot hybridization, was reduced approximately 40-50% in fetal mouse calvarial osteoblastic cells exposed to 1% ethanol for 48 hr while alkaline phosphatase was increased by 2-fold and bone morphogenetic protein showed essentially no change. Transcriptional activity of the msx2 promoter was specifically suppressed by alcohol in MC3T3-E1 osteoblasts. Taken together, these data demonstrate that fetal alcohol exposure decreases msx2 expression, a known regulator of osteoblast and myoblast differentiation, and suggest that one of the "putative" mechanisms for fetal alcohol syndrome is the inhibition of msx2 expression during key developmental periods leading to developmental retardation, altered craniofacial morphogenesis, and cardiac defects.

Comparison of 22-oxacalcitriol and 1,25(OH)2D3 on bone metabolism in young X-linked hypophosphatemic male mice.

Using a mouse model (Hyp) of human hypophosphatemic vitamin D-resistant rickets [X-linked hypophosphatemia (XLH)], we compared the effects of 22-oxa-1,25-dihydroxyvitamin D3 (OCT) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on restoring defects in mineral and skeletal metabolism. Hyp/Y mice received OCT or 1,25(OH)2D3 at doses of 0.05-0.25 micron.kg-1.day-1 for 4 wk. OCT normalized serum calcium levels, whereas 1,25(OH)2D3 produced hypercalcemia in Hyp/Y. OCT and 1,25(OH)2D3 also normalized serum phosphate levels and increased urinary calcium levels. Additionally, OCT and 1,25(OH)2D3 reduced elevated urinary pyridinoline levels and suppressed urinary adenosine 3',5'-cyclic monophosphate levels to normal. Bone ash content was low in Hyp/Y, and OCT was more effective than 1,25(OH)2D3 in reversing this defect. Histomorphometric analysis of bone turnover, mineralization rate, and osteoid content demonstrated comparable responses with OCT and 1,25(OH)2D3, although the highest dose of 1,25(OH)2D3 resulted in increased osteoid content and delayed mineralization. OCT appears to be more effective and definitely less toxic than 1,25(OH)2D3 in reversing skeletal lesions in Hyp/Y mice and may prove to be the drug of choice in the treatment of childhood XLH.

Production of interleukin-6 in human osteoblasts and human bone marrow stromal cells: evidence that induction by interleukin-1 and tumor necrosis factor-alpha is not regulated by ovarian steroids.

Studies in murine models of osteoporosis have suggested the hypothesis that ovarian steroids may control osteoclastic bone remodeling by limiting the production of interleukin-6 (IL-6) from osteoblasts and bone marrow stromal cells. To investigate this hypothesis in a human model, we have examined 12 separate strains of normal human osteoblasts (HOB) and 11 separate strains of human bone marrow stromal cells (HBMSC) and determined whether ovarian steroids regulate the induction of IL-6 by interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) or IL-1 + TNF. Treatment with IL-1, TNF or IL-1 + TNF resulted in the induction of IL-6 from both cell types with IL-1 + TNF inducing a synergistic induction of IL-6 in HOB (24- to 324-fold) and HBMSC (35-288 fold). Addition of 17 beta-estradiol or progesterone did not significantly alter IL-6 messenger RNA or protein levels in either HOB or HBMSC cultures stimulated with IL-1, TNF or IL-1 + TNF. Cultures incubated up to 96 h with the steroids did not affect IL-6 expression. Furthermore ovarian steroids did not affect IL-6 production in either HBMSC cultures representative of preosteoblasts or HOB cultures representative of highly differentiated osteoblasts. Specific chloramphenicol acetyl transferase assays and reverse transcriptase-polymerase chain reaction studies also demonstrated that the lack of an estrogen effect was not due to the failure of HOB to express functional estrogen receptors. Therefore, we conclude that the regulation of human osteoclastic bone remodeling by ovarian steroids does not occur through the direct regulation of IL-6 gene transcription or protein secretion in either early stages of osteoblast differentiation or the differentiated osteoblast.

Altered osteoblast gluconeogenesis in X-linked hypophosphatemic mice is associated with a depressed intracellular pH.

We have studied gluconeogenesis and intracellular pH levels in normal (+/Y) and X-linked hypophosphatemic (Hyp/Y) mice. Compared with +/Y littermates, Hyp/Y mouse osteoblasts showed a higher rate of glucose production from fructose (10-fold), glutamine, and malate, but no significant difference when alpha-ketoglutarate was used as substrate. The activities of the pentose cycle enzymes, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase, were not different in the two osteoblast preparations. Examination of intracellular pH (pHi) using the double excitation of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) revealed a significantly lower pHi in Hyp/Y mouse osteoblasts compared with +/Y mouse osteoblasts (7.01 +/- 0.03 n = 10 versus 7.15 +/- 0.04 n = 8, respectively; P < 0.05). These results show for the first time that osteoblasts are capable of glucose production and that glucose production is altered in the Hyp/Y mouse osteoblast. As altered gluconeogenesis has been associated with reduced intracellular pH in other systems, a similar mechanism may be operative in the Hyp/Y mouse osteoblast. The observed defects may be intrinsic to the Hyp phenotype as the alterations in intracellular pH and gluconeogenesis persisted in vitro, or they may represent impressed memory from the in vivo state and the presumed circulating factor that influences phosphate transport.

X-linked hypophosphatemic rickets and the murine Hyp homologue.

Recent studies have reported the cloning of several sodium-dependent phosphate cotransport proteins from the apical membrane of proximal tubules of several species. The human proximal tubule apical sodium-phosphate cotransport protein maps to chromosome 5 in the 5q35 region, indicating that this gene is not a candidate for the genetic defect leading to X-linked hypophosphatemia (XLH). Studies in what is thought to be the murine XLH homologue, Hyp, also indicate that the proximal tubular phosphate cotransporter gene does not map to the X chromosome. In Hyp, message levels for the apical membrane sodium cotransport protein are reduced by approximately 50%, similar to the reductions in the apical membrane protein levels of the transporter. This indicates a potential transcriptional defect in Hyp, leading to underexpression of the sodium-dependent phosphate transport protein. Recent studies in the Hyp osteoblast have characterized the intrinsic abnormalities of the cell leading to the osteomalacia characteristic of both Hyp and XLH. These studies demonstrate that the Hyp osteoblast expresses normal rates of phosphate transport, but altered gluconeogenesis similar to the proximal tubule, and that there is an underphosphorylation of an important matrix protein, osteopontin. Since osteopontin is involved in matrix mineralization, defective posttranslational modification of the protein could be a factor in producing the osteomalacia of the Hyp. Other recent studies have demonstrated improved modalities of treatment for Hyp and potentially for XLH. These involve the use of phosphate and nonhypercalcemic analogues of 1 alpha,25-dihydroxyvitamin D3. Thus, although the detection of the genetic defect producing XLH and Hyp is awaited, significant advances in the characterization of the phenotype and the bone abnormalities continue.

Phosphate transport in osteoblasts from normal and X-linked hypophosphatemic mice.

Human hypophophatemic vitamin D-resistant rickets (X-linked hypophosphatemia-XLH) is characterized by hypophosphatemia, a decreased tubular reabsorption of phosphate (P(i)) and defective skeleton mineralization. Utilizing a mouse model (Hyp) of XLH, which demonstrates biological abnormalities and skeletal defects of XLH, we analyzed sodium-dependent phosphate transport in isolated osteoblasts derived from the calvaria of normophosphatemic and hypophosphatemic mice. Initial rates of phosphate uptake by normal and Hyp osteoblasts showed similar slopes. Osteoblasts from both normal and Hyp mice exhibited saturable, sodium-dependent phosphate transport with apparent Vmax and Km values not significantly different (normal mice, Vmax = 24.30 +/- 3.45 nmol/mg prot. 10 min, Km = 349.49 +/- 95.20 mumol/liter; Hyp mice, Vmax = 23.03 +/- 3.41 nmol/mg prot. 10 min, Km = 453.64 +/- 106.93 mumol/liter, n = 24). No differences were found in the ability of normal and Hyp osteoblasts to respond to P(i) transport after 5 hours of P(i) deprivation. Both cell types exhibited a similar increase in cAMP in response to PTH. The accumulated results demonstrate that P(i) uptake and transport in normal and Hyp mouse osteoblasts is a sodium-dependent saturable process. As osteoblast P(i) uptake and transport is apparently normal in the Hyp mouse model of XLH, the "osteoblastic failure" described for the Hyp mouse should be attributed to other mechanism(s).

Expression of metalloproteinases and tissue inhibitors of metalloproteinases in human osteoblast-like cells: differentiation is associated with repression of metalloproteinase biosynthesis.

To more clearly define the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) within the human osteoblast (hOB) lineage, normal hOB and human osteogenic sarcoma cells possessing various levels of alkaline phosphatase (a marker of commitment to the osteoblast lineage) were treated with bone-resorbing agents to determine their effect on the production of interstitial collagenase, stromelysin, 72-kilodalton (kDa) gelatinase, 92-kDa gelatinase, TIMP-1, and TIMP-2. The results revealed that 1) normal hOB release copious amounts of 72-kDa gelatinase, TIMP-1, and TIMP-2; 2) hOB production of 72-kDa gelatinase and TIMP-2 is not regulated by agents that promote bone resorption (e.g. phorbol-12-myristate 13-acetate, recombinant human interleukin-1 beta, tumor necrosis factor-alpha, PTH, and vitamin D3); 3) normal hOB fail to secrete collagenase, stromelysin, or 92-kDa gelatinase when cultured on plastic or a type I collagen substratum, even in response to bone-resorptive agents or mononuclear cell-conditioned medium; 4) in contrast, certain of the osteogenic sarcoma cell populations produce collagenase, stromelysin, and 92-kDa gelatinase, especially when exposed to bone-resorbing stimuli; 5) in general, the capacity for metalloenzyme production by osteogenic sarcoma cell lines varies inversely with their alkaline phosphatase expression; and 6) the most committed (highest alkaline phosphatase) osteogenic sarcoma cell line, SAOS-2, precisely mimics the metalloproteinase profile of normal hOB. The results suggest that the expression of most metalloproteinases is under strict repression within the differentiated normal hOB, and cellular development is associated with diminished capacity to elaborate such enzymes.

Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone.

Human bone marrow stromal cells were examined for their osteogenic potential in an in vitro cell culture system. Dexamethasone (Dex) treatment induced morphological transformation of these cells from an elongated to a more cuboidal shape, increased their alkaline phosphatase activity and cAMP responses to PTH and prostaglandin E2, and was essential for mineralization of the extracellular matrix. Dex-induced differentiation of human bone marrow stromal cells was apparent after 2-3 days of treatment and reached a maximum at 7-14 days, as judged by alkaline phosphatase activity, although induction of osteocalcin by 1,25-dihydroxyvitamin D3 was attenuated by Dex. Withdrawal of Dex resulted in an enhancement of the 1,25-dihydroxyvitamin D3-induced secretion of osteocalcin, whereas alkaline phosphatase activity and the cAMP response to PTH remained at prewithdrawal levels. The steady state mRNA level of osteonectin was not affected by Dex. Our results, which demonstrate that Dex conditions the differentiation of human bone marrow osteogenic stromal cells into osteoblast-like cells, support the hypothesis of a permissive effect of glucocorticoids in ensuring an adequate supply of mature osteoblast populations. Furthermore, the established human bone marrow stromal cell culture provides a good model of an in vitro system to study the regulation of differentiation of human bone osteoprogenitor cells.

Monocytic secretion of interleukin-1 receptor antagonist in normal and osteoporotic women: effects of menopause and estrogen/progesterone therapy.

Interleukin-1 (IL-1), a potent stimulant of bone resorption, has been implicated in the pathogenesis of postmenopausal osteoporosis, because monocyte IL-1 bioactivity increases after the menopause and is decreased by estrogen and progesterone (EP) replacement. As IL-1 bioactivity reflect the production of both IL-1 and the IL-1 inhibitor, IL-1 receptor antagonist (IL-1ra), EP treatment could decrease IL-1 bioactivity by regulating the secretion of either IL-1 or IL-1ra. We now report that EP treatment in vivo decreased the secretion into the medium of cultured monocytes of IL-1ra and IL-1 beta as well as the IL-1 beta/IL-1ra ratio. We also found that in normal women the production of IL-1ra was within premenopausal levels in the first 7 yr after the menopause and increased linearly thereafter. In these women, monocyte IL-1 beta, IL-1 beta/IL-1ra ratio, and IL-1 bioactivity were all increased in the first 7 yr after the menopause and within the premenopausal range thereafter. In osteoporotic women, IL-1 beta, IL-1 beta/IL-1ra ratio, and IL-1 bioactivity increased after the menopause and returned to premenopausal levels after 15 yr from the menopause. In these women monocyte IL-1ra secretion was above the premenopausal range at all times after the menopause, but did not change with the passage of time since menopause. We conclude that hormone replacement decreases the in vitro secretion of both IL-1ra and IL-1 beta. The data also suggest that in normal women a progressive increase in the secretion of IL-1ra contributes to restore a normal IL-1/IL-1ra ratio after the menopause, a phenomenon which, in turn, may play a role in limiting postmenopausal bone loss.

Characterization of osteoblast-like cells from normal adult rat femoral trabecular bone.

Osteoblast-like cell cultures have been established from the trabecular surfaces of normal adult rat femoral trabecular bone. The cultured cells responded to stimulation by parathyroid hormone (rPTH), with a rise in intracellular cAMP in excess of 25-fold while failing to respond to incubation with sCT. Furthermore, the osteoblast-like cells exhibited a high level of alkaline phosphatase expression, both histochemically and biochemically. Incubations with 1,25(OH)2 vitamin D3 increased the alkaline phosphatase activity by 50% and stimulated bone Gla-protein (BGP) synthesis. When the cell layers were supplemented with both 50 micrograms/ml ascorbic acid and 10 mM beta-glycerophosphate and allowed to grow past confluency for 3 weeks, they formed calcified ridges and multilayered nodules. Confirmation of the mineralization of an extracellular matrix was made by von Kossa staining. This simple isolation technique now facilitates the availability of normal adult rat osteoblastic cells for investigation of bone and mineral metabolism.

Evidence for interleukin-1 beta production by cultured normal human osteoblast-like cells.

To determine if bone cells produce interleukin-1 beta (IL-1 beta), a potent bone resorption-stimulating agent, we studied well-characterized, nearly homogeneous cultures of normal human osteoblast-like (hOB) cells. With four strains of such cells, vehicle-treated cultures produced minimal IL-1 beta (mean +/- SEM, 1.3 +/- 0.3 pg/ml per 10(6) cells per 24 h) and showed dose-dependent (r = 0.99) increases to 2.2 +/- 0.7, 5.0 +/- 0.9, or 17.8 +/- 6.7 pg/ml, respectively, after treatment with lipopolysaccharide (LPS) at 3, 10, or 30 micrograms/ml (for increases after 10 and 30 micrograms/ml treatments, P less than 0.05). After treatment with tumor necrosis factor alpha (TNF-alpha) at 10 U/ml, IL-1 beta increased to 16.2 +/- 3.7 pg/ml (P less than 0.05). Neither 17 beta-estradiol nor bovine parathyroid hormone(1-34) (each at 10 nM), alone or in combination with LPS or TNF-alpha, affected IL-1 beta release. Northern blot analysis of total cellular RNA preparation revealed a single hybridization band at 1.9 kb when probed with a partially deleted cDNA for human IL-1 beta. The steady-state IL-1 beta mRNA levels showed a significant increase with LPS treatment and a lesser increase with TNF-alpha treatment in hOB cells. Moreover, TNF-alpha produced an even greater increase in IL-1 mRNA in HOBIT cells, a well-differentiated clonal cell line derived from normal hOB cells transfected with the SV40 large T antigen. We conclude that human cells of the osteoblast lineage produce IL-1 beta in response to well-recognized stimuli for IL-1 release from responsive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone matrix constituents stimulate interleukin-1 release from human blood mononuclear cells.

To test the hypothesis that mononuclear cells are stimulated to release interleukin 1 (IL-1) by bone fragments released in the bone microenvironment during the remodeling cycle, we have investigated the effects of bone matrix and some of its constituents on IL-1 secretin from peripheral blood mononuclear cells (PBMC). Increases in IL-1 activity were observed when either PBMC or adherent monocytes, but not lymphocytes depleted of monocytes, were co-cultured with either human or rat bone particles but not with latex particles of similar size. Co-culture of PBMC with bone particles in a transwell system where the cells were physically separated from the bone particles, or with osteoblast- or osteoclast-covered bone particles, did not stimulate IL-1 release, indicating that a physical contact between PBMC and the bone surface is required for eliciting IL-1 release. This was confirmed by the finding of a lower stimulatory effect of bone particles pretreated with etidronate, a bisphosphonate which decreases the bone binding capacity of PBMC. Constituents of bone matrix, such as collagen fragments, hydroxyproline, and, to a lesser extent, transforming growth factor-beta, but not osteocalcin, alpha 2HS glycoprotein, fragments of either bone sialoprotein or osteopontin, and fibronectin, stimulated PBMC IL-1 release in a dose-dependent fashion. Collagen-stimulated IL-1 release was partially and specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1-integrin cell surface collagen receptor. These data demonstrate that products of bone resorption, known to be chemotactic for mononuclear cells, stimulate PBMC IL-1 activity. These findings may help explain previous documentation of increased IL-1 secretion by circulating monocytes obtained from patients with high turnover osteoporosis.

Increased monocyte interleukin-1 activity and decreased vertebral bone density in patients with fasting idiopathic hypercalciuria.

Idiopathic hypercalciuria (IH) is a heterogeneous disorder frequently observed in patients with nephrolithiasis. At one extreme of its clinical spectrum is fasting hypercalciuria (FH), a condition characterized by increased bone resorption and turnover. In previous studies we have shown that monocytes from patients with high turnover osteoporosis and from women in early postmenopause elaborate increased amounts of interleukin-1 (IL-1), a cytokine that stimulates bone resorption in vitro and in vivo. Since IL-1 could also mediate the resorptive mechanism of FH and cause a clinically significant bone loss, we have studied the relationship of IH, vertebral mineral density, bone turnover, and monocyte IL-1 activity in 47 patients with absorptive hypercalciuria (AH), 23 with FH, and 38 nonhypercalciuric subjects with recurrent nephrolithiasis (controls). Vertebral mineral density, as measured by quantitative computer tomography, was decreased in each of the three patient groups, but was significantly lower in FH patients than in AH patients or control subjects. Twenty-four-hour total urinary hydroxyproline excretion was increased in FH patients compared to that in AH patients or controls, but blood levels of osteocalcin were not. Monocytes from FH subjects yielded significantly more IL-1 (alpha + beta) activity than those from AH patients or controls; levels of IL-1 activity in monocytes of AH and control patients were similar. In IH subjects, significant correlations were found between IL-1 and hydroxyproline (r = 0.70; P less than 0.0001), IL-1 and quantitative computer tomography values (r = -0.49; P less than 0.005), and IL-1 and urinary calcium (r = -0.36; P less than 0.05). Serum PTH levels were within normal limits in all subjects and were similar in the three study groups, 1,25-Dihydroxyvitamin D3 levels, although higher in IH patients than in controls, were not significantly different in FH and AH subjects. Increased IL-1 activity and decreased vertebral mineral density are features of a subset of patients with IH. Although a cause-effect relationship remains to be established, increased monocytic IL-1 activity, rather than elevated PTH or 1,25-dihydroxyvitamin D3 levels, could underlie the resorptive component of FH.