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Myeloma cells destroy bone by producing an osteoclast-stimulating factor that has chemical and biological characteristics similar to the bone-resorbing activity present in the supernatants of activated leukocyte cultures. Recently, a number of bone-resorbing leukocyte cytokines have been identified, including interleukin-1, lymphotoxin, and tumor necrosis factor. We have examined the products of human myeloma cells for the presence of these bone-resorbing cytokines. In a tumor cell line derived from a patient who had myeloma with osteolytic bone lesions and hypercalcemia, we found that the myeloma cells induced bone-resorbing activity and cytotoxic activity in vitro. Most of the bone-resorbing activity and all cytotoxic activity were suppressed by neutralizing antibodies to lymphotoxin. The myeloma cells expressed both lymphotoxin and tumor necrosis factor mRNA, but no tumor necrosis factor could be detected in the cell-culture medium. Interleukin-1 mRNA was not detected in the myeloma cells, and biologic activity of interleukin-1 was not measurable in the medium harvested from the cultured cells. The bone-resorbing activity induced by recombinant tumor necrosis factor and recombinant interleukin-1 was not affected by treatment with the lymphotoxin antibodies. When lymphotoxin was infused subcutaneously into normal mice (10 micrograms per day for three days), their plasma calcium levels increased. We also evaluated four established cell lines derived from three other patients with myeloma, and found a similar pattern of lymphotoxin expression in each. It appears that production of the bone-resorbing cytokine lymphotoxin is related to osteoclastic bone destruction and hypercalcemia in patients with myeloma.

Transforming growth factor beta (TGF-beta) is one of the most abundant of the known growth regulatory factors stored within the bone matrix. When bone is resorbed, TGF-beta is released in an active form and is a powerful bone growth stimulant. When injected into the subcutaneous tissue over the calvarial surface of rodents, it rapidly causes proliferation of the periosteal layer and accumulation of new woven bone. In this report, we describe the effects of TGF-beta 1 on first subcultures of fetal rat osteoblasts obtained from calvarial bones and cultured from confluence with ascorbic acid and beta-glycerophosphate. Under these conditions, nodules with characteristics of normal bone appear by day 8. Similar to experiments described by Antosz et al., TGF-beta added to confluent cultures inhibited the formation of bone nodules. Both the number and total area of the nodules were quantitated and shown to be completely inhibited by 2 ng/ml of TGF-beta 1. TGF-beta also impaired the expression of genes associated with bone formation, including type I collagen, alkaline phosphatase, osteopontin, and osteocalcin. TGF-beta also inhibited the expression of mRNA for the bone morphogenetic protein 2 (BMP-2). These results, showing suppression of markers representative of osteoblast differentiation, suggest that the effects of TGF-beta to stimulate bone formation in vivo are not likely a result of effects on differentiated mineralizing osteoblasts but, as suggested by previous studies, more likely are caused by effects on osteoblast precursors. These results also suggest that endogenous BMP-2 expression in fetal rat calvaria cells is important for bone cell differentiation.

The immune cell products interleukins la and -13, which stimulate osteoclast activity in vitro, are among the most potent bone resorbing factors so far described. Although it appears likely that these cytokines are involved in regulation of trabecular bone turnover, nothing is known of their effects on extracellular fluid calcium concentration. In this report, we show the effects of 72-hr subcutaneous infusions of interleukins la and -(3 on plasma calcium and bone morphology in mice. Both interleukins 1 caused a marked dosedependent increase in the plasma calcium. In higher doses, which cause the animals to die, the plasma calcium fell preterminally. Quantitative histomorphometry of bone sections showed evidence of increased numbers of osteoclasts and bone resorption surfaces. The effects were similar to those obtained with infusions of parathyroid hormone. These data suggest that in addition to its potential influence on trabecular bone volume, interleukin 1 may also modulate extracellular flulid calcium homeostasis under conditions in which it is produced excessively.Immune cell products cause alterations in bone cell function and are likely regulators of trabecular bone turnover. Recently, the cytokines interleukin 1 (IL-1) and tumor necrosis factor, both of which are produced by cells in the monocytemacrophage lineage, have been shown to resorb bone in vitro (1-4). Since production of bone resorbing cytokines has been linked to the hypercalcemia associated with hematologic malignancies, such as myeloma and some lymphomas (5, 6), we tested the effects of infusions of recombinant human IL-la and -/3 on calcium homeostasis in vivo. We found that both recombinant human IL-ls markedly increased the plasma calcium in vivo and caused a simultaneous increase in osteoclastic bone resorption. MATERIALS AND METHODSWe obtained recombinant and purified human IL-1 from a number of sources. Recombinant human IL-1l3 expressed in Escherichia coli was generously provided by Biogen (Geneva) via Jean-Michel Dayer and Jean-Phillippe Bonjour. The specific activity of this material was 2-4 x 107 units/mg in the thymocyte proliferation assay and mononuclear cell assay (7). This material was shown to be homogeneously pure by several analytical methods and partial amino acid sequence analysis (7). A separate preparation of recombinant human IL-1,B expressed in E. coli was kindly provided by D. R. Bertolini (Otsuka Pharmaceutical, Rockville, MD). The specific activity of this preparation was 2.5 x 108 units/mg in the thymocyte proliferation assay. We also verified the purity of this preparation by NaDodSO4/PAGE and immunoblotting with antisera specific for IL-1f3. Recombinant human IL-la expressed in E. coli was obtained from Peter Lomedico (Roche Research Institute, Hoffmann-LaRoche, Nutley, NJ). The protein was similarly shown to be pure by NaDodSO4/PAGE and had a specific activity of 5 X 106 units/mg in the thymocyte proliferation assay. Bacterial endotoxin for all three IL-1 preparations was measured by the Limulus amebocy...

Objective. To study the effects of a matrix metalloproteinase (MMP) inhibitor (S-34219) on osteoarthritis (OA) cartilage cultures and in the meniscectomized guinea pig model of OA.Methods. The inhibitory activity of S-34219 on MMPs and aggrecanase was studied by fluorimetry and immunoassay, respectively. The effects of S-34219 on proteoglycan and collagen degradation were studied in cultures of rabbit and human cartilage. Medial meniscectomy was performed on 29 Hartley male guinea pigs, and these animals were randomly allocated to 1 of 3 groups: a control meniscectomized group (MNXc) receiving the vehicle, or a meniscectomized group receiving either 10 mg/kg or 20 mg/kg S-34219, administered twice per day by oral gavage for 12 weeks from day 1 after surgery. An additional group comprised shamoperated animals. Tibial cartilage from the operated left knee was processed for histologic assessment of OA lesions.Results. The 50% inhibitory concentration (IC 50 ) of S-34219 on MMPs 1, 2, 3, 8, 9, and 13 was 55, 0.1, 0.5, 0.1, 0.03, and 0.2 nM, respectively; the IC 50 on aggrecanase 1 was 190 nM. In cultured rabbit cartilage, 100 nM S-34219 strongly inhibited MMP-dependent degradation of collagen and proteoglycans. A concentration 100 times higher was needed to inhibit aggrecanasedependent degradation. In cultures of human OA cartilage, 100 nM S-34219 inhibited spontaneous type II collagen degradation by 66% and proteoglycan degradation by only 22%. For in vivo studies, treated groups were compared with the MNXc group and the results, expressed as the percentage variation versus MNXc, were as follows: in the 10 and 20 mg/kg groups, a significant decrease (P < 0.05) in global histologic score (؊12% and ؊14%, respectively) was observed, and this was associated with a significant increase (P < 0.05) in cartilage thickness (؉19% and ؉18%, respectively). Neither dose level changed the proteoglycan content.Conclusion. In both treated animal groups, S-34219 significantly prevented the loss of cartilage thickness, probably by inhibiting collagen breakdown that normally leads to the erosion of fibrillated superficial areas. The absence of a protective effect on glycosaminoglycan loss, both in vitro and in vivo, suggests that aggrecanases may have an important role in cartilage loss. This study reinforces the relevance of these models for testing chondroprotective drugs, and the potential role of dual inhibitors of collagenase and aggrecanase as disease-modifying drugs in the management of OA.Osteoarthritis (OA) is a major joint disease in humans and various animals in which articular cartilage degenerates over a period of time and leads to denudation of the joint surface. This degeneration is mainly characterized by a progressive degradation of extracellular matrix (ECM) components, followed by chondrocyte death, tissue fibrillation, and erosion (1). Metalloproteinases of 2 families seem to play a major role in cartilage ECM breakdown. These are 1) aggrecanases, which, starting at an early stage of disease, specifically degrade ...