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There does not exist an entirely satsifactory drug for patients with established osteoporosis. No currently available drug is either universally acceptable or leads to substantial formation of new bone. Moreover, in spite of the recent introduction of newer bisphosphonates, intranasal calcitonin and estrogen-related compounds, there is still room for new preventive treatments for bone loss associated with the menopause and hormone withdrawal. There is an even greater need for acceptable and efficacious therapies for patients with established osteoporosis who have severe bone deficits. This is the reason that the pharmaceutical industry is now spending billions of dollars in a search for better modalities of treatment than those currently available. Specific problems can be found with all existing therapies, and drugs under development. The task that confronts the industry in identifying new therapies is particularly difficult. Although bringing a new drug to the market is very expensive in any pharmaceutical arena, it is particularly so in osteoporosis. This is because the Food and Drug Administration in the United States and many other regulatory agencies require evidence of beneficial effects on fracture rates. This problem is compounded somewhat by the expense involved in clinical studies in osteoporosis (requiring large numbers of patients from multiple centers), and the decline in research and development budgets associated with reduced profits due to managed care and price controls on drugs in some countries. Establishing practice guidelines for osteoporosis may also limit the use of drugs, particularly those that are expensive, or require treatment in many patients to see benefits in one. In the European Community, price controls on drugs already exist in many countries. A pharmaceutical company entering this field must consider the size of the therapeutic market, as well as the competition. For example, the bisphosphonate market is likely to be shared by a number of similar compounds during the next decade, and the same appears likely for estrogenrelated compounds. The lag time between discovery of a new lead for osteoporosis and the introduction of a new drug in the clinic may be 10–15 years. This certainly seems to have been the experience with resorption inhibitors such as alendronate and raloxifene. This means that there must be an enormous investment by the pharmaceutical company, possibly in the order of $500 million in today’s dollars, so that the eventual market must be substantial to accommodate this risk.

We have reported that P388D1 cell line murine macrophages stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans release interleukin-1 (IL-1) inhibitor. The IL-1 inhibitor was purified from conditioned media of P388D1 cells stimulated with A. actinomycetemcomitans LPS for 72 h to homogeneity by a four-step procedure: acetic acid extraction from conditioned media; Bio-Gel P-60 gel filtration chromatography; DEAE-Sepharose CL-6B column chromatography; and reverse-phase high-performance liquid chromatography on a C18 hydrophobic support. The purified IL-1 inhibitor gave a single band of protein with a molecular mass of 26 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified IL-1 inhibitor was a heat- and acid-stable protein that was inactivated by digestion with trypsin and reduction with dithiothreitol. This inhibitory factor suppressed the proliferation of C3H/HeJ mouse thymocytes and the proliferation of IL-1-dependent cell lines, D10.G4.1 and RPMI 1788, induced by IL-1. However, this inhibitor did not affect the proliferation of IL-2-dependent CTLL-2 cells induced by IL-2, the proliferation of C3H/HeJ mouse thymocytes stimulated with a mitogenic dose of concanavalin A, and the proliferation of IL-6-dependent B9 cells induced by IL-6. Furthermore, the IL-1 inhibitor significantly blocked stimulation of bone resorption in organ cultures of newborn mouse calvaria and inhibited the osteoclast-like cell formation in mouse marrow cultures. A monoclonal antibody prepared against the purified IL-1 inhibitor reacted with mouse recombinant IL-1 receptor antagonist (rIL-1ra), and a polyclonal antibody to mouse rIL-1ra reacted with the IL-1 inhibitor by Western blot (immunoblot) analysis. These results indicate that the IL-1 inhibitor is an identical molecule to rIL-1ra, suggesting that the IL-1 inhibitor (IL-1ra) released by macrophages stimulated with LPS from A. actinomycetemcomitans may play an important mediative role in the development of periodontal disease.

Bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used as a beneficial agent for the treatment of bone metastases in patients with breast cancer. It is well recognized that BP reduces osteolysis by promoting apoptosis in osteoclasts. However, recent animal and human data suggest that BPs not only reduce osteolysis associated with metastatic breast cancer, but also decrease tumor burden in bone. The mechanisms by which tumor burden is decreased following BP administration are unknown. Here we examined the effects of the BP ibandronate on MDA-231 human breast cancer cells in bone metastases in a well-characterized animal model of bone metastasis. Ibandronate, which was administered (s.c. daily; 4 microg/mouse/day) after bone metastases were established, inhibited the progression of established osteolytic bone metastases as assessed by radiographic analysis. Histological and histomorphometrical examination revealed that ibandronate reduced osteoclastic bone resorption, with increased apoptosis in osteoclasts. Furthermore, ibandronate also significantly decreased the MDA-231 tumor burden, with increased apoptosis in MDA-231 breast cancer cells in bone metastases. In contrast, ibandronate failed to inhibit MDA-231 tumor formation with no effects on apoptosis in MDA-231 breast cancer cells in the orthotopic mammary fat pads. These data suggest that the effects of ibandronate on apoptosis in MDA-231 breast cancer cells are restricted in bone in which ibandronate selectively deposits. Consistent with these in vivo results, a relatively high concentration of ibandronate (100 microM) increased caspase-3 activity and induced DNA fragmentation in MDA-231 breast cancer cells in culture. Moreover, a caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone, blocked ibandronate-induced DNA fragmentation in MDA-231 cells, suggesting an involvement of caspase-3 in ibandronate-induced apoptosis. Our results suggest that BP suppresses bone metastases through promotion of apoptosis in metastatic cancer cells as well as in osteoclasts. However, it still remains open whether BP has direct anticancer actions in vivo.

Myeloma is a unique hematologic malignancy that exclusively homes in the bone marrow and induces massive osteoclastic bone destruction presumably by producing cytokines that promote the differentiation of the hematopoietic progenitors to osteoclasts (osteoclastogenesis). It is recognized that neighboring bone marrow stromal cells influence the expression of the malignant phenotype in myeloma cells. This study examined the role of the interactions between myeloma cells and neighboring stromal cells in the production of osteoclastogenic factors to elucidate the mechanism underlying extensive osteoclastic bone destruction. A murine myeloma cell line 5TGM1, which causes severe osteolysis, expresses alpha(4)beta(1)-integrin and tightly adheres to the mouse marrow stromal cell line ST2, which expresses the vascular cell adhesion molecule-1 (VCAM-1), a ligand for alpha(4)beta(1)-integrin. Co-cultures of 5TGM1 with primary bone marrow cells generated tartrate-resistant acid phosphatase-positive multinucleated bone-resorbing osteoclasts. Co-cultures of 5TGM1 with ST2 showed increased production of bone-resorbing activity and neutralizing antibodies against VCAM-1 or alpha(4)beta(1)-integrin inhibited this. The 5TGM1 cells contacting recombinant VCAM-1 produced increased osteoclastogenic and bone-resorbing activity. The activity was not blocked by the neutralizing antibody to known osteoclastogenic cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor, or parathyroid hormone-related peptide. These data suggest that myeloma cells are responsible for producing osteoclastogenic activity and that establishment of direct contact with marrow stromal cells via alpha(4)beta(1)-integrin/VCAM-1 increases the production of this activity by myeloma cells. They also suggest that the presence of stromal cells may provide a microenvironment that allows exclusive colonization of myeloma cells in the bone marrow. (Blood. 2000;96:1953-1960)

Solid cancers metastasize to bone by a multistep process that involves interactions between tumor cells and normal host cells. Some tumors, most notably breast and prostate carcinomas, grow avidly in bone because the bone microenvironment provides a favorable soil. In the case of breast carcinoma, the final step in bone metastasis (namely bone destruction) is mediated by osteoclasts that are stimulated by local production of the tumor peptide parathyroid hormone-related peptide (PTH-rP), whereas prostate carcinomas stimulate osteoblasts to make new bone. Production of PTH-rP by breast carcinoma cells in bone is enhanced by growth factors produced as a consequence of normal bone remodeling, particularly activated transforming growth factor-beta (TGF-beta). Thus, a vicious cycle exists in bone between production by the tumor cells of mediators such as PTH-rP and subsequent production by bone of growth factors such as TGF-beta, which enhance PTH-rP production. The metastatic process can be interrupted either by neutralization of PTH-rP or by rendering the tumor cells unresponsive to TGF-beta, both of which can be accomplished experimentally. The osteoclast is another available site for therapeutic intervention in the bone metastatic process. Osteoclasts can be inhibited by drugs such as the new-generation bisphosphonates; as a consequence of this inhibition, there is a marked reduction in the skeletal events associated with metastatic cancer to bone, such as pain, fracture, and hypercalcemia. However and possibly even more importantly, there is also a reduction of tumor burden in bone. In experimental situations, this has clearly been shown to affect not only morbidity but also survival. The precise mechanism by which bisphosphonates inhibit osteoclasts is still unclear and may represent a combination of inhibition of osteoclast formation as well as increased apoptosis in mature osteoclasts. However, studies with potent bisphosphonates such as ibandronate, pamidronate, and risedronate have clearly documented that reduction of bone turnover and osteoclast activity leads to beneficial effects not only on skeletal complications associated with metastatic cancer, but also on tumor burden in bone.

Humoral hypercalcemia of malignancy results from the effects of tumor-produced factors on bone, kidney, and intestine that disrupt normal calcium homeostasis. Although parathyroid hormone-related protein (PTHrP) is a major mediator of the syndrome, tumors also produce other hypercalcemic factors, such as tumor necrosis factor (TNF), which may modulate the effects of PTHrP. It has been postulated that TNF may counteract the stimulatory effects of PTHrP on bone formation. To examine the effects of TNF on PTHrP-induced changes in calcium and bone metabolism, a murine tumor model of hypercalcemia was used. Nude mice were inoculated with Chinese hamster ovarian (CHO) cells expressing human TNF (CHO/TNF) or nontransfected CHO cells (CHO/-) and further treated with injections of human PTHrP(1-34) or vehicle. The effects of TNF, PTHrP, and the combination of the two factors on blood ionized calcium, osteoclast recruitment, and bone histomorphometry were evaluated. Mice bearing CHO/TNF tumors that were injected with PTHrP had significantly higher calcium concentrations, increased committed osteoclast progenitors, and mature osteoclasts as well as enhanced bone resorption compared with mice bearing CHO/TNF tumors injected with vehicle or those bearing CHO/- tumors injected with PTHrP or vehicle. A 2-fold increase in new woven bone formed in the calvaria at sites of previous bone resorption was observed in CHO/TNF mice treated with PTHrP. Bone formation rates in the vertebrae were similar in both CHO/- and CHO/TNF mice treated with PTHrP. These data demonstrate that the hypercalcemic effects of PTHrP are enhanced by TNF and that this effect is due to the increased production of committed osteoclast precursors with a subsequent increase in osteoclastic bone resorption. Furthermore, PTHrP caused a coupled increase in osteoclastic bone resorption and new bone formation that was not inhibited by TNF. These findings highlight the complex interactions that may occur between tumor-produced factors on bone that result in malignancy-associated hypercalcemia and suggest that TNF may not be responsible for the decreased bone formation seen in some patients with this condition.

Osteolytic bone destruction and its complications, bone pain, pathologic fractures, and hypercalcemia, are a major source of morbidity and mortality in patients with multiple myeloma. The bone destruction in multiple myeloma is due to increased osteoclast (OCL) activity and decreased bone formation in areas of bone adjacent to myeloma cells. The mechanisms underlying osteolysis in multiple myeloma in vivo are unclear. We used a human plasma cell leukemia cell line, ARH-77, that has disseminated growth in mice with severe combined immunodeficiency (SCID) and expresses IgG kappa, as a model for human multiple myeloma, SCID mice were irradiated with 400 rads and mice were injected either with 10(6) ARH-77 cells intravenously (ARH-77 mice) or vehicle 24 hours after irradiation. Development of bone disease was assessed by blood ionized calcium levels, x-rays, and histology. All ARH-77, but none of control mice that survived irradiation, developed hind limb paralysis 28 to 35 days after injection and developed hypercalcemia (1.35 to 1.46 mmol/L) a mean of 5 days after becoming paraplegic. Lytic bone lesions were detected using x-rays in all the hypercalcemic mice examined. No lytic lesions or hypercalcemia developed in the controls. Controls or ARH-77 mice, after developing hypercalcemia, were then killed and bone marrow plasma from the long bones were obtained, concentrated, and assayed for bone-resorbing activity. Bone marrow plasma from ARH-77 mice induced significant bone resorption in the fetal rat long bone resorption assay when compared with controls (percentage of total 45Ca released = 35% +/- 4% v 11% +/- 1%). Histologic examination of tissues from the ARH-77 mice showed infiltration of myeloma cells in the liver and spleen and marked infiltration in vertebrae and long bones, with loss of bony trabeculae and increased OCL numbers. Interestingly, cultures of ARH-77 mouse bone marrow for early OCL precursors (colony-forming unit-granulocyte-macrophage [CFU-GM]) showed a threefold increase in CFU-GM from ARH-77 marrow versus controls (185 +/- 32 v 40 +/- 3 per 2 x 10(5) cell plated). Bone-resorbing human and murine cytokines such as interleukin-6 (IL-6), IL-1 alpha or beta, TGF-alpha, lymphotoxin, and TNF alpha were not significantly increased in ARH-77 mouse sera or marrow plasma, compared with control mice, although ARH-77 cells produce IL-6 and lymphotoxin in vitro. Conditioned media from ARH-77 cells induced significant bone resorption in the fetal rat long bone resorption assay when compared with untreated media (percentage of total 45Ca released = 22% +/- 2% v 11% +/- 1%). This effect was not blocked by anti-IL-6 or antilymphotoxin (percentage of total 45Ca released = 19% +/- 1% and 22% +/- 1%, respectively). Thus, we have developed a model of human multiple myeloma bone disease that should be very useful to dissect the pathogenesis of the bone destruction in multiple myeloma.

Porcine pancreatic extracts (PXs) have previously been shown to decrease blood ionized calcium in BALB/c mice (T. Yoneda, Y. Takaoka, and G. R. Mundy. FEBS Lett., 278: 171-174, 1991). In the present study, we show that the PX is effective in preventing progression of hypercalcemia and decreasing osteoclastic bone resorption associated with a human squamous carcinoma in nude mice. PX inhibited osteoclast-like cell formation in mouse bone marrow cultures and bone resorption in organ cultures of fetal rat long bones which had been stimulated by serum-free culture supernatants of this cancer. In addition, PX increased food intake, decreased weight loss, and prevented development of cachexia. In parallel with these effects, PX prolonged survival of tumor-bearing animals. PX might have therapeutic potential for management of hypercalcemia and cachexia associated with malignancy.

Bone resorption requires cooperation between osteoclasts and mononuclear accessory cells by mechanisms which have not been elucidated. Since multinucleated cells in giant cell tumors of bone have many phenotypic and functional characteristics of normal osteoclasts, we have examined the interaction between the bone-resorbing multinucleated cells and the distinct mononuclear stromal cells from these tumors. We have found that these mononuclear cells produce an activity which stimulates both giant cells from giant cell tumors and rodent osteoclasts to resorb bone in vitro. We have identified the activity and found that it represents several products of the 5-lipoxygenase pathway of arachidonic acid metabolism, namely 5-hydroxyeicosatetraenoic acid and the leukotrienes. These data indicate that 5-lipoxygenase metabolites stimulate isolated osteoclasts to resorb bone in vitro and may represent a mechanism by which mononuclear stromal cells in human giant cell tumors communicate with the giant cells. In addition, these results may explain a possible mechanism for communication between accessory cells and osteoclasts involved in normal bone resorption.

The integrity of the skeleton is maintained by the continued cellular remodelling of bone that occurs throughout life and is characterized by an orderly sequence of events beginning with osteoclastic bone resorption followed by osteoblastic new bone formation to repair the localized defects made by osteoclasts. Bone resorption and formation are closely coupled by mechanisms which, until recently, have been poorly defined in both normal physiological and most pathological conditions, but are probably due to the generation during resorption of local 'coupling' factors. TGF-beta promises to be one of the key factors involved in coupling bone formation to previous bone resorption. This potent osteotropic polypeptide is abundant in the bone matrix, and is produced in response to factors that stimulate osteoclastic bone resorption. It is a very potent stimulator of osteoblastic bone formation, causing chemotaxis, proliferation and differentiation in committed osteoblasts. TGF-beta has complex effects on bone resorption: it inhibits osteoclast formation and osteoclast activity. TGF-beta is released from bone in a biologically inert state that is due to the presence of at least two proteins which appear to regulate TGF-beta activity. Release of active TGF-beta from these latent complexes occurs during bone resorption and is mediated by osteoclasts. Knowledge of the mechanisms responsible for these activation processes may be vital to understanding the role of TGF-beta in bone remodelling.

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Osteoinductive factor (OIF) is a glycoprotein in bone that induces ectopic bone formation. Implantation of OIF plus transforming growth factor beta (TGF-beta) type 1 or 2 into subcutaneous tissues of rats induces formation of bone at the implantation site. Since TGF-beta is also present in bone matrix and inhibits formation of multinucleated cells that express an osteoclast phenotype in long-term human marrow cultures, we tested the effects of OIF on formation of these osteoclast-like cells to determine the effects of OIF on cells in the osteoclast lineage. We found that OIF inhibited total multinucleated cell (MNC) formation in a dose-dependent fashion and preferentially inhibited formation of MNCs that react with monoclonal antibody 23c6 (23c6-positive MNCs), an antibody that identifies osteoclasts. In addition, low concentrations of OIF in combination with low concentrations of TGF-beta acted synergistically to inhibit 23c6-positive MNC formation. The inhibition of 23c6-positive MNC formation by OIF was not mediated by prostaglandin synthesis. These data suggest that regulatory growth factors, such as OIF or TGF-beta, that are stored within the bone matrix and released when bone is resorbed can serve as natural inhibitors of osteoclast activity by inhibiting osteoclast formation.

The calcitonin gene-related peptide (CGRP) is a peptide which normally circulates. It is encoded by the calcitonin gene, whose precise function is unknown. Since it has recently been shown that human CGRP (hCGRP) lowers plasma calcium levels in both the rat and the rabbit, we examined the in vitro effects of human synthetic CGRP on bone resorption (as measured by 45Ca release) stimulated by PTH, prostaglandin E2, and 1,25-dihydroxyvitamin D3. CGRP caused a dose-dependent inhibition of PTH-stimulated resorption, with 50% inhibition at approximately 5 X 10(-8) M CGRP. The inhibitory effects of CGRP on PTH-mediated bone resorption were not due to toxic effects, since bones preincubated with CGRP for 48 h were subsequently able to respond to PTH. The inhibitory activity of CGRP in the rat was approximately 3 orders of magnitude less potent than that of human calcitonin. In contrast to the effects of calcitonin, a marked inhibition of PTH-stimulated bone resorption was still observed after 96 h in the continued presence of CGRP. CGRP (10(-6)-10(-8) M) also inhibited resorption stimulated by prostaglandin and 1,25-dihydroxyvitamin D3 in a dose-dependent manner, but had no significant effect on basal bone resorption. In conclusion, these data show that hCGRP inhibits hormone-stimulated bone resorption in vitro. Although it is less potent than calcitonin in the rat, CGRP has been shown to have potency comparable to that of calcitonin in other species, and therefore, a role for CGRP as a therapeutic agent in states of increased bone resorption cannot be ruled out.

We developed a mouse bone marrow culture system to examine the process of osteoclast-like multinucleated cell formation from its progenitors. When mouse marrow cells were cultured for 8 days with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3, 10(-10) to 10(-7) M] or human PTH (1-34) (25-100 ng/ml), tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells formed. No TRACP-positive multinucleated cells appeared in the absence of these hormones. 1 alpha,25-(OH)2D3 and PTH also increased the number of the clusters of TRACP-positive mononuclear cells. Time course studies showed that these TRACP-positive mononuclear cell clusters appeared before the formation of TRACP-positive multinucleated cells, suggesting that the TRACP-positive mononuclear cells are precursors of the multinucleated cells. Salmon calcitonin markedly inhibited the formation of TRACP-positive multinucleated cells but not TRACP-positive mononuclear cell clusters induced by 1 alpha,25-(OH)2D3 or PTH. TRACP-positive mononuclear cells and multinucleated cells were rarely stained for nonspecific esterase, but some mononuclear cells were positively stained for both nonspecific esterase and TRACP. More that 90% of the TRACP-positive mononuclear cell clusters and multinucleated cells were found near colonies of alkaline phosphatase-positive mononuclear cells (possibly osteoblasts). When marrow mononuclear cells were cultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 or PTH, numerous resorption lacunae were formed. These results suggest that 1) TRACP-positive multinucleated cells formed in response to osteotropic hormones in mouse marrow cultures satisfy most of the criteria of osteoclasts, and 2) osteoblasts may play an important role in osteoclast formation.

A reliable method for obtaining osteoclast activating factor (OAF) in the culture medium of phytohemagglutinin-activated peripheral blood leukocytes is described. OAF was detected by its ability to stimulate resorption of fetal rat bone in organ culture. Most bone resorbing activity was released by activated leukocytes during the first 24 hours of culture, well before -3H-thymidine incorporation was increased. Cultures maintained for longer than 3 days showed a decrease in OAF activity. Stimulated leukocytes cultured in medium with as little as 0.01 per cent plasma showed increased -3H-thymidine incorporation and released as much OAF as leukocytes cultured with 20 per cent plasma. OAF release was stimulated by pokeweed mitogen and concanavalin A as well as by phytohemagglutinin.

Interferon-gamma (IFN-gamma) inhibits osteoclastic bone resorption in vitro, but the mechanism responsible for this inhibition is unknown. We have used a long-term human marrow culture system that forms multinucleated cells (MNC) with osteoclast characteristics to test the effect of recombinant human IFN-gamma on MNC formation. The addition of 1,25-dihydroxy-vitamin D3 (1,25D3) at 10(-8) M to these cultures significantly increased both MNC formation and the number of nuclei per MNC. IFN-gamma at 100 U/ml strongly inhibited both of these effects of 1,25D3 in this system. IFN-gamma significantly inhibited MNC formation at very low concentrations (4 U/ml), with 10 U/ml inhibiting 1,25D3-stimulated MNC formation by 50%. In contrast, 100 U/ml of IFN-gamma were required to inhibit the growth of granulocyte-macrophage colony-forming cells, the probable progenitor for MNC, by 50%. Treatment of cultures with IFN-gamma for only the first or last week of culture significantly inhibited MNC formation stimulated by 1,25D3. Autoradiographic studies with [3H]thymidine showed that IFN-gamma did not inhibit proliferation of precursors for MNC. Additionally, IFN-gamma inhibited MNC formation stimulated by parathyroid hormone or interleukin 1. These results suggest that IFN-gamma inhibits MNC formation, and that IFN-gamma inhibits bone resorption in part by inhibiting osteoclast formation.

Human peripheral blood cells, when cultured in vitro, release bone-resorbing factors, which have been called osteoclast-activating factors (OAF) but remain unidentified. We showed previously that a monocyte product, similar to interleukin 1 (IL 1), is a powerful stimulator of bone resorption in vitro. However, the possibility remained that other immune cell products may contribute to OAF activity. We have therefore tested three recombinant cytokines; IL 1, interleukin 2 (IL 2), and interferon-gamma (IFN-gamma) for their activity in a neonatal mouse bone resorption assay. We report here that purified recombinant murine IL 1 is a potent and powerful stimulator of bone resorption in vitro, active over a concentration range of 0.14 to 33 U/ml (1.3 X 10(-12) to 3.1 X 10(-10) M). IL 1-stimulated bone resorption was unaffected by cyclooxygenase inhibition but was inhibited by calcitonin and IFN-gamma. IL 2 had no effect on bone resorption.

The pathogenesis of hypercalcemia and mode of action of glucocorticoid therapy was examined in a patient with lymphosarcoma cell leukemia. Circulating neoplastic cells were cultured in vitro and secreted a bone-resorbing factor. The bone-resorbing factor was partially purified with the use of a bioassay for bone resorption, and was found to be chromatographically and pharmacologically similar to osteoclast activiating factor (OAF), which is produced by normal mitogen-activated peripheral blood lymphocytes. Other factors which stimulate bone resorption, such as parathyroid hormone, prostaglandins and the vitamin D metabolites, were excluded by criteria which included dose-response curves, radioimmunoassays, extraction in organic solvents and failure of glucocorticoids to inhibit bone-resorbing activity. The patient's hypercalcemia responded rapidly to prednisone therapy. The effects of the bone-resorbing factor secreted by the neoplastic cells on bone cultures to which cortisol was added were examined. Cortisol inhibited bone resorption directly at low doses (10(-8) M), which suggests that prednisone may have lowered the serum calcium in this patient by direct inhibition of bone resorption.

Antigen- or mitogen-stimulated leukocytes release bone-resorbing activity into culture supernatants in vitro. Among the agents likely to be present in such supernatants are monocyte-derived tumor necrosis factor (TNF-alpha) and lymphocyte-derived tumor necrosis factor (TNF-beta) (lymphotoxin), both of which have recently been shown to stimulate bone resorption in organ culture. To identify the mechanism of action of these agents, we compared bone resorption by isolated osteoclasts with bone resorption by osteoclasts cocultured with osteoblastic cells, and with bone resorption by osteoclasts incubated with supernatants from osteoblastic cells, in the presence and absence of recombinant TNF-alpha and TNF-beta. We found that neither TNF-alpha nor TNF-beta had any significant effect on bone resorption by isolated osteoclasts, but in the presence of osteoblasts the agents caused a twofold to threefold stimulation of bone resorption. A similar degree of stimulation was achieved by supernatants from osteoblasts incubated with TNF before addition to osteoclasts, compared with supernatants to which TNF were added after osteoblast incubation. These experiments suggest that TNF-alpha and TNF-beta stimulate bone resorption through a primary effect on osteoblastic cells, which are induced by TNF to produce a factor that stimulates osteoclastic resorption. Half-maximal stimulation of resorption occurred at 1.5 X 10(-10) M and 2.5 X 10(-10) M for TNF-alpha and TNF-beta, respectively. This degree of potency is comparable to that of parathyroid hormone, the major physiologic systemic regulator of bone resorption, and suggests that the TNF may exert a significant influence on osteoclastic bone resorption in vivo.