Stimulation of prostaglandin E2 and bone resorption by recombinant human interleukin 1 alpha in fetal mouse bones

Recombinant human interleukin 1 alpha (rhIL-1 alpha) stimulates prostaglandin E2 and bone resorption in cultured forearm bones of fetal mouse in a dose-dependent manner: the minimal rhIL-1 alpha to elicit a significant bone resorption was 1.6 ng/ml (89 pM). The half maximal concentrations to elicit bone resorption and thymocyte proliferation were 3.3 ng/ml (183 pM) and 0.31 ng/ml (17 pM), respectively. The bone resorbing activity induced by IL-1 was partially inhibited by indomethacin and hydrocortisone, and completely inhibited by anti-IL 1-antibody. There was a good correlation between PGE2 production and bone resorption induced by IL-1 alpha. These results suggest that rhIL-1 alpha stimulates bone resorption at approximately 10 times the concentrations necessary for thymocyte proliferation and that PGE2 produced in the bone is at least in part involved in osteoclastic bone resorption.     

Gamma interferon inhibits basal and interleukin 1-induced prostaglandin production and bone resorption in neonatal mouse calvaria

Production of the osteolytic arachidonic acid metabolites, prostaglandin (PG) E2, PGI2 and PGF2 alpha, by neonatal mouse calvariae was quantitated by gas chromatography/mass spectrometry. Mouse recombinant interleukin 1 (rIL-1) raised medium levels of PGE2 and PGI2 (measured as 6-keto-PGF1 alpha) in the dose range tested (1.0-10.0 U/ml culture medium), while an effect on PGF2 was only observed at 10 U/ml. Bone resorption in response to rIL-1 reached a plateau at 3.0 U/ml. Mouse recombinant gamma-interferon (rIFN-gamma) between 100-500 U/ml suppressed basal PG synthesis and spontaneous resorption of cultured bone. In addition, IFN-gamma at 100 U/ml prevented stimulation of PG synthesis by 3.0 U/ml rIL-1 and thereby reduced the bone resorbing activity of the cytokine by at least 60%. 5 X 10(-7) M indomethacin was equally effective in suppression of PG synthesis and bone resorption. The present study provides evidence that IFN-gamma inhibits PG synthesis and consequently resorption of cultured bone.     

Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.     

Epidermal growth factor stimulates prostaglandin production and bone resorption in cultured mouse calvaria.

Murine epidermal growth factor (EGF) stimulated the production of prostaglandin E₂ (PGE₂) and bone resorption in neonatal mouse calvaria in organ culture. The effect of EGF on bone resorption occurred at low concentrations of the polypeptide (half-max stimulation = 0.4 ng/ml, 6.6 × 10^?11 M). All concentrations of EGF which stimulated resorption also stimulated the production of PGE₂ by bone; concentrations of EGF which did not stimulate resorption did not enhance PGE₂ production. EGF-induced formation of PGE₂ and bone resorption were inhibited completely by indomethacin (200 ng/ml) and hydrocortisone (3 × 10^?6 M). Indomethacin did not inhibit the bone resorption-stimulating activity of exogenous PGE₂. The time courses of action of EGF, parathyroid hormone and exogenous PGE₂ on bone resorption were similar. Brief exposure (15 or 60 min) to EGF (10 ng/ml) did not cause bone resorption or an increase in PGE₂ accumulation in a subsequent 48-h incubation in the absence of EGF. High concentrations (30 to 100 ng/ml) of bovine fibroblast growth factor (FGF) also stimulated the production of PGE₂ and bone resorption. We conclude that concentrations of EGF equal to or less than those present in mouse plasma stimulate the resorption of mouse bone in organ culture by a mechanism that involves the enhanced local production of PGE₂.     

Stimulation of human chondrocyte prostaglandin E2 production by recombinant human interleukin-1 and tumour necrosis factor

In this study we have examined the effects of recombinant cytokine preparations on the production of prostaglandin E2 (PGE2) by human articular chondrocytes in both chondrocyte monolayer and cartilage organ cultures. The cytokines chosen for this study included only those reported to be present in rheumatoid synovial fluids and which therefore could conceivably play a role in chondrocyte activation in inflammatory arthritis. Of the cytokines tested, interleukin-1 (IL-1; alpha and beta forms) consistently induced the highest levels of PGE2 production followed, to a lesser extent, by tumour necrosis factor (TNF; alpha and beta forms). The IL-1s were effective at concentrations 2-3 orders of magnitude less than the TNFs, with each cytokine demonstrating a dose-dependent increase in PGE2 synthesis for the two culture procedures. The increased PGE2 production by the chondrocytes exhibited a lag phase of 4-8 h following the addition of the IL-1 or TNF and was inhibited by actinomycin D and cycloheximide, indicating a requirement for de novo RNA and protein synthesis, respectively. Our results suggest that IL-1 may be the key cytokine involved in modulating chondrocyte PGE2 production in inflammatory arthritis; they further extend the list of human chondrocyte responses which are affected by both IL-1 and TNF.     

Involvement of PKC-beta in PTH, TNF-alpha, and IL-1 beta effects on IL-6 promoter in osteoblastic cells and on PTH-stimulated bone resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-beta, LY379196, was used to determine the role of the PKC-beta isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) induced translocation of PKC-alpha and -beta(I) to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-beta(I) translocation by PTH, TNF-alpha or IL-1 beta was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-alpha-, or IL-1 beta-stimulated translocation of PKC-alpha. PTH, TNF-alpha, and IL-1 beta increased luciferase expression in UMR-106 cells transiently transfected with a -224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-beta(I) is a component of the signaling pathway that mediates PTH-, TNF-alpha-, and IL-1 beta-stimulated IL-6 expression and PTH-stimulated bone resorption.     

IL-1 as adjuvant. Role of T cells in the augmentation of specific antibody production by recombinant human IL-1 alpha

Human rIL-1 alpha significantly enhanced splenic plaque-forming cells (PFC) to SRBC in vitro and in vivo. A single i.p. injection was sufficient to produce a fivefold or greater increase in the generation of PFC in a primary response. IL-1 treatment resulted in an increased production of Ag-specific PFC, both in vitro and in vivo, in combination with suboptimal doses of Ag. When IL-1 was given with a primary dose of Ag in vivo, an enhanced IgG response occurred. IL-1 enhanced in vivo carrier priming for an anti-hapten PFC response, indicating increased Th activity. Furthermore, T cells from spleens of mice treated with IL-1 provided significantly more help in both carrier (SRBC)- and hapten (TNP)- specific PFC. The enhancement of PFC by IL-1 in vitro occurred even in the presence of an excess of neutralizing anti-IL-2 antibody. These results suggest that IL-1 may enhance T cell-dependent antibody production in part by increasing Th activity, and that the mechanism of IL-1 action in increasing antibody production involves pathways in addition to the induction of IL-2 secretion.     

Modulation of human chondrocyte metabolism by recombinant human interferon gamma: in-vitro effects on basal and IL-1-stimulated proteinase production, cartilage degradation and DNA synthesis

Human articular chondrocytes in monolayer culture and fragments of human articular cartilage were treated with recombinant human interferon gamma (IFN-gamma) both alone and in combination with interleukin 1 (IL-1). IFN-gamma alone inhibits metalloproteinase production, as measured in the caseinase assay, and decreases glycosaminoglycan release from cartilage fragments in culture. The synthesis of DNA, as measured by [3H]thymidine incorporation, is stimulated by IFN-gamma. Similar effects are seen in the presence of IL-1. Thus, IFN-gamma opposes the stimulatory effect of IL-1 on caseinase production and decreases IL-1-stimulated cartilage degradation, as measured by glycosaminoglycan release. In contrast, IFN-gamma has no effect on IL-1-stimulated prostaglandin production, and acts synergistically with IL-1 to cause a large stimulation of DNA synthesis. These results show that IFN-gamma has a number of effects on articular chondrocytes in-vitro and suggest a possible role for IFN-gamma in limiting cartilage degradation in inflammatory joint conditions.     

Actions of recombinant interleukin 1, interleukin 2, and interferon-gamma on bone resorption in vitro

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.     

Short- and long-term effects of IL-1 and TNF antagonists on periodontal wound healing

The present study tested the effects of local injection of IL-1 and TNF soluble receptors on a periodontal wound-healing model in nonhuman primates. In this model, periodontal lesions were developed for 16 wk, followed by open flap surgery. Starting at the time of surgery, groups of animals received localized injections of both soluble cytokine receptors or else PBS three times per week for 3, 14, or 35 days. Periodontal wound healing was analyzed for each group at the end of the treatment regimen. Fourteen days after surgery, a significant decrease was observed between the animals treated with soluble receptors and the untreated group with respect to recruitment of inflammatory cells in deep gingival connective tissue. Concurrent apoptosis of inflammatory cells in those tissues increased significantly in treated animals compared with untreated animals. All other outcome parameters of periodontal wound healing were likewise significantly improved in treated animals compared with untreated animals. In marked contrast, however, 35 days after surgery, there was a significant increase in the number of inflammatory cells that had infiltrated into deep gingival connective tissue in treated compared with untreated animals. Outcome parameters of periodontal wound healing worsened in treated animals when compared with untreated. These results indicate that proinflammatory cytokines may play different functional roles in early vs late phases of periodontal wound healing. Short-term blockade of IL-1 and TNF may facilitate periodontal wound healing, whereas prolonged blockade may have adverse effects.     

IL-21 effects on human IgE production in response to IL-4 or IL-13

In human atopic disease, IgE sensitizes the allergic response, while IgG4 is protective. Because IL-4 and IL-13 trigger switch recombination to both IgE and IgG4, additional agents must regulate the balance between these isotypes to influence susceptibility or tolerance to atopy. In this report, we define in vitro conditions leading to activation or inhibition of human IgE and IgG4 production by IL-21. IL-21 reduced IL-4-driven IgE synthesis by mitogen-stimulated human PBMC. IL-21 inhibition of human IgE production was not a direct effect on B cells, was not seen following B cell activation with IL-13, and was overcome by CD40 ligation. Neither IFN-gamma, IL-10, IL-12, CD40L expression, nor apoptosis was responsible for the inhibitory effect. In contrast, IL-21-stimulated secretion of IgG4 from PBMC. Our findings indicate that IL-21 may influence the production of both human IgE and IgG4, and thus contribute to the regulation of atopic reactions.     

IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.     

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by ⁴⁵Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10⁻⁷ M). Prostaglandin F and prostaglandin E₂ production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10⁻⁶M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10⁻⁷M) both PGE₂ and PGF_2α stimulated resorption in a dose-dependent manner, with PGE₂ being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE₂ had a marked inhibitory action at a higher concentration (10⁻⁶M). In combination, the effects of PGE₂ and PGF_2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE₂ production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.     

Parathyroid hormone temporal effects on bone formation and resorption

Parathyroid hormone (PTH) paradoxically causes net bone loss (resorption) when administered in a continuous fashion, and net bone formation (deposition) when administered intermittently. Currently no pharmacological formulations are available to promote bone formation, as needed for the treatment of osteoporosis. The paradoxical behavior of PTH confuses endocrinologists, thus, a model bone resorption or deposition dependent on the timing of PTH administration would de-mystify this behavior and provide the basis for logical drug formulation. We developed a mathematical model that accounts for net bone loss with continuous PTH administration and net bone formation with intermittent PTH administration, based on the differential effects of PTH on the osteoblastic and osteoclastic populations of cells. Bone, being a major reservoir of body calcium, is under the hormonal control of PTH. The overall effect of PTH is to raise plasma levels of calcium, partly through bone resorption. Osteoclasts resorb bone and liberate calcium, but they lack receptors for PTH. The preosteoblastic precursors and preosteoblasts possess receptors for PTH, upon which the hormone induces differentiation from the precursor to preosteoblast and from the preosteoblast to the osteoblast. The osteoblasts generate IL-6; IL-6 stimulates preosteoclasts to differentiate into osteoclasts. We developed a mathematical model for the differentiation of osteoblastic and osteoclastic populations in bone, using a delay time of 1 hour for differentiation of preosteoblastic precursors into preosteoblasts and 2 hours for the differentiation of preosteoblasts into osteoblasts. The ratio of the number of osteoblasts to osteoclasts indicates the net effect of PTH on bone resorption and deposition; the timing of events producing the maximum ratio would induce net bone deposition. When PTH is pulsed with a frequency of every hour, the preosteoblastic population rises and decreases in nearly a symmetric pattern, with 3.9 peaks every 24 hours, and 4.0 peaks every 24 hours when PTH is administered every 6 hours. Thus, the preosteoblast and osteoblast frequency depends more on the nearly constant value of the PTH, rather than on the frequency of the PTH pulsations. Increasing the time delay gradually increases the mean value for the number of osteoblasts. The osteoblastic population oscillates for all intermittent administrations of PTH and even when the PTH infusion is constant. The maximum ratio of osteoblasts to osteoclasts occurs when PTH is administered in pulses of every 6 hours. The delay features in the model bear most of the responsibility for the occurrence of these oscillations, because without the delay and in the presence of constant PTH infusions, no oscillations occur. However, with a delay, under constant PTH infusions, the model generates oscillations. The osteoblast oscillations express limit cycle behavior. Phase plane analysis show simple and complex attractors. Subsequent to a disturbance in the number of osteoblasts, the osteoblasts quickly regain their oscillatory behavior and cycle back to the original attractor, typical of limit cycle behavior. Further, because the model was constructed with dissipative and nonlinear features, one would expect ensuing oscillations to show limit cycle behavior. The results from our model, increased bone deposition with intermittent PTH administration and increased bone resorption with constant PTH administration, conforms with experimental observations and with an accepted explanation for osteoporosis.     

Effects of selective prostaglandin EP2 and EP4 receptor agonists on bone resorption and formation in fetal rat organ cultures

Prostaglandin E2 (PGE2) can stimulate bone resorption and formation through receptors which activate adenylyl cyclase. We examined the effects of selective EP2 and EP4 agonists (EP2A and EP4A) on the release of previously incorporated 45Ca from fetal rat long bones and the incorporation of [3H]-proline or [3H]-thymidine (TdR) in fetal rat calvaria to assess the relative effects of these selective agonists on bone formation and resorption. Only EP4A was effective in increasing 45Ca release. Both agonists increased [3H]-TdR incorporation and [3H]-proline incorporation into calvariae, particularly in the presence of cortisol.