R.A.D. Bunning

The effects of recombinant human interleukin-1β on cellular proliferation and the production of prostaglandin E2, plasminogen activator, osteocalcin and alkaline phosphatase by osteoblast-like cells derived from human bone

There is mounting evidence implicating cytokines such as interleukin-1 in the local regulation of bone homeostasis. In this report we show that recombinant human interleukin-1 beta (rhIL-1 beta) influences several activities of osteoblast-like cells derived from human trabecular bone explants in vitro. rhIL-1 beta stimulated cellular proliferation and the synthesis of prostaglandin E2 and plasminogen activator activity in the cultured human osteoblast-like cells in a dose-dependent manner. However, the induction of osteocalcin synthesis and alkaline phosphatase activity in response to 1,25(OH)2D3, two characteristics of the osteoblast phenotype, were antagonized by rhIL-1 beta over a similar dose range. This study adds further support to the potential role of interleukin-1 in the physiological and pathological modulation of bone cell metabolism.

Interleukin 1 preferentially stimulates the production of tissue-type plasminogen activator by human articular chondrocytes

Interleukin 1, derived from human placenta, stimulates plasminogen activator activity in human articular chondrocytes. The stimulation of plasminogen activator activity can be abolished by preincubation of placental interleukin 1 with an antiserum to homogeneous 22K factor, a species of interleukin 1 beta, indicating that the stimulation of plasminogen activator activity is due to interleukin 1 and not contaminating factors. Chondrocytes produce three species of plasminogen activator, with apparent Mr approximately 50,000, 65,000 and 100,000 as determined after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis with gels containing casein and plasminogen. Both placental interleukin 1 and 22K factor enhance the production of the species of Mr approximately 65,000 and 100,000. Comparison of the mobility of the plasminogen activator species on SDS-polyacrylamide gel electrophoresis with human urokinase (u-PA) and human melanoma tissue-type plasminogen activator (t-PA) and studies with antibodies to these enzymes indicate that the Mr approximately 50,000 species is a u-PA and the Mr approximately 65,000 a t-PA. The Mr approximately 100,000 species is possibly an enzyme-inhibitor complex. Interleukin 1 therefore appears to enhance the production of t-PA and a putative enzyme-inhibitor complex. Abolition of plasminogen activator activity in the fibrin plate assay with antibodies to t-PA and u-PA also confirms enhanced t-PA production on interleukin 1 stimulation, though there is also evidence for increased cell-associated production of u-PA.

Characterization of granulocyte chemotactic activity from human cytokine-stimulated chondrocytes as interleukin 8

Human articular chondrocytes, when stimulated with interleukin 1 beta (IL 1 beta), tumor necrosis factor-alpha (TNF-alpha), or with the double stranded RNA poly (rI).poly (rC), produce a chemotactic activity for granulocytes. The induction with IL 1 beta could be abolished by an antibody to IL 1 beta but not by an antibody to interleukin 6 (IL 6), indicating that the latter is not a mediator for the production of chemotactic activity. The inducers had no direct chemotactic effect on granulocytes. The granulocyte chemotactic factor from chondrocytes was characterized with a specific antibody against leukocyte-derived interleukin 8 (IL 8). The specificity of this antibody was demonstrated by immunochemical and biological criteria such that it could immunoprecipitate only the 6-7 kDa IL 8 protein from fibroblasts, and that it did not neutralize a structurally related monocyte chemotactic protein. This antibody against IL 8 completely neutralized the granulocyte chemotactic activity from stimulated chondrocytes. This demonstrates the identity of chondrocyte IL 8 with leukocyte- and fibroblast-derived IL 8. Our data show that leukocyte chemotaxis into the inflamed joint can be mediated by IL 8, induced in both synovial fibroblasts and chondrocytes by the inflammatory cytokines IL 1 and TNF-alpha.

Bradykinin stimulates the production of prostaglandin E2 and interleukin-6 in human osteoblast-like cells

The effect of bradykinin (BK) on proteinase activity, prostaglandin synthesis, and the production of interleukin-6 (IL-6) was investigated in cultures of human osteoblast-like cells. Bradykinin had no effect on stromelysin activity and plasminogen activator activity produced by human osteoblast-like cells. However, BK stimulated the production of prostaglandin E2, an effect that was markedly enhanced by pre-incubation with recombinant interleukin-1 alpha (rhIL-1 alpha), but was apparently unaffected by BK receptor antagonists types 1 and 2. Bradykinin stimulated the intracellular accumulation of total inositol phosphates suggesting that its effects were mediated by stimulation of phosphoinositide metabolism. Bradykinin within the dose range of 10(-11)-10(-5) M also significantly stimulated the production of IL-6. Bradykinin may, therefore, mediate a variety of responses in bone under both physiological and pathological conditions.

Natural human IL-1β exhibits regulatory actions on human bone-derived cells invitro

We have shown that natural homogenous IL-1 beta exhibits regulatory activities on human bone-derived osteoblast-like cells in vitro. IL-1 beta stimulated cellular proliferation and the synthesis of prostaglandin E2 and plasminogen activator activity by the cultured human osteoblast-like cells. In contrast to these stimulatory actions, IL-1 beta antagonised the stimulatory effects of 1.25(OH)2 D3 on the production of alkaline phosphatase and osteocalcin, two markers of the osteoblast phenotype. These studies indicate that this cytokine may therefore have potential physiological and pathological effects on bone metabolism.

The effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on human osteoblast-like cells

The activity of human osteoblast-like cells cultured in vitro is regulated by a number of factors, which include systemic hormones as well as agents that can be produced locally within bone. Several cytokines and growth factors have been demonstrated to be produced by osteoblasts themselves, and this includes granulocyte-macrophage colony-stimulating factor (GM-CSF). In this report we show that recombinant human GM-CSF (rhGM-CSF) modulates the activities of osteoblast-like cells derived from human trabecular bone in vitro. rhGM-CSF stimulated the proliferation of the cultured human osteoblast-like cells, but antagonised the induction by 1,25(OH)2D3 of osteocalcin synthesis and alkaline phosphatase activity, two characteristic products of osteoblasts. rhGM-CSF however, had no appreciable effect on the production of prostaglandin E2, or on the plasminogen activator activity associated with human osteoblast-like cells. These results are the first report of which we are aware of an apparently direct action of GM-CSF on cells of the osteoblast phenotype. These studies indicate that GM-CSF represents another haematological factor that can potentially exert regulatory actions on human osteoblast-like cells. GM-CSF may therefore be a potential paracrine/autocrine regulator of osteoblast activity.

Independent induction of interleukin 6 and prostaglandin E2 by interleukin 1 in human articular chondrocytes

Interleukin 6 is a cytokine with growth and differentiation activities on a number of cell types. Human articular chondrocytes produce interleukin 6 and this production appears to be constitutive but can be stimulated in a dose-dependent manner by interleukin 1. Other stimulators of interleukin 6 production in chondrocytes include tumour necrosis factor-alpha, polyriboinosinic: polyribocytidylic acid and bacterial lipopolysaccharide. Interleukin 6 production is not inhibited by prostaglandin E2 but may be partially dependent on prostaglandin E2 production. Using an antiserum to interleukin 6 we have demonstrated that the production of prostaglandin E2 under basal conditions and in response to interleukin 1 is probably not mediated by interleukin 6.

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.

Retinoids and synovial factor(s) stimulate the production of plasminogen activator by cultured human chondrocytes. A possible role for plasminogen activator in the resorption of cartilage in vitro.

Agents such as retinol, interleukin 1 and catabolin stimulate resorption of cultured cartilage. This process seems to be mediated by chondrocytes, but the mechanism by which breakdown occurs remains unknown. We have found that (10(-6)-10(-8) M) retinoic acid and (1 X 10(-6) M) retinol, in the presence or absence of a factor derived from cultured synovium (synovial factor), stimulate the degradation of fibrin by human chondrocytes in culture. Plasminogen was required for the enhancement of fibrinolysis, suggesting that the breakdown depended upon the production of plasminogen activators and subsequent liberation of plasmin. However, the chondrocytes did not release significant amounts of plasminogen activator, and the effects of the synovial factor and retinoids resulted from augmentation of the production or activity of enzymes which remained bound to the cell layer. The role of plasminogen in the resorption of cultured cartilage was also investigated. In the presence of plasminogen, (1 X 10(-8) M) retinoic acid or synovial factor stimulated the breakdown of cultured bovine nasal cartilage, but in the absence of plasminogen, the effect of synovial factor was abolished and that of retinoic acid reduced. However, in cultures containing both retinoic acid and synovial factor the resorption process was not affected by removal of plasminogen. Thus, the resorption of cartilage matrix in vitro may be partially mediated by plasminogen activators and plasmin.

The regulation of connective tissue metabolism by vasoactive intestinal polypeptide

Immunohistochemical studies have confirmed the innervation of bone with neuropeptidergic neurons containing vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP). In this study, we report effects of VIP on connective tissue cell metabolism. VIP stimulated PGE2 production in human articular chondrocytes, human osteoblast-like cells and human synovial cells, however, stromelysin production was unaffected. VIP also stimulated cAMP production in human osteoblast-like cells, but not in human articular chondrocytes or synovial cells. These findings are suggestive of a role of VIP in connective tissue cell metabolism which may contribute to the inflammatory processes of arthritis.