Judith E. Meats

Stimulation by human interleukin 1 of cartilage breakdown and production of collagenase and proteoglycanase by human chondrocytes but not by human osteoblasts in vitro

Human articular chondrocytes in culture synthesise collagenase and neutral proteoglycanase in response to addition of a 12-17 kDa protein produced by cultured human monocytes. This factor copurifies with interleukin 1, as assessed by lymphocyte activating factor activity, on gel filtration chromatography and isoelectric focusing. The interleukin 1 and chondrocyte-stimulating activities are destroyed by pretreatment of the material with phenylglyoxal. The same materials also promote the release of glycosaminoglycans from cultures of intact bovine nasal cartilage. The proteoglycanase activity release from chondrocytes appears to be a metalloproteinase because it is inhibited by EDTA and not by phenylmethylsulphonyl fluoride (PMSF), and because detection of its activity is dependent on the presence of 4-aminophenylmercuric acetate. Human osteoblast-like cells do not respond to this factor by increased proteinase production, but are stimulated to produce prostaglandins. These results suggest that interleukin 1 has activities upon non-immune cells which promote the degradation of connective tissue matrices. Human osteoblasts do not synthesise neutral collagen- and proteoglycan-degrading enzymes and thus are unlikely to be directly responsible for the matrix degradation which occurs during bone resorption.

Modulation of connective tissue metabolism by partially purified human interleukin 1

We have investigated the relationship between the monokine interleukin 1 (IL-1) and the connective tissue-stimulating activities produced by monocytes such as mononuclear cell factor (MCF). Using almost exclusively human tissue we have monitored a wide range of MCF-like activities through the partial purification of IL-1 by gel filtration and isoelectric focusing. Activities measured include stimulation of chondrocytes to produce prostaglandins, plasminogen activator and proteoglycanase, enhancement of synovial cell proliferation, and stimulation of cartilage resorption, in addition to IL-1 (lymphocyte activating factor) activity. The activities described show the same molecular heterogeneity; the active material has similar potencies in the different systems, and removal of IL-1 activity by pretreatment with phenylglyoxal also results in loss of the connective tissue-stimulating activities. These results show that the factors responsible for this wide range of activities are very closely related to IL-1 and give further evidence in support of the possible involvement of IL-1 in the processes of joint destruction occurring in chronic inflammatory conditions such as rheumatoid arthritis.

Effects of retinol and dexamethasone on cytokine-mediated control of metalloproteinases and their inhibitors by human articular chondrocytes and synovial cells in culture

Human articular chondrocytes in culture produced large amounts of specific mammalian collagenase, gelatinase and proteoglycanase when exposed to dialysed supernatant medium derived from cultured human blood mononuclear cells (mononuclear cell factor) or to conditioned medium, partially purified by fractionation with ammonium sulphate (60-90% fraction), from cultures of human synovial tissue (synovial factor). Human chondrocytes and synovial cells also released into culture medium an inhibitor of collagenase of apparent molecular weight about 30 000, which appeared to be similar to the tissue inhibitor of metalloproteinases synthesised by tissues in culture. The amounts of free collagenase inhibitor were reduced in culture media from chondrocytes or synovial cells exposed to mononuclear cell factor or synovial factor. While retinol inhibited the production of collagenase brought about by mononuclear cell factor or synovial factor, it restored the levels of inhibitor, which were reduced in the presence of mononuclear cell factor or synovial factor. Dexamethasone markedly reduced the production of collagenase by synovial cells, while only partially inhibiting factor-stimulated collagenase production by chondrocytes. Addition of puromycin as an inhibitor of protein synthesis reduced the amounts of both collagenase and inhibitor to control or undetectable levels.

Cell-cell interactions in the rheumatoid joint

Although rheumatoid synovium has been extensively studied in organ culture, particularly with respect to the synthesis of prostaglandins and proteinases, the behaviour of normal human synovium in culture has been much less well characterized. In this study, cultures of fragments of normal synovial tissue produced significantly less prostaglandin E (PGE) than cultures of rheumatoid synovium. The difference, however, did not persist when synovial cells obtained by enzymatic dispersion of normal and rheumatoid tissue were compared in monolayer culture. Production of PGE could be reactivated in both normal and rheumatoid synovial cells by products of mononuclear blood cells and also by factors in culture medium obtained after incubation of fragments of either normal or rheumatoid synovial tissue. These products of mononuclear cells and of synovial tissue also stimulated the production of PGE by human articular chondrocytes in monolayer culture. If these types of cellular interactions observed in vitro also occur in the arthritic joint as a result of the failure of normal control mechanisms, they could play a part in the irreversible destruction of joint structures.

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.

Messenger function of prostaglandins in cell to cell interactions and control of proteinase activity in the rheumatoid joint.

Destruction of joint structures in arthritis may result from failure of normal mechanisms controlling interactions among cells of the various tissues of the joint. Normal synovium in culture produces less prostaglandin E (PGE) and collagenase than rheumatoid. When rheumatoid synovium is dissociated into cells, the adherent cell cultures rapidly lose the ability to synthesize large amounts of PGE and collagenase and become indistinguishable from normal synovial cells. A mononuclear cell factor (MCF) derived from supernatant media of cultured human blood mononuclear cells and a synovial factor(s) (SF) from cultures of either normal or rheumatoid synovial fragments both stimulate production of PGE and proteinase by cells derived from human synovium, cartilage and bone. The activities of factors which may be present in these stimulatory supernatants may be unmasked in vitro when they are removed from the normal control present in vivo. Normal synovium probably contains cells which, with the appropriate stimulus, may be recruited to participate in joint tissue degradation. Normal connective tissue turnover may also be controlled by a neutral metallo-proteinase inhibitor (TIMP), which is produced in considerable amounts by normal synovium, but which cannot be detected in cultures of rheumatoid synovium. While corticosteroids inhibit the production and action of MCF and SF, they stimulate production of TIMP by normal or rheumatoid synovial tissue in vitro and may contribute to the endogenous control mechanisms. PGE may also have a modulatory role in these cellular interactions.

Properties of rheumatoid and normal synovial tissue in vitro and cells derived from them

SummaryUsing organ and cell culture techniques for tissues and cells derived from human sources, we have investigated cellular interactions involving synovial tissue. Normal synovium in culture produced less prostaglandin E (PGE) and collagenase than cultures of rheumatoid synovial fragments. When synovial tissue was dissociated by enzymatic digestion, monolayers of adherent cells were established in primary culture. The adherent cells rapidly lost the ability to synthesize large amounts of PGE and collagenase and rheumatoid synovial cells became indistinguishable from normal synovial cells. Supernatants from cultured human mononuclear blood cells contained activities (Mononuclear cell factor(s)=MCF) which stimulated PGE and collagenase production by either normal or rheumatoid synovial cells. Conditioned medium from cultures of either normal or rheumatoid synovial fragments (Synovial factor(s)=SF) also stimulated production of PGE and collagenase by these human cells. Both MCF and SF also stimulated the production of PGE by cells isolated from human trabecular bone. Since both normal and rheumatoid synovial cells respond similarly to these factors, there appears to be little specificity with regard to whether the target cells are derived from normal or pathological sources. Furthermore, since both normal and rheumatoid synovium are able to produce similar amounts of stimulatory activity, inflammatory cells are not solely responsible for these phenomena. Normal synovium must therefore contain cells which can be recruited to participate in these potential cellular interactions. Destruction of joint structures may be mediated by factors of the type studied here, which may be produced when there is failure of the mechanisms that prevent them from being synthesised or released.

Partial purification of a factor from human synovium that possesses interleukin 1, chondrocyte stimulating and catabolin-like activities

Human synovial explants in culture release material that stimulates the production of prostaglandin E2 (PGE2) and several extracellular enzymes by human chondrocytes. Fractionation of conditioned medium by gel filtration revealed a protein of approx. 15 kDa, which in addition to stimulating production of PGE2 and plasminogen activator by human articular chondrocytes, possessed interleukin 1 activity and induced cartilage degradation. Further purification using iso‐electric focussing again showed co‐elution of these activities with a major pI of 6.9 and a minor pI of 5.1–5.3. This study indicated that human synovium releases a facor that is closely related to or identical with interleukin 1 and suggests that this protein may participate in cellular interactions that occur within the rheumatoid joint.

Intercellular Messengers in Joint Tissues in Rheumatoid Arthritis

Although the cause of rheumatoid arthritis is uncertain, the mechanisms by which destruction of joint tissues may occur have been studied extensively. The inflammatory responses in rheumatoid arthritis are probably mediated by a variety of different agents which include prostaglandins, leukotrienes, kinins and other peptide mediators, complement components, and immune complexes. The ultimate destruction of proteoglycans and collagen within cartilage similarly depend upon the release of the appropriate degradative enzymes. At one time these were thought to be predominantly lysosomal acid proteinases but emphasis has recently shifted to neutral metallo-proteinases which include specific enzymes capable of degrading collagen or proteoglycans at neutral pH. Under normal conditions these proteinases are in latent form due in part to the presence of a tissue inhibitor of metallo-proteinases (TIMP). During studies of human joint tissues in culture, it has become apparent that products of one cell type may influence the behaviour of other cells. Thus, monocytes and macrophages may produce mediators, such as interleukins, one of which has been called mononuclear cell factor (MCF), which when added to cultures of human articular chondrocytes or synovial cells, markedly enhances production of prostaglandins and metallo-proteinases while depressing the amount of TIMP. Cultured human synovial tissue produces factors with similar properties, which may in turn be related to mediators such as catabolin, which can be produced by synovium and other connective tissues and which stimulate chondrocytes to degrade their own matrix. The production of these mediators may not only be relevant to rheumatoid arthritis but also to other diseases. Thus, MCF is capable of stimulating prostaglandin production by gingival cells and cells derived from human bone. Moreover MCF is itself capable of inducing bone resorption. Since both normal and diseased tissues are capable of producing and responding to these mediators, these potential degradative interactions must be kept in check in vivo. Glucocorticosteroids may play a role in the natural suppression of these mechanisms, since in vitro they are capable of inhibiting the production of factors as well as their effects on target tissues. Since these factors probably have anabolic activity as well, they may be involved in connective tissue repair after injury. Such intercellular mediators may play important roles in the control of connective tissue turnover, not only in disease states but also in the normal processes of growth and differentiation.

Enhanced production of prostaglandins and plasminogen activator during activation of human articular chondrocytes by products of mononuclear cells

SummaryWe have examined the way in which products of cultured human blood mononuclear cells activate human articular chondrocytes. Conditioned medium from mononuclear cells enhanced the production of prostaglandin E by cultured human chondrocytes and also stimulated fibrinolytic activity in these cultures. These two effects may be interrelated, since the increased fibrinolysis in response to products of mononuclear cells was partially inhibited by indomethacin, an inhibitor of prostaglandin biosynthesis. The increased fibrinolysis is probably attributable to plasminogen activator, since it was strongly dependent on the presence of plasminogen. Increased amounts of PGE and chondroitin sulphate were also released from intact fragments of cartilage exposed to medium from cultured mononuclear cells. The time course and dose dependence of these effects were studied. The addition of exogenous arachidonic acid markedly enhanced production of PGE2.Ultrogel AcA54 was used to fractionate medium from cultured mononuclear cells and the chondrocyte-stimulating activity eluted with an apparent molecular weight between 12 000 and 25 000 daltons. Adherent and non-adherent mononuclear blood cells were also partially separated and conditioned medium from each was assayed for chondrocyte-stimulating factors. Both populations released factor(s) which increased the production of prostaglandin E by chondrocytes, but more activity came from the adherent mononuclear cells. The possible interrelationship between the chondrocyte activating factor studied here and others described in the literature is discussed.