M. K. B. McGuire

Mode of action of dipyridamole on human platelets

Abstract The effects of dipyridamole were investigated on platelet aggregation, (14C)-5 hydroxytryptamine release, cyclic nucleotide metabolism and malonyldialdehyde and thromboxane B2 production. Pre-incubation of platelet-rich plasma with dipyridamole inhibited platelet aggregation in response to ADP, epinephrine, an epoxymethano prostaglandin endoperoxide analogue and collagen, and potentiated the inhibitory effect of prostacyclin on platelet aggregation. A small inhibition of 5-hydroxytryptamine release was also found. Dipyridamole potentiated the stimulation of platelet cyclic AMP accumulation by prostacyclin, and also potentiated the stimulatory effect of both arachidonic acid and dihomo-γ-linoleic acid on cyclic GMP accumulation. An inhibitory effect of the drug was observed on both cyclic AMP and cyclic GNP phosphodiesterase activities, particularly at high substrate concentrations. In addition, a slight stimulation of platelet membrane adenylate cyclase activity was found. The inhibition of platelet aggregation induced by collagen and arachidonic acid by the drug was accompanied by a reduction in the thromboxane B2 production. The inhibition of thromboxane B2 formation was also apparent in human platelet-rich plasma containing EDTA and in an isolated bovine platelet microsome preparation indicating that dipyridamole has a dose-dependent inhibitory effect on thromboxane synthesis which was independent of aggregation. Dipyridamole also inhibited malonyldialdehyde production in response to both thrombin and arachidonic acid. Thus dipyridamole may inhibit platelet function not only by increasing platelet cyclic AMP but may also exert a minor effect on platelet thromboxane biosynthesis.

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.

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.

Enhanced Breakdown of Bovine Articular Cartilage Proteoglycans by Conditioned Synovial Medium: The Effect of Serum and Dextran Sulphate

Medium from cultured bovine synovial membrane when applied to articular cartilage from the same animal enhanced proteoglycan breakdown, as measured by the release of [35S]sulphate from prelabelled cartilage, principally by activating chondrocytes to secrete or activate their own enzymes. This effect persisted whether or not the synovium was cultured in medium containing fetal calf serum. The release of proteoglycans from cartilage was markedly enhanced when the synovium was cultured in the presence of dextran sulphate (200 micrograms/ml), while dextran sulphate had no effect upon cartilage alone or when added together with medium cultured in the absence of dextran sulphate. Since the release of the proteoglycan breakdown products from frozen and thawed cartilage was not stimulated by dextran sulphate, this agent appeared to be enhancing the indirect chondrocyte-mediated effect of synovial medium rather than the direct proteolytic enzyme-induced effect. A possible mechanism for the production of a substance responsible for the chondrocyte-mediated matrix degradation is proposed, involving monocyte-like or macrophage-like cells, either resident in the synovial tissue or derived from the circulation.

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.

Enhanced breakdown of bovine articular cartilage proteoglycans by conditioned synovial medium in vitro. The effect of glucocorticoids and protein synthesis inhibitors.

Addition of conditioned synovial medium (SM) from cultured calf knee-joint synovium to cultures of articular cartilage from the same animal resulted in a significant increase in breakdown of cartilage proteoglycans. Culturing the synovium in the presence of glucocorticoids (hydrocortisone, dexamethasone, prednisolone) or protein synthesis inhibitors (cycloheximide or actinomycin D) reduced the breakdown effect. In contrast, enhancement of proteoglycan breakdown was observed when the cartilage was exposed to glucocorticoids in the presence of SM from synovium cultured without these drugs (control SM). The stimulatory effect on cartilage breakdown of control SM or control SM + glucocorticoids was markedly reduced in the presence of actinomycin D or cycloheximide. The authors conclude that glucocorticoids under certain conditions enhance cartilage degradation and therefore, although they exert the temporary anti-inflammatory effects, treatment of joint diseases with glucocorticoids may not be beneficial in the long-term.