Adherent Rheumatoid Synovial Cells Research Articles

Interleukin-4 Inhibits the Gene Expression and Biosynthesis of Cytosolic Phospholipase A2 in Lipopolysaccharide Stimulated U937 Macrophage Cell Line and Freshly Prepared Adherent Rheumatoid Synovial Cells

We recently reported that interleukin-4 (IL-4) inhibited prostanoid synthesis through inhibiting cyclooxygenase 2 biosynthesis. In the present study, we examined the effect of IL-4 on the expression of cytosolic phospholipase A2(cPLA2). The amounts of protein and mRNA of cPLA2were determined by western blotting and reverse transcription polymerase chain reaction (RT-PCR), respectively. Although interleukin-1α (IL-1α) and tumor necrosis factor α (TNFα) had little effect on the biosynthesis of cPLA2in phorbol myristate acetate (PMA)-differentiated U937 cells, lipopolysaccharide (LPS) increased the protein level of cPLA2in a dose-dependent manner. IL-4 inhibited the increased synthesis of cPLA2at the mRNA level. In addition, IL-4 inhibited the biosynthesis of cPLA2in untreated or LPS treated freshly prepared rheumatoid synovial cells at the mRNA level. These findings suggest that IL-4 inhibits prostanoid synthesis through inhibiting the expression of both cPLA2and cyclooxygenase 2.

Interleukin-4 inhibits prostaglandin E2 production by freshly prepared adherent rheumatoid synovial cells via inhibition of biosynthesis and gene expression of cyclo-oxygenase II but not of cyclo-oxygenase I.

To characterise the effect of interleukin-4 (IL-4) on the biosynthesis of cyclo-oxygenases I (COX I) and II (COX II), the rate limiting enzymes of the synthesis of prostaglandin E2 (PGE2), in freshly prepared rheumatoid synovial cells. Adherent synovial cells were obtained from rheumatoid synovium by collagenase digestion. The concentrations of PGE2 in culture supernatants were determined by enzyme linked immunosorbent assay. The protein and mRNA concentrations of COX I and COX II were determined by Western blotting and reverse transcription polymerase chain reaction, respectively. Freshly prepared synovial cells produced large amounts of PGE2. They also showed increased gene expression of COX I and COX II, and synthesised these proteins. IL-4 had suppressive effects on the production of PGE2 by untreated or lipopolysaccharide (LPS) stimulated synovial cells. In addition, IL-4 inhibited the biosynthesis of COX II at the mRNA level. In contrast, it did not modify the protein concentration of COX I. In tests of cell specificity, IL-4 did not reduce the mRNA concentration of COX II in interleukin-1 alpha (IL-1 alpha) stimulated cultured synovial fibroblasts at passages 3-6, but it reduced considerably the mRNA concentrations of COX II in an LPS or IL-1 alpha stimulated U937 monocyte/macrophage cell line. These results suggest that IL-4 might inhibit overproduction of PGE2 in rheumatoid synovia via selective inhibition of the biosynthesis of COX II, and that this inhibition might be specific to macrophage-like synovial cells.

Suppressive Effect of Interleukin-4 (IL-4) on IL-6 Production by Adherent Rheumatoid Synovial Cells

Interleukin-6 (IL-6) has recently been characterized as a mediator of multiple inflammatory responses. Excessive production of this cytokine has been demonstrated in the joints of patients with rheumatoid arthritis (RA). On the other hand, anti-inflammatory effects of IL-4 have recently been demonstrated. We therefore investigated the suppressive effect of IL-4 on IL-6 production by synovial cells in patients with RA. Freshly prepared adherent rheumatoid synovial cells expressed IL-6 mRNA and spontaneously produced a large amount of IL-6 in culture. This spontaneous production of IL-6 was significantly suppressed by IL-4. The suppressive effect of IL-4 on IL-6 production was demonstrated in all patients with RA tested in this study. On the other hand, IL-6 mRNA levels already expressed in adherent synovial cells were not reduced by 1L-4 in 24 hr of culture. The suppressive effect of IL-4 on IL-6 production suggests that IL-4 is an important physiological regulator of IL-6 production in synovial cells.

Effect of synthetic calcium pyrophosphate and hydroxyapatite crystals on the interaction of human blood mononuclear cells with chondrocytes, synovial cells, and fibroblasts

Synthetic calcium pyrophosphate dihydrate crystals and, to a lesser extent, synthetic hydroxyapatite crystals increased the amount of interleukin-1/mononuclear cell factor released by human blood monocytes, as measured by collagenase and prostaglandin E2 production by rabbit chondrocytes, human dermal fibroblasts, and adherent rheumatoid synovial cells. The same crystals also directly induced collagenase and prostaglandin E2 secretion by rabbit chondrocytes, and potentiated the action of interleukin-1/mononuclear cell factor on chondrocytes. These mechanisms may be important in the pathogenesis of the destructive arthropathies associated with these crystals.

Histamine H1 receptors on adherent rheumatoid synovial cells in culture: demonstration by radioligand binding and inhibition of histamine-stimulated prostaglandin E production by histamine H1 antagonists.

Histamine H1 receptors have been demonstrated on adherent rheumatoid synovial cells using biochemical and radioligand binding assays in vitro. The addition of histamine (17.8 mumol/l) to nine primary cultures of adherent rheumatoid synovial cells resulted in a two- to 21-fold increase in the production of prostaglandin E (PGE). This increase was inhibited by three H1 receptor antagonists (mepyramine, tripelennamine, and chlorpheniramine) in a dose related manner at concentrations below 10(-6) mol/l. Competitive binding assays with [3H]mepyramine gave ED50 values of approximately 10(-5) mol/l for the three H1 antagonists. H2 receptor antagonists (cimetidine and ranitidine) did not inhibit the histamine induced increase in PGE and did not compete effectively with the binding of H1 antagonists.

Comparative studies of adherent rheumatoid synovial cells in primary culture: characterisation of the dendritic (stellate) cell.

Primary cultures of adherent rheumatoid synovial cells contain variable proportions of fibroblasts, macrophages, and dendritic cells, as judged by morphological appearance. Comparative studies using various enzymic and histochemical staining procedures showed the dendritic cells to lack many of the characteristic features of macrophages, e.g. the failure to express HLA-DR (Ia) antigen. The dendritic cells and fibroblasts had several similarities, but differed to some extent in their nonspecific esterase activity, phagocytic and proliferative potential. As the proportions of dendritic cells and fibroblasts varied in relation to specific culture conditions, we examined the possibility that these morphologies might represent different functional states rather than distinct cellular origins. Using subcultured synovial fibroblasts with a uniform bipolar appearance, we have shown that exposure to interleukin-1 or mast cell products resulted in a transformation to dendritic morphology. This change in cell shape was prevented by the presence of indomethacin, but was subsequently achieved by the addition of exogenous PGE2. Thus it appears that the latter is the factor that modulates the morphological change of fibroblastic to dendritic cells. This study has also demonstrated the complete and reversible interchange of fibroblast/dendritic morphology, thereby confirming that these different shapes are manifest by the same cell. The changes in phenotypic expression associated with the dendritic appearance include increased production of collagenase, prostaglandin E, and nonspecific esterase, as well as an apparent inability to exhibit phagocytosis and to proliferate in culture. We conclude from our in vitro studies that the phenotypic behaviour of the synovial fibroblast (or synoviocyte) is very variable and dependent to a large extent upon local stimuli, but the identity and hierarchy of such stimulating and suppressive factors in relation to cellular interactions requires further study.

Histamine stimulates prostaglandin E production by rheumatoid synovial cells and human articular chondrocytes in culture

Histamine stimulates prostaglandin E (PGE) production by cultures of adherent rheumatoid synovial cells and human articular chondrocytes. When subcultured synovial fibroblasts or human articular chondrocytes were "primed" by preincubation with conditioned media from primary adherent rheumatoid synovial cell cultures (synovial factor), each produced even higher PGE levels upon histamine exposure. This histamine stimulation was prevented by histamine H1, but not H2, antagonists and was more marked if serum was absent from the culture media. Thus, histamine-induced PGE production by these cells is mediated via H1 receptor activation and subsequent arachidonic acid liberation.

Mononuclear Cell-Conditioned Medium Containing Mononuclear Cell Factor (MCF), Homologous With Interleukin 1, Stimulates Collagen and Fibronectin Synthesis by Adherent Rheumatoid Synovial Cells: Effects of Prostaglandin E2 and Indomethacin

Adherent rheumatoid synovial cells produce and release into supernatant culture medium latent collagenase and PGE2. The levels of collagenase and PGE2 can be increased by a soluble factor present in mononuclear cell-conditioned medium, partially purified by gel-filtration, which has homologies with interleukin 1, and is produced by mono cyte/macrophages. The synovial cell cultures produce collagens (procollagens) and fibronectin as well. The factor(s) present in the mononuclear cell conditioned a medium which increases medium levels of collagenase PGE2 also stimulates synthesis of total protein as well as types I and III procollagen by the synovial cells. This stimulation by the monocyte factor is augmented in the presence of indomethacin, which blocks endogenous PGE2 production. Medium levels of fibronectin parallel those of procollagen. The addition of exogenous PGE2 abolishes the effect of indomethacin on collagen and fibronectin synthesis. These observations of mononuclear cell-mediated increases in fibronectin synthesis may account for the high levels of fibronectin found by others in rheumatoid synovium and synovial fluids as the increases in collagen synthesis might also explain the fibrosis observed in some rheumatoid joints.

Interleukin 1 production by the human monocyte cell line U937 requires a lymphokine induction signal distinct from interleukin 2 or interferons.

A soluble product from cloned human T lymphocytes is capable of stimulating U937 cells, a line of human monocytes, to produce interleukin 1 (IL 1). We previously reported that U937 cells exposed to T lymphocyte-conditioned medium secrete mononuclear cell factor (MCF), which increases collagenase and prostaglandin E2 production by adherent rheumatoid synovial cells. Whereas structural and functional homologies between lymphocyte-activating factor (LAF, or IL 1) and MCF were described, previous attempts to measure LAF secretion by lymphokine-stimulated U937 cells were unsuccessful. Although the crude supernatants of cultured U937 cells exposed to medium from lectin-stimulated peripheral blood or cloned T lymphocytes contained MCF activity, no LAF activity was detected. After these crude supernatants were chromatographed on Ultrogel AcA54, however, and the fractions were individually assayed for IL 1, MCF and LAF activities were coeluted with apparent m.w. approximately 14,000 to 23,000. The inability to detect LAF activity in the unfractionated medium was accounted for by an inhibitor of lymphocyte proliferation present in fractions of higher m.w. The T lymphocyte product that stimulated U937 cell maturation and monokine production was secreted in response to lectin-stimulation in a dose-dependent fashion. Although we have previously demonstrated that the hormone 1,25-dihydroxyvitamin D3 caused maturational changes in U937 cells, and other investigators have reported effects of alpha and gamma interferon, these changes are dissociable from IL 1 production. Thus, a distinct lymphocyte-derived signal, necessary for the production of IL 1 by U937 cells, can be identified and dissociated from other biologic products that cause "maturational" changes. The detection of LAF activity in U937 cell supernatants requires the removal of an inhibitor of lymphocyte proliferation.

Effects of prostaglandin E 2, indomethacin, trifluoperazine and drugs affecting the cytoskeleton on collagenase production by cultured adherent rheumatoid synovial cells

Cultured adherent rheumatoid synovial cells with fibroblast properties release large amounts of collagenase and prostaglandin E 2 (PGE 2) into the medium. With age in culture and passage of the cells, the levels of collagenase and PGE 2 decrease, but can be increased by a factor (MCF; mononuclear cell factor) released by cultured human blood monocyte-macrophages. The magnitude of the stimulation varies with different synovial cell strains. To determine some of the mechanisms which regulate the collagenase response, synovial cells were exposed to a cyclooxygenase inhibitor (indomethacin) and substances which alter the cytoskeleton (cytochalasin B or colchicine) or interact with Ca 2+ · calmodulin (trifluoperazine). The collagenase response was retained even when PGE 2 synthesis was totally blocked with indomethacin. The collagenase response, however, was blunted at high indomethacin concentrations (> 10 μM) and paradoxically augmented at lower indomethacin concentrations (0.001 (μM). In some synovial cell strains, the blunting effect of 10 μM indomethacin was reversed by the addition of low concentrations of exogenous PGE 2 (10 ng/ml). Preincubation of synovial cells for 1 or 24 hr with colchicine or cytochalasin B (1–10 μM) resulted in an augmented collagenase and PGE 2 response to MCF. Cells preincubated or incubated with 1–50 μM trifluoperazine, a phenothiazine, also augmented collagenase stimulation by MCF, but, in contrast to colchicine or cytochalasin B, trifluoperazine suppressed the PGE 2 response. Thus, although PGE 2 and collagenase production by synovial cells may be dissociated, altering ambient PGE 2 levels affected basal collagenase production and modulated the collagenase response to MCF.

Chloroquine inhibits the production of a mononuclear cell factor by inhibition of lectin binding.

The effect of chloroquine (CQ) on the ability of normal human peripheral blood monocytes to produce a soluble mononuclear cell factor (MCF) capable of stimulating prostaglandins from adherent rheumatoid synovial cells was studied. CQ inhibited concanavalin A (Con A)- and bacterial peptidoglycan-induced MCF activity, but not colchicine-induced activity, in a dose-dependent manner. After incubation with CQ for 20 minutes, 3H Con A binding was reduced by 50%, and metabolism of pre-bound 3H Con A was inhibited. These data suggest that CQ can inhibit MCF production by inhibition of lectin binding, perhaps by inhibition of receptor recycling.

Heparin modulates intracellular cyclic AMP in human trabecular bone cells and adherent rheumatoid synovial cells.

Cells were cultured from explants of human trabecular bone excised from eight patients and incubated usually for 20 minutes with bovine parathyroid hormone, salmon calcitonin, prostaglandin E2, or heparin. The intracellular content of cyclic AMP was measured by radioimmunoassay and was significantly increased by parathyroid hormone in four, by calcitonin in two, by prostaglandin E2 in eight, and by heparin in seven out of eight cultures. In the two cultures containing calcitonin-responsive cells heparin inhibited the cyclic AMP response induced by calcitonin. Heparin did not affect the cyclic AMP response to parathyroid hormone or prostaglandin E2. Heparin also increased the cyclic AMP content of cultured adherent rheumatoid synovial cells. It is proposed that, in certain situations of focal pathological bone resorption, although concentrations of circulating hormones may be normal, the local release of products such as heparin may modify the effect of hormones which regulate connective tissue homoeostasis. local changes in hormone responses could contribute to the enhanced bone resorption associated with inflammatory processes such as rheumatoid arthritis.

Mast cell products stimulate collagenase and prostaglandin E production by cultures of adherent rheumatoid synovial cells

Mast cells were purified from histologically-confirmed dog mastocytomas and extracted for whole mast cell products (MCP). When added to cultures of human adherent rheumatoid synovial cells MCP induced a 50–400 fold increase in prostaglandin E synthesis and a 10–50 fold stimulation of collagenase production. The mast cell stimulatory factor has not been identified and was not due to histamine, heparin or prostaglandin E. These results indicate a novel way in which mast cells might interact with synovial cells to promote the production of inflammatory mediators and proteolytic enzymes which might contribute to connective tissue degradation.

Rheumatoid synovial cell hormone responses modulated by cell-cell interactions.

Cellular interactions within the rheumatoid synovium are likely to be responsible for the destructive properties of this tissue. The responses to hormones which regulate connective tissue metabolism may also be influenced by these cell-cell interactions. To determine the effect of cellular interactions on responses to hormones, human peripheral blood mononuclear cells were cocultured with adherent rheumatoid synovial cells. Coculture resulted in a decrease in response to prostaglandin E2 as assessed by a reduction in the magnitude of the acute prostaglandin-induced cyclic AMP response. In contrast, coculture resulted in an increase in the magnitude of the acute parathyroid hormone-induced cyclic AMP response. The decrease in response to prostaglandin was reversed by the presence of indomethacin during the preincubation, whereas indomethacin had no effect on the cyclic AMP response to parathyroid hormone. Increases in endogenous synovial cell prostaglandin production (stimulated by soluble factors released by the mononuclear cells) accounted for the subsequent decrease in the response to exogenous prostaglandin. The increase in cyclic AMP response to parathyroid hormone could not be explained by a soluble factor and was independent of ambient prostaglandin levels.

Studies of histamine in the rheumatoid joint.

Fragments of synovial membranes from three patients with rheumatoid arthritis were incubated in Dulbecco's modified Eagle's medium (DMEM). Histamine was released into the medium at rates up to 649.3 ng/g synovium/day. To investigate whether histamine in synovial fluid may have more than a vasoactive role, cells cultured from human trabecular bone adjacent to a rheumatoid joint and human adherent rheumatoid synovial cells were incubated with different concentrations of histamine for 5 min. Histamine increased the intracellular content of cyclic AMP up to three fold in a dose-related manner but had no effect on synovial cells. This offers preliminary evidence that cells grown from human trabecular bone have histamine receptors.

Rheumatoid synovial cell morphologic changes induced by a mononuclear cell factor in culture.

Adherent rheumatoid synovial cells in culture produce large amounts of prostaglandin E2 (PGE2) and collagenase. When exposed to a monocyte-derived factor, such cells exhibit marked increases in PGE2 and collagenase production. In addition, cellular morphology becomes more stellate. In the presence of this factor, indomethacin inhibits both PGE2 production and the stellate changes, whereas collagenase production usually continues at a high rate. Addition of PGE2 to cultures reproduces the stellate change as does the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP. Colchicine inhibits morphologic transformation induced by the monocyte-derived factor, whereas cytochalasin B has no effect. It appears that the stellate morphology is dependent upon PGE2-induced cAMP stimulation and is not related to collagenase production per se.

Immune functions of human synovial cells. Phenotypic and T cell regulatory properties of macrophage-like cells that express HLA-DR.

Normal and rheumatoid synovial cells have been analyzed in frozen sections and in suspension. HLA-DR-expressing, macrophage-like cells are demonstrated in normal synovial intima and in rheumatoid tissue. Suspended normal synoviocytes equaled peripheral blood non-T lymphocytes as stimulators of mixed lymphocyte reactions, whereas adherent rheumatoid synovial cells were extremely efficient as such stimulators and in presenting soluble antigens to autologous T lymphocytes. This HLA-DR-dependent T lymphocyte regulation might provide a cellular basis for the HLA-D haplotype-arthritis associations.

Stimulation of collagenase secretion from rheumatoid synovial tissue by human collagen peptides.

Dayer et al. demonstrated that mononuclear cells from human peripheral blood produce a mononuclear-cell factor that stimulates collagenase secretion from adherent rheumatoid synovial cells. The production of this mononuclear-cell factor can be stimulated by phytohemagglutinin, a T-cell mitogen. We have examined immune mechanisms by which collagenase secretion may be stimulated from explants of rheumatoid synovial tissue and from primary monolayer cultures of rheumatoid synovial cells. Conditioned media from cultures of normal peripheral-blood mononuclear cells that had been activated by phytohemagglutinin stimulated collagenase secretion from most explants and from all monolayer cultures that were examined. The direct addition of phytohemagglutinin stimulated collagenase secretion in explants from eight of fourteen patients and in monolayer cultures from three of six patients. These observations indicate the presence of responsive T lymphocytes in rheumatoid synovial tissue and implicate them in the stimulation of collagenase secretion from synovial cells. The direct addition of type-II collagen peptides to rheumatoid explants also stimulated collagenase secretion in explants from six to fifteen patients. Four of five of these patients displayed moderate or severe disease activity. Other patients showed a marginal stimulation of collagenase secretion on addition of type-II collagen peptides (four of fifteen patients) and type-I collagen peptides (three of twelve patients). None of the patients with mild or inactive disease responded to type-II collagen. With one exception, the addition of collagen peptides to monolayer cultures did not stimulate collagenase secretion. An increased production of factors that enhance or inhibit the migration of polymorphonuclear leukocytes was also observed in explant cultures in response to the addition of phytohemagglutinin (in seven of twelve patients), of type-I collagen peptides (in five of ten patients), and of type-II collagen peptides (in five of eleven patients). There was no correlation between production of these factors and stimulation of collagenase secretion in rheumatoid synovial tissue.

Stimulation of rheumatoid synovial cell collagenase and prostaglandin production by partially purified lymphocyte-activating factor (interleukin 1).

Human macrophages produce in culture a factor termed mononuclear cell factor (MCF) that increases the production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. A factor with similar biologic activity is also produced by the murine macrophage cell line P388D1. By using a sequential purification scheme involving ammonium sulfate fractionation; chromatography on DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose; and discontinuous polyacrylamide gel electrophoresis, the P388D1 cell-derived, synovial cell-stimulating factor was copurified with the lymphocyte-activating factor [LAF; interleukin 1 (IL 1)]. The specific activity of the partially purified LAF (IL 1) was approximately 15,000-fold higher than that of the LAF (IL 1) in the original P388D1 cell culture supernatant. On the basis of (i) the copurification of the P388D1 cell-derived LAF (IL 1) and synovial cell-stimulating factors; (ii) the similarity in cell of origin, molecular weight, and phenylglyoxal sensitivity of human MCF and murine LAF (IL 1); and (iii) the presence of LAF (IL 1) activity in preparations of partially purified human MCF, we have postulated that LAF (IL 1) may have effects on cell targets that are nonlymphoid in nature and also that human MCF may be similar to, or identical with human LAF (IL 1). The results of these studies have raised the possibility that LAF (IL 1) may play a role in macrophage-mediated activation of synovial cells and lymphocytes which are involved in the inflammatory responses associated with rheumatoid arthritis.

Effects of D-Penicillamine on lymphocyte modulation of synovial collagenase production

While D-Penicillamine is effective in the treatment of rheumatoid arthritis, its mechanism of action is unknown. In this study, effects of D-Penicillamine on collagenase production by adherent rheumatoid synovial cells were investigated. D-Penicillamine did not directly affect the synovial collagenase production. However, lymphocyte-free-supernatant (LFS) recovered from lymphocytes exposed to D-Penicillamine in vivo and in vitro significantly reduced collagenase production by adherent synovial cells. LFS from lymphocytes of normal subjects and from non-D-Penicillamine treated rheumatoid patients stimulated collagenase production. These investigations indicate that D-Pencillamine indirectly affects collagenase production by cultured synovial cells and suggests beneficial effects on controlling the primary disease process.