Diverse morphological responses of normal human synovial fibroblasts to mononuclear leukocyte products: relationship to prostaglandin production and plasminogen activator activities,and comparison with the effects of purified interleukin 1

Summary Supernatant media from cultured human mononuclear blood leukocytes (MCCM) induced morphological changes in normal human synovial fibroblasts in culture, including the formation of cells with a dendritic or stellate morphology and, less frequently, cells with a striking fenestrated appearance. These changes were fully reversed within 1 h of removing the MCCM. They were inhibited by indomethacin, the glucocorticoids hydrocortisone, prednisolone and dexamethasone, and by colcemid, but not by actinomycin D and only weakly by cycloheximide. The morphological responses to MCCM could be reproduced by MCCM fractions containing interleukin 1-like activity and by purified forms of human interleukin 1 (IL-1), including monocyte-derived IL-1 and recombinant IL-1. These responses were also inhibited by indomethacin, indicating a link with prostanoid production. However, the morphological responses were not related to the stimulation of plasminogen activator activity due to MCCM, MCCM fractions, or IL-1.     

Evidence that interleukin-1 induction of synovial cell plasminogen activator is mediated via prostaglandin E2 and cAMP

Addition of the cyclooxygenase inhibitor indomethacin to human synovial cells in culture, at concentrations which completely block prostaglandin E2 (PGE2) synthesis, reversibly inhibited the interleukin-1 (IL-1) stimulation of cell-associated and extracellular plasminogen activator (PA) production. Results of mixing experiments suggested that the inhibition by indomethacin was not due to stimulation of production and/or activation of a PA inhibitor, but reflected inhibition of PA synthesis. Simultaneous addition of PGE2 or dibutyryl cAMP prevented the inhibition by indomethacin. Addition of the phosphodiesterase inhibitor, theophylline, the adenylate cyclase stimulator, forskolin, or dibutyryl cAMP caused an enhancement of the IL-1 induction of synovial cell PA. These results suggest that the IL-1 induction of synovial cell PA occurs via generation of endogenous PGE2 and cAMP.     

Peripheral blood mononuclear cells stimulate N-acetyl-β-glucosaminidase levels of human synovial fibroblast-like cells

Summary Fibroblast-like synovial cells isolated from intact joints of non-arthritic human donors released up to nine-times higher activity of the lysosomal acid hydrolase N-acetyl--glucosaminidase (NAG) than controls when incubated in conditioned medium from homologous peripheral blood mononuclear cells (MCCM). This increase occurred without decrease in cell numbers or other evidence of cytotoxicity. An increase in cell-associated NAG activity was also suggested, but this was not statistically significant. Indomethacin present during production of MCCM or added with MCCM to fibroblast cultures did not alter the response to MCCM, indicating that the effect of MCCM was not due to the presence of products from the cyclo-oxygenase pathway. At a concentration known to block protein synthesis in most cells (10^–5 M), cycloheximide markedly suppressed the NAG releasing response to MCCM. The secretion of NAG due to MCCM was not affected by all-trans retinoic acid (10^–6 M) but was suppressed by the corticosteroid, dexamethasone.     

Interleukin-1beta induces elevation of spermidine/spermine N1-acetyltransferase activity and an increase in the amount of putrescine in synovial adherent cells from patients with rheumatoid arthritis

OBJECTIVE: To investigate polyamine metabolism in rheumatoid synovial adherent cells stimulated by interleukin- 1beta (IL-1beta). METHODS: Synovial adherent cells obtained from patients with rheumatoid arthritis (RA) were cultured and incubated in the presence or absence of human recombinant IL-1beta at a concentration of 10 ng/ml for 24 h. The cellular contents of polyamines as well as the activities of spermidine/spermine N1-acetyltransferase (SAT) and ornithine decarboxylase (ODC) were measured. RESULTS: Polyamines in synovial adherent cells decreased significantly after 24 h incubation in the absence of IL-1beta. However, in the presence of IL-Ibeta, putrescine and N'-acetylspermidine increased significantly. No significant difference was observed between the amount of spermidine in synovial adherent cells incubated with and without IL-1beta. Spermine and N8-acetylspermidine in synovial adherent cells incubated with IL-1beta decreased significantly more than in synovial adherent cells incubated without. SAT activity reached a peak 12 h after the addition of IL-1beta and then decreased, while the ODC activity did not increase. SAT activity was elevated by the addition of IL-1beta in a dose dependent manner. CONCLUSION: An increase in the putrescine level in rheumatoid synovial adherent cells as a result of the elevation of SAT activity induced by IL-1beta may play a role in RA.     

Stimulation of the hyaluronic acid levels of human synovial fibroblasts by recombinant human tumor necrosis factor alpha, tumor necrosis factor beta (lymphotoxin), interleukin-1 alpha, and interleukin-1 beta

Monocyte/macrophage polypeptides (monokines) alter the properties of synovial cells. This interaction could explain some of the properties of the inflamed synovium in rheumatic disease. Only recently has it been possible to test the action of purified monokines on the target synovial cells. We report here that recombinant human tumor necrosis factor alpha, tumor necrosis factor beta (lymphotoxin), interleukin-1 alpha, and interleukin-1 beta stimulate the hyaluronic acid (HA) levels of human synovial fibroblast-like cells. The effect of monokines was generally inhibited by indomethacin, suggesting the involvement of an endogenous cyclooxygenase product in the stimulation, and by the glucocorticoid, dexamethasone. In contrast, all-trans-retinoic acid stimulated synovial cell plasminogen activator activity but did not increase the HA levels. These findings could help to explain the raised HA levels found in the joint fluids and in the circulation of patients with rheumatic disease.     

Stimulation of the hyaluronic acid levels of human synovial fibroblasts by recombinant human tumor necrosis factor α, tumor necrosis factor β (lymphotoxin), interleukin-1α, and interleukin-1β

Monocyte/macrophage polypeptides (monokines) alter the properties of synovial cells. This interaction could explain some of the properties of the inflamed synovium in rheumatic disease. Only recently has it been possible to test the action of purified monokines on the target synovial cells. We report here that recombinant human tumor necrosis factor α, tumor necrosis factor β (lymphotoxin), interleukin-1α, and interleukin-1β stimulate the hyaluronic acid (HA) levels of human synovial fibroblast-like cells. The effect of monokines was generally inhibited by indomethacin, suggesting the involvement of an endogenous cyclooxygenase product in the stimulation, and by the glucocorticoid, dexamethasone. In contrast, all-trans-retinoic acid stimulated synovial cell plasminogen activator activity but did not increase the HA levels. These findings could help to explain the raised HA levels found in the joint fluids and in the circulation of patients with rheumatic disease.     

Independent regulation of plasminogen activator inhibitor 2 and plasminogen activator inhibitor 1 in human synovial fibroblasts

Objective. To study the plasminogen activator inhibitor(s) (PAI) produced in vitro by human synovial fibroblast-like cells. Methods. Human synovial cell explant cultures were established using cells from nonrheumatoid donors. PAI-2 and PAI-1 antigens were measured by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) levels were determined by Northern blot. Results. The synovial fibroblasts produced both PAI-2 and PAI-1. Interleukin-1 (IL-1) increased PAI-2 but decreased PAI-1 formation, both at the protein and the mRNA levels. Using cyclooxygenase inhibitors, evidence was obtained that an endogenous cyclooxygenase product(S) in the IL-1—treated cultures inhibited formation of both PAIs; exogenous prostaglandin E₂ (10⁻⁷M) reversed the effect of cyclooxygenase inhibition. The glucocorticoid dexamethasone (10⁻⁶ to 10⁻⁷M) inhibited IL-1—stimulated PAI-2 formation but reversed the suppressive effect of IL-1 on PAI-1 production. Conclusion. PAI-2 formation and PAI-1 formation can be regulated independently in human synoviocytes, illustrating the complexity of the modulation of the net PA activity expressed by these cells.     

Independent regulation of plasminogen activator inhibitor 2 and plasminogen activator inhibitor 1 in human synovial fibroblasts.

OBJECTIVE. To study the plasminogen activator inhibitor(s) (PAI) produced in vitro by human synovial fibroblast-like cells. METHODS. Human synovial cell explant cultures were established using cells from nonrheumatoid donors. PAI-2 and PAI-1 antigens were measured by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) levels were determined by Northern blot. RESULTS. The synovial fibroblasts produced both PAI-2 and PAI-1. Interleukin-1 (IL-1) increased PAI-2 but decreased PAI-1 formation, both at the protein and the mRNA levels. Using cyclooxygenase inhibitors, evidence was obtained that an endogenous cyclooxygenase product(s) in the IL-1-treated cultures inhibited formation of both PAIs; exogenous prostaglandin E2 (10(-7) M) reversed the effect of cyclooxygenase inhibition. The glucocorticoid dexamethasone (10(-6) to 10(-7) M) inhibited IL-1-stimulated PAI-2 formation but reversed the suppressive effect of IL-1 on PAI-1 production. CONCLUSION. PAI-2 formation and PAI-1 formation can be regulated independently in human synoviocytes, illustrating the complexity of the modulation of the net PA activity expressed by these cells.     

Hormonal control of plasminogen activator secretion in ZR-75-1 human breast cancer cells in culture

Activity of secreted plasminogen activator (PA) by ZR-75-1 human breast cancer cells in culture is shown to be altered by the addition of physiologically relevant concentrations of the hormones 17 beta-estradiol (E2), insulin, and dexamethasone. After 48 h, E2 stimulated PA activity 6-fold at concentrations as low as 10(-12) M. This stimulation was prevented by the addition of actinomycin D and cycloheximide. The antiestrogen tamoxifen reduced estrogen stimulation of PA, but had slight stimulatory effects on PA secretion by itself. Insulin (5 X 10(-10) M) induced a 2-fold increase in PA activity. Effects of insulin and E2 were additive, suggesting independent sites of control of PA production. Dexamethasone (10(-8) M) decreased PA activity by 20%, but did not inhibit cell growth at the concentration tested. These data suggest that secreted PA activity is differentially regulated by hormones and that effects of PA and growth do not occur in parallel.     

Interleukin-1β and interleukin-1α stimulate the plasminogen activator activity and prostaglandin E2 levels of human synovial cells

Monocyte—macrophage polypeptides (monokines) cause synovial cells to increase the levels of putative mediators of destruction and inflammation. This interaction may account for some of the properties of rheumatoid pannus. We report here that samples of purified human interleukin-1β(IL-1β) and recombinant IL-1β stimulate both the plasminogen activator activity and prostaglandin E₂ levels of human synovial fibroblast-like cells. The same holds true for purified pig IL-1 (catabolin) and recombinant murine IL-1. The elevation in plasminogen activator activity was inhibited by indomethacin, and this suggests that endogenous prostanoids are important in the IL-1-mediated stimulation of proteinase activity.     

Bone matrix degradation by the plasminogen activation system. Possible mechanism of bone destruction in arthritis

The observed increase in urokinase-type plasminogen activator (u-PA) and its receptor (u-PAR) in synovial tissue of patients with rheumatoid arthritis (RA) suggests pathophysiological involvement of the plasminogen activation (PA) system in inflammatory joint disease. In the present study, we investigated the capacity of the PA system to degrade non-mineralized and mineralized bone-like matrix in vitro as a model for bone destruction. Transfected mouse LB6 cell lines, that expressed either human u-PA or u-PAR, were cultured separately and simultaneously on radiolabelled bone matrix in the presence of plasminogen. Osteoblast-like murine calvarial MC3T3-E1 cells were used to produce a well-characterized, highly organized bone-like matrix, that could be mineralized in the presence of beta-glycerol phosphate. Bone matrix degradation was followed by the release of radioactivity in the culture medium. u-PA-producing cells, in contrast to u-PAR- producing cells, degraded both non-mineralized and mineralized bone matrix. This effect could be inhibited by anti-u-PA antibodies, as well as by tranexamic acid and by aprotinin, indicating that the degrading activity is u-PA mediated and plasmin dependent. Co-cultivation of a small portion of u-PA-producing cells with u-PAR-expressing cells resulted in a marked increase in degradation activity. Reduction of this potentiating effect by suramin or the amino-terminal fragment of u- PA, both competitive inhibitors of u-PA receptor binding, shows that this synergistic effect is due to binding of u-PA to u-PAR. u-PAR must be cell associated, as binding of u-PA to a soluble u-PAR prevented this enhancement. The capability of the PA system to degrade bone matrix in vitro, and the previously demonstrated increased expression of u-PA and u-PAR in synovial tissue of patients with RA, further support a role for the PA system in the development of bone erosions.      

Oncostatin M stimulates monocyte chemoattractant protein-1- and interleukin-1-induced matrix metalloproteinase-1 production by human synovial fibroblasts in vitro

Objective. To measure levels of oncostatin M (OSM) in the synovial fluid of rheumatoid arthritis (RA) patients and to examine the activities of human OSM in the regulation of human synovial fibroblast (HSF) production of chemokines and matrix metalloproteinases (MMP-1 and MMP-3) in vitro. Methods. We examined the levels of OSM in the synovial fluids of patients with arthritis by an enzymelinked immunosorbent assay (ELISA). ELISA of cell culture supernatants and Northern blots were used to assess responses of HSF to interleukin-1α (IL-1α), OSM, and other members of the IL-6/leukemia inhibitory factor (IL-6/LIF) family of cytokines. Results. We detected variable levels of OSM antigen in 9 of 10 RA patient synovial fluids, but levels were not detectable in 9 of 10 osteoarthritis (OA) patient fluids. Upon examining the responses of HSF in culture, OSM stimulated monocyte chemoattractant protein 1 (MCP-1), whereas RANTES secretion (regulated upon activation, normal T expressed and presumably secreted) was not altered by OSM alone. In IL-1α induced cells, OSM costimulation further enhanced MCP-1 release, but inhibited the release of RANTES and IL-8. Other members of the IL-6/LIF family of cytokines did not show these effects. OSM induced a small elevation of MMP-1 production over 2 and 3 days of stimulation (2-fold), and acted significantly to enhance IL-1α-induced production of MMP-1 (to 8-fold and 9-fold at 48 and 72 hours, respectively). No effect of OSM was seen on MMP-3 secretion, either alone or in IL-1α-costimulated cells. Conclusion. These results suggest that OSM has potentially important functions in the modulation of chemokine and metalloproteinase production by synovial cells of the joint.     

Interleukin-1β inhibits luteinizing hormone-induced plasminogen activator activity in rat preovulatory folliclesin vitro

The effects of interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα) on ovulation-associated plasminogen activator (PA) activity were investigated using preovulatory follicles excised 48h after equine chorionic gonadotrophin (16IU)-priming of immature rats. Follicles were incubated for 6 and 14h with a single dose of LH (1 μg/ml) only, or various cytokine doses in the presence or absence of LH. PA activity in follicular homogenates was determined by a radioactively labelled fibrin-coated plate method and secreted levels of the ovulatory mediators progesterone (P) and prostaglandin E (PGE) were measured by radioimmunoassay. LH induced timedependent rises in PA (2.5-fold over control at 6h and fourfold over control at 14h), while IL-1β and TNFα alone had no effect over either time period. LH and cytokine coincubations over 14h revealed that IL-1β dosedependently inhibited the LH-induced increase in PA activity, up to 85%. The effects of TNFα on LH-induced PA activity were not significant. Both IL-1β and TNFα increased P and PGE secretion time- and dose-dependently. In summary, IL-1β dose-dependently inhibits the LH-induced increase in PA activity in rat preovulatory folliclesin vitro while, as with TNFα, increasing P and PGE synthesis. This study, shows that the earlier reported pro-ovulatory action of IL-1β is not likely to be mediated by activation of the PA-system and suggests that IL-1β may mediate a regulatory loop controlling the extent and distribution of LH-induced PA activity in rat preovulatory follicles.     

Transforming growth factor beta stimulates urokinase-type plasminogen activator and DNA synthesis, but not prostaglandin E2 production, in human synovial fibroblasts.

Transforming growth factor beta (TGF-beta) is usually associated with matrix formation and tissue repair; in contrast, cellular expression of the serine proteinase, urokinase-type plasminogen activator (u-PA) is often correlated with tissue remodeling, as well as with cell migration and transformation. We report here that purified recombinant human TGF-beta (greater than or equal to 300 pg/ml) can stimulate rapidly (within 2 h) the u-PA activity of nonrheumatoid synovial fibroblast-like cells. As for interleukin 1 (IL-1), u-PA mRNA levels are raised in response to TGF-beta, but unlike IL-1, no increase in prostaglandin E2 levels occurs. In contrast to a number of other examples in the literature, in which these two cytokines have opposing actions, TGF-beta can potentiate the action of optimal concentrations of IL-1 in enhancing u-PA expression. These effects of TGF-beta are similar to those of all-trans-retinoic acid. In addition, synovial fibroblast DNA synthesis was stimulated by TGF-beta. Because TGF-beta has been detected in the synovia of patients with rheumatoid arthritis and has been shown to reduce the collagenase levels and proliferation of synovial fibroblast-like cells, it has been proposed by others to be involved beneficially in the reparative processes occurring in arthritic lesions. However, on the basis of our findings, we propose alternative functions for this cytokine--namely, roles in the destructive events as well as in the synovial hyperplasia observed in rheumatoid joints.     

Interleukin 4 increases human synovial cell expression of VCAM-1 and T cell binding.

OBJECTIVE--The effects were studied of interleukin 4 (IL-4) on T cell-synovial cell adhesion and on the expression of adhesion molecules on the surface of synovial fibroblast-like cells. METHODS--The adhesion of T cells toward the synovial cells were measured by 51chromium-labelled adhesion assay. The expression of adhesion molecules on synovial cells were analysed by flowcytometry. RESULTS--Stimulation of synovial cells with IL-4 increased T cell-synovial cells adhesion in a time- and dose-dependent manner. IL-4 considerably enhanced the expression of VCAM-1 on the surface of synovial cells, but not the expression of ICAM-1 and ELAM-1. The combination of IL-1 beta and IL-4 had no effect on the expression of ICAM-1 or VCAM-1 on the surface of synovial cells. The increased adhesion of T cells to IL-4 stimulated synovial cells was inhibited significantly by adding anti-VCAM-1 or anti-CD29 monoclonal antibody. Furthermore, anti-VLA-4 alpha or the combination of anti-VLA-4 alpha and anti-VCAM-1 antibodies blocked completely T-cell binding to IL-4 stimulated synovial cells. CONCLUSIONS--These results suggest that the increased adhesion of T cells to IL-4-stimulated synovial cells is mediated by VLA-4/VCAM-1 pathway.     

Increased expression of IL-1 receptors in response to IL-1β may produce more IL-6, IL-8, VEGF, and PGE2 in senescent synovial cells induced in vitro than in presenescent cells

Primary synovial cells with high passage number demonstrate increased production of proinflammatory mediators in response to inflammatory stimuli compared with cells with low passage number. This study used synovial cells to learn how different numbers of serial subculture passages affect the production of proinflammatory mediators in response to interleukin (IL)-1β. Synovial cells were serially subcultured in flasks until passage 7. During cell passage, synovial cells were treated with IL-1β for 24?h. Levels of proinflammatory mediators were analyzed by ELISA, real-time PCR, and Western blot. Synovial cells at passage 7 had elongated morphology and higher activity of β-galactosidase, a marker of senescence, than those at passage 3. Production of IL-6, IL-8, vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE₂) in response to IL-1β was significantly increased in cells at passage 7 compared with passage 3. To evaluate the mechanism of this different response, IL-1 receptor expression was studied in cells stimulated with IL-1β. Compared with cells at passage 3, cells at passage 7 had stronger expression of IL-1 receptors 1 and 2, as well as significantly increased expression of both receptors, in response to IL-1β. This finding suggests that differential production in response to inflammatory stimuli associated with cell passages should be considered when in vitro experiments are used to evaluate the production levels of proinflammatory mediators.     

Peripheral blood mononuclear cells stimulate prostacyclin levels of human synovial fibroblast-like cells

Summary Fibroblast-like synovial cells, isolated from intact joints of non-arthritic human donors and from explants of rheumatoid and non-rheumatoid synovial tissue, released prostacyclin (PGI₂) when incubated in conditioned medium from human peripheral blood mononuclear cells (MCCM). The effect of MCCM on the rate of PGI₂ synthesis (measured by radioimmunoassay as the stable product, 6-keto-PGF₁ ) was clearly established within 2 h and appeared to require RNA and protein synthesis as judged by inhibition with actinomycin D and cycloheximide, respectively. Low concentrations of dexamethasone suppressed the increase in PGI₂ levels. Prostaglandin E₂ (PGE₂) levels were also raised by the MCCM and reduced by dexamethasone. All-trans retinoic acid did not stimulate the levels of either prostanoid. These findings offer an explanation for some of the inflammatory events occurring in rheumatoid lesions.     

Characterization of the newly established human type B synovial lining cell line, RAMAK-1: expression of adhesion molecules and cytokine production

RAMAK-1, a new spontaneously immortalized synovial cell line consisting of type B lining cells, was recently derived from a patient with rheumatoid arthritis (RA). The present study was designed to characterize RAMAK-1 cells in terms of expression of adhesion molecules and cytokine production. RAMAK-1 cells constitutively express intercellular adhesion molecule-1. In contrast, vascular cell adhesion molecule-1 (VCAM-1) expression by the cells was not observed in the absence of stimulation. However, when stimulated by tumor necrosis factor-α, VCAM-1 expression was readily apparent and sustained over 48 h. RAMAK-1 cells spontaneously produced interleukin (IL)-6, IL-8 and granulocyte macrophage colony stimulating factor, but not IL-1α nor IL-1β. However, when stimulated with IL-1β, the cells expressed IL-1α and IL-1β mRNA. This cell line presents functional responses when stimulated with pro-inflammatory cytokines and may be useful in elucidating the initial lesions of the synovial lining layer of RA.