Plasminogen activator activity of rheumatoid and nonrheumatoid synovial fibroblasts

Extracellular and cell associated plasminogen activator (PA) activities were measured in a series of synovial fibroblast cell lines, derived by outgrowth and passage from both rheumatoid and nonrheumatoid tissue. In early passages of these cell lines under standardized culture conditions, the enzyme activity was low for most lines but relatively high in a few. There was no significant difference in the PA levels of the cell lines from either source. In addition, cells from both groups increased their PA activity in response to conditioned medium from peripheral blood mononuclear cells. It is concluded that the presence of fibrin-like material in rheumatoid joints cannot be simply explained by a relative defect in PA production by cells obtained after outgrowth and passage from rheumatoid explant material.     

Activated monocytes induce arthritis-associated changes in mitochondria of cultured synovial fibroblasts

We have recently shown that synovial fibroblasts cultured from patients with reactive or rheumatoid arthritis exhibit increased autofluorescence when compared with controls. Morphological studies suggested that this increase was related to the anomalous structure of mitochondria in cells cultured from rheumatoid or non-rheumatoid inflammatory synovial tissue. The present study describes attempts to find an explanation for these observations. The effects of conditioned media of cultured mononuclear cells were tested on normal synovial fibroblasts. Conditioned media of monocytes stimulated with lipopolysaccharide or poly-IC induced an increase in the cellular autofluorescence and changes in the morphology of mitochondria in normal fibroblasts. These changes were indistinguishable from those seen in synovial fibroblasts cultured from various arthritides. Indomethacin or gold salts did not abolish the effects of monocyte-conditioned media. Abnormal mitochondria could not be induced in the presence of cycloheximide. This study describes a new aspect of monocyte-fibroblast interactions during rheumatoid and non-rheumatoid inflammation of synovial tissue.     

Comparison of the inhibitory effects of two types (90 kDa and 190 kDa) of hyaluronic acid on the expression of fibrinolytic factors in human synovial fibroblasts

 Hyaluronic acid (HA) has been shown to be clinically effective, and is currently used for the treatment of arthropathy. We previously reported that HA of molecular weight 90 kDa (90-HA) inhibits the fibrinolytic factors in human synovial fibroblasts. In the present study, we investigated the effect of high molecular weight (190 kDa) HA (190-HA) compared with 90-HA on the pericellular fibrinolytic system of human synovial fibroblasts in osteoarthritis (OA) and rheumatoid arthritis (RA). Human synovial fibroblasts were obtained from synovial tissues of OA and RA, and were cultured in the presence and absence of 90-HA and 190-HA. Antigens of urokinase-type plasminogen activator (u-PA) and PA inhibitor-1 (PAI-1) were measured by ELISA, and the u-PA activity of the cell surface fraction was evaluated by electrophoretic enzymography. The binding assay of u-PA and the immunocytochemical analysis of u-PA were performed to detect u-PA receptor (u-PAR). HA inhibited the secretion of both u-PA and PAI-1 antigens from the synovial fibroblasts of OA to their conditioned medium, and the degree of inhibition was more effective in OA than in RA. The u-RA binding assay to these cells showed that both 90-HA and 190-HA slightly decreased the maximal number of binding sites (Bmax) in OA. However, in RA, stimulation with 90-HA and 190-HA decreased Bmax by a half and a quarter, respectively. Immunohistochemical analysis showed that u-PAR was constitutively expressed in both synovial fibroblasts, but if these cells were treated with HA, the decrease in the staining of u-PAR was more pronounced in RA than in OA. Furthermore, the degree was more effective with 190-HA than with 90-HA. HA inhibited the pericellular fibrinolytic activity mediated by the u-PA/u-PAR system in synovial fibroblasts of OA and RA, and 190-HA inhibited it more effectively than 90-HA. Received: August 17, 2001 / Accepted: December 13, 2001     

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.     

Rheumatoid pannus formation: synovial cell attachment to the surface of cartilage

Abstract

As a part of the rheumatoid synovial tissue reaction, proliferating synovial cells penetrate the cartilage in the form of a pannus, and cartilage destruction takes place in the zone between the cells and cartilage. The cellular origin of rheumatoid pannus has been debated by many investigators, and it is accepted that fibroblast proliferation, endothelial cells proliferation and monocyte chemotaxis are probably involved. It has been observed that fibroblasts in the pannus share properties of the fibroblasts and chondrocytes. Mast cell activation is frequently associated with proinflammatory cytokine and metalloprotease expression, suggesting an important role for the mast cell in mediating matrix degradation. The mechanism of interaction of polymorphonuclear leukocytes with immune complexes trapped in rheumatoid cartilage resembles that associated with phenomenon of frustrated phagocytosis. Increased levels of IL-1 in the rheumatoid joint may play an important role in joint destruction by stimulation of pannus formation through induction of synovial cell attachment to the articular surface. Synovial cell attachment to cartilage may be the initial step in pannus formation. We have recently shown that the increased expression of VLA-5 and ICAM-1 at the cartilage-pannus junction may result from the interaction of synovial mononuclear cells with matrix proteins. Therefore, we have raised the possibility that VLA-5 may facilitate the growth of pannus by virtue of its ability to react with fibronectin with resulting proliferation of the synoviocytes of the pannus. Further study will be needed in order to understand the precise mechanism of cartilage erosion and pannus formation in the various arthritic disorders.     

Nitric oxide induced cell death in human osteoarthritic synoviocytes is mediated by tyrosine kinase activation and hydrogen peroxide and/or superoxide formation

OBJECTIVE: To investigate the regulation of osteoarthritis (OA) synovial fibroblast nitric oxide (NO) induced cell death. METHODS: Cultured synovial fibroblasts from human OA synovium were incubated with NO donor sodium nitroprusside (SNP) in the absence or presence of specific inhibitors of different protein kinases, cyclooxygenase-2 (COX-2), caspase-3 and caspase-9, inducible NO synthase, and in the absence or presence of prostaglandin E2 (PGE2). Experiments were also performed using scavengers of NO (carboxy-PXTO), peroxynitrite (uric acid), and superoxide (taxifolin). The level of cell death was measured by MTT and DNA fragmentation. RESULTS: Human OA synovial fibroblasts incubated with SNP decreased cell viability and increased DNA fragmentation in a dose dependent manner. This was associated with increased levels of both COX-2 and PGE2 production. Selective inhibition of COX-2 by NS-398 significantly inhibited SNP induced cell death, even in the presence of exogenously added PGE2. Experiments revealed that SNP treated cells expressed increased levels of active caspase-3 and caspase-9, while Bcl-2 was downregulated. Incubation of these treated cells with inhibitors of caspase-3 (Z-DEVD-FMK) or caspase-9 (Z-LEHD-FMK) protected viability of SNP treated OA synovial fibroblasts, indicating that NO mediated cell death was mainly related to apoptosis. This was also confirmed by measuring the DNA fragmentation (TUNEL method) and the level of active caspase-3 (immunocytochemistry) in these cells. Data also showed that SNP induces the activation of kinases MEK 1/2, p38, and tyrosine kinases. Specific inhibition of tyrosine kinases completely abrogated the SNP induced cell death. In turn, this cell death protection was associated with a marked inhibition of caspase-3 and caspase-9 activities, as well as COX-2/PGE2 production. Moreover, data showed that the NO donor SNP induced cell death was not solely related to the production of NO or peroxynitrite, but to the generation of reactive oxygen species (ROS) such as hydrogen peroxide and/or superoxide. CONCLUSION: Our results provided strong evidence of the role of tyrosine kinase and mitogen activated protein kinase activation, by upregulation of COX-2 expression, in NO induced OA synovial fibroblast death. The generation of ROS such as hydrogen peroxide and superoxide appeared to be a major factor in the death of these cells.     

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

Summary Using 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.     

Rheumatoid pannus formation: synovial cell attachment to the surface of cartilage

As a part of the rheumatoid synovial tissue reaction, proliferating synovial cells penetrate the cartilage in the form of a pannus, and cartilage destruction takes place in the zone between the cells and cartilage. The cellular origin of rheumatoid pannus has been debated by many investigators, and it is accepted that fibroblast proliferation, endothelial cells proliferation and monocyte chemotaxis are probably involved. It has been observed that fibroblasts in the pannus share properties of the fibroblasts and chondrocytes. Mast cell activation is frequently associated with proinflammatory cytokine and metalloprotease expression, suggesting an important role for the mast cell in mediating matrix degradation. The mechanism of interaction of polymorphonuclear leukocytes with immune complexes trapped in rheumatoid cartilage resembles that associated with phenomenon of frustrated phagocytosis. Increased levels of IL-1 in the rheumatoid joint may play an important role in joint destruction by stimulation of pannus formation through induction of synovial cell attachment to the articular surface. Synovial cell attachment to cartilage may be the initial step in pannus formation. We have recently shown that the increased expression of VLA-5 and ICAM-1 at the cartilage-pannus junction may result from the interaction of synovial mononuclear cells with matrix proteins. Therefore, we have raised the possibility that VLA-5 may facilitate the growth of pannus by virtue of its ability to react with fibronectin with resulting proliferation of the synoviocytes of the pannus. Further study will be needed in order to understand the precise mechanism of cartilage erosion and pannus formation in the various arthritic disorders.     

Glucocorticoid inhibition of urokinase-like plasminogen activators in cultured human lymphoblasts

Two human lymphoblast cell lines, LICR-LON-HMy2 (HMy2 cells) and GM4672A cells, are moderately growth inhibited by dexamethasone (1,4-pregnadien-9-fluoro-16 alpha-methyl-11 beta, 17 alpha, 21-triol-3,20-dione) (Dex). Both cell types secrete a urokinase (UK)-like plasminogen activator (PA). Treatment of both HMy2 and GM4672A cells with Dex for 1-4 days inhibits extracellular PA activity in a concentration-dependent manner, being half-maximal at approximately 1 X 10(-9)M. Inhibition of PA in both cell types is specific for active glucocorticoids, and this specificity parallels the ability of various steroids to bind to glucocorticoid receptors. HMy2 cell PA is fully suppressible by Dex, whereas up to one third of the activator expressed by GM4672A cells is resistant to glucocorticoid inhibition. Mixing experiments using a UK standard and conditioned media from Dex-treated cells suggest an absence of glucocorticoid-inducible inhibitors to UK or plasmin in both cell types. However, conditioned media from Dex-treated GM4672A cells inhibits a portion of the homologous cellular activator in conditioned media from control GM4672A cells. Thus, low levels of glucocorticoid-inducible inhibitors may contribute to, but cannot fully account for, Dex inhibition of GM4672A PA activity. Glucocorticoid-inducible inhibitors in HMy2 cells are either totally absent or are present at undetectable levels. Thus, regulation of UK-like PAs in HMy2 and GM4672A cells differs with respect to the extent to which glucocorticoids inhibit constitutively expressed activator levels, as well as the possible contribution of glucocorticoid-inducible inhibitors to the regulatory process in GM4672A cells.     

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.     

Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by toll-like receptor signaling

OBJECTIVE: To test the hypothesis that bacterial products acting as adjuvants, such as CpG oligodeoxynucleotides (ODNs) and peptidoglycans (PGs), are able to activate synoviocytes, and to determine the involvement of Toll-like receptors (TLRs) in this activation process. METHODS: Cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) were stimulated with CpG ODNs or PGs. The expression of various integrins was determined by fluorescence-activated cell sorting. TLR and matrix metalloproteinase (MMP) messenger RNA (mRNA) was measured by real-time polymerase chain reaction. Additionally, levels of interleukin-6 (IL-6) and IL-8 in the culture supernatants were assessed by enzyme-linked immunosorbent assay. Blocking experiments were performed by adding anti-TLR-2 and anti-TLR-4 monoclonal antibodies to cultures stimulated with bacterial PGs. RESULTS: Incubation of synovial fibroblasts with CpG ODNs resulted in neither up-regulation of the expression of integrins on the cell surface, up-regulation of MMP mRNA expression, nor IL-6 and IL-8 production. However, incubation of RA synovial fibroblasts as well as OA synovial fibroblasts with staphylococcal PGs led to an up-regulation of CD54 (ICAM-1) surface expression and to increased expression of MMP-1, MMP-3, and MMP-13 mRNA. Furthermore, production of the proinflammatory cytokines IL-6 and IL-8 was increased by treatment with PGs. We demonstrated that cultured synovial fibroblasts express low levels of TLR-2 and TLR-9 mRNA. TLR-2 was up-regulated after stimulation with PGs, whereas TLR-9 mRNA remained at baseline levels after stimulation with CpG ODNs. Anti-TLR-2 monoclonal antibodies significantly inhibited production of IL-6 and IL-8 induced by stimulation with PGs. CONCLUSION: We demonstrate that bacterial PGs activate synovial fibroblasts, at least partially via TLR-2, to express integrins, MMPs, and proinflammatory cytokines. Inhibition of TLR signaling pathways might therefore have a beneficial effect on both joint inflammation and joint destruction.     

Actin cytoskeleton dynamics linked to synovial fibroblast activation as a novel pathogenic principle in TNF-driven arthritis

Rheumatoid arthritis is a chronic inflammatory disorder whose origin of defect has been the subject of extensive research during the past few decades. While a number of immune and non-immune cell types participate in the development of chronic destructive inflammation in the arthritic joint, synovial fibroblasts have emerged as key effector cells capable of modulating both joint destruction and propagation of inflammation. Ample evidence of aberrant changes in the morphology and biochemical behaviour of rheumatoid arthritis synovial fibroblasts have established the tissue evading and "transformed" character of this cell type. We have recently demonstrated that actin cytoskeletal rearrangements determine the pathogenic activation of synovial fibroblasts in modelled TNF-mediated arthritis, a finding correlating with similar gene expression changes which we observed in human rheumatoid arthritis synovial fibroblasts. Here, we show that pharmacological inhibition of actin cytoskeleton dynamics alters potential pathogenic properties of the arthritogenic synovial fibroblast, such as proliferation, migration and resistance to apoptosis, indicating novel opportunities for therapeutic intervention in arthritis. Recent advances in this field of research are reviewed and discussed.     

Pro-apoptotic effect of nonsteroidal anti-inflammatory drugs on synovial fibroblasts

Abstract

Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the articular synovial tissues. Although the etiology of RA has not yet been elucidated, physical and biochemical inhibition of synovial hyperplasia, which is the origin of articular destruction, may be an effective treatment for RA. Nonsteroidal anti-inflammatory drugs (NSAIDs) have long been used for the treatment of RA. The mechanism of action of NSAIDs generally involves the inhibition of cyclooxygenase (COX) at sites of inflammation. Thus, NSAIDs were not generally considered to have a so-called anti-rheumatic effect, including inhibition of progressive joint destruction and induction of remission. However, certain conventional NSAIDs and celecoxib, a selective COX-2 inhibitor, have been reported to inhibit synovial hyperplasia by inducing the apoptosis of human synovial fibroblasts. Therefore, it has been suggested that such NSAIDs may not only have an anti-inflammatory effect but also an anti-rheumatic effect. In this review, we summarize findings about the pro-apoptotic effect, in other words, anti-proliferative effect of NSAIDs on synovial fibroblasts from patients with RA.     

Rheumatoid fibroblast-like synoviocytes overexpress the chemokine stromal cell-derived factor 1 (CXCL12), which supports distinct patterns and rates of CD4+ and CD8+ T cell migration within synovial tissue

OBJECTIVE: A characteristic feature of the inflammatory infiltrate in rheumatoid arthritis is the segregation of CD4 and CD8 T lymphocyte subsets into distinct microdomains within the inflamed synovium. The aim of this study was to test the hypothesis that chemokines in general and stromal cell-derived factor 1 (SDF-1; CXCL12) in particular are responsible for generating this distinctive microcompartmentalization. METHODS: We examined how synovial CD4/CD8 T cell subsets interacted in coculture assays with fibroblasts derived from chronic inflammatory synovial lesions and normal synovial tissue as well as from fetal lung and adult skin. We used the ability of T cells to migrate beneath fibroblasts (a process called pseudoemperipolesis) as an in vitro marker of T cell accumulation within synovial tissue. RESULTS: Rheumatoid fibroblast-like synoviocytes (FLS) displayed a unique ability to support high levels of CD4 and CD8 T cell pseudoemperipolesis. Nonrheumatoid FLS as well as fetal lung fibroblasts supported low levels of pseudoemperipolesis, while skin-derived fibroblasts were unable to do so. CD8 T cells migrated under fibroblasts more efficiently and at a higher velocity than CD4 T cells, a feature that was intrinsic to CD8 T cells. Rheumatoid fibroblasts constitutively produced high levels of SDF-1 (CXCL12), which was functionally important, since blocking studies showed reductions in T cell pseudoemperipolesis to levels seen in nonrheumatoid FLS. Rheumatoid fibroblasts also constitutively produced high levels of vascular cell adhesion molecule 1 (VCAM-1; CD106), but this did not contribute to T cell pseudoemperipolesis, unlike the case for B cells, which require SDF-1 (CXCL12)-CXCR4 and CD49d-VCAM-1 (CD106) interactions. Importantly, only combinations of rheumatoid FLS and rheumatoid-derived synovial fluid T cells supported pseudoemperipolesis when examined ex vivo, confirming the in vivo relevance of these findings. CONCLUSION: These studies demonstrate that features intrinsic to both fibroblasts (the production of SDF-1) and CD8/CD4 T cells (the expression of CXCR4) are responsible for the characteristic pattern of T lymphocyte accumulation seen in the rheumatoid synovium. These findings suggest that the SDF-1/CXCR4 ligand/receptor pair is likely to play an important functional role in T lymphocyte accumulation and positioning within the rheumatoid synovium.     

Interrelationship between adenylate cyclase activity, adenosine 3':5' cyclic monophosphate phosphodiesterase activity, adenosine 3':5' cyclic monophosphate levels, and growth of cells in culture

To investigate how cell population density influences the intracellular concentration of cyclic AMP we have measured adenylate cyclase and cyclic AMP phosphodiesterase activities and cyclic AMP levels at various stages of cell density in normal rat-kidney (NRK) cells, which exhibit contact-inhibition of growth, and in normal chick-embryo fibroblasts (CEF), which do not show contact inhibition of growth under our conditions. Until NRK cells reach confluency, both activities increase with increasing cell population and cyclic AMP levels are low. As NRK cells reach confluency, cyclic AMP phosphodiesterase activity decreases somewhat whereas adenylate cyclase activity continues to rise. This increase in synthetic ability is accompanied by the increase in cyclic AMP levels which occurs in these cells at confluency. In CEF grown in 5% serum where density-dependent inhibition of growth is not observed, both adenylate cyclase and cyclic AMP phosphodiesterase activities increase proportionately with increasing cell population density. No significant alteration occurs in the ratio between these two enzyme activities and no change is observed in cyclic AMP levels.The NaF-stimulated activity in NRK cells increases with increasing cell density until the cells reach confluency; thereafter the NaF-stimulated activity remains constant. In contrast, the NaF-stimulated activity observed in CEF does not vary appreciably between light and heavy density.The observed changes in the enzymes of cyclic AMP metabolism accurately reflect the changes in cyclic AMP concentration as a function of cell population density. The data indicate that these two enzyme activities respond to increasing cell density to elicit a rise in intracellular cyclic AMP levels. The elevated cyclic AMP levels are thought to be involved in the regulation of cellular growth rate and the mediation of contact inhibition of growth.     

Interleukin-1 stimulation of phospholipase activity in rat synovial fibroblasts. Possible regulation by cyclooxygenase products

Tritiated arachidonic acid (3H-AA)-labeled rat synovial fibroblasts stimulated with human recombinant interleukin-1 beta (rIL-1 beta) released incorporated radiolabel in a time-dependent and dose-dependent manner, with labeled prostaglandins representing 29% of the released radiolabel. Treatment of the cells with dibutyryl cAMP or prostaglandin E2 enhanced both spontaneous and rIL-1 beta-induced 3H-AA release; treatment with indomethacin or naproxen inhibited the response. The effects of these cyclooxygenase inhibitors on 3H-AA release were not reversed by the addition of prostaglandin E2. The activities of phospholipase A, phospholipase C, and diglyceride lipase were detected in the homogenates of rat synovial fibroblasts. Pretreatment of synovial cells with rIL-1 beta resulted in a threefold stimulation of phospholipase A activity and a slight increase in phospholipase C activity in cell homogenates. These data show that rIL-1 beta stimulates phospholipase activities in rat synovial fibroblasts and that at least one of these activities may be regulated by either prostaglandins or cAMP.     

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.