Independent regulation of plasminogen activator inhibitor 2 and plasminogen activator inhibitor 1 in human synovial fibroblasts.
код для вставкиСкачать1526 INDEPENDENT REGULATION OF PLASMINOGEN ACTIVATOR INHIBITOR 2 AND PLASMINOGEN ACTIVATOR INHIBITOR 1 IN HUMAN SYNOVIAL FIBROBLASTS JOHN A. HAMILTON, DAISY CHEUNG, ENRICO L. FILONZI, DIANA S. PICCOLI, JOHANN WOJTA, MARISA GALLICHIO, KATHERINE McGRATH, and KARENA LAST 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 PAL1 antigens were measured by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) levels were determined by Northern blot. Results. The synovial fibroblasts produced both PA13 and PAI-1. Interleukin-1 (IL-1) increased PA13 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 E, (10-7M) reversed the effect of cyclooxygenase inhibition. The to 10q7M) inhibglucocorticoid dexamethasone ( From the Department of Medicine, University of Melbourne, and the Department of Diagnostic Haematology, Royal Melbourne Hospital, Parkville, Victoria, Australia. Supported by a program grant from the National Health and Medical Research Council of Australia and by the National Heart Foundation of Australia. Dr. Wojta's work was supported by grant 7617 from the Austrian Fund for the Promotion of Scientific Research. John A. Hamilton, PhD, DSc: Department of Medicine; Daisy Cheung, BSc (Hons): Department of Medicine; Enrico L. Filonzi, BSc (Hons): Department of Medicine; Diana S. Piccoli, BSc (Hons): Department of Medicine; Johann Wojta, PhD: Department of Diagnostic Haematology; Marisa Gallichio, BSc (Hons): Department of Diagnostic Haematology; Katherine McGrath, MBBS, FRACP: Department of Diagnostic Haematology; Karena Last, BSc (Hons): Department of Medicine. Address reprint requests to John A. Hamilton, PhD, DSc, Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Parkville 3050,Victoria, Australia. Submitted for publication March 26, 1992; accepted in revised form August 10, 1992. Arthritis and Rheumatism, Vol. 35, No. 12 (December 1992) ited IL-l-stimulated PAL2 formation but reversed the suppressive effect of IL-1 on PAI-1 production. Conclusion. PA13 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. The extent of tissue remodeling and cell migration that occurs during, for example, inflammatory reactions may be dependent on the net cellular proteolytic activity (1). For instance, such activity in the joint tissue of rheumatoid arthritis patients is likely to be determined in part by the balance between the levels of proteases and those of their corresponding inhibitors. One such proteolytic system of potential significance to these events is the plasminogen activator (PA)-PA inhibitor (PAI) system. There are 2 mammalian PAS, urokinase-type PA (uPA) and tissue-type PA (tPA), which are serine proteases capable of cleaving plasminogen to generate the less specific protease, plasmin (2). Urokinase-type PA is considered important in tissue remodeling, cell migration, and neoplasia (3), while tPA is generally viewed as being critical in fibrin resolution (4). Two inhibitors of both PAS are PAI-1 and PAI-2 (5); however, PAI-2 shows a preference for uPA and PAI-1 for tPA, leading to the view that PAI-1 is the primary physiologic inhibitor of fibrinolysis mediated by tPA (5). The physiologic role of PAI-2 is unclear, although its tissue distribution suggests that it may function locally during inflammation and wound healing (6). However, the roles of the 2 PAIs have not been thoroughly defined as yet. Both PAIs are members of the serine protease inhibitor (serpin) superfamily (9, PA1 IN SYNOVIAL FIBROBLASTS the gene structures for the 2 human molecules having now been determined (7-10). Activated synovial fibroblast-like cells have been implicated in the invasive properties of the inflamed synovium of rheumatoid lesions (11). It is likely that their properties in such lesions are being modified by locally generated cytokines; it has been shown, for example, that interleukin-1 (IL-1) elicits a number of responses in such cells (12-14). Elevated PA levels have been found in rheumatoid tissue explant cultures and synovial fluids when compared with their noninflamed counterparts (15,16). Also, plasmin has been shown to degrade cartilage (17). Cytokineactivated synovial fibroblasts could be a source of uPA activity in rheumatoid lesions, and the control of its expression by these cells could be important in understanding the pathogenesis of such lesions, which includes prominent deposition of fibrin in the synovial tissue (18). We have proposed that enhanced PA activity in these cells, resulting from the action of monocyte/macrophage cytokines, could be one manifestation of the expression of an immature, invasive, “tumor-like’’ phenotype, which could help to explain some of the features of the erosive synovial “pannus” in rheumatoid joints (19). PAI-1 is produced by many cultured cells, particularly those of mesodermal origin (5,6); PAI-2, which was first isolated from placenta, appears to have a more restricted distribution. Of particular relevance to joints, human monocytes produce PAI-2 and PAI-1 in vitro (20,21), while PAI-1 is produced by human chondrocytes and cartilage tissue in vitro (22,23). It was heretofore not known whether human synovial fibroblast-like cells produce PAI-2 and/or PAI-1. We report herein that they can produce both PAI-2 and PAI-1. In this particular cell type, PAI-2 levels are increased by IL-1, but levels of PAI-1 are decreased by the cytokine; treatment with a glucocorticoid elicited changes opposite to those brought about by IL-1. MATERIALS AND METHODS Synovial cell cultures. Human synovial cell explant cultures were established from different nonrheumatoid donors as previously described (18). Passaged cells were functionally and morphologically similar to the synovial fibroblast-like cells (18). Cells were plated for 8 hours at 5 x lo4 cells/0.5 mywell in 48-well plates (Costar, Cambridge, MA) in a-modified minimal essential medium (a-MEM; Commonwealth Serum Laboratories, Parkville, Australia) supplemented with 10% heat-inactivated (56°C for 30 minutes) fetal calf serum (FCS). The cells were then incubated overnight in 1527 fresh medium, usually containing 1% FCS depleted of plasminogen (18) (this allows concomitant measurement of PA activity [18]). For experiments, incubation was in 500 pl a-MEM plus 1% FCS (or 10% FCS) in the presence or absence of cytokine or drugs, usually for 24 hours. Supernatants and cell lysates were collected and assayed for PAI-2 and PAI- 1. PAL2 enzyme-linked immunosorbent assay (ELISA). PAI-2 was quantitated using an ELISA developed at Biotech Australia Pty. Ltd. (Roseville, Australia). In this assay, 96-well plates (Nunc-Immuno Plate; Nunc, Roskilde, Denmark) were coated overnight at room temperature with monoclonal murine anti-human PAT-2 IgG (MAI-21,2 pg/ml; Biopool, Umea, Sweden). Samples or standard at C200 ng/ml (either native human PAI-2 [24] supplied by E. Kruithof [Lausanne, Switzerland] or recombinant human material [25] [supplied by Biotech Australia]) were added to the well for 2 hours, followed by goat anti-PAI-2 IgG (2.0 pg/ml; Biotech Australia) and then by peroxidaseconjugated rabbit anti-goat IgG (1 23,000 dilution; Dakopatts, Copenhagen, Denmark). The sensitivity of the assay was 3 ng/ml, and absorbance was linearly related to PAI-2 concentration up to 50 ng/ml. The PAI-2 assay recognizes both free PAI-2 and PAI-2 complexed with uPA. PAI-1 ELISA. PAI-1 antigen in the samples was quantitated by ELISA (26,27). Briefly, a monoclonal antiPAI-1 antibody (5PAI12; obtained from B. R. Binder, University of Vienna), which recognizes active PAI-1, latent PAI-1, and PAI-1 in complex with tPA immobilized to a micro-ELISA plate was used to bind the PAI-1 contained in the sample. A second peroxidase-labeled monoclonal antiPAI-1 antibody (3PAI5; obtained from B. R. Binder), which recognizes active and latent PAI-1 as well as PAI-1 in complex with tPA, was used to quantitate the amount of bound PAI-1. Purified melanoma PAI-1 (26) was used as a calibration standard (27). The sensitivity of the assay was 0.2 ng/ml, and absorbance was linearly related to PAI-1 concentration up to 40 ng/ml. Detection of messenger RNA (mRNA). Synovial fibroblasts were plated at 5 x lo5 cells in 100 mm-diameter tissue culture dishes (Corning Labware, Corning, NY) and treated as described previously (28). Total RNA was prepared as Table 1. Effect of interleukin-1 (IL-1) on human synovial fibroblast PAL2 and PAI-1 levels* PAI-2 (ng/ml) Addition IL-1 Cellassociated 23 62 f2 f 3t PAI- 1 (ng/ml) Extracellular Cellassociated Extracellular 4.8 f 1 10.3 f lb 1.7 f 0.2 ND 111 2 10 64 2 5$ * Sixth-passage synovial cells from donor MC, cultured (5 X lo4 cells/well) in triplicate as described in Materials and Methods, were incubated for 24 hours at 37°C with or without IL-la (20 pM). Cell lysates and supernatants were collected and assayed for plasminogen activator inhibitor 2 (PAI-2) and PAI-1 content by enzymelinked immunosorbent assay. ND = not detected. t P < 0.001 versus control. $ P < 0.02 versus control. HAMILTON ET AL 1528 reported earlier (28), using the method of Chirgwin et a1 (29). Up to 10 &ane of RNA was fractionated on a formaldehydecontaining 1% agarose gel before transfer to Hybond-Nf membrane (Amersham, Sydney, Australia). The filter was hybridized overnight at 42°C in a standard hybridization buffer (29) containing >2 x lo6 counts per minute of 32P-labeled PAI-2 complementary DNA (cDNA) (10). After hybridization, the membrane was washed several times and exposed to Kodak X A R J film (Eastman-Kodak, Rochester, NY) at -70°C (28). Reagents. Recombinant human IL-1 a (specific activity 5.7 x lo7 unitshg) was obtained as a gift from P. Lomedico (Hoffmann-La Roche, Nutley, NJ). Dexamethasone, indomethacin, and prostaglandin E, were from Sigma (St. Louis, MO). Human cDNA probes were obtained from the following sources: PAI-2 from T-C. Wun (Monsanto, St. Louis, MO), PAI-1 from D. Loskutoff (Scripps Clinic and Research Foundation, La Jolla, CA), and GAPDH from 0. Bernard (Walter and Eliza Hall Institute, Parkville, Australia). Statistical analysis. Measurements in supernatants and cell lysates are presented as the mean 2 SEM values from triplicate cultures. The significance of differences was assessed using Student's 2-tailed t-test; P values less than 0.05 were considered significant. RESULTS Production of PAL2 and PAI-1 by synovial fibroblasts. The conditioned media and cell lysates of human synovial fibroblast-like cells were examined for both PAI-2 and PAI-1, by ELISA. In Table 1, it can be seen that the cells produced both PAIs, with PAI-2 being predominantly found in the cell lysates while PAI-1 was mostly in the conditioned medium. This relative distribution was observed in a total of 30 human synovial fibroblast populations, irrespective of passage number. Table 2. Effect of FCS concentration on IL-1 down-modulation of PAI-1 levels* Extracellular PAL1 (ng/ml) Experiment 2 Experiment 1 Addition 1% FCS 10% FCS 1% FCS 10% FCS IL-1 73 f 2 64 +. 2t 133 +. 6 88 2 2t 66 +. 5 205 +. 11 115 2 41 72 2 3 * Seventh-passage synovial cells from donor GE, cultured (5 x lo4 cells/well) in triplicate in a-modified minimal essential medium containing 1% fetal calf serum (FCS) or 10% FCS as described in Materials and Methods, were incubated for 24 hours at 37°C with or without IL-la (20 pM). Supernatants were collected and assayed for PAI-1 content by enzyme-linked immunosorbent assay. Values are the mean 2 SEM. See Table 1 for other definitions. t P < 0.05 versus control. t P < 0.01 versus control. § P < 0.002 versus control. 160{ z ;;a c v c;' 140 - 120100- "1 7r""/ 60 0 0.02 0.2 2 IL-1 (pM) I 20 Figure 1. Human synovial fibroblast plasminogen activator inhibitor 2 (PAI-2) levels after treatment with interleukin-1 (IL-1). Fifthpassage synovial cells from donor JT, cultured (5 x lo4 celldwell) in triplicate as described in Materials and Methods, were incubated for 24 hours at 37°C with increasing concentrations of IL-la. Cell lysates were collected and assayed for PAI-2 content by enzymelinked immunosorbent assay. Values are the mean f SEM. Effect of IL-1 on synovial fibroblast PAI-2 and PAI-1 levels. IL-1 has been found to stimulate the formation of a number of synovial products (for examples, see refs. 12-14). As demonstrated in Table 1, IL-1 was shown in the present study to raise levels of PAI-2. The stimulation of PAI-2 by IL-1 was observed in 15 synovial cell lines studied. In contrast, Table 1 shows that there was a reduction in levels of PAI-1 in the presence of IL-1. Under the same culture conditions (i.e., in 1% FCS),a slight reduction in PAI-1 levels induced by IL-1 was noted with 14 other synovial lines but not observed with 4; this small effect of IL-1 was not due to a redistribution of PAI-1 between extracellular and cellassociated compartments. The data in Table 1 were obtained from cultures performed in low serum concentrations, in order to reduce the contribution of serum. It was reasoned that raising the FCS level to 10% might stimulate the cells to produce PAI-1 and the inhibitory action of IL-1 might therefore be more readily apparent, since it presumably can suppress only newly formed PAI-1. As can be observed in 2 separate experiments (Table 2), the basal PAI-1 level was higher for cultures in 10% FCS than for cultures in 1% FCS, and the inhibitory action of IL-1 could be demonstrated more easily. The dose-responsiveness of the effect of IL-1 on synoviocyte PAI-2 levels in cell lysates is presented PA1 IN SYNOVIAL FIBROBLASTS 1529 h 3 200 \ bn c v z 100 U 0 4 8 12 24 Time (h) Figure 2. Kinetics of the IL-1 effect on human synovial fibroblast PAI-2 levels. Second-passage synovial cells from donor AB, cultured (5 X lo4 cells/well) in triplicate as described in Materials and Methods, were incubated at 37°C for various time periods in the absence (0)or presence (0)of IL-la (20 pM). Cell lysates were collected and assayed for PAL2 by content by enzyme-linked immunosorbent assay. Values are the mean f SEM (where not shown, error bars are smaller than the symbol). See Figure 1 for definitions. in Figure 1; in this and other experiments, the IL-1 was active at concentrations of -0.2 pM. The kinetics with IL-1 at an optimal concentration are depicted in Figure 2. The first changes in the cell lysates were measurable between 2 and 4 hours after addition of the cytokine. Under all conditions, the majority of the PAI-2 remained associated with the cell. The kinetics of the suppressive effect of IL-1 at an optimal concentration on supernatant PAI-1 levels under the same culture conditions are presented in Figure 3. Differences were observed within 3 hours. PA1 mRNA levels. Northern blot analysis (Figure 4A) indicated that IL-1 could raise the level of PAI-2 mRNA, possibly reflecting an increase in PAI-2 gene transcription. Consistent with this, we have found that the RNA synthesis inhibitor actinomycin D (2 pglml) blocked the increased formation of PAI-2 due to IL-1 in an 8-hour experiment (results not shown). Protein synthesis also appeared to be required, since cycloheximide (10 pglml) inhibited the increase in antigen expresshn (not shown). The reduction in PAI-1 transcripts by IL-1 is presented in Figure 4B; PAI-1 mRNA is usually found as a doublet (79,23). Effect of a cyclooxygenase inhibitor. Endogenous cyclooxygenase products, including prostaglandin E, (PGE,), are produced in IL-1-treated synovial cell lines and can, in turn, regulate the levels of certain products under the control of IL-1 (12,13,30). As can be seen in Table 3, the cyclooxygenase inhibitor indomethacin potentiated the action of IL-1 in enhancing PAI-2 formation and exogenous PGE, reversed the effect of indomethacin on PAI-2 expression. The effect of indomethacin was noted in 8 other experiments and was confirmed using another cyclooxygenase inhibitor, naproxen (lO-'A4). These data suggest that an endogenous cyclooxygenase product(s) is quite critical in determining the increase in PAI-2 levels brought about by IL-1. Likewise, the inhibitory effect of IL-1 on PAI-1 levels could be reversed, at least partially, by indomethacin and restored by exogenous PGE,; data from an experiment with cells cultured in both 1% FCS and 10% FCS are presented in Table 4. This effect of indomethacin was found for 9 of 12 synovial cell lines cultured in 1% FCS and 5 of 5 lines cultured in 10% FCS. With some cell lines, indomethacin (lO-'A4) raised the basal PAI-1 levels. Effect of glucocorticoids. A glucocorticoid, such as dexamethasone, can often have an effect opposite to that of IL-1 in many cell types, including synoviocytes (29-31). In Table 5, it is shown that dexamethasone could suppress the action of IL-1 in enhancing h 2 -. bn cl v c o r * ' . ' . ' . ' . ' . ' . ' " 0 3 12 6 24 Time (h) Figure 3. Kinetics of the IL-1 effect on human synovial fibroblast PAL1 levels. Sixth-passage synovial cells from donor PL, cultured (5 x lo4 cells/well) in triplicate as described in Materials and Methods, were incubated at 37°C for various time periods in the absence (0)or presence (0)of IL-1a (20 pM) in a-modified minimal essential medium containing 1% fetal calf serum. Supernatants were collected and assayed for PAI- 1 by enzyme-linked immunosorbent assay. Values are the mean SEM (where not shown, error bars are smaller than the symbol). See Figure 1 for definitions. * 1530 HAMILTON E T AL Figure 4. Effect of IL-1 on steady-state PA1 messenger RNA levels. Cytoplasmic RNA was isolated from &hour cultures of human synoviocytes, and Northern blot analysis was performed as described in Materials and Methods. A, Studies using fifth-passage cells from donor LL. The blot was probed with 32P-labeled PAI-2 complementary DNA (cDNA). Lane 1, Control; lane 2, IL-la (20 pM). Ethidium bromide-stained ribosomal RNA bands indicated even loading in the lanes. B, Studies using second-passage cells from donor FH. The blot was probed with "P-labeled PAI-1 cDNA and GAPDH cDNA (the latter as a housekeeping gene to check RNA loading). Lane 1, Control; lane 2, IL-la (20 pM). See Figure 1 for other definitions. PAI-2 formation, an observation confirmed under the same culture conditions with 5 other lines using lop610p7M dexamethasone. With 3 of 5 lines, treatment with the steroid slightly reduced basal PAI-2 levels (Table 5 ) . Dexamethasone (lO-'it4) also suppressed the increase in PAI-2 mRNA levels resulting from IL-I action, as measured by Northern blot analysis (results not shown). When PAI-1 levels were monitored in the presence of glucocorticoid, it was found that the steroid did not suppress these levels and reversed the inhibitory action of IL-1 (Table 6). When this experiment was carried out with 12 additional lines cultured in 1% FCS or 10% FCS, dexamethasone (lop6 to 10-7M) enhanced basal PAI-1 levels in 5 cases but had no significant effect in 7; the opposing action of the steroid on the IL-1-mediated changes was demonstrated in 11 of 12 lines. DISCUSSION This is the first report on the nature of the PAIs produced by cultured human synovial fibroblast-like cells. We have shown that this cell population can PA1 IN SYNOVIAL FIBROBLASTS 1531 Table 5. Effect of glucocorticoid on human synovial fibroblast PAL2 levels* Table 3. Cyclooxygenase inhibition and PAI-2 regulation* PAI-2 (ng/ml) Addition Iedo Indo + PGE, IL-1 IL-1 IL-1 + Indo + Indo + PGE, Cell-associated Extracellular 23 f 2 16 t 2 10 t 2 521 ND ND 62 t 3 165 f 8 t 52 f 4 10 f 1 27 f 2f 12 f 1 * Sixth-passage synovial cells from donor MC, cultured lo4 cells/well) in triplicate in 1% fetal calf serum as described in Materials and Methods, were incubated for 24 hours at 37°C with or without IL-la (20 pM). Some cultures also contained indomethacin (Indo) (W5M) in the presence or absence of prostaglandin E, (PGE,) (10-7M). Cell lysates and supernatants were collected and assayed for PAL2 content by enzyme-linked immunosorbent assay. Values are the mean f SEM. See Table 1 for other definitions. t P < 0.001 versus treatment with IL-1 alone. t. P < 0.002 versus treatment with IL-1 alone. (5 X produce both PAI-2 and PAI-1. As has been generally reported for other cell types (6,32), PAI-2 was mainly cell associated and presumably in a nonglycosylated form (33) (although when monocyte/macrophages are treated in certain ways, they can secrete PAI-2 that is mostly in a glycosylated form [33]). The converse is true for the distribution of PAI-1 in the synovial cell cultures, as is usually the case for this PA1 (5,6). It was also shown that IL-la stimulates levels of PAI-2 protein and mRNA, but reduces those for PAI-1. Purified recombinant human IL-lp gave similar results (data not shown). These observations indicate PAL2 (ng/ml) Addition Dex IL- 1 IL-1 + Dex Cell-associated Extracellular 18 2 0.2 13 2 1 521 422 83 2 2 32f I t 30 t 2 12 f 1f * Sixth-passage synovial cells from donor GD, cultured (5 x lo4 cells/well) in triplicate as described in Materials and Methods, were incubated for 24 hours at 37°C with or without IL-la (20 pM) in the presence or absence of dexamethasone (Dex) (10-7M). Cell lysates and supernatants were collected and assayed for PAI-2 content by enzyme-linked immunosorbent assay. Values are the mean f SEM. See Table 1 for other definitions. t P < 0.001 versus treatment with IL-1 alone. t. P < 0.002 versus treatment with IL-1 alone. that the synthesis of these 2 PAIs can be modulated independently, as has been found in other cells (6). IL-1 has been shown in many studies to enhance PAI-1 synthesis (3,although in human chondrocytes we showed for the first time that it could have the reverse effect (23). It is not that the synovial cells cannot produce PAL1 in response to a cytokine, since transforming growth factor p can stimulate them to do so (Hamilton JA et al: submitted for publication). In human umbilical vein endothelial cells, IL-1 did not affect PAI-2 production, but increased the amount of PAI-1 produced by the cells (6). Others have shown Table 6. Effect of glucocorticoid on human synovial fibroblast PAI-1 levels* Table 4. Cyclooxygenase inhibition and PAI- 1 regulation* PAI-I (ng/ml) Extracellular PAI- 1 (ng/ml) Addition - 1% FCS 10% FCS 7.0 f 0.5 8.5 f 0.4 1.8 t 0.2 1.9 +. 0.2 2.9 5.7 2.5 Indo Indo + PGE, 4.1 t 0.2 4.8 t 0.7 0.6 2 0.1 IL-1 IL-1 IL-1 + Indo + Indo + PGE, 3.2 0.4 f 0.3t f 0.1 f 0.1 f 0.3.t f 0.1 ~~ * Seventh-passage synovial cells from donor FH, cultured (5 x lo4 cells/well) in triplicate in a-modified minimal essential medium containing 1% fetal calf serum (FCS) or 10% FCS as described in Materials and Methods, were incubated for 24 hours at 37°C with or without IL-la (20 pM). Some cultures also contained indomethacin (Indo) (10-5M) in the presence or absence of prostaglandin E, (PGE,) Supernatants were collected and assayed for PAI-1 content by enzyme-linked immunosorbent assay. Values are the mean f SEM. See Table 1 for other definitions. t P < 0.05 versus treatment with IL-1 alone. $ P < 0.001 versus treatment with IL-1 alone. 1% FCS 10% FCS Dex PGE, Dex + PGE, 5.5 f 0.6 7.0 t 0.4 2.2 t 0.5 2.9 f 0.4 11.3 f 0.2 13.8 f 0.4 6.5 f 0.1 8.5 f 0.9 IL- 1 IL-1 IL-1 IL-1 3.2 2 0.3 4.4 f 0.2t 1.7 f 0.4 2.5 f 0.2 7.1 ? 0.3 10.1 2 0.4f 5.2 2 0.1 7.1 f 0.5 Addition + Dex + PGE, + Dex + PGE, * Third-passage synovial cells from donor WD, cultured (5 x lo4 celldwell) in triplicate in 1% fetal calf serum (FCS) or 10% FCS as described in Materials and Methods, were incubated for 24 hours at 37°C with or without IL-la (20 pM) in the presence or absence of dexamethasone (Dex) ( 10-7M), prostaglandin E, (PGE,) (10-7M), or both. Supernatants were collected and assayed for PAI-1 content by enzyme-linked immunosorbent assay. Values are the mean f SEM. See Table 1 for other definitions. t P < 0.05 versus treatment with IL-1 alone. f P < 0.01 versus treatment with IL-1 alone. 1532 that IL-1 stimulates PAI-2 mRNA and PAI-1 mRNA expression in human MRC-5 fetal lung fibroblasts, but induces an increase in only the latter mRNA species in an SV40-transformed human fibroblast cell line, XP12RO (34). Thus, the cellular PA1 response to IL-1 is cell type specific, as can occur for cytokine effects in a number of vitro systems. The diverse findings for PA1 regulation with the various human cell populations, including fibroblasts, highlight the importance of using fibroblasts isolated from synovial tissue if one is to be able to relate the findings to arthritic disease. Evidence was also given above for downmodulation of the IL-1-mediated stimulation of PAI-2 formation by an endogenous cyclooxygenase product(s), since cyclooxygenase inhibitors could potentiate the action of IL-1. Exogenous PGE, reversed the effect of indomethacin. Likewise, the inhibition of PAI-1 formation by IL-1 was partially due to the formation of a cyclooxygenase product(s). Regulation of the metabolism of IL- I-treated synovial fibroblasts by an endogenous cyclooxygenase product(s) has been noted before (12,13,30,35) and demonstrates that this type of product can influence the degree of response elicited by IL-1. We and others have previously shown that enhancement of uPA activity in human synoviocytes by IL- 1 and mononuclear cell-conditioned medium was partially dependent on the generation of a cyclooxygenase product (13,35,36). It has been postulated that the potential down-regulation of synoviocyte uPA by cyclooxygenase inhibitors could represent a significant portion of the clinical action of these drugs in the treatment of inflammatory arthritides (13); in light of the findings reported herein, it could be that this modulation of uPA activity may be occurring in part by prevention of suppression of PA1 levels produced by endogenous prostanoids in the IL-1-treated synoviocyte cultures. In other words, part of the action of this class of nonsteroidal antiinflammatory drug in joint disease may be to maintain PA1 levels by blocking the formation of endogenous PGE,. Given the evidence that IL-1 induction of synovial cell PA activity is somewhat dependent on the generation of endogenous PGE, and, subsequently, on intracellular elevation of CAMP (13,36), it is likely that endogenous PGE, is functioning via this mechanism in down-regulating PAL2 and PAI-1 expression. In support of this contention, there is evidence for 2 putative binding sites for a cAMP response element in the promoter region of the human PAI-2 gene (37), and cholera toxin, an activator of adenylate cyclase, in- HAMILTON ET AL duced PAL2 in cultured murine macrophages (38); treatment of UCT/gl-1 or endothelial cells with exogenous cAMP or forskolin caused a decrease in PAI-I activity (3p-41). The glucocorticoid, dexamethasone, while usually having slight and varied effects on basal PA1 levels in the synoviocytes, inhibited IL- 1-stimulated PAL2 formation but reversed the suppressive effect of IL-1 on PAI-1 levels. These results provide yet another example of the contrasting pattern of regulation of the 2 PAIs and also provide further examples of the opposing actions of glucocorticoids and IL-1 (31). It has been reported that, in other cells, dexamethasone can reduce PAL2 formation while potentiating that of PAI-1 (32). Dexamethasone has been shown to reduce PAI-2 activity in murine macrophages (38), while 1 study showed down-regulation of secreted PAI-2 antigen and mRNA expression in the human fibrosarcoma line HT1080 (32) whereas another showed no reduction in the amounts of secreted PAL2 in this line (6). Dexamethasone can increase PAI-1 activity in a number of cell types, but fails to do so in a variety of other cell lines (5). Net uPA activity and mRNA expression in human synovial cells have previously been shown to be down-regulated by glucocorticoids (28,42,43); part of the loss of activity could be due to enhanced PAI-I expression. This down-regulation of uPA activity has been proposed as providing a partial explanation for the antiinflammatory action of glucocorticoids in rheumatic disease (28,42,43). The enhancement of PAI-I formation by this class of drug in these cells would provide extra protection against any putative neighboring matrix breakdown in inflamed joints arising from the uPA activity of the synoviocytes; however, the inhibition of PAI-2 formation in the same cell type would favor the expression of net uPA activity. We have reported previously that IL-1 stimulates low and transient uPA activity and uPA mRNA expression in the same synoviocytes (13,28,44). The varied regulation of the 2 PAIs described above, including the influence of endogenous cyclooxygenase products, indicates the complexity in the regulation of this protease activity by the synovial cells. It is intriguing that this cell type, which is believed to play a part in some of the destructive processes evident in inflammatory joint disease (ll), should be producing both types of PAI, one (PAI-1) often believed to be more important in fibrinolysis and the other (PAI-2) in inflammation, pregnancy, and extravascular events (6). However, it is possible that the functions of these PA1 IN SYNOVIAL FIBROBLASTS 2 PAIs may not be as clearly delineated as is generally assumed and it may be, for example, that PAI-1 has a role in controlling inflammation and tissue destruction. The suppression by IL-1 of synoviocyte PAI-1 formation may therefore favor the possibility of local PAmediated proteolysis in inflamed joints. Human monocytes produce PAI-1 and PAI-2 (20), and chondrocytes produce PAI-1 (22,23). Perhaps the prominent fibrin deposition noted in rheumatoid synovial tissue (45) owes its presence in part to PAIs in joints (presumably PAI-I [5]), and based on our findings above, the synoviocyte could be making a contribution. PAI-2 and PAI-1 have been found in rheumatoid synovial fluids (Hamilton JA et al: unpublished observations) and could be derived in part from the synoviocytes. ACKNOWLEDGMENTS The authors thank Biotech Australia Pty. Ltd. for the supply of recombinant human PAI-2 and goat anti-PAI-2 IgG and for support and contributions to this program, J. Bartlett for the supply of synovial tissue, and S. Wong for typing the manuscript. REFERENCES 1. Reich E: Activation of plasminogen: a general mechanism for producing localised extracellular proteolysis, Molecular Basis of Biological Degradative Processes. Edited by RD Berlin, M Herrman, IH Lepow. New York, Academic Press, 1978 2. Saksela 0: Plasminogen activation and regulation of pericellular proteolysis. 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