Cartilage-Derived Chondrocytes Inducible Nitric Oxide Synthase in ...

SB 203580 Inhibits p38 Mitogen-Activated Protein Kinase, Nitric Oxide Production, and Inducible Nitric Oxide Synthase in Bovine Cartilage-Derived Chondrocytes Alison M. Badger,1* Michael N. Cook,* Michael W. Lark,* Tonie M. Newman-Tarr,* Barbara A. Swift,* Allen H. Nelson,† Frank C. Barone,† and Sanjay Kumar* Nitric oxide (NO) is implicated in a number of inflammatory processes and is an important mediator in animal models of rheumatoid arthritis and in in vitro models of cartilage degradation. The pyridinyl imidazole SB 203580 inhibits p38 mitogenactivated protein (MAP) kinase in vitro, blocks proinflammatory cytokine production in vitro and in vivo, and is effective in animal models of arthritis. The purpose of this study was to determine whether SB 203580 could inhibit p38 MAP kinase activity, NO production, and inducible NO synthase (iNOS) in IL-1 stimulated bovine articular cartilage/chondrocyte cultures. The results indicated that SB 203580 inhibited both IL-1 stimulated p38 MAP kinase activity in isolated chondrocytes and NO production in bovine chondrocytes and cartilage explants with an IC50 value of approximately 1 mM. To inhibit NO production, SB 203580 had to be present in cartilage explant cultures during the first 8 h of IL-1 stimulation, and activity was lost when it was added 24 h following IL-1. SB 203580 did not inhibit iNOS activity, as measured by the conversion of arginine to citrulline, when added directly to cultures where the enzyme had already been induced, but had to be present during the induction period. Using a 372-bp probe for bovine iNOS we demonstrated inhibition of IL-1-induced mRNA by SB 203580 at both 4 and 24 h following IL-1 treatment. The iNOS mRNA levels were consistent with NO levels in 24-h cell culture supernatants of the IL-1-stimulated bovine chondrocytes used to obtain the RNA. The Journal of Immunology, 1998, 161: 467– 473. ytokines such as IL-1 and TNF-a play a predominant role during inflammatory responses and autoimmune disease (1). Much attention has been paid to the role of cytokines in rheumatoid arthritis (RA),2 and there may be a role for these cytokines in the development of osteoarthritis (OA) (2– 4). It is now generally accepted that cytokines can induce proteoglycan degradation, inhibit proteoglycan synthesis, and induce matrix metalloproteinases (5–10), resulting in damage to both cartilage and bone. One of the mechanisms by which cytokines elicit their proinflammatory effects is by the stimulation of the production of nitric oxide. Nitric oxide (NO) is a multifunctional messenger molecule generated by a family of enzymes termed the NO synthases (NOS). The inducible form of nitric oxide synthase (iNOS) releases high levels of NO in response to IL-1 and TNF. IL-1 has been shown to induce the release of NO from chondrocytes and cartilage of a number of species (11–14), and NO may activate matrix metalloproteinases responsible for proteoglycan degradation in articular cartilage (15). Stimulation of NO production in

C

Departments of *Bone and Cartilage Biology and †Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406 Received for publication October 6, 1997. Accepted for publication March 6, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Alison M. Badger, Department of Bone and Cartilage Biology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., P.O. Box 1539, King of Prussia, PA 19406 – 0939. E-mail address: alison— m—[email protected] 2 Abbreviations used in this paper: RA, rheumatoid arthritis; OA, osteoarthritis; NO, nitric oxide; iNOS, inducible nitric oxide synthase; SB 203580, 4-(4-fluorophenyl)2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole; CSBP, CSAID binding protein; RK, reactivating kinase; GST, glutathione-S-transferase; ATF2, activating transcription factor 2; L-NMMA, NG-monomethyl-L-arginine; MAPK, mitogen-activated protein kinase; MAPKAPK, mitogen-activated protein kinase-activated protein kinase; hsp, heat shock protein.

Copyright © 1998 by The American Association of Immunologists

bone by proinflammatory cytokines indicates that NO may be involved as a mediator of bone disease in RA and postmenopausal osteoporosis (16). SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5(4-pyridyl)imidazole; Fig. 1) is a member of a new series of pyridinyl imidazole compounds that inhibit IL-1 and TNF-a production from LPS-stimulated human monocytes and the human monocyte cell line THP-1 with IC50 values of 50 to 100 nM (17, 18). The term CSAID has been coined for these compounds, and they have shown activity in a number of animal models of acute and chronic inflammation, including collagen- and adjuvant-induced arthritis (19, 20). The molecular target of SB 203580 has been identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSAID binding proteins (CSBPs) (17), p38 (21), or RK (22). Binding of the pyridinyl imidazole compounds to CSBP in THP-1 cytosol correlates with their ability to inhibit cytokine synthesis in response to various stimuli (17). It has been shown that several cell types express p38 kinase, and of particular relevance is the finding that human articular chondrocytes express p38 proteins that are time-dependently activated by IL-1 (23). In the studies described here, we show that IL-1 stimulation of bovine articular cartilage-derived chondrocytes results in the induction of p38 MAP kinase, which is inhibited in a dose-dependent manner by SB 203580, and that this compound is able to inhibit IL-1-stimulated NO prodution from both cartilage and chondrocyte cultures. In addition, we show that inhibition of NO production is the result of transcriptional regulation of iNOS.

Methods and Materials Reagents SB 203580 was synthesized at SmithKline Beecham Pharmaceuticals (Fig. 1). Recombinant human IL-1a (which was used in all the experiments), the metalloproteinase inhibitor BB94, and the GST-ATF2 were also prepared 0022-1767/98/$02.00

468

SB 203580 INHIBITS p38 KINASE AND iNOS IN BOVINE CHONDROCYTES

FIGURE 1. SB 203580: 4-(4-fluorophenyl)-2-(4-methylsulfinyl-phenyl)5-(4-pyridyl)imidazole.

at SmithKline Beecham. DMEM and Ham’s F-12 medium were obtained from Life Technologies (Grand Island, NY). FBS was obtained from HyClone (Logan, UT). The media were supplemented with 100 U/ml of penicillin, 100 mg/ml of streptomycin, 2.5 mg/ml amphotericin B, and 2 mM L-glutamine (Life Technologies). Dulbecco’s PBS was obtained from Life Technologies and contained 23 antibiotics. L-ascorbic acid, NG-monomethyl-L-arginine (L-NMMA), pronase E from Streptomyces griseus, and hyaluronidase type V were obtained from Sigma (St. Louis, MO); collagenase D from Clostridium histolyticum was purchased from Boehringer Mannheim (Indianapolis, IN). [3H]arginine was obtained from Amersham (Arlington Heights, IL). SeaPlaque agarose was obtained from FMC Bioproducts (Rockland, ME). Other standard buffer components and chemicals, unless specifically indicated otherwise, were obtained from Sigma.

Cartilage and chondrocyte cultures Carpal metacarpal joints of calves (0 –3 mo old) were obtained from Covance (Denver, PA). Full thickness articular cartilage slices were aseptically collected and placed in Dulbecco’s PBS with 23 antibiotics for 30 min. Cartilage disks (5–7 mg) were dissected from the cartilage using a sterile leather punch (Libertyville Saddle Shop, Libertyville IL). Disks were transferred to 96-well flat-bottom plates (Nunc, Copenhagen, Denmark) containing DMEM supplemented with antibiotics and 10% FCS. This medium was changed 48 to 72 h later to DMEM with 0.5% FCS containing 25 mg/ml ascorbic acid, and samples to be tested were added 24 h later. NO levels in the cartilage explant supernatants were determined 72 h following the addition of the samples being evaluated. Bovine chondrocytes were isolated from bovine cartilage as described previously for human chondrocytes (24, 25). Cartilage was cut into small pieces, and chondrocytes were liberated by sequential treatment with hyaluronidase (0.2% in DMEM without FCS) for 30 min, pronase E (0.25% in DMEM without FCS) for 30 min, and collagenase D (0.2% in DMEM with 10% FCS) for 20 h at 37°C. No effort was made to separate different layers of cartilage. Cells were washed twice in DMEM with 10% FCS. Viability was determined by trypan blue exclusion. For NO production and isolation of RNA, monolayer cultures of chondrocytes were used. Chondrocytes were seeded into six-well tissue culture grade plates (Corning, Cambridge, MA) at a concentration of 2 3 106 cells/ml (4 ml/well) in Ham’s F-12 containing 10% FCS, 50 mg/ml ascorbic acid, and antibiotic/antimycotic. Cells were allowed to adhere and were cultured for 72 h at 37°C in 5% CO2. The medium was then replenished, and the chondrocytes were treated with log doses of SB 203580 for 30 min followed by stimulation with IL-1. The cells were incubated at 37°C in a 5% CO2 atmosphere for either 4 or 24 h. At the end of the incubation, the extracellular medium was removed and saved for evaluation of nitrite levels, and the cells were homogenized in Triazol reagent (Molecular Research Center, Cincinnati, OH; 0.5 ml/well) for the isolation of RNA. For culture of chondrocytes in agarose, a previously described method was used (26). Briefly, a 2% (w/v) solution of SeaPlaque agarose in Ham’s F-12 medium containing antibiotics but without FCS was prepared by heating to 80°C. The agarose was cooled and mixed with an equal volume of Ham’s F-12 with 20% FCS. A chondrocyte suspension was added to the agarose to a final density of 8 3 105 cells/ml, and 0.5-ml aliquots were distributed into 24-well plates. Plates were incubated at 37°C for 30 min to allow the cells to settle and then at room temperature until the agar gelled. Liquid nutrient (1 ml of Ham’s F-12 containing 20% FCS and 25 mg/ml ascorbic acid) was added and was replaced every other day.

Immune complex kinase assays Bovine chondrocytes were established in 10-cm tissue culture petri dishes in DMEM with 10% FCS (10 ml, 1 3 106/ml) and stimulated with 20 ng/ml of IL-1 for varying periods of time. The cells were washed twice in PBS, solubilized on ice in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM

NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 25 mM b-glycerophosphate, 20 mM NaF, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1 mM PMSF, 10 mg/ml leupeptin, and 5 U/ml aprotinin), and centrifuged at 15,000 3 g for 20 min at 4°C. Endogenous kinases were precipitated from cell lysates using anti-p38 (17) or anti-MAPKAP kinase-2 Abs (supplied by Dr. Jacques Landry, Quebec, Canada) bound to protein A-agarose for 4 h at 4°C. The beads were washed twice with lysis buffer and twice with kinase buffer (25 mM HEPES (pH 7.4), 25 mM MgCl2, 25 mM b-glycerophosphate, 100 mM sodium orthovanadate, and 2 mM DTT). The immune complex kinase assays were initiated by the addition of 25 ml of kinase buffer containing 2 mg of GST-ATF2 for p38 or 2 mg of the small heat shock protein 27 (hsp27; StressGen Biotechnology, Ontario, Canada) for MAPKAP kinase-2 as substrate and 50 mM [g-32P]ATP (20 Ci/mmol; Amersham). After 30 min at 30°C, the reaction was stopped by the addition of SDS sample buffer, and the phosphorylated products were resolved by SDS-PAGE and visualized by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For the in vitro inhibition experiment, SB 203580 was added directly to the immune complexes before the kinase assay was performed. For inhibition in intact cells, the cells were pretreated with SB 203580 before stimulation with IL-1. Escherichia coli expressing GST-ATF2 was provided by Dr. Roger Davis, Worcester, MA). Recombinant GST-ATF2 was expressed in E. coli and purified over glutathione Sepharose 4B (Pharmacia, Uppsala, Sweden) chromatography according to the manufacturer’s instructions.

Nitrite determination NO production was measured by estimating the stable NO metabolite, nitrite, in conditioned medium using a spectrophotometric method based on the Griess reaction (27). Following culture of the cartilage explants/chondrocytes for the times indicated, 100 ml of the culture supernatants or sodium nitrite standard dilutions were mixed with 50 ml of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride, and 1.25% H3PO4) and incubated for 10 min at room temperature. Nitrite concentrations were determined by measuring absorbance at 550 nm in an ELISA reader (Molecular Devices, Sunnyvale, CA). The detection limit of the test was 2 mM NO2 2 . Values are expressed as micromolar concentrations of nitrite released per 106 chondrocytes or per milligram of cartilage.

Determination of iNOS activity Inducible NOS activity was evaluated in monolayer cultures of bovine chondrocytes. The cells were plated (4 3 106 in 2 ml) in 12-well plastic dishes in Ham’s F-12 with 10% FCS containing 50 mg/ml ascorbic acid. The cells were incubated for 72 h, at which time the medium was replenished. IL-1 (20 ng/ml) and varying concentrations of SB 203580 were then added for an additional 24 h. Following the incubation period, the cells were washed twice in medium and then incubated in fresh medium for a further 20 min. During this period a second set of wells that had been treated with IL-1 alone was treated with similar doses of SB 203580 to determine the compound’s effect on the already induced enzyme. [3H]arginine (3 mCi/ml) was added to the cultures for 20 min, and incorporation of labeled arginine was terminated by rapid aspiration of extracellular medium and two washes (1 ml) using ice-cold PBS. Cells were lysed by addition of 1 ml 0.4 N HClO4, and the intracellular extract was neutralized with K2CO3 and 50 mM Tris-HCl buffer (pH 7.4). Aliquots (100 ml) of the neutralized intracellular extract were used to measure [3H]arginine uptake into the cells. To separate [3H]citrulline from [3H]arginine, 400-ml aliquots of the extracts were applied to 1-ml columns of Dowex AG50WX-8 (Na1 form) as described previously (28, 29). The columns were washed three times with 1 ml of water, and the radioactive material in the flow-through fractions (i.e., that containing almost exclusively [3H]citrulline) was quantified by scintillation spectroscopy. All measurements were made in duplicate, and the data are presented as the recovery of radiolabeled [3H]citrulline. The disintegrations per minute are corrected for the number of counts per 4 3 106 cells.

Isolation of chondrocyte RNA and Northern blot analysis Total RNA was isolated from chondrocytes treated with IL-1 and SB 203580 by a modified guanidine isothiocyanate extraction using Tri-Reagent (MRC, Cincinnati, OH) according to the manufacturer’s instructions. RNA concentration and purity were determined spectrophotometrically. All RNA samples had an A260/A280 ratio .1.8. Approximately 10 mg of the RNA samples were electrophoresed in 1% agarose gels containing 2.5% formaldehyde, 20 mM 3-(N-morpholino)propanesulfonic acid, 5 mM sodium acetate, and 1 mM EDTA, pH 7.0. After electrophoresis, the RNA was transferred to positively charged nylon membranes (Bio-Rad, Hercules, CA). Membranes were prehybridized for 1 to 2 h at 68°C with

The Journal of Immunology

FIGURE 2. A, IL-1 activation of CSBP/p38 and MAPKAP kinase-2 by IL-1. Chondrocytes (1 3 107 cells in 10 ml of medium) were plated in 100-mm plates and allowed to adhere for 24 h. Cells were treated with 20 ng/ml of IL-1 for different time periods as indicated. At the end of the incubation, cells were harvested, p38 or MAPKAP kinase-2 was immunoprecipitated, and the immune complex kinase assay was performed as described in Materials and Methods. The upper and lower panels show the time course of activation of p38 and MAPKAP kinase-2, respectively. Lane 1, Control untreated; lanes 2 through 5, IL-1 treated at 5, 15, 30, and 60 min. B, SB 203580 inhibits CSBP/p38 and MAPKAP kinase-2 activity. Upper panel, Chondrocytes were treated with IL-1 for 5 min and harvested. CSBP was immunoprecipitated from the cell lysates, and a kinase assay was performed in the absence (lane 1) or the presence (lanes 2–5) of SB 203580. Lower panel, Chondrocytes were pretreated with the indicated concentrations of SB 203580 and then exposed to IL-1 for 5 min. Cells were harvested, and MAPKAP kinase-2 was assayed with hsp27 as a substrate as described in Materials and Methods.

ExpressHyb (Clontech, Palo Alto, CA). Hybridization was performed under identical conditions as prehybridization with addition of 32P-labeled (Amersham) specific probe for bovine iNOS. The 372-bp probe was provided by Dr. T. Jungi (35) (University of Berne, Berne, Switzerland) and corresponds to nucleotides 682 to 1053 of the human iNOS cDNA (30). Radiolabel on the blots was analyzed and integrated using phosphorimaging.

Statistical analysis Comparisons between treated and control groups were determined by Student’s t test, with p , 0.05 considered significant. All experiments were performed at least three times, and the results shown are those from representative experiments.

469

FIGURE 3. SB 203580 inhibits NO production in bovine articular cartilage explant culture. Cartilage disks were established in 96-well plates in DMEM with 10% FCS for 48 h. Medium was then changed to DMEM with 0.5% FCS for 24 h, and IL-1 and compound were added. NO production was measured in the explant supernatants 72 h later. Data are the pooled results of two experiments showing the mean 6 SE of 12 replicates/group. * indicates p , 0.05; *** indicates p , 0.001.

substrate. As shown in Figure 2B, SB 203580 inhibited the activity of p38 in a dose-dependent manner, with an IC50 value of about 0.1 mM. Complete inhibition occurred at 1 and 10 mM. While SB 203580 inhibits the activity of p38 MAP kinase, it has no effect on the activation of p38 by upstream kinase in response to IL-1 or any other stimuli (31). It has been shown that MAPKAP kinase-2 is a physiologic substrate of p38 in vivo (32). The activity of MAPKAP kinase-2 itself is not inhibited by SB 203580, but activation of the enzyme by p38 MAP kinase in cells is inhibited by SB 203580 (31). Therefore, to show that SB 203580 inhibits p38 MAP kinase in bovine chondrocytes, we pretreated the chondrocytes with SB 203580 at different concentrations before activation with IL-1 and then assayed for MAPKAP kinase-2 activity using hsp27 as substrate. As shown in Figure 2B, SB 203580 inhibited the activation of MAPKAP kinase-2 in a dose-dependent manner with an IC50 value of about 0.4 mM, suggesting that SB 203580 treatment results in the inhibition of p38 MAP kinase activity within the cell. Almost complete inhibition was achieved at 1 and 10 mM. Similar IC50 values have been reported in several earlier reports for other cell types (31–33). NO production by bovine articular cartilage and chondrocytes

Results Activation of p38 MAP kinase activity by IL-1 and inhibition by SB 203580 IL-1 induced a rapid activation of p38 MAP kinase in the chondrocyte cultures (Fig. 2A). A fourfold activation of p38 was achieved by a 5-min treatment with IL-1. At 60 min postactivation, p38 activity was still increased threefold over the basal level. We also analyzed the activation of MAPKAP kinase-2, a physiologic substrate of p38, by IL-1 in the same cell lysate. MAPKAP kinase-2 was activated fivefold within 5 min after treatment with IL-1, but unlike P38, its activity level fell rapidly. At 60 min, MAPKAP kinase-2 activity was only twofold over the basal level (Fig. 2A). Immunoblots verified that the same amount of kinase was immunoprecipitated from different samples (data not shown). To determine the inhibition of p38 MAP kinase activity by SB 203580, different concentrations of the compound were added directly to the p38 immune complex purified from IL-1-activated chondrocytes and were assayed for activity using GST-ATF2 as

Bovine articular cartilage produces large amounts of NO following stimulation with IL-1 (34). In these studies cartilage explants were cultured for 48 h in DMEM with 10% FCS and then transferred to medium containing 0.5% FCS. Twenty-four hours later the cultures were stimulated with 20 ng/ml of IL-1, and SB 203580 was added at doses ranging from 0.15 to 10 mM. The explants were incubated for an additional 72 h, and secreted NO was determined in the supernatants. In the presence of IL-1 there was a two- to threefold increase in NO production compared with that in the untreated control cultures. This increase was inhibited by SB 203580 with an IC50 value that ranged from 0.6 to 1 mM, and maximal inhibition was observed at 5 to 10 mM. The pooled results of two experiments are shown in Figure 3. For comparison, in this culture system, IL-1-induced NO production was inhibited by LNMMA at 500 mM (L-NMMA is a specific competitive inhibitor of all isoforms of NOS) and by SB 203580 (5 mM), but not by the metalloproteinase inhibitor BB94 (Fig. 4). For SB 203580 to significantly inhibit IL-1 induced NO production in cartilage explants,

470


FIGURE 4. L-NMMA and SB 203580, but not BB94, inhibit IL-1-stimulated NO production in bovine articular cartilage explant cultures. Cartilage disks were established in 96-well plates in DMEM with 10% FCS for 48 h. Medium was then changed to DMEM with 0.5% FCS for 24 h, and IL-1 and compounds were added. NO production was measured in the explant supernatants 72 h later. Data are the mean 6 SD of six replicates per group. *** indicates p , 0.001.

the compound had to be present early during the incubation period. Maximum inhibition was obtained when the compound was added during the first 4 h of culture with IL-1. The effect became weaker over the next 4 h and was lost by 24 h (Fig. 5). To demonstrate that SB 203580 was equally active on isolated chondrocytes as in the cartilage explants, bovine chondrocytes were isolated from the cartilage by enzymatic digestion and established either as monolayer cultures or in agarose. In agarose the chondrocytes maintain their chondrocyte phenotype for several weeks. The effects of SB 203580 were examined on chondrocytes that had been incubated in agarose culture for 2 wk. At this time the chondrocytes have formed large aggregates that can be stimulated with IL-1 (20 ng/ml) to produce NO, which accumulates in the supernatant. IL-1 (20 ng/ml) and varying doses of SB 203580 were added on day 14 of culture, and the NO accumulated in the

FIGURE 6. Chondrocytes cultured in agarose for 2 wk can be stimulated with IL-1 to produce NO, which is inhibitable by SB 203580. Following 2 wk in culture, IL-1 (20 ng/ml) and log doses of SB 203580 were added, and NO was measured in the agarose supernatants 72 h later. v is the untreated control; Œ is the IL-1-treated control; f indicates the SB 203580- plus IL-1-treated groups. Data are the mean 6 SD of four replicates per group. * indicates p , 0.05; ** indicates p , 0.01; *** indicates p , 0.001.

supernatant was determined 72 h later. There was a dose-related inhibitory effect with SB 203580, with an IC50 value of approximately 1 mM (Fig. 6). Effect of SB 203580 on iNOS activity of chondrocytes To show that the activity of SB 203580 was due to its ability to inhibit the synthesis of iNOS and not to a direct effect on NOS activity, we examined the ability of the compound to inhibit IL1-induced conversion of radiolabeled arginine to citrulline during or after the iNOS synthetic phase. As shown in Figure 7, SB 203580 inhibited nitrite production when the compound was added to the cell cultures in combination with IL-1 (Fig. 7A), but not when it was added at the time of iNOS assay (Fig. 7B). No significant effects of SB 203580 on arginine uptake into cells were observed (data not shown). This would indicate that the compound has no direct effect on NOS enzyme activity directly, but has a dose-related effect on its induction. Expression of iNOS RNA

FIGURE 5. Time course of inhibition of NO production by SB 203580. Explants were prepared, and IL-1 was added as described in Materials and Methods and Figure 4. SB 203580 was added with IL-1 (time zero) or at different time points after IL-1 (2, 4, 6, 8, and 24 h). NO was measured in the supernatants 72 h following the addition of IL-1. Data are the mean 6 SD of six replicates per group. * indicates p , 0.05; *** indicates p , 0.001.

To determine the level at which SB 203580 regulated iNOS, the effect on iNOS mRNA was examined using Northern analysis. Chondrocytes were established in monolayers in six-well tissue culture plates (for 72 h), treated with SB 203580, and stimulated with IL-1 (100 ng/ml) for 4 or 24 h, and then RNA was isolated as described in Materials and Methods. Following electrophoresis of the RNA and blotting onto nylon membranes, the blots were hybridized with the labeled iNOS probe and analyzed using the PhosphorImager. Figure 8A shows that there was a large increase in expression of iNOS mRNA following treatment with IL-1 and that this was inhibited in a dose-related manner by SB 203580. Similar results were obtained at both 4 and 24 h, with slightly more inhibition being obtained with SB 203580 at the 4 h point as measured by the PhosphorImager counts (Fig. 8B). Normalization of the PhosphorImager counts based on the 28S ribosomal RNA were within 10% of the data shown in Figure 8B. The inhibition of induced message was in agreement with the NO levels in the 24-h cell supernatants of the IL-1-stimulated bovine chondrocytes used to obtain the RNA (Fig. 8C).

The Journal of Immunology

FIGURE 7. SB 203580 inhibits iNOS activity in IL-1-stimulated chondrocytes (A), but does not inhibit iNOS when added directly to the already induced enzyme (B). Cells were established in monolayer cultures (4 3 106 cells in 2 ml) in 12-well plates. Cells were incubated for 72 h, then the medium was replenished, and IL-1 (20 ng/ml) and log doses of SB 203580 were added for 24 h (A). In a second set of wells that had been pretreated with IL-1 for 24 h, SB 203580 was added to determine its effect on the already induced enzyme (B). The conversion of [3H]arginine to [3H]citrulline was used as a measure of iNOS activity. Disintegrations per minute for control unstimulated cells averaged 1000 6 100. Data are the mean 6 SD of triplicate cultures. * indicates p , 0.05; ** indicates p , 0.01.

Discussion Pyridinyl imidazoles have been shown to inhibit the p38 kinasemediated synthesis of cytokines such as IL-1 and TNF-a (IC50 5 0.1–1 mM) (17, 18). SB 203580 as well as other members in this series of compounds have shown efficacy in several animal models of inflammation where cytokines play a definitive role. Examples are collagen-induced arthritis in the DBA/LacJ mouse (20, 36); adjuvant-induced arthritis in the Lewis rat, in which protective effects on both bone and cartilage have been observed (20); and mouse models of endotoxin shock (37, 38). In addition, these compounds are active in the fetal rat long bone resorption assay, thus demonstrating a direct therapeutic effect on bone integrity (39). The role of cytokines in these models is well documented, and inhibition of TNF-a and IL-1 by the CSAID compounds is well established. SB 203580 inhibits TNF-a synthesis at the translational rather than the transcriptional level (40, 41), and the target of this and other CSAID compounds is known as CSBP/p38 kinase (17). SB 203580 is a highly selective and potent inhibitor of p38 MAP kinase (32). One of the physiologic substrates of CSBP/p38 is MAPKAP kinase-2, which, in turn, phosphorylates hsp27. SB 203580 inhibits the activation of MAPKAP kinase-2 with an IC50 of about 0.4 mM, and subsequently the phosphorylation of hsp27

471

FIGURE 8. Expression of IL-1-stimulated iNOS message in bovine chondrocytes is inhibited in a dose-related manner by SB 203580. Chondrocytes were established in monolayer culture (8 3 106 cells in 4 ml) in six-well plates for 72 h. Cells were then stimulated with IL-1 (100 ng/ml) and were treated with SB 203580 for 4 or 24 h and RNA isolated. A, upper panel, is a representative Northern blot showing that the radiolabeled iNOS probe is associated with the 4.2-kb message and dose-related inhibition by SB 203580 at both 4 and 24 h. The ethidium bromide-stained agarose gel following electrophoresis is shown as an indicator of total RNA loading (A, lower panel). Lane 1, Control untreated; lane 2, IL-1 control; lanes 3 through 6, IL-1 plus SB 203580 at 0.01, 0.1, 1, and 10 mM. B shows the PhosphorImager counts for this Northern blot. Open bars indicate 4 h after IL-1 treatment; closed bars indicate 24 h after IL-1 treatment. C shows the nitrite levels in the pooled 24-h supernatants of cells used to prepare the RNA.

in stress-stimulated cells is inhibited by this compound (32). In this study we report for the first time that there is clearly an elevation in specific p38 kinase activity in bovine chondrocytes treated with IL-1 and that the CSAID inhibitor SB 203580 inhibits this activity and blocks the production of the proinflammatory enzyme iNOS. One of the documented proinflammatory actions of IL-1 and TNF-a is the induction of NO from a variety of tissues and cell types, including bone, cartilage, and cartilage-derived chondrocytes (12, 34, 42). In fact, the induction of iNOS by IL-1 results in copious amounts of NO that can contribute to tissue regulation and damage, and the spontaneous production of NO has been observed in OA-affected chondrocytes (43). NO has also been shown to mediate the IL-1-induced inhibition of proteoglycan synthesis in rat articular cartilage in vitro (44). In recent studies, the knockout of iNOS has been shown to block both the IL-1-induced inhibition

472


of proteoglycan biosynthesis and the release of cartilage proteoglycan in the zymosan-induced arthritis model (W. van den Berg, unpublished observations). The role of NO in vivo in inflammatory disease has also been well documented, and inhibitors of iNOS have been shown to protect against both inflammation and cartilage matrix loss in a number of experimental models, including inflammatory arthritides (45– 48). Together, these data suggest that direct inhibition of iNOS or inhibition of iNOS production could be therapeutically beneficial in diseases such as OA and RA, in which there is both significant cartilage proteoglycan loss as well as inflammatory components. In addition to inducing iNOS and the production of NO in cartilage/chondrocytes, IL-1 has been shown to induce the activation of p38 kinase in both human (23) and rabbit (49) articular cartilage. In the studies described here we have investigated the effect of IL-1 on the p38 kinase in bovine articular chondrocytes and also its effects on NO production and iNOS expression. IL-1 activated CSBP/p38 kinase within 5 min of treatment, and the level of activation was maintained over a 60-min period. In contrast, activation of MAPKAP kinase-2 peaked 5 min following the addition of IL-1 and then rapidly decreased. SB 203580 inhibited the activity of p38 MAP kinase in vitro when added directly to the p38 kinase assay (Fig. 2B, upper panel). It also inhibited the activity of p38 in cells; this was shown by the inhibition of activation of MAPKAP kinase-2 by p38 when SB 203580 was added to the cell cultures (Fig. 2B, lower panel). The IC50 value of inhibition of p38 MAP kinase when the compound was either added directly to the kinase assay or to the cells was about 0.1 to 0.4 mM, similar to that described in previous reports (31–33). To profile the effect of SB 203580 further in the bovine cartilage/chondrocyte assays, we examined its effects on NO production. The compound effectively inhibited IL-1-stimulated NO release into the culture medium with an IC50 value of approximately 0.6 mM (Fig. 3). SB 203580 appeared to inhibit only the inducible levels of NO (Figs. 3–5) (A. Badger, unpublished observations). To effectively inhibit NO production, SB 203580 needed to be added to the cultures within 4 h of IL-1. After this time the compound began to lose its inhibitory activity, indicating that its site of action is probably during the enzyme induction period (Fig. 5). The fact that IL-1 effects are still inhibited 2 h following induction may well be due to transport and availability in explant cultures. However, in separate experiments, a significant amount of p38 remained active for up to 4 to 6 h after IL-1 treatment (data not shown). A similar profile of drug action was obtained in the case of HIV-1 LTR transcriptional activation, where the key role of p38 activity occurred some 2 to 4 h after cytokine and UV stimulation (33). Measurement of p38 kinase activity and its inhibition by SB 203580 could not be performed in cartilage explants due to the technical difficulty in quickly isolating the active p38 enzyme from this tissue. However, as chondrocytes are the only cell type residing in cartilage, we used cartilage-derived chondrocytes for these experiments. Modulation of p38 MAP kinase (Fig. 2) and the inhibitory effects of SB 203580 on IL-1-induced NO production were clearly demonstrated in cartilage-derived chondrocytes grown in agarose (Fig. 6) and monolayer culture (Fig. 8C). Experiments to determine whether SB 203580 affected iNOS activity in chondrocytes, measured by the conversion of arginine to citrulline, resulted in the finding that inhibition occurred with an IC50 between 0.1 and 1 mM, and that the compound was inactive when added to the assay mixture, indicating inhibition of enzyme induction but not its activity (Fig. 7). We extended this observation to show that IL-1-induced expression of iNOS mRNA was inhibited in a dose-dependent manner by SB 203580 at both 4 and 24 h following activation with IL-1 (Fig. 8). Inhibition of iNOS mRNA

at the 24 h point paralleled the inhibition of NO production in the supernatant of the cells used to prepare the RNA (Fig. 8). Since both p38 activity and iNOS expression are inhibited with comparable IC50 values (;0.5 mM), we hypothesize that p38 MAP kinase resides in the signal transduction pathway of IL-1-mediated NO induction. Activation of p38 MAP kinase appears to be an early event, whereas NO induction is a downstream late event mediated by IL-1. Our results are consistent with those of a similar report in which the spontaneous activation of iNOS in chondrocytes was inhibited by SB 203580 (A. Amin, unpublished observations). In contrast to our findings described here, Guan et al. (50) found that an inhibitor of p38 MAP kinase, SC68376, enhanced NO biosynthesis in rat primary mesangial cells by increasing iNOS mRNA expression, protein expression, and nitrite production. A difference in the cell system (rat mesangial cells vs bovine articular chondrocytes) may explain the discrepancy between the two results. In addition, SC68376 is a relatively less well-characterized p38 inhibitor, with an IC50 value of 2 to 5 mM, compared with SB 203580, which has been extensively characterized and exhibits an IC50 value of at least 10-fold lower. It is possible that SC 68376 has other inhibitory activity and thus complicates the interpretation of the results. Inhibition of the transcriptional regulation of iNOS and the subsequent production of NO by cartilage-derived chondrocytes by SB 203580 indicate that the CSAID compounds may be useful for therapy of disease states in which NO has been shown to play a proinflammatory role. In addition to the experimental models already discussed, additional evidence for the role of NO is provided by studies showing spontaneous production of this mediator by primary synovial cultures from both RA and OA patients, which may contribute to the pathology of these diseases (51). In addition, an increased expression of blood mononuclear cell iNOS has been described in RA patients (52). Thus, the CSAID compounds could be efficacious therapeutic agents by way of their ability to inhibit cytokines such as TNF and IL-1 and by inhibiting the downstream signal transduction pathways initiated by IL-1 and or TNF, including their ability to modulate iNOS expression. Given the central role that IL-1 plays in the pathology of RA and OA, an inhibitor of both IL-1 synthesis as well as IL-1 signal transduction and action would be of immense utility in the management of these debilitating diseases by controlling the inflammatory contribution as well as the effects on cartilage matrix biosynthesis and degradation.

Acknowledgments We acknowledge the expert technical assistance of Annalisa Hand, and Drs. S. Kassis, U. Prabhakar, and M. Gowen for review of the manuscript.

References 1. Dinarello, C. A. 1991. Inflammatory cytokines: interleukin-1 and tumor necrosis factor as effector molecules in autoimmune diseases. Curr. Opin. Immunol. 3:941. 2. Loyau, G., and J. P. Punol. 1990. The role of cytokines in the development of osteoarthritis. Scand. J. Rheumatol. 81:8. 3. Kirkham, B. 1991. Interleukin-1, immune activation pathways, and different mechanisms in osteoarthritis and rheumatoid arthritis. Ann. Rheum. Dis. 50:395. 4. Westacott, C. I., and M. Sharif. 1996. Cytokines in osteoarthritis: mediators or markers of joint destruction. Semin. Arthritis Rheum. 25:254. 5. Dingle, J. T., J. Saklatvala, R. M. Hembry, J. A. Tyler, H. B. Fell, and R. N. Jubb. 1979. A cartilage catabolic factor from synovium. Biochem. J. 184:177. 6. Gowen, M., D. D. Wood, E. J. Ihrie, J. E. Meats, and R. G. G. Russell. 1984. Stimulation by human IL-1 of cartilage breakdown and production of collagenase and proteoglycanase by human chondrocytes but not by human osteoblasts in vitro. Biochim. Biophys. Acta 797:186. 7. Krakauer, T., J. J. Oppenheim, and H. E. Jasin. 1985. Human interleukin-1 mediates cartilage matrix degradation. Cell. Immunol. 91:92. 8. Tyler, J. A. 1985. Chondrocyte-mediated depletion of articular cartilage in vitro. Biochem. J. 224:461.

The Journal of Immunology 9. Saklatvala, J. 1992. Regulation of chondrocytes by cytokines. In: Biological Regulation of the Chondrocytes. M. Adolphe, ed. CRC Press, Boca Raton, p. 191. 10. Verbruggen, G., and E. M. Veys. 1992. Regulation of chondrocytes in rheumatoid arthritis. In: Biological Regulation of the Chondrocytes. M. Adolphe, ed. CRC Press, Boca Raton, p. 275. 11. Stadler, J., M. Stefanovic-Racic, T. R. Billiar, R. D. Curran, L. A. McIntyre, H. L. Georgescu, R. L. Simmons, and C. H. Evans. 1991. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide. J. Immunol. 147:3915. 12. Charles, I. G., R. M. J. Palmer, M. S. Hickery, M. T. Bayliss, A. P. Chubb, V. S. Hall, D. W. Moss, and S. Moncada. 1993. Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte. Proc. Natl. Acad. Sci. USA 90:11419. 13. Palmer, R. M. J., M. S. Hickery, I. G. Charles, S. Moncada, and M. T. Bayliss. 1993. Induction of nitric oxide synthase in human chondrocytes. Biochem. Biophys. Res. Commun. 197:1431. 14. Rediske, J. J., C. F. Koehne, B. Zhang, and M. Lotz. 1994. The inducible production of nitric oxide by articular cell types. Osteoarthritis Cartilage 2:199. 15. Murrell, G. A. C., D. Jang, and R. J. Williams. 1995. Nitric oxide activates metalloprotease enzymes in articular cartilage. Biochem. Biophys. Res. Commun. 206:15. 16. Evans, D. M., and S. H. Ralston. 1996. Nitric oxide and bone. J. Bone Miner. Res. 11:300. 17. Lee, J. C., J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S. Kumar, D. Green, D. McNulty, M. J. Blumenthal, J. R. Heys, S. W. Landvatter, J. E. Strickler, M. M. McLaughlin, I. R. Siemens, S. M. Fisher, G. P. Livi, J. R. White, J. L. Adams, and P. R. Young. 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739. 18. Gallagher, T. F., S. M. Fier-Thompson, R. S. Garigipati, M. E. Sorenson, J. M. Smietana, D. Lee, P. E. Bender, J. C. Lee, J. T. Laydon, D. E. Griswold, M. D. Chabot-Fletcher, J. J. Breton, and J. L. Adams. 1995. Triarylimidazole inhibitors of IL-1 biosynthesis. Bioorg. Med. Chem. Lett. 5:1171. 19. Lee, J. C., A. M. Badger, D. E. Griswold, D. Dunnington, A. Truneh, B. Votta, J. R. White, P. R. Young, and P. E. Bender. 1993. Bicyclic imidazoles as a novel class of cytokine biosynthesis inhibitors. Ann. NY Acad. Sci. 696:149. 20. Badger, A. M., J. N. Bradbeer, B. Votta, J. C. Lee., J. L. Adams, and D. E. Griswold. 1996. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J. Pharmacol. Exp. Ther. 279:1453. 21. Han, J., J. D. Lee., S. L. Bibb, and R. J. Ulevitch. 1994. A MAP kinase targeted by endotoxin and in mammalian cells. Science 265:808. 22. Rouse, J., P. Cohen, S. Trigon, M. Morange, A. Alonso-Llamazares, D. Zamanillo, T. Hunt, and A. R. Nebreda. 1994. Identification of a novel protein kinase cascade stimulated by chemical stress and heat shock which activates MAP kinase-activated protein MAPKAP kinase-2 and induces phosphorylation of the small heat shock proteins. Cell 78:1027. 23. Geng, Y., J. Valbracht, and M. Lotz. 1996. Selective activation of the mitogenactivated protein kinase subgroups c-Jun NH2 terminal kinase and p38 by IL-1 and TNF in human articular chondrocytes. J. Clin. Invest. 98:2425. 24. Kuettner, K. E., B. U. Pauli, G. Gall, V. A. McMemoli, and R. K. Schenk. 1982. Synthesis of cartilage matrix by mammalian chondrocytes in vitro: isolation, culture characteristics and morphology. J. Cell Biol. 93:743. 25. Mulfait, A. M., G. Verbruggen, E. M. Veys, J. Lambert, L. D. Ridder, and M. Cornelissen. 1994. Comparative and combined effects of interleukin 6, interleukin 1b, and tumor necrosis factor a on proteoglycan metabolism of human articular chondrocytes cultured in agarose. J. Rheumatol. 21:314. 26. Aydelotte, M. B., and K. E. Kuettner. 1988. Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production. Connect. Tissue Res. 18:205. 27. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and 15N-nitrate in biological fluids. Anal. Biochem. 126:131. 28. Brendt, D. S., and S. H. Snyder. 1989. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc. Natl. Acad. Sci. USA 86:9030. 29. Brendt, D. S., and S. H. Snyder. 1990. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc. Natl. Acad. Sci. USA 87:682. 30. Geller, D. A., C. J. Lowenstein, R. A. Shapiro, A. K. Nussler, M. Di Silvio, S. G. Wang, D. K. Nakayama, R. L. Simmons, S. H. Snyder, and T. R. Billiar. 1993. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc. Natl. Acad. Sci. USA 90:3491. 31. Young, P. R., M. M. McLaughlin, S. Kumar, S. Kassis, M. L. Doyle, D. McNulty, T. F. Gallagher, S. Fisher, P. C. McDonnell, S. A. Carr, M. J. Huddleston, G. Seibel, T. G. Porter, G. P. Livi, J. L. Adams, and J. C. Lee. 1997. Pyridinyl

473

32.

33.

34.

35.

36.

37.

38.

39. 40.

41.

42. 43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site. J. Biol. Chem. 272:12116. Cuenda, A., J. Rouse, Y. N. Doza, R. Meier, P. Cohen, T. F. Gallagher, P. R. Young, and J. C. Lee. 1995. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364:229. Kumar, S., M. Orsini, J. C. Lee, P. C. McDonnell, C. M. Debouck, and P. R. Young. 1996. Activation of the HIV-1 long terminal repeat by cytokines and environmental stress requires an active CSBP/p38 MAP kinase. J. Biol. Chem. 271:30864. Stefanovic-Racic, M., T. I. Morales, D. Taskiran, L. A. McIntyre, and C. H. Evans. 1996. The role of nitric oxide in proteoglycan turnover by bovine articular cartilage organ cultures. J. Immunol. 156:1213. Adler, H., Frech, B., Thony, M., Pfister, H., Peterhans, E., and T. W. Jungi. 1995. Inducible nitric oxide synthase in cattle: differential cytokine regulation of nitric oxide synthase in bovine and murine macrophages. J. Immunol. 154:4710. Griswold, D. E., L. M. Hillegass, P. C. Meunier, M. J. DiMartino, and N. Hanna. 1988. Effect of inhibitors of eicosanoid metabolism in murine collagen-induced arthritis. Arthritis Rheum. 31:1406. Badger, A. M., D. Olivera, J. E. Talmadge, and N. Hanna. 1989. Protective effect of SK&F 86002, a novel dual inhibitor of arachidonic acid metabolism, in murine models of endotoxin shock: inhibition of tumor necrosis factor as a possible mechanism of action. Circ. Shock 27:51. Olivera, D. L., K. M. Esser, J. Lee, R. G. Greig, and A. M. Badger. 1992. Beneficial effects of SK&F 105809, a novel cytokine-suppressive agent, in murine models of endotoxin shock. Circ. Shock 37:301. Votta, B. J., and D. R. Bertolini. 1994. Cytokine suppressive anti-inflammatory compounds inhibit bone resorption in vitro. Bone 15:533. Lee, J. C., B. Votta, B. J. Dalton, D. E. Giswold, P. E. Bender, and N. Hanna. 1990. Inhibition of human monocyte IL-1 production by SK&F 86002. Int. J. Immunother. 6:1. Young, P. R., P. McDonnell, D. Dunnington, A. Hand, J. Laydon, and J. C. Lee. 1993. Bicyclic imidazoles inhibit IL-1 and TNF production at the protein level. Agents Actions 39:C67. Stefanovic-Racic, M., J. Stadler, and C. H. Evans. 1993. Nitric oxide and arthritis. Arthritis Rheum. 36:1036. Amin, A. R., P. E. Di Cesare, P. Vyas, M. Attur, E. Tzeng, T. R. Billiar, S. A. Stuchin, and S. B. Abramson. 1995. The expression and regulation of nitric oxide synthase in human osteoarthritis-affected chondrocytes: evidence for upregulated neuronal nitric oxide synthase. J. Exp. Med. 182:2097. Jarvinen, T. A. H., T. Moilanen, T. L. N. Jarvinen, and E. Moilanen. 1995. Nitric oxide mediates interleukin-1 induced inhibition of glycosaminoglycan synthesis in rat articular cartilage. Mediators Inflammation 4:107. Stefanovic-Racic, M., K. Meyers, C. Meschter, J. W. Coffey, R. A. Hoffman, and C. H. Evans. 1994. N-monomethyl arginine, an inhibitor of nitric oxide synthase, suppresses the development of adjuvant arthritis in rats. Arthritis Rheum. 37: 1062. Weinberg, J. B., D. L. Granger, D. S. Pisetsky, M. F. Seldin, M. A. Misukonis, S. N. Mason, A. M. Pippen, P. Ruiz, E. R. Wood, and G. S. Gilkeson. 1994. The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine. J. Exp. Med. 179:651. Connor, J. R., P. T. Manning, S. L. Settle, W. M. Moore, G. M. Jerome, R. K. Webber, F. S. Tjoeng, and M. G. Currie. 1995. Suppression of adjuvantinduced arthritis by selective inhibition of inducible nitric oxide synthase. Eur. J. Pharmacol. 273:15. Brenner, T., S. Brocke, F. Szafer, R. A. Sobel, J. F. Parkinson, D. H. Perez, and L. Steinman. 1997. Inhibition of nitric oxide synthase for treatment of experimental autoimmune encephalomyelitis. J. Immunol. 158:2940. Scherle, P. A., M. A. Pratta, W. S. Feeser, E. J. Tancula, and E. C. Arner. 1997. The effects of IL-1 on mitogen-activated protein kinases in rabbit articular chondrocytes. Biochem. Biophys. Res. Commun. 230:573. Guan, Z., L. D. Baier, and A. R. Morrison. 1997. p38 Mitogen-activated protein kinase down-regulates nitric oxide and up-regulates prostaglandin E2 biosynthesis stimulated by interleukin-1b. J. Biol. Chem. 272:8083. McInnes, I. B., B. P. Leung, M. Field, X. Q. Wei, F.-P. Huang, R. D. Sturrock, A. Kinninmonth, J. Weidner, R. Mumford, and F. Y. Liew. 1996. Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthritis patients. J. Exp. Med. 184:1519. St. Clair, E. W., W. E. Wilkinson, T. Lang, L. Sanders, M. A. Misukonis, G. S. Gilkeson, D. S. Pisetsky, D. L. Granger, and J. B. Weinberg. 1996. Increased expression of blood mononuclear cell nitric oxide synthase type 2 in rheumatoid arthritis patients. J. Exp. Med. 184:1173.