of Metalloproteinases and Enhances Synthesis of Tissue Inhibitors ...

2 downloads 0 Views 480KB Size Report
The American Association of Immunologists, Inc., is published twice each ... IL-1 induced the synthesis of IL-6, IL-8, and matrix metalloproteinases (MMP)-1 and -3. .... directly proportional to the number of target mRNA transcripts present in the total RNA ..... levels are indicated by bar histogram: IL-1Я (f); IL-1Я. LXA4 (D). The.
Lipoxin A4 Inhibits IL-1␤-Induced IL-6, IL-8, and Matrix Metalloproteinase-3 Production in Human Synovial Fibroblasts and Enhances Synthesis of Tissue Inhibitors of Metalloproteinases1 Snezna Sodin-Semrl, Brunella Taddeo, Daniel Tseng, John Varga, and Stefano Fiore2 Lipoxins are a novel class of endogenous eicosanoid mediators that potently inhibit inflammatory events by signaling via specific receptors expressed on phagocytic cells. Animal models have shown that lipoxin A4 (LXA4) down-regulates inflammation in vivo. Here we demonstrate, for the first time, the expression of LXA4 receptors, and their up-regulation by IL-1␤, in normal human synovial fibroblasts (SF). We examined whether exogenous LXA4 abrogated IL-1␤ stimulation of SF in vitro. IL-1␤ induced the synthesis of IL-6, IL-8, and matrix metalloproteinases (MMP)-1 and -3. At nanomolar concentrations, LXA4 inhibited these IL-1␤ responses with reduction of IL-6 and IL-8 synthesis, by 45 ⴞ 7% and 75 ⴞ 11%, respectively, and prevented IL-1␤-induced MMP-3 synthesis without significantly affecting MMP-1 levels. Furthermore, LXA4 induced a 2-fold increase of tissue inhibitor of metalloproteinase (TIMP)-1 and a ⬃3-fold increase of TIMP-2 protein levels. LXA4 inhibitory responses were dose dependent and were abrogated by pretreatment with LXA4 receptor antiserum. LXA4-induced changes of IL-6 and TIMP were accompanied by parallel changes in mRNA levels. These results indicate that LXA4 in activated SF inhibits the synthesis of inflammatory cytokines and MMP and stimulates TIMP production in vitro. These findings suggest that LXA4 may be involved in a negative feedback loop opposing inflammatory cytokine-induced activation of SF. The Journal of Immunology, 2000, 164: 2660 –2666.

A

lthough its exact etiology is unknown, increased production of inflammatory cytokines seems to be a critical alteration in the pathogenesis of rheumatoid arthritis (RA)3 (1, 2). For example, TNF-␣ and IL-1 are potent agonists of human synovial fibroblast (SF) cell activation, a central mechanism of tissue damage in the inflamed joint (3, 4). The active role of SF in the formation of the invasive rheumatoid pannus has been recently stressed by groups indicating the relevance of the synoviocyte synthesis of matrix metalloproteinase (5–7) and release of IL-6 (1, 8 –11), a cytokine that plays an essential pathogenetic role in animal models of RA (12). Immunomodulatory cross-interactions existing between lipid and cytokine networks (13, 14) have led us to address the role of a novel class of antiinflammatory eicosanoids, the lipoxins, in reg-

Section of Rheumatology, Department of Medicine, University of Illinois College of Medicine, Chicago, IL 60607 Received for publication August 19, 1999. Accepted for publication December 13, 1999. 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 This work was supported in part by the Arthritis Foundation, Greater Chicago Chapter, and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR-44998) to S.F. 2 Address correspondence and reprint requests to Dr. Stefano Fiore, Section of Rheumatology, M/C 733, Molecular Biology Research Building, Room 1252, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, IL 60607-7171. E-mail address: [email protected] 3

Abbreviations used in this paper: RA, rheumatoid arthritis; EMEM, Eagle’s MEM; LXA4, lipoxin A4, 5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; LXA4R, lipoxin A4 receptor; ␣LXA4R, rabbit serum raised against the lipoxin A4 receptor; MMP, matrix metalloproteinases; MMP-1, collagenase, matrix metalloproteinase-1; MMP-3, stromelysin, matrix metalloproteinase-3; FPR, formyl peptiderelated receptor; ORF, open reading frame; SF, synovial fibroblast; TIMP-1, tissue inhibitor of matrix metalloproteinase-1; TIMP-2, tissue inhibitor of matrix metalloproteinase-2; DRB, 5,6-dichlorobenzimidazole riboside; PMN, polymorphonuclear cells; AhR, aryl hydrocarbon receptor. Copyright © 2000 by The American Association of Immunologists

ulating SF activation by IL-1␤. Lipoxins are trihydroxytetraenecontaining eicosanoids that modulate leukocyte function (reviewed in Ref. (15)) and play a role in inflammation and wound healing (16). Lipoxin A4 (LXA4), the most potent isomer generated in mammalian cells (16), causes inhibition of neutrophil activation (17, 18), antagonism toward peptido-leukotrienes (19), and decrease of inflammatory infiltrates and edema in vivo (20). These antiinflammatory effects are exerted via specific high affinity receptors and a G protein, pertussis toxin-sensitive mechanism of signal transduction (21–23). We report for the first time the identification of functional LXA4 receptors on human SF. These receptors, detected by RT-PCR and nested PCR using oligonucleotide primers derived from the sequence of the LXA4R expressed in cells of myeloid lineage (24), mediate LXA4 suppression of IL-1␤-induced activation of human SF.

Materials and Methods Materials Synthetic LXA4 was obtained from Cayman Chemical (Ann Arbor, MI). DMEM and cell culture reagents were from BioWhittaker (Walkersville, MD), with the exception of vitamin supplement obtained from Life Technologies (Grand Island, NY). Plasticware, agarose, and other biochemicals were from Fisher Scientific (Houston, TX). Reverse transcription and PCR enzymes were purchased from Promega (Madison, WI). Human rIL-1␤ was purchased from Boehringer Mannheim (Indianapolis, IN). Rabbit LXA4 receptor antiserum (␣LXA4R) was prepared as previously reported (17).

Cell culture Human primary SF were obtained from arthroscopic knee biopsies and grown under standard 5% CO2 humidified atmosphere at 37°C. Eagle’s MEM (EMEM) or DMEM containing 10% FBS, penicillin (100 U/ml), streptomycin (100 ␮g/ml), and vitamin supplement were used for cell culture. After trypsinization, cells were propagated in 162-cm2 flasks until reaching passage 6. For experiments, SF were seeded in either 25-cm2 flasks or six-well plates and allowed to reach confluence. Twenty-four hours before each experiment, fresh medium containing 5% FBS was 0022-1767/00/$02.00

The Journal of Immunology added to cultures. Only SF passages 7–11 were used in experiments to assure that cultures would be free of contaminating macrophages. Results were confirmed with monocyte-macrophage-depleted, fibroblast-enriched cultures, obtained by sequential negative plus positive selection steps involving a ferrit-bound microbead magnetic system using specific Abs directed against monocytes-macrophages and fibroblast cell surface markers, respectively (Miltenyi Biotec, Auburn, CA).

Measurement of IL-6, IL-8, MMP, and TIMP proteins SF supernatants were collected at 24 h after the beginning of treatments, centrifuged (1500 rpm, 5 min), aliquoted and stored at ⫺20°C. Samples were thawed at room temperature and, when necessary, diluted in 5% FBScomplete EMEM (Biowhittaker). Triplicate sample determinations using ELISA kits for human IL-6, IL-8 purchased from Endogen (Woburn, MA) were performed following manufacturer’s instructions. Human collagenase (matrix metalloproteinase-1 (MMP-1)), stromelysin (matrix metalloproteinase-3 (MMP-3)), tissue inhibitor of matrix metalloproteinase-1 (TIMP1), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) ELISA kits were from Amersham Pharmacia Biotech (Arlington Heights, IL). In all experiments, a final vehicle ethanol concentration of ⬍0.01% was used.

2661 and the enzyme cleavase VII, which clips off the 5⬘-fragment of the signal probe, recognizes this overlapping. The released fragment accumulation is directly proportional to the number of target mRNA transcripts present in the total RNA sample. A biotinylated oligonucleotide captures (capture oligo) the signal probe fragments on a streptavidin-coated plate and forms a primer-template substrate for DNA polymerase, which extends the signal probe fragment with fluorescein-dUTP. Final detection is achieved with an anti-fluorescein alkaline phosphatase-conjugated Ab and a chemifluorescent substrate. This novel method of mRNA detection was preferred to quantitative PCR techniques on the basis of its independence from RT amplification of template RNA and its direct quantitation of target RNA molecules. In addition, two other advantages for this new Xplore technology are the very low amounts of total RNA required for the measurements (as little as ⬃100 ng) and the very high sensitivity of the assay (with lower detection limits in the ⬃5-amol range). Known amounts of nucleic acid standards are used to define a standard curve for the signal of interest, whereas availability of ␤-actin and GAPDH assays allow for normalization of results. Densitometric analysis of PCR data was performed using a Gel

Actinomycin D pulse experiments Transcription inhibition was achieved with actinomycin D or with 5,6dichlorobenzimidazole riboside (DRB), both from Sigma (St. Louis, MO). SF were treated with IL-1␤, LXA4, or vehicle for 24 h before the addition of actinomycin D (0.5 ␮g/ml) or DRB (10 ␮g/ml). At 0, 3, 6, 12, and 24 h after actinomycin D addition, supernatants were used for ELISA, and RNA was extracted from cell pellets for mRNA.

RNA isolation, reverse transcription, and RT-PCR Total RNA extraction was performed using TriZol reagent (Life Technologies) followed by DNase treatment (RQ1 RNase-free DNase, Promega), phenol-chloroform extraction, and ethanol precipitation (Fisher Scientific, Houston, TX). RNA purity was checked by spectrophotometry, and RNA integrity was confirmed by visualization of 28S and 18S bands on agarose gel. Next, 1 ␮g of RNA was transcribed using the reverse transcription system (avian myeloblastosis virus RT, Promega). RT-PCR analyses were performed with the following sets of primers and conditions: for LXA4R, set 1: forward, 5⬘-CAC CAG GTG CTG CTG GCA AG-3⬘ (bp ⫺20 to ⫺1), reverse, 5⬘-AAT ATC CCT GAC CCC ATC CTC A-3⬘ (bp 1055–1076) amplifying a 1095-bp fragment including the full open reading frame (ORF) (annealing temperature, 60°C); set 2: forward, 5⬘-TGC TTG GGG TCA CCT TTG TC-3⬘ (bp 95–114), reverse, 5⬘-TGA AGC AGA ATT GGC AGC CG-3⬘ (bp 1004 –1023) generating a 928-bp fragment (annealing temperature, 58°C). The ␤-actin signal was determined to verify equal loading and consistency of the RT-PCR amplification. The following specific primers were used: forward, 5⬘-CAT GTG CAA GGC CGG CTT CG-3⬘ (bp 86 –105), reverse, 5⬘-GAA GGT GTG GTG CCA GAT TT-3⬘ (bp 292–311), amplifying a 226-bp fragment (annealing temperature, 56°C). Primer set 1 for the LXA4 receptor gene has been used previously for the amplification of LXA4R mRNA from myeloid cell types (25). The second set of LXA4 receptor primers has been selected by software-aided analysis (Vector NTI version 5.0, Informax, Bethesda, MD) of the formyl peptide receptor (FPR)-related gene family of receptors. This has allowed us to identify optimal PCR primers within the ORF region of the intronless LXA4R gene carrying minimal homology to other members of the formyl peptide receptor (FPR) family. BLAST homology searches with the GenBank databases were also performed with all primers to exclude random matching with known genomic sequences. The IL-6 RT-PCR was done with the following set of primers: forward, 5⬘-GCC TTC GGT CCA GTT GCC TT-3⬘ (bp 1398 –1418 within exon 1) and reverse, 5⬘-AGT GCC TCT TTG CTG CTT TCA C-3⬘ (bp 2677–2655 within exon 2) at an annealing temperature of 51°C. The IL-6 fragment amplified was 232 bp long. TIMP-1 and TIMP-2 RT-PCR primers were all 25-mers. The sequence of the TIMP-1 forward was 5⬘-ATC CTG TTG TTG CTG TGG CTG ATA G-3⬘ and reverse, 5⬘-TGC TGG GTG GTA ACT CTT TAT TTC A-3⬘ and generated a 629-bp fragment. The TIMP-2 forward primer, 5⬘-AAA CGA CAT TTA TGG CAA CCC TAT C-3⬘ and reverse, 5⬘-ACA GGA GCC GTC ACT TCT CTT GAT G-3⬘ generated a 430-bp fragment (annealing temperature, 64°C).

Quantification of mRNA transcripts Quantitation of IL-6 and ␤-actin mRNA transcripts was also performed using the Xplore TM system (Endogen). Briefly, two oligonucleotide probes (invader probe and signal probe) hybridize to a specific mRNA target splice junction. They overlap with one base at the hybridization site,

FIGURE 1. LXA4R mRNA expression in human synovial fibroblasts. A, After 24 h incubation with IL-1␤ (1 ng/ml) or indicated concentrations of LXA4, RT-PCR amplification of a 1095-bp fragment from human synovial fibroblasts was performed using primers spanning the LXA4R gene full length ORF (see Materials and Methods). After 28 cycles of PCR amplification (top), 2 ␮l of the resulting PCR reaction were used for a nested PCR step (25 cycles) using a second set of internal LXA4R primers amplifying a 928-bp fragment (middle PCR). Samples were resolved in 1% agarose gel. Lane 1, control cells; lane 2, LXA4 (10⫺7 M); lane 3, IL-1␤ ⫹ LXA4 (10⫺9 M); lane 4, IL-1␤ ⫹ LXA4 (10⫺7 M); lane 5, IL-1␤ alone; lane 6, PCR mix-DNA template; lane 7, PCR with RNA from lane 5; lane 8, retinoic acid-differentiated HL-60 cells. B, Densitometric analysis of results shown in A, top PCR. Results are from a representative experiment of three separate ones.

2662

LXA4 MODULATES SF CELL ACTIVATION

FIGURE 2. LXA4 inhibits IL-1␤-induced release of IL-6, IL-8, and MMP-3 protein in a dose-dependent manner via functional LXA4R. Human synovial fibroblasts were incubated (24 h, 37°C) with vehicle (ethanol ⬍0.01%, u), LXA4 (10⫺9 M, 䡺), or IL-1␤ (500 pg/ml, f) alone or IL-1␤ in presence of the indicated LXA4 concentrations (j). IL-6 (A), IL-8 (B), and MMP-3 (C) were measured in supernatants by ELISA. Insets, Samples were kept for 24 h in the presence of IL-1␤ (f), IL-1␤ plus LXA4 (j), or IL-1␤ plus LXA4 after 30 min of incubation with ␣LXA4R (hatched bars). Treatments in presence of LXA4 (j) and the ␣LXA4R sera (o) were compared with IL-1␤-stimulated samples (f) to perform a paired Student t test. Results with p ⬍ 0.01 (ⴱ), p ⬍ 0.005 (ⴱⴱ) or without statistical significance are indicated. Results are the mean ⫾ SD of triplicate determinations from of a representative experiment of three.

Doc 1000 system integrated with the Molecular Analyst software (BioRad, Hercules, CA).

Results High affinity LXA4 receptors are expressed in human SF and up-regulated by IL-1␤ Expression of specific high affinity LXA4R is required for LXA4induced modulation of leukocyte functions (17). We examined whether LXA4R is expressed in human SF using primers specific

for LXA4R. LXA4R mRNA expression was demonstrated by RTPCR amplification of SF-derived total RNA (Fig. 1, lane 1). mRNA expression was markedly stimulated by IL-1␤ (lane 5), LXA4 (lane 2), or combinations of IL-1␤ and LXA4 (lanes 3 and 4). Retinoic acid-differentiated HL-60 cells (lane 8), from which LXA4R has been cloned, were used as positive control (24). Semiquantitative PCR amplification of LXA4R mRNA, with primers encompassing the full length ORF, yielded a mix of PCR products resolved in multiple bands of molecular size ranging from ⬃1350 to

The Journal of Immunology

2663

FIGURE 3. LXA4 up-regulates TIMP-1 and TIMP-2 synthesis. LXA4 and IL-1␤ in the indicated combinations were added to SF with or without preincubation with ␣LXA4R (50 ␮l/ml, 30 min at 37°C). After 24 h of incubation, supernatants were collected, and ELISA performed for TIMP-1 (A) and TIMP-2 (B). Vehicle (u); LXA4 (10⫺9 M, 䡺); LXA4 after ␣LXA4R preincubation (dashed bars); IL-1 ␤ (500 pg/ml, f); IL-1 ␤ and LXA4 (j); LXA4 plus IL-1 ␤ after ␣LXA4R preincubation (o). Results are the mean ⫾ SD of triplicate determinations from of a representative experiment of three.

350 bp (Fig. 1A, top). The HL-60 cell-derived signal was consistent with previous RT-PCR results obtained with use of these primers in myeloid cells (25). A nested PCR step was used to resolve the specificity of the full length amplimers obtained from our first PCR step. A second set of LXA4R primers (designed to minimize homology to other known related genes) yielded a single band of the expected 928-bp size (Fig. 1A, middle PCR). Also, a third set of LXA4R primers, yielding a smaller 359-bp fragment, was designed to achieve the lowest homology with other sequences in the FPR gene cluster and used in a nested PCR step to confirm the expression of a true LXA4R on human SF (data not shown). Densitometric analysis of the semiquantitative PCR results indicates that IL-1␤ can up-regulate LXA4R mRNA expression by ⬃3-fold (Fig. 1B). LXA4 inhibits IL-1␤-induced synthesis of IL-6, IL-8, and MMP-3 We next investigated SF responses to LXA4. Because IL-6 has been implicated in the development of inflammatory synovitis, we determined the effect of LXA4 on IL-6 production. As shown in Fig. 2A, no effect was observed with LXA4 alone, whereas IL-1␤induced IL-6 release was inhibited by LXA4 at nanomolar concentrations (Fig. 2A). Inhibition by LXA4 was dose dependent and was abrogated by specific ␣LXA4R sera (Fig. 2A, inset). These results indicate that LXA4 inhibits IL-1␤-induced IL-6 release in human SF via functional LXA4R. After IL-1␤ addition, release of IL-8, a potent chemokine that stimulates angiogenesis and induces leukocyte recruitment at inflammatory sites of events, was also inhibited by LXA4 in a dose-dependent manner (Fig. 2B). The inhibition was reversed by ␣LXA4R (Fig. 2B, inset) Next, we evaluated the effect of LXA4 on IL-1␤-induced release of enzymes involved in tissue destruction. Results in Fig. 2C indicate that at subnanomolar concentrations, LXA4 potently inhibits MMP-3 synthesis. LXA4 modulation of MMP-3 synthesis was effectively prevented by ␣LXA4R serum (Fig. 2C, inset). Results also indicated that LXA4 did not significantly modify the expression of MMP-1 (data not shown). LXA4 is a potent agonist of TIMP synthesis During physiological tissue remodeling, activation of enzymes such as MMP is controlled by specific inhibitors. TIMP-1 and TIMP-2 regulation by LXA4 was next determined. As shown in Fig. 3, LXA4 stimulated the synthesis of TIMP-1 and TIMP-2,

FIGURE 4. LXA4 up-regulates TIMP-1 and TIMP-2 mRNA levels. LXA4 (10⫺9 M) was added to SF with or without preincubation with ␣LXA4R (50 ␮l/ml, 30 min at 37°C). After 24 h of incubation, total RNA was used for semiquantitative RT-PCR. Lane 1, control; lane 2, vehicle; lane 3, LXA4; lane 4, LXA4 ⫹ ␣LXA4R. Densitometric signals, normalized for ␤-actin, are indicated in the lower panel for TIMP-1 (䡺) and TIMP-2 (f). Results are from a representative experiment of three.

reversed the slight inhibition of TIMP-2 by IL-1␤ stimulation and induced up to 5-fold increase in TIMP-2 protein levels (Fig. 3B). As shown in Figs. 3 and 4, ␣LXA4R serum only partially affected LXA4 stimulation of TIMP synthesis, resulting in a modest reduction of both mRNA and protein levels (Figs. 3 and 4). These results are in contrast to complete abrogation of LXA4 inhibition of IL-6, IL-8, and MMP-3 release by ␣LXA4R (Fig. 2), suggesting that in SF LXA4 modulatory activities might involve novel signaling pathways in addition to those triggered by activation of functional LXA4R. LXA4 regulates TIMP and IL-6 mRNA levels in human synovial fibroblasts Next, TIMP-1, TIMP-2, and IL-6 mRNA expression was examined to determine whether LXA4 effect involved pre- or posttranscriptional events. Results shown in Figs. 4 and 5 confirmed those obtained for protein synthesis (Fig. 3), with IL-1␤ inducing a slight increase of TIMP-1 and a modest reduction of TIMP-2. LXA4 alone, or in combination with IL-1␤, increased mRNA expression of both TIMPs. Notably, LXA4 up-regulation of TIMP-2 mRNA was in the order of 3- to 6-fold compared with control cells, whereas a 2-fold increase was measured for TIMP-1 (Figs. 4 and 5). As indicated in Fig. 5, basal TIMP-1 mRNA expression in unstimulated cells was higher than TIMP-2 with a significant increase for TIMP-2 mRNA expression occurring 24 h after LXA4

2664

LXA4 MODULATES SF CELL ACTIVATION cin D or DRB, two inhibitors of nucleic acid synthesis, were used to investigate the transcriptional step potentially modulated by LXA4. Actinomycin D was added 24 h after SF incubation with the indicated combinations of IL-1␤ and LXA4 to block RNA synthesis. Results indicated a mRNA half-life of ⬃3.7 h for both IL-1␤ and IL-1␤ plus LXA4-treated cells (Fig. 6). The decrease of mRNA levels observed for the 24 h after actinomycin D additions was not reflected on IL-6 protein levels that continued to increase slowly (Fig. 6A). As shown in Fig. 6, B and C, use of semiquantitative RT-PCR analysis confirmed the results obtained with the Xplore assay. RT-PCR data also indicate similar LXA4 activities toward IL-8 and MMP-3 (data not shown). These data indicate that in human SF LXA4 dose-dependent inhibition of IL-1␤-induced IL-6, IL-8, and MMP-3 mRNA levels does not involve posttranscriptional regulation steps such as modulation of mRNA stability, suggesting that earlier transcriptional events are affected by LXA4. Overall LXA4 modulation of IL-1␤-induced SF activation resulted in a concerted reduction of proinflammatory mediators, such as IL-6, IL-8, and MMP-3, along with increased synthesis of TIMP, molecules that protect extracellular matrix from excessive degradation. These results clearly suggest that LXA4 may activate an antiinflammatory signaling pathway during inflammatory synovitis.

Discussion FIGURE 5. Time course for LXA4 up-regulation of TIMP-1 and TIMP-2. SF were treated with IL-1␤, LXA4 or the combination. Total RNA was extracted after 4h (lanes 1–7) and 24 h of incubation (lanes 8 –12). mRNA for TIMP-1 and 2 was amplified by semiquantitative RTPCR and densitometry performed. Lane 1, control cells; lanes 2 and 8, vehicle; lanes 3 and 9, IL-1␤ (500 pg/ml); lanes 4 and 10, IL-1␤ plus LXA4 10⫺7 M; lanes 5 and 11, IL-1␤ plus LXA4 10⫺9 M; lanes 6 and 12, LXA4 10⫺7 M; lane 7, LXA4 10⫺9 M. Densitometry results shown in the lower panel indicate TIMP-1 mRNA (䡺) and TIMP-2 mRNA (f). Results are representative of three separate experiments.

The present results demonstrate, for the first time, that human SF express functional LXA4R and that antiinflammatory actions arise by its interaction with the cognate endogenous ligand LXA4. Namely, LXA4 was found to inhibit IL-1␤-induced MMP-3 expression, IL-6 and IL-8 release while stimulating the synthesis of TIMP proteins. Synthesis of these mediators by SF has been recognized as a pivotal step in the pathophysiology of inflammatory arthritides (3, 26). The ability of LXA4 to modify these processes was tested after triggering SF cell activation by use of IL-1␤, a typical proinflammatory cytokine. Results indicate that SF expression of LXA4R mRNA, and presumably protein expression, is upregulated up to ⬃5-fold upon stimulation with IL-1␤ when compared with control cells (Fig. 1). Because several LXA4 antiinflammatory activities are mediated via signaling pathways involving LXA4R cell surface expression (17, 22), the noted IL-1␤ up-regulation suggests that this cytokine is activating potential negative feedback pathways along with the proinflammatory modification that it exerts on SF. Among IL-1␤ effects on LXA4R expression the appearance during LXA4R RT-PCR amplification of multiple bands, with sizes above and below the expected amplimer, was also addressed. These signals cannot represent immature nuclear forms of LXA4R mRNA transcripts because the human LXA4R ORF is encoded by an intronless gene; rather, they suggest the presence of LXA4R related sequences. In view of these

addition. Conversely, lower basal TIMP-2 mRNA levels in unstimulated cells were accompanied by significant increase after 4 h of exposure to LXA4, and a substantial 4- to 6-fold increase was reached after 24 h. As indicated for IL-6 (Table I), increased levels of TIMP protein and mRNA were observed until reaching plateau at ⬃120 h (TIMP-1 ⬃1 ␮g/ml and TIMP-2 ⬃50 ng/ml, after LXA4 stimulation). whereas relative ratios among different treatments were comparable with those observed at 24 h (data not shown). To reveal the effect of LXA4 on IL-6 mRNA levels, a novel quantitative assay was used. LXA4 inhibition was observed at 24 and 120 h of incubation, with a ⬃25– 40% reduction of the IL-6 protein and mRNA levels induced by IL-1␤ (Table I). Actinomy-

Table I. Effect of LXA4 on IL-6 protein and mRNA synthesis by synovial fibroblasts treated with IL-1␤a IL-6 Protein (ng/ml) (ELISA)

IL-6 mRNA (amol/200 ng RNA) (Xplore)

Agonists

24 h

5 days

24 h

5 days

Ethanol (⬍0.01%) LXA4 (10⫺9 M) IL-1␤ (500 pg/ml) IL-1␤ ⫹ LXA4

0.14 ⫾ 0.01 0.17 ⫾ 0.01 64.5 ⫾ 4.4 42.5 ⫾ 6.2*

0.34 ⫾ 0.17 0.39 ⫾ 0.18 417.0 ⫾ 24.1 274.3 ⫾ 24.4**

0.6 ⫾ 0.08 0.5 ⫾ 0.06 155.3 ⫾ 12.8 121.3 ⫾ 5.4*

3.1 ⫾ 0.2 2.4 ⫾ 0.2 46.1 ⫾ 3.7 27.4 ⫾ 3.1**

a Confluent SF were cultured in EMEM with 10% FBS. Cells were placed in 5% FBS fresh media 24 h before agonist addition. Incubations were stopped at either 24 h or 5 days, and supernatants and cell pellets were collected and subjected to ELISA and Xplore assays. IL-6 ELISA results are the mean ⫾ SEM from three separate experiments with SF obtained from three different donors. IL-6 mRNA values are the mean ⫾ SD of a single experiment performed in triplicate and are representative of two separate experiments. Student’s t test analysis for the IL-1␤-stimulated samples was assessed comparing treatments in the presence or absence of LXA4. ⴱ, p ⬍ 0.1; ⴱⴱ, p ⬍ 0.05.

The Journal of Immunology

FIGURE 6. Effect of LXA4 on IL-6 synthesis and mRNA expression. SF were treated with IL-1␤ (500 pg/ml) in presence and absence of LXA4 (10⫺9 M) for 24 h before the addition of actinomycin D (0.5 ␮g/ml). After 0, 3, 6, 12, and 24 h of incubation, IL-6 was quantitated in supernatants by ELISA, whereas RNA was extracted from cell pellets for mRNA quantification with the Xplore assay (A) or RT-PCR analysis (B). A, IL-6 protein levels are indicated by bar histogram: IL-1␤ (f); IL-1␤ ⫹ LXA4 (䡺). The line plot indicates mRNA levels: IL-1␤ (f); IL-1␤ plus LXA4 (E). Student’s t test analysis for the IL-1␤-stimulated samples was assessed comparing treatments in presence or absence of LXA4. Results with P values of

2665 data, we next performed a nested PCR confirming the identity of the target RNA strands being investigated (Fig. 1). Detection of LXA4R mRNA by RT-PCR does not exclude that other pathways might be involved in mediating LXA4 antiinflammatory activities. In myeloid cells, we have previously demonstrated that LXA4 inhibition of homotypic aggregation and ␤2 integrin up-regulation in activated polymorphonuclear cells (PMN) is abrogated by use of LXA4R antisense oligonucleotides and specific blocking ␣LXA4R sera (17). In the present study (Fig. 2, insets), ␣LXA4R serum was used to test the consequence of blocking LXA4R-mediated signaling in SF. Results confirm that LXA4R molecules are functionally expressed in human SF and that their activation is responsible for the inhibitory actions of LXA4 on IL-1␤-induced cell activation. Specifically, LXA4 potently inhibits IL-1␤-induced synthesis of IL-8 and MMP-3 and reduces by ⬃35% IL-6 production, activities that are almost completely abrogated if cells are exposed to the blocking ␣LXA4R serum (Fig. 2, insets). Noticeably, LXA4 selectively impacted MMP-3 production without statistically significant alteration of MMP-1 levels (data not shown). Although LXA4 inhibition of SF responses resemble events previously described in human PMN (17, 18, 27), stimulation of TIMP-1 and TIMP-2 is a novel observation (Fig. 3). For the first time, our results show that LXA4 has agonist actions on human synovial fibroblasts expressing the specific LXA4R receptor. The significance of these bioactivities is corroborated by recent studies in human epithelial cells, neutrophils, and murine inflammatory models where LXA4 agonist functions lead to regulation of the chemokine-cytokine axis (28). As previously noted, exposing SF to ␣LXA4R caused ⬃90% reduction of LXA4 effects on IL-6, IL-8, and MMP-3 but only a ⬃30% inhibition of LXA4-induced TIMP stimulation. These data suggest that multiple signaling pathways are activated and required for the expression of the full array of LXA4 antiinflammatory activities in SF. Several models, such as internalization of the receptor-ligand complex or presence of specific LXA4 transporter proteins, as we have previously documented in PMN (29), can be hypothesized that would allow exogenous LXA4 to reach the critical intracellular compartment where it may interact directly with transcriptional apparatuses. To support a role for the direct interaction of LXA4 with transcription factors, recent studies have reported that LXA4 interacts directly with the aryl hydrocarbon receptor (AhR) determining its activation (30). Our results would, however, indicate that LXA4-positive regulation of TIMP (Figs. 3–5) occurs at nanomolar concentrations of the agonist in contrast with the micromolar amounts required for the activation of AhR-dependent transcription. Thus, unless potential mechanisms for intracellular accumulation of LXA4 in SF will be experimentally verified, the present data would suggest that in human SF, transcriptional mechanisms other than AhR may be involved in LXA4 stimulation of TIMP synthesis. Inhibition of IL-6 release by LXA4 is of interest because significant elevation of IL-6 has been detected both systemically and in the joint tissues in RA patients (8, 11). Experimental and clinical observations support a pathogenetic role for IL-6 in RA (8, 11, 12, 31, 32). For example, a functional IL-6 gene is necessary for development of collagen type II-induced arthritis in mice (12), and a

⬍0.05 (ⴱ) have been indicated. B, Vehicle, lanes 1, 5, 9, 13, and 17; IL-1␤, lanes 2, 6, 10, 14, and 18; IL-1␤ ⫹ LXA4, lanes 3, 7, 11, 15, and 19; LXA4 alone, lanes 4, 8, 12, 16, and 20. C, Histogram indicates densitometric results (after ␤-actin normalization) for IL-6 mRNA signals obtained from the RT-PCR shown in B. Vehicle (u), IL-1␤ (f), IL-1␤ ⫹ LXA4 (j), and LXA4 alone (䡺). Results are representative of two separate experiments.

2666 link with IL-6 levels has been established for an Ag-induced arthritis model (31). In the above-cited models, abrogated or reduced IL-6 levels block the onset of RA. Results shown in Fig. 6 and Table I indicate that nanomolar concentrations of LXA4 can effectively antagonize IL-1␤-induced synthesis of IL-6 mRNA. Time course studies shown in Table I indicate that LXA4 caused a reduction of IL-6 mRNA apparent half-lives (from ⬃2.3 and 1.8 days, IL-1␤ vs. IL-1␤ plus LXA4, respectively). However, actinomycin D pulse experiments show that after IL-1␤ stimulation the IL-6 mRNA half-life of ⬃3.7 h is not significantly affected by LXA4 (Fig. 6), suggesting that LXA4 triggered events can impact the transcriptional regulation of the IL-6 gene without altering its mRNA stability. Overall, LXA4 modulation of SF activation could greatly impact the progression of inflammatory processes where reduction of SFderived IL-8 can lead to reduced migration of inflammatory leukocytes in the joint tissues whereas lower levels of MMP-3 could limit the extent of tissue destruction. Increased synthesis of TIMP-1 and TIMP-2 would further enhance LXA4-mediated mechanisms of tissue protection. The in vivo significance of the observed responses to LXA4 by SF remains to be determined. However, the concerted inhibition of inflammatory cytokine-induced MMP expression, IL-6 and IL-8 release, combined with stimulation of TIMP-1 and TIMP-2 expression strongly suggest that the immunomodulatory actions of LXA4 may play a major role in counteracting the development of inflammatory arthritides. In vivo studies have recently demonstrated that LXA4 significantly decreases inflammatory infiltrates and edema in a mouse model of inflammation (20). In this model, stable LXA4 analogues interact with a mouse homologue of the human LXA4R, leading to antiinflammatory activities that are more potent than those observed with equimolar concentrations of dexamethasone (20). Similarly, our results with human SF strongly suggest that LXA4 possesses antiinflammatory effects that may be involved in regulating critical pathophysiological processes in the development of inflammatory arthritides.

Acknowledgments We thank Dr. Yong Kang for his skilled technical contribution to the experiments, Dr. Mark Hutchinson (Arthroscopy Service, University of Illinois at Chicago Hospital, Chicago, IL) for the biopsy material, and Dr. Marilyn Olson (Third Wave Technologies, Madison, WI) and Dr. Mark Moody (Endogen) for the assistance provided for the Xplore assays.

References 1. Arend, W. P., and J. M. Dayer. 1990. Cytokines and cytokine inhibitors or antagonists in rheumatoid arthritis. Arthritis Rheum. 33:305. 2. Castor, C. W., E. M. Smith, M. C. Bignall, and P. A. Hossler. 1997. Connective tissue activation. XXXVII. Effects of cytokine combinations, implications for an integrated cytokine network. J. Rheumatol. 24:2080. 3. Bucala, R., C. Ritchlin, R. Winchester, and A. Cerami. 1991. Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts. J. Exp. Med. 173:569. 4. Mussener, A., M. J. Litton, E. Lindroos, and L. Klareskog. 1997. Cytokine production in synovial tissue of mice with collagen-induced arthritis (CIA). Clin. Exp. Immunol. 107:485. 5. Migita, K., K. Eguchi, Y. Kawabe, Y. Ichinose, T. Tsukada, T. Aoyagi, H. Nakamura, and S. Nagataki. 1996. TNF-␣ -mediated expression of membranetype matrix metalloproteinase in rheumatoid synovial fibroblasts. Immunology 89:553. 6. MacNaul, K. L., N. Chartrain, M. Lark, M. J. Tocci, and N. I. Hutchinson. 1990. Discoordinate expression of stromelysin, collagenase, and tissue inhibitor of metalloproteinases-1 in rheumatoid human synovial fibroblasts: synergistic effects of interleukin-1 and tumor necrosis factor-␣ on stromelysin expression. J. Biol. Chem. 265:17238.

LXA4 MODULATES SF CELL ACTIVATION 7. Firestein, G. S., and M. M. Paine. 1992. Stromelysin and tissue inhibitor of metalloproteinases gene expression in rheumatoid arthritis synovium. Am. J. Pathol. 140:1309. 8. Uson, J., A. Balsa, D. Pascual-Salcedo, J. A. Cabezas, J. M. Gonzalez-Tarrio, E. Martin-Mola, and G. Fontan. 1997. Soluble interleukin 6 (IL-6) receptor and IL-6 levels in serum and synovial fluid of patients with different arthropathies. J. Rheumatol. 24:2069. 9. Jarvis, J. N., W. Wang, H. T. Moore, L. Zhao, and C. Xu. 1997. In vitro induction of proinflammatory cytokine secretion by juvenile rheumatoid arthritis synovial fluid immune complexes. Arthritis Rheum. 40:2039. 10. Kotake, S., K. Sato, K. J. Kim, N. Takahashi, N. Udagawa, I. Nakamura, A. Yamaguchi, T. Kishimoto, T. Suda, and S. Kashiwazaki. 1996. Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation. J. Bone Miner. Res. 11:88. 11. Okamoto, H., M. Yamamura, Y. Morita, S. Harada, H. Makino, and Z. Ota. 1997. The synovial expression and serum levels of interleukin-6, interleukin-11, leukemia inhibitory factor, and oncostatin M in rheumatoid arthritis. Arthritis Rheum. 40:1096. 12. Alonzi, T., E. Fattori, D. Lazzaro, P. Costa, G. K. Probert, F. De Bendetti, V. Poli, and G. Ciliberto. 1998. Interleukin-6 is required for the development of collagen induced arthritis. J. Exp. Med. 187:461. 13. Serhan, C. N., J. Z. Haeggstrom, and C. C. Leslie. 1996. Lipid mediator networks in cell signalling: update and impact of cytokines. FASEB J. 10:1147. 14. Das, U. N. 1991. Interaction(s) between essential fatty acids, eicosanoids, cytokines, growth factors and free radicals: relevance to new therapeutic strategies in rheumatoid arthritis and other collagen vascular diseases. Prostaglandins Leukotrienes Essent. Fatty Acids 44:201. 15. Samuelsson, B., S. E. Dahle´n, J. A. Lindgren, C. A. Rouzer, and C. N. Serhan. 1987. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects: review. Science 237:1171. 16. Serhan, C. N. 1994. Lipoxin biosynthesis and its impact in inflammatory and vascular events: review. Biochim. Biophys. Acta 1212:1. 17. Fiore, S., and C. N. Serhan. 1995. Lipoxin A4 receptor activation is distinct from that of the formyl peptide receptor in myeloid cells: inhibition of CD11/18 expression by lipoxin A4-lipoxin A4 receptor interaction. Biochemistry 34:16678. 18. Lee, T. H., C. E. Horton, A. U. Kyan, D. Haskard, A. E. Crea, and B. W. Spur. 1989. Lipoxin A4 and lipoxin B4 inhibit chemotactic responses of human neutrophils stimulated by leukotriene B4 and N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Clin. Sci. 77:195. 19. Badr, K. F., D. K. DeBoer, M. Schwartzberg, and C. N. Serhan. 1989. Lipoxin A4 antagonizes cellular and in vivo actions of leukotriene D4 in rat glomerular mesangial cells: evidence for competition at a common receptor. Proc. Natl. Acad. Sci. USA 86:3438. 20. Takano, T., S. Fiore, J. F. Maddox, H. R. Brady, N. A. Petasis, and C. N. Serhan. 1997. Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors. J. Exp. Med. 9:1693. 21. Fiore, S., M. Romano, E. M. Reardon, and C. N. Serhan. 1993. Induction of functional lipoxin A4 receptors in HL-60 cells. Blood 81:3395. 22. Fiore, S., S. W. Ryeom, P. F. Weller, and C. N. Serhan. 1992. Lipoxin recognition sites: specific binding of labeled lipoxin A4 with human neutrophils. J. Biol. Chem. 267:16168. 23. Fiore, S., S. Nigam, and C. N. Serhan. 1991. Lipoxins trigger the release but not the oxygenation of arachidonic acid in human neutrophils: dissociation between lipid remodeling and adhesion. Adv. Prostaglandin Thromboxane Leukotriene Res. 21B:553. 24. Fiore, S., J. F. Maddox, H. D. Perez, and C. N. Serhan. 1994. Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor. J. Exp. Med. 180:253. 25. Maddox, J. F., M. Hachicha, T. Takano, N. A. Petasis, V. V. Fokin, and C. N. Serhan. 1997. Lipoxin A4 stable analogs are potent mimetics that stimulate human monocytes and THP-1 cells via a G-protein-linked lipoxin A4 receptor. J. Biol. Chem. 272:6972. 26. Firestein, G. S. 1996. Invasive fibroblast-like synoviocytes in rheumatoid arthritis: passive responders or transformed aggressors? Arthritis Rheum. 39:1781. 27. Grandordy, B. M., H. Lacroix, E. Mavoungou, S. Krilis, A. E. Crea, B. W. Spur, and T. H. Lee. 1990. Lipoxin A4 inhibits phosphoinositide hydrolysis in human neutrophils. Biochem. Biophys. Res. Commun. 167:1022. 28. Hachicha, M., Pouliot, M., Petasis, N. A., and Serhan, C. N. 1999. Lipoxin (LX)A4 and aspirin-triggered 15-epi-LXA4 inhibit tumor necrosis factor 1␣-initiated neutrophil responses and trafficking: regulators of a cytokine-chemokine axis. J. Exp. Med. 189:1923. 29. Simchowitz, L., S. Fiore, and C. N. Serhan. 1994. Carrier-mediated transport of lipoxin A4 in human neutrophils. Am. J. Physiol. 267:C1525–34. 30. Schaldach, C. M., J. Riby, and L. F. Bjeldanes. 1999. Lipoxin A4: a new class of ligand for the Ah receptor. Biochemistry 38:7594. 31. Baumann, H., and I. Kushner. 1998. Production of interleukin-6 by synovial fibroblasts in rheumatoid arthritis. Am. J. Pathol. 152:641. 32. Ohshima, S., Y. Saeki, T. Mima, M. Sasai, K. Nishioka, S. Nomura, M. Kopf, Y. Katada, T. Tanaka, M. Suemura, and T. Kishimoto. 1998. Interleukin-6 plays a key role in the development of antigen-induced arthritis. J. Immunol. 95:8222.