Reversal of autocrine and paracrine effects of interleukin 1 (IL-1) in human arthritis by type II IL-1 decoy receptor. Potential for pharmacological intervention.

Interleukin 1 (IL-1), produced by both synovial cells and chondrocytes, plays a pivotal role in the pathogenesis of cartilage destruction in osteoarthritis (OA). We examined the specific expression and function of IL-1 receptor family-related genes in human joint tissues. Gene array analysis of human normal and OA-affected cartilage showed mRNA expression of IL-1 receptor accessory protein (IL-1RAcp) and IL-1 type I receptor (IL-1RI), but not IL-1 antagonist (IL-1ra) and IL-1 type II decoy receptor (IL-1RII). Similarly, human synovial and epithelial cells showed an absence of IL-1RII mRNA. Functional genomic analyses showed that soluble (s) IL-1RII, at picomolar concentrations, but not soluble TNF receptor:Fc, significantly inhibited IL-1beta-induced nitric oxide (NO) and/or prostaglandin E(2) production in chondrocytes, synovial and epithelial cells. In OA-affected cartilage, the IC(50) for inhibition of NO production by sIL-1RII was 2 log orders lower than that for sIL-1RI. Human chondrocytes that overexpressed IL-1RII were resistant to IL-1-induced IL-1beta mRNA accumulation and inhibition of proteoglycan synthesis. In osteoarthritis, deficient expression by chondrocytes of innate regulators or antagonists of IL-1 such as IL-1ra and IL-1RII (soluble or membrane form) may allow the catabolic effects of IL-1 to proceed unopposed. The sensitivity of IL-1 action to inhibition by sIL-1RII has therapeutic implications that could be directed toward correcting this unfavorable tissue(s) dependent imbalance.

thritis, characterized by a limited infiltration of neutrophils into the synovial space and, in general, an absence of the classical signs of inflammation. Chondrocytes embedded within articular cartilage that is avascular and aneural reside in a sequestered environment, perhaps more than other cell types, whereby cellular metabolism is regulated by an autocrine mechanism responsive to biomechanical and pericellular signals. Recent observations by this and other laboratories indicate that, despite the general absence of clinical signs of inflammation, chondrocytes derived from patients with OA, show superinduction of proinflammatory genes typically associated with the products of synovial tissues in rheumatoid arthritis, including nitric-oxide synthase, cyclooxygenase-2, TNF␣, IL-6, and IL-8. The spontaneous production of the corresponding gene products and inflammatory mediators promotes a catabolic state, which leads to progressive cartilage damage in OA (1)(2)(3)(4). This intraarticular inflammatory response in OA-affected cartilage, which may be considered as an in situ "molecular inflammation," is partially dependent on autocrine IL-1␤ production, which induces and sustains an imbalance of cartilage homeostasis and extracellular matrix synthesis (5). The autocrine production of IL-1 in OA-affected cartilage is amplified by engagement of integrins such as ␣ 5 ␤ 1 by abnormally expressed extracellular matrix proteins, including proteolytic fragments of fibronectin (5,6).
Prolonged exposure of articular tissue to IL-1␤ can provoke a variety of cellular and inflammatory responses. For example, constitutive intraarticular expression of an adenoviral IL-1␤ transgene in rabbit joints induces multiple intraarticular manifestations, which include intense inflammation, leukocytosis, synovial hypertrophy, hyperplasia, highly aggressive pannus formation, and erosion of articular cartilage and bone. It also induces systemic effects including diarrhea and fever. Following the loss of the IL-1␤ transgene, which occurs after 28 days, most of the pathophysiological symptoms described above regress within 4 weeks (7). These results suggest that the pathophysiological effects of IL-1␤ in the local and systemic environment are reversible. The effects of IL-1 are not limited to inflammation, as bone formation, insulin secretion, appetite regulation, fever induction, and neuronal phenotype development are also regulated by this cytokine (8).
The action of IL-1 requires signaling through the cytoplasmic domain of the IL-1 type I receptor, which associates with a receptor accessory protein, IL-1RAcp. In vivo, the action of IL-1 is attenuated by at least two endogenous antagonists, the IL-1 receptor antagonist (IL-1ra), a naturally occurring IL-1 inhib-* 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.
ʈ Supported by the National Arthritis Foundation (Basic Sciences itor that binds to IL-1R without inducing biological responses (8), and the non-signaling type II IL-1 receptor (IL-1RII), which is devoid of a cytoplasmic domain and serves as a decoy, or activity down-modulating, receptor (9). In view of this background, we performed studies that examined the relative expression of IL-1 and associated proteins in OA and normal articular cartilage. We sought to determine whether an imbalance exists between signaling and inhibitory molecules that could favor the unchecked catabolic action of IL-1 in cartilage. We also examined the question of whether pharmacological intervention using the decoy IL-1RII receptor could reverse the catabolic events involved in OA cartilage degradation. The data indicate that future therapeutic strategies in osteoarthritis could involve restoring deficient IL-1 antagonists in diseased tissue.
Isolation of Bovine and Human Chondrocytes-Bovine cartilage was obtained from young calves, and chondrocytes were isolated from hooves by standard methods with minor modifications (11,12). The released cells were suspended in RPMI 1640 ϩ 10% FBS ϩ antibiotics and plated in a 24-well plate (Becton Dickinson, Lincoln Park, NJ) at a density of 5 ϫ 10 5 cells/2.0 cm 2 for 48 h. Human chondrocytes were isolated from OA-affected cartilage. The cartilage was cut into small pieces and digested with Pronase (0.1%) for 30 min in phosphatebuffered saline, followed by digestion with collagenase P (0.1%) for 12-16 h in Ham's F-12 medium (12).
Organ Culture of OA Cartilage and Analysis of Mediators-Organ culture was carried out as described previously (11,13). Briefly, knee articular cartilage from patients undergoing knee replacement surgery was obtained and cut in 3-mm discs, and four to six discs (ϳ100 mg) were placed in organ culture in 2 ml of Ham's F-12 medium ϩ 0.1% human albumin for 24 -72 h, with or without modulators. The medium was analyzed for nitric oxide (NO) as nitrite release and prostaglandin E 2 (PGE 2 ) (13) (radioimmunoassay, Sigma).
Chondrocyte Culture in Alginate Beads-Bovine or human chondrocytes were immobilized in alginate beads according to the method of Hauselmann et al. (14). Briefly, cells were suspended in filter-sterilized, low viscosity alginate solution (1.2%) at a concentration of 6 ϫ 10 6 cells/ml and slowly passed through a 22-gauge needle into a 102 mM CaCl 2 solution. After instantaneous gelation, the beads were further allowed to polymerize for 10 min in CaCl 2 solution. After two washes in 0.15 M NaCl and one wash in Ham's F-12 medium (supplemented with L-glutamine (2 mM), penicillin and streptomycin (100 IU/ml and 100 g/ml, respectively), gentamicin (50 g/ml), heat-inactivated FBS (10%) (Life Technologies, Inc.) and ascorbic acid (25 g/ml)), the beads (5 beads/well; 40,000 cells/bead) were finally incubated in complete culture medium for 6 days in a humidified atmosphere of 5% CO 2 at 37°C before further experiments.
Isolation of Synovial Cells-Synovial cells were retrieved from joints of patients undergoing knee replacement surgery. The cells were released and cultivated as described (15) in Ham's F-12 medium containing 10% FBS. The cells were also stained for the presence of type B (fibroblast-like) synovial cells using anti-fibronectin antibody (anti-CD68).
Proteoglycan Synthesis-Chondrocytes immobilized in alginate beads were incubated in Ham's F-12 culture medium (supplemented with L-glutamine (2 mM), gentamicin (50 g/ml), amphotericin B (0.25 g/ml), heat-inactivated FBS (0.5%), ascorbic acid (25 g/ml)) with 10 Ci/ml sodium sulfate (Na 2 35 SO 4 ) for 4 h at 37°C in a 5% CO 2 atmosphere. Alginate beads were washed five times with 0.15 M NaCl and solubilized in 0.5 ml of Soluene 350 (Packard) in scintillation counting tubes. After addition of 4.5 ml of liquid scintillation counting fluid (Hionic Fluor, Packard), the [ 35 S]-labeled proteoglycan content was measured using a Beckman LS 7000 counter and normalized with total genomic DNA content of the cells.
Isolation of RNA from Cells and OA Cartilage-The method was basically as described by Adams et al. (16) with minor modifications (11). Briefly, the cartilage was milled into fine powder in liquid nitrogen and was extracted with 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% sodium dodecyl sarcosine, and 0.1 M 2-mercaptoethanol for 4 h on a rocker. It was then extracted with water-saturated phenol, followed by phenol and chloroform. The aqueous phase was layered onto a cesium trifluoroacetate gradient for ultracentrifugation (24,000 rpm/24 h). The RNA pellet was dissolved in guanidium thiocyanate and precipitated with alcohol in the presence of acetic acid.
DNA Estimation-The cells from the calcium alginate beads were released as previously reported (14). Genomic DNA was isolated using a QIAamp kit (Qiagen Inc. Valencia, CA) as described by the manufacture and estimated at 260/280 nm using a spectrophotometer (Beckman).
Northern Analysis-Total RNA was isolated using TRI reagent (MRC Inc., Cincinnati, OH). Briefly, 20 g of RNA was subjected to electrophoresis in 1% agarose formaldehyde gel. RNA from the gel was then transferred by capillary action onto a nylon membrane (Zeta Probe, Bio-Rad). The membrane was hybridized with [ 32 P]dATP-labeled full length human IL-1RII cDNA or IL-1␤ cDNA. After hybridization, the blot was exposed to Kodak x-ray film (Eastman Kodak Co.) for 24 -48 h with intensifying screens at Ϫ70°C.
Hybridization of Expression Array-A human cytokine expression array (catalog no. GA001) from R&D Systems was used for this study. Total RNA was isolated from four normal and four OA cartilage samples, as described above. Equal amounts of RNA from each normal and OA cartilage sample were pooled separately. cDNA labeling reactions were performed using [␣-33 P]dATP (PerkinElmer Life Sciences) as described by R&D Systems. Labeled cDNA using both cytokine-specific primers and oligo(dT) primers were pooled and hybridized with expression array blot as recommended by the manufacturer.
Isolation of Peripheral Blood Mononuclear Cells (PBMC)-PBMCs were isolated from heparanized human blood using a Ficoll gradient as described previously (17). The buffy layer of cells was stimulated with LPS in RPMI 1640 ϩ 10% FCS.
Cloning and Expression of Soluble Type II Human IL-1 Receptor in SV40 T Antigen Immortalized Chondrocytes-The full-length cDNA for type II IL-1R was amplified using PCR primers, cloned, and expressed in the C-20/A4 chondrocytes after stable transfection of a mammalian expression vector pCB7 coding for hygromycin resistance as reported previously (5). A clone, designated C-20/A4 IL-1RII ϩ , was isolated after hygromycin selection.
Determination of PGE 2 and Nitrite-PGE 2 was determined in the culture supernatant using a radioimmunoassay, as reported previously (11), with a detection limit of 1.0 pg/ml. NO production was measured by estimating the stable NO metabolite, nitrite, in conditioned medium by a modified Griess reaction (11). The values were expressed as micromolar nitrite or nanograms per milliliter PGE 2 released per 100 mg wet weight of cartilage or 5 ϫ 10 5 chondrocytes.
Statistics-Data are expressed as mean Ϯ standard deviation. Each value is the mean of at least three samples. The results were analyzed by Student's t test. Differences with p Ͻ 0.05 were considered as significant.

Expression of IL-1 Family Receptors in Human
Cartilage-We and others observed previously that human OA-affected cartilage (but not normal cartilage) produced IL-1 in an autocrine fashion, which regulates the production of other inflammatory mediators (1,11,13,18). This regulation was also dependent on the concentration of the IL-1 antagonizing molecules, IL-1ra and IL-1RII, which can also exist in soluble forms together with sIL-1RI. We therefore analyzed the expression of mRNA encoding members of the IL-1 receptor family in human normal and OA cartilage that regulate IL-1 signaling and responses. Total RNA was extracted from four normal and four OA cartilage samples. Equal amounts of RNA from each sample were pooled together as either normal or OA RNA and labeled, as described under "Experimental Procedures." A nylon membrane representing specific DNA oligonucleotide sequences, including those for IL-1 receptor accessory protein (IL-1RAcP), IL-1RI, IL-1RII, and IL-1ra, was probed with the RNA from either normal or OA cartilage (Fig. 1A). The signals were normalized against the housekeeping genes, GAPDH and ␤-actin. Both normal and OA cartilage showed a relatively high expression of IL-RAcP and IL-1RI mRNA, whose translated protein receptors are prerequisite for signal transduction of IL-1 family proteins (19). The mRNA analysis of normal and OA cartilage samples did not show detectable signals above background for IL-1ra and IL-1RII.
In view of the role of IL-1 in the pathophysiology of human osteoarthritis, we examined the ability of sIL-1RII and membrane IL-1RII (mIL-1RII) to attenuate IL-1-induced effects in chondrocytes, cartilage, and synovial cells. We examined the expression of IL-1RII by reverse transcription-PCR in RNA obtained from human normal and OA-affected cartilage ob-tained from eight different patients (of which three are represented) in Fig. 1B (panel a) in synovial cells, A549 epithelial, and HEK293 cells (transfected or not with IL-1RII cDNA). As observed with the cartilage samples, human epithelial and synovial cells also did not show expression of IL-1RII mRNA. However, HEK293 cells transfected with IL-1RII cDNA showed expression of IL-1RII.
Expression of Biologically Active IL-1RII in Human Chondrocytes-The above observations led us to examine the role of IL-1RII in chondrocyte function. We selected an immortalized human chondrocyte cell line (C-20/A4) for the present study, as these cells do not release NO or PGE 2 in the presence or absence of IL-1 (data not shown). Human IL-1RII cDNA was transfected in C-20/A4 cells, and a clone, C-20/A4 IL-1RII ϩ , that stably expressed IL-1RII was isolated after hygromycin selection. Northern blot analysis of nontransfected C-20/A4 cells did not express detectable amounts of IL-1RII ϩ mRNA similar to normal and OA cartilage (Fig. 2). However, C-20/A4 IL-1RII ϩ cells expressed significant levels of IL-1RII mRNA, similar to LPS-stimulated PBMC (20). C-20/A4 and C-20/A4IL-1RII ϩ cells were stained with anti-␣ v ␤ 3 integrin (CD51 and CD61 complex) antibodies and anti-IL-1RII antibodies. As expected, C-20/A4 and C-20/A4IL-1RII ϩ cells showed similar expression of ␣ v ␤ 3 integrin, and there was a significant expression of IL-1RII on the cell surface of C-20/A4IL-1RII ϩ cells as compared with nontransfected cells (Fig. 3).
Regulation of Nitric Oxide and PGE 2 Production by IL-1RII in Bovine and Human OA Chondrocytes-The biological activity of the recombinant soluble IL-1RII was tested both in bovine and OA chondrocytes, which are known to respond to human IL-1␤ (21). Bovine and human chondrocytes grown in monolayers were stimulated with human IL-1␤ in the presence of supernatants from C-20/A4 and C-20/A4IL-1RII ϩ cells, which contained 500 pg/ml human soluble recombinant IL-1RII, and commercially available, purified sIL-1RII from R&D Systems. An equivalent amount of total protein from C-20/A4 control supernatant (constituting mostly serum albumin) was added accordingly. Both IL-1RII preparations at similar concentrations had the ability to block IL-1␤-induced NO and PGE 2 production by Ն80% in both cell types (Fig. 4, A and B). The biological activities of the commercial source of sIL-1RII FIG. 1. A, expression of human interleukin-1 receptor mRNA in normal and OA-affected cartilage. Articular cartilage from the knee was obtained from four normal and four osteoarthritic patients. Total RNA was isolated as described under "Experimental Procedures." Equal amounts of total RNA from normal and OA-affected cartilage were pooled, and first strand cDNA synthesis using [ 33 P]dATP was carried out. The radiolabeled cDNA was purified using a G25 Sephadex column, and a cytokine expression array was probed at 65°C for 16 -18 h. The remaining protocol was carried out as suggested by the manufacturer (R&D Systems). The membrane was exposed to a PhosphorImager, and the spots (shown above the bars) were quantified using Quant Imager (Molecular Dynamics). Duplicate spots were normalized with two housekeeping genes (GAPDH and ␤-actin). B, reverse transcription-PCR analysis of full-length IL-1RII in various cell types. Total RNA from PBMC Ϯ LPS (1 g/ml for 24 h), normal human cartilage (NC), and OA cartilage (OAc), human synovial cells (OAs), A549 human epithelial cells, HEK293, and HEK293 transfected with IL-1RII (HEK293 IL-1RII ϩ ) was isolated as described under "Experimental Procedures." The first strand cDNA synthesis and PCR were performed using specific primers for IL-1RII (panel a) as described previously (5). Panel b shows amplification of GAPDH. and chondrocyte-derived sIL-1RII were similar. There was no detectable IL-1RII in the supernatants of nontransfected C-20/A4 cells. All of the subsequent experiments were performed with the chondrocyte-derived sIL-1RII, unless otherwise specified.
Specificity of IL-1RII-The specificity of the effects of IL-1 and the neutralizing activity of sIL-1RII were examined in primary bovine chondrocytes, which respond to both recombinant human IL-1␤ and TNF␣ with respect to induction of NO. Ten ng/ml human IL-1␤ increased NO, which was significantly inhibited in dose-dependent (0.5-1.5 ng/ml) fashion by soluble IL-1RII; in contrast, IL-1-induced NO production was not inhibited by up to 25 g/ml soluble p75 TNF␣ receptor fused to Fc region of Ig (TNFR:Fc) (Fig. 4C). Furthermore, 1000 units/ml TNF␣ induced NO production, which could be inhibited by more than 90% by 25 g/ml TNFR:Fc, but not by 1 ng/ml IL-1RII, a concentration sufficient to block IL-1-dependent responses in the cells (Fig. 4C). These experiments indicate that the inhibitory effects of soluble IL-1RII are specific to IL-1 stimulation and are not related to activation by different ligands (e.g. TNF␣).
Regulation of Proteoglycan Synthesis by IL-1RII in Chondrocytes-Primary chondrocytes, when grown in monolayer for even 1 week, show significant loss of chondrocyte phenotype and dedifferentiate into fibroblast-like cells (22). Therefore, bovine and human chondrocytes were immobilized in alginate beads to avoid dedifferentiation of chondrocytes. IL-1␤ has been reported to inhibit proteoglycan synthesis in chondrocytes in relatively long term cultures of about 3 days (23). The immobilized human and bovine cells were stimulated with 5 ng/ml human IL-1␤ in the absence and presence of various concentrations of sIL-1RII. IL-1-stimulated cells produced NO at 72 h, which could be inhibited by Ͼ50% with 250 and 500 pg/ml sIL-1RII (Fig. 5). At the same time point, IL-1␤ significantly inhibited the incorporation of [ 35 S]sulfates into proteoglycans in immobilized bovine and human chondrocytes as compared with uninduced cells (p Ͻ 0.01). IL-1RII at 500 pg/ml, but not at 250 pg/ml, significantly reversed the effect of IL-1 on proteoglycan synthesis. IL-1RII at 1 ng/ml completely blocked the actions of IL-1. These experiments suggested that the synthesis of proteoglycan was equally sensitive to the effects of sIL-1RII as compared with NO production. These experiments also showed that relatively low levels of sIL-1RII were sufficient to significantly block the effects of IL-1 in both bovine and human chondrocytes.
We compared the regulation of proteoglycan synthesis by IL-1 in immobilized cultures of C-20/A4 and C-20/A4IL-1RII ϩ cells. As immobilized C-20/A4IL-1RII ϩ cells make low amounts of sIL-1RII, we chose to stimulate the cells with a low concentration (0.5 ng/ml) of IL-1. Addition of IL-1 to C-20/A4 cells showed a significant decrease (23%) in the incorporation of [ 35 S]sulfates into the proteoglycans as compared with noninduced cells. However, stimulation of C-20/A4IL-1RII ϩ cells with 0.5 ng of IL-1 showed no significant decrease in proteoglycan synthesis under similar experimental conditions. Increasing the concentration of IL-1 to 1 ng/ml showed a significant decrease in proteoglycan synthesis in both C-20/A4 and C-20/A4IL-1RII ϩ cells (Fig. 6). The susceptibility of chondrocytes to IL-1 is directly dependent on the amount of IL-1 and IL-1RII.
Regulation of IL-1 mRNA by IL-1RII-Recent studies have shown that production of inflammatory mediators such as NO, PGE 2 , IL-6, and matrix metaloproteinase in chondrocytes is induced by autocrine IL-1 (5,13,24). In view of these observations, we examined the induction of IL-1 mRNA by relatively high concentrations of IL-1 (5-10 ng/ml) in nontransfected and IL-1RII-transfected cells that expressed membrane IL-1RII and also released sIL-1RII. C-20/A4 and C-20/A4IL-1RII ϩ cells (10 6 ) were exposed to 5-10 ng/ml IL-1 for 4 h and analyzed for IL-1 mRNA by Northern analysis (Fig. 7). IL-1 induced IL-1 mRNA in C-20/A4 cells, however, there was no detectable expression of IL-1 mRNA in C-20/A4IL-1RII ϩ cells, even at concentrations of IL-1 as high as 10 ng/ml. These experiments suggest that transgene expression of IL-1RII inhibits the induction of IL-1 mRNA and probably the autocrine production of IL-1.
Regulation of NO and PGE 2 in Human OA Cartilage in ex Vivo Conditions-Based on the above observations, the ability of IL-1RI and IL-1RII to inhibit the spontaneous production of NO and PGE2 by autocrine IL-1 was compared under identical conditions. IL-1RI (1 mg/ml) has been reported previously to inhibit the spontaneous production of NO and PGE 2 by ϳ50% in human OA-affected cartilage in ex vivo (13). In this experiment, the IC 50 required to inhibit NO accumulation in OAaffected cartilage was 1 mg/ml for IL-1RI and 125 pg/ml for IL-1RII (Fig. 8). These experiments suggested that sIL-1RII was more effective than sIL-1RI in neutralizing the action of endogenous autocrine IL-1 in OA cartilage.
Human OA cartilage was also incubated in basal F12 media in ex vivo conditions to examine the effect of sIL-1RII on the effects of exogenously added IL-1. The human OA cartilage cultures spontaneously released NO and PGE 2 , which was further augmented by addition of IL-1, as previously reported. Increasing concentrations of sIL-1RII receptor showed a dosedependent inhibition of spontaneous production of NO and PGE 2 as shown above and in Table I. Addition of 1 ng/ml IL-1 to these cartilage cultures resulted in up-regulation of both NO and PGE 2 production, which was inhibited significantly by 250 pg of sIL-1RII (Table I). These experiments again showed that IL-1RII can significantly neutralize both the effects of endogenous and exogenous IL-1 in ex vivo conditions.

Regulation of PGE 2 Production by Synovial Cells by sIL-1RII-
The synovial cells of the joint play an important role in the pathophysiology of arthritis and are highly sensitive to IL-1 (1,2). Similar to chondrocytes, synovial cells and epithelial cells do not express detectable amounts of IL-1ra and IL-1RII mRNA (Fig. 1B). We tested the effects of IL-1 on PGE 2 production in rabbit and human synovial and human epithelial cells in the presence and absence of IL-1RII. The cells were grown in monolayer cultures for 48 h and stimulated with optimum concentrations of IL-1 (5-10 ng/ml) in the absence and presence of sIL-1RII (Fig. 9). The production of PGE 2 was then estimated at 72 h. As expected, IL-1 induced the production of PGE 2 in all three cell types, and IL-1RII (500 pg/ml) inhibited PGE 2 production by Ն50% in rabbit synovial cells and human epithelial cells. Human synovial cells incubated with 1 ng/ml sIL-1RII, ϳ50% inhibition of PGE 2 production was seen. These experi-ments showed that relatively low concentrations of IL-1RII can attenuate the inflammatory responses in synovial cells and other human fibroblast-like cells. DISCUSSION The regulation and the effect of the IL-1 family of proteins including its receptors and antagonists is complex (25). IL-1 activity seems to be a tightly regulated event, involving IL-1RI, IL-1RII, and IL-1ra in addition to receptor accessory proteins and multiple kinase-regulated signaling pathways (26). IL-1RII is up-regulated in various pathophysiological conditions, such as sepsis, meningococcal infections, Alzheimer's disease, and anorexia (27). In contrast, patients with OA show decreased accumulation of IL-1RII in the serum with increasing severity of the disease (28). IL-1RII can also be induced in vitro by cytokines, chemoattractants, and other modulators (29). FIG. 4. A and B, comparison and biological activity of human chondrocyte-derived sIL-1RII compared with commercially available sIL-1RII in bovine and human chondrocytes. Primary bovine (A) and human (B) OA chondrocytes isolated from cartilage and grown for 48 h in monolayer cultures were stimulated with IL-1␤ (5 ng/ml) in the presence of control supernatant (SN) obtained from nontransfected cells, and transfected SN from IL-1RII ϩ -transfected cells containing 500 pg/ml or purified sIL-1RII at 500 pg/ml. NO and PGE 2 production was estimated at the end of the experiment (72 h) as described under "Experimental Procedures." The p values were compared between control SN and sIL-1RII-treated cells. *, p Յ 0.01; **, p Յ 0.001. C, primary bovine chondrocytes that were grown for 48 h in monolayer cultures were stimulated with 10 ng of human IL-1␤ or 1000 units/ml human TNF␣, in the presence of 0.5 -1.5 ng/ml IL-1RII or 25 g/ml soluble p75 TNF receptor fused to Fc portion of Ig as reported previously (13). Nitrite was estimated 72 h after stimulation. The data represent one of two similar experiments. The p values (n ϭ 3) were compared between IL-1-or TNF-treated cells in the presence of sIL-1RII or TNFR:Fc. *, p Յ 0.01 Recent studies have also shown that IL-1RII can be induced in vivo by anti-inflammatory drugs such as aspirin (30) and dexamethasone (31).
We showed previously that 100 mg of OA cartilage released ϳ10 -50 pg/ml IL-1. This concentration was sufficient to stim-ulate NO and PGE 2 production in ex vivo conditions, and these effects were blocked by sIL-1RI (13) or IL-1 neutralizing mAbs (data not shown). However, exogenous addition of 10 -50 pg/ml recombinant IL-1 under the same experimental conditions did not induce or augment NO or PGE 2 production in normal or OA cartilage in vitro (13).
In human OA-affected cartilage, detectable levels of mRNA for IL-1 and IL-1RI (13,32,33) and undetectable levels of IL-1RII and IL-1ra have been observed despite superinduction of inflammatory mediators (1, 2, 5, 11, 13). This suggests that the chondrocytes and synovial cells may not be the source of IL-1RII, which is seen in serum as well as synovial fluids of OA patients (28,34). Low concentrations of autocrine IL-1␤, in the absence of a decoy receptor and a receptor antagonist, may therefore exacerbate an inflammatory response and imbalance cartilage homeostasis in a long term disease such as osteoarthritis. Thus, the human articular chondrocyte may lack a naturally occurring defense mechanism against the insults of low levels of endogenous IL-1. This assumption is supported by the recent finding that IL-1ra-deficient mice spontaneously develop arthritis (35). Our hypothesis is further based on the observations that overexpression of IL-1ra in mice had significant reduction in the incidence and severity of collagen-induced arthritis. Furthermore, mice injected with neutralizing IL-1ra antibodies had a significantly earlier onset of collageninduced arthritis with increased inflammation and severity (36). Some of the beneficial effects of IL-1ra have been observed in humans, where infusion of IL-1ra significantly inhibited cartilage and bone damage (37). Similarly, overexpression of IL-1RII in mice showed resistance to inflammatory response to IL-1 but not allergic responses induced by collagen (38). Injection of IL-1RII in rabbits with antigen-induced arthritis showed dose-dependent inhibition of joint diameter, plasma PGE 2 , and synovial fluid IL-1. Significant inhibitory effects were also seen histologically on soft tissue swelling and joint damage (39). In vitro studies have suggested that blockade of both IL-1RII and IL-1ra activity enhances cellular responses to IL-1 (40). Our hypothesis is further strengthened by the recent report, which suggests that lung epithelial cells that lack IL-1RII are more susceptible to IL-1 and inflammatory mediators released by them (41).
We provide evidence that the effects of the soluble IL-1RII on activated chondrocytes is specific, since it inhibited NO production induced by IL-1, but not by TNF. We have shown previously that IL-1 and TNF have synergistic effects in chondrocytes when used in combination; the current experiments would indicate that inhibition of the individual actions of these cytokines in cartilage will not be achieved by IL-1 blockade alone (13). Furthermore, the effects of IL-1 and IL-1RII with respect to NO and PGE 2 production are not influenced by exogenous addition of other mediators such as IL-6 (data).  IL-1RII, both as a cell-surface receptor and a soluble protein, has unique IL-1 antagonist properties due to its ability to be a functional decoy receptor, thereby making it a promising candidate for pharmacological intervention and gene therapy (42). In order to understand the biology of IL-1RII in inflammatory responses, we stably transfected IL-1RII cDNA in a human chondrocyte cell line (C-20/A4) for expression of this receptor. The lack of IL-1RII expression observed in the C-20/A4 cells may be similar to that observed in OA chondrocytes. The transgene expression of IL-1RII in C-20/A4IL-1RII ϩ cells could attenuate the effects of IL-1 in both monolayer and immobilized cultures with respect to inhibition of proteoglycan synthesis.
The phenotypic characteristics of differentiated chondrocytes are complex and critical for its cartilage-specific metabolism (22). Chondrocytes grown in monolayer cultures in vitro lose their morphological and biochemical characteristics within two weeks. This includes loss of gene expression of extracellular matrix proteins such as aggregan and type II collagen and increased synthesis of type I collagen (43). IL-1 accelerates the loss of phenotype (44). Periodically, they are also less responsive to IL-1 with respect to production of NO and matrix metaloproteinase-9 (45). Dedifferentiated chondrocytes can restore phenotype when cultivated in alginate beads for a period of at least 1 week. Irrespective of the low expression of IL-1RII in stably transfected human chondrocytes in immobilized beads, we observed significant protection to exogenous IL-1 in terms of proteoglycan synthesis. The reason for the lowered production of sIL-1RII in alginate beads as compared with monolayer cultures is not clear. It is possible that recombinant IL-1RII was entrapped in the beads or that expression of transgene from the CMV promoter may be poor under non-proliferating conditions.
Comparing the effects of IL-1RI and IL-1RII in human OA cartilage explants showed that IL-1RII was more effective than IL-1RI in inhibiting the production of NO. It is important to note that only superficial chondrocytes as compared with deep zone chondrocytes in OA cartilage show enhanced expression of IL-1 and IL-1RI (2). The ability of recombinant sIL-1RII to attenuate the effects of IL-1 in ex-vivo conditions suggests the feasibility of the 47-kDa protein to effectively diffuse into the superficial layer of OA cartilage. IL-1RII could also attenuate the IL-1-induced expression of both the early response gene (cyclooxygenase-2) and the intermediate gene (inducible nitricoxide synthase). These results suggest that IL-1RII corrects chondrocyte metabolism and cartilage homeostasis by attenuating the effects of IL-1. This potent IL-1 neutralizing property may be due to the multifunctional effects of IL-1RII as compared with IL-1RI. The human IL-1RII possesses activity across species, since it was effective in bovine chondrocytes, rabbit synovial cells, and rodent epithelial cells.
The stably transfected human chondrocytes expressing IL-1RII also showed a feedback effect on the expression of IL-1 mRNA. Overexpression of IL-1RII in the C-20/A4 cells blocked the induction of IL-1 mRNA and, subsequently, the autocrine effects of IL-1. This may be partially attributed to the recent observation that intracellular cytosolic proIL-1, but not secreted proIL-1, complexes with IL-1RII. Thus, it is possible that (a) IL-1RII sequesters intracellular proIL-1 and blocks its processing (46), (b) IL-1RII has higher affinity for IL-1␤ than IL-1RI, and (c) IL-1 and IL-1RII complex competitively competes with IL-1Acp protein (47), thus blocking IL-1RI from actively engaging IL-1Acp for downstream signaling. These multiple effects of IL-1RII may contribute to the break in the autocrine loop involving induction of IL-1␤ by IL-1␤.
Synovial cells are highly susceptible to IL-1 (48). Similarly, epithelial cells have been known to lack IL-1RII expression (41). Our experiments show that IL-1RII attenuates the effects of IL-1 in various cell types including rabbit and mouse synovial cells and human epithelial cells. Since either endogenous or transgene IL-1RII shows significant protection to IL-1␤induced insults in chondrocytes and synovial cells of the joint, it may be a promising therapeutic candidate for slowing or reversing cartilage degeneration and maintaining cartilage homeostasis.