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Functional Genomic Analysis of Type II IL-1 Decoy Receptor: Potential for Gene Therapy in Human Arthritis and Inflammation¹

Mukundan G. Attur^*, Mandar N. Dave^*, Mary Y. Leung^*, Christine Cipolletta^*, Marcia Meseck, Savio L. C. Woo and Ashok R. Amin^2,*,,

^* Laboratory for Functional and Pharmacogenomics, Hospital for Joint Diseases, New York, NY 10003; Departments of Pathology and Medicine, New York University Medical Center, New York, NY 10016; Kaplan Cancer Center, New York, NY 10016; and Institute for Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, New York, NY 10029

	Abstract

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Gene expression arrays show that human epithelial cells and human arthritis-affected cartilage lack detectable amounts of mRNA for IL-1 antagonizing molecules: IL-1Ra and IL-1RII, but constitutively express IL-1. Functional genomic analysis was performed by reconstituting human IL-1RII expression in various IL-1RII-deficient cell types to examine its antagonist role using gene therapy approaches. Adenovirus-expressing IL-1RII when transduced into human and bovine chondrocytes, human and rabbit synovial cells, human epithelial cells, and rodent fibroblasts expressed membrane IL-1RII and spontaneously released functional soluble IL-1RII. The IL-1RII⁺ (but not IL-1RII^-) cells were resistant to IL-1-induced, NO, PGE₂, IL-6, and IL-8 production or decreased proteoglycan synthesis. IL-1RII inhibited the function of IL-1 in chondrocytes and IL-1- and TNF--induced inflammatory mediators in human synovial and epithelial cells. IL-1RII⁺ chondrocytes were more resistant to induction of NO and PGE₂ by IL-1 compared with IL-1RII^- cells incubated with a 10-fold (weight) excess of soluble type II IL-1R (sIL-1RII) protein. In cocultures, IL-1RII⁺ synovial cells released sIL-1RII, which in a paracrine fashion protected chondrocytes from the effects of IL-1. Furthermore, IL-1RII⁺ (but not IL-1RII^-) chondrocytes when transplanted onto human osteoarthritis-affected cartilage in vitro, which showed spontaneous release of sIL-1RII for 20 days, inhibited the spontaneous production of NO and PGE₂ in cartilage in ex vivo. In summary, reconstitution of IL-1RII in IL-1RII^- cells using gene therapy approaches significantly protects cells against the autocrine and paracrine effects of IL-1 at the signaling and transcriptional levels.

	Introduction

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Classically, osteoarthritis (OA),³ unlike rheumatoid arthritis (RA), is considered an inherently noninflammatory disorder of movable joints characterized by deterioration of articular cartilage and the formation of new bone at the joint surfaces and margins. The synovial fluid in OA, in contrast to that in RA, typically contains few neutrophils (<3000/mm³), and except in advanced disease, the synovium itself does not exhibit significant cellular proliferation or infiltration by inflammatory leukocytes. OA also differs from RA in that it is not a systemic disease (1, 2, 3).

Recent studies indicate that despite the general absence of clinical signs of inflammation, chondrocytes derived from OA-affected cartilage show superinduction of proinflammatory genes (typically associated with the products of synovial tissues in RA) including NO synthase, cyclooxygenase 2 (COX-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). This intra-articular inflammatory response in OA-affected cartilage, which may be considered an in situ molecular inflammation, is partially dependent on constitutive production of IL-1 that sustains an imbalance of cartilage homeostasis and extracellular matrix synthesis (2). The autocrine production of IL-1 in OA-affected cartilage is amplified by engagement of integrins such as ₅₁ by abnormally expressed extracellular matrix proteins, including proteolytic fragments of fibronectin (4, 5). Gene array analysis of human (normal and OA-affected) cartilage showed mRNA expression of IL-1 receptor accessory protein and IL-1 type I receptor (IL-1RI), but not IL-1 receptor antagonist (IL-1Ra) or IL-1 type II decoy receptor (IL-1RII). Similarly, human synovial and epithelial cells showed an absence of IL-1RII mRNA (6). Functional analysis using soluble IL-1RII (sIL-1RII) at pico/nanogram concentrations (but not soluble TNFR, Fc) showed that it significantly inhibited IL-1-induced NO and/or PGE₂ production in chondrocytes, synovial cells, and epithelial cells. In OA-affected cartilage, the IC₅₀ for inhibition of spontaneous production of NO by sIL-1RII was 2 log orders lower than that for sIL-1RI.

Prolonged exposure to IL-1 in articular tissue provokes a variety of cellular and inflammatory responses. For example, constitutive intra-articular expression of an adenoviral IL-1 transgene in rabbit joints induces multiple intra-articular 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 revert within 4 wk (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. Bone formation, insulin secretion, appetite regulation, fever induction, and neuronal phenotype development are also regulated by this cytokine (8).

In OA, deficient expression of innate antagonist regulators of IL-1 by chondrocytes such as IL-1Ra and IL-1RII may allow the catabolic effects of IL-1 to proceed unopposed (6). The inhibition of IL-1 action by sIL-1RII has therapeutic implications that could be directed toward correcting this unfavorable tissue-dependent imbalance.

In the present study, we show that reconstituting the functional expression of IL-1RII (using adenoviral vectors) in IL-1RII^- cells such as chondrocytes, epithelial cells, and synovial cells 1) reduces and reverses the susceptibility of cells to the effects of IL-1 with respect to induction of NO, PGE₂, IL-6, IL-8, and IL-1 production and proteoglycan synthesis; and 2) releases sIL-1RII, which protects other susceptible cells in coculture and transplantation against the effects of IL-1 (in ex vivo and vitro). Furthermore, membrane IL-1RII is more potent than sIL-1RII alone in neutralizing the effects of IL-1.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents and cell lines

All media and FBS were purchased from Life Technologies (Gaithersburg, MD), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Cytokines were obtained from PeproTech (Rocky Hill, NJ), and ELISA kits were purchased from R&D Systems (Minneapolis, MN). Human epithelial cells (A549) and rabbit synovial cells (HIG82) were obtained from American Type Culture Collection (Manassas, VA). The mouse lung fibroblasts were provided by Leslie Ballou (9). Rat anti-human IL-1RII Ab (M25) was provided by Immunex (Seattle, WA).

Procurement of human cartilage

Cartilage slices were taken from the knees of patients diagnosed with advanced OA (ages 50–70 years) who were undergoing knee replacement surgery. The OA patients had been free of nonsteroidal anti-inflammatory drugs for at least 2 wk before surgery. Nonarthritic knee cartilage was obtained from patients with fractures or from accident victims after knee amputation, the same day. The use of discarded human cartilage was approved by appropriate institutional review boards, and patient consent was obtained.

Isolation of bovine and human chondrocytes

Bovine cartilage was obtained from young calves. Isolation of bovine chondrocytes from hooves was conducted by standard methods with minor modifications (10). The released cells were suspended in RPMI 1640, 10% FBS, and antibiotics and plated in a 24-well plate (BD Labware, Lincoln Park, NJ) at a density of 5 x 10⁵ cells/2.0 cm² for 48 h. Human chondrocytes were isolated from OA-affected cartilage. The cartilage was cut into small pieces and digested with Trypsin (0.2%) for 30 min in PBS, followed by digestion with collagenase II (0.1%) for 12–16 h in Ham’s F-12 medium.

Organ culture of OA cartilage and analysis of inflammatory mediators

Organ culture was conducted as described previously (10). Briefly, knee articular cartilage from patients undergoing knee replacement surgery was obtained and cut into 3-mm discs, and four to six discs (100 mg) were placed in organ culture in 2 ml of Ham’s F-12 medium and 0.1% human albumin for 24–72 h with or without modulators. The medium was analyzed for nitrite (10) and PGE₂ (RIA, Sigma).

Chondrocyte culture in alginate beads

Chondrocytes were immobilized in alginate beads, as reported previously (6). Briefly, cells were suspended in filter-sterilized low-viscosity alginate solution (1.2%) at a concentration of 6 x 10⁶ cells/ml and slowly passed through a 22-gauge needle into a 102 mM CaCl₂ solution. After instantaneous gelation, the beads were further allowed to polymerize for 10 min in CaCl₂ 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-streptomycin (100 IU/ml to 100 µg/ml), gentamicin (50 µg/ml), heat-inactivated FBS (10%; Life Technologies), and ascorbic acid (25 µg/ml)), beads (5 beads/well; 40,000 cells/bead) were finally maintained in complete culture medium for 6 days in a humidified atmosphere of 5% CO₂ at 37°C before additional experiments.

Chondrocyte transplantation

Genetically modified chondrocytes were grown in vitro. The cells were trypsinized and suspended in complete Ham’s F-12 medium. They were slowly added to the articular surface of OA-affected cartilage (with the underlying subchondral bone) in organ cultures as previously reported (11). The production of IL-1RII NO, PGE₂, proteoglycan metabolism, and other parameters was estimated at different time periods in the presence and the absence of IL-1.

Isolation of synovial cells

Synovial cells were retrieved from joints of patients undergoing knee replacement surgery. The cells were released and cultivated as previously described (12) in Ham’s F-12 medium containing 10% FBS. The cells were also stained for type B (fibroblast-like) synovial cells using anti-fibronectin Ab (anti-CD68).

Cloning and expression of soluble type II human IL-1R in baculovirus

Full-length sIL-1RII was cloned from human neutrophils using RT-PCR and expressed in pFAST BAC-1 BACMID as previously reported (4, 13). The recombinant baculovirus-expressed sIL-1RII was estimated by ELISA (R&D Systems). The biological activity of sIL-1RII released from Sf9 insect cells was compared with that of commercially available sIL-1RII from R&D Systems.

Preparation of adenoviral vectors

The full-length human IL-1RII was subcloned from pFAST-BAC1 (4) to pAd1/Rous sarcoma virus (RSV) vector at the HindIII-NotI site. The adenoviral vector used in this study is deleted of E1A and E1B and a portion of the E3 region. This impairs the ability of the virus to replicate and transform nonpermissive cells. The transgene transcription was driven by the RSV long terminal repeat. The recombinant clone was screened in A549 cells for the synthesis of functional sIL-1RII. The viral vector was rescued by calcium phosphate coprecipitation with the plasmid pBHG10. Individual plaques were picked and amplified for analysis of functional activity and DNA restriction pattern. Clone 7 was chosen for further amplification for large-scale production in 293 cells. The final cell harvest was purified on a cesium chloride gradient and dialyzed against the final formulation buffer, 10 mM Tris (pH 7.5)/1 mM MgCl₂/150 mM NaCl/10% glycerol. The virus preparation was filtered through a 0.2-µm pore size filter unit and tested for the presence of endotoxin and mycoplasma. The virus was also quantified by measuring the absorbance at A₂₆₀ to determine virus particle concentration and by plaque assay to determine the infectious titer. The AdMock (clone Ad. DL312) was prepared similarly. The AdRSVRII and AdMock had 2.0 x 10¹² and 2.7 x 10¹² viral particles/ml, respectively. The endotoxin levels in both preparations were <0.1 EU/ml.

Transduction of various cell types

Both primary cells and cell lines were grown in monolayer cultures for 48 h and washed twice with serum-free DMEM. The cells were then infected with 10³ multiplicity of infection (moi)/cell (unless mentioned otherwise) for 24 h at 37°C, followed by washing with serum-free DMEM twice. The cells were allowed to grow in low serum-containing medium (0.5% FBS) for another 24 h. The cells were then stimulated with recombinant human IL-1 (10 ng/ml) for 24–72 h.

Immunostaining of IL-1RII

Primary cultures (5,000–10,000 cells/well) of human and bovine chondrocytes and A549 epithelial cells were grown in four-well chamber slides (Fisher, Pittsburgh, PA) for 48 h. The cultures were transduced with either AdMock or AdRSVRII (10³ moi/cell) as described above. Twenty-four hours after transduced cells were fixed with 2% paraformaldehyde in PBS containing 0.1% BSA (pH 7.0), for 20 min at 4°C and washed twice with PBS before permeabilizing the cells with 0.2% saponin in PBS for 20 min at 4°C. The cells were washed thoroughly with PBS twice, treated with 10 µg/ml anti-IL-1RII Abs (M25), and counterstained with Hoechst stain (Molecular Probes, Eugene, OR) for 20 min at 4°C. The cells were washed twice and stained with goat anti-rat Texas Red (10 µg/ml)-labeled secondary Ab (Molecular Probes) for 20 min at 4°C. The cells were washed and fixed with 0.4% paraformaldehyde. Conventional brightfield and fluorescence microscopies were performed on a Zeiss Axiphot microscope (New York, NY). The cells were observed at an excitation wavelength of 405 nm for Hoechst stain and at 620 nm for Texas Red. The images were captured using MetaPhor imaging system 4.5.

Proteoglycan synthesis

Chondrocytes immobilized in alginate beads were incubated in Ham’s F-12 culture medium (supplemented with L-glutamine (2 mM), gentamicin (50 µg/ml), amphotericin B (0.25 µg/ml), heat-inactivated FBS (0.5%), and ascorbic acid (25 µg/ml)) with 10 µCi/ml sodium sulfate (Na₂³⁵SO₄) for 4 h at 37°C in a 5% CO₂ atmosphere. Alginate beads were washed five times with 0.15 M NaCl and solubilized in 0.5 ml of Soluene 350 (Packard Instrument, Downers Grove, IL) in scintillation counting tubes. After addition of 4.5 ml of liquid scintillation counting fluid (Hionic Fluor; Packard Instrument) the ³⁵S-labeled proteoglycan content was measured using a Beckman LS 7000 counter (Beckman, Palo Alto, CA) (6).

Isolation of RNA from OA-affected cartilage

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-ME for 4 h on a rocker (10). It was then extracted with water-saturated phenol, followed by phenol and chloroform. The aqueous layer was layered onto cesium trifluoroacetate gradient for ultracentrifugation (24,000 rpm/24 h). The RNA pellet was dissolved in guanidium thiocyanate and was precipitated with alcohol in the presence of acetic acid. The RNA pellet was resuspended in a total volume of 50 µl of diethylpyrocarbonate-treated water.

RT-PCR analysis of IL-1

Total RNA from A549 epithelial cells was isolated using TRIreagent (MRC, Cincinnati, OH) as described previously (6). The total RNA was treated with DNase to remove contaminating genomic DNA and was purified using a Qiagen RNeasy Minicolumn (Qiagen, Valencia, CA). Five micrograms of total RNA isolated from A549 cells was used for first-strand cDNA synthesis using the Superscript Reverse Transcriptase II system (Life Technologies) and PCR analysis. The sense primer 5'-GCGCCTGGTCACCAGGGCTGC-3' and the antisense primer 5'-GGATCTCGCTCCTGGAAGATC-3' were used for amplification of GAPDH (for 30 cycles). PCR analysis of IL-1 was performed using the sense primer 5'- ATGGCAGAAGTACCTAAGCTCGC-3' and the antisense primer 5'-ACACAAATTGCATGGTGAAGTCAGTT-3', and PCR of IL-1RII was preformed using sense primer 5'-CGGGATCCATGTTGCGCTTGTACGTGT-3' and the antisense primer 5'-TAAAGCGGCCGCTCACTTGGGATAGAATTG-3' in a PerkinElmer thermal cycler (94°C for 30 s, 60°C for 1 min, and 72°C for 2 min for 30 cycles). The IL-1 (800 bp) and IL-1RII (1200 bp) fragments were cloned and sequenced as previously reported (6). The signal was quantitated as previously described using a densitometer (Molecular Dynamics, Piscataway, NJ). For RT-PCR control, cDNA synthesis was conducted (from total RNA isolated from AdMock- or AdRSVRII-treated cells) without the addition of reverse transcriptase.

Real-time PCR

cDNA synthesis using Superscript II was conducted according to the manufacturer’s protocol (Life Technologies) from 0.25 µg of DNase-treated total RNA. Primers and probes for IL-1 (X56087) were designed using the primer express algorithm (PE Applied Biosystems, Foster City, CA). Real-time PCR was conducted using fluorogenic probes for quantification. The forward and reverse primers were 5'-ATTTGAGTCTGCCCAGTTCCC-3' and 5'-TCAGTTATATCCTGGCCGCCT-3', respectively, and the fluorogenic probe was 5'-Fam-ACCTCTCAAGCAGAAAACATGCCCGTCT-Tamra-3'. The PCR was conducted using AmpliTaq Gold DNA polymerase according to the manufacturer’s protocol (PE Applied Biosystems). Human genomic DNA (Clontech Laboratories, Palo Alto, CA) was used as a standard, and the number of copies of cDNA in the samples were calculated from the intensity of the fluorescence.

Determination of PGE₂ and nitrite

PGE₂ was determined in the culture supernatant using an RIA, as reported previously, 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 (10). The values were expressed as micromolar concentrations of nitrite or nanograms per milliliter of PGE₂ released per 100 mg wet weight of cartilage or 5 x 10⁵ chondrocytes.

Statistics

Data are expressed as the mean ± SD. Each value is the mean of at least three samples. The results were analyzed using Student’s t test. Differences with p 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lack of expression of IL-1RII in human chondrocytes and epithelial cells

We have recently shown, using gene expression arrays, that human cartilage and epithelial cells lack the expression of endogenous IL-1-antagonizing molecules such as IL-1RII and IL-1Ra (6, 14). Both cell types play a profound role in the inflammation and pathophysiology of arthritis, thus making the cartilage and joints susceptible to low concentrations of endogenous IL-1.

Regulation of IL-1 in human cartilage

Total RNA was isolated directly from human normal and OA-affected cartilage to quantitate the expression of mRNA for IL-1 by real-time PCR. Fig. 1A shows low expression of mRNA transcripts of IL-1 in normal cartilage and a 10-fold up-regulation in human OA-affected cartilage. Incubation of normal and OA-affected cartilage in ex vivo showed spontaneous production of IL-1. There was at least 10-fold up-regulation of IL-1 protein in OA-affected cartilage (Fig. 1B) compared with normal cartilage, similar to that observed at the level of IL-1 mRNA expression.

View larger version (53K): [in this window] [in a new window]   FIGURE 1. A and B, Expression of IL-1 mRNA and IL-1 protein in normal and OA-affected cartilage. Total RNA was extracted from cartilage as described in Materials and Methods. Equal amounts of DNase-treated total RNA was used for the estimation of mRNA transcripts. cDNA of normal and OA-affected cartilage were PCR-amplified in triplicate using the TaqMan method (50 ). Fluorescence was measured during cycling reactions (increase in fluorescence subtracted from the baseline fluorescence signal) was plotted against the cycle number, as shown in A. The mRNA transcripts were estimated using specific primers (PDARs, fluoresochromes). The transcript levels were normalized with GAPDH, as shown in B. Human normal and OA cartilage (10 patients) were incubated in ex vivo conditions in triplicate as described in Materials and Methods. IL-1 protein released in ex vivo was estimated by ELISA at 48 h. The data represent one of two similar experiments. The p values were compared between normal and OA-affected cartilage. n = 3. **, p 0.01. C–E, Expression and production of adenovirus-mediated IL-1 type II receptor in various cell types. Human (C) and bovine (D) chondrocytes and human epithelial cells (E) were transduced with AdMock or AdRSVRII as described in Materials and Methods. Cells were stained with rat-antiIL-1RII Abs (M25) followed by goat anti-rat Texas Red-labeled secondary Ab (shown in red). The cells were counterstained with Hoechst nuclear stain, which is shown in blue or green. Soluble IL-1RII was estimated from spent medium 48 h posttransduction using ELISA. The data represent one of two similar experiments and the mean sIL-1RII protein in picograms per milliliter of triplicate determinations in culture supernatant.

Reconstitution of IL-1RII in various IL-1RII^- cell types

The human IL-1RII was cloned from human neutrophils as previously reported (4). IL-1RII-expressing adenovirus (AdRSVRII) was prepared, as described in Materials and Methods. Human and bovine chondrocytes as well as epithelial cells were transduced with the mock virus (AdMock) and stained with Abs to IL-1RII as shown in the upper panel of Fig. 1, C–E. These cells lacked the expression of intracellular IL-1RII and demonstrated only nuclear staining. As expected, there was an undetectable amount of sIL-1RII in cell supernatants of mock-transduced cells. Cells transduced with the AdRSVRII showed significant expression of intracellular IL-1RII in >90% of the cells. These cells also spontaneously released a significant amount of sIL-1RII in the medium. The production of sIL-1RII in human OA-affected chondrocytes was significantly higher than in bovine chondrocytes and epithelial cells; this observation was made consistently in this study.

Regulation of NO and PGE₂ production by IL-1RII⁺ human OA-affected chondrocytes and synovial cells

The biological activity of rIL-1RII was initially tested in primary IL-1RII⁺ human chondrocytes and synovial cells. Nontransduced OA-affected chondrocytes and synovial cells showed spontaneous production of PGE₂ as previously reported (1).

Transduction of chondrocytes with the AdMock virus demonstrated the presence of IL-1RII in the medium similar to spontaneous production of PGE₂. Transduction of these cells with adenovirus (AdRSVRII; at moi of 100, 1,000, and 10,000) released sIL-RII at concentrations of 60 ± 10, 594 ± 100, and 630 ± 70 pg/ml, respectively, in the supernatant (Fig. 2). Stimulation of untransduced cells with IL-1 showed a significant augmentation in the accumulation of PGE₂. Human chondrocytes transduced with AdRSVRII showed inhibition of IL-1-induced PGE₂ accumulation. These experiments show that transfection of cells at an moi of 100 (which released 60 pg/ml sIL-1RII) was sufficient to significantly inhibit the activity of 10 ng/ml human IL-1.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Transduction of human chondrocytes and synovial fibroblasts with AdRSVRII inhibited IL-1-mediated PGE2 production. Human chondrocytes or synovial cells isolated from OA-affected cartilage or joints, respectively, were seeded in 24-well plates at a density of 10,000 cells/well. The cells were allowed to grow for 48 h and were transduced with both AdMock (103) and AdRSVRII (102, 103, and 104 moi/cells). The sIL-1RII was estimated in the supernatant 24 h posttransduction by ELISA. The cells were stimulated with 10 ng/ml human IL-1, and PGE2 production was followed 48 h poststimulation. The data represent one of three similar experiments. Values shown represent the mean sIL-1RII protein (picograms per milliliter) in triplicate culture supernatants. The p values were compared with IL-1-stimulated cells. n = 3. *, p 0.05; **, p 0.01

Regulation of IL-6 and IL-8 production by human IL-1RII⁺ human epithelial cells

Previous studies have shown that IL-1 induces the production of IL-6 and IL-8 in A549 cells (14). We also compared the sensitivity of IL-1RII⁺ A549 epithelial cells to that of nontransduced cells with respect to the production of IL-6 and IL-8. AdMock-transduced cells showed a significant increase in the production of IL-6 (from 96 ± 36 to 3127 ± 504 pg/ml) and IL-8 (from 63 ± 41 to 1335 ± 300 pg/ml) when treated with IL-1 for 24 h. As expected, there was also a significant increase in the induction of IL-6 (2000–3000 pg) and IL-8 (1500–1800 pg) in nontransduced cells in the presence of IL-1. AdRSVRII-transduced cells showed significant inhibition (p < 0.001) of IL-1-induced IL-6 (down to 952 ± 354 pg/ml) and IL-8 (down to 228 ± 106 pg/ml) production in the same experiment. These experiments show that multiple inflammatory mediators, which are induced by IL-1, can be inhibited in IL-1RII⁺ A549 cells.

Regulation of PGE₂ production in human IL-1RII⁺ murine fibroblasts

We also tested the biological activity of human IL-1RII in murine cells. Murine fibroblast cells that were transduced with moi of 100 and 1000 viral particles of AdRSVRII released 574 ± 70 and 1685 ± 50 pg sIL-1RII in the medium. Exposure of the nontransduced cells to IL-1 induced 25 ± 2 ng/ml PGE₂. IL-1RII⁺ cells exposed to human IL-1 (10 ng/ml) showed significant inhibition (by 25–50%) of PGE₂ production compared with IL-1RII^- cells (data not shown).

Specificity of IL-1RII in bovine chondrocytes, rabbit synovial cells, and human A549 epithelial cells

We examined the ability of IL-1RII to neutralize the effects of IL-1 and TNF- in various cell types. Bovine chondrocytes respond to both human IL-1 and TNF- (6). As previously observed with the human chondrocytes, AdMock-transduced bovine chondrocytes did not show any effect on the expression of NO and PGE₂ (Fig. 3). However, cells that were stimulated with IL-1 or TNF- showed equivalent amounts of up-regulation of both NO and PGE₂ after 72 h in culture. Bovine chondrocytes transduced with AdRSVRII were significantly resistant to the effects of IL-1, but not TNF-. This suggests that human IL-1RII is not only biologically active in bovine cells, but is specific for inhibiting IL-1-induced, and not TNF--induced, NO and PGE₂ expression.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Regulation of NO and PGE2 by IL-1 and TNF in genetically modified bovine chondrocytes. Primary bovine chondrocytes were grown in monolayer culture in 24-well plates for 48 h and transduced with both AdMock and AdRSVRII virus. The sIL-1RII was estimated (picograms per milliliter) in triplicate in the supernatant 24 h after transduction. The cells were stimulated with 10 ng/ml human IL-1 or 1000 U TNF-. PGE2 and NO production were estimated 72 h poststimulation. The data represent one of two similar experiments and are the mean ± SD (n = 3). The p values were compared with IL-1- or TNF--stimulated cells. **, p 0.01.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Regulation of PGE2 by IL-1 and TNF in genetically modified rabbit synovial cells (HIG82) and human epithelial cells (A549). Cells were grown in monolayer and transduced with either AdMock or AdRSVRII virus. The sIL-1RII (picograms per milliliter) was estimated in the supernatant 24 h after transduction as shown. The cells were stimulated with 10 ng/ml human IL-1 or 1000 U TNF-. PGE2 production was estimated from the supernatant 72 h poststimulation. The data represent one of two similar experiments and are the mean ± SD (n = 3). The p values were compared with IL-1- or TNF--stimulated cells. **, p 0.01.

Regulation of IL-1 mRNA by IL-1RII

Recent studies have shown that the production of inflammatory mediators such as NO, PGE₂, IL-6, and MMP in arthritis-affected chondrocytes is induced by autocrine production of IL-1 (4, 16, 17). In view of these observations, we examined the induction of IL-1 mRNA by IL-1 (5–10 ng/ml) in IL-1RII⁺ cells. IL-1RII^+/- A549 human epithelial cells were exposed to IL-1 for 4 h and analyzed for IL-1 mRNA by RT-PCR analysis (Fig. 5). IL-1 induced IL-1 mRNA in nontransduced and mock virus-transduced cells. However, the induction of IL-1 mRNA in IL-1RII⁺ cells (which accumulated 1 ng/ml sIL-1RII) was significantly inhibited (70%). These experiments suggest that transgene expression of sIL-1RII "sponges" the IL-1. Furthermore, the membrane form of IL-1RII (which lacks a cytoplasmic tail) may compete with IL-1RI with respect to signal transduction apparatus and molecules, and subsequent induction of IL-1 mRNA.

View larger version (68K): [in this window] [in a new window]   FIGURE 5. Regulation of IL-1 transcription by IL-1RII+/- in A549 epithelial cells. A549 cells were transduced with AdMock and AdRSVRII as described in Materials and Methods. After 24 h transduction the sIL-1RII from the supernatant was estimated by ELISA and induced with IL-1 (10 ng/ml) for an additional 24 h in low serum (0.5%) medium. Total RNA was isolated and used for cDNA synthesis. PCR analysis of IL-1RII, IL-1, and GAPDH and quantitation was performed as described in Materials and Methods. STD, molecular size standards. The data represent one of two similar experiments.

To dissect the role of soluble and membrane IL-1RII, bovine chondrocytes were transduced with AdMock and AdRSVRII for 24 h. The cells were washed and resuspended in fresh medium with and without various concentrations of sIL-1RII. The cells were then induced with 10 ng/ml IL-1 as shown in Fig. 6. Levels of NO, PGE₂, and sIL-1RII were estimated at 72 h after IL-1 stimulation. Untransduced and AdMock-transduced cells showed up-regulation of NO and PGE₂ by IL-1. Cells stimulated with 10 ng IL-1 and incubated with 1–10 ng/ml sIL-1RII showed a dose-dependent inhibition of IL-1-induced NO and PGE₂ production. However, IL-1RII-transduced cells, which accumulated 80–100 pg/ml sIL-1RII in the medium under identical experimental conditions showed significant inhibition of NO and PGE₂ production, similar to cells treated with 10 ng/ml purified sIL-1RII protein. These experiments suggest that in IL-1RII⁺ cells, the membrane (or soluble) form of IL-1RII (independently or in conjunction) has a profound impact on blocking IL-1 signaling.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Comparison of cell surface IL-1RII and sIL-1RII in the regulation of inflammatory mediators in bovine chondrocytes. Primary chondrocytes were grown in monolayer culture and transduced with AdMock or AdRSVRII virus. The sIL-1RII was estimated in the supernatant 24 h after transduction (n = 3). The cells were also incubated with different concentrations of sIL-1RII (1–10 ng) and stimulated with IL-1 (10 ng/ml). NO and PGE2 production was estimated 72 h poststimulation. The data represent the mean ± SD (n = 3). The p values were compared with IL-1 stimulated cells. *, p 0.05; **, p 0.01. The data represent one of two similar experiments.

The paracrine effects of inflammatory mediators by the synovium have detrimental effects on cartilage in OA and RA. To examine the paracrine effects of sIL-1RII, we transduced primary human synovial cells with AdMock and AdRSVRII and allowed them to attach to the surface of the plate for 24 h. Human OA-affected cartilage was placed in buoyant chamber inserts that were separated from the synovial cells by 1–1.5 cm. The accumulation of PGE₂ (by cartilage and synovial cells) was estimated at the end of 72 h in the presence and the absence of IL-1 (Fig. 7). Cartilage incubated with IL-1RII⁺ synovial cells (but not IL-1RII^- cells) showed inhibition of spontaneous and IL-1-mediated production of PGE₂.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. Regulation of NO and PGE2 in cocultures of synovial cells and OA-affected cartilage. Primary synovial fibroblast isolated from OA patients undergoing joint replacement surgery were grown as monolayer culture and transduced with either AdMock or AdRSVRII virus. The sIL-1RII was estimated 24 h posttransduction. The OA cartilage, which was cut into small pieces, was placed in cell culture inserts with a pore size 0.2 µm and inserted into 24-well plates. The coculture was stimulated in the absence and the presence of IL-1 (1 ng/ml). NO and PGE2 were estimated 72 h poststimulation. The data represent the mean ± SD (n = 3). The p values were compared with uninduced or IL-1-stimulated cultures. *, p 0.05; **, p 0.01. The data represent one of two similar experiments.

Cartilage samples were also labeled with ³⁵S to examine proteoglycan synthesis induced by IL-1. The experiments show a decrease in proteoglycan synthesis by IL-1, which could be reversed by paracrine sIL-1RII released by IL-1RII-transduced synovial cells. These experiments also show that IL-1RII⁺ synovial cells released sIL-1RII, which protected the cartilage against the insults of IL-1.

Regulation of matrix synthesis influencing factors and proteoglycan synthesis in IL-1RII-transduced bovine chondrocytes

Previous studies have shown that IL-1, NO, and PGE₂ influence matrix (collagen and proteoglycan) homeostasis directly and indirectly in cartilage (18). We also examined the regulation of NO, PGE₂, and proteoglycan synthesis in the presence of IL-1 in IL-1RII^- and IL-1RII⁺ bovine chondrocytes (Fig. 8). The chondrocytes were immobilized in Ca²⁺ alginate beads to maintain their dedifferentiated phenotypes and matrix synthesis in long-term (>7-day) cultures (19, 20). Addition of IL-1 induced NO and PGE₂ in control and AdMock-transduced immobilized cells. Estimation of NO and PGE₂ in IL-1RII⁺ with or without IL-1 cell supernatants showed that immobilized bovine chondrocytes behaved similar to the short-term monolayer cultures with respect to susceptibility to IL-1, as shown in Fig. 5. Addition of IL-1 to chondrocytes showed a significant decrease in the incorporation of ³⁵S into proteoglycans compared with uninduced and AdMock-transduced cells as previously reported (6). In summary, these experiments showed that IL-1RII⁺ chondrocytes are resistant to direct and indirect factors (induced by IL-1) that influence cartilage homeostasis.

View larger version (20K): [in this window] [in a new window]   FIGURE 8. Regulation of NO, PGE2, and proteoglycan synthesis in bovine chondrocytes encapsulated in alginate beads. Primary bovine chondrocytes transduced with either AdMock or AdRSVRII virus were encapsulated in alginate beads (40,000 cells/bead). Five or six beads per well in 24-well plates were incubated in the absence or the presence of IL-1 (10 ng/ml). NO, PGE2 (as shown in the upper panel), and PG synthesis were estimated 72 h poststimulation. The data represent the mean ± SD (n = 3). The p values were compared with IL-1- or IL-1 AdMock-stimulated cells. *, p 0.05; **, p 0.01.

Previous studies have shown that autologous transplantation of chondrocytes in vivo and in vitro has a significant effect on cartilage repair (21). We have modified the approach by transplanting genetically modified IL-1RII⁺ chondrocytes in vitro. Human OA-affected cartilage obtained from surgery was divided into two parts. One part was used to isolate chondrocytes that were transduced with AdMock or AdRSVRII. The IL-1RII AdMock- or AdRSVRII-transfected chondrocytes were then transplanted onto the other piece of cartilage that was incubated in F-12 medium as described in Materials and Methods. Cartilage chips transplanted with IL-1RII^- cells showed spontaneous production of NO, PGE₂, IL-6, and IL-8, which could be augmented by IL-1 (Table I). Cartilage chips transplanted with IL-1RII⁺ cells showed inhibition of spontaneous production of NO, PGE₂, IL-6, and IL-8, which may be due to autocrine production of IL-1 by the OA cartilage. Furthermore, there was also inhibition of exogenously added IL-1-induced NO, PGE₂, IL-6, and IL-8 in cartilage slices transplanted with IL-1RII⁺ cells compared with IL-1RII^- cells. In a similar experiment (as shown in Fig. 8), IL-1 inhibited proteoglycan synthesis in OA cartilage slices resurfaced with AdMock-transduced chondrocytes. OA-affected cartilage that was resurfaced with AdRSVRII-transduced chondrocytes significantly inhibited IL-1-induced inhibition of proteoglycan synthase.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The IL-1 family of proteins, including its receptor and antagonist, exhibit complex regulations in biological systems. The recent identification of closely related ligands that also bind to the IL-1R contributes to the complexity of this family of proteins. These include new receptor antagonists for IL-1, such as IL-1HY1 and IL-1HY2, and other novel ligands, such as IL-1H1-H4 (22, 23, 24, 25). IL-1 activity, which is usually imparted by IL-1 and IL-1 via the IL-1R, seems to be a tightly regulated process. This also involves a balance of IL-1 accessory proteins and IL-1 antagonist, which are mainly IL-1RII and IL-1Ra (26). Some intriguing observations suggest that soluble IL-1RI, which we have shown to have IL-1 antagonist activity in human OA cartilage (6), also induces intracellular signaling by a phenomenon called reverse signaling by binding membrane IL-1 (27).

Among various antagonist molecules, the IL-1 type II decoy receptor is of interest due to its up-regulation under various pathophysiological conditions, such as sepsis, meningococcal infections, Alzheimer’s disease, and anorexia (26, 28). This receptor also shows decreased expression under various pathophysiological conditions, including human endometriosis, osteoporosis, and OA, where IL-1 plays a major role in the disease process (29, 30). Furthermore, it has been shown to be up-regulated during pharmacological intervention with aspirin, dexamethasone, and estrogen therapy, which change the balance of the effects of IL-1 in pathophysiologic conditions.

Human OA-affected cartilage shows 10-fold up-regulation of IL-1 mRNA (by real-time PCR) and proteins (by ELISA) compared with normal cartilage, as it is quite possible that the up-regulation of IL-1 mRNA and protein could be a result of IL-1 gene polymorphisms (31). Recently, chondrocytes in the superficial and medial zone in OA cartilage have been reported to be positive for IL-1 and TNF- compared with normal cartilage using immunohistologic methods (32). We have also shown functional up-regulation of TNF- and TNF- convertase activity in human OA- and RA-affected cartilage compared with normal (33). Although both IL-1 and TNF- are up-regulated in OA cartilage, the precise role of TNF- in cartilage homeostasis remains elusive, although they share many biological activities. This observation is strengthened by the following observations: 1) the spontaneous occurrence of arthritis in TNF--transgenic mice can be prevented by a blockade of IL-1RI (34); 2) although both IL-1- and TNF--transgenic mice show spontaneous occurrence of arthritis, the rapid destruction of cartilage is more pronounced in IL-1-transgenic mice than in TNF--transgenic mice (35); 3) in the animal model of CIA, Joosten et al. (36), has shown that blockage of IL-1 and is more effective (than anti-TNF-) in decreasing cartilage and bone destruction (36); and 4) spontaneous production of inflammatory mediators by human OA-affected cartilage in ex vivo could be inhibited by soluble IL-1R, but not soluble TNF receptor (6, 16). In the present study we have advanced this observation by neutralizing the effects of IL-1 by IL-1RII as an antagonist molecule.

The reconstitution of IL-1RII by recombinant adenovirus in human and bovine chondrocytes, synovial cells, and epithelial cells that lack IL-1RII led to the functional expression of both membrane and soluble IL-1RII proteins, thus suggesting that the defect in IL-1RII expression in these cells may be at the level of the gene.

The up-regulation of relatively low concentrations of IL-1 in conjunction with unopposing IL-1 antagonizing molecules can exacerbate the disease process in long-term diseases such as OA. This hypothesis is further supported by a recent study in which IL-1Ra knockout mice spontaneously develop arthritis (37). Overexpression of IL-1Ra transgene in mice also significantly reduces inflammation (38). Additionally, mice injected with IL-1Ra or IL-1RII had a significant decrease in the early onset of Ag-induced arthritis with a decrease in inflammation (39, 40).

In summary, our studies suggest that IL-1RII⁺ cells were immune to the insults of IL-1 with respect to the production of PGE₂, NO, IL-6, and IL-8, which have been implicated in inflammation and cartilage destruction. The human IL-1RII, unlike IL-1Ra, was biologically active across species (human, bovine, rodent, and rabbit), although there is only 59% amino acid identity between the human IL-1RII and other species. Furthermore, human IL-1RII derived from neutrophils was active in various cell types, including chondrocytes, synovial cells, and epithelial cells, which were defective in IL-1RII expression.

We also examined the specificity of IL-1RII with respect to inhibition of IL-1 and TNF- effects in chondrocytes, synovial cells, and epithelial cells. As discussed above, although exogenous IL-1 and TNF- induces the common inflammatory mediators such as NO, PGE₂, IL-6, and IL-8 in OA chondrocytes (in vitro), the spontaneous production of these inflammatory mediators in human OA-affected cartilage (ex vivo) is mediated predominantly by IL-1. IL-1RII exclusively inhibited IL-1 effects in chondrocytes, but inhibited IL-1- and TNF--induced PGE₂ production in epithelial and synovial cells. This may be due to the ability of TNF- to induce autocrine IL-1 in these cell types. The important role of TNF- in the pathogenesis of human RA synovium is well documented, where anti-TNF- therapy down-regulated not only TNF- expression, but also IL-1 and IL-1 in the synovium (41). The possibility of IL-1RII directly modulating TNF--induced responses cannot be ruled out in the long term.

Overall, our present study shows that adenovirus-mediated expression of IL-1RII decoy receptor was significantly effective in neutralizing the effects of IL-1 in various cell types.

The consensus for anti-inflammatory gene therapy approaches for arthritis is that local expression of therapeutic proteins by transduced chondrocytes or synovial cells would circumvent the problem associated with systemic delivery to joints. The coculture and transplantation experiments designed in this study allowed interaction of both chondrocyte/synovial and chondrocyte/chondrocyte cells to examine the effects of autocrine and paracrine mediators in in vivo-like conditions compared with monolayer cultures. Our studies suggest that sIL-1RII released by synovial cells not only inhibits the autocrine induction of NO and PGE₂ by IL-1 in the synovial cells, but also in a paracrine fashion inhibits the spontaneous production of NO and PGE₂ in OA cartilage. Therefore, the strategy to directly transduce synovial cells in joints, as described for IL-1Ra gene therapy (42), may also be a viable strategy for IL-1RII gene therapy.

We also examined the effects of transplanted IL-1RII⁺ chondrocytes in human cartilage. These experiments suggest that cells transduced with this receptor continued to express IL-1RII for at least 2 wk (in ex vivo) and protected the cartilage against the effects of IL-1 with respect to the production of NO and PGE₂ and imbalancing proteoglycan synthesis. The cartisel approach (trade name by Genzyme, Cambridge, MA) of transplantation of autologous chondrocytes proprogated in vitro and transplanted into the same joint has been a feasible approach for cartilage repair in sports medicine (43). IL-1RII⁺ chondrocytes (as a modified cartisel approach) may be significantly effective for cartilage repair when the transplanted chondrocytes and those in its vicinity may be protected against the insults of IL-1. This is further supported by the observation showing that transgene mice overexpressing IL-1RII in keratinocytes had a predominant effect locally and did not influence systemic IL-1 responses (44).

There are several reports in the literature that adenovirus may induce an inflammatory and/or immune response in cells. Transduction of the mock virus in chondrocytes in this study did not show any significant increase in inflammatory mediators such as NO, PGE₂, IL-6, and IL-8. However, the possibility of an immune response to adenovirus in vivo cannot be ruled out. This can be avoided by using adeno-associated virus as a vector for future in vivo studies (45).

These experiments also give insight into the mechanism of action of IL-1RII in joint cells. The potent IL-1-neutralizing property may be due to the multifunctional effects of IL-1RII compared with IL-1RI. IL-1 inhibitory functions of IL-1RII have been demonstrated for the soluble and membrane forms of the receptor. This study shows similar observations, where the presence of both soluble and membrane forms of IL-1RII significantly inhibited IL-1 effects vs the soluble form alone. Furthermore, our studies suggest that the IL-1RII expressed in these cells was at least 2 logs more potent in its ability to inhibit the effects of IL-1 compared with the soluble receptor. This may be due to various possibilities: 1) intracellular IL-1RII blocks induction of IL-1 mRNA and subsequent autocrine effects of IL-1; 2) intracellular cytosolic pro-IL-1 complexes with IL-1RII and results in inhibited secretion of IL-1 (46); 3) IL-1RII has a higher affinity for IL-1 than IL-1RI (47); and 4) IL-1 and IL-1RII complex competes for IL-1 accessory protein, which is required for IL-1RI signaling (48, 49). These multiple effects of intracellular IL-1RII may reverse the autocrine loop involving induction of IL-1 by IL-1 itself.

	Acknowledgments

 
We thank Andrea L. Barrett for preparation of this manuscript, Sonali Trivedi for preparation of some of the figures, and Dr. Smita Palejwala for critically reviewing the manuscript. We also thank Dr. Leslie Ballou (Vanderbilt University, Memphis, TN) for providing us with the mouse fibroblast cells. We thank Immunex Corp. for the generous gift of (M25) receptor Abs, and National Disease Research Interchange (Philadelphia, PA) and the Anatomic Gift Foundation (Laurel, MD) for some of thecartilage samples.

	Footnotes

 
¹ A.R.A. is the recipient of the Young Investigator Award from the Arthritis Foundation, New York Chapter.

² Address correspondence and reprint requests to Dr. Ashok R. Amin, Department of Rheumatology and Medicine, Laboratory for Functional and Pharmacogenomics, New York University School of Medicine and Hospital for Joint Diseases, 301 East 17th Street, Room 1600, New York, NY 10003. E-mail address: amina01{at}popmail.med.nyu.edu

³ Abbreviations used in this paper: OA, osteoarthritis; COX-2, cyclooxygenase 2; hIL-1, human IL-1; sIL-1RII, soluble type II IL-1R; IL-1Ra, IL-1R antagonist; IL-1RI, type I IL-1R; IL-1RII, type II IL-1R; IL-1RII⁺, type II IL-1R-transduced cells; IL-1RII^-, Admock-transduced cells; moi, multiplicity of infection; RA, rheumatoid arthritis; RSV, Rous sarcoma virus.

Received for publication May 31, 2001. Accepted for publication December 11, 2001.

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