PMC:17821 / 16673-30249 JSONTXT

{"project":"2_test","denotations":[{"id":"11056679-9739057-4381687","span":{"begin":221,"end":223},"obj":"9739057"},{"id":"11056679-4111070-4381688","span":{"begin":878,"end":880},"obj":"4111070"},{"id":"11056679-1605310-4381689","span":{"begin":1441,"end":1443},"obj":"1605310"},{"id":"11056679-3052092-4381690","span":{"begin":2450,"end":2452},"obj":"3052092"},{"id":"11056679-90108-4381691","span":{"begin":4242,"end":4244},"obj":"90108"},{"id":"11056679-6501574-4381692","span":{"begin":7410,"end":7412},"obj":"6501574"},{"id":"11056679-3494788-4381693","span":{"begin":7866,"end":7868},"obj":"3494788"},{"id":"11056679-8594005-4381694","span":{"begin":9258,"end":9260},"obj":"8594005"}],"text":"Materials and methods\n\nAnimals\nMale and female C57BL/6 mice were obtained from Jackson (Bar Harbor, Maine, USA). DBA/1 mice were obtained from Bomholdgard (Rye, Denmark). FcR γ-chain-/- mice were kindly given by T Saito [10]. FcR γ-chain-/- mice were backcrossed to C57BL/6 mice for 12 generations. FcR γ-chain-/- were also backcrossed to a DBA/1 background. Mice were fed a standard diet and tap water ad libitum. Mice were used between the ages of 8 and 12 weeks and weighed 25–30 g.\n\nChemicals\nPoly-L-lysine (PLL), lysozyme, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), and zymosan A (from Saccharomyces cerevisiae) were obtained from Sigma Chemical Company, St Louis, MO, USA. N,N-dimethyl-1,3-propanediamine (DMPA) was obtained from BDH Chemicals Ltd, Poole, UK.\n\nLysozyme coupling to PLL\nLysozyme was coupled to PLL in accordance with the method of Danon et al [36]. As described by those authors, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide was used as an activator and PLL as a nucleophil. Free carboxyl groups of the protein were then coupled to amino groups of PLL. The molecular mass was raised whereas the isoelectric point remained high, as was determined in a 5% polyacrylamide slab gel with 0.8% ampholines (pH gradient 3.5-9.5). The molecular mass appeared to be 74 kD on SDS-PAGE.\n\nInduction of arthritis by immune complexes, zymosan, or SCW\nSpecific rabbit anti-lysozyme antisera, made as described elsewhere [35] and made complement-free by heating at 56°C for 30 min, were injected (0.2 ml) intravenously into mice. ICA was then induced by injecting 3 μg of PLL-coupled lysozyme into the right knee joint. The left knee joint was injected with saline solution and used as a control. As an additional control, some mice were given normal rabbit serum instead of specific anti-lysozyme. When PLL-lysozyme was injected into the knee joint without prior administration of specific anti-lysozyme antibodies, no inflammation or cartilage damage developed, in either C57BL/6 or DBA/1 mice.\nZymosan (30 mg) was dissolved in 1 ml saline solution by heating up to 100°C twice and was then sonicated to obtain a homogeneous suspension. Arthritis was induced in other mice by injecting 180 μg zymosan into both left and right knee joints. Fcγ-chain knockout mice and the control strain (C57BL/6) were matched for age and sex.\nSCW arthritis was induced by injecting 25 μg SCWs (rhamnose content), prepared as described elsewhere [37] and dissolved in 6 μl saline, into the right knee joint of C57BL/6 and DBA/1 mice.\n\nHistology\nTotal knee joints of mice were isolated 3 days after induction of arthritis in Fcγ-chain deficient mice and at days 3, 7, and 14 in C57BL/6 and DBA/1 mice. For standard histology, tissue was fixed in 4% formaldehyde, decalcified in formic acid, and subsequently dehydrated and embedded in paraffin. Paraffin sections were cut at 7 μm and mounted on gelatine-coated slides. Haematoxylin/eosin (H\u0026E) staining was performed to study the inflammatory cells.\nInfiltrate and exudate were scored separately. The severity was determined by two blinded observers, using an arbitrary score (0–3): 0 = none, 1 = mild, 2 = moderate, and 3 = maximal cellularity. To study PG depletion from the cartilage matrix, sections were stained with safranin O and then coun-terstained with fast green. PG depletion from several cartilage surfaces was scored (patella + adjacent femur surface, lateral condyle femur + adjacent surface tibia plateau, and medial femoral condyle + adjacent surface tibia plateau).\nThe severity of depletion was scored by two blinded observers, using an arbitrary score reflecting the level of destaining: 0 = none, 1 = mild, 2 = moderate, and 3 = maximal destaining of cartilage. Chondrocyte death was scored using H\u0026E-stained sections. The amount of empty lacunae was given as a percentage of total amount of cells (empty lacunae + viable chondrocytes). Cartilage erosion was scored by expressing the amount of eroded cartilage as a percentage of the cartilage surface.\n\nDetection of Fcγ receptors\nTo compare expression of FcγRII/RIII by DBA/1 and C57BL/6 mice, we performed immunohistochemistry using a rat antibody against murine FcγRII/RIII (2.4G2, Pharmin-gen, San Diego, CA, USA) [38]. Briefly, cryostat sections were fixed in acetone vapour for 3 min and endogenous peroxidase was blocked using 1% H2O2. Sections were incubated overnight with 2.4G2 (1 μg/ml) in phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA). Sections were washed in PBS and then incubated with rabbit anti-rat IgG coupled to biotin (Vector Laboratories, Burlingame, CA, USA). Then the sections were incubated with avidin biotin complexes (ABC; Vectastain Elite-kit, Vector Laboratories) and developed using diaminobenzidine (Sigma Chemical Company, St Louis, MO, USA).\n\nDetection of IgG and C3c\nIgG deposited in the tissue by immune-complex formation was detected on paraffin-embedded knee-joint sections. Sections were incubated with biotinylated rat anti-rabbit antibodies (Vector Laboratories) for 1 h. Sections were washed thoroughly with PBS, incubated with ABC complexes, and developed using the method described above.\nC3c was detected using cryostat sections. Sections were incubated with rat anti-murine C3c (Nordic, Tilburg, The Netherlands) after acetone fixation and inhibition of endogenous peroxidase. Isotype-matched antibodies directed against an irrelevant epitope (Vector) were used as a negative control. Staining was scored using image analysis by measuring the mean amount of blue light that passed through a well-defined location in the tissue section near the cruciate ligaments.\n\nAnalysis by fluorescence-activated cell sorter (FACS)\nPeritoneal macrophages of different strains were isolated from the peritoneal cavity by lavage with ice-cold DMEM/ 10% fetal calf serum (FCS)/1% pyruvate. These cells (5 × 106/100 μl) were incubated with 2.4G2 (5 μg/ml). They were then washed and incubated with mouse-adsorbed hamster anti-rat F(ab')2 fragments labelled withfluorescein isothiocyanate (FITC). FACS analysis was performed using a Coulter Epics XL/XL-MCL (Coulter Electronics Ltd, Mijdrecht, The Netherlands). Omitting the first antibody and substitution of 2.4G2 for isotype-matched irrelevant antibodies (DAKO, Glostrup, Denmark) were used as negative controls. The window was set so that \u003e95% of cells were F4/80-positive, indicating that \u003e95% of cells were macrophages.\n\nStimulation of peritoneal macrophages with heat-aggregated gamma globulin\nPeritoneal macrophages were isolated by peritoneal lavage using ice-cold DMEM/10% FCS. Cells were put into 24-well plates (Costar, Acton, MA, USA) at a concentration of 1 × 106 cells/ml. After a 4-day adjustment period, the culture medium was changed to one containing heat-aggregated gamma globulin (HAGG) at 100 μg/ml, or a control medium. After 24 or 48 h, the culture medium was collected. HAGGs were obtained by heating 10 mg/ml rabbit IgG (Sigma Chemicals, St Louis, MO, USA) to 63°C for 30 min. After heating, the solution was centrifuged at 12 000 \t\t\t\t\t\tg \t\t\t\t\t for 10 min. The concentration of the HAGG in the supernatant was determined by reading the absorbance at 280 nm.\n\nProduction of inflammatory mediators by synovial tissue\nSynovial tissue was isolated by dissection of patellar tendon and patellar plate containing the patella, tendons, and synovium, as described earlier [39]. Mediators were obtained by elution from synovial specimens derived from six knee joints in 2 ml Roswell Park Memorial Institute (RPMI) medium for 1 h at room temperature. Washouts were tested for their bioactive IL-1 levels in an IL-1-sensitive bioassay (NOB-1 assay) and in a radioimmunoassay (RIA) to determine the total protein levels.\n\nBioassay for IL-1\nIL-1 activity was measured in the one-stage bioassay for IL-1 as described by Gearing et al [40]. The assay was performed as a culture of the IL-1-specific thymoma cell EL-4, designated NOB-1, which produces IL-2 and IL-4 in response to IL-1, in co-culture with the lymphokine-responder CTLL line. Briefly, EL-4 cells were washed twice and resuspended at 2.5 × 105 cells per ml RPMI medium containing 5% FCS. The cells were distributed into 96-well microtitre plates at 2.5 × 104 cells per well in 100-μl volumes. CTLL cells (4 × 103) suspended in 50 μl RPMI medium were added, followed by appropriate dilutions of test sample to a final volume of 200 μl. After 20 h, 0.5 μCi 3H-thymidine (specific activity 20 Ci/mmol; Amersham Nederland BV, 's Hertogenbosch, The Netherlands) was added to each well. After a 3-h incubation, the contents were harvested, and incorporated activity was determined. The EL-4 line, from which NOB-1 was derived, does not incorporate thymidine, because it is deficient in thymidine kinase, and therefore the incorporation of 3H-thymidine is a measure of only CTLL proliferation. Maximal 3H-thymidine incorporation in the bioassay in the presence of IL-1 was between 15 000 and 20 000 cpm. Culture media contained only minor concentrations of IL-2 or IL-4, as was assessed by testing samples with CTLL alone.\n\nMeasurement of IL-1α and IL-1β protein levels\nIL-1 culture supernatants were measured in duplicate by a nonequilibrium RIA as described elsewhere [41]. Briefly, 100 μl polyclonal rabbit anti-murine IL-1α and IL-1β (diluted in RIA buffer, pH 7.4) was added to 100 μl of samples and standards and kept on ice. After vortexing, the tubes were incubated at 4°C. After 24 h, 100 μl of the appropriate 125I-labelled IL-1α and β containing approximately 10 000 cpm was added to each tube, and incubation was continued for a further 24 h at 4°C. RIA buffer (750 μl) containing 9% (w/v) polyethylene glycol 6000 (Merck Diagnostica, Darmstadt, Germany) and 3% (w/v) goat anti-rabbit serum was added, to separate bound and free tracer. The tubes were incubated for 20 min at room temperature. After centrifugation at 1500 × \t\t\t\t\t\tg \t\t\t\t\t for 15 min, supernatants were quickly drained on adsorbent paper. A gamma-counter was used to count the radioactivity remaining on the paper. The radioactivity in control tubes (the nonspecific binding activity) was subtracted from samples and standards. The detection limit of the assay was 20 pg/ml for both IL-1α and IL-1β.\n\nMeasurement of IL-1 receptor antagonist\nProtein levels of IL-1 receptor antagonist (IL-1Ra) in synovial washout specimens were detected using a specific sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, microtitre plates were coated with unconjugated first antibody (anti-IL1Ra: MAB480, R\u0026D Systems, Abingdon, UK) overnight at 4°C. Subsequently, wells were blocked using 1% BSA followed by a 3-h incubation with patella washouts at 37°C. Wells were then incubated with a biotinylated specific antibody (IL-1Ra: BAF480, R\u0026D Systems) and a signal enhancement step was performed by incubating with PolyHRP (CLB, Amsterdam, The Netherlands). Microtitre plates were developed using o-phenylene-diamine and optical density was read at 492 nm. Between all steps, a washing episode was included using PBS/0.1% Tween 20.\n\nSemiquantitative detection of mRNA using reverse-transcription polymerase chain reaction\nLevels of mRNA for four different cytokines/chemokines were detected using a semiquantitative method. In brief, mice were killed by cervical dislocation, immediately followed by dissection of the patella with adjacent synovial tissue. Six patellae per group were obtained. Two biopsies with a diameter of 3 mm were punched from the synovial tissue, using a biopsy punch (Stiefel, Wachtersbach, Germany): one from the medial and one from the lateral aspect of the patella. Three lateral and three medial biopsies were pooled (six total), to obtain two samples per group. The samples were immediately frozen in liquid nitrogen. Thereafter samples were ground to powder using a Micro-dismembrator II (B Braun, Melsungen, Germany) and total RNA was extracted using 1 ml TRIzol reagent.\nOne microgram of RNA was used for reverse-transcription polymerase chain reaction (RT-PCR). Reverse transcription of mRNA was done using oligoDT primers, and 1/20 of the cDNA was used in one amplification by polymerase chain reaction (PCR). PCR was performed at a final concentration of 200 μM dNTPs, each primer at 0.1 μM, and 1unit Taq polymerase (Life Technologies, Breda, The Netherlands) in standard PCR buffer. Message for glycer-aldehyde-3-phosphate dehydrogenase (GAPDH), IL-1β, IL-1Ra, MCP-1, and MIP-2 was amplified using specific primers. The primer sequences for IL-1β were: 5'-TTGACGGACCCCAAAAGATG-3' (sense) and 5'-AGAAGGTGCTCATGTCCTCA-3' (antisense), for IL-1ra: 5'-TGCTGGGGACCCTACAGTCAC-3' (sense) and 5'-GCAAGTGCATCATCGTTGTTC-3' (antisense), for MCP-1: 5'-CTCACCTGCTGCTACTCATTC-3' (sense) and 5'-GCATGAGGTGGTTGTGAAAA-3' (antisense) and for MIP-2: 5'-GCTGGCCACCAACCACCAGG-3' (sense) and 5'-AGCGAGGCACATCAGGTACG-3' (antisense). The PCR-reaction was paused after 20, 23, 26, 29, 32, 35, 38, 41, and 44 cycles at the very end of the extension phase (72°C) and 5-μl samples were taken. PCR products were separated on 1.6% agarose gel and stained with ethidium bromide. The results are presented as cycle number in which the first detectable amount of DNA appears on the agarose gel. Samples were compared after correction for GAPDH content for each individual sample, to rule out confounding by variation of cellularity in the biopsies.\n\nStatistical analysis\nDifferences between experimental groups were tested for significance using the Wilcoxon rank test. P values \u003c0.05 were considered significant.\n"}

{"project":"Colil","denotations":[{"id":"T38","span":{"begin":221,"end":223},"obj":"9739057"},{"id":"T39","span":{"begin":878,"end":880},"obj":"4111070"},{"id":"T40","span":{"begin":2450,"end":2452},"obj":"3052092"},{"id":"T41","span":{"begin":1441,"end":1443},"obj":"1605310"},{"id":"T42","span":{"begin":4242,"end":4244},"obj":"90108"},{"id":"T43","span":{"begin":7410,"end":7412},"obj":"6501574"},{"id":"T44","span":{"begin":7866,"end":7868},"obj":"3494788"},{"id":"T45","span":{"begin":9258,"end":9260},"obj":"8594005"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Materials and methods\n\nAnimals\nMale and female C57BL/6 mice were obtained from Jackson (Bar Harbor, Maine, USA). DBA/1 mice were obtained from Bomholdgard (Rye, Denmark). FcR γ-chain-/- mice were kindly given by T Saito [10]. FcR γ-chain-/- mice were backcrossed to C57BL/6 mice for 12 generations. FcR γ-chain-/- were also backcrossed to a DBA/1 background. Mice were fed a standard diet and tap water ad libitum. Mice were used between the ages of 8 and 12 weeks and weighed 25–30 g.\n\nChemicals\nPoly-L-lysine (PLL), lysozyme, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), and zymosan A (from Saccharomyces cerevisiae) were obtained from Sigma Chemical Company, St Louis, MO, USA. N,N-dimethyl-1,3-propanediamine (DMPA) was obtained from BDH Chemicals Ltd, Poole, UK.\n\nLysozyme coupling to PLL\nLysozyme was coupled to PLL in accordance with the method of Danon et al [36]. As described by those authors, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide was used as an activator and PLL as a nucleophil. Free carboxyl groups of the protein were then coupled to amino groups of PLL. The molecular mass was raised whereas the isoelectric point remained high, as was determined in a 5% polyacrylamide slab gel with 0.8% ampholines (pH gradient 3.5-9.5). The molecular mass appeared to be 74 kD on SDS-PAGE.\n\nInduction of arthritis by immune complexes, zymosan, or SCW\nSpecific rabbit anti-lysozyme antisera, made as described elsewhere [35] and made complement-free by heating at 56°C for 30 min, were injected (0.2 ml) intravenously into mice. ICA was then induced by injecting 3 μg of PLL-coupled lysozyme into the right knee joint. The left knee joint was injected with saline solution and used as a control. As an additional control, some mice were given normal rabbit serum instead of specific anti-lysozyme. When PLL-lysozyme was injected into the knee joint without prior administration of specific anti-lysozyme antibodies, no inflammation or cartilage damage developed, in either C57BL/6 or DBA/1 mice.\nZymosan (30 mg) was dissolved in 1 ml saline solution by heating up to 100°C twice and was then sonicated to obtain a homogeneous suspension. Arthritis was induced in other mice by injecting 180 μg zymosan into both left and right knee joints. Fcγ-chain knockout mice and the control strain (C57BL/6) were matched for age and sex.\nSCW arthritis was induced by injecting 25 μg SCWs (rhamnose content), prepared as described elsewhere [37] and dissolved in 6 μl saline, into the right knee joint of C57BL/6 and DBA/1 mice.\n\nHistology\nTotal knee joints of mice were isolated 3 days after induction of arthritis in Fcγ-chain deficient mice and at days 3, 7, and 14 in C57BL/6 and DBA/1 mice. For standard histology, tissue was fixed in 4% formaldehyde, decalcified in formic acid, and subsequently dehydrated and embedded in paraffin. Paraffin sections were cut at 7 μm and mounted on gelatine-coated slides. Haematoxylin/eosin (H\u0026E) staining was performed to study the inflammatory cells.\nInfiltrate and exudate were scored separately. The severity was determined by two blinded observers, using an arbitrary score (0–3): 0 = none, 1 = mild, 2 = moderate, and 3 = maximal cellularity. To study PG depletion from the cartilage matrix, sections were stained with safranin O and then coun-terstained with fast green. PG depletion from several cartilage surfaces was scored (patella + adjacent femur surface, lateral condyle femur + adjacent surface tibia plateau, and medial femoral condyle + adjacent surface tibia plateau).\nThe severity of depletion was scored by two blinded observers, using an arbitrary score reflecting the level of destaining: 0 = none, 1 = mild, 2 = moderate, and 3 = maximal destaining of cartilage. Chondrocyte death was scored using H\u0026E-stained sections. The amount of empty lacunae was given as a percentage of total amount of cells (empty lacunae + viable chondrocytes). Cartilage erosion was scored by expressing the amount of eroded cartilage as a percentage of the cartilage surface.\n\nDetection of Fcγ receptors\nTo compare expression of FcγRII/RIII by DBA/1 and C57BL/6 mice, we performed immunohistochemistry using a rat antibody against murine FcγRII/RIII (2.4G2, Pharmin-gen, San Diego, CA, USA) [38]. Briefly, cryostat sections were fixed in acetone vapour for 3 min and endogenous peroxidase was blocked using 1% H2O2. Sections were incubated overnight with 2.4G2 (1 μg/ml) in phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA). Sections were washed in PBS and then incubated with rabbit anti-rat IgG coupled to biotin (Vector Laboratories, Burlingame, CA, USA). Then the sections were incubated with avidin biotin complexes (ABC; Vectastain Elite-kit, Vector Laboratories) and developed using diaminobenzidine (Sigma Chemical Company, St Louis, MO, USA).\n\nDetection of IgG and C3c\nIgG deposited in the tissue by immune-complex formation was detected on paraffin-embedded knee-joint sections. Sections were incubated with biotinylated rat anti-rabbit antibodies (Vector Laboratories) for 1 h. Sections were washed thoroughly with PBS, incubated with ABC complexes, and developed using the method described above.\nC3c was detected using cryostat sections. Sections were incubated with rat anti-murine C3c (Nordic, Tilburg, The Netherlands) after acetone fixation and inhibition of endogenous peroxidase. Isotype-matched antibodies directed against an irrelevant epitope (Vector) were used as a negative control. Staining was scored using image analysis by measuring the mean amount of blue light that passed through a well-defined location in the tissue section near the cruciate ligaments.\n\nAnalysis by fluorescence-activated cell sorter (FACS)\nPeritoneal macrophages of different strains were isolated from the peritoneal cavity by lavage with ice-cold DMEM/ 10% fetal calf serum (FCS)/1% pyruvate. These cells (5 × 106/100 μl) were incubated with 2.4G2 (5 μg/ml). They were then washed and incubated with mouse-adsorbed hamster anti-rat F(ab')2 fragments labelled withfluorescein isothiocyanate (FITC). FACS analysis was performed using a Coulter Epics XL/XL-MCL (Coulter Electronics Ltd, Mijdrecht, The Netherlands). Omitting the first antibody and substitution of 2.4G2 for isotype-matched irrelevant antibodies (DAKO, Glostrup, Denmark) were used as negative controls. The window was set so that \u003e95% of cells were F4/80-positive, indicating that \u003e95% of cells were macrophages.\n\nStimulation of peritoneal macrophages with heat-aggregated gamma globulin\nPeritoneal macrophages were isolated by peritoneal lavage using ice-cold DMEM/10% FCS. Cells were put into 24-well plates (Costar, Acton, MA, USA) at a concentration of 1 × 106 cells/ml. After a 4-day adjustment period, the culture medium was changed to one containing heat-aggregated gamma globulin (HAGG) at 100 μg/ml, or a control medium. After 24 or 48 h, the culture medium was collected. HAGGs were obtained by heating 10 mg/ml rabbit IgG (Sigma Chemicals, St Louis, MO, USA) to 63°C for 30 min. After heating, the solution was centrifuged at 12 000 \t\t\t\t\t\tg \t\t\t\t\t for 10 min. The concentration of the HAGG in the supernatant was determined by reading the absorbance at 280 nm.\n\nProduction of inflammatory mediators by synovial tissue\nSynovial tissue was isolated by dissection of patellar tendon and patellar plate containing the patella, tendons, and synovium, as described earlier [39]. Mediators were obtained by elution from synovial specimens derived from six knee joints in 2 ml Roswell Park Memorial Institute (RPMI) medium for 1 h at room temperature. Washouts were tested for their bioactive IL-1 levels in an IL-1-sensitive bioassay (NOB-1 assay) and in a radioimmunoassay (RIA) to determine the total protein levels.\n\nBioassay for IL-1\nIL-1 activity was measured in the one-stage bioassay for IL-1 as described by Gearing et al [40]. The assay was performed as a culture of the IL-1-specific thymoma cell EL-4, designated NOB-1, which produces IL-2 and IL-4 in response to IL-1, in co-culture with the lymphokine-responder CTLL line. Briefly, EL-4 cells were washed twice and resuspended at 2.5 × 105 cells per ml RPMI medium containing 5% FCS. The cells were distributed into 96-well microtitre plates at 2.5 × 104 cells per well in 100-μl volumes. CTLL cells (4 × 103) suspended in 50 μl RPMI medium were added, followed by appropriate dilutions of test sample to a final volume of 200 μl. After 20 h, 0.5 μCi 3H-thymidine (specific activity 20 Ci/mmol; Amersham Nederland BV, 's Hertogenbosch, The Netherlands) was added to each well. After a 3-h incubation, the contents were harvested, and incorporated activity was determined. The EL-4 line, from which NOB-1 was derived, does not incorporate thymidine, because it is deficient in thymidine kinase, and therefore the incorporation of 3H-thymidine is a measure of only CTLL proliferation. Maximal 3H-thymidine incorporation in the bioassay in the presence of IL-1 was between 15 000 and 20 000 cpm. Culture media contained only minor concentrations of IL-2 or IL-4, as was assessed by testing samples with CTLL alone.\n\nMeasurement of IL-1α and IL-1β protein levels\nIL-1 culture supernatants were measured in duplicate by a nonequilibrium RIA as described elsewhere [41]. Briefly, 100 μl polyclonal rabbit anti-murine IL-1α and IL-1β (diluted in RIA buffer, pH 7.4) was added to 100 μl of samples and standards and kept on ice. After vortexing, the tubes were incubated at 4°C. After 24 h, 100 μl of the appropriate 125I-labelled IL-1α and β containing approximately 10 000 cpm was added to each tube, and incubation was continued for a further 24 h at 4°C. RIA buffer (750 μl) containing 9% (w/v) polyethylene glycol 6000 (Merck Diagnostica, Darmstadt, Germany) and 3% (w/v) goat anti-rabbit serum was added, to separate bound and free tracer. The tubes were incubated for 20 min at room temperature. After centrifugation at 1500 × \t\t\t\t\t\tg \t\t\t\t\t for 15 min, supernatants were quickly drained on adsorbent paper. A gamma-counter was used to count the radioactivity remaining on the paper. The radioactivity in control tubes (the nonspecific binding activity) was subtracted from samples and standards. The detection limit of the assay was 20 pg/ml for both IL-1α and IL-1β.\n\nMeasurement of IL-1 receptor antagonist\nProtein levels of IL-1 receptor antagonist (IL-1Ra) in synovial washout specimens were detected using a specific sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, microtitre plates were coated with unconjugated first antibody (anti-IL1Ra: MAB480, R\u0026D Systems, Abingdon, UK) overnight at 4°C. Subsequently, wells were blocked using 1% BSA followed by a 3-h incubation with patella washouts at 37°C. Wells were then incubated with a biotinylated specific antibody (IL-1Ra: BAF480, R\u0026D Systems) and a signal enhancement step was performed by incubating with PolyHRP (CLB, Amsterdam, The Netherlands). Microtitre plates were developed using o-phenylene-diamine and optical density was read at 492 nm. Between all steps, a washing episode was included using PBS/0.1% Tween 20.\n\nSemiquantitative detection of mRNA using reverse-transcription polymerase chain reaction\nLevels of mRNA for four different cytokines/chemokines were detected using a semiquantitative method. In brief, mice were killed by cervical dislocation, immediately followed by dissection of the patella with adjacent synovial tissue. Six patellae per group were obtained. Two biopsies with a diameter of 3 mm were punched from the synovial tissue, using a biopsy punch (Stiefel, Wachtersbach, Germany): one from the medial and one from the lateral aspect of the patella. Three lateral and three medial biopsies were pooled (six total), to obtain two samples per group. The samples were immediately frozen in liquid nitrogen. Thereafter samples were ground to powder using a Micro-dismembrator II (B Braun, Melsungen, Germany) and total RNA was extracted using 1 ml TRIzol reagent.\nOne microgram of RNA was used for reverse-transcription polymerase chain reaction (RT-PCR). Reverse transcription of mRNA was done using oligoDT primers, and 1/20 of the cDNA was used in one amplification by polymerase chain reaction (PCR). PCR was performed at a final concentration of 200 μM dNTPs, each primer at 0.1 μM, and 1unit Taq polymerase (Life Technologies, Breda, The Netherlands) in standard PCR buffer. Message for glycer-aldehyde-3-phosphate dehydrogenase (GAPDH), IL-1β, IL-1Ra, MCP-1, and MIP-2 was amplified using specific primers. The primer sequences for IL-1β were: 5'-TTGACGGACCCCAAAAGATG-3' (sense) and 5'-AGAAGGTGCTCATGTCCTCA-3' (antisense), for IL-1ra: 5'-TGCTGGGGACCCTACAGTCAC-3' (sense) and 5'-GCAAGTGCATCATCGTTGTTC-3' (antisense), for MCP-1: 5'-CTCACCTGCTGCTACTCATTC-3' (sense) and 5'-GCATGAGGTGGTTGTGAAAA-3' (antisense) and for MIP-2: 5'-GCTGGCCACCAACCACCAGG-3' (sense) and 5'-AGCGAGGCACATCAGGTACG-3' (antisense). The PCR-reaction was paused after 20, 23, 26, 29, 32, 35, 38, 41, and 44 cycles at the very end of the extension phase (72°C) and 5-μl samples were taken. PCR products were separated on 1.6% agarose gel and stained with ethidium bromide. The results are presented as cycle number in which the first detectable amount of DNA appears on the agarose gel. Samples were compared after correction for GAPDH content for each individual sample, to rule out confounding by variation of cellularity in the biopsies.\n\nStatistical analysis\nDifferences between experimental groups were tested for significance using the Wilcoxon rank test. P values \u003c0.05 were considered significant.\n"}