Insulin-like growth factor 1induced interleukin-1 receptor II overrides the activity of interleukin-1 and controls the homeostasis of the extracellular matrix of cartilage.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 48, No. 5, May 2003, pp 1281–1291 DOI 10.1002/art.11061 © 2003, American College of Rheumatology Insulin-Like Growth Factor 1–Induced Interleukin-1 Receptor II Overrides the Activity of Interleukin-1 and Controls the Homeostasis of the Extracellular Matrix of Cartilage Jun Wang, Dirk Elewaut, Eric M. Veys, and Gust Verbruggen Objective. We examined the effect of the insulinlike growth factor 1 (IGF-1)/IGF receptor I (IGFRI) autocrine/paracrine anabolic pathway on the extracellular matrix (ECM) of human chondrocytes and the mechanism by which IGF-1 reverses the catabolic effects of interleukin-1 (IL-1). Methods. Phenotypically stable human articular cartilage cells were obtained from normal cartilage and maintained in culture in alginate beads for 1 week to reach equilibrium of accumulated cell-associated matrix (CAM) compounds. Levels of CAM components aggrecan and type II collagen (CII) and levels of intracellular IGF-1, IL-1␣, and IL-1␤ and their respective plasma membrane–bound receptors IGFRI, IL-1 receptor I (IL-1RI), and the decoy receptor IL-1RII were assayed using flow cytometry to investigate the relationship between the autocrine/paracrine pathways and the homeostasis of ECM molecules in the CAM. The effects of IGF-1 on the expression of IGF-1, IL-1␣, and IL-1␤ and their respective receptor systems, the aggrecan core protein, and CII were determined by flow cytometry. Results. Cause–effect relationship experiments showed that IGF-1 up-regulates the levels of IGF-1, IGFRI, aggrecan, and CII in the CAM. No effects on the expression of IL-1␣ and IL-1␤ and their signaling receptor IL-1RI were observed. However, IGF-1 was able to reverse IL-1␤–mediated degradation of aggrecan and the repression of the aggrecan synthesis rate. Interest- ingly, levels of aggrecan and CII in the CAM strongly correlated not only with IGF-1, but also with IL-1RII, which acts as a decoy receptor for IL-1␣ and IL-1␤. This suggests that IGF-1 and IL-1RII may cooperate in regulating ECM homeostasis. Additional experiments demonstrated that IGF-1 up-regulated IL-1RII, thereby overriding the catabolic effects of IL-1. Conclusion. These findings reveal a new paradigm by which IGF-1 influences chondrocyte metabolism, by reversing the IL-1–mediated catabolic pathway through up-regulation of its decoy receptor. Homeostasis of the extracellular matrix (ECM) of articular cartilage is dependent on the responses of articular cartilage cells to autocrine and paracrine anabolic and catabolic pathways. The most relevant growth factors and cytokines known to be involved in cartilage metabolism are produced by the chondrocytes themselves (1,2) Synthesis and accumulation of the ECM is regulated by locally produced growth factors, such as the insulin-like growth factors (IGFs) and transforming growth factor ␤. A large body of experimental data has substantiated the importance of IGFs 1 and 2 as promoters of growth and matrix synthesis by articular cartilage cells. Both IGFs enhance aggrecan synthesis by human articular cartilage cells cultured in serum-free medium (3,4). The two forms of IGF, however, differentially affect chondrocyte metabolism. It has been suggested that in cartilage, IGF-1 is the regulator of growth and differentiation, whereas IGF-2 may be an important regulator of glucose metabolism (5). IGF-1 has been shown to enhance the synthesis of aggrecan and type II collagen (CII) by chondrocytes (6–9) and to directly decrease both basal and cytokine-stimulated degradation of proteoglycan in cartilage (10). Although it has been proposed that the action of IGF-1 is predominantly as an endocrine factor, whereas IGF-2 interacts Supported by an FWO grant (3G013201). Studies were performed in collaboration with the Institut de Recherches Servier, Courbevoie, France. Jun Wang, MD, Dirk Elewaut, MD, PhD, Eric M. Veys, MD, PhD, Gust Verbruggen, MD, PhD: Ghent University Hospital, Ghent, Belgium. Address correspondence and reprint requests to Gust Verbruggen, MD, PhD, Polikliniek Reumatologie, 0K12, Universitair Hospitaal, De Pintelaan 185, Ghent B-9000, Belgium. E-mail: firstname.lastname@example.org. Submitted for publication May 29, 2002; accepted in revised form January 24, 2003. 1281 1282 WANG ET AL as an autocrine/paracrine factor with the chondrocyte (11), several reports suggest that IGF-1 produced locally in cartilage may be as physiologically important as the circulating hormone (12,13). IGF-2 was shown to be less potent than IGF-1 in the same batches of articular cartilage evaluated for biosynthesis and catabolism of proteoglycans (14). Both IGFs act through their respective receptors (15–17). IGF-1 interacts with its specific membrane receptor IGFRI, which also interacts with IGF-2 and insulin, although with 10–500 times lower affinity. It has been suggested that most of the known effects of IGFs 1 and 2 are mediated by IGFRI (18). IGF binding proteins (IGFBPs), which are produced by chondrocytes and bind to IGF, are important controlling factors of IGF activity (19–21). The secretion of IGFBPs is controlled by the cytokines interleukin-1␣ (IL-1␣) and tumor necrosis factor ␣ (20,22). Turnover and degradation of the matrix are dependent on the responsiveness of the articular cartilage cell to catabolic cytokines, of which IL-1␣ and IL-1␤ are the main agonists (23,24). Besides its capability to induce degradation of articular cartilage, IL-1 has been shown to suppress the synthesis of aggrecan and collagen by the chondrocyte (25,26). This reduced production of ECM compounds is mediated in part by an IL-1– induced generation of nitric oxide (27). The effects of IL-1 are mediated through the high-affinity cell surface receptor IL-1 receptor I (IL-1RI) (28,29). Important controlling factors of IL-1 activity are IL-1R family– related proteins, among which is the decoy receptor IL-1RII, which is expressed on the chondrocyte plasma membrane and binds IL-1␣ and IL-1␤, but does not transmit the IL-1 signals (30,31). In the present study, we examined the autocrine/ paracrine anabolic/catabolic pathways that are potentially relevant to ECM homeostasis. We focused on the effects of IGF-1 and IL-1 on the accumulation of aggrecan and CII in the cell-associated matrix (CAM) of phenotypically stable articular cartilage chondrocytes cultured in alginate. The results indicate that IGF-1 is able to reverse the IL-1–mediated depression and degradation of the ground substance of articular cartilage. Furthermore, we found that this is mediated by a novel mechanism whereby IGF-1 reverses IL-1 activity through up-regulation of its decoy receptor. MATERIALS AND METHODS Isolation of chondrocytes. Human articular chondrocytes were isolated as described elsewhere (32), with a few modifications (33). Briefly, articular cartilage was obtained at autopsy (performed within 24 hours of death). All donors had died as a result of trauma or a brief illness (cerebrovascular or cardiovascular accident), and none of them had been receiving corticosteroids or cytostatic drugs. Visually intact cartilage was obtained from the femoral condyles, diced into small fragments, and chondrocytes were isolated by sequential enzymatic digestion (hyaluronidase, Pronase, collagenase) of the ECM, as described in detail elsewhere (33). The procedure resulted in the liberation of ⬃150 ⫻ 106 chondrocytes from the femoral condyles of each subject. Trypan blue exclusion revealed that ⬎95% of the cells were viable after isolation. Culture of chondrocytes in alginate gel. Chondrocyte cultures in alginate beads were prepared as described elsewhere (34), with some modifications. Chondrocytes suspended in 1 volume of double-concentrated Hanks’ balanced salt solution (HBSS; Gibco) without calcium and magnesium were carefully mixed with an equal volume of 4% alginate (lowviscosity alginate from Macrocystis pyrifera; Sigma-Aldrich, Bornem, Belgium) in HBSS and autoclaved for 15 minutes. The final chondrocyte concentration was 5 ⫻ 106/ml in 2% alginate. The chondrocyte–alginate suspension was then slowly dripped through a 23-gauge needle into a 102-mM solution of calcium chloride, and the beads were allowed to polymerize for 10 minutes at room temperature. Calcium chloride was then removed, and the beads were washed 3 times with 0.15M sodium chloride. Beads were maintained in a 6-well plate containing 4 ml of Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY) with 10% fetal calf serum (FCS; Gibco BRL) and 50 g of ascorbate/ml and incubated at 37°C in an atmosphere of 5% CO2. Nutrient medium was replaced twice weekly for 7–14 days. The chondrocyte cultures consisted of 1 ⫻ 106 cells per culture. Preparation of chondrocytes for flow cytometry. After the respective culture periods, the culture medium was aspirated, and the alginate beads were washed and dissolved by a 10-minute incubation with 3 ml of 55 mM trisodium citrate dihydrate, pH 6.8, 0.15M NaCl at 25°C. The resulting suspension was centrifuged at 1,500 revolutions per minute for 10 minutes to separate cells with their CAM (35) from the constituents of the interterritorial matrix (ITM). IGFRI, IL1RI, and IL-1RII on the cell membrane and aggrecan and CII in the CAM were tested immediately after incubation with the appropriate antibodies for 30 minutes at 4°C in the dark. Twenty microliters of a 50-g/ml preparation of fluorescein isothiocyanate (FITC)–labeled antibodies was used to react with 2 ⫻ 105 cells that had been resuspended in 100 l of phosphate buffered saline. In order to evaluate the expression of IGF-1, IL-1␣, and IL-1␤ inside the cells, chondrocytes in culture were incubated with monensin (GolgiStop; PharMingen, San Diego, CA) at 4 l/6 ml of medium, for 5 hours to block protein transport from the Golgi apparatus. The cells were then isolated from the alginate and permeabilized using a Cytofix/ Cytoperm Plus kit (PharMingen) according to the manufacturer’s instructions. The procedure was followed by incubation with monoclonal antibodies (mAb). Antibodies used for flow cytometry. Mouse anti-human mAb (IgG1 subclass) against IGFRI (clone 33255.111), IL-1RI (clone 35730.111), IL-1RII (clone 34141.11), and IL-1␤ (clone CONTROL OF NORMAL CARTILAGE HOMEOSTASIS BY IL-1RII 8516.311) and the mouse IgG1 negative control were obtained from R&D Systems (Abingdon, UK). Mouse anti-human IGF-1 mAb (clone AHG0014) and IL-1␣ mAb (clone 624B3F2) were purchased from BioSource Europe (Nivelles, Belgium). Mouse anti-human chondrocyte-specific aggrecan mAb (clone 4D11-2A9; BioSource Europe), which specifically reacts with the G1 domain of the invariable hyaluronanbinding region of the human aggrecan molecule, was used to detect the aggrecan in the chondrocyte CAM. Mouse antihuman CII mAb (clone II-4C11; ICN Biomedicals, Aurora, OH) was chosen to detect CII. All antibodies (except aggrecan and CII) were conjugated with FITC (isomer I; Sigma-Aldrich) as previously described (36). The antiaggrecan and anti–CII mAb were conjugated with phycoerythrin (PE; Sigma-Aldrich) as described elsewhere (37). The conjugated mAb were used in a direct immunofluorescent staining protocol for flow cytometry. Appropriate FITC-labeled or PE-labeled isotype-matched mouse or rabbit IgG1 (clone X40; Becton Dickinson, San Jose, CA) was used as a negative control. Flow cytometric analysis. Stained cells were analyzed with a flow cytometer (FACSort) using CellQuest software (both from Becton Dickinson). For each sample, 15,000 events were analyzed. Cells were gated on forward and side scatter to exclude dead cells, debris, and aggregates. Propidium iodide was additionally used to exclude dead cells when the epitopes outside the cells (i.e., IGFRI, IL-1RI, and IL-1RII) and the ECM molecules were analyzed (36,38). The mean fluorescence intensity (MFI) of the positive cell population, which is due to the binding of the conjugated antibodies to the specific antigen, was used to quantify the presence of IGFRI, IL-1RI, and IL-1RII on the plasma membrane, the ECM molecules in the CAM, and the accumulation of IGF-1, IL-1␣, and IL-1␤ inside the cells. MFI values were obtained by subtracting the MFI of the negative control population from the MFI of the positive stained population. For comparison between experiments, a Quantum Simply Cellular Microbead kit (Sigma-Aldrich) was used to calibrate the fluorescence scale of the flow cytometer (39). The microbeads were stained and processed in parallel with the cell samples, using the same amount of FITC-labeled antibodies and the same incubation time. The fluorescence scale of the cytometer was adapted before every experiment in order to keep identical MFIs for the 4 peaks of the calibration beads. The MFI of the cell samples was then analyzed without changing any of the instrument settings. The reproducibility and reliability of this procedure have been demonstrated previously (36,38). Effect of exogenous IGF on the expression of ECM molecules and on the levels of intracellular cytokine and growth factor. Chondrocytes were obtained from 3 donors and cultured in alginate. The culture medium consisted of DMEM supplemented with 2.5% FCS. IGF-1 (R&D Systems) was added at concentrations of 0 and 100 ng/ml (3) beginning on day 1. Medium was replaced every 3 days. After 7 days of culture, chondrocytes were harvested, and CAM aggrecan and CII, plasma membrane–bound IGFRI, IL-1RI, and IL-1RII, and intracellular IGF-1, IL-1␣, and IL-1␤ levels were measured. An identical protocol with increasing concentrations of IGF-1 was used in studies of chondrocytes from 3 additional 1283 Figure 1. Expression of extracellular matrix compounds (aggrecan, type II collagen) and components of the insulin-like growth factor (IGF)/interleukin-1 (IL-1) pathways by chondrocytes (n ⫽ 6 donors) cultured in alginate. Shown are the results of flow cytometric analyses (chondrocyte mean fluorescence intensity) after 3, 7, and 14 days of culture. Values are the mean and SD for the 6 batches of chondrocytes; F ⫽ mean of triplicate experiments for each batch of chondrocytes. IL-1RII ⫽ IL-1 receptor II; IGFRI ⫽ IGF receptor I. donors to find the most efficient dose of IGF-1 that would affect the IL-1 regulatory pathways. To confirm that IGF-1–induced IL-1RII repressed IL-1 catabolic activity, chondrocytes from 3 additional donors were cultured in DMEM supplemented with 2.5% FCS and increasing amounts (0, 25, 100 and 200 ng/ml) of IGF-1. After 3 days of culture, 100 pg/ml of IL-1␤ was added. To the chondrocytes cultured with 200 ng/ml of IGF-1, we added 100 g/ml of neutralizing anti–IL-1RII mAb (or an isotypematched nonspecific control antibody). After 7 days of culture, the nutrient media were removed from the chondrocyte cultures. The alginate beads were dissolved with 55 mM trisodium citrate dihydrate, and the suspension was centrifuged. The pellet with the chondrocytes was recovered to assay CAM aggrecan and CII as well as plasma membrane IL-1RII by flow 1284 WANG ET AL Table 1. MFI values for growth factors and cytokines, as well as their receptors and cell-associated matrix compounds, produced by chondrocytes from macroscopically intact cartilage obtained from normal donors* Sex/age IGFRI IGF-1 IL-1RI IL-1RII IL-1␣ IL-1␤ CII Aggrecan M/28 F/16 M/54† F/34 M/59† M/54† M/44 M/47 M/56 M/59† F/40 M/38 M/18 M/59† M/57 M/18 F/67 F/73 Overall mean ⫾ SD Average CV 3.26 ⫾ 0.16 4.28 ⫾ 0.05 7.70 ⫾ 0.94 4.92 ⫾ 0.09 3.01 ⫾ 0.05 4.58 ⫾ 0.20 2.36 ⫾ 0.05 4.25 ⫾ 0.15 1.96 ⫾ 0.19 2.50 ⫾ 0.32 5.41 ⫾ 0.15 4.36 ⫾ 0.08 2.64 ⫾ 0.10 1.82 ⫾ 0.05 2.77 ⫾ 0.08 0.95 ⫾ 0.09 0.46 ⫾ 0.02 0.47 ⫾ 0.03 3.20 ⫾ 1.90 4.92 – – – – – – 40.27 ⫾ 6.10 42.20 ⫾ 0.55 19.86 ⫾ 2.81 27.90 ⫾ 1.70 44.50 ⫾ 3.85 39.15 ⫾ 0.94 27.03 ⫾ 0.26 21.96 ⫾ 4.30 40.60 ⫾ 1.21 21.03 ⫾ 3.00 21.80 ⫾ 1.03 32.10 ⫾ 0.47 31.53 ⫾ 9.37 7.64 1.43 ⫾ 0.05 0.61 ⫾ 0.03 1.06 ⫾ 0.01 2.44 ⫾ 0.09 1.58 ⫾ 0.19 2.86 ⫾ 0.09 1.86 ⫾ 0.11 2.55 ⫾ 0.18 1.53 ⫾ 0.07 2.11 ⫾ 0.08 1.57 ⫾ 0.08 1.63 ⫾ 0.14 1.75 ⫾ 0.05 2.98 ⫾ 0.48 1.51 ⫾ 0.06 0.91 ⫾ 0.21 0.39 ⫾ 0.06 0.24 ⫾ 0.03 1.60 ⫾ 0.80 7.62 1.16 ⫾ 0.07 2.01 ⫾ 0.11 5.70 ⫾ 0.32 2.36 ⫾ 0.06 3.94 ⫾ 0.11 4.66 ⫾ 0.22 1.71 ⫾ 0.11 4.50 ⫾ 0.52 1.70 ⫾ 0.08 3.28 ⫾ 0.63 4.64 ⫾ 0.24 2.26 ⫾ 0.08 3.14 ⫾ 0.10 1.09 ⫾ 0.13 5.02 ⫾ 0.21 0.68 ⫾ 0.14 0.57 ⫾ 0.03 0.14 ⫾ 0.03 2.70 ⫾ 1.70 8.02 – – – – – – 26.76 ⫾ 0.40 22.22 ⫾ 0.42 21.38 ⫾ 4.80 46.14 ⫾ 6.95 20.40 ⫾ 1.85 15.28 ⫾ 0.73 22.14 ⫾ 0.68 42.41 ⫾ 9.37 12.35 ⫾ 1.13 8.81 ⫾ 1.10 6.80 ⫾ 0.50 14.60 ⫾ 1.36 21.60 ⫾ 12.10 9.85 – – – – – – 44.00 ⫾ 7.10 60.64 ⫾ 1.98 5.60 ⫾ 0.25 56.48 ⫾ 7.41 36.27 ⫾ 3.20 30.06 ⫾ 0.52 56.05 ⫾ 0.05 72.90 ⫾ 4.56 50.16 ⫾ 3.00 6.39 ⫾ 0.56 5.10 ⫾ 0.48 12.20 ⫾ 0.84 36.30 ⫾ 24.1 7.08 – – – – – – 4.32 ⫾ 0.17 6.22 ⫾ 0.14 1.03 ⫾ 0.09 4.31 ⫾ 0.18 10.74 ⫾ 1.24 8.70 ⫾ 0.28 5.07 ⫾ 0.31 4.78 ⫾ 0.69 14.85 ⫾ 1.43 1.33 ⫾ 0.11 1.80 ⫾ 0.17 1.10 ⫾ 0.19 5.40 ⫾ 4.30 8.24 – – – – – – 14.07 ⫾ 1.84 18.20 ⫾ 0.48 8.46 ⫾ 0.09 13.48 ⫾ 1.50 31.94 ⫾ 1.90 22.57 ⫾ 2.10 21.66 ⫾ 1.16 5.13 ⫾ 0.69 38.16 ⫾ 0.61 2.16 ⫾ 0.04 3.60 ⫾ 0.15 4.10 ⫾ 0.12 15.30 ⫾ 11.60 6.04 * Values are the mean ⫾ SD mean fluorescence intensity (MFI) of triplicate samples from each donor. MFI values were obtained by subtracting the MFI of the negative control population from the MFI of the positive population. MFI values for insulin-like growth factor 1 (IGF-1), interleukin-1␣ (IL-1␣), IL-1␤, type II collagen (CII), and aggrecan were not measured in the first 6 donors. IGFRI ⫽ insulin-like growth factor receptor I; IL-1RI ⫽ interleukin-1 receptor I. The average coefficient of variation (CV) of the 18 triplicate samples illustrates the reliability of the technique; values are percentages. † There were 2 different 54-year-old male donors and 3 different 59-year-old male donors. cytometry. The resulting supernatant was harvested, and a commercial enzyme amplified-sensitivity immunoassay (EASIA) kit (PG-EASIA kit; BioSource Europe) was used to assess the production and deposition of newly synthesized aggrecan in the artificial interterritorial alginate matrix. Fractions of the supernatant containing the newly synthesized macromolecules were used for gel-permeation chromatography on Sepharose CL-2B (Pharmacia, Brussels, Belgium) in 0.067M phosphate buffer. Aliquots of the eluted fractions were used to determine the molecular size of the aggrecans by EASIA. The aggrecan aggregates and monomeric aggrecan were shown as 2 distinct peaks (3). Statistical analysis. Values obtained for the different variables in the entire donor population as well as for the MFI results (triplicate cell cultures) are expressed as the mean ⫾ SD. Spearman’s correlation coefficients were used to examine correlations between the mean values of different parameters for the chondrocyte cultures in equilibrium. Since the classic Bonferroni correction is not applicable to large-number comparisons, P values less than 0.05 were considered statistically significant. Such an approach has been used in previous studies of large numbers of comparisons (40). Student’s t-test was used to examine whether variables for the chondrocyte cultures became significantly different over time in culture or after IGF treatment. P values of 0.05 were considered significant. RESULTS Expression of ECM and IGF-1/IL-1 pathways by chondrocytes in alginate. Chondrocyte samples obtained from 6 donors (5 males, 1 female; age range 37–60 years) were used to define the optimum stage for investigating homeostasis of the ECM molecules and the autocrine pathways controlling this equilibrium. Results of flow cytometric analysis after 3, 7, and 14 days of culture are summarized in Figure 1. Chondrocytes rapidly reexpressed membrane-bound IGFRI, IL-1RI, and IL-1RII and accumulated CAM aggrecan and CII. Expression of the plasma membrane receptors reached an optimum after 1 week in culture. When plasma membrane receptor expression on days 3 and 7 was compared, MFI values for IGFRI, IL-1RI, and IL-1RII raised, on average, by 48% (P ⫽ 0.048), 77% (P ⫽ 0.133), and 172% (P ⫽ 0.026), respectively, and then dropped. CAM aggrecan and CII accumulated during the first week of culture to level off during further culture in alginate (average changes in MFI on day 7 versus day 3, ⫹298% for aggrecan [P ⫽ 0.083] and ⫹47% for CII [P ⫽ 0.008]). Intracellular levels of IGF-1, IL-1␣, and IL-1␤ in chondrocytes were high immediately after the isolation procedure (day 3), but decreased and reached the lowest levels after 2 weeks of in vitro culture. Comparison of intracellular levels of IGF-1, IL-1␣, and IL-1␤ on days 3 and 14 showed decreases in MFI values to 41% (P ⫽ 0.0002), 46% (P ⫽ 0.002), and 25% (P ⫽ 0.0003) of the baseline values, respectively. CONTROL OF NORMAL CARTILAGE HOMEOSTASIS BY IL-1RII 1285 Table 2. Correlations between IGFRI, IL-1RI, and IL-1RII expression on the cell membrane, IGF-1, IL-1␣, and IL-1␤ inside the cells, and extracellular matrix molecules in the cell-associated matrix* IGFRI IGFRI IGF-1 IL-1RI IL-1RII IL-1␣ IL-1␤ CII Aggrecan – r ⫽ 0.7358 P ⫽ 0.0064† r ⫽ 0.2724 P ⫽ 0.2742 r ⫽ 0.7404 P ⫽ 0.0004† r ⫽ 0.1586 P ⫽ 0.6226 r ⫽ 0.4532 P ⫽ 0.1390 r ⫽ 0.7002 P ⫽ 0.0112† r ⫽ 0.8117 P ⫽ 0.0013† IGF-1 IL-1RI IL-1␣ IL-1RII IL-1␤ CII Aggrecan r ⫽ 0.9110 P ⫽ 0.00001† – – r ⫽ 0.2254 P ⫽ 0.4812 r ⫽ 0.6701 P ⫽ 0.0171† r ⫽ 0.0039 P ⫽ 0.9904 r ⫽ 0.4005 P ⫽ 0.1970 r ⫽ 0.7140 P ⫽ 0.0091† r ⫽ 0.7971 P ⫽ 0.0019† – r ⫽ 0.3615 P ⫽ 0.1405 r ⫽ 0.7550 P ⫽ 0.0045† r ⫽ 0.8558 P ⫽ 0.0004† r ⫽ 0.3071 P ⫽ 0.3316 r ⫽ 0.3246 P ⫽ 0.3033 – r ⫽ 0.1389 P ⫽ 0.6669 r ⫽ 0.5584 P ⫽ 0.0592 r ⫽ 0.8135 P ⫽ 0.0013† r ⫽ 0.9409 P ⫽ 0.0000† – r ⫽ 0.6990 P ⫽ 0.0114† r ⫽ ⫺0.0408 P ⫽ 0.8999 r ⫽ ⫺0.0659 P ⫽ 0.8388 – r ⫽ 0.4799 P ⫽ 0.1143 r ⫽ 0.4718 P ⫽ 0.1215 – * Values are correlation coefficients and their P values. IGFRI ⫽ insulin-like growth factor receptor I; IL-1RI ⫽ interleukin-1 receptor I; IGF-1 ⫽ insulin-like growth factor 1; IL-1␣ ⫽ interleukin-1␣; CII ⫽ type II collagen. † Difference is significant. Based on these data, we decided to study the homeostasis of the ECM after 1 week of culture. Intracellular cytokine and growth factor levels, expression of the respective plasma membrane receptors, and homeostasis of CAM molecules. Chondrocytes were obtained from 18 donors (13 males, 5 females; age range 16–73 years). Expression of different antigens was quantified using chondrocyte MFI values after staining with the specific conjugated antibodies. Table 1 shows the mean ⫾ SD values for the different variables, as obtained in triplicate samples from each donor. The average coefficient of variation (CV) for each variable was calculated from these data and illustrated the reliability of the technique. The MFI values reflect the number of FITC- or PE-conjugated antibodies fixed on the chondrocytes and, since mAb were used, the number of epitopes detected. The MFI values for each of these items varied widely for the 18 donors. The mean chondrocyte MFIs for IGFRI, IL-1RI, and IL-1RII were 3.20 ⫾ 1.90 (CV 58%), 1.60 ⫾ 0.80 (CV 49%), and 2.70 ⫾ 1.70 (CV 64%), respectively. These low MFI values for the receptors on the cell membrane reflected the low numbers of receptor molecules on the cell surface. IGF-1, IL-1␣, and IL-1␤ levels inside the cells, which directly indicate their production by the chondrocytes, were ⬃10 times higher: 31.53 ⫾ 9.37 (CV 30%), 21.60 ⫾ 12.10 (CV 56%), and 36.30 ⫾ 24.10 (CV 67%), respectively. The mean chondrocyte MFI values for aggrecan and CII in the CAM were 15.30 ⫾ 11.60 (CV 76%) and 5.40 ⫾ 4.30 (CV 80%), respectively. Correlations between the different parameters. Correlations between the expression of the receptors on the cell membrane, IGF-1, IL-1␣, and IL-1␤ levels inside the cells, and the accumulation of CAM molecules are shown in Table 2. IGFRI correlated significantly with its ligand IGF-1 (r ⫽ 0.7358, P ⫽ 0.0064), aggrecan (r ⫽ 0.8117, P ⫽ 0.0013), and CII (r ⫽ 0.7002, P ⫽ 0.0112). In addition, there was significant correlation between IGFRI and the presence of IL-1RII (r ⫽ 0.7404, P ⫽ 0.0004), the decoy receptor for IL-1. Besides its receptor, IGF-1 strongly correlated with the 2 ECM molecules in the CAM: aggrecan (r ⫽ 0.7971, P ⫽ 0.0019) and CII (r ⫽ 0.7140, P ⫽ 0.0091). Similar degrees of correlation were found between IL-1RII and the 2 ECM molecules in the CAM. Correlations were observed between the functional IL-1RI on the plasma membrane and the intracellular IL-1␣ and IL-1␤ levels (r ⫽ 0.7550, P ⫽ 0.0045 and r ⫽ 0.8558, P ⫽ 0.0004) and between the 2 IL-1 isoforms (r ⫽ 0.6990, P ⫽ 0.0114). Levels of CAM ECM molecules did not correlate with the agonists of the IL-1 pathway. Finally, there was a strong correlation between the accumulation of CII and aggrecan in the CAM (r ⫽ 0.9110, P ⫽ 0.00001). Effect of exogenous IGF on the expression of CAM molecules and on levels of intracellular cytokines and growth factors. Chondrocytes were obtained from 3 additional donors (1 male, 2 females; age range 30–67 years). The cells were harvested for flow cytometric analysis after 7 days in culture. In these 3 donors, 100 ng/ml of IGF-1 significantly up-regulated the expression 1286 Figure 2. A–C, Percentage change in the expression of insulin-like growth factor 1 (IGF-1), IGF receptor I (IGFRI), interleukin-1 receptor I (IL-1RI), IL-1RII, IL-1␣, IL-1␤, aggrecan (AGGR), and type II collagen (COLL) after exposure of chondrocytes obtained from 3 different donors to 100 ng/ml of IGF-1. Values are the mean and SD mean fluorescence intensity (MFI) of triplicate cultures. Gray portions of the bars are results of IGF-stimulated cultures; solid portions are results of control cultures. Numbers across the bottom are baseline MFI values. ⴱ ⫽ P ⬍ 0.05 versus baseline. of IGFRI on the cell membrane (11–57%) and increased the intracellular levels of IGF-1 (9–38%) (Figure 2). Furthermore, the expression of the decoy receptor IL1RII increased by 31–63%. Exogenous IGF-1 had no effect on cell membrane IL-1RI or on the concentration of IL-1␣ and IL-1␤ inside the chondrocytes. The accumulation of aggrecan and CII in the CAM significantly WANG ET AL increased during exposure to 100 ng/ml IGF-1. The magnitude of the effect varied among the batches of donor chondrocytes. In another 3 chondrocyte samples (3 females; age range 26–61 years), dose-response experiments with increasing concentrations of IGF-1 showed that a concentration as low as 12.5 ng/ml significantly affected the expression of IL-1RII in 2 of the donors (Table 3). In the third donor, a significant up-regulation was obtained at an IGF-1 concentration of 50 ng/ml. This effect leveled off at 100–200 ng/ml of IGF-1. Up-regulation of IL-1RII correlated well with the accumulation of aggrecan in the CAM. Plasma membrane IGFRI was less effectively influenced by IGF-1. Figure 3 shows the changes in the MFI for cell membrane IGFRI, IL-1RI, IL-1RII, and CAM aggrecan induced by increasing doses of IGF-1 in a representative donor. Repression of IL-1 activity by IGF-1–induced IL-1RII. Three samples of chondrocytes (1 female, 2 males; age range 37–59 years) were cultured in 2.5% FCS. IL-1␤ at a concentration of 100 pg/ml reduced the accumulation of aggrecan in the CAM to ⬃70% of the baseline value in the 3 control cultures (Figure 4B). Increasing amounts of IGF-1 (25–100 ng/ml) restored CAM aggrecan levels in a dose-dependent manner, leveling off at 100 ng/ml of IGF-1 (130–190% of the baseline values in the 3 chondrocyte samples). Addition of 100 g/ml of neutralizing anti–IL-1RII mAb (but not an isotype-matched nonspecific IgG) to the chondrocytes cultured with IL-1␤ and 200 ng/ml of IGF-1 significantly reduced aggrecan accumulation in the chondrocyte CAM, nearing the levels reached in the presence of IL-1 alone. Plasma membrane IL-1RII followed the same trend under the different test conditions used, proving that IGF-1 restored the IL-1␤– induced down-regulation of IL-1RII (Figure 4A). IGF-1–induced IL-1RII repression of IL-1 activity was confirmed when synthesis and accumulation of aggrecan in the artificial ECM of the chondrocytes were assayed by EASIA (Figure 4C). IL-1␤ depressed the synthesis and deposition of aggrecan, and IGF-1 at 100 ng/ml completely abolished this effect. This upregulation of depressed aggrecan synthesis induced by IL-1␤ was not seen in the presence of 100 g/ml of the neutralizing anti–IL-1RII mAb. Study of the molecular size of the aggrecans synthesized under the experimental conditions validated the IGF-1/IL-1RII/IL-1␤ pathway. Gel-permeation chromatography patterns showed that control chondrocytes synthesized mainly aggrecan aggregates that eluted in the void volume (Kav ⫽ 0.0). IL-1␤ caused the CONTROL OF NORMAL CARTILAGE HOMEOSTASIS BY IL-1RII 1287 Table 3. Percentage of change in mean fluorescence intensity for IGFRI, IL-1RI, IL-1RII, and aggrecan after exposure of normal chondrocytes to increasing doses of IGF-1* Donor sex/ age, IGF-1 dose F/26 None 12.5 ng/ml 25.0 ng/ml 50.0 ng/ml 100.0 ng/ml 200.0 ng/ml F/51 None 12.5 ng/ml 25.0 ng/ml 50.0 ng/ml 100.0 ng/ml 200.0 ng/ml F/61 None 12.5 ng/ml 25.0 ng/ml 50.0 ng/ml 100.0 ng/ml 200.0 ng/ml IL-1RI IL-1RII IGF-1R Aggrecan 100.0 ⫾ 5.0 110.0 ⫾ 17.5 125.0 ⫾ 22.5 115.0 ⫾ 20.0 117.5 ⫾ 10.0 137.5 ⫾ 30.0 100.0 ⫾ 15.5 178.1 ⫾ 11.3† 264.9 ⫾ 34.0† 312.1 ⫾ 12.8‡ 394.7 ⫾ 12.8§ 418.1 ⫾ 6.8§ 100.0 ⫾ 8.5 100.0 ⫾ 11.0 113.4 ⫾ 9.8 115.8 ⫾ 13.4 129.3 ⫾ 11.0 128.0 ⫾ 15.8 100.0 ⫾ 17.5 267.7 ⫾ 9.3‡ 283.0 ⫾ 10.8‡ 421.2 ⫾ 40.1‡ 421.6 ⫾ 5.4§ 475.0 ⫾ 14.7§ 100.0 ⫾ 2.6 102.1 ⫾ 11.9 88.6 ⫾ 11.9 109.8 ⫾ 10.9 97.9 ⫾ 21.8 100.0 ⫾ 6.7 100.0 ⫾ 4.26 308.8 ⫾ 11.9§ 385.1 ⫾ 16.4§ 428.8 ⫾ 27.2‡ 435.6 ⫾ 4.9§ 431.8 ⫾ 17.4§ 100.0 ⫾ 6.45 176.1 ⫾ 22.6¶ 164.5 ⫾ 16.8¶ 185.2 ⫾ 23.9¶ 267.1 ⫾ 20.0† 254.8 ⫾ 17.4‡ 100.0 ⫾ 19.6 154.3 ⫾ 4.5¶ 176.9 ⫾ 5.4† 194.3 ⫾ 14.3† 208.8 ⫾ 10.0† 216.9 ⫾ 11.8† 100.0 ⫾ 21.2 98.5 ⫾ 15.1 101.5 ⫾ 10.6 100.0 ⫾ 22.7 109.1 ⫾ 30.3 89.4 ⫾ 15.1 100.0 ⫾ 10.9 123.4 ⫾ 18.7 126.6 ⫾ 12.5 143.8 ⫾ 6.2¶ 170.3 ⫾ 25.0¶ 176.6 ⫾ 20.3¶ 100.0 ⫾ 13.0 87.0 ⫾ 18.5 113.0 ⫾ 18.5 127.8 ⫾ 37.0 135.3 ⫾ 18.5 140.7 ⫾ 11.1¶ 100.0 ⫾ 19.6 102.2 ⫾ 26.1 230.4 ⫾ 41.3¶ 358.7 ⫾ 50.0† 556.5 ⫾ 17.4§ 565.2 ⫾ 52.2‡ * Values are the mean ⫾ SD percentage of change from baseline. IGFRI ⫽ insulin-like growth factor receptor I; IL-1RI ⫽ interleukin-1 receptor I; IGF-1 ⫽ insulin-like growth factor 1. † P ⬍ 0.01 versus baseline. ‡ P ⬍ 0.001 versus baseline. § P ⬍ 0.0001 versus baseline. ¶ P ⬍ 0.05 versus baseline. aggrecans to be enzymatically degraded and the molecular size of this population to drop. Hence, the degradation products were retarded (Kav ⫽ 0.18) during chromatography (Figure 5). IGF-1 completely eliminated the IL-1␤–induced degradation of the aggrecans, and the population once more eluted with a Kav of 1.0. Neutralization of the IGF-1–induced IL-1RII again enabled IL-1 ␤ activity. The same changes in gelpermeation chromatography elution profiles were observed when the experiments were performed on the aggrecan populations of 2 other donors (data not shown). DISCUSSION The intercellular matrix of cartilage is composed of 2 compartments: the CAM, which lies close to the chondrocyte, and, adjacent to the CAM, the ITM (41). The CAM is a constant part of the ECM (42,43), the macromolecular compounds of which are metabolized or turned over in a particular way (44). Newly synthesized aggrecans have been shown to reside in the CAM for short periods of time, with a higher rate of aggrecan turnover here than in the ITM (45). The neosynthesized CAM macromolecules leave the territorial matrix at a later stage to diffuse to the ITM (44). The ITM forms the largest domain of the intercellular matrix. One of the advantages of chondrocyte culture in alginate is the reversibility of the gelled condition of this matrix, allowing the study of the different intercellular compartments surrounding the chondrocyte in vitro. Our studies on the homeostasis of the ECM of articular cartilage were conducted on chondrocytes that maintained their original phenotype in vitro when cultured in alginate. Although data about intracellular and pericellular metabolic events may not provide an accurate representation of the export of matrix macromolecules to the extracellular environment, they allow the biologist to estimate the processes of synthesis and turnover that lead to homeostasis of the ECM. We focused on the accumulation of aggrecan and CII in the CAM of phenotypically stable articular cartilage chondrocytes. Immediately after their isolation and after initiation of the culture procedure, the chondrocytes showed high levels of intracellular growth factors 1288 WANG ET AL Figure 3. Effects of increasing concentrations of insulin-like growth factor 1 (IGF-1) on chondrocyte mean fluorescence intensity for cell membrane interleukin-1 receptor I (IL-1RI), IL-1RII, IGF receptor I (IGFRI), and the cell-associated matrix component aggrecan in a representative donor (51-year-old woman). Values are the mean and SD of triplicate experiments. and cytokines. Mechanical stress, sustained by the cells during the process of their isolation, is thought to have initiated these metabolic changes. Similar processes of activation of cells by mechanical forces, known as mechanotransduction, have been described for different cells, including skeletal muscle cells (46), chondrocytes (47,48), and endothelial cells (49), and are poorly understood. However, elevated levels of IGF-1, IL-1␣, and IL-1␤ inside the cells decreased and stabilized within the Figure 4. Dose-response effect of insulin-like growth factor 1 (IGF-1) on interleukin-1␤ (IL-1␤)–depressed plasma membrane levels of IL-1 receptor II (IL-1RII) (A) and on the accumulation of aggrecan in the cell-associated matrix (CAM) (B) and in the interterritorial matrix (ITM) (C). Note the reversal of the IGF-1–mediated effects by anti–IL-1RII neutralizing monoclonal antibody (anti–IL-1RII). Values are the mean ⫾ SD of 3 experiments performed on chondrocytes from 3 different donors (E ⫽ male donor age 59; ⫽ male donor age 37; F ⫽ female donor age 51). CO ⫽ control culture; contr ab ⫽ isotype-control antibody. MFI ⫽ mean fluorescence intensity. CONTROL OF NORMAL CARTILAGE HOMEOSTASIS BY IL-1RII Figure 5. Elution profiles of aggrecan, as determined by Sepharose CL-2B gel-permeation chromatography, in chondrocytes from a representative donor, a 59-year-old man. Top, F ⫽ control chondrocytes; E ⫽ interleukin-1␤ (IL-1␤)–depressed chondrocytes. Bottom, F ⫽ IL-1␤–depressed chondrocytes after exposure to 200 ng of insulin-like growth factor 1 (IGF-1); E ⫽ IL-1␤–depressed/IGF-1–up-regulated chondrocytes exposed to IL-1 receptor II neutralizing monoclonal antibody (anti–IL-1RII). first 2 weeks in culture. The cells were thus allowed to recuperate from the isolation procedure, to restore their repertoire of plasma membrane receptor proteins, and to rebuild a cell-associated ECM. The new equilibriums were reached after 1–2 weeks in culture, and it was decided to perform the experiments after a 1-week culture period. The objective of these studies was to investigate which of the autocrine/paracrine pathways directs the homeostasis of the ECM in an in vitro system where normal articular cartilage cells rebuild the ECM. One would theoretically assume that in an IGF-driven system, the amounts of ECM ground substance in the CAM of chondrocytes would correlate positively with the strength of that IGF/IGFR pathway. In a system in which the IL-1/IL-1RI pathway controls the metabolic events in a given tissue, the amounts of ECM molecules in a chondrocyte CAM would be expected to correlate negatively with the levels of this catabolic cytokine system. After 1 week in culture, significant correlations were found between the main actors of both the cata- 1289 bolic and anabolic pathways: IGFRI significantly correlated with its ligand IGF-1, and likewise, correlations were observed between the signal-transducing IL-1RI on the plasma membrane and the intracellular IL-1␣ and IL-1␤ levels. The strength of the IGF-1/IGFRI pathway significantly correlated with the amounts of aggrecan and CII accumulated in the CAM. In addition, there was a significant correlation between IGF-1/IGFRI and the presence of IL-1RII, the decoy receptor for IL-1. Additionally, the same degree of correlation was found between IL-1RII and the ECM molecules in the CAM. Levels of CAM ECM molecules did not correlate with the agonists of the IL-1 pathway. The accumulation of a series of ECM macromolecules in the CAM of chondrocytes cultured in an artificial environment was used in these studies to explore the effects of IGF-1 on the synthesis and turnover of the ECM. The results of our cause–effect relationship experiments showed that 25–100 ng/ml of IGF-1 enhanced the accumulation of aggrecan and CII in the chondrocyte CAM. These results supported the observations of other investigators who have shown that growth factors, especially IGF, direct the production and accumulation of ECM by chondrocytes in normal and diseased cartilage (6–10). These observations have been confirmed in studies of isolated cartilage cells in different in vitro culture systems (3,4). Exogenous IGF-1 induced its own production and the expression of IGFRI on the plasma membrane in our studies. Other investigators have also assessed the modulation of IGF-1 gene expression by chondrocytes following exogenous IGF-1 supplementation. Persistent exposure of chondrocytes to 100 ng/ml of IGF-1 resulted in a maximum IGF-1 messenger RNA response after 24 hours (50). In vivo, IGF-1 and growth hormone increased levels of IGF-1 messenger RNA and immunoreactivity of chondrocytes in the proliferative zone of the growth plate of hypophysectomized rats (51). Our data suggest that IGF-1 induces an autoinductive IGF-1 autocrine/paracrine transcriptional response. The mechanism of this autoinduction is, at present, unclear and may result from complex interferences of other growth and differentiation factors that are present in this experimental system. Although the baseline levels of IGFRI expression on the plasma membrane and of aggrecan in the CAM tended to decrease with age, and some differences in the IGF-1 response between different donors were obvious, the cause–effect experiments showed the same type of response regardless of the age of the donor. Furthermore, exogenous IGF-1 was shown to induce the expression of IL-1RII on the chondrocyte 1290 WANG ET AL plasma membrane. IL-1RII binds and neutralizes IL-1␤ in bioassays, but not (or almost not) IL-1␣ (52,53). IL-1RII acts as a molecular trap for the IL-1 agonist without participating in its signaling. Through the upregulation of IL-1RII, IGF-1 can thus protect cartilage cell ECM against IL-1–induced destruction. This was illustrated in our in vitro experiments in which IGF-1 countered the biologic effects of IL-1␤, for example, the deficient synthesis and degradation and inadequate deposition of aggrecan in the CAM and in the ECM of IL-1␤–depressed chondrocytes. This protective effect was shown to be modulated through the up-regulation of plasma membrane IL-1RII levels, since an IL-1RII– neutralizing IgG abolished the supporting activity of IGF-1. A decrease in both the basal level and the cytokine-stimulated degradation of proteoglycan by IGF-1 in cartilage explant cultures, as demonstrated previously (10), is consistent with these findings. Additionally, human chondrocytes that overexpressed IL1RII were previously shown to be resistant to IL-1– induced inhibition of proteoglycan synthesis, and soluble IL-1RII significantly inhibited a series of IL-1␤–induced effects in chondrocytes (31). Along with chondrocytes, many other specialized interstitial tissue cells express IL-1RII, and the expression of this decoy receptor has been reported to be up-regulated by different inflammatory cytokines and growth factors (54–56). This is the first study to show that IGF-1 induces IL-1RII in articular cartilage chondrocytes. In summary, the findings of the present study indicate that human articular chondrocytes rebuild their ECM in vitro, with obvious synthesis and turnover of this ECM as early as 2 weeks. At that moment, homeostasis of the ECM is under the control of the IGF-1/IGFRI autocrine pathway. The pathway overrides the catabolic effects of the IL-1/IL-1RI pathway by up-regulating IL-1RII. 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