Platelet-derived growth factor potentiates cellular responses of articular chondrocytes to interleukin-1

Recombinant human interleukin-1 alpha (IL-1 alpha) induced a time-dependent (0-72 hours) and concentration-dependent (0.01-10 ng/ml) production of metalloproteinases (collagenase, gelatinase, stromelysin) and prostaglandin E2 (PGE2) in rabbit articular chondrocytes (RAC). Exposure of RAC to recombinant human platelet-derived growth factor homodimer BB (PDGF-BB; 2-200 ng/ml) in the presence of stimulatory and substimulatory concentrations of IL-1 alpha resulted in a marked augmentation of metalloproteinase and PGE2 production. PDGF-BB exerted no agonist effects on RAC responsiveness. PDGF-BB up-regulated the number of IL-1 receptors per chondrocyte but had no effect on receptor affinity. Cycloheximide and actinomycin D caused a concentration-dependent suppression of the PDGF-BB-mediated potentiation of radiolabeled IL-1 alpha binding to RAC and cell responsiveness to IL-1 alpha. Similarly, IL-1 increased the number of PDGF receptors on RAC without changing receptor affinity. These data are discussed within the context of cytokine-growth factor interactions as components of the pathogenesis of arthritic diseases.     

Effects of recombinant human interleukin-1 beta on rabbit articular chondrocytes. Stimulation of prostanoid release and inhibition of cell growth

Using an in vitro model, we characterized the production of prostaglandin E2, 6-keto-prostaglandin F1 alpha, and thromboxane B2 by normal rabbit articular chondrocytes stimulated by interleukin-1 beta. The prostanoids were produced in a dose- and time-dependent manner, which was sensitive to actinomycin D and cycloheximide. Interleukin-1 also had a pronounced, but reversible, cytostatic effect on chondrocytes, which was not attributable to prostanoid or polyamine synthesis.     

Hsp90 mediates insulin-like growth factor 1 and interleukin-1beta signaling in an age-dependent manner in equine articular chondrocytes

OBJECTIVE: Many metabolic processes in chondrocytes thought to contribute to age-related changes in the extracellular matrix are influenced by known roles of Hsp90. Age-related decreases in the level of Hsp90 have been documented in numerous cell types and could contribute to cartilage degeneration. The aim of this study was to investigate the roles of age and Hsp90 in insulin-like growth factor 1 (IGF-1) and interleukin-1beta (IL-1beta) signaling in chondrocytes. METHODS: Levels of Hsp90 messenger RNA (mRNA) and protein, with respect to age, were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. The Hsp90 inhibitor geldanamycin (50 nM, 100 nM, or 500 nM) was used to assess age-related responses to Hsp90 with concurrent IGF-1 or IL-1beta stimulation of chondrocytes. Quantitative real-time PCR was used to measure COL2A1 and matrix metalloproteinase 13 (MMP13) gene expression; Western blot analysis was performed to determine the phosphorylation status of p42/44 and Akt/protein kinase B. RESULTS: The effects of Hsp90 inhibition with geldanamycin were concentration dependent. Inhibition of Hsp90 with 100 nM or 500 nM geldanamycin blocked IGF-1-induced cell proliferation, Akt and p42/44 activation, and COL2A1 expression. Basal and IL-1beta-induced up-regulation of MMP13 mRNA was blocked by all concentrations of geldanamycin tested. Gain-of-function assays with Hsp90 resulted in increased expression of MMP13 mRNA. CONCLUSION: These results suggest that Hsp90 is involved in opposing signaling pathways of cartilage homeostasis, and that catabolic responses are more sensitive to Hsp90 inhibition than are anabolic responses. Further studies are needed to determine the role of Hsp90 inhibition in osteoarthritis in order to assess its potential as a therapeutic target.     

Effects of recombinant human interleukin-1β on rabbit articular chondrocytes

Using an in vitro model, we characterized the production of prostaglandin E₂, 6-keto-prostaglandin F_1α, and thromboxane B₂ by normal rabbit articular chondrocytes stimulated by interleukin-1β. The prostanoids were produced in a dose- and time-dependent manner, which was sensitive to actinomycin D and cycloheximide. Interleukin-1 also had a pronounced, but reversible, cytostatic effect on chondrocytes, which was not attributable to prostanoid or polyamine synthesis.     

Inhibition of interleukin-1-induced collagenase production in human articular chondrocytes in vitro by recombinant human interferon-gamma

The production of collagenase by human articular chondrocytes in response to interleukin-1 beta is inhibited in a dose-dependent manner by interferon-gamma (1-1,000 units/ml). The analysis of culture medium samples by Western blotting and the measurement of levels of tissue inhibitor of metalloproteinases suggest that the decrease in measurable collagenase activity is primarily due to the inhibition of procollagenase production. These results provide evidence of a role for interferon-gamma in limiting connective tissue degradation.     

Influence of human recombinant interleukin-1 beta on human articular cartilage. Mitotic activity and proteoglycan metabolism

The effects of human recombinant interleukin-1 beta (IL-1 beta) were investigated on recently isolated (1 to 3 weeks) and on well-established (older than 3 weeks) monolayer cultured human articular chondrocytes. IL-1 beta was found to depress 35S-proteoglycan synthesis rates and to enhance prostaglandin E2 (PGE2) production in these monolayer cultured chondrocytes. Induction of 35S-proteoglycan-degradative activity by these cells also occurred in IL-1 beta treated cultures. These "catabolin" -IL-1 activities were observed in recently isolated as well as in well-established "old" cultures. IL-1 beta increased 3H-thymidine incorporation rates in the "old" cultures. However, in recently isolated chondrocytes a dose-dependent reduction of the 3H-thymidine incorporation occurred. The depression of mitotic activity in these cells was partially abolished by indomethacin, indicating that this depression was a PGE2 effect. However, supplementing IL-1 beta with indomethacin did not raise the 3H-thymidine incorporation rates above the control levels. It can be concluded that IL-1 beta in itself is unable to induce proliferation in recently isolated cartilage cells. Our results suggest the possible existence of two different receptors for the different IL-1 beta activities. Hence, human articular chondrocytes respond differently to in vitro IL-1 beta exposure at different stages of differentiation.     

Effects of interleukin-1 beta on insulin-like growth factor-I autocrine/paracrine axis in cultured rat articular chondrocytes.

OBJECTIVE--To clarify the interaction of tissue destruction and repair of articular cartilage during inflammation, the effects of interleukin-1 beta (IL-1 beta) on the expression of insulin-like growth factor I (IGF-I), its receptor, and its binding proteins were examined. METHODS--Articular chondrocytes from five week rats were cultured in serum free medium treated with IL-1 beta (1-100 U/ml) for 24 hours. The concentration of IGF-1 in the conditioned medium was measured by RIA, and IGFBP were analysed by immunoligand blotting method. IGF-I receptors were also examined by [125I]IGF-I binding study. RESULTS--IL-1 beta induced the secretion of IGF-I and IGF-binding protein in chondrocytes; this was not inhibited by indomethacin (5 micrograms/ml). IL-1 beta also increased the number of IGF-I receptors but had no effect on receptor affinity. IL-1 beta inhibited chondrocyte proliferation, while exogenous IGF-I and growth hormone stimulated chondrocyte cell growth. IL-1 beta did not change IGF-I mRNA levels. CONCLUSION--IL-1 beta up-regulated the IGF-I autocrine/paracrine axis in cultured articular chondrocytes. These observations provide insight into the critical role played by IL-1 beta in tissue destruction and repair, and into the direct interaction between cytokines and growth factors associated with inflammatory arthropathy.     

Integrin-mediated adhesion of human articular chondrocytes to cartilage

OBJECTIVE: To determine 1) the kinetics and strength of adhesion of human articular chondrocytes to a cut cartilage surface, and 2) the role of specific integrins in mediating such adhesion, using an in vitro model. METHODS: Human articular chondrocytes isolated from cadaveric donors (mean +/- SD age 38 +/- 13 years) were cultured in high-density or low-density monolayer. Following release from culture with trypsin and a 2-2.5-hour recovery period, chondrocytes were analyzed either for adhesion to cartilage or for integrin expression by flow cytometry. RESULTS: Following culture in monolayer, adhesion of chondrocytes to cartilage increased with time, from 6-16% at 10 minutes to a maximum of 59-82% at 80-320 minutes. After 80 minutes of adhesion, the resistance of cells to flow-induced shear stress (50% detachment) was approximately 21 Pa. Chondrocyte adhesion to cartilage decreased with pretreatment of cells with monoclonal antibodies that bound to and blocked certain integrins. After an 80-minute incubation time, adhesion of chondrocytes cultured in high-density monolayer decreased from the value of IgG1-treated controls (55%) with blocking of the beta1 integrin subunit (to 23%) or with blocking of alpha 5 beta 1 (to 36%). Following expansion of chondrocytes in low-density monolayer, the mechanisms of adhesion to cartilage were generally similar. After an 80-minute incubation time, adhesion of chondrocytes cultured in low-density monolayer decreased from the value of IgG1-treated controls (62%) with blocking of the beta1 integrin subunit (to 30%) or with blocking of alpha 5 beta 1 (to 44%). Additionally, adhesion of these cells decreased to 46% by blocking of alpha v beta 5, with a similar trend in effect for chondrocytes cultured in high-density monolayer. Blocking of the alpha 1 or alpha 3 integrin subunits or alpha v beta 3 had no detectable effect on adhesion, even though these receptors were detected by flow cytometry. CONCLUSION: Under the culture and seeding conditions studied, beta1, alpha 5 beta 1, and alpha v beta 5 integrins mediate human chondrocyte adhesion to cartilage. These chondrocyte integrins have a potential role in the initial adhesion and retention of chondrocytes at a cartilage defect site following clinical procedures of chondrocyte transplantation.     

Isolation and cultivation of human articular chondrocytes

Adult human articular chondrocytes were isolated from surgically excised articular cartilage from 15 patients suffering from trauma or disease of the knee. Cells were isolated with an enzymatic digestion method and cultured with F12 medium supplemented with serum. The effects of serum and basic fibroblast growth factor (bFGF) on the growth of cultured chondrocytes were studied. Serum stimulated the growth of chondrocytes at concentrations from 1-30%. bFGF significantly stimulated the growth of chondrocytes in a dose-dependent manner at concentrations from 1-100 ng/ml. Chondrocytes grew well in F12 medium supplemented with 10% fetal bovine serum (FBS). These cultured chondrocytes could be passaged for many generations and revealed an average of 2,702-fold increase of cell numbers during 2-6 months period (cumulative population doublings = 11 times). Cumulative population doublings increased to 24 times in cell cultured with medium supplemented with bFGF (10 ng/ml). Immunocytochemical study using anti-S-100 antibodies demonstrated that these cultures were pure cell cultures of chondrocytes. We have demonstrated that cell cultures of adult human articular chondrocytes can be established with these methods. Cultured chondrocytes provide an in vitro model system for studying the physiology and pathology of human articular chondrocytes and may be used for autologous transplantation of chondrocytes to treat articular cartilage defects.     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.

     

Production of leukemia inhibitory factor by human articular chondrocytes and cartilage in response to interleukin-1 and tumor necrosis factor α

Objective. To investigate the production of leukemia inhibitory factor (LIF) by human articular chondrocytes and cartilage. Methods. Chondrocytes and cartilage were cultured with and without added cytokines, and the conditioned media assayed for LIF by a specific radioreceptor competition assay. Results. Interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) stimulated LIF production by chondrocyte monolayers and cartilage. LIF was generally not detected in unstimulated cultures. Northern blot analysis showed increased amounts of LIF messenger RNA in IL-1– and TNFα-treated chondrocyte cultures. Conclusion. Chondrocytes, stimulated by IL-1 and/or TNFα, are potential contributors to the elevated levels of LIF observed in the synovial fluids of patients with rheumatoid arthritis and other inflammatory arthritides.