The effect of interleukin-1β and transforming growth factor β on cathepsin B activity in human articular chondrocytes

Cathepsin B is a lysosomal cysteine proteinase, thought to be involved in the degradation of connective tissue breakdown products internalized by endocytosis. It has also been implicated in the extracellular matrix degradation of collagens and proteoglycans in disease states such as tumour invasion, rheumatoid arthritis and osteoarthritis. To date it is still unclear which factors can potentially modulate the release and/or activation of this enzyme.The aim of this study was to investigate the effect of interleukin-1 and transforming growth factor on cathepsin B activity in both cell lysates and cell supernatants, using first passage cultures of human articular chondrocytes. Enzyme activity was determined using a specific synthetic substrate for cathepsin B, in a fluorimetric assay.     

Anti-arthritic effects of crocin in interleukin-1β-treated articular chondrocytes and cartilage in a rabbit osteoarthritic model

Objective

Interleukin-1β-mediated production of matrix metalloproteinases (MMPs) plays a pivotal role in the process of osteoarthritis. Crocin, a pharmacologically active component of Crocus sativus L. (saffron), has been used in Chinese traditional medicine. In this study, we aimed to investigate the effects of crocin on MMP-1, MMP-3 and MMP-13 expression in rabbit chondrocytes induced by interleukin-1β (IL-1β) and in an experimental rabbit model induced by anterior cruciate ligament transection.

Methods

Chondrocytes isolated from the articular cartilage of 4-week-old rabbits were cultured and passaged. Confluent chondrocytes were treated with various concentrations of crocin in the presence or absence of IL-1β (10 ng/ml) for 24 h. Quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting were used to investigate the expression of inducible MMP-1, MMP-3 and MMP-13. In addition, the in-vivo effects of crocin were assessed by morphological and histological analysis.

Results

IL-1β markedly upregulated the expression of MMP-1, -3 and -13 in chondrocytes, and this activation was inhibited by co-incubation with crocin in a dose-dependent manner, in contrast with the control group. Moreover, crocin inhibited IL-1β-induced activation of the nuclear factor kappa B pathway through suppressing degradation of inhibitory-kappa-B-α. In-vivo investigations showed that crocin ameliorated cartilage degeneration and that expression of the MMP-1, -3 and -13 genes in cartilage was significantly inhibited by crocin.

Conclusion

Taken together, our findings suggest that the anti-inflammatory activity of crocin may be of potential value in the prevention and treatment of osteoarthritis.     

Inhibition of histone deacetylases antagonized FGF2 and IL-1β effects on MMP expression in human articular chondrocytes

Fibroblast growth factor-2 (FGF2) and interleukin-1β (IL-1β) stimulate the expression of matrix metalloproteinases (MMPs) in articular chondrocytes, which may contribute to cartilage degradation and development of osteoarthritis. Histone deacetylases (HDACs) have recently been implicated in the regulation of MMP gene expression. To investigate the functional involvement of HDACs in the signaling pathway of FGF2 and IL-1β, we examined the effects of HDAC inhibition on activities of FGF2 or IL-1β on gene expression of MMP-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5), collagen type II, and aggrecan. Human articular chondrocyte cultures were treated with FGF2 or IL-1β in the presence or absence of HDAC inhibitor (trichostatin A, TSA). Gene expression levels after treatments were assessed using quantitative real time PCR. Results showed that FGF2 and IL-1β both increased MMP-1 and -13 expression, while IL-1β also increased MMP-3 mRNA levels. These effects were attenuated in the presence of TSA in a dose dependent manner. In contrast to the effects on MMPs, FGF2 decreased mRNA levels of ADAMTS-5, which was not affected by HDAC inhibition. FGF2, IL-1β, and TSA inhibited expression of aggrecan, while TSA also decreased mRNA levels of collagen type II. These findings showed that HDAC inhibition antagonized FGF2 and IL-1β induced MMP expression. Combination of FGF2 and the HDAC inhibitor decreases both anabolic and catabolic genes, which may slow the cartilage turnover and be beneficial for maintaining cartilage integrity.     

Synergism of basic fibroblast growth factor and interleukin-1β to induce articular cartilage-degradation in the rabbit

Interleukin-1 (IL-1) and basic fibroblast growth factor (bFGF) synergistically induce proteasesin vitro. To investigate this synergyin vivo, we injected IL-1 and bFGF alone and in combination into the lapine knee. Three days later, we compared the glycosaminoglcan (GAG) content of tibial cartilage of cytokinetreated and contralateral control-knees. IL-1 caused significant increases of granulocytes and GAG in the synovial fluid but minor cartilage-GAG losses of 11, 11 & 16% at 5kU, 10 kU and 100 kU IL-1/knee, respectively. bFGF at 2 and 10 g/knee caused no changes. 10 g bFGF in combination with 10 kU IL-1 induced a 33% GAG loss (p<0.01) that lasted 21 days. IL-1/bFGF induced cartilage degradation may be useful to 1) evaluate agents which modulated proteoglycan catabolism and 2) assess factors that accelerate cartilage-repair.     

Elevated serum levels of interleukin-6, interleukin-1β and human chorionic gonadotropin in pre-eclampsia

Citation Kalinderis M, Papanikolaou A, Kalinderi K, Ioannidou E, Giannoulis C, Karagiannis V, Tarlatzis BC. Elevated serum levels of interleukin‐6, interleukin‐1β and human chorionic gonadotropin in pre‐eclampsia. Am J Reprod Immunol 2011; 66: 468–475 Problem Pre‐eclampsia (PE) is a pregnancy‐specific syndrome of unknown aetiology. It is believed to involve an inflammatory process. The aim of the study was to investigate and compare the concentrations of two proinflammatory cytokines interleukin‐6 (IL‐6) and interleukin‐1β (IL‐1β) and to evaluate the possible interaction between them and human chorionic gonadotropin (hCG) in women with normotensive pregnancy and PE. Method of study A prospective case–control study was carried out in 30 women with PE and 30 normotensive controls. Serum IL‐1β, IL‐6 and hCG levels were determined by enzyme‐linked immunosorbent assay (ELISA) and automated immunofluorescent assay, respectively. Results Serum IL‐6, IL‐1β and hCG levels were significantly increased in women with PE compared to controls (P < 0.001 for each); however, no correlation was found between IL‐6, IL‐1β and hCG. Conclusion Our results highlight the inflammatory origin of PE and reinforce the possible role of hCG in the complex aetiology of its pathogenesis.     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.     

Comparison of abilities of recombinant interleukin-1α and -β and noninflammatory IL-1β fragment 163–171 to upregulate C3b receptors (CR1) on human neutrophils and to enhance their phagocytic capacity

Both recombinant IL-1 and - caused an upregulation of C3b receptors (CR1) on human neutrophils and caused a receptor-mediated enhancement of phagocytosis of C3b·IgG-coated microspheres by these leukocytes. The and forms of the recombinant cytokine were of comparable potency regarding CR1 upregulation, although both generally had less than 25% of the potency of FMLP in this respect. Recombinant IL-1 was slightly more potent than the form of the cytokine regarding phagocytosis of opsonized microspheres and, again, both forms were less potent than FMLP in causing an enhancement of phagocytosis by neutrophils. The synthetic noninflammatory immunostimulatory nonapeptide corresponding to residues 163–171 of IL-1 was completely inert with respect to upregulation of CR1 on neutrophils and the enhancement of phagocytosis by these cells. Thus this domain in the intact IL-1 molecule apparently is not involved in CR1 upregulation and the ensuing enhancement in phagocytosis by neutrophils, although it is apparently important in the immunostimulatory activity regarding the proliferation of lymphocytes.     

Dose- and time-dependent effects of histamine on hypothalamic levels of interleukin-1β in rats

Some recent studies give support to the potential interaction between histamine (HA) and interleukin-1 (IL-1) in the central nervous system (CNS). At the peripheral level, HA acts as an immune modulator, but little is known on the neuroimmune role of this biogenic amine in the CNS. In the present study we have investigated the effects of HA, mepyramine, famotidine, thioperamide, and L-histidine on hypothalamic IL-1. HA induced a time- and dose-dependent decrease in the concentration of IL-1. The maximum effect was obtained 30 minutes after injection. The HA-induced IL-1 response in the hypothalamus was not inhibited by either mepyramine, famotidine or thioperamide. In addition, L-histidine exerted the same effect as HA. The interaction between HA and IL-1 in the CNS might be linked to neuroendocrine regulation as well as to neurotrophic activity and neuroimmune function.     

Senescence of chondrocytes in aging articular cartilage: GADD45β mediates p21 expression in association with C/EBPβ in senescence-accelerated mice

Growth arrest and DNA damage-inducible protein 45β (GADD45β) is expressed in normal and early osteoarthritic articular cartilage. We recently reported that GADD45β enhances CCAAT/enhancer binding protein β (C/EBPβ) activation in vitro. This study was undertaken in order to determine whether GADD45β is expressed with C/EBPβ in aging articular cartilage. We also investigated whether the synergistic expression of GADD45β and C/EBPβ may be involved in the mechanism of chondrocyte senescence. Senescence-accelerated mice (SAMP1) were used as a model of aging. GADD45β, C/EBPβ, and p21 were analyzed by immunohistochemistry. A luciferase reporter assay using ATDC5 cells was performed in order to examine p21 as a target gene of the GADD45β/C/EBPβ cascade. GADD45β exhibited increased expression in the aging articular cartilage of SAMP1 mice compared to that in control mice. The co-localization of GADD45β and C/EBPβ was confirmed by double immunostaining. The synergistic mechanisms of GADD45β and C/EBPβ on the gene regulation of p21, a molecule related to cellular senescence, were verified by a p21-luciferase reporter assay. Co-expression of C/EBPβ and p21 was confirmed. These observations suggest that the synergism between GADD45β and C/EBPβ may play an important role in cellular senescence in the aging articular cartilage.     

Disruption of the integrity of human peritoneal mesothelium by interleukin-1β and tumor necrosis factor-α

Inflammatory and neoplastic disease processes of the abdominal cavity are frequently associated with disruption of the integrity of the peritoneal mesothelium. In the present study, we analyzed the effects of the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) on the morphology and expression of adhesion molecules of human peritoneal mesothelial cells (HPMC). Treatment of HPMC with IL-1 and TNF- resulted in a time- and dose-dependent alteration of the normal cobblestone morphology of the mesothelium with loss of polarization, cellular retraction and exposure of the submesothelial matrix. The effect was already observable after 6 h of treatment and was most pronounced at a dose of 10 ng/ml of IL-1 or TNF-. These morphological alterations were associated with a significant rearrangement of the expression of mesothelial adhesion molecules as detected by flow cytometry. IL-1 and TNF- both led to a loss of the expression of the hemidesmosomal integrin subunits 6 (P<0.01 and P<0.001) and 4 (P<0.01) and an increased expression of the integrin subunit 5 (P<0.001 and P<0.01). IL-1 furthermore upregulated the expression of the integrin subunits 1, 2 and the adhesion molecule CD44 while the latter was downregulated by TNF-. Our data indicate that IL-1 and TNF- may significantly affect disease processes of the abdominal cavity by their potential to disrupt the mesothelial basal cell-matrix adhesion and, thus, the integrity of the peritoneal mesothelial cell lining.     

TGF-β1 and IGF-1 influence the re-differentiation capacity of human chondrocytes in 3D pellet cultures in relation to different oxygen concentrations

To prevent de-differentiation of chondrocytes in vitro, the 3D environment, growth factors and different oxygen concentrations were considered. In this in vitro study, we quantified the influence of insulin-like growth factor (IGF)-1 and/or transforming growth factor (TGF)-β1 under differing oxygen (5/21% O(2)) levels on the proliferation and synthesis rates of hyaline extracellular matrix (ECM) components in chondrogenic pellet cultures. Human chondrocytes isolated from articular cartilage were transferred into conical tubes to form pellets. Pellets were stimulated with TGF-β1 and/or IGF-1. After 2 and 5 weeks of cultivation the DNA concentration and expression of pro-collagen type 1, type 2 and aggrecan were analysed. Under hypoxia the DNA content remained stable. In contrast, under normoxia, cells showed an increase of DNA concentration after stimulation with TGF-β1/IGF-1 and TGF-β1. Nevertheless, DNA contents under normoxia did not reach the values of hypoxic-cultivated cells. Under both culture conditions a reduced synthesis of pro-collagen type 1 could be determined. Although the expression of pro-collagen type 2 was significantly higher under normoxia, a decrease in the case of TGF-β1/IGF-1- and IGF-1-stimulated cells was observed. Under hypoxia pro-collagen type 2 contents remained stable or increased for TGF-β1/IGF-1-stimulated cells. Furthermore, incubation with growth factors resulted in aggrecan accumulation under hypoxia, while a reduced expression under normoxia could be determined for TGF-β1/IGF-1- and IGF-1-stimulated cells. Our results demonstrate that the treatment with growth factors causes differences in the expression of ECM compounds within pellet cultures. While under normoxia TGF-β1 alone leads to a positive effect of the expression of hyaline cartilage-specific ECM components, an additive effect of both growth factors was only determined under hypoxia.     

Decreased expression of P-glycoprotein in interleukin-1β and interleukin-6 treated rat hepatocytes

OBJECTIVE AND DESIGN: As acute inflammation is known to cause a reduction in hepatic P-Glycoprotein (PGP) expression and activity in rats, we tested the hypothesis that the pro-inflammatory cytokines interleukin (IL-)1beta and IL-6 also mediate reductions in PGP. METHODS: Hepatocytes were incubated with 0-50 ng/ml of cytokine for 24-72 h. PGP/mdr expression was examined by immunodetection and quantitative RT-PCR analysis and PGP efflux activity was assayed. RESULTS: PGP protein was significantly reduced in cells treated for 3 days with IL-1beta and 24 h with IL-6 (p < 0.05), maximal effects occurring at 5 ng/ml for each cytokine. PGP activity was reduced in both IL-1beta and IL-6 treated cells (p < 0.05). mdr1 mRNA was decreased in cells treated with IL-6, but not IL-1beta. spgp and mdr2 were not affected. CONCLUSIONS: Our data indicate that IL-6 and IL-1beta have suppressive effects on the expression and activity of PGP in cultured hepatocytes, likely occurring through distinct mechanisms. These cytokines may have a potential role in PGP regulation during inflammatory responses.     

Effects of recombinant human IL-Iβ on production of prostaglandin E2, leukotriene B4, NAG,and superoxide by human synovial cells and chondrocytes

The effects of recombinant human IL-1 on the production of prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄),N-acetyl--D-glucosaminidase (NAG), and superoxide by synovial cells and chondrocytes derived from osteoarthritis patients were determined. IL-1 markedly enhanced PGE₂ production in chondrocytes and, to the lesser extent, in synovial cells. Synovial cells and chondrocytes spontaneously released LTB₄ into culture medium and IL-1 significantly inhibited LTB₄ production by these cells. IL-1 significantly suppressed the release of NAG and superoxide by synovial cells, whereas it significantly enhanced the production of NAG and superoxide by chondrocytes. Production of intracellular superoxide dismutase by synovial cells was significantly enhanced on incubation with IL-1, but that of chondrocytes was not altered. IL-6, unlike IL-1, significantly suppressed the production of NAG and superoxide by synovial cells and chondrocytes.These results suggest that IL-1 has differing effects on the release of mediators by synovial cells and chondrocytes and that these cells also vary in their responses to IL-1 and IL-6.     

Pathogenetic role and clinical significance of interleukin-1β in cancer

In recent years, pro-oncogenic mechanisms of the tumour microenvironment (ТМЕ) have been actively discussed. One of the main cytokines of the TМЕ is interleukin-1 beta (IL-1β), which exhibits proinflammatory properties. Some studies have shown an association between an increase in IL-1β levels and tumour progression. The purpose of this review is to analyse the pathogenic mechanisms induced by IL-1β in the TМЕ, as well as the diagnostic significance of the presence of IL-1β in patients with cancer and the efficacy of treatment with IL-1β inhibitors. According to the literature, IL-1β can induce an increase in tumour angiogenesis due to its effects on the differentiation of epithelial cells, pro-angiogenic molecule secretion and expression of adhesion molecules, thus increasing tumour growth and metastasis. IL-1β is also involved in the suppression of anti-tumour immune responses. The expression and secretion of IL-1β has been noted in various types of tumours. In some clinical studies, an elevated level of IL-1β was found to be associated with low efficacy of anti-cancer therapy and a poor prognosis. In most experimental and clinical studies, the use of IL-1β inhibitors contributed to a decrease in tumour mass and an increase in the response to anti-tumour drugs.     

In vivo morphometry and functional analysis of human articular cartilage with quantitative magnetic resonance imaging – from image to data, from data to theory

Analyses of form-function relationships and disease processes in human articular cartilage necessitate in vivo assessment of cartilage morphology and deformational behavior. MR imaging and advanced digital post-processing techniques have opened novel possibilities for quantitative analysis of cartilage morphology, structure, and function in health and disease. This article reviews work on three-dimensional post-processing of MR image data of articular cartilage, summarizing studies on the accuracy and precision of quantitative analyses in human joints. It presents normative values on cartilage volume, thickness, and joint surface areas in the human knee, and describes the correlation between different joints and joint surfaces as well as their association with gender, body dimensions, and age. The article summarizes ongoing work on functional adaptation of articular cartilage to mechanical loading, analyses of in situ cartilage deformation in intact joints in vivo and in vitro, and the quantitative evaluation of cartilage tissue loss in osteoarthritis. We describe evolving techniques for assessment of the structural/biochemical composition of articular cartilage, and discuss future perspectives of quantitative cartilage imaging in the context of joint mechanics, mechano-adaptation, epidemiology, and osteoarthritis research. Specifically, we show that fat-suppressed gradient echo sequences permit valid analysis of cartilage morphology, both in healthy and severely osteoarthritic joints, as well as highly reproducible measurements (CV%=1 to 3% in the knee, and 2 to 10% in the ankle). Relatively small differences in cartilage morphology exist between both limbs of the same person (～5%), but large differences between individuals (CV% ～20%). Men display only slightly thicker cartilage then women (～10%), but significantly larger joint surface areas (～25%), even when accounting for differences in body weight and height. Weight and height represent relatively poor predictors of cartilage thickness (r² <15%), but muscle cross section areas display more promising correlations (r² >40%). The level of physical exercise (sportive activity) does not account for interindividual differences in cartilage thickness. The thickness appears to decrease slightly in the elderly – in particular in women, even in the absence of osteoarthritic cartilage lesions. Strenuous physical exercises (e.g., knee bends) cause a 6% patellar cartilage deformation in young individuals, but significantly less deformation in elderly men and women (<3%). The time required for full recovery after exercise (fluid flow back into the matrix) is relatively long (～90 min). Static in situ compression of femoropatellar cartilage with 150% body weight produces large deformations after 4 h (～30% volume change), but only very little deformation during the first minutes of loading. Quantitative analyses of magnetization transfer and proton density hold promise for biochemical evaluation of articular cartilage, and are shown to be related to the deformational behavior of the cartilage. Application of these techniques to larger cohorts of patients in epidemiological and clinical studies will establish the role of quantitative cartilage imaging not only in basic research on form-function relationships of articular cartilage, but also in clinical research and management of osteoarthritis.     

Change of inflammatory cytokines after co-culture of inflamed cartilage blocks and chondrocytes transfected by interleukin-β1 receptor antagonist gene and transforming growth factor-β1 gene in vitro

Objective To evaluate the in vitro effects of recombinant human interluekin-β1 receptor antagonist(IL-1Ra) gene and transforming growth factor-β1 (TGF-β1) gene on rabbit osteoarthritis (OA).Methods Articular cartilages were extracted from mature New Zealand rabbits and by enzyme digestion,isolated for chondrocytes which were then identified with specific extracellular matrix collagen type Ⅱ stained immunocytochemistry.The chondrocytes were divided into IL-1Ra-transfected group (group A), TGF-β1?transfected group (group B) , combined IL-1Ra- and TGF-β1-transfected group (group C) , untransfected group (group D) and the blank control group (group E).LipofectamineTM 2000 Reagent was used as the vehicle for transfection among groups A, B and C.All the groups of chondrocytes were co-cultured with fragmented articular cartilages and added with 20 ng IL-β 1?expect for group E.The transgenic expression of chondrocytes was detected under fluorescence microscope at 12h,24h,2d,4d and 6 d after transfection and co-culture.In addition, radioimmunoassay (RIA) was used to determine the levels of IL-1βand TNF-α in each group at 2 d, 4 d and 6 d after transfection and co-culture.Results The chondrocytes were successfully isolated and cultured.Collagen type Ⅱ stained immunocytochemistry showed the brownish - yellow cytoplasm and unstained chromophobic nuclei.Under fluorescence microscope, the expression of enhanced green fluorescent protein was observed in groups A, BandC, which peaked at 24 hours after transfection (16.16±2.71)% vs (16.54±2.91)% vs (17.20±2.39)% and gradually declined 2 d later.At any time spots, the IL-1βevel was highest in group D, followed by group B, group A, group C, and group E.The level of TNF-a in each group was ordered by group D＞group A＞group B＞group C＞group E on days 2 and 6, and by group E＞group A＞group B＞group C＞group D on day 4.The level of TNF-α in group A was slightly higher than that of group B, but the difference was not statistical significance.There were statistical difference among the other groups.The expressions of IL-1βand TNF-α in groups A, B and C were significantly lower on day 6 than those on days 2 and 4.The level of IL-1βin groups D and E did not change with time, while the level of TNF -α was the lowest in group D and highest in group E on day 4.ConclusionsTransfection with IL-1Ra or TGF-β1 can reduce the level of inflammatory cytokines.Combined use of IL-1Ra and TGF-β1 genes may show control of inflammatory response and provide evidences for gene therapy of osteoarthritis.     

Endogenous nitric oxide and prostaglandin E2 do not regulate the synthesis of each other in interleukin-1β-stimulated rat articular cartilage

Increased levels of nitric oxide (NO) and prostaglandins (PG) are present in the synovial fluid from patients with rheumatoid arthritis and osteoarthritis. Interleukin-1 (IL-1) has been shown to induce the synthesis of both of these mediators. The present work was designed to study the interactions of NO and PGE₂ synthesis induced by IL-1 in rat articular cartilage. Incubation of intact cartilage with IL-1 resulted in different dose response curves for NO and PGE₂ synthesis. Two inhibitors of nitric oxide synthase N-monomethyl-L-arginine (L-NMMA) and L-N-iminoethyl-ornithine, (L-NIO), abolished the IL-1-induced nitrite production but failed to have any influence on the PGE₂ synthesis. Exogenous NO, produced by two chemically different NO-releasing compounds (SIN-1 and GEA 3175) had no effect on PGE₂ synthesis in articular cartilage. Dexamethasone and ketoprofen inhibited IL-1 induced PGE₂ production, while nitrite synthesis remained unaltered. Acetylsalisylic acid (ASA) reduced PGE₂ synthesis and had a slight inhibitory action also on NO production. In conclusion, our results show, that IL-1 induces the synthesis of both PGE₂ and NO in articular cartilage but these two inflammatory mediators are not mediating the synthesis of one another.     

Effects of high mobility group box protein-1, interleukin-1β, and interleukin-6 on cartilage matrix metabolism in three-dimensional equine chondrocyte cultures

The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1β, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1β treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1β-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1β, whereas slight anabolic effects were induced by IL-6 and HMGB-1.