Complement C1s activation in degenerating articular cartilage of rheumatoid arthritis patients: immunohistochemical studies with an active form specific antibody.

OBJECTIVE: The first complement component C1s was reported to have novel functions to degrade matrix components, besides its activities in the classic complement pathway. This study explores participation of C1s in articular cartilage degradation in rheumatoid arthritis (RA). METHODS: Normal articular cartilage (n = 6) and cartilage obtained from joints with RA (n = 15) and osteoarthritis (OA, n = 10) were immunostained using anti-C1s monoclonal antibodies PG11, which recognises both active and inactive C1s, and M241, which is specifically reactive to activated C1s. The effects of inflammatory cytokines on C1s production by human articular chondrocytes were also examined by sandwich ELISA. RESULTS: In normal articular cartilage, C1s was negative in staining with both PG11 and M241. In contrast, degenerating cartilage of RA was stained with PG11 (14 of 15 cases), and in most of the cases (13 of 15 cases) C1s was activated as revealed by M241 staining. In OA, C1s staining was restricted in severely degrading part of cartilage (5 of 10 cases), and even in that part C1s was not activated. In addition, C1s production by chondrocytes in vitro was increased by an inflammatory cytokine, tumour necrosis factor alpha. CONCLUSION: These results suggest that C1s activated in degenerative cartilage matrix of RA but not in that of OA. C1s is thought to participate in the pathogenesis of RA through its collagenolytic activity in addition to the role in the classic cascade.     

Prevention of cartilage destruction with intraarticular osteoclastogenesis inhibitory factor/osteoprotegerin in a murine model of osteoarthritis

OBJECTIVE: To investigate the effect of osteoclastogenesis inhibitory factor/osteoprotegerin (OPG) on chondrocytes in the development of osteoarthritis (OA) in vivo. METHODS: To determine the role of endogenous OPG in the progression of OA, OA was surgically induced in OPG+/- mice and their wild-type (WT) littermates. To determine the role of exogenous OPG, knee joints of C57BL/6J mice with surgically induced OA were injected intraarticularly with recombinant human OPG (rHuOPG) or vehicle 5 times a week. All mice were euthanized 4 weeks after OA induction; joints were harvested and evaluated immunohistochemically. RESULTS: Although OA changes were induced in both WT and OPG+/- mice, the degenerative changes in the articular cartilage were significantly enhanced in OPG+/- mice. In C57BL/6J mice with surgically induced OA, intraarticular OPG administration protected the articular cartilage from the progression of OA. The Mankin and cartilage destruction scores in OPG-treated animals were approximately 50% of those seen in the control group. Furthermore, OPG administration significantly protected articular cartilage thickness. Findings of the TUNEL assay indicated that rHuOPG prevented chondrocyte apoptosis in joints with surgically induced OA. Results of immunostaining indicated that OPG protein was detected in the synovium and in resident chondrocytes at higher levels in the OPG-treated group than in the control group. CONCLUSION: These data indicate that endogenous OPG had a protective effect against the cartilage destruction that occurs during OA progression. Furthermore, direct administration of rHuOPG to articular chondrocytes prevented cartilage destruction in an experimental murine model of OA via prevention of chondrocyte apoptosis.     

Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage

OBJECTIVE: To determine the presence of mesenchymal progenitor cells (MPCs) in human articular cartilage. METHODS: Primary cell cultures established from normal and osteoarthritic (OA) human knee articular cartilage were analyzed for the expression of CD105 and CD166, cell surface markers whose coexpression defines mesenchymal stem cells (MSCs) in bone marrow and perichondrium. The potential of cartilage cells to differentiate to adipogenic, osteogenic, and chondrogenic lineages was analyzed after immunomagnetic selection for CD105+/CD166+ cells and was compared with bone marrow-derived MSCs (BM-MSCs). RESULTS: Up to 95% of isolated cartilage cells were CD105+ and approximately 5% were CD166+. The mean +/- SEM percentage of CD105+/CD166+ cells in normal cartilage was 3.49 +/- 1.93%. Primary cell cultures from OA cartilage contained significantly increased numbers of CD105+/CD166+ cells. Confocal microscopy confirmed the coexpression of both markers in the majority of BM-MSCs and a subpopulation of cartilage cells. Differentiation to adipocytes occurred in cartilage-derived cell cultures, as indicated by characteristic cell morphology and oil red O staining of lipid vacuoles. Osteogenesis was observed in isolated CD105+/CD166+ cells as well as in primary chondrocytes cultured in the presence of osteogenic supplements. Purified cartilage-derived CD105+/CD166+ cells did not express markers of differentiated chondrocytes. However, the cells were capable of chondrocytic differentiation and formed cartilage tissue in micromass pellet cultures. CONCLUSION: These findings indicate that multipotential MPCs are present in adult human articular cartilage and that their frequency is increased in OA cartilage. This observation has implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these progenitor cells might be involved in the pathogenesis of arthritis.     

Anti-Fas-induced apoptosis in chondrocytes reduced by hyaluronan: evidence for CD44 and CD54 (intercellular adhesion molecule 1) invovement

OBJECTIVE: To investigate the in vitro effect of therapeutic hyaluronan (HA) of 500-730 kd on anti-Fas-induced apoptosis of chondrocytes from osteoarthritis (OA) patients, and to assess its mechanism of action by analyzing the role of the 2 HA receptors, CD44 and CD54 (intercellular adhesion molecule 1 [ICAM-1]). METHODS: Chondrocytes isolated from human OA knee cartilage were cultured and the effect of HA on both spontaneous and anti-Fas-induced apoptosis was evaluated. Apoptosis was analyzed by JAM test (for quantitative analysis of fragmented DNA), cell death detection immunoassay (for quantitative analysis of oligonucleosome), TUNEL assay, and electron microscopy. Blocking experiments with anti-CD44 and anti-CD54 alone or in combination were performed to investigate the HA mechanism of action. RESULTS: Both quantitative tests demonstrated that anti-Fas significantly induced apoptosis of isolated OA chondrocytes. HA at 1,000 microg/ml significantly reduced the anti-Fas-induced apoptosis of chondrocytes but did not affect spontaneous chondrocyte apoptosis. These data were also confirmed by TUNEL staining and by electron microscopy morphologic evaluation. The antiapoptotic effects of HA on anti-FAS-induced chondrocyte apoptosis were significantly decreased by both anti-CD44 (mean +/- SD 57 +/- 12% inhibition) and anti-ICAM-1 (31 +/- 22% inhibition). The mixture of the 2 antibodies had an additive effect, since the rate of inhibition increased to 87 +/- 13%. CONCLUSION: These data demonstrate that 500-730-kd HA exerts an antiapoptotic effect on anti-FAS-induced chondrocyte apoptosis by binding its specific receptors (CD44 and ICAM-1). Furthermore, this HA fraction may be able to slow down chondrocyte apoptosis in OA by regulating the processes of cartilage matrix degradation.     

High CCL18/PARC expression in articular cartilage and synovial tissue of patients with rheumatoid arthritis

OBJECTIVE: We studied the role of CCL18/pulmonary and activation-regulated chemokine (PARC) in rheumatoid arthritis (RA). METHODS: Human cartilage tissues and synovial membranes were obtained from patients with RA and with osteoarthritis (OA). Sera samples were obtained from RA patients, OA patients, healthy controls, and patients with flu, and synovial fluid (SF) from patients with RA and OA. Real-time PCR was performed with RNA from cartilage samples. Immunohistochemical analysis of CCL18/PARC was done with RA and OA cartilage and synovial tissue. Levels of CCL18/PARC in serum and SF were evaluated by ELISA. RESULTS: CCL18/PARC mRNA was expressed at significantly higher levels in RA cartilage than in OA (p = 0.0001) and control (p < 0.0001) samples. CCL18/PARC mRNA expression was much higher in RA synovial membrane than OA samples (p = 0.0001). All RA cartilage and synovial tissue samples exhibited medium to strong staining for CCL18/PARC. Serum levels of CCL18/PARC were higher in RA patients (156.21 +/- 125.73 ng/ml, n = 71) than in OA patients (64.54 +/- 40.90 ng/ml, n = 12) and controls (28.04 +/- 10.96 ng/ml, n = 20). Levels of CCL18/PARC in RA SF (275.20 +/- 228.16 ng/ml, n = 15) were higher than in OA (33.13 +/- 14.84 ng/ml, n = 6; p = 0.0198). CCL18/PARC levels correlated significantly with rheumatoid factor levels (r = 0.431, p = 0.0040), but not with matrix metalloproteinase-3, erythrocyte sedimentation rate, and C-reactive protein. CONCLUSION: CCL18/PARC was highly expressed in RA articular cartilage and synovial tissue compared with OA samples. Our data indicated that CCL18/PARC levels are not related to the conditions of generalized inflammation, but are related to the pathogenesis of RA.     

Interactions of synovial fluid immunoglobulins with chondrocytes.

OBJECTIVE. To study the interaction of synovial fluid (SF) immunoglobulins with living chondrocytes, and to evaluate the relative contribution of type II collagen (CII) antibodies. METHODS. SF of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and gout were incubated with isolated bovine articular chondrocytes. Ig binding was measured by flow cytometry and by quantitation with 125I-labeled anti-IgG and anti-IgM. Complement-dependent cytotoxicity was determined by 51Cr release. Immunoglobulin binding and cytotoxicity were compared between chondrocytes obtained from the superficial and from the deep cartilage zones. RESULTS. Significantly greater IgG and IgM binding was found with RA SF compared with OA or gout SF. Chondrocytes bound more Ig than did fibroblasts. The relative contribution of anti-CII antibodies to Ig binding was studied following absorption of the SF with bovine CII, and by incubation with bacterial collagenase-treated chondrocytes. There was a small but significant reduction in IgG and IgM binding with SF samples that were positive for anti-CII. RA SF exhibited modest, but significantly greater complement-dependent cytotoxicity than OA SF. Gel chromatography fractionation indicated that IgM antibodies were responsible for the cytotoxic activity. Additional studies showed that SF IgM antibodies bound preferentially to, and killed chondrocytes obtained from, the superficial layers of cartilage. CONCLUSION. Anti-CII antibodies contained in RA SF represent one of many antibody specificities reacting with chondrocyte membrane antigens. Chondrocyte-reactive SF antibodies may play an important pathogenic role in the processes leading to irreversible cartilage damage in RA. These deleterious effects appear to be exerted particularly on chondrocytes located near the articular surface of cartilage.     

Increased Bcl-2/p53 ratio in human osteoarthritic cartilage: a possible role in regulation of chondrocyte metabolism

OBJECTIVE: To determine whether Bcl-2, p53, and Fas/CD95 help to control cartilage metabolism. METHODS: Six normal and 14 osteoarthritic (OA) cartilage samples were examined, and two zones from each sample showing the least (Min) and most (Max) anatomical damage were selected. Chondrocytes were isolated by sequential enzymatic digestion and freshly processed. Bcl-2, p53, and Fas/CD95 expression was evaluated by immunofluorescence and FACS analysis; the cell cycle was analysed using propidium iodide, and chondrocyte proliferation assessed by [(3)H]thymidine incorporation. RESULTS: Intracellular levels of Bcl-2 were significantly higher in Max (27.5%) than in Min (21%, p<0.01) OA or normal chondrocytes (18.5%, p<0.01). Intracellular p53 expression was significantly decreased in Max (25.5%) compared with Min (37%, p<0.01) OA or normal cartilage (41.5%, p<0.05). Fas/CD95 receptor expression on surface chondrocytes did not significantly differ between OA and normal cartilage. Cell cycle analysis showed that the proportion of activated chondrocytes in the S phase was significantly higher in Max (69%) than in Min (49%) OA or normal cartilage (43%). The prevalence of proliferating chondrocytes progressively increased according to the degree of OA damage (mean (SEM) Min 1247 (260), Max 2423 (460), p<0.05). Chondrocyte [(3)H]thymidine uptake correlated positively with Bcl-2 (r(s) = 0.62, p = 0.009) and correlated inversely with p53 levels (r(s) = -0.55, p = 0.02). CONCLUSIONS: Bcl-2 and p53 play a part in apoptosis, but also help to regulate chondrocyte growth and differentiation. Whereas Bcl-2 promotes cell survival, p53 can arrest cell cycle. The data confirm that chondrocyte activity is enhanced in OA and suggest that the increased Bcl-2/p53 ratio sustains the metabolic boost of chondrocytes.     

Interactions of synovial fluid immunoglobulins with chondrocytes

Objective. To study the interaction of synovial fluid (SF) immunoglobulins with living chondrocytes, and to evaluate the relative contribution of type II collagen (CII) antibodies. Methods. SF of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and gout were incubated with isolated bovine articular chondrocytes. Ig binding was measured by flow cytometry and by quantitation with ¹²⁵I-labeled anti-IgG and anti-IgM. Complement-dependent cytotoxicity was determined by ⁵¹Cr release. Immunoglobulin binding and cytotoxicity were compared between chondrocytes obtained from the superficial and from the deep cartilage zones. Results. Significantly greater IgG and IgM binding was found with RA SF compared with OA or gout SF. Chondrocytes bound more Ig than did fibroblasts. The relative contribution of anti-CII antibodies to Ig binding was studied following absorption of the SF with bovine CII, and by incubation with bacterial collagenase-treated chondrocytes. There was a small but significant reduction in IgG and IgM binding with SF samples that were positive for anti-CII. RA SF exhibited modest, but significantly greater complement-dependent cytotoxicity than OA SF. Gel chromatography fractionation indicated that IgM antibodies were responsible for the cytotoxic activity. Additional studies showed that SF IgM antibodies bound preferentially to, and killed chondrocytes obtained from, the superficial layers of cartilage. Conclusion. Anti-CII antibodies contained in RA SF represent one of many antibody specificities reacting with chondrocyte membrane antigens. Chondrocyte-reactive SF antibodies may play an important pathogenic role in the processes leading to irreversible cartilage damage in RA. These deleterious effects appear to be exerted particularly on chondrocytes located near the articular surface of cartilage.     

Lectin-like oxidized low-density lipoprotein receptor 1 mediates matrix metalloproteinase 3 synthesis enhanced by oxidized low-density lipoprotein in rheumatoid arthritis cartilage

OBJECTIVE: To investigate for the presence of oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor 1 (LOX-1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox-LDL with LOX-1 can induce matrix metalloproteinase 3 (MMP-3) in articular cartilage explant culture. METHODS: Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX-1 and ox-LDL by confocal fluorescence microscopy. The association between ox-LDL and LOX-1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox-LDL, with or without preincubation with neutralizing anti-LOX-1 antibody. MMP-3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme-linked immunosorbent assay. RESULTS: The majority of the RA chondrocytes stained positively with both anti-LOX-1 and anti-ox-LDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti-LOX-1 antibody suppressed the binding of DiI-labeled ox-LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX-1. In contrast to native LDL, ox-LDL induced MMP-3 synthesis by articular chondrocytes in association with the induction of LOX-1, which resulted in enhanced secretion of MMP-3 into the culture medium. Anti-LOX-1 antibody reversed ox-LDL-stimulated MMP-3 synthesis to control levels. CONCLUSION: Ox-LDL, principally mediated by LOX-1, enhanced MMP-3 production in articular chondrocytes. Increased accumulation of ox-LDL with elevated expression of LOX-1 in RA cartilage indicates a specific role of the receptor-ligand interaction in cartilage pathology in RA.     

Enhancement of cartilage matrix protein synthesis in arthritic cartilage

Objective. To investigate whether the synthesis of cartilage matrix protein (CMP) is enhanced in arthritic cartilage. Methods. The content of CMP in human and pig cartilage was determined by immunoblotting, and CMP-producing chondrocytes in osteoarthritic (OA) and rheumatoid arthritic (RA) joints were immunostained. Results. CMP was undetectable in the condylar cartilage and disc of pigs, whereas it was abundant in the rib and tracheal cartilage of the same animals. By immunohistochemical analysis, CMP was localized in only a few chondrocytes (5%) in normal human joints, whereas numerous chondrocytes (>60%) were immunostained in RA joints. The number of CMP-producing cells was also increased in OA cartilage (>40%). Immunoblotting analyses confirmed that the CMP content in the cartilage from OA and RA patients was much higher than that in normal cartilage. Conclusion. These findings demonstrate that articular chondrocytes can synthesize CMP, although it is suppressed under physiologic conditions. The results also suggest that articular chondrocytes express CMP in response to arthritic stimuli.     

The serine protease inhibitor trappin-2 is present in cartilage and synovial fluid in osteoarthritis

OBJECTIVE: Trappins are small serine protease inhibitors bound to extracellular matrix (ECM) through the actions of transglutaminase (TGase) enzymes. Trappin-2 is present in many tissues and is upregulated at sites of injury. In osteoarthritis (OA), serine proteases contribute to articular cartilage destruction, and TGase activity is increased. Yet little is known about matrix-bound serine protease inhibitors or TGase substrates in articular cartilage. Our purpose was to determine if trappin-2 was present in OA cartilage and synovial fluid (SF). METHODS: OA knee articular cartilage and SF were assayed for trappin-2 protein by Western blotting, ELISA, and immunohistochemistry. Trappin-2 mRNA was detected with RT-PCR. The ECM components bound to trappin-2 were identified by 2-D gel electrophoresis and peptide fingerprinting. RESULTS: Trappin-2 was detectable in OA articular cartilage extracts, cultured chondrocytes, conditioned media, and SF by Western blotting. OA cartilage protein extracts contained significantly higher quantities of trappin-2 than normal cartilage protein extracts (22.98 +/- 1.28 ng/mg wet weight vs 14.97 +/- 1.92 ng/mg wet weight; p < 0.01). RT-PCR confirmed the presence of trappin-2 mRNA in OA chondrocytes. Immunohistochemical studies of OA cartilage revealed trappin-2 protein in chondrocytes. Peptide mapping of trappin-2 binding partners showed that fibromodulin was bound to trappin-2 in cartilage. CONCLUSION: We confirmed the presence of trappin-2 in OA cartilage and SF. Elevated levels of TGase activity in OA cartilage may increase levels of this serine protease inhibitor in response to injury.     

Inhibition of interleukin-1beta-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

OBJECTIVE: To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin-1beta (IL-1beta)-stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage. METHODS: IL-1beta was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800-kd HA to assess its effect on IL-1beta-induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti-CD44 antibody were performed to investigate the mechanism of action of HA. RESULTS: Treatment and pretreatment with 800-kd HA at 1 mg/ml resulted in significant suppression of IL-1beta-stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA-binding blocking experiments using anti-CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti-CD44 antibody, which suppressed IL-1beta-stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL-1beta in normal and OA cartilage. CONCLUSION: This study demonstrates that HA effectively inhibits IL-1beta-stimulated production of MMP-1, MMP-3, and MMP-13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL-1beta may involve direct interaction between HA and CD44 on chondrocytes.     

Increased apoptosis in human osteoarthritic cartilage corresponds to reduced cell density and expression of caspase-3

OBJECTIVE: Chondrocyte apoptosis has been described in both human and experimentally induced osteoarthritis (OA), but its importance in the etiopathogenesis of OA is uncertain. The aims of this study were to determine the rate of chondrocyte apoptosis using different methods, and to investigate the relationship between this process and cartilage cellularity, expression of proapoptotic molecules, and expression of antiapoptotic molecules in articular cartilage obtained from patients with OA and from nonarthritic controls. METHODS: We examined the extent of apoptosis in OA and nonarthritic control cartilage using expression of caspase-3, an enzyme that mediates the final stage of cell death by apoptosis, as well as the TUNEL method. We used immunohistochemistry to analyze the expression of a panel of proapoptotic and antiapoptotic molecules that regulate apoptosis in articular cartilage, in order to determine whether the rate of apoptosis is associated with the expression of these molecules. RESULTS: The median (range) percentage of TUNEL-positive chondrocytes in knee OA cartilage (n = 10 specimens), hip OA cartilage (n = 9), and control cartilage (n = 7) was 3.11 (1.67-3.67), 1.86 (1.22-2.89), and 0.39 (0.00-1.78), respectively. When all cartilage samples were pooled, apoptosis showed a strong inverse correlation with cellularity (r = -0.74, P < 0.0001). The percentage (range) of cells expressing caspase-3 in the 3 groups was 15.70 (7.40-20.50), 15.77 (7.42-20.5), and 7.40 (5.90-8.00), respectively. One-way analysis of variance showed that the differences between groups for both TUNEL-positive cells and expression of caspase-3 were statistically significant (P < 0.0001). There was a significant positive correlation between TUNEL-positive cells and expression of caspase-3 (r = 0.654, P< 0.01). CONCLUSION: The data suggest that apoptosis is increased, on average, 2-4-fold in OA cartilage. Considering that OA develops over many years, such an increase in the rate of apoptosis in the articular cartilage could play an important role in the disease process.