Distinct signaling pathways are involved in hypoxia- and IL-1-induced VEGF expression in human articular chondrocytes.

Recent data suggest that vascular endothelial growth factor (VEGF) is involved in the pathogenesis of osteoarthritis (OA). Cartilage is an avascular tissue, leading to a low cartilage O2 level. Thus, in a variety of pathologic or physiologic conditions, VEGF is partly regulated by hypoxic stress. The implications of hypoxia for VEGF expression by OA chondrocytes, however, are not known. We investigated the regulatory system of VEGF in OA chondrocytes under hypoxic conditions. Chondrocytes were obtained from articular cartilage of patients with OA. Cells were cultured and then incubated under hypoxic (95% N2, 5% CO2) or normoxic conditions, with or without interleukin (IL)-1 (10 ng/mL) stimulation. The mitogen activated protein kinase (MAPK) inhibitors were also used. VEGF levels in the culture supernatants were measured using an enzyme-linked immunosorbent assay. Western blot analysis was used to examine the expression of hypoxia inducible factor (HIF)-1alpha. Hypoxia significantly increased VEGF levels (p<0.05). Hypoxia-induced VEGF secretion was abolished by p38MAPK inhibitor, but not by JNK inhibitor. In contrast, IL-1-induced VEGF secretion was blocked by JNK inhibitor, and not by p38MAPK inhibitor. Both hypoxia and IL-1-induced HIF-1alpha were attenuated by p38 MAPK and JNK inhibitors. We demonstrate that hypoxia and IL-1 induce VEGF production in chondrocytes through distinct MAPK signaling pathways, indicating that VEGF is induced in a HIF-1-dependent or -independent manner in chondrocytes.

Comparative analysis of gene expression profiles in intact and damaged regions of human osteoarthritic cartilage.

OBJECTIVE: To analyze the differences in gene expression profiles of chondrocytes in intact and damaged regions of cartilage from the same knee joint of patients with osteoarthritis (OA) of the knee. METHODS: We compared messenger RNA expression profiles in regions of intact and damaged cartilage (classified according to the Mankin scale) obtained from patients with knee OA. Five pairs of intact and damaged regions of OA cartilage were evaluated by oligonucleotide array analysis using a double in vitro transcription amplification technique. The microarray data were confirmed by real-time quantitative polymerase chain reaction (PCR) amplification and were compared with previously published data. RESULTS: About 1,500 transcripts, which corresponded to 8% of the expressed transcripts, showed > or = 2-fold differences in expression between the cartilage tissue pairs. Approximately 10% of these transcripts (n = 151) were commonly expressed in the 5 patient samples. Accordingly, 114 genes (35 genes expressed in intact > damaged; 79 genes expressed in intact < damaged) were selected. The expression of some genes related to the wound-healing process, including cell proliferation and interstitial collagen synthesis, was higher in damaged regions than in intact regions, similar to the findings for genes that inhibit matrix degradation. Comparisons of the real-time quantitative PCR data with the previously reported data support the validity of our microarray data. CONCLUSION: Differences between intact and damaged regions of OA cartilage exhibited a similar pattern among the 5 patients examined, indicating the presence of common mechanisms that contribute to cartilage destruction. Elucidation of this mechanism is important for the development of effective treatments for OA.

Catabolic stress induces features of chondrocyte senescence through overexpression of caveolin 1: possible involvement of caveolin 1-induced down-regulation of articular chondrocytes in the pathogenesis of osteoarthritis.

OBJECTIVE: Articular chondrocyte senescence is responsible, at least in part, for the increased incidence of osteoarthritis (OA) with increased age. Recently, it was suggested that caveolin 1, a 21-24-kd membrane protein, participates in premature cellular senescence. Caveolin 1 is the principal structural component of caveolae, vesicular invaginations of the plasma membrane. This study was undertaken to investigate whether the catabolic factors oxidative stress and interleukin-1beta (IL-1beta) induce features of premature senescence of articular chondrocytes through up-regulation of caveolin 1 expression. METHODS: Caveolin 1 expression was investigated in human OA cartilage by real-time polymerase chain reaction and in rat OA cartilage by immunohistologic analysis. We studied whether IL-1beta and H2O2 induce caveolin 1 expression in OA chondrocytes and analyzed the relationship between cellular senescent phenotypes and caveolin 1 expression in human chondrocytes. RESULTS: In human and rat OA articular cartilage, caveolin 1 positivity was associated with cartilage degeneration. Both IL-1beta and H2O2 up-regulated caveolin 1 messenger RNA and protein levels, and both treatments induced marked expression of senescent phenotypes: altered cellular morphology, cell growth arrest, telomere erosion, and specific senescence-associated beta-galactosidase activity. Caveolin 1 overexpression induced p38 MAPK activation and impaired the ability of chondrocytes to produce type II collagen and aggrecan. In contrast, down-regulation of caveolin 1 with antisense oligonucleotide significantly inhibited the features of chondrocyte senescence induced by catabolic factors. Caveolin 1 induction and stresses with both IL-1beta and H2O2 up-regulated p53 and p21 and down-regulated phosphorylated retinoblastoma (Rb), suggesting that the p53/p21/Rb phosphorylation pathway, as well as prolonged p38 MAPK activation, may mediate the features of chondrocyte senescence induced by stress. CONCLUSION: Our findings suggest that IL-1beta and oxidative stress induce features of premature senescence in OA chondrocytes, mediated, at least in part, by stress-induced caveolin 1 expression. This indicates that caveolin 1 plays a role in the pathogenesis of OA via promotion of chondrocyte down-regulation.

Enhanced production of MMP-1, MMP-3, MMP-13, and RANTES by interaction of chondrocytes with autologous T cells.

It has been reported that T cells and chondrocytes interact through cell surface molecules such as MHC, CD4 or CD8 in osteoarthritis (OA) and T cells are activated. The objective of this study is to investigate the responses of chondrocyte-T cell interaction in terms of metalloprotease (MMP) and chemokine production. Articular cartilage and autologous blood were obtained from patients with OA and fracture who under went prosthetic surgery. Synovial fluid (SF) was collected from OA patients. Isolated chondrocytes were co-cultured with autologous T cells. SF cells were analyzed by immunostaining or Alcian blue staining. The production of MMP-1, MMP-3, MMP-13, and regulated on activation, normal T expressed and secreted (RANTES) was enhanced by direct co-culture compared to indirect co-culture using Transwell. Production ratio of RANTES in OA was significantly higher than non-arthritic samples. CD3 positive mononuclear cells and chondrocyte-like cells were found in SF. Chondrocyte-T cell contact was more adhesive in OA samples. These results showed the production of MMPs and RANTES was enhanced by the interaction and that chondrocyte-T cell contact was possible in vivo.

Fibulin-4 is a target of autoimmunity predominantly in patients with osteoarthritis.

Autoimmunity to chondrocyte-producing proteins has been reported in patients with osteoarthritis (OA) as well as in those with rheumatoid arthritis (RA). To answer whether or not OA-specific autoimmunity exist, we performed screening of chondrocyte-producing autoantigens by two-dimensional electrophoresis and Western blotting with each of 20 OA and 20 RA serum samples. We identified an apparently OA-specific autoantigen spot with a molecular mass of 52 kDa and a Isoelectric point of 4.1 as fibulin-4 by mass fingerprinting. By preparing recombinant proteins of fibulin-4, we determined prevalence of the autoantibodies to fibulin-4 in 92 patients with OA, 67 patients with RA, 40 patients with systemic lupus erythematosus, and 43 patients with systemic scleroderma. As a result, the IgG type anti-fibulin-4 autoantibodies were detected in 23.9% of sera from patients with OA, in 8.9% of sera from patients with RA, in 2.5% of sera from patients with systemic lupus erythematosus, and in 9.3% of sera from patients with systemic scleroderma. Furthermore, we immunized DBA/1J, ICR, BALB/c, and C57BL/6 mice with the recombinant fibulin-4 proteins to investigate arthritogenecity of fibulin-4. As a result, mild synovitis was detected in all of the four strains. In addition, we demonstrated expression of fibulin-4 in chondrocytes at both mRNA and protein levels in vivo and in vitro by RT-PCR, Western blotting, and immunohistochemistry. Taken together, fibulin-4, expressed in chondrocytes and recognized as an autoantigen mainly in OA rather than in RA, may play pathogenic roles in OA.

Suppressive effects of hyaluronan on MMP-1 and RANTES production from chondrocytes.

The purpose of the study was to examine the effects of hyaluronan (HA) on human chondrocytes in terms of production of MMP-1 and RANTES. Chondrocytes were obtained from patients with osteoarthritis (OA) or femoral neck fracture (control). Chondrocytes in monolayer culture were treated with various molecular weights (1.2, 50, 800 and 1,900 kD) of HA and then stimulated with IL-1beta. Production and expression of MMP-1 and RANTES were quantified by ELISA and real-time polymerase chain reaction (PCR). The response was blocked by anti-CD44 antibody. Production of MMP-1 was significantly suppressed by both 800- and 1,900-kD HA, while production of RANTES was suppressed by 1,900-kD HA. Expression of MMP-1 and RANTES mRNA was inhibited by 1,900-kD HA. Suppressive effects of HA on production of MMP-1 were canceled by treatment of anti-CD44 antibody. Higher CD44 expression was found in OA chondrocytes than in those of control. High-molecular-weight HA suppressed MMP-1 and RANTES production, mediated partly by CD44-HA interaction.

Catabolic stress induces expression of hypoxia-inducible factor (HIF)-1 alpha in articular chondrocytes: involvement of HIF-1 alpha in the pathogenesis of osteoarthritis.

Transcription factor hypoxia-inducible factor (HIF)-1 protein accumulates and activates the transcription of genes that are of fundamental importance for oxygen homeostasis - including genes involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation, and matrix production - under hypoxic conditions. We speculated that HIF-1alpha may have an important role in chondrocyte viability as a cell survival factor during the progression of osteoarthritis (OA). The expression of HIF-1alpha mRNA in human OA cartilage samples was analyzed by real-time PCR. We analyzed whether or not the catabolic factors IL-1beta and H2O2 induce the expression of HIF-1alpha in OA chondrocytes under normoxic and hypoxic conditions (O2 <6%). We investigated the levels of energy generation, cartilage matrix production, and apoptosis induction in HIF-1alpha-deficient chondrocytes under normoxic and hypoxic conditions. In articular cartilages from human OA patients, the expression of HIF-1alpha mRNA was higher in the degenerated regions than in the intact regions. Both IL-1beta and H2O2 accelerated mRNA and protein levels of HIF-1alpha in cultured chondrocytes. Inhibitors for phosphatidylinositol 3-kinase and p38 kinase caused a significant decrease in catabolic-factor-induced HIF-1alpha expression. HIF-1alpha-deficient chondrocytes did not maintain energy generation and cartilage matrix production under both normoxic and hypoxic conditions. Also, HIF-1alpha-deficient chondrocytes showed an acceleration of catabolic stress-induced apoptosis in vitro. Our findings in human OA cartilage show that HIF-1alpha expression in OA cartilage is associated with the progression of articular cartilage degeneration. Catabolic-stresses, IL-1beta, and oxidative stress induce the expression of HIF-1alpha in chondrocytes. Our results suggest an important role of stress-induced HIF-1alpha in the maintenance of chondrocyte viability in OA articular cartilage.

The prevalence of autoantibodies against cartilage intermediate layer protein, YKL-39, osteopontin, and cyclic citrullinated peptide in patients with early-stage knee osteoarthritis: evidence of a variety of autoimmune processes.

We studied arthritis-related autoantibodies in 136 patients with knee osteoarthritis (OA) and 67 age- and sex-matched healthy individuals in the Shanghai District of China. Serum antibody titers for recombinant fusion proteins of cartilage intermediate layer protein (CILP) and YKL-39 were analyzed using enzyme-linked immunosorbent assay (ELISA) and Western blot. Serum antibody titers against recombinant osteopontin (OPN) and cyclic citrullinated peptide (CCP) antibodies were measured also using ELISA. Anti-CILP antibodies were detected in 25/136 OA patients but only 1/67 controls. Anti-YKL-39, anti-OPN, and anti-CCP antibodies were detected in 9/136, 11/136, and 7/136 of the OA patients, respectively, and 0/67 controls. There was rarely overlap of these antibodies in a single patient, suggesting distinct antigen specificity in each case. The antibodies were detected in patients with OA of grades II and III but not grade IV. The prevalence of autoantibodies to various arthritis-related proteins in early-stage knee OA supports the involvement of a specific immune response in initial cartilage degeneration in OA.

Induction of vascular endothelial growth factor and matrix metalloproteinase-3 (stromelysin) by interleukin-1 in human articular chondrocytes and synoviocytes.

We investigated the effects of interleukin (IL)-1 on vascular endothelial growth factor (VEGF) and matrix metalloproteinase-3 (MMP-3) (stromelysin) secretion from chondrocytes and synoviocytes in different clinical conditions. Specifically, cells obtained from osteoarthritic (OA) (n = 7), rheumatoid arthritic (RA) (n = 5), and post-traumatic (PT) (n = 5) patients were stimulated in vitro with IL-1beta in the presence or absence of an IL-1 receptor antagonist (IL-1ra) (anakinra). Levels of secreted MMP-3 and VEGF were measured by enzyme-linked immunosorbent assay. The VEGF mRNA expression was analyzed quantitatively. Interleukin-1 induced both VEGF and MMP-3 secretion from all of the samples tested, and VEGF mRNA expression was also upregulated. Interleukin-1ra significantly suppressed the enhancing effect of IL-1 on MMP-3 and VEGF in both cell types. In conclusion, IL-1 simultaneously induces MMP-3 and VEGF production from chondrocytes and synoviocytes in inflammatory, degenerative, and post-traumatic joints. Therefore, IL-1ra might be beneficial for protection from VEGF-mediated alterations of cartilage metabolism in pathologic and physiologic conditions.

Up-regulation of microsomal prostaglandin E synthase 1 in osteoarthritic human cartilage: critical roles of the ERK-1/2 and p38 signaling pathways.

OBJECTIVE: Microsomal prostaglandin E synthase 1 (mPGES-1) is the final enzyme of the cascade that produces prostaglandin E(2) (PGE(2)), a key actor in arthritis. To study mPGES-1 synthesis in human cartilage and its regulation by interleukin-1beta (IL-1beta), we used human cartilage and an immortalized human chondrocyte cell line. Furthermore, we investigated the signaling pathways involved in mPGES-1 expression. METHODS: We used real-time quantitative reverse transcription-polymerase chain reaction, Northern blotting, and Western blotting to measure mPGES-1 messenger RNA (mRNA) and protein expression in human chondrocytes. PGE(2) production was measured by enzyme-linked immunosorbent assay. RESULTS: Cartilage specimens from osteoarthritis (OA) patients contained far greater amounts of mPGES-1 and cyclooxygenase 2 (COX-2) mRNA than did normal cartilage. Incubation with IL-1beta markedly increased mPGES-1 mRNA and protein in a dose-dependent and time-dependent manner, in parallel with an increase in PGE(2) levels. Both PD98059, an ERK pathway inhibitor, and SB203580, a p38alpha/beta MAPK inhibitor, abolished the increases in mPGES-1 mRNA and protein in response to IL-1beta. The specific p38alpha MAPK inhibitor SC906 suppressed IL-1beta-induced COX-2 expression but not IL-1beta-induced mPGES-1 expression, suggesting preferential involvement of p38beta MAPK in IL-1beta-induced mPGES-1 expression. CONCLUSION: This study is the first to show that mPGES-1 is stimulated in human chondrocytes by the proinflammatory cytokine IL-1beta via activation of both ERK-1/2 and p38 MAPK in an isoform-specific manner. We postulate that mPGES-1 may be a novel target for OA therapy.

A potential role of 15-deoxy-delta(12,14)-prostaglandin J2 for induction of human articular chondrocyte apoptosis in arthritis.

The cyclopentenone prostaglandin (PG) J2 is formed within the cyclopentenone ring of the endogenous prostaglandin PG D2 by a nonenzymatic reaction. The PG J family is involved in mediating various biological effects including the regulation of cell cycle progression and inflammatory responses. Here we demonstrate the potential role of 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PG J2) in human articular chondrocyte apoptosis. 15d-PG J2 was released by human articular chondrocytes and found in joint synovial fluids taken from osteoarthritis or rheumatoid arthritis patients. Proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) up-regulated chondrocyte release of 15d-PG J2. PG D2 synthase mRNA expression was up-regulated by IL-1beta, TNF-alpha, or nitric oxide. 15d-PG J2 induced apoptosis of chondrocytes from osteoarthritis or rheumatoid arthritis patients as well as control nonarthritic subjects in a time- and dose-dependent manner and in a peroxisome proliferator-activated receptor gamma-dependent manner. Peroxisome proliferator-activated receptor gamma expression was up-regulated by IL-1beta and TNF-alpha. Inhibition of NF-kappaB, and the activation of p38 MAPK were also found to be involved in 15d-PG J2-induced chondrocyte apoptosis. Such signal pathways led to the activation of the downstream pro-apoptotic molecule p53 and caspase cascades. Together, these results suggest that 15d-PGJ2 may play an important role in the pathogenesis of arthritic joint destruction via a regulation of chondrocyte apoptosis.

Virus-associated arthritis.

The occurrence of arthritis in patients who were infected by a virus has been widely observed. In some cases, the clinical appearance seems to resemble that of rheumatoid arthritis. The mechanism by which the viral infection proceeds to the arthritic manifestation is, however, still to be investigated. Several biological and immunological pathways are suggested to be involved in the pathogenesis. The representatives of such potentially 'arthritogenic' viruses include human T-cell lymphotropic virus type I (HTLV-I), which causes destructive inflammatory arthritis in model animals. Other examples are hepatitis C virus and rubella virus. Clinical and pathological features of these virus-induced forms of arthritis are discussed.

Autoimmunity against YKL-39, a human cartilage derived protein, in patients with osteoarthritis.

OBJECTIVE: Our previous study revealed that some patients with rheumatoid arthritis (RA) possessed autoantibodies to YKL-39, a cartilage related protein. We investigated whether patients with osteoarthritis (OA) also displayed autoimmunity to YKL-39. METHODS: Autoantibodies to recombinant YKL-39 as well as human cartilage glycoprotein-39 were detected by ELISA and Western blotting. The tested serum samples were derived from 117 patients with OA, 94 patients with RA, and 2 groups of 50 arthropathy-free healthy donors who matched the OA and RA groups for age and sex. We determined autoepitopes on YKL-39 using 3 overlapping fragments of YKL-39 (designated F1, F2, F3). T cell proliferation response to YKL-39 was analyzed using the 3H-thymidine incorporation assay. RESULTS: Autoantibodies to YKL-39 were detected in 13 (11.1%) patients with OA and 11 (11.8%) with RA. In the epitope mapping, all the 3 fragments of YKL-39 were found to carry autoepitopes, but F1 was recognized most frequently. Proliferative responses of peripheral blood mononuclear cells against YKL-39 were detected in 6 (46%) of the 13 OA patients who were positive for the anti-YKL-39 autoantibodies and in 2 (17%) of the 11 antibody positive RA patients. CONCLUSION: These results show that autoimmunity to YKL-39 in patients with OA was present at equal or somewhat higher frequency than in patients with RA. The cellular and humoral immune responses to YKL-39 may be involved in the pathological process of OA as well as RA.

Expression of the anaphylatoxin receptor C5aR (CD88) by human articular chondrocytes.

Although the complement system is implicated in the inflammatory process in arthritic diseases, a direct interaction between chondrocytes and complement has not been demonstrated. In this study, we investigated expression of the C5a receptor (C5aR) on chondrocytes of cartilage from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and bone fracture as normal controls by reverse transcriptase polymerase chain reaction (RT-PCR), flow cytometry, and immunohistochemistry. The RT-PCR detected mRNA for C5aR in most or all of the tested samples (73% in OA, 100% in RA, 89% in normal). The FACS analysis revealed different expression ratios between individuals varying from 0.7% to 77.1%; however, expression ratios of C5aR were significantly higher in RA than in controls (26.0% in RA, 9.0% in OA, 6.9% in normal). The expression of C5aR was upregulated significantly by addition of IL-1beta in RA and normal samples but not in OA. In addition, the C5aR-positive chondrocytes were confirmed by immunohistochemistry. In conclusion, expression of C5aR and the effect of IL-1beta on the expression were different between RA and OA. The C5aR may contribute to chondrocyte metabolism and the pathogenesis of arthritis differently between in RA and OA.

T-cell clonotypes specific for Dermatophagoides pteronyssinus in the skin lesions of patients with atopic dermatitis.

T-cell mediated immune response, toward the house dust mite (HDM) antigens in particular, has been reported to be involved in the pathogenesis of atopic dermatitis (AD). On the other hand, studies on the infiltrating lymphocytes in the skin lesion of AD revealed oligoclonal T-cell accumulation. However, it is not clear exactly what antigen(s) the accumulating T cells are exactly recognize in situ. Therefore, this study attempted to determine whether or not the clonally expanded T-cell clones in the diseased skin recognize HDM. Specifically, peripheral blood mononuclear cells (PBMC) obtained from six patients with AD, who revealed high titers of anti-HDM IgE, were stimulated with HDM antigens purified from Dermatophagoides pteronyssinus (Dp). T-cell clonotypes expanded by the stimulation were then identified by the analysis of their T-cell receptor (TCR) B-gene sequences using a combination of the reverse transcription-polymerase chain reaction and subsequent single strand conformation polymorphism separation. The Dp-responding T-cell clonotypes were compared with those that accumulated in the AD skin lesion in vivo. Nucleotide sequences of the TCR were also determined. As a result, the Dp stimulation induced oligoclonal T-cell expansion from the originally heterogeneous peripheral T-cell population of AD patients. However, only a small part of the Dp-reacting T-cell clonotypes detected in PBMC was identical to those accumulated in the AD skin lesion in vivo, and vice versa. This indicates that the frequency of the clonal expansion of Dp-specific T-cell clonotypes in the skin lesion of AD would be rather limited.