Clock gene expression in different synovial cells of patients with rheumatoid arthritis and osteoarthritis.

Patients with rheumatoid arthritis (RA) show modulated circadian rhythms of inflammatory cytokines and cortisol, which may be associated with a modified expression of clock genes. The expression of major clock genes was previously studied in synovial tissues and fibroblasts of patients with RA and osteoarthritis (OA). We therefore especially aimed to examine the localization of clock genes at the cellular level in synovial tissue. Furthermore we were interested in studying the expression of the D site of albumin promoter (albumin D-box) binding protein (DBP) at the immunohistochemical level in human samples. Methods used include the in situ expression of the clock genes Brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal 1), Circadian Locomotor Output Cycles Kaput (Clock), Period 1 and 2 (Per 1 and Per 2), and DBP was examined by immunohistochemistry in synovial tissues of patients with RA or OA. Additionally, expression profiles of different clock genes were determined over 24h by real time PCR in synovial fibroblasts (SFs) after a 2h serum shock or TNF-α. Results show that all clock genes investigated were found to be expressed both in RA and OA synovial tissues. Double staining against cell specific markers revealed that clock proteins were especially seen in macrophages, SFs and B-lymphocytes. Cell counting showed that clock proteins were found in approximately 5-20% of cells. Additionally, preliminary cell culture experiments showed that TNF-α treatment resulted in differential 24h expression profiles between RA and OA samples and also compared to the results obtained from the serum shock experiments. From our study we conclude that the major clock genes, including DBP, are expressed in samples from RA and OA patients, especially in macrophages and synovial fibroblasts, but also in B-lymphocytes. Preliminary experiments suggest that TNF-α seems to be able to modify clock gene expression in synovial fibroblasts.

Autoantibodies to the translational suppressors T cell intracytoplasmic antigen 1 and T cell intracytoplasmic antigen 1-related protein in patients with rheumatic diseases: increased prevalence in systemic lupus erythematosus and systemic sclerosis and correlation with clinical features.

OBJECTIVE: T cell intracytoplasmic antigen 1 (TIA-1) and TIA-1-related protein (TIAR) are involved in posttranscriptional regulation of the expression of tumor necrosis factor alpha (TNFalpha) and other proteins. Given the pivotal role of TNFalpha in chronic inflammatory diseases, this study was undertaken to analyze sera from patients with systemic autoimmune diseases for the presence of autoantibodies to TIA proteins and to investigate the expression of these proteins in inflamed tissue. METHODS: The presence of autoantibodies to TIA proteins in sera from 385 patients with rheumatic diseases and healthy controls was determined by immunoblotting using recombinant antigens. Expression of TIA proteins in skin and kidney tissue was analyzed by immunohistochemistry. Serum levels of TNFalpha were measured by enzyme-linked immunosorbent assay. RESULTS: Autoantibodies to TIA-1 and/or TIAR were detected in 61% of patients with systemic lupus erythematosus (SLE), 42% of patients with systemic sclerosis (SSc), 15-31% of patients with other rheumatic diseases, and 6% of healthy controls. Compared with patients negative for anti-TIA antibody, anti-TIA antibody-positive SLE patients had higher disease activity (P = 0.01), elevated antibodies to double-stranded DNA (P = 0.0003), and increased serum TNFalpha levels (P = 0.018). In SLE patients, anti-TIAR antibodies were associated with lupus nephritis (P = 0.02), while in patients with SSc, anti-TIA-1 was associated with lung involvement (P = 0.02). Immunohistochemical analysis of skin and kidney tissue revealed aberrant expression of TIA proteins in skin lesions from SLE and SSc patients, as well as in glomerular cells from SLE patients. CONCLUSION: Aberrant expression of TIA proteins in inflammatory tissue may lead to systemic autoantibody responses, particularly in SLE and SSc. Increased occurrence of anti-TIA autoantibodies in patients with severe organ involvement may point to a possible pathogenetic role.

Differentially regulated expression of growth differentiation factor 5 and bone morphogenetic protein 7 in articular cartilage and synovium in murine chronic arthritis: potential importance for cartilage breakdown and synovial hypertrophy.

OBJECTIVE: To examine whether the endogenous expression of growth differentiation factor 5 (GDF-5) and bone morphogenetic protein 7 (BMP-7) is altered in the cartilage and synovium of human tumor necrosis factor alpha (TNFalpha)-transgenic (hTNFtg) mice with chronic arthritis, and to investigate the response of hTNFtg chondrocytes as well as fibroblast-like synoviocytes (FLS) to these morphogens in vitro. METHODS: Analyses were performed in hTNFtg mice with chronic destructive arthritis and in wild-type (WT) mice as controls. Expression of GDF-5 and BMP-7 in the articular cartilage and synovium was examined by real-time polymerase chain reaction and immunohistochemistry. Human TNFtg cartilage explants, chondrocytes, and FLS monolayer cultures were assessed for basal matrix biosynthesis as well as growth factor responsiveness, using (35)S-sulfate incorporation assays. In addition, the DNA content/cell proliferation rate was measured. RESULTS: The expression of GDF-5 and BMP-7 was decreased in articular cartilage from hTNFtg mice, whereas expression of both morphogens was increased in arthritic synovium from hTNFtg mice, as compared with the levels in WT controls. Isotope incorporation revealed a marked reduction of matrix synthesis in hTNFtg cartilage as well as a decrease in responsiveness to GDF-5 and BMP-7. The DNA content did not change in arthritic cartilage as compared with WT cartilage. In hTNFtg FLS, growth factor stimulation increased the rate of cell proliferation and the production of extracellular matrix. CONCLUSION: In this murine model of TNFalpha-mediated arthritis, the expression of GDF-5 and BMP-7 is regulated differentially in articular cartilage and synovium. In articular cartilage, the down-regulation of GDF-5 and BMP-7, which function to maintain matrix integrity, could potentially compromise tissue repair, whereas in synovium, the increased expression of GDF-5 and BMP-7 might contribute to synovial hypertrophy.

The rheumatoid arthritis-associated autoantigen hnRNP-A2 (RA33) is a major stimulator of autoimmunity in rats with pristane-induced arthritis.

A single intradermal injection of the mineral oil pristane in susceptible DA.1F rats induces erosive arthritis closely mimicking rheumatoid arthritis (RA). Pristane-induced arthritis (PIA) is driven by autoreactive T cells but no autoantigen has been identified to date. We therefore analyzed B and T cell responses to autoantigens potentially involved in the pathogenesis of RA, including IgG, citrullinated proteins, stress proteins, glucose-6-phosphate isomerase, and heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (RA33). IgG and IgM autoantibodies to hnRNP-A2 were detectable in sera of pristane-primed DA.1F rats already 1 wk before disease onset, reached maximum levels during the acute phase, and correlated with arthritis severity. Apart from rheumatoid factor, autoantibodies to other Ags were not observed. CD4(+) lymph node cells isolated 10 days after pristane injection produced IFN-gamma but not IL-4 in response to stimulation with hnRNP-A2, whereas none of the other candidate Ags elicited cytokine secretion. Surprisingly, hnRNP-A2 also stimulated lymph node cells of naive animals to produce inflammatory cytokines in a MyD88-dependent manner. Furthermore, hnRNP-A2 was highly overexpressed in the joints of rats injected with pristane. Overexpression coincided with the appearance of anti-RA33 Abs and preceded the onset of clinical symptoms of PIA by several days. Taken together, these data suggest hnRNP-A2 to be among the primary inducers of autoimmunity in PIA. Therefore, this Ag might play a pivotal role in the pathogenesis of PIA and possibly also human RA.

Differential tissue expression and activation of p38 MAPK alpha, beta, gamma, and delta isoforms in rheumatoid arthritis.

OBJECTIVE: Activation of p38 MAPK is a key signaling step in chronic inflammation. Inhibition of p38 MAPK is considered to be a promising future strategy to control inflammatory diseases, but studies of compounds to inhibit this kinase have so far been limited to investigation of their side effects. We undertook the present study to investigate which specific molecule, among 4 different isoforms of p38 MAPK (alpha, beta, gamma, and delta), is predominantly expressed and activated in inflammation. Such knowledge could allow more specific targeting of p38 MAPK in inflammatory disease. METHODS: Studies were performed on inflamed tissue from patients with rheumatoid arthritis, as a prototype of inflammatory disease. The expression and activation of the alpha, beta, gamma, and delta isoforms of p38 MAPK were examined by immunoblotting, immunoprecipitation, and immunohistochemistry. RESULTS: Immunoblot analysis revealed that alpha and gamma were the predominantly expressed p38 MAPK isoforms, whereas the other 2 isoforms were less frequently present. By immunohistochemistry, the expression of all p38 MAPK isoforms was localized to the synovial lining layer as well as to blood vessels. Colabeling with cell-specific markers revealed that macrophages expressed the alpha and gamma isoforms, synovial fibroblasts the beta and gamma isoforms, and granulocytes the delta isoform, whereas T lymphocytes were rarely positive for any p38 MAPK isoform. Double-labeling with isoform-specific antibody and pan-p38 antibody against the phosphorylated form of p38 MAPK showed activation of the alpha and gamma isoforms. Occasional activation of the beta isoform was also noted in the synovial lining and the endothelium, whereas the delta isoform, although expressed in pericytes around blood vessels, was not phosphorylated. This phosphorylation pattern was confirmed in immunoprecipitation studies in which activated p38 MAPK from synovial tissue extracts was identified as p38 MAPKalpha and -gamma but not p38 MAPKbeta or -delta. CONCLUSION: These data show that the alpha and gamma isoforms of p38 MAPK dominate in chronic inflammation. Effective strategies to inhibit p38 MAPK should therefore aim to specifically target either or both of these isoforms.

Aberrant expression of the autoantigen heterogeneous nuclear ribonucleoprotein-A2 (RA33) and spontaneous formation of rheumatoid arthritis-associated anti-RA33 autoantibodies in TNF-alpha transgenic mice.

Human TNF-alpha transgenic (hTNFtg) mice develop erosive arthritis closely resembling rheumatoid arthritis (RA). To investigate mechanisms leading to pathological autoimmune reactions in RA, we examined hTNFtg animals for the presence of RA-associated autoantibodies including Abs to citrullinated epitopes (anti-cyclic citrullinated peptide), heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (anti-RA33), and heat shock proteins (hsp) (anti-hsp). Although IgM anti-hsp Abs were detected in 40% of hTNFtg and control mice, IgG anti-hsp Abs were rarely seen, and anti-cyclic citrullinated peptide Abs were not seen at all. In contrast, >50% of hTNFtg mice showed IgG anti-RA33 autoantibodies, which became detectable shortly after the onset of arthritis. These Abs were predominantly directed to a short epitope, which was identical with an epitope previously described in MRL/lpr mice. Incidence of anti-RA33 was significantly decreased in mice treated with the osteoclast inhibitor osteoprotegerin and also in c-fos-deficient mice lacking osteoclasts. Pronounced expression of hnRNP-A2 and a smaller splice variant was seen in joints of hTNFtg mice, whereas expression was low in control animals. Although the closely related hnRNP-A1 was also overexpressed, autoantibodies to this protein were infrequently detected. Because expression of hnRNP-A2 in thymus, spleen, brain, and lung was similar in hTNFtg and control mice, aberrant expression appeared to be restricted to the inflamed joint. Finally, immunization of hTNFtg mice with recombinant hnRNP-A2 or a peptide harboring the major B cell epitope aggravated arthritis. These findings suggest that overproduction of TNF-alpha leads to aberrant expression of hnRNP-A2 in the rheumatoid joint and subsequently to autoimmune reactions, which may enhance the inflammatory and destructive process.

CD44 is a determinant of inflammatory bone loss.

Chronic inflammation is a major trigger of local and systemic bone loss. Disintegration of cell-matrix interaction is a prerequisite for the invasion of inflammatory tissue into bone. CD44 is a type I transmembrane glycoprotein that connects a variety of extracellular matrix proteins to the cell surface. Tumor necrosis factor (TNF) is a major inducer of chronic inflammation and its overexpression leads to chronic inflammatory arthritis. By generating CD44(-/-) human TNF-transgenic (hTNFtg) mice, we show that destruction of joints and progressive crippling is far more severe in hTNFtg mice lacking CD44, which also develop severe generalized osteopenia. Mutant mice exhibit an increased bone resorption due to enhanced number, size, and resorptive capacity of osteoclasts, whereas bone formation and osteoblast differentiation are not affected. Responsiveness of CD44-deficient osteoclasts toward TNF is enhanced and associated with increased activation of the p38 mitogen-activated protein kinase. These data identify CD44 as a critical inhibitor of TNF-driven joint destruction and inflammatory bone loss.

Single and combined inhibition of tumor necrosis factor, interleukin-1, and RANKL pathways in tumor necrosis factor-induced arthritis: effects on synovial inflammation, bone erosion, and cartilage destruction.

OBJECTIVE: To investigate the efficacy of single and combined blockade of tumor necrosis factor (TNF), interleukin-1 (IL-1), and RANKL pathways on synovial inflammation, bone erosion, and cartilage destruction in a TNF-driven arthritis model. METHODS: Human TNF-transgenic (hTNFtg) mice were treated with anti-TNF (infliximab), IL-1 receptor antagonist (IL-1Ra; anakinra), or osteoprotegerin (OPG; an OPG-Fc fusion protein), either alone or in combinations of 2 agents or all 3 agents. Synovial inflammation, bone erosion, and cartilage damage were evaluated histologically. RESULTS: Synovial inflammation was inhibited by anti-TNF (-51%), but not by IL-1Ra or OPG monotherapy. The combination of anti-TNF with either IL-1Ra (-91%) or OPG (-81%) was additive and almost completely blocked inflammation. Bone erosion was effectively blocked by anti-TNF (-79%) and OPG (-60%), but not by IL-1Ra monotherapy. The combination of anti-TNF with IL-1Ra, however, completely blocked bone erosion (-98%). Inhibition of bone erosion was accompanied by a reduction of osteoclast numbers in synovial tissue. Cartilage destruction was inhibited by anti-TNF (-43%) and was weakly, but not significantly, inhibited by IL-1Ra, but was not inhibited by OPG monotherapy. The combination of anti-TNF with IL-1Ra was the most effective double combination therapy in preventing cartilage destruction (-80%). In all analyses, the triple combination of anti-TNF, IL-1Ra, and OPG was not superior to the double combination of anti-TNF and IL-1Ra. CONCLUSION: Articular changes caused by chronic overexpression of TNF are not completely blockable by monotherapies that target TNF, IL-1, or RANKL. However, combined approaches, especially the combined blockade of TNF and IL-1 and, to a lesser extent, TNF and RANKL, lead to almost complete remission of disease. Differences in abilities to block synovial inflammation, bone erosion, and cartilage destruction further strengthen the rationale for using combined blockade of more than one proinflammatory pathway.

Osteoclasts are essential for TNF-alpha-mediated joint destruction.

The detailed cellular and molecular mechanisms leading to joint destruction in rheumatoid arthritis, a disease driven by proinflammatory cytokines, are still unknown. To address the question of whether osteoclasts play a pivotal role in this process, transgenic mice that express human TNF (hTNFtg) and that develop a severe and destructive arthritis were crossed with osteopetrotic, c-fos-deficient mice (c-fos(-/-)) completely lacking osteoclasts. The resulting mutant mice (c-fos(-/-)hTNFtg) developed a TNF-dependent arthritis in the absence of osteoclasts. All clinical features of arthritis, such as paw swelling and reduction of grip strength, progressed equally in both groups. Histological evaluation of joint sections revealed no difference in the extent of synovial inflammation, its cellular composition (except for the lack of osteoclasts), and the expression of matrix metalloprotein-ase-3 (MMP-3) and MMP-13. In addition, cartilage damage, proteoglycan loss, and MMP-3, -9, and -13 expression in chondrocytes were similar in hTNFtg and c-fos(-/-)hTNFtg mice. However, despite the presence of severe inflammatory changes, c-fos(-/-)hTNFtg mice were fully protected against bone destruction. These data reveal that TNF-dependent bone erosion is mediated by osteoclasts and that the absence of osteoclasts alters TNF-mediated arthritis from a destructive to a nondestructive arthritis. Therefore, in addition to the use of anti-inflammatory therapies, osteoclast inhibition could be beneficial for the treatment of rheumatoid arthritis.

Characterization of autoreactive T cells to the autoantigens heterogeneous nuclear ribonucleoprotein A2 (RA33) and filaggrin in patients with rheumatoid arthritis.

The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33-induced cytokine secretion revealed high IFN-gamma and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 phenotype and secreted higher amounts of IFN-gamma than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA.

Tumor necrosis factor alpha-mediated joint destruction is inhibited by targeting osteoclasts with osteoprotegerin.

OBJECTIVE: To study the effects of osteoclast-targeted therapies, such as osteoprotegerin (OPG) and pamidronate, on joint inflammation and bone destruction using a tumor necrosis factor alpha (TNF alpha)-transgenic mouse model. METHODS: Mice were placed into 5 groups that received either OPG, pamidronate, a combination of both agents, infliximab as a positive control, or phosphate buffered saline as a negative control. Treatment was initiated at the onset of arthritis, continued over 6 weeks, and thereafter, the clinical, radiologic, and histologic outcomes were assessed. RESULTS: A significant improvement in clinical symptoms, as assessed by the reduction of paw swelling, was only found in the infliximab group, whereas all other treatment groups failed to show significant improvement. However, when assessing structural damage with radiographic analysis, a significant retardation of joint damage was evident in animals treated with OPG (55% reduction of erosions), pamidronate (50% reduction of erosions) the combination therapy of OPG and pamidronate (64% reduction of erosions), and with infliximab (66% reduction of erosions). Confirming these data, quantitative histologic analysis revealed a significant reduction in the size of bone erosions in all treatment groups (OPG 56%, pamidronate 53%, OPG and pamidronate 81%, and infliximab 46%) compared with the control group. Furthermore, a significant reduction of osteoclast numbers was seen in animals treated with OPG alone or in combination with pamidronate as well as in animals treated with infliximab. CONCLUSION: These data suggest that OPG alone or in combination with bisphosphonates is an effective therapeutic tool for the prevention of TNF alpha-mediated destruction of bone by reducing the number of bone-resorbing cells in the inflammatory tissue.