Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial hyperplasia. Inflammation and degradation within the joints of RA patients are generally accompanied by elevated levels of cytokines, chemokines, and matrix-degrading enzymes (1–5).
Originally thought to be solely responsible for energy storage and acting as structural connective tissue for organs and padding gaps, adipose tissue is increasingly being regarded as an immunoendocrine organ (3, 6–9). In addition, cytokine-like mediators produced by the major cell type of adipose tissue, the adipocyte, have been identified and termed adipocytokines or, for short, adipokines (7). In RA, the production of adipokines is increased, especially within, but not limited to, periarticular adipose tissue (10–14). However, the detailed biologic function of adipokines in RA still needs to be fully elucidated.
In this study, we focused on the adipokine adiponectin, a C1q/tumor necrosis factor α (TNFα) homolog (15, 16), which is increased in the synovial fluid of RA patients compared with osteoarthritis (OA) patients (11). Adiponectin is the adipokine with the highest concentration in human sera and synovial fluids (in the μg/ml range) (11, 17). Adiponectin was previously thought to be secreted by adipocytes only (18), but has been shown to be expressed by other cell types, including osteoblasts and synovial cells such as RA synovial fibroblasts (RASFs) (19, 20). Besides its numerous functions in energy metabolism, adiponectin plays a role in the cardiovascular system (21) and in the immune system (22, 23).
In this study, we investigated the differential effects that adiponectin might have within the RA joint, specifically on effector cells in RA pathophysiology. RASFs, aggressive cartilage-invading cells, play a central role in RA (24, 25). They are exposed in vivo to highly increased concentrations of adiponectin in the synovial tissue and in the synovial fluid of RA patients. We therefore investigated the effects of adiponectin on the transcriptome and secretome of RASFs with special interest in proinflammatory and matrix-degrading molecules involved in the pathophysiology of RA. In previous experiments (20), we showed that adiponectin strongly induced interleukin-6 (IL-6) and pro–matrix metalloproteinase 1 (proMMP-1) in RASFs, suggesting that adiponectin has proinflammatory and prodestructive properties in RA as opposed to the antiinflammatory and antiatherogenic properties observed in cardiovascular and metabolic diseases (21, 26–28). In addition, we studied the effects of adiponectin on other cell types relevant to RA pathophysiology, including lymphocytes, chondrocytes, and endothelial cells, to further elucidate the adiponectin-mediated effects in RA joints. Adiponectin-induced signaling in RASFs was also analyzed.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
In the present study, we closely analyzed the effect of adiponectin on the gene expression profile of RASFs (20) and investigated the effect of adiponectin on other key cell types involved in the pathogenesis of RA, namely, lymphocytes, chondrocytes, and endothelial cells. The aggressive phenotype of RASFs is characterized by driving inflammation, invasion, degradation, and migration (25, 36). The process of inflammation, primarily within the synovial tissue, is characterized by the recruitment of inflammatory cells and the release of proinflammatory mediators and chemokines. MMPs are key factors in perpetuating invasion into and degradation of articular cartilage (5, 37, 38). Chemokines cause migration of further inflammatory cells and RASFs into the synovial tissue and toward the invasion zone. The endogenously activated RASFs (39) are further stimulated by inflammatory immune cells and numerous cytokines.
In this study, we showed that adiponectin stimulated the synthesis of chemokines in particular, but also of cytokines, MMPs, genes involved in inflammation and bone metabolism, and receptors and growth factors in RASFs. Adiponectin, which is found at high concentrations in the synovial fluid of RA patients (11, 17), promotes proinflammatory and prodestructive pathways in RA as opposed to its “protective role” in metabolic and cardiovascular diseases (21, 26–28). Numerous changes were observed in RASFs upon adiponectin stimulation. Proinflammatory factors, including IL-6 and IL-11 (40, 41), were strongly induced in RASFs. IL-11, like IL-6, belongs to a cytokine family that shares the cytokine receptor subunit gp130. Soluble IL-6R forms a complex with membrane-bound gp130 and thus enables cells that do not express IL-6R themselves to respond to IL-6. It is hence assumed to act as an IL-6 agonist (41, 42). Activin A induces cell proliferation of RASFs (43) and is also involved in inflammatory processes (32, 44).
A very important group of genes and proteins that are regulated by adiponectin is the group of MMPs, specifically, MMP-1, MMP-3, MMP-10, and MMP-12. Protein secretion of proMMP-1, MMP-3, and MMP-10, as well as levels of MMP-12 mRNA, were considerably increased in RASFs by adiponectin stimulation. All of these MMPs are known to be involved in cartilage degradation in inflammatory arthritis (5, 37, 38). The direct involvement of MMPs in the aggressive phenotype of RASFs has been shown in previous studies and results in the invasion and destruction of adjacent cartilage by RASFs (1, 45).
Another crucial ontologic group that was highly regulated in RASFs by adiponectin consisted of chemokines. The chemokines GROα, ENA-78, GCP-2, IL-8, and MCP-1, which were produced by RASFs upon adiponectin stimulation, attract inflammatory cells to the synovium to further increase inflammation (4). Besides being a chemoattractant, IL-8 is a potent angiogenic factor (46–48). Of note, distinct chemokines that appeared to be highly regulated at the mRNA level, such as MIP-3α and I-TAC, were not secreted by RASFs, suggesting a posttranscriptional regulation mechanism. Cellular lysates of adiponectin-stimulated and unstimulated RASFs did not show detectable amounts of MIP-3α and I-TAC either, which excluded the possibility that the proteins are not secreted but instead retained within the cell.
Bone marrow stromal cell antigen 2 (BST2) has been suggested to be involved in pre–B cell growth and is expressed on RA-derived synovial cell lines and other fibroblast cell lines (49). However, its specific function has not yet been determined. Interestingly, adiponectin induced strong expression of BST2 in RASFs, underlining the potential pathophysiologic role in RA. Up-regulated fibroblast growth factors 10 and 13 may contribute to RASF proliferation.
Due to concerns that LPS contamination found in recombinantly produced proteins may contribute to the effects observed for adiponectin, we performed experiments to verify that the effects were adiponectin mediated. LPS, a polysaccharide, is unaffected by proteases, in contrast to adiponectin. Therefore, any effect left after proteolytic digestion of adiponectin can be attributed to LPS, provided that the digestion was effective, which we successfully confirmed. The abrogation of the adiponectin-induced IL-8 and IL-6 secretion by RASFs when using digested adiponectin showed that the effects were actually adiponectin mediated. Both treated and untreated LPS had a strong effect on IL-8 secretion by RASFs, demonstrating that LPS activity is not destroyed by the digestion procedure. Of note, the low LPS concentration (0.1 mg/ml) usually expected in recombinant proteins containing minor LPS contaminations caused only a minimal increase in IL-8 secretion, suggesting that the adiponectin used was not significantly contaminated.
Induction of gene expression and protein secretion by adiponectin is not restricted to RASFs. We showed that other central cell types involved in RA pathogenesis also respond to adiponectin. Factors that are induced in RASFs are similarly induced upon adiponectin stimulation in bovine and human chondrocytes. IL-6, RANTES, and MMP-3 were induced in bovine chondrocytes, and secretion of IL-6, IL-8, GROα, MCP-1, proMMP-1, and MMP-3 was increased in human chondrocytes. In primary human lymphocytes, adiponectin stimulation resulted in the synthesis of TNFα, IL-6, IL-8, and RANTES, indicating that the currently successfully targeted cytokines TNFα and IL-6 are at least in part regulated by adiponectin. TNFα, IL-6, and IL-8 contribute to the initiation and perpetuation of immune responses by activation of the cells of the immune system. RANTES secretion was up-regulated in activated but not in unactivated lymphocytes. Activation of lymphocytes may “unlock” cellular signaling pathways and thus allow adiponectin to affect the secretion of RANTES, a chemokine responsible for the recruitment of additional lymphocytes to the inflamed tissue.
Interestingly, none of the selected CD3+, CD4+, CD8+, and CD19+ lymphocyte subpopulations responded to adiponectin as strongly as total lymphocytes (at equal cell densities). CD8+ and CD19+ cells did not respond at all to stimulation with adiponectin, while CD3+ and CD4+ cells responded by significant up-regulation of TNFα, IL-6, and IL-8. The interaction of lymphocytes and/or further subpopulations in unseparated lymphocytes is most likely responsible for these findings. Endothelial cells that were stimulated with adiponectin displayed enhanced secretion of IL-6, IL-8, GROα, MCP-1, and RANTES. Also, mRNA for adhesion molecules and for the endothelial cell activation markers ICAM-1 and VCAM-1 were up-regulated. The adiponectin-induced changes may contribute to endothelial activation, leukodiapedesis, thus increasing the transmigration of leukocytes from the blood into RA synovium.
The aforementioned results indicate that adiponectin-induced chemokines from RASFs, chondrocytes, and lymphocytes contribute to the activation and recruitment of inflammatory cells into the synovial tissue. Adiponectin is predominantly expressed in the lining layer and sites of synovial invasion as well in the perivascular area and around leukocyte infiltrates (20). The additional adiponectin-induced chemokine expression around vessels and in leukocyte infiltrates may increase the influx of inflammatory cells into the synovium and recruit additional RASFs to the sites of invasion. Here, adiponectin is strongly expressed, and may increase production of MMPs by RASFs as well as chondrocytes, contributing to the perpetuation of inflammation and to its becoming chronic as well as to matrix destruction in RA.
Since adiponectin levels are generally higher in RA than in OA (20), we were interested in whether adiponectin contributes to the aggressive cellular phenotype of RASFs (25, 50). OASFs, which do not display such an activated phenotype, were used as a control. For all analyzed parameters, RASFs revealed a stronger response to adiponectin than did OASFs. Therefore, adiponectin probably plays a more significant role in RA than in OA, especially with regard to the chronic inflammation and the aggressive phenotype of RASFs.
Further analyses showed that p38 MAPK (20) and PKC were involved in adiponectin-mediated signaling in RASFs, in contrast to PKA and NF-κB. Hence, at least for IL-6 and MCP-1, the signaling molecules p38 MAPK and PKC are very likely to be part of adiponectin-induced signaling.
Our data show that adiponectin significantly affects the gene and protein expression profiles of RASFs, lymphocytes, chondrocytes, and endothelial cells in a manner that promotes inflammation and matrix destruction in RA. These findings indicate that adiponectin appears to be a major player in the pathogenesis of RA.