In patients with rheumatoid arthritis (RA), the synovium of the inflamed joints is the site of a chronic inflammatory reaction, in which leukocytes and macrophages infiltrate, synoviocytes proliferate, and several proinflammatory cytokines (especially, interleukin-6 [IL-6] and tumor necrosis factor α), autoantibodies, and immune complexes are produced (1). In this situation, infiltrating leukocytes and proliferating synoviocytes are thought to interact with each other. Accordingly, several in vitro studies have focused on the interaction between fibroblast-like synoviocytes (FLS) and leukocytes by studying the findings in cocultures of these cells (2–7). Coculture of FLS obtained from RA synovium with B lymphocytes causes adhesion of B lymphocytes to FLS, and consequently this interaction supports the survival of B lymphocytes and also enhances the production of cytokines and immunoglobulin (8–12). The former mechanism is dependent on vascular cell adhesion molecule 1 (VCAM-1) and very late activation antigen 4 (VLA-4) (10), whereas the latter is yet to be determined.
Osteopontin (OPN) is abundant in the bone matrix, where it acts as a bridge between hydroxyapatite and osteoclasts to support bone resorption (13–15). It is also secreted by T lymphocytes and assists in the maturation of B lymphocytes and the migration of macrophages (16–18). These diverse functions are partly explained by the several functional domains of OPN, including integrin-binding, calcium-binding, and heparin-binding sites. Several studies have revealed that the function of OPN depends on posttranslational modifications (19–24). These modifications, which vary between different OPN-expressing cells, consist of phosphorylation at dozens of serine and threonine residues, along with glycosylation (25), sialylation (21), transglutamination (26), and cleavage (27, 28). However, the association between the details of posttranslational modification and the function of OPN is still poorly understood.
As for the relationship between OPN and arthritis, OPN-null mice are protected against inflammatory joint destruction in collagen-induced arthritis (29). A blocking antibody directed against the thrombin-cleaved neoepitope of OPN, which cooperates with several integrin receptors as a ligand (30, 31), also shows a curative effect on induced arthritis in mice and monkeys (32, 33). Indeed, in patients with RA, OPN, especially in the thrombin-cleaved form, is strongly detected in the synovium and synovial fluid of inflamed joints (34–36). In vitro studies on the function of OPN in arthritis have revealed that OPN stimulates the production of several proinflammatory cytokines by mononuclear cells in patients with RA (37), and also that monocytes obtained from mice with induced arthritis show increased migration toward thrombin-cleaved OPN (32). However, these studies were performed using recombinant OPN, which lacks various posttranslational modifications, and thus the posttranslational modification–dependent functions were not taken into account.
Considering these results and the fact that OPN is also strongly expressed in FLS from the RA synovium (35), we hypothesized that RA FLS express a unique form of OPN that acts to stimulate production of cytokines by creating a bridge between FLS and B lymphocytes in cocultures of these cells. The purpose of this study was to analyze the role of OPN in the development of RA, by focusing on the interaction between FLS and B lymphocytes.
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The present study revealed a new role of OPN in the RA synovium, using primary cultured FLS derived from RA patients to analyze the function of FLS-expressed native OPN. A specifically modified 75-kd OPN was predominantly expressed by RA FLS and was associated with significantly elevated IL-6 production in FLS–B lymphocyte cocultures. The 75-kd OPN formed a >200-kd OPN/fibronectin–crosslinked molecule via transglutamination, which was detected on the synovial cell surface, resulting in exposure of its thrombin-cleaved neoepitope. This >200-kd cell surface OPN affected the interactions between FLS and B lymphocytes by supporting adhesion of B lymphocytes to FLS in FLS–B lymphocyte cocultures, and consequently IL-6 production was enhanced. This FLS–B lymphocyte interaction also appeared to occur in vivo.
OPN by itself weighs 37 kd, as determined previously using mass spectrometry (25), but due to its various posttranslational modifications, the migration of full-length OPN, detected on SDS-PAGE, differs among cell types within ∼45–80 kd (42). Accordingly, our results from Western blotting of OPN showed multiple bands with different molecular weights. In particular, a single band at 75 kd that was detected predominantly in RA FLS suggested that this specifically modified 75-kd OPN was worth investigating to determine the function of OPN in RA.
In fact, the localization of OPN was different between 75-kd OPN–negative and 75-kd OPN–positive FLS. The existence of >200-kd OPN in the cell surface fraction of 75-kd OPN–positive FLS on Western blotting, and the staining pattern of OPN on immunofluorescence assay of these FLS, showing the cell outline distribution, suggested that OPN was associated with the cell surface side of the plasma membrane. These distinct findings were considered attributable to 75-kd OPN, since they were negative in 75-kd OPN–negative FLS. Such posttranslational modification–dependent differences in subcellular localization of OPN have also been observed in previously published studies of OPN expressed by normal rat kidney (NRK) cells, which revealed that among phosphorylated and nonphosphorylated forms of OPN expressed by NRK cells, only phosphorylated OPN was associated with the cell surface (19, 23). Moreover, another study revealed that OPN was detected at >200 kd molecular weight and found to be localized on the surface of NRK cells when covalently crosslinked to fibronectin by a transglutaminase (26). Our results were consistent with that study, since the observations from immunoprecipitation and Western blotting of the FLS suggested that >200-kd OPN was an OPN/fibronectin-crosslinked molecule, and inhibition of a transglutaminase reduced the expression of >200-kd OPN. Considering these facts together, >200-kd OPN could be considered an OPN/fibronectin–covalently crosslinked molecule synthesized by a transglutaminase from 75-kd OPN.
In contrast, >200-kd cell surface OPN was poorly detected in 75-kd OPN–negative FLS. Moreover, overexpression of OPN by 75-kd OPN–negative FLS only increased the expression of 54-kd OPN, and did not induce expression of 75-kd OPN nor did it alter the expression of >200-kd OPN, suggesting that there might be an unknown enzyme in 75-kd OPN–positive FLS that performs the specific modifications of 75-kd OPN necessary for it to associate with the cell surface and to form the >200-kd OPN/fibronectin–crosslinked molecule.
To assess the relevance of 75-kd OPN in RA, we performed cocultures of FLS and B lymphocytes. Previous studies have revealed that FLS–B lymphocyte coculture allows the adhesion of B lymphocytes to FLS, which induces cell–cell interactions between FLS and B lymphocytes and, consequently, increases the production of several cytokines, including IL-6 (9, 12), but the mechanism has not been elucidated. We focused on IL-6 production in FLS–B lymphocyte cocultures and found that increased IL-6 production was associated with the existence of 75-kd OPN, which suggested that 75-kd OPN together with cell–cell interactions between FLS and B lymphocytes can enhance IL-6 production. We also revealed that among FLS and B lymphocytes, FLS were the dominant cell type for IL-6 production in coculture, as was shown in experiments involving knockdown of IL-6 in FLS.
To examine whether 75-kd OPN could enhance IL-6 production, we performed overexpression and knockdown of OPN in FLS, which demonstrated that IL-6 production was increased and decreased in accordance with the increase and decrease of 75-kd OPN and >200-kd OPN, respectively. Among these 2 OPN forms with different molecular weights, >200-kd OPN appeared to enhance IL-6 production, since the transglutaminase inhibitor that reduced the expression of only the >200-kd OPN significantly suppressed IL-6 production in 75-kd OPN–positive FLS–B lymphocyte cocultures (results not shown).
We then analyzed how 75-kd OPN or its crosslinked form, >200-kd OPN, enhanced IL-6 production in FLS–B lymphocyte cocultures. The >200-kd OPN on the cell surface exposed its thrombin-cleaved neoepitope, SVVYGLR (27), a ligand for integrin α4β1, also known as VLA-4, integrin α9β1, and integrin α4β7 (30). Considering the fact that VLA-4 is expressed by B lymphocytes and supports adhesion of B lymphocytes to FLS in FLS–B lymphocyte coculture (12), >200-kd OPN was indicated as the ligand for VLA-4, which acts as an adhesion molecule. This idea was supported by the results from our B lymphocyte adhesion assay with a blocking antibody against OPN, and by the fact that the transglutaminase inhibitor also suppressed adhesion of B lymphocytes to FLS (results not shown). Meanwhile, integrin α4β7, which was also expressed by B lymphocytes, did not mediate such adhesion (12).
VCAM-1, which was expressed by FLS and also supports B lymphocyte adhesion, possibly through VLA-4 (11), was detected in equal amounts among RA and non-RA FLS (results not shown). Therefore, we postulated that integrin α4β7 and VCAM-1 were not involved in the adhesion of B lymphocytes to FLS or subsequent IL-6 production, and that such adhesion was mediated by >200-kd OPN and VLA-4, which would further initiate the cell–cell interaction between FLS and B lymphocytes, leading to IL-6 production.
Our findings suggested that, in response to these FLS–B lymphocyte interactions, FLS boosted their IL-6 production. Similar studies on cell–cell interactions between FLS and T lymphocytes have shown that lymphocyte function–associated antigen 1, intercellular adhesion molecule 2, and the ezrin/Akt pathway are involved in their interaction, which also enhances IL-6 production (2). Thus, this pathway may also be involved in FLS–B lymphocyte interactions, although further investigation is needed to fully elucidate the mechanism.
In summary, the findings from these in vitro experiments showed that RA FLS are characterized by the expression of 75-kd OPN. This was the substrate of a transglutaminase that formed the >200-kd OPN/fibronectin–crosslinked molecule, a molecule localized on the surface of FLS in its thrombin-cleaved form. This surface OPN mediated cell–cell interactions between FLS and B lymphocytes, and enhanced IL-6 production (Figure 6). Moreover, such FLS–B lymphocyte interactions, or interactions between FLS-expressing OPN and B lymphocytes stimulating IL-6 production, appeared to take place in vivo, as shown by immunohistochemistry. Taking into account the previously reported findings on the pathogenic significance of IL-6 (45), B lymphocytes (46, 47), VLA-4 (48), and the thrombin-cleaved neoepitope of OPN (32, 33) in RA, the present study revealed a novel role of OPN in RA.
Figure 6. Diagram of the in vitro findings, showing cell–cell interactions between fibroblast-like synoviocytes (FLS) and B lymphocytes (BL). RA FLS were characterized by the expression of 75-kd OPN, from which a transglutaminase synthesized the >200-kd OPN/fibronectin (FN)–crosslinked molecule that was localized on the synovial cell surface in its thrombin-cleaved form. This cell surface OPN mediated cell–cell interactions between OPN and B lymphocytes by supporting adhesion of B lymphocytes to FLS through very late activation antigen 4 (VLA-4) and enhanced IL-6 production. An unknown link between FLS and B lymphocytes is also shown. See Figure 5 for other definitions.
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Regarding the weaknesses of this study, we need to mention the discrepancy in the findings between RA FLS and 75-kd OPN–positive FLS. Since 75-kd OPN was detected in all 10 RA FLS, but also in 3 non-RA FLS, it was not specific to RA FLS. There is no doubt that 75-kd OPN enhanced IL-6 production in FLS–B lymphocyte cocultures and appeared to aggravate chronic inflammation in vivo. However, it should not be considered the cause of RA; rather, we could postulate that it is one of several molecules with induced expression in arthritis and is involved in the chronic progression of arthritis by stimulating IL-6 production. This is supported by the observation that, in the joint tissue of a non-RA donor, the FLS expressed amounts of 75-kd OPN comparable with those in RA FLS, and showed severe synovitis at the time of surgery.
We also have to note that the mechanism involved in the modification of 75-kd OPN has not been defined, and the responsible transglutaminase has not been identified. Further investigations in this area would be required for better understanding of the pathology of RA. Nevertheless, our results show that a specifically modified 75-kd form of OPN was expressed by RA FLS. This form of OPN affected cell–cell interactions between FLS and B lymphocytes by supporting the adhesion of B lymphocytes to FLS. As a result, IL-6 production was enhanced in FLS–B lymphocyte cocultures.