The innate and adaptive immune systems are important in the pathogenesis of rheumatoid arthritis (RA). The presence of activated T cells and B cells, the rearrangement of their respective receptors, and their specificity for local antigens within the joint all support the role of the antigen-driven adaptive responses in the pathogenesis of RA (1). However, an increasing body of data supports the role of the innate immune system in RA and in experimental models of RA (2–4). Toll-like receptors (TLRs) may be critical for the generation of both innate and adaptive immunity. In response to pathogens, activation of the innate immune system through TLRs on macrophages and dendritic cells (DCs) results in the production of proinflammatory cytokines, including tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β). This activation promotes the development of the adaptive immune response involving T and B lymphocytes, which then contributes to clearance of the pathogenic stimulus and resolution of the inflammatory response. Despite the development of an adaptive immune response, expression of the early responders TNFα and IL-1β persists and may directly contribute to the pathogenesis of RA (2, 4–8).
At least 10 mammalian TLR family members have been identified. TLRs are pattern recognition receptors that may be found on a variety of cells and tissues (9), but they are particularly important on monocytes, macrophages, and DCs (10). Gene deletion studies have demonstrated that TLR-4 is principally responsible for lipopolysaccharide (LPS)–induced activation, while TLR-2–deficient mice were unresponsive to Staphylococcus aureus peptidoglycan (PG) (11, 12). TLR-2 and TLR-4 signaling lead to the activation of NF-κB (2) and the MAP kinases JNK (7) and p38 (13) through myeloid differentiation factor 88–dependent and –independent pathways (14, 15).
Recent studies have demonstrated the increased expression of TLR-2 and TLR-4 on peripheral blood (PB) monocytes from patients with RA (16, 17). In a study utilizing immunohistochemistry, both TLR-2 and TLR-4 were found to be expressed in the synovial tissue of patients with RA (16–18). Furthermore, in a study using reverse transcriptase–polymerase chain reaction (RT-PCR) and in situ hybridization, TLR-2 was found to be expressed in the rheumatoid joint and to be up-regulated in RA synovial fibroblasts by TNFα and IL-1β (8, 19).
While the expression of TLR-2 and TLR-4 on synovial tissue macrophages has been documented (16–18), quantitative studies have not been performed to examine the level of expression of TLR-2 and TLR-4 on synovial macrophages (the principal source of proinflammatory cytokines in RA) and to define the response to TLR ligands. These studies are important, because ligands for TLR-2 and TLR-4 have been identified in the rheumatoid joint. Bacterial PG was identified in RA synovial tissue by a monoclonal antibody, particularly in macrophages and antigen-presenting cells (20, 21). Additionally, endogenous mammalian TLR agonists, including fibrinogen, extra domain A (ED-A) of fibronectin, Hsp60 and Hsp70, low molecular weight fragments of hyaluronic acid, and high mobility group box chromosomal protein 1 (HMGB-1), a highly conserved nuclear protein that stabilizes nucleosome formation, are expressed in the RA joint, and each has been shown to activate NF-κB through TLR-4 and/or TLR-2 (22–29).
Macrophages, an important component of the innate immune system, are the principal source of TNFα, IL-1β, and other cytokines and chemokines, such as IL-8, that are pivotal in promoting inflammation and joint destruction in RA (30, 31). These observations demonstrate the critical role of macrophages in chronic synovitis, and they suggest that TLRs may be important not only for the initiation of this process, but also for its persistence and progression. The current study was undertaken to characterize the expression and function of TLR-2 and TLR-4 on CD14+ macrophages obtained from the joints of patients with RA. The expression of TLR-2 and TLR-4 was increased on macrophages isolated from the joints of patients with RA compared with control macrophages and monocytes; however, there was no significant difference in this expression between RA and other forms of inflammatory arthritis. Despite the lack of difference between cell surface TLR-2 and TLR-4 expression, the responses to PG and LPS were significantly greater with RA synovial macrophages compared with macrophages in other forms of inflammatory arthritis or compared with normal control in vitro–differentiated macrophages. Factors other than the level of cell surface expression of TLR-2 and TLR-4 were responsible for the increased response to TLR ligands by RA synovial macrophages.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Previous studies demonstrated that TLR-2 and TLR-4 are expressed in RA synovial tissue (8, 16, 18) and that this expression was increased compared with that in osteoarthritis or normal synovial tissue (18). TLR-2 was detected by in situ hybridization primarily in cells expressing fibroblast markers (8), while 2-color immunohistochemistry showed that TLR-2 colocalized with CD16+ macrophages in the lining (17). We have extended these observations, demonstrating that the expression of both TLR-2 and TLR-4 is increased on RA SF macrophages compared with that on normal in vitro–differentiated macrophages or normal monocytes. There was no difference in the expression of TLR-2 or TLR-4 on synovial macrophages from patients with RA and those from patients with other forms of inflammatory arthritis. Nonetheless, activation by both TLR-2 and TLR-4 ligands was greater with RA synovial macrophages than with those from patients with other forms of inflammatory arthritis or with control in vitro–differentiated macrophages. These observations suggest that activation through TLR-2 and TLR-4 might contribute to the ongoing inflammation in RA.
The mechanism contributing to the increased expression of TLR-2 and TLR-4 on macrophages obtained from the joints of patients with RA might be related to local factors, since there was no difference between RA and other forms of inflammatory arthritis. TLR-2 expression was increased on PB monocytes in response to LPS and IL-1β (40, 41). Further, treatment of monocytes with interferon-γ (IFNγ) resulted in increased cell surface expression of both TLR-2 and TLR-4 (18, 42), and IFNγ sensitized the monocytes to respond to LPS (43). Although the levels of IFNγ detected in SF of patients with established RA were low (44), IFNγ mRNA was present in synovial tissue lymphocytes (45), and it is possible that IFNγ contributes to sensitizing macrophages to express increased levels of TLR-2 and TLR-4 in vivo. Other cytokines expressed locally, including IL-10 and macrophage colony-stimulating factor, may also contribute to the increased TLR-2 expression in the RA joint (17). In summary, local factors may contribute to the increased expression of TLR-2 and TLR-4 in RA.
The effect of age and therapy on TLR expression was examined. We observed no effect of age or medications, including nonbiologic DMARDs or TNF inhibitors, on the expression of TLR-2 or TLR-4 in joints of RA patients or patients with other forms of inflammatory arthritis. A recent study demonstrated a decrease of TLR-2 and TLR-4 expression on PB monocytes and in the synovial tissue following therapy with TNFα blockers, supporting a role for inflammation in effecting the expression of TLRs (16). We did not observe an effect of therapy on TLR expression, probably because we obtained SF samples from patients with active disease despite therapy and did not examine the samples before and after the initiation of therapy, when disease activity was improved.
Prior studies have not examined the ability of RA synovial macrophages to respond to TLR ligands. A key observation in this study is that RA synovial macrophages demonstrated increased activation, employing both TLR-2 and TLR-4 ligands, compared with macrophages from the joints of patients with other forms of inflammatory arthritis and compared with control in vitro–differentiated macrophages. There are a number of potential explanations for the differences between these groups. We first explored the possibility that the level of TLR may control the response. When we used control macrophages, the cell surface expression of TLR-2 was highly associated with the level of cytokine induced by stimulation with the TLR-2 ligand PG. Further, the forced reduction of TLR-2 expression resulted in a decreased response to PG. These observations support the hypothesis that increased expression of TLR-2 may result in an enhanced response to TLR-2 ligands. Concordant with such a relationship for TLR-4, the level of TLR-4 expression in mice determined the responsiveness to the TLR-4 ligand LPS (46). In contrast, in the present study, no relationship between the cell surface expression of TLR-2 and response to PG, or between the cell surface expression of TLR-4 and response to LPS, was observed with SF macrophages in RA or other forms of inflammatory arthritis. This lack of association may be explained by several factors.
The effects of therapy may have contributed to these results, since PG-induced IL-8 expression by synovial macrophages of RA patients receiving nonbiologic DMARDs was reduced (P < 0.05) compared with that by synovial macrophages of RA patients who were not receiving these medications. There was no reduction of the response to LPS in patients receiving nonbiologic DMARDs. It is possible that a reduction of PG-induced IL-8 expression by RA synovial macrophages in patients receiving nonbiologic DMARDs may explain why this response was not significantly greater than that observed with RA PB monocytes. Also, in the absence of stimulation, IL-8 mRNA expression was 50-fold greater in normal monocytes than in normal in vitro–differentiated macrophages (data not shown), which may contribute to an increased PG-induced expression of IL-8 by monocytes. This explanation seems less likely, since the LPS-induced IL-8 expression by RA synovial macrophages was significantly greater than that observed with RA PB monocytes. Further, no effect on activation by PG or LPS was associated with anti-TNFα therapy. Therefore, although therapy may have modulated the degree of activation, the increased response to TLR ligands by RA synovial macrophages was not associated with the type of therapy.
It is possible that the RA synovial macrophages were inherently more responsive than those obtained from patients with other forms of inflammatory arthritis, due to conditioning within the joint. It is possible that activation through an endogenous TLR ligand, such as heat-shock proteins (HSPs) (17, 47), may predispose the macrophages to heightened activation by subsequent exposure to microbial TLR ligands (48, 49). Alternatively, it is also feasible that prior in vivo exposure to potential endogenous TLR-2 or TLR-4 ligands may have induced tolerance to repeat stimulation (50, 51), partially reducing the response expected for the level of TLR-2 or TLR-4 expression in some RA patients, and possibly accounting for the lack of association between TLR expression and response to TLR ligand in RA patients. In support of this possibility, the PG-induced responses by RA synovial macrophages, although increased compared with those by control in vitro–differentiated macrophages, were less than would have been expected by the control macrophages at comparable levels of TLR-2. This was also the case for macrophages from the joints of patients with other forms of inflammatory arthritis. Further, it is possible that the reduced response by macrophages in other forms of inflammatory arthritis (compared with that by RA macrophages) may be due to greater prior in vivo activation of these macrophages in response to endogenous TLR ligands, resulting in tolerance to a repeat challenge by microbial TLR ligands (50–52).
Differences between RA synovial macrophages and control in vitro–differentiated macrophages, examined by intracellular staining for cytokines, may relate to the fact that the control macrophages were purified, while the macrophages present in the RA SF samples contained a mixture of cells including T lymphocytes. It is possible that additional cytokines, such as IFNγ, IL-6, or granulocyte–macrophage colony-stimulating factor, secreted from other cell types may have increased the responsiveness of the macrophages obtained from the SF of RA patients compared with that of the control in vitro–differentiated macrophages. However, the SF from patients with other forms of inflammatory arthritis and the RA PB also contained a mixture of cell types, and their response to PG and LPS was generally reduced compared with the results obtained with RA synovial macrophages. The fact that purified RA synovial macrophages also demonstrated an increased response to PG compared with that of control macrophages supports the notion of a primary role for the RA synovial macrophage.
How might activation through TLR-2 or TLR-4 contribute to the pathogenesis of RA? We suggest that once inflammation within the joint causes joint destruction, a variety of molecules are released, such as ED-A of fibronectin, HSPs, and HMGB-1. Each of these molecules is highly expressed in the rheumatoid joint, and each is capable of activating TLR-2 and/or TLR-4 (22–29). Therefore, once joint damage occurs, a self-perpetuating process, mediated by TLR ligation by endogenous ligands, may be established that promotes the chronic, progressive destruction mediated by the continued activation of macrophages. In support of the notion of the role of potential endogenous TLR ligands, a recent study demonstrated that SF from RA patients activated a TLR-4–expressing line, suggesting that TLR-4 ligands may be present in RA SF (53). Further studies with animal models will be required to fully elucidate the potential mechanisms by which activation of macrophages through endogenous TLRs may contribute to the progression of inflammatory synovitis.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Pope had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Huang, Pope.
Acquisition of data. Huang, Ma, Adebayo.
Analysis and interpretation of data. Huang, Pope.
Manuscript preparation. Huang, Pope.
Statistical analysis. Huang, Pope.