Rheumatoid arthritis (RA) is a chronic autoimmune disease that results in the destruction of cartilage and bone in joints. Collagen-induced arthritis (CIA) is a well-established murine model of this disease and shares many features with RA (1, 2). Specifically, susceptibility to both CIA and RA is associated with the specific class II major histocompatibility complex allele (3, 4). In addition, autoantibodies to type II collagen have been detected in the synovial fluid of patients with RA, and these autoantibodies have an aggravating effect on CIA in mice (5–7). In addition, pathogenic contributions of CD4+ T helper cells have been reported in both CIA and RA (8, 9).
Interleukin-17 (IL-17) is a cytokine secreted by T cells, natural killer (NK) cells, and neutrophils (10), and it induces IL-6, IL-8, chemokine, and metalloproteinase production by target cells (11). Central pathogenic roles of IL-17 in CIA have been reported recently. For example, systemic or local IL-17 gene transfer aggravated CIA, whereas administration of an IL-17–blocking antibody ameliorated CIA even after the onset of arthritis (12, 13), and IL-17–deficient mice also showed reduced severity of CIA (14). Furthermore, IL-23–deficient mice, which show an impaired Th17 response, do not exhibit CIA, because IL-23 is an essential factor for the maintenance of Th17 cells (15).
Although Roark et al recently reported the infiltration of IL-17–producing γ/δ T cells together with IL-17–producing CD4+ T (Th17) cells in inflamed joints of mice with CIA (16), the precise predominance, distribution, kinetics, cytokine-production requirements, and characteristics of these cells, especially in the context of IL-17–producing γ/δ T versus Th17 cells, remain unclear. Elucidation of these factors will be critical in terms of understanding the pathogenesis of CIA, finding novel therapeutic targets associated with IL-17, and determining the optimal timing and site for therapeutic intervention in CIA.
In the current study, we performed spatiotemporal analysis of IL-17–producing cells in CIA and demonstrated that γ/δ T cells are the predominant source of IL-17 in swollen joints of mice with CIA. IL-17–producing γ/δ T cells were maintained by IL-23 but not by type II collagen in vitro. Furthermore, IL-17 production by γ/δ T cells was efficiently stimulated by inflammatory cytokines independently of T cell receptor (TCR). Contrary to the results observed in mice with CIA, IL-17–producing γ/δ T cells could not be detected in the affected joints of patients with RA.
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The present study first focused on IL-17–producing T cells in the swollen joints of mice with CIA. It was observed that γ/δ T cells were the predominant source of IL-17 and were more abundant than Th17 cells. DX5+ NK cells did not secrete IL-17 in swollen joints. A direct comparison of the absolute numbers of IL-17–producing γ/δ T cells with the absolute numbers of Th17 cells simultaneously in each joint of mice with CIA was performed for the first time. Although it is known that γ/δ T cells are not necessary for the induction of CIA, because γ/δ TCR–deficient mice can mount CIA (37), the present findings in the kinetics study and adoptive transfer experiments, together with previous reports (16, 18, 38), suggest that not only Th17 cells but also IL-17–producing γ/δ T cells contribute to the exacerbation of CIA. In contrast, α/β τ cells, especially Th17 cells, are essential for the induction of CIA, because α/β TCR–deficient mice cannot mount CIA (37). In addition, IL-17–producing invariant NK T cells in CIA have been reported recently (37), but these cells were not analyzed in the current study.
The origin and functions of IL-17–producing γ/δ T cells in physiologic and pathologic conditions have been elucidated recently. It was reported that a subset of γ/δ T cells acquired an IL-17–producing function in the thymus (26) and produced cytokines immediately in response to initial stimulation. In various murine infectious disease models, these γ/δ T cells predominantly produce IL-17 and eradicate pathogens (40–43). However, the precise requirements of IL-17 production by γ/δ T cells especially in CIA are unknown, although IL-23 was known as a sufficient stimulant of IL-17 production by γ/δ T cells in naive mice (42). Here, it was demonstrated that the combination of IL-23 and IL-1β synergistically stimulated IL-17 production, but stimulation via γ/δ TCR had a limited effect. Given the enhanced expression of IL-1β and IL-23 in the inflamed joints of mice with CIA (44, 45), these findings suggest that IL-17 production by γ/δ T cells in CIA might mainly be an inflammatory cytokine–driven process rather than a TCR signal–driven process.
The present study showed that IL-17–producing γ/δ T cells were CCR6 positive, and CCR6 was already expressed on IL-17–producing γ/δ T cells in the thymus of naive mice. CCL20, the only chemokine known to interact with CCR6, is physiologically expressed at epithelial surfaces (46) and fibroblast-like synoviocytes (29) and is up-regulated in inflammatory conditions (30, 46). These findings suggest that CCR6 might have some roles in determining the physiologic distribution of IL-17–producing γ/δ T cells. In fact, it was found that a small number of CCR6+ IL-17–producing γ/δ T cells were present in the joints of naive mice.
Next, we focused on the differences between IL-17–producing γ/δ T cells and Th17 cells. IL-17–producing γ/δ T cells were maintained by IL-23 but not by a specific antigen (type II collagen, in this case). In contrast, Th17 cells responded to type II collagen and IL-23. Furthermore, IL-17–producing γ/δ T cells were induced equivalently in response to stimulation by IFA plus solution in the absence of type II collagen. Together with results from the previous study demonstrating that IL-17–producing γ/δ T cells are induced equally by CFA plus type II collagen and CFA (16), the present data suggest that IL-17–producing γ/δ T cells do not recognize the specific antigen (type II collagen) but rather proliferate in response to IL-23, which may be produced locally by synovial cells (44). The ligands of γ/δ T cells are largely unknown, and further analysis of possible antigens of IL-17–producing γ/δ T cells in CIA could be difficult (47). However, the present study confirmed the diverse usage of γ/δ TCR in IL-17–producing γ/δ T cells in CIA (Figure 4C), which supported the present conclusion that IL-17–producing γ/δ T cells are antigen independently induced by inflammatory cytokines.
In summary, it is speculated that the sequence of pathology of CIA is as follows. First, type II collagen–specific Th17 cells are induced by type II collagen plus CFA, which then infiltrate into the joints and cause primary inflammation. Although antigen-independent IL-17–producing γ/δ T cells could be induced simultaneously by CFA, they are not essential for the induction of arthritis. Next, primary inflammation induces local production of IL-23 from synoviocytes and increases the expression of IL-1β in joint cartilage and pannus (45). Locally produced IL-23 induces the proliferation of resident IL-17–producing γ/δ T cells. These γ/δ T cells, stimulated by IL-1β and IL-23, produce enhanced amounts of IL-17 and exacerbate the arthritis of CIA. Another, but not mutually exclusive, possibility is that primary inflammation enhances CCL20 expression in vascular endothelial cells and fibroblast-like synoviocytes (30) in inflamed joints and recruits CCR6+ IL-17–producing cells. In the ankylosing phase, the burned-out tissue does not produce inflammatory cytokines, and the activities and the number of IL-17–producing γ/δ T cells decrease to the basal level.
Finally, the cytokine profiles of T cells in the inflamed joints of SKG mice and patients with RA were compared with those in mice with CIA. In contrast to what was observed in mice with CIA, IL-17–producing γ/δ T cells were not detected in the swollen joints of SKG mice. A lack of IL-17–producing γ/δ T cells in SKG mice was not caused by the differences in strain or adjuvant. It was also observed that IL-17–producing γ/δ T cells are hardly induced in immunized joints, their DLNs, non-DLNs, and spleens of SKG mice (data not shown) 10 days after immunization with CFA plus type II collagen. Given that TCR signals in SKG mice are attenuated because of a point mutation in ZAP-70 (21), and differentiation of γ/δ T cells needs a strong signal via the TCR (48, 49), there may be some defects in γ/δ T cell differentiation in SKG mice. This speculation was supported by data showing impaired development of specific subsets of γ/δ T cells in ZAP-70–knockout mice (50). Furthermore, IL-17 production from γ/δ T cells in the synovial tissue of patients with RA has not yet been detected. In contrast to IL-17–producing γ/δ T cells, IFNγ-producing γ/δ T cells were present. In addition, among CD4+ T cells, Th1 cells were predominant; this finding was consistent with a previous report (51).
These results suggest that IFNγ-producing cells, but not IL-17–producing cells including γ/δ T cells, play predominant pathogenic roles in RA. These distinct pathogenic cell populations may result from differences between CIA and RA such as species and age-related susceptibility. Alternatively, IL-17–producing γ/δ T cells may play an important role in RA as well but are suppressed by the effects of medical treatment. It should be noted that in the present study, we could access joint materials only from patients with progressed stages of RA. Therefore, further studies with patients with recent-onset RA who have not received medical treatment are necessary to determine whether IL-17–producing γ/δ T cells are present.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Usui 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 conception and design. Y. Ito, Usui.
Acquisition of data. Y. Ito, Usui, Kobayashi, Iguchi-Hashimoto, H. Ito, Yoshitomi, Nakamura, Shimizu, Kawabata, Yukawa, Hashimoto, N. Sakaguchi, S. Sakaguchi, Yoshifuji, Nojima, Ohmura, Fujii, Mimori.
Analysis and interpretation of data. Y. Ito, Usui.