Paul D. Doodes

Antigen-Specific B Cells Are Required as APCs and Autoantibody-Producing Cells for Induction of Severe Autoimmune Arthritis

B cells play an important role in rheumatoid arthritis, but whether they are required as autoantibody-producing cells as well as APCs has not been determined. We assessed B cell autoantibody and APC functions in a murine model of autoimmune arthritis, proteoglycan (PG)-induced arthritis, using both B cell-deficient mice and Ig-deficient mice (mIgM) mice that express an H chain transgene encoding for membrane-bound, but not secreted, IgM. The IgH transgene, when paired with endogenous λ L chain, recognizes the hapten 4-hydroxy-3-nitro-phenyl acetyl and is expressed on 1–4% of B cells. B cell-deficient and mIgM mice do not develop arthritis after immunization with PG. In adoptive transfer of PG-induced arthritis into SCID mice, T cells from mIgM mice immunized with PG were unable to transfer disease even when B cells from PG-immunized wild-type mice were provided, suggesting that the T cells were not adequately primed and that Ag-specific B cells may be required. In fact, when PG was directly targeted to the B cell Ig receptor through a conjugate of 4-hydroxy-3-nitrophenyl acetyl-PG, T cells in mIgM mice were activated and competent to transfer arthritis. Such T cells caused mild arthritis in the absence of autoantibody, demonstrating a direct pathogenic role for T cells activated by Ag-specific B cells. Transfer of arthritic serum alone induced only mild and transient arthritis. However, both autoreactive T cells and autoantibody are required to cause severe arthritis, indicating that both B cell-mediated effector pathways contribute synergistically to autoimmune disease.

IL-27 Induces a Th1 Immune Response and Susceptibility to Experimental Arthritis

IL-27 is the newest member of the cytokine family comprised of IL-12 and IL-23. IL-27 was originally described as a cytokine that along with IL-12 induces the differentiation of naive precursor T cells into Th1 effector cells. This activity has been called into question based on evidence in infectious disease and autoimmune models in which IL-27 is not absolutely required for the generation of IFN-γ, and IL-27 plays a regulatory role in controlling inflammation. We have previously reported in proteoglycan-induced arthritis (PGIA), a model of rheumatoid arthritis, that severe arthritis is dependent on the production of IFN-γ. In this study, we report that IL-27 was expressed in spleen and joint tissues of arthritic mice. We determined the involvement of IL-27 in PGIA by assessing the progression of arthritis in IL-27R−/− mice. Development of arthritis in IL-27R−/− mice was delayed and severity reduced in comparison with IL-27R+/+ littermate controls. Histology confirmed a reduction in joint cellularity, cartilage destruction, and bone erosion. Diminished arthritis was associated with fewer T cells producing IFN-γ and decreased IFN-γ secretion overtime. Moreover, the frequency of IL-4- and IL-17-expressing T cells and the production of IL-4 and IL-17 were similar in IL-27R−/− mice and controls. Our results indicate that IL-27 is critically involved in the induction of inflammation in PGIA. IL-27 functions by inducing the differentiation of IFN-γ-producing T cells in vivo that are essential for the development of arthritis.

Development of Proteoglycan-Induced Arthritis Is Independent of IL-17

IL-17 is the hallmark cytokine for the newly identified subset of Th cells, Th17. Th17 cells are important instigators of inflammation in several models of autoimmune disease; in particular, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE), which were previously characterized as Th1-mediated diseases. Although high levels of IFN-γ are secreted in CIA and EAE, disease is exacerbated in IFN-γ- or IFN-γ receptor-deficient mice due to the ability of IFN-γ to suppress IL-17 secretion. However, in proteoglycan-induced arthritis (PGIA), severe arthritis is dependent on the production of IFN-γ. We were therefore interested in determining the role of IL-17 in PGIA. We assessed the progression of arthritis in IL-17-deficient (IL-17−/−) mice and found the onset and severity of arthritis were equivalent in wild-type (WT) and IL-17−/− mice. Despite evidence that IL-17 is involved in neutrophil recruitment, synovial fluid from arthritic joints showed a comparable proportion of Gr1+ neutrophils in WT and IL-17−/− mice. IL-17 is also implicated in bone destruction in autoimmune arthritis, however, histological analysis of the arthritic joints from WT and IL-17−/− mice revealed a similar extent of joint cellularity, cartilage destruction, and bone erosion despite significantly reduced RANKL (receptor activator of NK-κB ligand) expression. There were only subtle differences between WT and IL-17−/− mice in proinflammatory cytokine expression, T cell proliferation, and autoantibody production. These data demonstrate that IL-17 is not absolutely required for autoimmune arthritis and that the production of other proinflammatory mediators is sufficient to compensate for the loss of IL-17 in PGIA.

Expression of CD80/86 on B Cells Is Essential for Autoreactive T Cell Activation and the Development of Arthritis

Depletion of B cells in rheumatoid arthritis is therapeutically efficacious. Yet, the mechanism by which B cells participate in the inflammatory process is unclear. We previously demonstrated that Ag-specific B cells have two important functions in the development of arthritis in a murine model of rheumatoid arthritis, proteoglycan (PG)-induced arthritis (PGIA). PG-specific B cells function as autoantibody-producing cells and as APCs that activate PG-specific T cells. Moreover, the costimulatory molecule CD86 is up-regulated on PG-specific B cells in response to stimulation with PG. To address the requirement for CD80/CD86 expression on B cells in the development of PGIA, we generated mixed bone marrow chimeras in which CD80/CD86 is specifically deleted on B cells and not on other APC populations. Chimeras with a specific deficiency in CD80/CD86 expression on B cells are resistant to the induction of PGIA. The concentration of PG-specific autoantibody is similar in mice sufficient or deficient for CD80/86-expressing B cells, which indicates that resistance to PGIA is not due to the suppression of PG-specific autoantibody production. CD80/86-deficient B cells failed to effectively activate PG-specific autoreactive T cells as indicated by the failure of T cells from PG-immunized CD80/86-deficient B cell chimeras to transfer arthritis into SCID mice. In vitro secondary recall responses to PG are also dependent on CD80/86-expressing B cells. These results demonstrate that a CD80/86:CD28 costimulatory interaction between B cells and T cells is required for autoreactive T cell activation and the induction of arthritis but not for B cell autoantibody production.

Suppression of Proteoglycan-Induced Arthritis by Anti-CD20 B Cell Depletion Therapy Is Mediated by Reduction in Autoantibodies and CD4+ T Cell Reactivity

B cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) since the discovery of RA as an autoimmune disease. There is renewed interest in B cells in RA based on the clinical efficacy of B cell depletion therapy in RA patients. Although, reduced titers of rheumatoid factor and anti-cyclic citrullinated peptide Abs are recorded, the mechanisms that convey clinical improvement are incompletely understood. In the proteoglycan-induced arthritis (PGIA) mouse model of RA, we reported that Ag-specific B cells have two important functions in the development of arthritis. PG-specific B cells are required as autoantibody-producing cells as well as Ag-specific APCs. Herein we report on the effects of anti-CD20 mAb B cell depletion therapy in PGIA. Mice were sensitized to PG and treated with anti-CD20 Ab at a time when PG-specific autoantibodies and T cell activation were evident but before acute arthritis. In mice treated with anti-CD20 mAb, development of arthritis was significantly reduced in comparison to control mAb-treated mice. B cell depletion reduced the PG-specific autoantibody response. Furthermore, there was a significant reduction in the PG-specific CD4+ T cell recall response as well as significantly fewer PG-specific CD4+ T cells producing IFN-γ and IL-17, but not IL-4. The reduction in PG-specific T cells was confirmed by the inability of CD4+ T cells from B cell-depleted mice to adoptively transfer disease into SCID mice. Overall, B cell depletion during PGIA significantly reduced disease and inhibited both autoreactive B cell and T cell function.

IFN-γ Regulates the Requirement for IL-17 in Proteoglycan-Induced Arthritis

The contribution of the proinflammatory cytokines IFN-γ and IL-17 to the pathogenesis of experimental arthritis is controversial. In proteoglycan (PG)-induced arthritis (PGIA), severe arthritis is dependent on the production of IFN-γ, whereas IL-17 is dispensable. In collagen-induced arthritis and Ag-induced arthritis, although high levels of IFN-γ are secreted, disease is exacerbated in IFN-γ or IFN-γ receptor-deficient mice due to the ability of IFN-γ to suppress IL-17 expression. In the current study, we investigated the effect of IFN-γ on the IL-17 response and its consequences in PGIA. In PG-immunized IFN-γ−/− mice, despite reduction in arthritis, the PG-specific CD4+ T cell IL-17 response was significantly increased. Elevated IL-17 contributed to development of arthritis, as disease in IFN-γ/IL-17−/− was significantly reduced in comparison with either IFN-γ−/− or IL-17−/− mice. A contribution of IFN-γ and IL-17 to the development of arthritis was also identified in T-bet−/− mice. PG-specific CD4+ T cells from T-bet−/− mice produced reduced IFN-γ and elevated concentrations of IL-17. Both IFN-γ and IL-17 contribute to arthritis, as T-bet−/− mice lacking IL-17 (T-bet/IL-17−/−) were resistant, whereas wild-type, T-bet−/−, and IL-17−/− mice were susceptible to PGIA. T cell proliferation and autoantibody production did not correlate with development of disease; however, expression of cytokines and chemokines in joint tissues demonstrate that IFN-γ and IL-17 cooperatively contribute to inflammation. These results demonstrate that both IFN-γ and IL-17 have the potential to induce PGIA, but it is the strength of the IFN-γ response that regulates the contribution of each of these Th effector cytokines to disease.

CCR5 is involved in resolution of inflammation in proteoglycan-induced arthritis

OBJECTIVE CCR5 and its ligands (CCL3, CCL4, and CCL5) may play a role in inflammatory cell recruitment into the joint. However, it was recently reported that CCR5 on T cells and neutrophils acts as a decoy receptor for CCL3 and CCL5 to assist in the resolution of inflammation. The aim of this study was to determine whether CCR5 functions as a proinflammatory or antiinflammatory mediator in arthritis, by examining the role of CCR5 in proteoglycan (PG)-induced arthritis (PGIA). METHODS Arthritis was induced by immunizing wild-type (WT) and CCR5-deficient (CCR5(-/-)) BALB/c mice with human PG in adjuvant. The onset and severity of PGIA were monitored over time. Met-RANTES was used to block CCR5 in vivo. Arthritis was transferred to SCID mice, using spleen cells from arthritic WT and CCR5(-/-) mice. The expression of cytokines and chemokines was measured by enzyme-linked immunosorbent assay. RESULTS In CCR5(-/-) mice and WT mice treated with the CCR5 inhibitor Met-RANTES, exacerbated arthritis developed late in the disease course. The increase in arthritis severity in CCR5(-/-) mice correlated with elevated serum levels of CCL5. However, exacerbated arthritis was not intrinsic to the CCR5(-/-) lymphoid cells, because the arthritis that developed in SCID mouse recipients was similar to that in WT and CCR5(-/-) mice. CCR5 expression in the SCID mouse was sufficient to clear CCL5, because serum levels of CCL5 were the same in SCID mouse recipients receiving cells from either WT or CCR5(-/-) mice. CONCLUSION These data demonstrate that CCR5 is a key player in controlling the resolution of inflammation in experimental arthritis.

Pathways for interleukin‐1–driven arthritis

T cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) on the basis of an observed genetic association with MHC class II alleles, the high numbers of T cells in the inflamed synovial tissues in RA, and the requirement for T cells in rodent models of RA. The effector functions of CD4+ T cells are, in general, carried out by the production of cytokines. Synovial CD4+ T cells could contribute directly to synovitis by the production of inflammatory cytokines. Originally, CD4+ effector T cells were divided into two subsets based on their preferential production of cytokines (Figure 1). Th1 cells are potent activators of cell-mediated immunity and produce interferon-γ (IFN-γ), lymphotoxinβ (LTβ), and tumor necrosis factor (TNF) whereas Th2 cells are potent activators of B cell IgE production, eosiniphil recruitment and secretion of interleukin (IL)-4, IL-5, and IL-13 (1). More recently, T cells expressing IL-17 have been recognized as a distinct T cell subset, Th17, which produce IL-17, IL-17F, IL-21, and IL-22 (2). In several animal models of RA, IL-17 has been implicated in promoting disease based on the observation that arthritis is ameliorated by neutralizing IL-17 or in IL-17-deficient mice. Innate immune signals from dendritic cells and macrophages drive the differentiation of T cell subsets. Differentiation of Th17 cells is determined by the production of several cytokines, most predominantly transforming growth factor β-1 (TGFβ-1), IL-6 and IL-1β, although there is some disagreement about the requirement for IL-1β (3). Evidence in support of IL-1β as a Th17 differentiation factor showed that IL-1β alone induced CD4+ differentiation into Th17 cells and that blocking IL-1β disables Th17 T cells differentiation mediated by TGFβ-1 and IL-6 (4). Also IL-1β enhances Th17 cell differentiation in autoimmune encephalomyelitis (5) and in Bordetella infection (6). Furthermore, spontaneous arthritis develops in the interleukin-1 receptor antagonist (IL-1ra)-deficient mice that is IL-17-dependent (7). Figure 1 Overview of T helper cell differentiation and cytokine produced. Mechanisms by which IL-1 and IL-17 reciprocally activate each others function. IL-1 is a major mediator of inflammation and is produced by monocytes, macrophages, and synovial lining cells. Members of the IL-1 family IL-1α, IL-1β, and IL-1ra bind to the IL-1 receptor. IL-1α and IL-1β are pro-inflammatory cytokines whereas IL-1receptor antagonist (IL-1ra) is a naturally occurring inhibitor of IL-1 and competes with IL-1α and IL-1β for binding to the IL-1R. IL-1 is a central mediator of the inflammatory process in RA acting to stimulate monocytes, recruit inflammatory cells into the joint and induce secretion of factors that degrade cartilage (8). Inhibition of IL-1 with IL-1ra in RA is moderately efficacious. In IL-1ra-deficient mice on the BALB/c background, inflammatory and erosive arthritis develops spontaneously due to induction of autoimmunity. Autoimmunity in these mice is characterized by excess production of pro-inflammatory cytokines IL-1, IL-6, TNF and IL-17 and autoantibodies; IgG rheumatoid factor, anti-type II collagen and anti-DNA (7, 9). Further studies show that arthritis is T cell dependent and that arthritis does not develop in IL-1ra-deficient mice also deficient in either IL-17 or TNF highlighting the importance of these cytokines in the development of disease (10). In this issue of Arthritis & Rheumatism, Koenders and colleagues report new findings regarding the role of cytokines in IL-1ra-deficient mice. The objective of this study was to use the IL-1ra-deficient mice to assess the importance of TNF and IL-17 in chronic progression of arthritis that is IL-1-dependent. After arthritis was established in IL-1ra-deficient mice, IL-1, IL-17 and TNF were neutralized. The results from these studies were quite different from those in which IL-1ra-deficient mice were also deficient in either TNF or IL-17. In the Koenders study, TNF blockade had no effect on arthritis severity. These results are contrary to those observed for the IL-1ra and TNF double-deficient mice in which arthritis is significantly suppressed (10). At face value the data indicate that TNF is critical for the development of arthritis but not for the chronic phase of disease. It would have been helpful in the Koenders study to test TNF blocked with the soluble TNFRI before the development of arthritis to more directly recapitulate the Horai study in the TNF/IL-1ra double-deficient mice. There may be some differences between complete ablation of TNF at the gene level and neutralization of TNF with soluble TNFRI. The IL-1ra-deficient mice may be analogous to the group of RA patients refractory to anti-TNF therapy where IL-1 may dominate the drive toward joint inflammation What is most interesting about the Koenders study is the relationship between IL-1 and IL-17 in this model of spontaneous arthritis. Inhibition of IL-1 had a dramatic effect on all aspects of arthritis pathology. There was almost complete suppression of the macroscopic arthritis score, cellular infiltration into the joint, proteoglycan-depletion, and bone erosion. In addition, blockade of IL-1 reduced the number of IL-17+ cells in the lymph nodes. In comparison, the effect of IL-17 blockade was modest. Progression of disease was reduced but not arrested and cellular infiltration and bone erosion were suppressed although not as dramatically as IL-1 blockade. Interestingly, staining for IL-1 in the joint was reduced with anti-IL-17 antibody treatment. In the chronic progression of arthritis in the IL-1ra-deficient mice, IL-1 appears to drive the disease through at least two interrelated mechanisms. The primary mechanism is inflammation driven by excess IL-1 completely independent of TNF and partially independent of IL-17. A secondary mechanism is the IL-1 induced differentiation of Th17 cells. Although it is not apparent if other cytokines such as TGF-β1 are upregulated in IL-1ra deficient mice and act in concert with IL-1, clearly IL-1 plays a pivotal role. The Koenders study is in agreement with studies in autoimmune encephalomyelitis, an IL-17-dependent autoimmune disease, where disease is suppressed and IL-17 production reduced in IL-1 receptor-1-deficient mice (5). The reduced efficacy of IL-17 blockade in comparison to gene ablation of IL-17 in the IL-1ra-deficient mice may indicate that IL-17 is acting at the induction phase. Once Th17 cells are activated, IL-17 can induce the secretion of TNF and IL-1 having both additive and synergistic effects on cytokine production, neutrophil recruitment and tissue destruction in particular bone erosion (11). Thus, in the chronic phase of arthritis in IL-1ra-deficient mice, IL-1 and IL-17 form an inflammatory loop enhancing tissue damage. It is important to point out, although several models of rodent arthritis are IL-17-dependent and considered Th17-autoimmune diseases, proteoglycan-induced arthritis (PGIA) is Th1-dependent requiring IFN-γ but not IL-17 for arthritis development. In PGIA, a deficiency in IL-17 neither inhibits arthritis, neutrophil recruitment nor bone erosion indicating that other inflammatory mediators such as IL-1, IL-6, and TNF are sufficient to induce disease (12, 13). It is clear from the rodent models of RA that very similar pathology can be induced by completely different mechanisms. Despite evidence for IL-17 in some models of arthritis, data supporting a role for IL-17 in RA is inconclusive. Evidence can be cited for the expression of both IL-17 and IFN-γ in RA synovial fluids and in the T cell areas of RA synovial tissue (14-17). Since RA is a very a heterogeneous disease it is possible the Th1/Th17 spectrum represents different subtypes of RA. Another possibility is that Th1/Th17 cells may act at different stages of disease or synergistically to induce pathogenesis. The role of IFN-γ and IL-17 in RA remains the subject of intense research.