Myeloid cells, including macrophages and neutrophils, are critical to the pathogenesis of rheumatoid arthritis (RA) through the release of proinflammatory cytokines, chemokines, and other mediators such as prostaglandins and matrix metalloproteinases ([1-3]). Monocytes, macrophages, and granulocytes express the death receptor Fas (). RA synovial tissue () and RA synovial fluid macrophages () also express both Fas and FasL. We previously demonstrated that RA synovial fluid macrophages were resistant to FasL-mediated apoptosis, due to increased expression of the antiapoptotic molecule FLIP (). Further, RA peripheral blood monocytes and synovial fluid macrophages are resistant to FasL-mediated apoptosis induced by activated CD4+CD25− responder T cells ().
There is mounting evidence that Fas also has a nonapoptotic role, which is dependent on cell type and context (). Fas signaling has been implicated in T cell proliferation and activation, liver regeneration after partial hepatectomy, and nerve regeneration after crush injury (). The severity of collagen-induced arthritis (CIA) has been shown to be reduced in DBA/J mice that expressed a mutant Fas receptor (lpr) (). Interruption of Fas–FasL interactions on human macrophages resulted in reduced Toll-like receptor 4 (TLR-4) and interleukin-1 receptor (IL-1R) signaling, mediated at least in part by damping of myeloid differentiation factor 88 (MyD88) signaling due to the increased availability of FADD, which was no longer recruited to the Fas receptor (). To specifically examine the role of Fas on myeloid cells in vivo, we recently generated a myeloid-lineage Fas-deficient line by crossing Fasflox/flox and CreLysM mice (). These mice exhibited no phenotypic abnormalities at 2–4 months of age (young); however, when they reached 6–8 months of age (old) they developed a systemic lupus erythematosus–like disease associated with leukocytosis, splenomegaly, antinuclear antibodies, and glomerulonephritis.
In order to determine the role of myeloid-specific Fas in the effector phase of arthritis, we studied young CreLysMFasflox/flox mice, using the immune complex–mediated K/BxN serum–transfer model of arthritis. These young CreLysMFasflox/flox mice exhibited no alteration in the initial induction of arthritis; however, once the clinical arthritis peaked, amelioration of disease was more rapid in CreLysMFasflox/flox mice. This improvement was associated with reduced inflammation and neutrophil infiltration. While there was no difference in levels of proinflammatory IL-1β or IL-6 within the involved joints, levels of IL-10 were significantly increased, and levels of the neutrophil chemotactic chemokine CXCL5 and the endogenous TLR-2 ligand gp96 were significantly reduced. There was no significant difference in the ability of IL-1β to induce IL-6 or CXCL5 in CreLysMFasflox/flox versus control mouse macrophages, but the level of IL-1β- and gp96–induced IL-10 was significantly increased in CreLysMFasflox/flox macrophages. Further, IL-10 suppressed the synergistic induction of IL-6 and CXCL5 in macrophages in response to IL-1β and gp96. These observations demonstrate that intact macrophage Fas signaling promotes ongoing inflammation by lessening the expression of IL-10 and enhancing the expression of the endogenous TLR-2 ligand gp96.
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This study demonstrates that Fas expression on myeloid cells promotes anti-GPI–positive serum transfer–induced arthritis. The initial phase of the clinical course of arthritis in CreLysMFasflox/flox mice was comparable with that observed in control mice. However, the CreLysMFasflox/flox mice exhibited more rapid amelioration during the chronic phase of the disease, which was confirmed histologically by the findings of reduced inflammation and decreased neutrophils in the joints (Figure 1). At the time the mice were killed (day 11), levels of IL-10 were significantly increased, and levels of the neutrophil chemotactic chemokine CXCL5 and the endogenous TLR-2 ligand gp96 were significantly reduced, in the ankles of the CreLysMFasflox/flox mice (Figure 3). IL-1β is critical for the initiation of serum transfer–induced arthritis (), while local expression of gp96 within the joint promotes progression of disease after peak clinical activity (). Activation of Fas-deficient macrophages with IL-1β or gp96 resulted in significantly higher concentrations of IL-10 compared with the levels in control macrophages, and this was associated with increased phosphorylation of ERK. These observations suggest that the absence of intact Fas signaling in macrophages results in reduced gp96 and increased IL-10, promoting resolution of the arthritis. This may be mediated, at least in part, by reduction of chemokines such as CXCL5.
A recent study demonstrated that myeloid cell FasL promotes central nervous system inflammation following injury (). Mice with deficiency of FasL in myeloid cells exhibited reduced spinal cord damage, reduced neutrophil infiltration, and reduced neutrophil and monocyte recruitment to the peritoneum following injection with thioglycollate (). In contrast, in the current study, although the number of neutrophils was decreased in the inflamed joints of CreLysMFasflox/flox mice, thioglycollate-induced recruitment of Fas-deficient neutrophils was not reduced. Neutrophil apoptosis is crucial to the resolution of inflammation (), and reduction of neutrophil apoptosis exacerbates inflammatory arthritis (). Fas signaling may promote neutrophil apoptosis, which is inhibited by the antiapoptotic proteins Mcl-1 and Bcl-2 (), while Mcl-1 is essential for neutrophil survival in the absence of a death signal ([28, 29]). We observed no difference in spontaneous apoptosis by Fas-deficient neutrophils. Therefore, although neutrophil numbers were reduced in the ankles of CreLysMFasflox/flox mice, there was no evidence of an intrinsic defect in neutrophil migration and no spontaneous increase of neutrophil cell death.
Neutrophils are critical to the pathogenesis of anti-GPI–positive serum transfer–induced arthritis (). The neutrophil chemotactic chemokines CXCL1, CXCL2, and CXCL5 and the receptor for these chemokines CXCR2 are increased in the joints of mice with serum transfer–induced arthritis (). Mice deficient in CXCR2 exhibit attenuated serum transfer–induced arthritis, with an onset similar to that observed in control mice (), consistent with the course observed in the CreLysMFasflox/flox mice in the present study. We chose to examine CXCL5 because it was previously demonstrated that anti-CXCL5 ameliorates IL-17–induced arthritis but anti-CXCL1 does not (). Reduced levels of CXCL5 were observed in the joints of the CreLysMFasflox/flox mice, and at the time of tissue acquisition the concentration of CXCL5 was highly correlated with joint swelling, which also correlated with the number of neutrophils in the joints. Since there was no intrinsic defect in the recruitment or survival of Fas-deficient neutrophils, these findings indicate that reduced CXCL5 contributed to the observed disease amelioration during the chronic phase of arthritis in the CreLysMFasflox/flox mice.
Consistent with the results of an earlier study in DBA/llpr/lpr mice with CIA (), there was no reduction in levels of the proinflammatory cytokines IL-1β or IL-6 in the joints of the CreLysMFasflox/flox mice. Therefore, reduced IL-1β, which is necessary for induction of the arthritis, does not appear to be the cause of the enhanced amelioration of the disease in the CreLysMFasflox/flox mice. Further, the induction of CXCL5 expression by IL-1β was not reduced in Fas-deficient mouse macrophages, suggesting that reduced IL-1β–induced CXCL5 was not responsible for the amelioration of arthritis. However, we observed that levels of the endogenous TLR-2 ligand gp96 were significantly reduced in the joints of the CreLysMFasflox/flox mice on day 11 (Figure 3D). We previously demonstrated that gp96 concentrations in the ankles were increased during serum transfer–induced arthritis, peaking at the time of maximal joint swelling, and that neutralizing anti-gp96 antibody ameliorated the clinical course (). Since gp96 induced the expression of CXCL5 comparably in Fas-deficient and control macrophages, the reduction of gp96 in the CreLysMFasflox/flox mice may have contributed to the reduced CXCL5 observed in the arthritic joints. In support of this interpretation, reduced levels of CXCL5, but not IL-10, were observed on day 4 in the ankles of the CreLysMFasflox/flox mice compared with control mice in additional experiments using 300 μl of K/BxN serum (data not shown).
The mechanism for the induction of gp96 in arthritis is unknown, although IL-2 and interferon-γ are known to promote its expression ([33, 34]). Gp96 is highly expressed in RA synovial tissue and synovial fluid, and is capable of activating macrophages through TLR-2 ([12, 16]). These features identify a pivotal role of gp96 in the persistent activation of macrophages, leading to the self-perpetuating inflammatory process observed in RA (). It is possible that other endogenous TLR ligands, such as tenascin C or high mobility group box chromosomal protein 1, may also contribute to the progression of arthritis ([36-38]). These observations suggest that a reduction of the endogenous TLR-2 ligand gp96 may contribute to the amelioration of arthritis later in the clinical course in the CreLysMFasflox/flox mice.
In a previous study using human macrophages that express both Fas and FasL, interruption of Fas signaling resulted in suppressed IL-1R1 and TLR-4 signaling which was mediated by the interaction of FADD with MyD88, resulting in reduced expression of IL-6 (). Further, macrophages from lpr and gld mice exhibited reduced TLR-4–induced IL-6. Consistent with these observations, in the current study Fas-deficient murine macrophages exhibited reduced levels of IL-6 at 4 hours in response to the endogenous TLR-2 ligand gp96 (Figure 4A), as well as to microbial TLR-2 and TLR-4 ligands (data not shown). However, Fas-deficient murine macrophages exhibited no reduction in the induction of IL-6 or CXCL5 in response to IL-1β. Nevertheless, in human macrophages, interruption of Fas–FasL signaling via an antagonistic anti-FasL antibody resulted in increased IL-1β–induced IL-6 expression and NF-κB activation (). These differences may be due to the fact that we were not able to definitively detect FasL on the surface of murine macrophages (data not shown), consistent with the earlier observation that expression of FasL on the surface of unmanipulated macrophages is quite low (). Therefore, the influence of Fas–FasL interactions between macrophages may be less dramatic in mice than in humans. Nonetheless, DBA/llpr/lpr mice exhibited no reduction of cellular or humoral immunity to collagen (), consistent with the important role of myeloid-expressed Fas shown in the current study.
IL-10 is important in the pathogenesis of rheumatoid and experimental arthritis. It is expressed in the joints of patients with RA (). In studies using ex vivo RA synovial tissue cultures, neutralization of IL-10 promoted, and the addition of exogenous IL-10 suppressed, the spontaneous expression of proinflammatory cytokines (), supporting the notion that it plays a key role in controlling inflammation in RA. Further, the absence of IL-10 exacerbates CIA ([41, 42]), while treatment with IL-10 suppresses it (). Additionally, the severity of K/BxN serum transfer–induced arthritis is increased in IL-10–deficient mice (), demonstrating that IL-10 suppresses, but does not prevent, arthritis in this model. In the present study IL-10 levels were significantly higher in the joints of CreLysMFasflox/flox mice than in controls, suggesting that increased IL-10 may contribute to the amelioration of arthritis during the chronic phase of the disease in these mice.
Other mechanisms for the reduced severity of arthritis during the chronic phase, in addition to reduced levels of gp96, are also possible. Concentrations of IL-1R antagonist (IL-1Ra) are also increased in RA and, although only modestly effective therapeutically, IL-1Ra is approved for the treatment of the disease. IL-1Ra–deficient mice spontaneously develop arthritis () and develop more severe K/BxN serum transfer–induced arthritis, while IL-1Ra–transgenic mice are largely protected (). Type I interferon-β is also expressed by macrophages and other cell types and is capable of suppressing anti-GPI–mediated arthritis (), although it was not effective in RA (). Overall, these observations indicate that the enhanced expression of IL-10, and possibly other antiinflammatory cytokines, contributed to the amelioration of arthritis observed in CreLysMFasflox/flox mice.
In experiments to examine the mechanism for the increased concentrations of IL-10 in the joints of CreLysMFasflox/flox mice, we found that incubation of Fas-deficient murine macrophages with either IL-1β or gp96 resulted in increased IL-10 expression. Activation of ERK and activation of p38 are important for the induction of IL-10 by microbial TLR ligands and immune complexes, while activation of Akt exerts a permissive effect by reducing the activity of GSK-3 (). In response to IL-1β, only ERK pathway expression was differentially regulated between macrophages from CreLysMFasflox/flox mice and from control mice. There was no difference in the expression of markers of alternative macrophage differentiation, including Fizz1, Arg1, Ym1, or IL-10, in GM-CSF–differentiated bone marrow macrophages (data not shown). In addition, macrophages from aged CreLysMFasflox/flox mice exhibited no increase in the constitutive activation of ERK, p38, or Akt (). These results provide evidence that increased ERK activation in response to IL-1β, rather than a difference in the pattern of macrophage differentiation, contributed to the enhanced expression of IL-10 in CreLysMFasflox/flox mice.
Taken together, the findings reported herein suggest that in the presence of intact Fas signaling in macrophages, the expression of IL-10, and possibly other suppressive cytokines, is restrained, permitting the full expression of inflammation. Further, the induction of the endogenous TLR-2 ligand gp96 is enhanced in the presence of intact macrophage Fas signaling, promoting disease progression and further joint destruction. These observations also provide a rationale for therapeutic strategies in RA that reduce the expression of endogenous TLR ligands and promote enhanced expression of IL-10.
- Top of page
- 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. 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 conception and design. Huang, Birkett, Koessler, Cuda, Haines, Jin, Pope.
Acquisition of data. Huang, Birkett, Koessler, Cuda, Jin.
Analysis and interpretation of data. Huang, Haines, Perlman, Pope.