Protease‐activated receptor 2 mediates the proinflammatory effects of synovial mast cells

Objective

Mast cells are hypothesized to play a role in the pathogenesis of rheumatoid arthritis (RA) by mechanisms requiring elucidation. Tryptase released from these cells can activate protease‐activated receptor 2 (PAR‐2), which was recently shown to have proinflammatory actions. The purpose of this study was to examine the relationship between synovial mast cells and PAR‐2. Mast cell proximity to PAR‐2–expressing cells was investigated in RA synovium. In murine studies, we assessed the capacity of mast cell tryptase to mediate synovial proinflammatory responses via PAR‐2 and whether degranulating mast cells induced synovial hyperemia by PAR‐2 activation.

Methods

RA synovial tissue was examined by immunohistochemistry. PAR‐2^+/+ and PAR‐2^−/− C57BL/6J mice were used to investigate the PAR‐2 dependence of compound 48/80–induced synovial hyperemia, as measured by laser Doppler imaging, and joint swelling and hyperemic responses to recombinant human β‐tryptase.

Results

Mast cells and synovial lining cells staining for PAR‐2 were colocalized in RA articular tissue. Compound 48/80 administration resulted in vasodilatation in PAR‐2^+/+ mice but not in PAR‐2^−/− mice, which showed a vasoconstrictor response. Eliminating the 5‐hydroxytryptamine–mediated component of this response with methysergide unveiled an enhanced PAR‐2–mediated vasodilatation to compound 48/80 in PAR‐2^+/+ mice and ablated the vasoconstrictor response in PAR‐2^−/− mice. Treatment with β‐tryptase resulted in dose‐dependent knee joint swelling and synovial vasodilatation in PAR‐2^+/+ mice but not PAR‐2^−/− mice.

Conclusion

This in vivo study is the first to explore the relationship between synovial mast cells and PAR‐2. Our results support the hypothesis that mast cells contribute to the pathogenesis of inflammatory arthritis through PAR‐2 activation via release of mast cell tryptase.

     

Antiinflammatory effects of tumor necrosis factor on hematopoietic cells in a murine model of erosive arthritis

Objective

To investigate the mechanisms leading to the influx of inflammatory hematopoietic cells into the synovial membrane and the role of tumor necrosis factor receptor I (TNFRI) and TNFRII in this process in an animal model of rheumatoid arthritis (RA).

Methods

We performed bone marrow transplantations in human TNF–transgenic mice using hematopoietic cells from wild‐type, TNFRI^−/−, TNFRII^−/−, and TNFRI/II^−/− mice as donors and assessed the severity of arthritis histologically. Generation of osteoclasts from the different genotypes was analyzed in vitro and in vivo. Apoptosis was analyzed using annexin V staining as well as TUNEL assays.

Results

Despite lacking responsiveness to TNF in their hematopoietic compartment, mice not only developed full‐blown erosive arthritis but even showed increased joint destruction when compared with mice with a TNF‐responsive hematopoietic compartment. We demonstrated different roles of the 2 different TNFRs in the regulation of these processes. The absence of TNFRI on hematopoietic cells did not affect joint inflammation but markedly attenuated erosive bone destruction via reduced synovial accumulation of osteoclast precursors. In contrast, the absence of TNFRII on hematopoietic cells increased joint inflammation as well as erosive bone destruction via the regulation of osteoclast precursor apoptosis.

Conclusion

Our findings indicate that selective blockade of TNFRI, leaving the antiinflammatory effects of TNFRII unaltered instead of unselectively blocking TNF, might be advantageous in patients with RA.

     

Cyclin‐dependent kinase inhibitor p21, via its C‐terminal domain,is essential for resolution of murine inflammatory arthritis

Objective

The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin‐dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21‐mediated suppression of experimental inflammatory arthritis.

Methods

Experimental arthritis was induced in wild‐type or p21^−/− (C57BL/6) mice, using the K/BxN serum–transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide‐induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex‐based assay, flow cytometry, or enzyme‐linked immunosorbent assay.

Results

Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21^−/− mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21‐intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages.

Conclusion

These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.

     

CXCR5‐ and CCR7‐dependent lymphoid neogenesis in a murine model of chronic antigen‐induced arthritis

Objective

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease with unknown etiology and only partially defined pathogenesis. The aim of this study was to establish a murine model of chronic arthritis in which the development of tertiary lymphoid tissue, a hallmark of human RA, is locally induced, and to characterize the roles of the homeostatic chemokine receptors CXCR5 and CCR7 in this process.

Methods

We developed a modified model of chronic antigen‐induced arthritis (AIA) in mice with a strong bias toward inflammation. Disease pathology was assessed up to 9 months in wild‐type, CXCR5‐deficient, and CCR7‐deficient mice by determination of knee joint swelling and cellular and humoral immune responses, as well as by histologic analysis of arthritic knee joints.

Results

In this novel model of AIA, mice developed organized ectopic lymphoid follicles with topologically segregated B cell and T cell areas, high endothelial venules, and germinal center formation within the chronically inflamed synovial tissue. Analysis of the initiation and progression of AIA in wild‐type, CXCR5^−/−, and CCR7^−/− mice revealed a reduction of acute inflammatory parameters in both knockout strains as well as significantly reduced joint destruction in CXCR5^−/− mice. Most importantly, the development and organization of tertiary lymphoid tissue were significantly impaired in CXCR5‐deficient and CCR7‐deficient mice.

Conclusion

Our results suggest that an inflammatory microenvironment efficiently triggers lymphoid neogenesis in autoimmune diseases such as RA. Moreover, the generation of autoreactive tertiary lymphoid tissues, which is entirely dependent on homeostatic chemokines, may in turn maintain local aberrant chronic immune responses.

     

Deletion of either CD55 or CD97 ameliorates arthritis in mouse models

Objective

CD55 (decay‐accelerating factor) is best known for its role in the negative regulation of the complement system. Indeed, lack of this molecule leads to disease aggravation in many autoimmune disease models. However, CD55 is abundantly present on fibroblast‐like synoviocytes and is also a ligand of the adhesion‐class heptahelical receptor CD97, which is expressed by infiltrating macrophages. Treatment with antibodies to CD97 ameliorates the collagen‐induced model of rheumatoid arthritis (RA) in DBA/1 mice, but the net contribution of CD55 is unknown. This study was undertaken to investigate the role of CD55 in experimental RA.

Methods

Arthritis was induced in wild‐type, CD55^−/−, and CD97^−/− mice using collagen‐induced and K/BxN serum–transfer models. Incidence of arthritis was monitored over time, and disease activity was assessed by clinical and immunohistochemical evaluation.

Results

In contrast to observations in many inflammatory disease models, lack of CD55 resulted in decreased arthritis in experimental models of RA. Consistent with the previously reported effects of anti‐CD97 antibody treatment, CD97^−/− mice had reduced arthritis activity compared with wild‐type controls.

Conclusion

Our findings indicate that the lack of CD55 or CD97 in 2 different models of arthritis increases resistance to the disease. These findings provide insight into a role for CD55 interaction with CD97 in the pathogenesis of RA and suggest that therapeutic strategies that disrupt CD55/CD97 may be clinically beneficial.

     

Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen‐induced arthritis

Objective

Annexin 1 (Anx‐1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx‐1 in experimental arthritis and in GC‐mediated inhibition of inflammation, using antigen‐induced arthritis (AIA) in Anx‐1 knockout (Anx‐1^−/−) mice.

Methods

Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti‐mBSA IgG were determined by enzyme‐linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real‐time polymerase chain reaction.

Results

A significant exacerbation of arthritis was observed in the Anx‐1^−/− mice compared with wild‐type (WT) mice. This was associated with increased mRNA expression of synovial interleukin‐1β, tumor necrosis factor α, interleukin‐6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx‐1^−/− mice, and also significantly reduced the serum levels of anti‐mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx‐1^−/− mice.

Conclusion

Anx‐1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.

     

C5a receptor enables participation of mast cells in immune complex arthritis independently of Fcγ receptor modulation

Objective

Mast cells are tissue‐resident immune sentinels that are implicated in the pathogenesis of inflammatory joint disease. The aim of this study was to test our hypothesis that complement fragments could be key activators of synovial mast cells in autoimmune arthritis.

Methods

In vivo studies used the murine K/BxN arthritis model, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR^−/− and control mast cells were engrafted into mast cell–deficient WBB6 F1‐Kit^w/Kit^W‐v (W/Wv) mice to examine the requirement for this receptor in arthritis. C5aR‐dependent activation of mast cells was investigated in C5aR^−/− animals and in murine and human mast cell cultures.

Results

Murine synovial mast cells express functional C5aR. Unlike their wild‐type counterparts, C5aR^−/− mast cells adoptively transferred into W/Wv mice were not competent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR^−/− mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG‐mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate the expression of Fcγ receptors (FcγR) or to otherwise alter the activation threshold. In human mast cells, C5a promoted the production of the neutrophil chemotaxin interleukin‐8, and recruitment of neutrophils at 24 hours after serum administration was impaired in C5aR^−/− mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis.

Conclusion

Stimulation via C5aR is required to unleash the proinflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism that is distinct from C5a‐modulated expression of FcγR.

     

Bim–Bcl‐2 homology 3 mimetic therapy is effective at suppressing inflammatory arthritis through the activation of myeloid cell apoptosis

Objective

Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies that activate the apoptotic cascade may have potential for use in RA; however, few therapeutic agents fit this category. The purpose of this study was to examine the potential of Bim, an agent that mimics the action of Bcl‐2 homology 3 (BH3) domain–only proteins that have shown success in preclinical studies of cancer, in the treatment of autoimmune disease.

Methods

Synovial tissues from RA and osteoarthritis patients were analyzed for the expression of Bim and CD68 using immunohistochemistry. Macrophages from Bim^−/− mice were examined for their response to lipopolysaccharide (LPS) using flow cytometry, real‐time polymerase chain reaction analysis, enzyme‐linked immunosorbent assay, and immunoblotting. Bim^−/− mice were stimulated with thioglycollate or LPS and examined for macrophage activation and cytokine production. Experimental arthritis was induced using the K/BxN serum–transfer model. A mimetic peptide corresponding to the BH3 domain of Bim (TAT‐BH3) was administered as a prophylactic agent and as a therapeutic agent. Edema of the ankles and histopathologic analysis of ankle tissue sections were used to determine the severity of arthritis, its cellular composition, and the degree of apoptosis.

Results

The expression of Bim was reduced in RA synovial tissue as compared with controls, particularly in macrophages. Bim^−/− macrophages displayed elevated expression of markers of inflammation and secreted more interleukin‐1β following stimulation with LPS or thioglycollate. TAT‐BH3 ameliorated arthritis development, reduced the number of myeloid cells in the joint, and enhanced apoptosis without inducing cytotoxicity.

Conclusion

These data demonstrate that BH3 mimetic therapy may have significant potential for the treatment of RA.

     

Essential role of microRNA‐155 in the pathogenesis of autoimmune arthritis in mice

Objective

MicroRNAs (miRNA) are a new class of regulatory elements. Altered expression of miRNA has been demonstrated in the inflamed joints of patients with rheumatoid arthritis (RA). The aim of this study was to examine the role of miRNA in the pathogenesis of autoimmune arthritis, using 2 murine models.

Methods

Collagen‐induced arthritis (CIA) and K/BxN serum‐transfer arthritis were induced in wild‐type (WT) and miR‐155–deficient (miR‐155^−/−) mice. The severity of arthritis was determined clinically and histologically. Anticollagen antibodies and cytokines were measured by enzyme‐linked immunosorbent assay. The cellular composition of the draining lymph nodes after induction of CIA was measured by flow cytometry.

Results

The miR‐155^−/− mice did not develop CIA. Deficiency in miR‐155 prevented the generation of pathogenic autoreactive B and T cells, since anticollagen antibodies and the expression levels of antigen‐specific T cells were strongly reduced in miR‐155^−/− mice. Moreover, Th17 polarization of miR‐155^−/− mouse T cells was impaired, as shown by a significant decrease in the levels of interleukin‐17 (IL‐17) and IL‐22. In the K/BxN serum‐transfer arthritis model, which only depends on innate effector mechanisms, miR‐155^−/− mice showed significantly reduced local bone destruction, attributed to reduced generation of osteoclasts, although the severity of joint inflammation was similar to that in WT mice.

Conclusion

These results demonstrate that miR‐155 is essentially involved in the adaptive and innate immune reactions leading to autoimmune arthritis, and therefore miR‐155 might provide a novel target for the treatment of patients with RA.

     

SIGIRR/TIR‐8 is an inhibitor of toll‐like receptor signaling in primary human cells and regulates inflammation in models of rheumatoid arthritis

Objective

Single‐immunoglobulin interleukin‐1 receptor–related (SIGIRR), which is also known as Toll/interleukin‐1 receptor 8 (TIR‐8), is a member of the TIR domain–containing family of receptors and was first characterized as an inhibitor of interleukin‐1 receptor (IL‐1R) and Toll‐like receptor (TLR) signaling. In the Dextran sulfate sodium–induced colitis model, SIGIRR^−/− mice were shown to have increased inflammation and to be more susceptible to endotoxin challenge. Increasing evidence implicates TLR and IL‐1R signaling in the pathology of rheumatoid arthritis (RA). Therefore, the purpose of this study was to investigate the involvement of SIGIRR in regulating inflammation in disease‐relevant models.

Methods

Primary human monocyte‐derived macrophages and dendritic cells (DCs) were used to overexpress SIGIRR as well as to knock down endogenously expressed SIGIRR using small interfering RNAs. SIGIRR was also overexpressed in synovial cells derived from RA patients. To investigate the role of SIGIRR in vivo, zymosan‐induced arthritis (ZIA) and collagen antibody–induced arthritis (CAIA) were induced in SIGIRR‐knockout mice.

Results

SIGIRR overexpression inhibited TLR‐induced cytokine production in macrophages and DCs, while SIGIRR knockdown resulted in increased cytokine production following TLR stimulation. Moreover, SIGIRR overexpression inhibited the spontaneous release of cytokines by human RA synovial cells. The role of SIGIRR as an inhibitor of inflammation was confirmed in vivo, since SIGIRR^−/− mice developed a more severe disease in both the ZIA and CAIA models.

Conclusion

Our study is the first to show the expression pattern and function of SIGIRR in primary human cells. Furthermore, this investigation defines the role of SIGIRR in disease‐relevant cell types and demonstrates that SIGIRR is a potential therapeutic target for RA.

     

Evaluation of protease‐activated receptor 2 in murine models of arthritis

Objective

Protease‐activated receptor 2 (PAR‐2) activation has been linked to pro‐ and antiinflammatory cellular responses. We undertook this study to explore the importance of PAR‐2 activation in 4 murine models of arthritis and to analyze the expression of PAR‐2 in human arthritic synovium.

Methods

Zymosan‐induced arthritis (ZIA), K/BxN serum–induced arthritis, and Freund's complete adjuvant (CFA)–induced arthritis were generated in naive PAR‐2^−/− mice and PAR‐2^+/+ littermates. Antigen‐induced arthritis (AIA) was generated in immunized mice using methylated bovine serum albumin (mBSA). The severity of arthritis was assessed by clinical scoring, technetium uptake measurement, and histologic analysis. Immune responses to mBSA were also evaluated from AIA. The expression of PAR‐2 in synovial tissues from rheumatoid arthritis (RA) and osteoarthritis (OA) patients was compared.

Results

In AIA, arthritis was significantly decreased in PAR‐2–deficient mice and was associated with decreased levels of anti‐mBSA IgG antibodies and lymph node cell proliferation. No difference in arthritis severity was seen in mice with ZIA, K/BxN serum–induced arthritis, and CFA‐induced arthritis. Synovial biopsy specimens from RA patients demonstrated significantly increased expression of PAR‐2 compared with those from OA patients.

Conclusion

PAR‐2 deficiency was found to modulate articular inflammation in murine models of arthritis that require prior immunization and was associated with reduced levels of anti‐mBSA IgG and lymph node cell proliferation in AIA. Expression of PAR‐2 in RA synovium was significantly higher than that in OA synovium, and this suggests that PAR‐2 is implicated in the pathogenesis of immune‐mediated forms of arthritis.

     

Suppression of autoimmune arthritis in interleukin‐1‐deficient mice in which T cell activation is impaired due to low levels of CD40 ligand and OX40 expression on T cells

Objective

To elucidate the roles of interleukin‐1 (IL‐1) in the development of 2 etiologically different rheumatoid arthritis (RA) models: the type II collagen (CII)–induced arthritis (CIA) model and the human T cell leukemia virus type I transgenic (HTLV‐I Tg) mouse model.

Methods

For the CIA model, DBA/1J‐background IL‐1α^−/−, IL‐1β^−/−, IL‐1α/β^−/−, and wild‐type littermate mice were immunized with CII. For the HTLV‐I Tg model, BALB/c IL‐1β^−/− or IL‐1α/β^−/− mice were crossed with HTLV‐I Tg mice. The effects of IL‐1 deficiency were assessed as follows: Development of arthritis was assessed both macroscopically and microscopically. Serum antibody titer was measured by enzyme‐linked immunosorbent assay. Proliferative response of lymph node cells was assayed by measurement of ³H‐thymidine incorporation. Expression of T cell surface molecule CD40 ligand (CD40L) and OX40 was determined by multicolor flow cytometric analysis.

Results

The development of arthritis was markedly suppressed in IL‐1α/β^−/− mice in both models, although the effect was less prominent in HTLV‐I Tg mice. Deficiency of only IL‐1α or only IL‐1β was also associated with disease suppression. Antibody production after immunization with CII was normal in IL‐1α/β^−/− mice, while autoantibody production was suppressed in IL‐1α/β^−/− HTLV‐I Tg mice. In IL‐1α/β^−/− mice, the T cell proliferative response against CII was greatly reduced in both the CIA and the HTLV‐I Tg models, suggesting inefficiency of T cell activation. Furthermore, expression of CD40L and OX40 on T cells was greatly reduced in IL‐1α/β^−/− mice.

Conclusion

These observations suggest that T cell activation by IL‐1 is important for the development of autoimmunity and arthritis in these mice.

     

Vegfb gene knockout mice display reduced pathology and synovial angiogenesis in both antigen‐induced and collagen‐induced models of arthritis

Objective

To determine the role of vascular endothelial growth factor B (VEGF‐B) in 2 mouse models of arthritis, antigen‐induced arthritis (AIA) and collagen‐induced arthritis (CIA).

Methods

For AIA studies, monarticular AIA was induced by methylated bovine serum albumin (mBSA) priming of Vegfb gene knockout ( Vegfb ^−/−) and wild‐type ( Vegfb ^+/+) mice, followed by intraarticular injection of mBSA or saline control 8 days later. CIA was induced in Vegfb ^−/− and Vegfb ^+/+ mice by intradermal injection of chick type II collagen in adjuvant. Arthritis was monitored in both models using defined criteria (clinical and histologic). Angiogenesis was measured by synovial vessel density in diseased and control joints.

Results

In AIA studies, Vegfb ^+/+ mice displayed significant knee joint swelling and synovial inflammation 7 days after intraarticular injection of antigen. Synovial inflammation was associated with angiogenesis, since vessel density in AIA synovium was significantly higher in arthritic than in control joints from the same animal. Knee joint swelling, synovial inflammation, and inflammation‐associated vessel density in arthritic joints were reduced in Vegfb ^−/− mice compared with arthritic joints from Vegfb ^+/+ mice. Similarly, in CIA, both disease incidence and mean clinical severity scores were significantly reduced in Vegfb ^−/− mice compared with Vegfb ^+/+ mice. Mean histologic severity scores and mean synovial vessel density were reduced in diseased joints from Vegfb ^−/− mice when compared with diseased joints from Vegfb ^+/+ mice.

Conclusion

The reduction in inflammation‐associated synovial angiogenesis in Vegfb ^−/− mice implicates VEGF‐B in pathologic vascular remodeling in inflammatory arthritis. VEGF‐B may be an attractive target in the design of anti‐angiogenic therapies for rheumatoid arthritis.

     

Antiviral gene expression in rheumatoid arthritis: Role of IKKϵ and interferon regulatory factor 3

Objective

The rheumatoid synovium displays characteristics of Toll‐like receptor (TLR) activation and antiviral gene expression, including production of RANTES and interferon‐β (IFNβ). The mechanism of this activation in rheumatoid synovial tissue is unknown. This study was designed to investigate the role of the IKK‐related kinase IKKϵ and IFN regulatory factor 3 (IRF‐3) in the activation of antiviral genes in rheumatoid arthritis (RA).

Methods

Kinase assay and immunostaining were performed on synovial tissue. Dominant‐negative (DN) IKKϵ adenoviral infection of human fibroblast‐like synoviocytes (FLS) was followed by poly(I‐C) stimulation and Western blotting. Quantitative polymerase chain reaction was performed on DN IKKϵ–infected FLS and IKKϵ^−/− and IKKϵ^+/+ mouse FLS.

Results

Western blotting showed that IKKϵ phosphorylation was significantly greater in RA synovium compared with osteoarthritis synovium. Kinase assay confirmed that IKKϵ was activated in RA synovium, and immunostaining showed localization of pIKKϵ to the intimal lining. Western blot analysis demonstrated that activation of IRF‐3 was also increased in RA synovium. Poly(I‐C), lipopolysaccharide, and tumor necrosis factor α (TNFα) activated phosphorylation of IKKϵ and IRF‐3 in FLS. DN IKKϵ inhibited IRF‐3 phosphorylation as well as RANTES and IFNβ protein production in synoviocytes. Antiviral gene expression was also reduced in FLS from IKKϵ^−/− mice compared with IKKϵ^+/+ mice.

Conclusion

Antiviral gene expression in RA, especially due to TLR ligands and TNFα, is dependent on IKKϵ and IRF‐3, and this pathway plays a key role in the production of type I IFNs and chemokines such as RANTES. These findings indicate that the IKKϵ pathway may have potential as a therapeutic target in RA.

     

Regulation of systemic and local myeloid cell subpopulations by bone marrow cell–derived granulocyte–macrophage colony‐stimulating factor in experimental inflammatory arthritis

Objective

Even though there are clinical trials assessing granulocyte–macrophage colony‐stimulating factor (GM‐CSF) blockade in rheumatoid arthritis (RA), questions remain as to how GM‐CSF acts as a proinflammatory cytokine. The aims of this study on the regulation of arthritis progression by GM‐CSF were to determine the source of the GM‐CSF, whether there are systemic effects, the changes in synovial tissue leukocyte populations, and the arthritis model dependence on GM‐CSF.

Methods

Bone marrow chimeras were used to determine the source of GM‐CSF required for the development of collagen‐induced arthritis (CIA). The K/BxN serum–transfer model of arthritis was tested in GM‐CSF^−/− mice and using anti–GM‐CSF monoclonal antibodies. Cell populations from arthritic mice were assessed by differential staining and flow cytometry.

Results

In the CIA model, GM‐CSF produced by bone marrow–derived cells was required for arthritis development. GM‐CSF blockade, while ameliorating the development of CIA, was found to have systemic effects, limiting the increase in circulating Ly‐6C^high monocytes and neutrophils. GM‐CSF blockade led to fewer synovial macrophages (both Ly‐6C^high and Ly‐6C^low), neutrophils, and lymphocytes. In the absence of GM‐CSF, K/BxN serum–transfer arthritis initially developed normally; however, the numbers of Ly‐6C^high monocytes and synovial macrophages (both Ly‐6C^high and Ly‐6C^low) were again reduced, along with the peak disease severity and maintenance.

Conclusion

GM‐CSF is a key player in two arthritis models, participating in interactions between hemopoietic cells, both locally and systemically, to control myeloid cell numbers as well as presumably to “activate” them. These results could be useful for the analysis of current clinical trials targeting GM‐CSF in patients with RA.

     

The programmed death 1/programmed death ligand 1 inhibitory pathway is up‐regulated in rheumatoid synovium and regulates peripheral T cell responses in human and murine arthritis

Objective

T cells play a major role in the pathogenesis of rheumatoid arthritis (RA). The programmed death 1 (PD‐1)/programmed death ligand 1 (PDL‐1) pathway is involved in peripheral tolerance through inhibition of T cells at the level of synovial tissue. The aim of this study was to examine the role of PD‐1/PDL‐1 in the regulation of human and murine RA.

Methods

In synovial tissue and synovial fluid (SF) mononuclear cells from patients with RA, expression of PD‐1/PDL‐1 was examined by immunohistochemistry and flow cytometry, while PD‐1 function was assessed in RA peripheral blood (PB) T cells after stimulation of the cells with anti‐CD3 and PDL‐1.Fc to crosslink PD‐1. Collagen‐induced arthritis (CIA) was induced in PD‐1^−/− C57BL/6 mice, and recombinant PDL‐1.Fc was injected intraperitoneally to activate PD‐1 in vivo.

Results

RA synovium and RA SF were enriched with PD‐1+ T cells (mean ± SEM 24 ± 5% versus 4 ± 1% in osteoarthritis samples; P = 0.003) and enriched with PDL‐1+ monocyte/macrophages. PD‐1 crosslinking inhibited both T cell proliferation and production of interferon‐γ (IFNγ) in RA patients; PB T cells incubated with RA SF, as well as SF T cells from patients with active RA, exhibited reduced PD‐1–mediated inhibition of T cell proliferation at suboptimal, but not optimal, concentrations of PDL‐1.Fc. PD‐1^−/− mice demonstrated increased incidence of CIA (73% versus 36% in wild‐type mice; P < 0.05) and greater severity of CIA (mean maximum arthritis score 5.0 versus 2.3 in wild‐type mice; P = 0.040), and this was associated with enhanced T cell proliferation and increased production of cytokines (IFNγ and interleukin‐17) in response to type II collagen. PDL‐1.Fc treatment ameliorated the severity of CIA and reduced T cell responses.

Conclusion

The negative costimulatory PD‐1/PDL‐1 pathway regulates peripheral T cell responses in both human and murine RA. PD‐1/PDL‐1 in rheumatoid synovium may represent an additional target for immunomodulatory therapy in RA.

     

Unmasking of a protective tumor necrosis factor receptor I–mediated signal in the collagen‐induced arthritis model

Objective

To examine the relative importance of tumor necrosis factor receptor I (TNFRI) signaling in the hematopoietic tissue compartment in the progression of collagen‐induced arthritis (CIA), a model of rheumatoid arthritis (RA).

Methods

DBA/1 mice were administered a lethal radiation dose and were then rescued with bone marrow derived from either DBA/1 or TNFRI^−/− mice. CIA was then induced, and disease progression was characterized.

Results

Surprisingly, mice with CIA that received TNFRI^−/− donor marrow developed increased disease severity as compared with control mice with CIA. This could not be attributed to an increased primary response to collagen or to the contribution of a non‐DBA genetic background. In mice that received TNFRI^−/− bone marrow, histologic markers of advanced disease were evident shortly after initiation of the immune response to collagen and long before clinical evidence of disease. Serum TNFα was undetectable, whereas serum interleukin‐12 p40 levels were increased, at the end point of the study in mice that received TNFRI^−/− bone marrow.

Conclusion

These data raise the intriguing possibility of the existence of an antiinflammatory, TNFRI‐mediated circuit in the hematopoietic compartment. This circuit bears a resemblance to the switch in TNFα function that has been observed during the resolution of bacterial infections. These data suggest that TNFRI‐mediated signals in the radioresistant tissues contribute to disease progression, whereas TNFRI‐mediated signals in the radiosensitive tissues can contribute to protection from disease. We thus put forward the hypothesis that the degree of response to TNFα blockade in RA is dependent in part on the relative genetic strengths of these 2 pathways.

     

Antiinflammatory functions of p38 in mouse models of rheumatoid arthritis: Advantages of targeting upstream kinases MKK‐3 or MKK‐6

Objective

Inhibitors of p38 demonstrate limited benefit in rheumatoid arthritis (RA), perhaps due to the antiinflammatory functions of p38α. This study was performed to determine if selective deletion of p38α in macrophages affects the severity of arthritis and whether blocking upstream kinases in the p38 pathway, such as MKK‐3 or MKK‐6, avoids some of the limitations of p38 blockade.

Methods

Wild‐type (WT) mice and mice with selective deletion of p38α in macrophages (p38α^ΔLysM) were injected with K/BxN sera. Antigen‐induced arthritis was also induced in p38α^ΔLysM mice. Mouse joint extracts were evaluated by enzyme‐linked immunosorbent assay, quantitative polymerase chain reaction (qPCR), and Western blot analysis. Bone marrow–derived macrophages (BMMs) were stimulated with lipopolysaccharide (LPS) and were evaluated by qPCR and Western blotting. Bone marrow chimeras were generated using MKK‐3^−/− and MKK‐6^−/− mice, and K/BxN serum was administered to induce arthritis.

Results

Compared to WT mice, p38α^ΔLysM mice had increased disease severity and delayed resolution of arthritis, which correlated with higher synovial inflammatory mediator expression and ERK phosphorylation. In contrast to WT BMMs cultured in the presence of a p38α/β inhibitor, LPS‐stimulated MKK‐6– and MKK‐3–deficient BMMs had suppressed LPS‐mediated interleukin‐6 (IL‐6) expression but had normal IL‐10 production, dual‐specificity phosphatase 1 expression, and MAPK phosphorylation. WT chimeric mice with MKK‐6– and MKK‐3–deficient bone marrow had markedly decreased passive K/BxN arthritis severity.

Conclusion

Inhibiting p38α in a disease that is dominated by macrophage cytokines, such as RA, could paradoxically suppress antiinflammatory functions and interfere with clinical efficacy. Targeting an upstream kinase that regulates p38 could be more effective by suppressing proinflammatory cytokines while preventing decreased IL‐10 expression and increased MAPK activation.