Discrepancy between the in vitro and in vivo effects of murine mesenchymal stem cells on T-cell proliferation and collagen-induced arthritis

INTRODUCTION: The goal of this study is to analyze the potential immunosuppressive properties of mesenchymal stem cells (MSC) on T cell proliferation and in collagen-induced arthritis (CIA). An additional aim is to investigate the role of interferon-gamma (IFN-gamma) in these processes. METHODS: MSC were isolated from bone marrow of DBA/1 wild type and IFN-gamma receptor knock-out (IFN-gammaR KO) mice and expanded in vitro. Proliferation of anti-CD3-stimulated CD4+ T cells in the presence or absence of MSC was evaluated by thymidine incorporation. CIA was induced in DBA/1 mice and animals were treated with MSC by intravenous or intraperitoneal injections of wild type or IFN-gammaR KO MSC. RESULTS: Purity of enriched MSC cultures was evaluated by flow cytometry and their ability to differentiate into osteoblasts and adipocytes. In vitro, wild type MSC dose-dependently suppressed anti-CD3-induced T cell proliferation whereas IFN-gammaR KO MSC had a significantly lower inhibitory potential. A role for inducible nitric oxide (iNOS), programmed death ligand-1 (PD-L1) and prostaglandin E2 (PGE2), but not indoleamine 2,3-dioxigenase (IDO), in the T cell inhibition was demonstrated. In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II. CONCLUSIONS: Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA. Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.status: publishe

Effector mechanisms of interleukin-17 in collagen-induced arthritis in the absence of interferon-γ and counteraction by interferon-γ

Introduction Interleukin (IL)-17 is a pro-inflammatory cytokine in rheumatoid arthritis (RA) and collagen-induced arthritis ( CIA). Since interferon (IFN)-gamma inhibits Th17 cell development, IFN-gamma receptor knockout (IFN-gamma R KO) mice develop CIA more readily. We took advantage of this model to analyse the mechanisms of action of IL-17 in arthritis. The role of IFN-gamma on the effector mechanisms of IL-17 in an in vitro system was also investigated. Methods IFN-gamma R KO mice induced for CIA were treated with anti-IL-17 or control antibody. The collagen type II (CII)-specific humoral and cellular autoimmune responses, myelopoiesis, osteoclastogenesis, and systemic cytokine production were determined. Mouse embryo fibroblasts (MEF) were stimulated with IL-17, tumor necrosis factor (TNF)-alpha and the expression of cytokines and chemokines were determined. Results A preventive anti-IL-17 antibody treatment inhibited CIA in IFN gamma R KO mice. In the joints of anti-IL-17-treated mice, neutrophil influx and bone destruction were absent. Treatment reduced the cellular autoimmune response as well as the splenic expansion of CD11b(+) cells, and production of myelopoietic cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-6. IL-17 and TNF-alpha synergistically induced granulocyte chemotactic protein-2 (GCP-2), IL-6 and receptor activator of NF kappa B ligand (RANKL) in MEF. This induction was profoundly inhibited by IFN-gamma in a STAT-1 (signal transducer and activator of transcription-1)dependent way. Conclusions In the absence of IFN-gamma, IL-17 mediates its proinflammatory effects mainly through stimulatory effects on granulopoiesis, neutrophil infiltration and bone destruction. In vitro IFN-gamma profoundly inhibits the effector function of IL-17. Thus, aside from the well-known inhibition of the development of Th17 cells by IFN-gamma, this may be an additional mechanism through which IFN-gamma attenuates autoimmune diseases

Pro-inflammatory properties of stromal cell-derived factor-1 (CXCL12) in collagen-induced arthritis

CXCL12 (stromal cell-derived factor 1) is a unique biological ligand for the chemokine receptor CXCR4. We previously reported that treatment with a specific CXCR4 antagonist, AMD3100, exerts a beneficial effect on the development of collagen-induced arthritis (CIA) in the highly susceptible IFN-γ receptor-deficient (IFN-γR KO) mouse. We concluded that CXCL12 plays a central role in the pathogenesis of CIA in IFN-γR KO mice by promoting delayed type hypersensitivity against the auto-antigen and by interfering with chemotaxis of CXCR4(+ )cells to the inflamed joints. Here, we investigated whether AMD3100 can likewise inhibit CIA in wild-type mice and analysed the underlying mechanism. Parenteral treatment with the drug at the time of onset of arthritis reduced disease incidence and modestly inhibited severity in affected mice. This beneficial effect was associated with reduced serum concentrations of IL-6. AMD3100 did not affect anti-collagen type II antibodies and, in contrast with its action in IFN-γR KO mice, did not inhibit the delayed type hypersensitivity response against collagen type II, suggesting that the beneficial effect cannot be explained by inhibition of humoral or cellular autoimmune responses. AMD3100 inhibited the in vitro chemotactic effect of CXCL12 on splenocytes, as well as in vivo leukocyte infiltration in CXCL12-containing subcutaneous air pouches. We also demonstrate that, in addition to its effect on cell infiltration, CXCL12 potentiates receptor activator of NF-κB ligand-induced osteoclast differentiation from splenocytes and increases the calcium phosphate-resorbing capacity of these osteoclasts, both processes being potently counteracted by AMD3100. Our observations indicate that CXCL12 acts as a pro-inflammatory factor in the pathogenesis of autoimmune arthritis by attracting inflammatory cells to joints and by stimulating the differentiation and activation of osteoclasts

Mast cells protect from post-traumatic spinal cord damage in mice by degrading inflammation-associated cytokines via mouse mast cell protease 4

AbstractMast cells (MCs) are found abundantly in the central nervous system and play a complex role in neuroinflammatory diseases such as multiple sclerosis and stroke. In the present study, we show that MC-deficient KitW-sh/W-sh mice display significantly increased astrogliosis and T cell infiltration as well as significantly reduced functional recovery after spinal cord injury compared to wildtype mice. In addition, MC-deficient mice show significantly increased levels of MCP-1, TNF-α, IL-10 and IL-13 protein levels in the spinal cord. Mice deficient in mouse mast cell protease 4 (mMCP4), an MC-specific chymase, also showed increased MCP-1, IL-6 and IL-13 protein levels in spinal cord samples and a decreased functional outcome after spinal cord injury. A degradation assay using supernatant from MCs derived from either mMCP4−/− mice or controls revealed that mMCP4 cleaves MCP-1, IL-6, and IL-13 suggesting a protective role for MC proteases in neuroinflammation. These data show for the first time that MCs may be protective after spinal cord injury and that they may reduce CNS damage by degrading inflammation-associated cytokines via the MC-specific chymase mMCP4

Proinflammatory role of the Th17 cytokine interleukin-22 in collagen-induced arthritis in C57BL/6 mice

OBJECTIVE: To investigate the role of interleukin-22 (IL-22) in collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. METHODS: C57BL/6 mice were immunized with type II collagen (CII) in Freund's incomplete adjuvant with added Mycobacterium tuberculosis, and levels of IL-22 and its specific receptor, IL-22 receptor type I (IL-22RI), were measured in sera and tissue by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction analysis. Clinical and histologic signs of arthritis were recorded and compared with those in C57BL/6 mice deficient in the IL-22 gene (IL-22(-/-)). Humoral and cellular immune responses against CII were analyzed. In vitro osteoclastogenesis assays were performed on splenocytes. RESULTS: Upon immunization with CII in Freund's incomplete adjuvant plus heat-killed Mycobacterium tuberculosis, sera from C57BL/6 mice were found to contain high levels of IL-22, and the specific IL-22RI was expressed in lymphoid tissue, including splenocytes. IL-22(-/-) mice were less susceptible to CIA than were wild-type mice, as evidenced by their decreased incidence of arthritis and decreased pannus formation. Remarkably, the less severe form of arthritis in IL-22(-/-) mice was associated with increased production of CII-specific and total IgG antibodies, whereas cellular CII responses were unchanged. In vitro, IL-22 was found to promote osteoclastogenesis, a process that might contribute to its proinflammatory activity in CIA. CONCLUSION: Endogenous IL-22 plays a proinflammatory role in CIA in C57BL/6 mice. Our data also indicate that IL-22 promotes osteoclastogenesis and regulates antibody production.status: publishe

Proinflammatory Role of the Th17 Cytokine Interleukin-22 in Collagen-Induced Arthritis in C57BL/6 Mice

Objective. To investigate the role of interleukin-22 (IL-22) in collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. Methods. C57BL/6 mice were immunized with type II collagen (CII) in Freund's incomplete adjuvant with added Mycobacterium tuberculosis, and levels of IL-22 and its specific receptor, IL-22 receptor type I (IL-22RI), were measured in sera and tissue by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction analysis. Clinical and histologic signs of arthritis were recorded and compared with those in C57BL/6 mice deficient in the IL-22 gene (IL-22(-/-)). Humoral and cellular immune responses against CII were analyzed. In vitro osteoclastogenesis assays were performed on splenocytes. Results. Upon immunization with CII in Freund's incomplete adjuvant plus heat-killed Mycobacterium tuberculosis, sera from C57BL/6 mice were found to contain high levels of IL-22, and the specific IL-22RI was expressed in lymphoid tissue, including splenocytes. IL-22(-/-) mice were less susceptible to CIA than were wild-type mice, as evidenced by their decreased incidence of arthritis and decreased pannus formation. Remarkably, the less severe form of arthritis in IL-22(-/-) mice was associated with increased production of CII-specific and total IgG antibodies, whereas cellular CII responses were unchanged. In vitro, IL-22 was found to promote osteoclastogenesis, a process that might contribute to its proinflammatory activity in CIA. Conclusion. Endogenous IL-22 plays a proinflammatory role in CIA in C57BL/6 mice. Our data also indicate that IL-22 promotes osteoclastogenesis and regulates antibody production

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outcome, lesion development and axonal plasticity after spinal cord injur