Hilde Kelchtermans

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

OBJECTIVEnTo investigate the role of interleukin-22 (IL-22) in collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis.nnnMETHODSnC57BL/6 mice were immunized with type II collagen (CII) in Freunds 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.nnnRESULTSnUpon immunization with CII in Freunds 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.nnnCONCLUSIONnEndogenous 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.

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

IntroductionThe 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-γ (IFN-γ) in these processes.MethodsMSC were isolated from bone marrow of DBA/1 wild type and IFN-γ receptor knock-out (IFN-γR 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-γR KO MSC.ResultsPurity 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-γR 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-γR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.ConclusionsWhereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-γ, 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.

Activated CD4+CD25+ regulatory T cells inhibit osteoclastogenesis and collagen-induced arthritis

Objectives: Patients with rheumatoid arthritis (RA) have defective CD4+CD25+ regulatory T (Treg) cells and increased osteoclastogenesis. A similar situation has been described in collagen-induced arthritis (CIA). In this study, it was investigated whether a single transfer of polyclonally activated Treg cells inhibits CIA and osteoclastogenesis. Methods: Purified Treg cells were expanded in vitro with anti-CD3 and anti-CD28 antibody-coated beads and injected into DBA/1 mice. Mice were immunised with collagen type II (CII) in complete Freund adjuvant (CFA) and scores of arthritis were recorded. In vitro osteoclastogenesis assays were performed on splenocytes by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)κB ligand (RANKL). Levels of anti-CII antibody and cytokines were determined in the supernatant using ELISA and Bio-Plex protein array system. Results: It was found that 106 activated Treg cells significantly counteracted the development of CIA, which was accompanied by decreased serum levels of TNFα and IL6, but not by inhibition of autoimmune antibody responses. The differentiation of osteoclasts in splenocyte cultures was significantly reduced in the presence of prestimulated Treg cells. Expression of cytokines that are described to inhibit osteoclastogenesis, including granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)γ, interleukin (IL)5 and IL10, were dramatically increased upon addition of Treg cells. Furthermore, splenocytes from mice that had been treated with Treg cells displayed an impaired capacity to develop into mature osteoclasts, suggesting that Treg cells abrogated osteoclastogenesis in vivo. Conclusions: Activated CD4+CD25+ Treg cells improve clinical symptoms of CIA, regulate cytokine production and inhibit osteoclastogenesis in vitro and in vivo.

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.

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

IntroductionInterleukin (IL)-17 is a pro-inflammatory cytokine in rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). Since interferon (IFN)-γ inhibits Th17 cell development, IFN-γ receptor knockout (IFN-γR 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-γ on the effector mechanisms of IL-17 in an in vitro system was also investigated.MethodsIFN-γR 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)-α and the expression of cytokines and chemokines were determined.ResultsA preventive anti-IL-17 antibody treatment inhibited CIA in IFNγR 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-α synergistically induced granulocyte chemotactic protein-2 (GCP-2), IL-6 and receptor activator of NFκB ligand (RANKL) in MEF. This induction was profoundly inhibited by IFN-γ in a STAT-1 (signal transducer and activator of transcription-1)-dependent way.ConclusionsIn the absence of IFN-γ, IL-17 mediates its pro-inflammatory effects mainly through stimulatory effects on granulopoiesis, neutrophil infiltration and bone destruction. In vitro IFN-γ profoundly inhibits the effector function of IL-17. Thus, aside from the well-known inhibition of the development of Th17 cells by IFN-γ, this may be an additional mechanism through which IFN-γ attenuates autoimmune diseases.

Protective role of IFN‐γ in collagen‐induced arthritis conferred by inhibition of mycobacteria‐induced granulocyte chemotactic protein‐2 production

Mice with a disrupted IFN‐γ system are remarkably susceptible to experimental autoimmune diseases, such as collagen‐induced arthritis (CIA), which rely on the use of CFA. The inflammatory lesions of these IFN‐γ knockout (KO) mice are characterized by an excessive proportion of neutrophils. Here, we show that the increased severity of CIA in IFN‐γR KO as compared with wild‐type mice is accompanied by increased levels of the CXC chemokine granulocyte chemotactic protein‐2 (GCP‐2), a major neutrophil‐attracting chemokine in mice. We demonstrated that the heat‐killed mycobacteria present in CFA elicited production of GCP‐2 in mouse embryo fibroblast cultures and that this production was inhibited by IFN‐γ. Inhibition of GCP‐2 production by IFN‐γ was STAT‐1‐dependent. IFN‐γ receptor KO mice treated with neutralizing anti‐GCP‐2 antibodies were protected from CIA, indicating the in vivo importance of GCP‐2 in the pathogenesis of CIA. Our data support the notion that one of the mechanisms whereby endogenous IFN‐γ mitigates the manifestations of CIA consists of inhibiting production of GCP‐2, thereby limiting mobilization and infiltration of neutrophils, which are important actors in joint inflammation. These results may also be applicable to other experimental models of autoimmunity that rely on the use of CFA.

Validity of the global anti-phospholipid syndrome score to predict thrombosis: a prospective multicentre cohort study

OBJECTIVEnTo investigate the validity of the global APS score (GAPSS) to predict thrombosis in patients with autoimmune diseases.nnnMETHODSnThis prospective cohort study included consecutive patients with aPL or SLE. aPL, aPS-PT and GAPSS were determined. A Cox proportional hazards model assessed the validity of GAPSS and identified other potential independent predictors of thrombosis.nnnRESULTSnOne hundred and thirty-seven patients [43.5 (s.d. 15.4) years old; 107 women] were followed up for a mean duration of 43.1 (s.d. 20.7) months. Mean GAPSS was significantly higher in patients who experienced a thrombotic event compared with those without [10.88 (s.d. 5.06) vs 8.15 (s.d. 5.31), respectively, P = 0.038]. In univariate analysis, age [hazard ratio (HR) = 1.04 (95% CI 1.01, 1.08)] and GAPSS above 16 [HR = 6.86 (95% CI 1.90, 24.77)] were each significantly associated with thrombosis during follow-up, while history of arterial thrombosis [HR = 2.61 (95% CI 0.87, 7.82)] failed to reach significance. Among aPL assays, IgG aPS/PT--a component of the GAPSS--was significantly associated with thrombosis [HR = 2.95 (95% CI 1.02, 8.51)]. In multivariate analysis, GAPSS above 16 remained the only significant predictor of thrombosis [HR = 6.17 (95% CI 1.70, 22.40)].nnnCONCLUSIONnThis first external validation study confirmed that GAPSS can predict thrombosis in patients with aPL and associated autoimmune diseases.

From antibody to clinical phenotype, the black box of the antiphospholipid syndrome: pathogenic mechanisms of the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is diagnosed by the combination of vascular thrombosis and/or pregnancy morbidity and the detection of antiphospholipid antibodies (aPLs) in plasma. In the last few years, a great effort has been made to unravel the mechanism by which aPLs cause thrombosis and a vast amount of mechanisms have been proposed. aPLs were proposed to induce a prothrombotic state by influencing the cellular blood compartment, the plasma compartment, the vascular wall and even metabolic pathways beyond the hemostatic system. However, due to the diversity in the mechanisms and the differences in the methodology, the focus of the mechanistical studies in this field seems to be largely diffused. It is hard to imagine that aPLs can exert such a diversity of effects, resulting in either thrombosis and/or pregnancy morbidity and the relationship between aPLs and the clinical manifestations remains to be a mysterious black box. In an attempt to get insight in what takes place inside the black box, we have analyzed 126 mechanistical studies on aPLs and discussed differences in the type of antibodies that were used, the involvement of beta2-glycoprotein I (β2GPI), and the criteria used to diagnose APS patients.

Variability in Exposure of Epitope G40-R43 of Domain I in Commercial Anti-Beta2-Glycoprotein I IgG ELISAs

Background A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-β2glycoprotein I (β2GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces β2GPI opens up, thereby exposing G40-R43. Objectives To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays. Methods Two patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes β2GPI irrespective of its conformation. These antibodies were tested in commercial anti-β2GPI assays (A–E). Results In assay A, both antibodies showed equal reactivity towards β2GPI, indicating that all the β2GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43. Conclusions Exposure of epitope G40-R43 on β2GPI is highly variable between commercial anti-β2GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43.

Mouse Cytomegalovirus Infection in BALB/c Mice Resembles Virus-Associated Secondary Hemophagocytic Lymphohistiocytosis and Shows a Pathogenesis Distinct from Primary Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immunological disorder that is characterized by systemic inflammation, widespread organ damage, and hypercytokinemia. Primary HLH is caused by mutations in granule-mediated cytotoxicity, whereas secondary HLH occurs, without a known genetic background, in a context of infections, malignancies, or autoimmune and autoinflammatory disorders. Clinical manifestations of both HLH subtypes are often precipitated by a viral infection, predominantly with Herpesviridae. Exploiting this knowledge, we established an animal model of virus-associated secondary HLH by infecting immunocompetent wild-type mice with the β-herpesvirus murine CMV. C57BL/6 mice developed a mild inflammatory phenotype, whereas BALB/c mice displayed the clinicopathologic features of HLH, as set forth in the Histiocyte Society diagnostic guidelines: fever, cytopenia, hemophagocytosis, hyperferritinemia, and elevated serum levels of soluble CD25. BALB/c mice also developed lymphadenopathy, liver dysfunction, and decreased NK cell numbers. Lymphoid and myeloid cells were in a hyperactivated state. Nonetheless, depletion of CD8+ T cells could not inhibit or cure the HLH-like syndrome, highlighting a first dissimilarity from mouse models of primary HLH. Immune cell hyperactivation in BALB/c mice was accompanied by a cytokine storm. Notably, plasma levels of IFN-γ, a key pathogenic cytokine in models of primary HLH, were the highest. Nevertheless, murine CMV–infected IFN-γ–deficient mice still developed the aforementioned HLH-like symptoms. In fact, IFN-γ–deficient mice displayed a more complete spectrum of HLH, including splenomegaly, coagulopathy, and decreased NK cell cytotoxicity, indicating a regulatory role for IFN-γ in the pathogenesis of virus-associated secondary HLH as opposed to its central pathogenic role in primary HLH.