Influence of anticardiolipin and anti-β2 glycoprotein I antibody cutoff values on antiphospholipid syndrome classification

Background: Anticardiolipin (aCL) and anti-beta 2 glycoprotein I (a beta 2GPI) immunoglobulin (Ig) G/IgM antibodies are 2 of the 3 laboratory criteria for classification of antiphospholipid syndrome (APS). The threshold for clinically relevant levels of antiphospholipid antibodies (aPL) for the diagnosis of APS remains a matter of debate. The aim of this study was to evaluate the variation in cutoffs as determined in different clinical laboratories based on the results of a questionnaire as well as to determine the optimal method for cutoff establishment based on a clinical approach.Methods: The study included samples from 114 patients with thrombotic APS, 138 patients with non-APS thrombosis, 138 patients with autoimmune disease, and 183 healthy controls. aCL and a beta 2GPI IgG/IgM antibodies were measured at 1 laboratory using 4 commercial assays. Assay-specific cutoff values for aPL were obtained by determining 95th and 99th percentiles of 120 compared to 200 normal controls by different statistical methods.Results: Normal reference value data showed a nonparametric distribution. Higher cutoff values were found when calculated as 99th rather than 95th percentiles. These values also showed a stronger association with thrombosis. The use of 99th percentile cutoffs reduced the chance of false positivity but at the same time reduced sensitivity. The decrease in sensitivity was higher than the gain in specificity when 99th percentiles were calculated by methods wherein no outliers were eliminated.Conclusions: We present cutoff values for aPL determined by different statistical methods. The 99th percentile cutoff value seemed more specific. However, our findings indicate the need for standardized statistical criteria to calculate 99th percentile cutoff reference values.Background: Anticardiolipin (aCL) and anti-beta 2 glycoprotein I (a beta 2GPI) immunoglobulin (Ig) G/IgM antibodies are 2 of the 3 laboratory criteria for classification of antiphospholipid syndrome (APS). The threshold for clinically relevant levels of antiphospholipid antibodies (aPL) for the diagnosis of APS remains a matter of debate. The aim of this study was to evaluate the variation in cutoffs as determined in different clinical laboratories based on the results of a questionnaire as well as to determine the optimal method for cutoff establishment based on a clinical approach.Methods: The study included samples from 114 patients with thrombotic APS, 138 patients with non-APS thrombosis, 138 patients with autoimmune disease, and 183 healthy controls. aCL and a beta 2GPI IgG/IgM antibodies were measured at 1 laboratory using 4 commercial assays. Assay-specific cutoff values for aPL were obtained by determining 95th and 99th percentiles of 120 compared to 200 normal controls by different statistical methods.Results: Normal reference value data showed a nonparametric distribution. Higher cutoff values were found when calculated as 99th rather than 95th percentiles. These values also showed a stronger association with thrombosis. The use of 99th percentile cutoffs reduced the chance of false positivity but at the same time reduced sensitivity. The decrease in sensitivity was higher than the gain in specificity when 99th percentiles were calculated by methods wherein no outliers were eliminated.Conclusions: We present cutoff values for aPL determined by different statistical methods. The 99th percentile cutoff value seemed more specific. However, our findings indicate the need for standardized statistical criteria to calculate 99th percentile cutoff reference values.A

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

Defective CD4(+)CD25(+ )regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4(+)CD25(+ )T(reg )cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of T(reg )cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4(+)CD25(+ )T cells in the central and peripheral lymphoid tissues. In addition, CD4(+)CD25(+ )T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4(+)CD25(+ )T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for T(reg )cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both T(reg )cells and accessory cells. Our results demonstrate that the decrease in T(reg )cell activity in CIA is counter-regulated by endogenous IFN-γ

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

Detection of anti-cardiolipin and anti-β2glycoprotein I antibodies differs between platforms without influence on association with clinical symptoms

Background: The anti-phospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with persistent presence of anti-phospholipid antibodies (aPL). Laboratory criteria include aPL detection by coagulation tests for lupus anticoagulant (LAC) or solid phase assays measuring anti-beta 2 glycoprotein I (a beta 2GPI) or anticardiolipin (aCL) immunoglobulin (Ig) G/IgM antibodies. External quality control programs illustrate that commercially available aPL assays produce variable results. Objective: We aimed to investigate the agreement and diagnostic accuracy of solid phase assays. Materials and Methods: In thismulti-centre study, 1,168 patient samples were tested on one site for aCL and a beta 2GPI IgG/IgM antibodies by four solid phase test systems. Samples included APS patients, controls and monoclonal antibodies (MoAB) against different epitopes of beta 2GPI. LAC was determined by the local centre. Results: aCL IgM assays resulted in the most discrepancies (60%), while aCL IgG and a beta 2GPI IgM assays resulted in lower discrepancies (36%), suggesting better agreement. Discrepant samples displayed lower median aPL titers. Dependent on the solid phase test system, odds ratios (ORs) for thrombosis and pregnancy morbidity ranged from 1.98 to 2.56 and 3.42 to 4.78, respectively. Three platforms showed lower sensitivity for MoAB directed against the glycine (Gly) 40-arginine (Arg) 43 epitope of domain I of beta 2GPI. Conclusion: Poor agreement was observed between different commercially available aCL and a beta 2GPI IgG/IgM assays, hampering uniformity in the identification of aPL-positive patients. Clinical association was globally concordant between solid phase test systems considering results of the four aPL together. An assay sensitive in detecting the MoAB against Gly40-Arg43 of domain I of beta 2GPI reached the highest OR for thrombosis

Immunoregulatory properties of interferon-γ in collagen-induced arthritis : effects on CD4+ CD25+ regulatory T cell function and neutrophil infiltration

Over a period of several years, work in our laboratory has addressed the role of interferon-g (IFN-g) in collagen-induced arthritis (CIA), a well-characterized experimental model for rheumatoid arthritis (RA) in humans. Mice lacking a functional IFN-g receptor (IFN-gR KO) were found to be more susceptible to CIA than wild-type mice: disease onset is accelerated and arthritic symptoms are more severe. CD4+CD25+ regulatory T (Treg) cells have proven to be important in the control of various autoimmune diseases. In addition, aggrevated disease in IFN-gR KO mice is associated with a high proportion of neutrophils in the joints. In this thesis we therefore investigated the possibilities that IFN-g exerts its protective effect in CIA through stimulation of Treg cells or through inhibition of neutrophil-specific chemokines. By rendering wild-type mice deficient in Treg cells and by a single transfer of Treg cells in wild-type mice, we demonstrated the importance of Treg cells in the pathogenesis of CIA. Wild-type mice treated with depleting anti-CD25 antibodies developed a significantly more severe arthritis, comparable to the disease course in IFN-gR KO mice. Thus, we proposed that the higher susceptibility of IFN-gR KO mice to CIA might be ascribed to defects in the production or function of these Treg cells. Having demonstrated that IFN-g receptor deficiency did not affect the number of CD4+CD25+ cells in the central and peripheral lymphoid tissues, nor their potential to suppress effector T (Teff) cell proliferation in vitro, we examined the effect of immunization on Treg cell numbers and function. After immunization with collagen type II (CII) in complete Freund’s adjuvant (CFA), the capacity of Treg cells to suppress TCR-triggered proliferation of Teff cells was significantly lower as compared to that of naive Treg cells. In addition, suppressive activity of Treg cells became significantly more impaired in the absence of IFN-g signaling. Accordingly, expression of Foxp3, a highly specific marker for Treg cells, was lower in Treg cells obtained from immunized IFN-gR KO mice. We completed this part of the study by showing that the effect of endogenous IFN-g, which accounts for the more suppressive activity in immunized wild-type as compared to IFN-gR KO mice, concerns both Treg cells and accessory cells. Together, these data demonstrate that the decrease in Treg cell activity in CIA is counter-regulated by endogenous IFN-g. Since a single transfer of Treg cells significantly improved clinical symptoms of arthritis without affecting humoral responses, we investigated whether the effects of Treg cells in CIA may rely on the inhibition of osteoclastogenesis, a major pathogenic process in CIA. Osteoclast differentiation was found to be significantly decreased in the presence of Treg cells. Inhibition of osteoclastogenesis was accompanied by increased expression levels of cytokines inhibiting osteoclastogenesis, including granulocyte macrophage colony-stimulating factor (GM-CSF), IL-5 and IL-10. These data provide an explanation for the beneficial effect of Treg cells in CIA and suggest that Treg cells may be used for the treatment of RA. In a subsequent part of our work we investigated the mechanism underlying the excessive proportion of neutrophils in the inflammatory lesions of IFN-gR KO mice. Neutrophils are considered important in the pathogenesis of CIA, for example by their ability to destroy cartilage. We documented significantly increased levels of myeloperoxidase and matrix metalloproteinase-9 in synovia of arthritic IFN-gR KO mice as compared to wild-type counterparts, thereby quantificating the higher infiltration of neutrophils in the joints of IFN-gR KO mice. The excessive proportion of neutrophils in synovia of arthritic IFN-gR KO mice could be explained by significantly increased levels of the CXC chemokine granulocyte chemotactic protein-2 (GCP-2), a major neutrophil-attracting chemokine in mice that is considered to be the functional equivalent of IL-8 in humans. We further demonstrated that heat-killed mycobacteria present in CFA elicit production of GCP-2 in mouse embryo fibroblast (MEF) cultures, and that this production is inhibited by IFN-g. Inhibition of GCP-2 production by IFN-g was found to be signal transducer and activator of transcription-1 (STAT-1) dependent. In addition we found that IL-17, known to be important in the pathogenesis of arthritis, synergizes with mycobacteria for the production of GCP-2. Again, IFN-g was found to inhibit this production of GCP-2. IFN-gR KO mice treated with neutralizing anti-GCP-2 antibodies were completely protected from CIA, indicating the in vivo importance of GCP-2 in the pathogenesis of CIA. These data support the notion that one of the mechanisms whereby endogenous IFN-g mitigates the manifestations of CIA consists in inhibiting production of GCP-2, thereby limiting mobilisation and infiltration of neutrophils, which are important actors in joint inflammation. To summarize our investigations, we can state that IFN-g exerts its protective effect in CIA in part by inhibition of the decrease in Treg cell activity elicited by immunization with CII in CFA. The protective effect of Treg cells in CIA could be explained by inhibition of osteoclastogenesis. In addition, IFN-g was found to limit the infiltration of neutrophils at the site of inflammation through downregulation of the production of GCP-2. Our data may provide an explanation for the protective effect of IFN-g in other autoimmune models that rely on the use of CFA.status: publishe

The Significance of Antibodies against Domain I of Beta-2 Glycoprotein I in Antiphospholipid Syndrome

The antiphospholipid syndrome (APS) is characterized by vascular thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPLs). Progress is being made in understanding the pathogenesis of the syndrome, but difficulties persist in the identification of patients at risk for thrombosis and/or pregnancy morbidity. Beta-2 glycoprotein I ( (2) GPI), a plasma protein consisting of five sushi domains, is thought to be the main antigenic target of aPLs. Antibodies recognizing domain I of (2) GPI are predominantly present in patients with an elevated risk of thrombosis, whereas antidomain IV/V antibodies are found in nonthrombotic autoimmune diseases. Indeed, domain I antibodies proved to be pathogenic in multiple studies. Retrospective studies have provided evidence for an added clinical value of antidomain I antibodies in the risk stratification of patients with APS. Still, wide ranges of odds ratio exist between studies, probably due to differences in the study and control population, and detection methods used. Despite the proven pathogenicity of antidomain I antibodies and their correlations with clinical manifestations of APS, heterogeneity of the current studies has prohibited their acceptance in the official diagnostic criteria. Well-designed large longitudinal prospective studies with available and new, preferentially functional, assays for the risk stratification of patients with APS are required

Thrombin generation in low plasma volumes

Abstract Accurate thrombin generation determination by calibrated automated thrombinography can be sustained when reducing the plasma and reagent volumes up to half, but not for higher reductions or plasma dilutions