Val247Leu β2-glycoprotein-I allelic variant is associated with antiphospholipid syndrome: systematic review and meta-analysis

Introduction

Previous studies have suggested that the possession of the Val/Val genotype of the Val247Leu polymorphism of the β₂-glycoproteinI (β₂-GPI) gene may be associated with antiphospholipid syndrome (APS), and, among patients with APS, with the production of anti-β₂-GPI antibodies or the development of thrombosis. Given the controversial results reported, the aim of this work is to combine previous findings by means of a systematic review and a meta-analysis.

Methods

We retrieved studies analyzing the genotype of the above-mentioned polymorphism among patients with APS by means of electronic database search. A meta-analysis was conducted in a random effects model and calculations of odds ratio (OR) and confidence intervals (CI) were done. Sensitivity analysis and tests for heterogeneity of the results were performed.

Results

Eight previous studies analyzed the association of APS, anti-β₂-GPI antibodies and/or thrombosis with the Val247Leu polymorphism. After meta-analysis, patients with APS had a significantly higher prevalence of the Val/Val genotype of this genetic variant when compared with controls (OR = 2.04; 95% CI: 1.12, 3.73; P = 0.02). Among patients with APS, those with anti-β₂-GPI antibodies had a higher prevalence of this genotype (OR = 1.73; 95% CI: 1.04, 2.87; P = 0.03). No significant results were found for the presence of arterial or venous thrombosis.

Conclusions

Val/Val genotype of β₂-GPI gene is associated with a significant excess risk to suffer from APS and, among patients with APS, to have anti-β₂-GPI antibodies. No definite conclusions can be made regarding the association of this polymorphism with thrombosis among APS patients.     

Cellular immune response to β2-glycoprotein-I valine/leucine247 phenotypes in Mexican patients with primary antiphospholipid syndrome

Homozygote genotype V²⁴⁷ of the β₂-glycoprotein-I (β₂GP-I) gene has been associated with anti-β₂GP-I and thrombosis in patients with primary anti-phospholipid syndrome APS (PAPS). However, the cellular immune response to β₂GP-I²⁴⁷ has been little studied.

Role of dendritic cells in Sjögren's syndrome

Sjögren's syndrome (SS) is a chronic inflammatory and lymphoproliferative autoimmune disease of unknown aetiology. It is characterised by progressive mononuclear cell infiltration of the salivary and lacrimal glands and a decreased glandular secretion, resulting in dryness of the mouth and eyes (xerostomia and keratoconjunctivitis sicca, respectively). Dendritic cells (DC) are considered to be the most potent antigen‐presenting cells. Because of their central role in initiating an immune response while maintaining tolerance, impaired function of these cells might lead to the break of peripheral tolerance and initiation of immune responses to self‐antigens. This review will focus on the possible role of DC in SS.     

Immune complex enhances tolerogenecity of immature dendritic cells via FcγRIIb and promotes FcγRIIb-overexpressing dendritic cells to attenuate lupus

A balance of inhibitory and activating signals determines the function of dendritic cells (DCs) in the immune response, which may be regulatory or stimulatory. Defects of inhibitory receptor FcγRIIb are involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE), in which high levels of circulating immune complexes (IC) exist. Our previous study showed that IC/Ig can suppress TLR4-triggered inflammatory responses in macrophages via FcγRIIb. This led us to question whether IC/Ig can polarize FcγRIIb-overexpressing DCs (DC-FcγRIIb) to be tolerogenic, thus attenuating lupus progression once infused in vivo. First, we found that IC/Ig markedly inhibited LPS- or CpG-induced DC maturation, enhanced tolerogenicity of DCs via FcγRIIb, and induced massive prostaglandin E2 (PGE2) secretion from DCs, both contributing to T-cell hyporesponsiveness. Endogenous Ig and lupus-derived IC also exhibited the same effect. DC-FcγRIIb, transfected with recombinant adenovirus encoding FcγRIIb, displayed enhanced tolerogenic function and produced more PGE2 in the presence of IC, thus further inhibiting T-cell responses. Importantly, in vivo infusion with DC-FcγRIIb significantly reduced kidney damage and prolonged the survival of lupus-prone MRL/lpr mice either before or after the onset of clinic lupus. Therefore, administration of DC-FcγRIIb may be a new approach to attenuate lupus progression.     

Research around β2-glycoprotein I: A major target for antiphospholipid antibodies

β2-Glycoprotein I (β2GPI), a phospholipid-binding protein, is one of the major target antigens for antiphospholipid antibodies (aPL) found in patients with antiphospholipid syndrome (APS). Thrombophilic disorders in APS patients are strongly associated with aPL, and their pathogenic properties depend on the presence of β2GPI. Procoagulant cell stimulation by aPL, via β2GPI, is one of the most plausible mechanisms of thrombosis in APS, and p38 mitogen activated protein kinase (MAPK) pathway plays a crucial role in such activation. β2GPI is proteolytically cleaved in domain V by activated factor X or plasmin, leading to the generation of the nicked form of β2GPI. Recently, increasing attention is focused on the role of nicked-β2GPI as a regulator of extrinsic fibrinolysis pathway.     

Entry sites for oral vaccines and drugs: A role for M cells,enterocytes and dendritic cells?

M cells have long been considered as the unique entry site of macromolecules and pathogens in the intestine, allowing delivery to antigen-presenting cells in the Peyer's patches. Therefore, antigen formulation for the development of oral vaccines has been based on administration of antigens in the form of live replicating pathogens or soluble antigen vectorized into biodegradable microspheres. However, progress in the understanding of the biology of dendritic cells, as well as identification of their localization at different sites of the intestine, suggest that they may capture antigen directly from the lumen of mucosal tissues or from epithelial cells of the intestine. Besides, a role for the absorptive epithelium in antigen presentation through both classical or non-classical MHC elements suggests that PP may not be the exclusive inductive site of the immune response in the gut. Thus, depending on the nature of the antigen (soluble or infectious) there may be different sites of antigen entry through the intestine, and each site may have distinct efficiency to promote a protective immune response, depending on the presence and function of dendritic cells. Cross talk between M cells, epithelial cells and dendritic cells may play an important role in determining the outcome of tolerance versus immunity.     

Murine autoimmune hearing loss mediated by CD4+ T cells specific for β-tubulin

Autoimmune inner ear disease is described as progressive, bilateral although asymmetric, sensorineural hearing loss and can be improved by immunosuppressive therapy. We showed that the inner ear autoantigen β-tubulin is capable of inducing experimental autoimmune hearing loss (EAHL) in mice. Immunization of BALB/c mice with β-tubulin resulted in hair cell loss and hearing loss, effects that were not seen in animals immunized with control peptide. Moreover, the EAHL model showed that β-tubulin responsiveness involved CD4(+) T cells producing IFN-γ, and T cell mediation of EAHL was determined by significantly increased auditory brainstem response after adoptive transfer of β-tubulin-activated CD4(+) T cells into naive BALB/c recipients. The potential mechanisms responsible for the observed pathology of EAHL can be attributed to decreased frequency and impaired suppressive function of regulatory T cells. Our study suggests that EAHL may be a T cell-mediated organ-specific autoimmune disorder of the inner ear.     

Post-translational oxidative modification of β2-glycoprotein I and its role in the pathophysiology of the antiphospholipid syndrome

Vascular thrombosis and/or recurrent miscarriages are the main characteristics defining Antiphospholipid Syndrome (APS). Currently there is no well-defined clinical features and/or laboratory tests that predicts the risk of adverse prognostic outcomes in APS. In this short review, we report the importance of posttranslational modification of beta2 glycoprotein I, the major autoantigen in the APS beta2 glycoprotein I that may, in part, explain possible mechanisms for the generation of auto antibodies to beta2 glycoprotein I. A specific ELISA measuring the level of oxidised beta2 glycoprotein I could be used as a potential new laboratory test - along with other laboratory tests - to more accurately predict the risk of having a clinical event in patients with APS.     

The clinical value of assays detecting antibodies against domain I of β2-glycoprotein I in the antiphospholipid syndrome

As the clinical symptoms of the antiphospholipid syndrome (APS) frequently occur irrespective of the syndrome, diagnosis predominantly depends on the laboratory assays measuring the level or function of antiphospholipid antibodies (aPLs). β2-glycoprotein I (β2GPI) is increasingly accepted as the most important target of aPLs. Anti-β2GPI antibodies constitute a heterogeneous population, but current in vivo and in vitro evidence show that especially the first domain (DI) of β2GPI contains an important pathogenic epitope. This epitope containing Glycine40-Arginine43 (G40-R43) has proven to be cryptic and only exposed when β2GPI is in its open conformation. A previous study demonstrated a highly variable exposure of the cryptic epitope in commercial anti-β2GPI assays, with implications on correct patient classification. Unexpectedly, recent unpublished data revealed impaired exposure of the pathogenic epitope in the commercially available anti-DI chemiluminescence immunoassay (CIA) assay detecting specific antibodies directed to DI.In this review we summarize the laboratory and clinical performance characteristics of the different anti-DI assays in published data and conclude with inconsistent results for both the correlation of anti-DI antibodies with clinical symptoms and the added value of anti-DI antibodies in the classification criteria of APS. Additionally, we hypothesize on possible explanations for the observed discrepancies. Finally, we highly advise manufacturers to use normal pooled plasma spiked with the monoclonal anti-DI antibodies to verify correct exposure of the cryptic epitope.     

Should we include anti-prothrombin antibodies in the screening for the antiphospholipid syndrome?

Anti-prothrombin antibodies belong to the family of the antiphospholipid antibodies. Their prevalence ranges from 50 to 90% of antiphospholipid-positive patients, depending on the laboratory methodology employed for their detection. ELISA techniques are the most commonly used methods for their measurements, which allow a quick determination of their titer and isotype(s). Unfortunately, no well standardized assays are yet commercially available. The clinical relevance of anti-prothrombin antibodies as risk factors for thromboembolic events has been investigated by a number of retrospective and case-control studies. We reviewed 11 studies: data on 1,440 patients and 371 sex- and age-matched controls were available. Seven studies reported a significant association between anti-prothrombin antibodies and thrombosis by univariate analysis. Multivariate analysis was performed in five studies: only two of them confirmed the association. The sensitivity and specificity of anti-prothrombin antibodies for thrombosis were analysed according to the antibody isotype and to the arterial and/or venous site of thrombosis. The sensitivities are disappointingly low, particularly when the M isotype and arterial thrombosis are considered, whereas the specificities are somewhat better, even though they range widely. These data do not allow to recommend the measurement of anti-prothrombin antibodies in the routine laboratory workout of antiphospholipid-positive patients in order to define their thrombotic risk.     

Systemic increase in type I interferon activity in Sjögren's syndrome: a putative role for plasmacytoid dendritic cells

In the salivary glands of primary Sj?gren's syndrome (pSjS) patients, type I IFN activity is increased, but systemic levels of type I IFN proteins are rarely detected. This study focused on the systemic activity of type I IFN in pSjS, as well as the role of peripheral plasmacytoid dendritic cells (pDC). Monocytes obtained from pSjS patients showed an increased expression of 40 genes. Twenty-three of these genes (58%), including IFI27, IFITM1, IFIT3 and IFI44, were inducible by type I IFN. pSjS serum had an enhanced capability of inducing IFI27, IFITM1, IFIT3 and IFI44 in the monocytic cell line THP-1, likely due to the action of IFN-beta. This effect could be inhibited by blocking the type I IFN receptor, supporting a high type I IFN bioactivity in pSjS serum. In addition, circulatory pDC showed increased expression of CD40. This expression was correlated to the expression level of the type I IFN-regulated genes IFI27 and IFITM1 in monocytes of the same individual. This study indicates that the increased type I IFN activity observed in pSjS patients is not only a local but also a systemic phenomenon and points to pDC as a possible source of this activity.     

Application of dendritic cells stimulated with Trichinella spiralis excretory–secretory antigens alleviates experimental autoimmune encephalomyelitis

The parasitic nematode, Trichinella spiralis (T. spiralis), exerts an immunomodulatory effect on the host immune response through excretory–secretory products (ES L1) released from encysted muscle larvae. Our model of combined T. spiralis infection and experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats demonstrated a significant reduction in EAE severity in infected animals. Recently, we have created an immune status characteristic for the live infection by in vivo application of dendritic cells (DCs) stimulated with ES L1 products of T. spiralis muscle larvae. Moreover, these cells were able to ameliorate EAE when applied 7 days before EAE induction. ES L1-stimulated DCs increased production of IL-4, IL-10 and TGF-β, and decreased production of IFN-γ and IL-17, both at the systemic level and in target organs. A significant increase in the proportion of CD4+CD25+Foxp3+ T cells was found among spleen cells, and CNS infiltrates from DA rats treated with ES L1-stimulated DCs before EAE induction, compared to controls injected with unstimulated DCs. Regulatory T cells, together with elevated levels of IL-10 and TGF-β, are most likely involved in restraining the production of Th1 and Th17 cytokines responsible for autoimmunity and thus are responsible for the beneficial effect of ES L1-educated DCs on the course of EAE. Our results show that ES L1 antigen-stimulated DCs are able not only to provoke, but also to sustain anti-inflammatory and regulatory responses regardless of EAE induction, with subsequent amelioration of EAE, or even protection from the disease.     

Varying effects of different β-glucans on the maturation of porcine monocyte-derived dendritic cells

β-Glucans are well known for their immunomodulatory capacities in humans and mice. For this reason, together with the European ban on growth-promoting antibiotics, β-glucans are intensively used in pig feed. However, as shown in the present study, there is much variation in the stimulatory capacities of β-glucans from different sources. Since dendritic cells (DCs) are the first cells that are encountered after an antigen is taken up by the intestinal epithelial cell barrier, we decided to investigate the effect of two concentrations (5 and 10 μg/ml) of five commercial β-glucan preparations, differing in structure and source, on porcine monocyte-derived dendritic cells (MoDCs). Although all β-glucans gave rise to a significant reduction of the phagocytic activity of DCs, only Macrogard induced a significant phenotypic maturation. In addition to Macrogard, zymosan, another β-glucan derived from Saccharomyces cerevisiae, and curdlan also significantly improved the T-cell-stimulatory capacity of MoDCs. Most interesting, however, is the cytokine secretion profile of curdlan-stimulated MoDCs, since only curdlan induced significant higher expression levels of interleukin-1β (IL-1β), IL-6, IL-10, and IL-12/IL-23p40. Since the cytokine profile of DCs influences the outcome of the ensuing immune response and thus may prove valuable in intestinal immunity, a careful choice is necessary when β-glucans are used as dietary supplement.     

Improved protocol for the isolation of naïve follicular dendritic cells

Follicular dendritic cells (FDCs) in lymphoid organs play an important role in the humoral immune response. However, because the isolation of FDCs is difficult due to their very small population size and fragility under mechanical and chemical stresses, the genetic and biochemical characteristics of FDCs remain unclear. Previously, we identified FDCs as ICAM-1⁺ cells in the CD45⁻ non-hematopoietic cell fraction from naïve mouse spleen after cell separation by means of digestion with a combination of enzymes. In the present study, using a new combination of enzymes, we found that FDCs are highly enriched in the CD45⁻ICAM-1⁺CD21/35⁺ cell fraction. CD45⁻ICAM-1⁺CD21/35⁺ cells in the mouse spleen retained an antigen administered in vivo for more than 7 days. Moreover, CD45⁻ICAM-1⁺CD21/35⁺ cells isolated from the spleen of mice administered with a cognate antigen enhanced the survival and proliferation of antigen-specific B cells in vitro. Our improved protocol for the isolation of naïve FDCs will be useful for the analysis of FDCs in vitro and in vivo.     

α1-Antitrypsin modifies general natural killer cell interactions with dendritic cells and specific interactions with islet β-cells in favour of protection from autoimmune diabetes

The autoimmune destruction of pancreatic β-cells is the hallmark of type 1 diabetes (T1D). Failure of anti-CD3 antibodies to provide long-lasting reversal of T1D and the expression of a natural killer (NK) cell ligand on β-cells suggest that NK cells play a role in disease pathogenesis. Indeed, killing of β-cells by NK cells has been shown to occur, mediated by activation of the NK cell activating receptor, NKp46. α1-Antitrypsin (AAT), an anti-inflammatory and immunomodulatory glycoprotein, protects β-cells from injurious immune responses and is currently evaluated as a therapeutic for recent onset T1D. Although isolated T lymphocytes are not inhibited by AAT, dendritic cells (DC) become tolerogenic in its presence and other innate immune cells become less inflammatory. Yet a comprehensive profile of NK cell responses in the presence of AAT has yet to be described. In the present study, we demonstrate that AAT significantly reduces NK cell degranulation against β-cells, albeit in the whole animal and not in isolated NK cell cultures. AAT-treated mice, and not isolated cultured β-cells, exhibited a marked reduction in NKp46 ligand levels on β-cells. In related experiments, AAT-treated DC exhibited reduced inducible DC-expressed interleukin-15 levels and evoked a weaker NK cell response. NK cell depletion in a T1D mouse model resulted in improved β-cell function and survival, similar to the effects observed by AAT treatment alone; nonetheless, the two approaches were non-synergistic. Our data suggest that AAT is a selective immunomodulator that retains pivotal NK cell responses, while diverting their activities away from islet β-cells.     

A possible coagulation-independent mechanism for pregnancy loss involving β(2) glycoprotein 1-dependent antiphospholipid antibodies and CD1d

PROBLEM β(2) glycoprotein1 (β(2) GP1)-dependent antiphospholipid antibodies (aPL) increase the risk for recurrent pregnancy loss. We address whether anti-β(2) GP1 antibodies can interact with phosphatidylserine (PS)-bearing CD1d on trophoblast cells and induce local inflammation. METHODS CD1d-bearing choriocarcinoma cells were used in flow cytometry and immunoprecipitation experiments. CD1d-mediated cytokine induction was assessed using antibody cross-linking. Cytokine production during co-culture of decidual lymphocytes with CD1d-bearing cells was also examined. RESULTS Trophoblast surface-expressed CD1d forms a complex with PS-bound β(2) GP1. Anti-β(2) GP1 mAb cross-linking causes IL12p70 release from CD1d-bearing cells. IL12p70 release from CD1d-bearing trophoblast cells was also induced during co-culture with human decidual lymphocytes. The addition of anti-β2GP1 mAb to co-cultures resulted in a three-fold increase in IL12p70 secretion. IFNγ secretion from decidual lymphocytes was also induced during co-culture with anti-β2GP1 mAbs. CONCLUSIONS β(2) GP1-dependent IL12 release from CD1d-bearing trophoblast in the presence of aPL may link the antiphospholipid syndrome to pregnancy loss via an inflammatory mechanism.     

Activated plasmacytoid dendritic cells regulate type 2 innate lymphoid cell–mediated airway hyperreactivity

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