interleukin-8 and Antiphospholipid-Syndrome

The role of inflammation in thrombotic complications of primary antiphospholipid syndrome (PAPS) is controversial. The aim of this study was to evaluate levels of inflammation and coagulation markers in patients with thrombotic PAPS (t-PAPS). Patients with t-PAPS and individuals with no history of thrombosis were enrolled. The association of t-PAPS with levels of tumor necrosis factor (TNF)-α, C-reactive protein (hs-CRP), interferon (IFN)-α, interleukins (IL)-6, -8, factor VIII (FVIII), von Willebrand factor (VWF) and tissue factor (TF) was evaluated by regression models. The levels of these markers were also compared between controls and subgroups of t-PAPS patients with triple positivity, recently diagnosed thrombosis, recurrent thrombosis and venous thrombosis. Patients with t-PAPS (n = 101) had a 8.6-fold increased levels of TNF-α, 90% increased levels of hs-CRP, 80% increased levels of IL-6, 30% increased levels of FVIIIAg, 50% increased levels of VWF and 66% increased levels of TF as compared to controls (n = 131), and the differences did not change after adjustments for sex, age and cardiovascular risk factors. Inflammatory markers were elevated in t-PAPS regardless of the aPL profile, number of previous thrombosis or time elapsed since diagnosis. TNF-α and IL-8 levels were higher in t-PAPS patients with venous thrombosis, in comparison with those with arterial thrombosis and controls. Patients with t-PAPS presented with increased levels of inflammatory and coagulation markers, which suggests that t-PAPS is associated not only with hypercoagulability but also with a persistent inflammatory state.

Topics: Adult; Antiphospholipid Syndrome; Biomarkers; Blood Coagulation; C-Reactive Protein; Case-Control Studies; Factor VIII; Female; Humans; Inflammation; Interferon-alpha; Interleukin-6; Interleukin-8; Male; Middle Aged; Thrombosis; von Willebrand Factor

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by recurrent thromboembolism and/or pregnancy morbidity in the presence of Antiphospholipid antibodies, mainly anti-β2 glycoprotein I (anti-β2GPI). The autoantibodies lead to monocyte and endothelial cell activation and subsequent secretion of tissue factor (F3) and proinflammatory cytokines, like interleukins 6 (IL6) and 8 (IL8). The etiology of the syndrome remains largely unknown, with the contribution of environmental, genetic and epigenetic factors considered significant.. We aimed to identify epigenetic changes and factors potentially implicated in the pathophysiology of APS. To this end, we compared DNA methylation levels of the IL8 and F3 genes between healthy donors (HDs) and APS patients, using whole blood as a source.. Methylation was significantly reduced in the IL8 promoter and significantly increased in the F3 gene body in APS patients compared to HDs and correlated with specific clinical parameters. In an ex vivo model partially mimicking APS, stimulation of monocytes with a mixture of β2GPI, anti-β2GPI and CXCL4 also induces DNA methylation changes in the above genes, along with increase of their expression. Stimulation of human umbilical vein endothelial cells (HUVECs) with the same mixture also results in transcriptional upregulation of epigenetic factors, including MΕCP2, DNMT3, TET1, HDAC9 and ARID5B.. The above data support that epigenetic alterations could be implicated in the pathophysiology of APS and prompt further investigation of their potential diagnostic or therapeutic utility.

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Cells, Cultured; CpG Islands; DNA Methylation; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-8; Promoter Regions, Genetic; Thromboplastin

While diabetes and APS are individually associated with increased risk of poor perinatal outcomes, in particular preeclampsia, recent studies have demonstrated an association between concurrent aPL and diabetes leading to an increased risk of pregnancy morbidity. Hyperglycemia and aPL have independently been shown to alter human trophoblast function by inducing a pro-inflammatory, anti-angiogenic, and antimigratory response. However, little is known about the effects of concurrent hyperglycemia and aPL on trophoblast function.. A human first-trimester extravillous trophoblast cell line was exposed to glucose at 5 mmol/L (normoglycemia) or 25 mmol/L (hyperglycemia), all in the presence or absence of low-dose aPL or control IgG. For some experiments, the TLR4 antagonist, LPS-RS, was included. Cell culture supernatants were measured for inflammatory IL-1β and IL-8, and angiogenic PlGF, sFlt-1, and sEndoglin by ELISA. Inflammasome-associated uric acid was measured using a bioassay; caspase-1 was measured using an activity assay. Trophoblast migration was quantified using a two-chamber colorimetric assay.. Compared to excess glucose alone, combination excess glucose and low-dose aPL (a) further augmented trophoblast inflammatory IL-1β, inflammasome-associated uric acid and caspase-1, and pro-angiogenic PlGF; (b) dampened trophoblast inflammatory IL-8, anti-angiogenic sEndoglin, and sFlt-1; and (c) further reduced trophoblast migration.. Our findings indicate that while concurrent aPL and hyperglycemia are overall detrimental to trophoblast function, the presence of two simultaneous insults triggers some protective effects.

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Cell Line; Cell Movement; Diabetes Mellitus; Endoglin; Female; Glucose; Humans; Hyperglycemia; Interleukin-1beta; Interleukin-8; Membrane Proteins; Pregnancy; Pregnancy Complications; Toll-Like Receptor 4; Trophoblasts; Vascular Endothelial Growth Factor Receptor-1

What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation?. aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8.. Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated.. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20).. Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was measured by ELISA.. aPL significantly increased trophoblast cellular and exosome expression of miR-146a-5p, miR-146a-3p, miR-155 and miR-210. aPL-induced up-regulation of trophoblast miR-146a-5p, miR-146a-3p and miR-210, but not miR-155, was inhibited by the TLR4 antagonist, LPS-RS. While inhibition or overexpression of miR-146a-5p had no effect on aPL-induced trophoblast IL-8 secretion, miR-146a-3p inhibition significantly reduced this response. aPL-induced trophoblast IL-8 secretion was inhibited by the presence of the TLR8-DN. In the absence of aPL, transfection of trophoblast cells with a miR-146a-3p mimic significantly increased IL-8 secretion and this was inhibited by the presence of the TLR8-DN. Patients with aPL and adverse pregnancy outcomes (APOs) expressed significantly higher levels of circulating miR-146a-3p compared with healthy pregnant controls with no pregnancy complications (P < 0.05).. While the enrichment of miR-146a-3p in trophoblast-derived exosomes support the role of this miR acting in a paracrine or endocrine manner through exosome delivery, this has not been demonstrated. However, miR-146a-3p may also exert its pro-inflammatory effect intracellularly within the same trophoblast cell targeted by aPL.. These findings provide a novel mechanism of trophoblast inflammation through miRs activating RNA-sensing receptors. Furthermore, circulating exosomal-associated miR-146a-3p in APS patients may serve clinically as a biomarker for related APOs.. This study was supported in part by grants from the American Heart Association (#10GRNT3640032 to V.M.A.), the March of Dimes Foundation (Gene Discovery and Translational Research Grant #6-FY12-255 to V.M.A.), NICHD, NIH (R01HD049446 to V.M.A.), the Gina M. Finzi Memorial Student Summer Fellowship from the Lupus Foundation of America (to S.M.G.), and the Yale University School of Medicine Medical Student Fellowship (to S.M.G.). The authors declare no competing financial interests.. N/A.

Topics: Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Female; Humans; Interleukin-8; MicroRNAs; Pregnancy; Pregnancy Trimester, First; Toll-Like Receptor 4; Toll-Like Receptor 8; Trophoblasts

Our previous study demonstrated that Toll-like receptor 4 (TLR4) could act as a co-receptor with annexin A2 (ANX2) mediating anti-β2-glycoprotein I/β2- glycoprotein I (anti-β2GPI/β2GPI) -induced tissue factor (TF) expression in human acute monocytic leukaemia cell line THP-1. In the current study, we further explored the roles of TLR4 and its adaptors, MD-2 and MyD88, as well as nuclear factor kappa B (NF-κB), in anti-β2GPI/β2GPI-induced the activation of THP-1 cells, especially on the expression of some proinflammatory molecules. The results showed that treatment of THP-1 cells with anti-β2GPI (10μg/ml)/β2GPI (100μg/ml) complex could increase IL-6 (interleukin-6), IL-8 (interleukin-8) as well as TNF-α (tumor necrosis factor alpha) expression (both mRNA and protein levels). These effects could be blocked by addition of TAK-242 (5μM), a blocker of signaling transduction mediated by the intracellular domain of TLR4, and also by NF-κB inhibitor PDTC (20μM). Overall, our results indicate that anti-β2GPI/β2GPI complex induced IL-6, IL-8 and TNF-α expression involving TLR4/MD-2/MyD88 and NF-κB signaling pathways and this might be associated with pathological mechanisms of antiphospholipid syndrome (APS).

Topics: Antigen-Antibody Complex; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cell Line; Humans; Interleukin-6; Interleukin-8; Lymphocyte Antigen 96; Myeloid Differentiation Factor 88; NF-kappa B; Phosphorylation; Signal Transduction; Toll-Like Receptor 4

The anti-phospholipid syndrome (APS) is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL). aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN) and Toll-like receptors (TLR) to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonist LPS monitoring neutrophil effector functions, namely the oxidative burst, phagocytosis, L-Selectin shedding and IL-8 production. aPL alone were only able to induce minor activation of PMN effector functions at high concentrations. However, in the additional presence of LPS the activation threshold was markedly lower indicating a synergistic activation pathway of aPL and TLR in PMN. In summary, our results indicate that PMN effector functions are directly activated by aPL and boosted by the additional presence of microbial products. This highlights a role for PMN as important innate immune effector cells that contribute to the pathophysiology of APS.

Topics: Antiphospholipid Syndrome; Apoptosis; CD11b Antigen; Flow Cytometry; Humans; Interleukin-8; L-Selectin; Lipopolysaccharides; Neutrophil Activation; Neutrophils; Phagocytosis; Respiratory Burst; Signal Transduction; Toll-Like Receptor 4

It has been shown that stimulation of endothelial cells and monocytes by antiphospholipid antibodies leads to a prothrombotic state involving upregulation of tissue factor (TF). We examined the in vitro effects of IgG fractions from patients with antiphospholipid syndrome (APS) and of a beta-2-glycoprotein 1-independent human monoclonal antiphospholipid antibody (HL-5B) on human umbilical vein endothelial cells (HUVEC) in comparison to untreated cell controls and to exposure to monoclonal IgG control antibody. We also examined the effect of recombinant monocyte chemoattractant protein-1 (MCP-1) on peripheral blood monocytes. Stimulation of endothelial cells with APS IgG fractions or HL-5B resulted in time-dependent upregulation of MCP-1 mRNA and protein expression. Stimulation with HL-5B also led to time-dependent upregulation of interleukin (IL)-8 and intracellular adhesion molecule-1 (ICAM-1) mRNA and IL-8 protein expressions. Stimulation of monocytes with recombinant MCP-1 resulted in an upregulation of TF mRNA and TF protein. In conclusion these results might represent a mechanism for antiphospholipid antibody-mediated thrombosis in APS patients.

Topics: Antibodies, Antiphospholipid; Antibodies, Monoclonal; Antiphospholipid Syndrome; Cells, Cultured; Chemokine CCL2; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Immunoblotting; Immunoglobulin G; Intercellular Adhesion Molecule-1; Interleukin-8; Monocytes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Time Factors; Umbilical Veins

To determine the effects of primary antiphospholipid syndrome (PAPS)-derived anti-beta(2)GPI antibodies on gene expression in human umbilical vein endothelial cells (HUVEC) by gene profiling using microarrays.. Anti-beta(2)GPI antibodies purified from sera of patients with PAPS or control IgG isolated from normal subjects were incubated with HUVEC for 4 h before isolation of RNA and processing for hybridisation to Affymetrix Human Genome U133A-2.0 arrays. Data were analysed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. For selected genes microarray data were confirmed by real-time PCR analysis or at the protein level by ELISA.. A total of 101 genes were found to be upregulated and 14 genes were downregulated twofold or more in response to anti-beta(2)GPI antibodies. A number of novel genes not previously associated with APS were induced, including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL-18 receptor 1, and growth factors CSF2, CSF3 IL-6, IL1beta and FGF18. The majority of downregulated genes were transcription factors/signalling molecules including ID2. Quantitative real-time RT-PCR analysis confirmed the microarray results for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C).. This study reveals a complex gene expression response in HUVEC to anti-beta(2)GPI antibodies with multiple chemokines, pro-inflammatory cytokines, pro-thrombotic and pro-adhesive genes regulated by these antibodies in vitro. Some of these newly identified anti-beta(2)GPI antibody-regulated genes could contribute to the vasculopathy associated with this disease.

Topics: Adult; Antigen-Antibody Reactions; Antiphospholipid Syndrome; Autoantibodies; beta 2-Glycoprotein I; Case-Control Studies; Down-Regulation; E-Selectin; Endothelial Cells; Female; Gene Expression Profiling; Humans; Immunoglobulin G; Interleukin-8; Middle Aged; Oligonucleotide Array Sequence Analysis

Platelet factor 4 heparin enzyme immunoassay, platelet aggregation test, and serotonin release assay are commonly used to diagnose and confirm heparin-induced thrombocytopenia. We describe a case of recurrent thrombocytopenia appearing in a few hours after each heparin administration and who tested negative for the three assays. Further analysis revealed anti-interleukin (IL)-8 antibodies and IL-8-dependent platelet activation facilitated by heparin, which may explain this unusual case of heparin-induced thrombocytopenia.

Topics: Anticoagulants; Antiphospholipid Syndrome; Female; Heparin; Humans; Immunoenzyme Techniques; Interleukin-8; Middle Aged; Platelet Activation; Platelet Count; Thrombocytopenia