Konstantinos Kambas1, Akrivi Chrysanthopoulou1, Dimitrios Vassilopoulos2, Eirini Apostolidou1, Panagiotis Skendros3, Andreas Girod4, Stella Arelaki1, Marios Froudarakis5, Lydia Nakopoulou6, Alexandra Giatromanolaki7, Prodromos Sidiropoulos8, Maria Koffa9, Dimitrios T Boumpas10, Konstantinos Ritis11, Ioannis Mitroulis12. 1. Laboratory of Molecular Haematology, Democritus University of Thrace, Alexandroupolis, Greece. 2. Second Department of Medicine, Athens University School of Medicine, Hippokration General Hospital, Athens, Greece. 3. First Department of Internal Medicine, University General Hospital of Alexandroupolis, Alexandroupolis, Greece. 4. Life Sciences Research Unit-FSTC, University of Luxembourg, Walferdange, Luxembourg. 5. Department of Pneumonology, University General Hospital of Alexandroupolis, Alexandroupolis, Greece. 6. First Department of Pathology, Medical School, University of Athens, Athens, Greece. 7. Department of Pathology, University General Hospital of Alexandroupolis, Alexandroupolis, Greece. 8. Department of Rheumatology, Clinical Immunology and Allergy, University Hospital, Medical School, University of Crete, Heraklion, Greece. 9. Laboratory of Cellular and Molecular Biology, Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece. 10. Third Department of Internal Medicine, National University of Athens Medical School, Sotiria Hospital, Athens, Greece Biomedical Research Foundation of Academy of Athens, Centre for Immunology and Transplantations, Athens, Greece. 11. Laboratory of Molecular Haematology, Democritus University of Thrace, Alexandroupolis, Greece First Department of Internal Medicine, University General Hospital of Alexandroupolis, Alexandroupolis, Greece. 12. Laboratory of Molecular Haematology, Democritus University of Thrace, Alexandroupolis, Greece First Department of Internal Medicine, University General Hospital of Alexandroupolis, Alexandroupolis, Greece Department of Internal Medicine III, Division of Vascular Inflammation, Diabetes and Kidney, University Clinic Carl-Gustav-Carus, University of Dresden, Dresden, Germany.
Abstract
OBJECTIVES: Antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is characterised by neutrophil activation. An elevated prevalence of venous thromboembolic events has been reported in AAV. Because of the critical role of neutrophils in inflammation associated thrombosis, we asked whether neutrophil tissue factor (TF) may be implicated in the thrombotic diathesis in AAV. METHODS: Neutrophils from four patients and sera from 17 patients with ANCA associated vasculitis with active disease and remission were studied. TF expression was assessed by immunoblotting and confocal microscopy. Circulating DNA levels were evaluated. TF expressing microparticles (MPs) were measured by flow cytometry and thrombin-antithrombin complex levels by ELISA. RESULTS: Peripheral blood neutrophils from four patients with active disease expressed elevated TF levels and released TF expressing neutrophil extracellular traps (NETs) and MPs. TF positive NETs were released by neutrophils isolated from the bronchoalveolar lavage and were detected in nasal and renal biopsy specimens. Elevated levels of circulating DNA and TF expressing neutrophil derived MPs were further observed in sera from patients with active disease. Induction of remission attenuated the aforementioned effects. Control neutrophils treated with sera from patients with active disease released TF bearing NETs and MPs which were abolished after IgG depletion. Treatment of control neutrophils with isolated IgG from sera from patients with active disease also resulted in the release of TF bearing NETs. TF implication in MP dependent thrombin generation was demonstrated by antibody neutralisation studies. CONCLUSIONS: Expression of TF in NETs and neutrophil derived MPs proposes a novel mechanism for the induction of thrombosis and inflammation in active AAV. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
OBJECTIVES: Antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is characterised by neutrophil activation. An elevated prevalence of venous thromboembolic events has been reported in AAV. Because of the critical role of neutrophils in inflammation associated thrombosis, we asked whether neutrophil tissue factor (TF) may be implicated in the thrombotic diathesis in AAV. METHODS: Neutrophils from four patients and sera from 17 patients with ANCA associated vasculitis with active disease and remission were studied. TF expression was assessed by immunoblotting and confocal microscopy. Circulating DNA levels were evaluated. TF expressing microparticles (MPs) were measured by flow cytometry and thrombin-antithrombin complex levels by ELISA. RESULTS: Peripheral blood neutrophils from four patients with active disease expressed elevated TF levels and released TF expressing neutrophil extracellular traps (NETs) and MPs. TF positive NETs were released by neutrophils isolated from the bronchoalveolar lavage and were detected in nasal and renal biopsy specimens. Elevated levels of circulating DNA and TF expressing neutrophil derived MPs were further observed in sera from patients with active disease. Induction of remission attenuated the aforementioned effects. Control neutrophils treated with sera from patients with active disease released TF bearing NETs and MPs which were abolished after IgG depletion. Treatment of control neutrophils with isolated IgG from sera from patients with active disease also resulted in the release of TF bearing NETs. TF implication in MP dependent thrombin generation was demonstrated by antibody neutralisation studies. CONCLUSIONS: Expression of TF in NETs and neutrophil derived MPs proposes a novel mechanism for the induction of thrombosis and inflammation in active AAV. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
Authors: Santhosh V R Kumar; Onkar P Kulkarni; Shrikant R Mulay; Murthy N Darisipudi; Simone Romoli; Dana Thomasova; Christina R Scherbaum; Bernd Hohenstein; Christian Hugo; Susanna Müller; Helen Liapis; Hans-Joachim Anders Journal: J Am Soc Nephrol Date: 2015-02-02 Impact factor: 10.121