CLINICAL IMMUNOLOGY doi: 10.1111/j.1365-3083.2011.02600.x .................................................................................................................................................................. The Isotype of Autoantibodies Influences the Phagocytosis of Antibody-Coated Platelets in Autoimmune Thrombocytopenic Purpura M. Hoemberg* , D. Stahlà§, P. Schlenke*, W. Sibrowski*, U. Pachmann– & U. Cassens** Abstract *Institute of Transfusion Medicine and Autoimmune thrombocytopenic purpura (AITP) is an acquired autoimmune Transplantation Immunology, University bleeding disorder, characterized by isolated thrombocytopenia because of Hospital Muenster, Germany; Department of destruction of auto-antibody-coated platelets by Fc-receptor-mediated phagocy- Pediatric Oncology and Hematology, University Children’s Hospital, Cologne, Germany; tosis. The destruction of autoantibody-sensitized platelets by FccR-bearing àUniversity Clinic for Blood Group Serology phagocytic cells and the following antigen presentation are considered to play and Transfusion Medicine, Paracelsus Medical a key role for the pathophysiology of AITP. Although different isotypes of University, Salzburg, Austria; §Haema AG, AITP-mediating autoantibodies, e.g. IgG, IgM and IgA, are frequently found Leipzig, Germany; –Center of Transfusion in AITP patients, their role in the pathophysiology of AITP remains unclear. Medicine, Bayreuth, Germany; and **Institute of Transfusion Medicine, Laboratory Medicine Using a flow cytometric monocyte-based phagocytosis assay, we investigated and Medical Microbiology, Klinikum Dortmund, the impact of disease-associated autoantibody isotype in antibody-mediated Dortmund, Germany phagocytosis of platelets. Platelets, labelled with 5-chloromethyl fluorescein diacetate (CMFDA), were incubated with AITP patients’ serum characterized by pure IgG or IgM antiplatelet autoantibodies. Labelled platelets were incu- Received 23 November 2010; Accepted in bated with monocytes. Phagocytosis was defined as the product of percentage revised form 9 June 2011 of CMFDA-positive monocytes and mean fluorescence intensity of CMFDA. Correspondence to: M. Hoemberg, Institute of Adherence of platelets to monocytes was quantified by anti-CD61-PerCp in a Transfusion Medicine and Transplantation CMFDA+ CD14+ gate. IgG-coated platelets showed a significantly higher Immunology, University Hospital Muenster, phagocytic index than IgM-coated platelets (mean 796 ± 157 versus Domagkstrasse 11, D-48149 Mu¨nster, Germany. 539 ± 78, P < 0.01). There were no significant differences regarding platelet E-mail: [email protected] adherence to monocytes. The isotype of autoantibodies influences the quantity of in vitro phagocytosis of autologous platelets by monocytes. Therefore, the AITP-mediating autoantibody isotype should be considered more carefully in pathophysiologic models and furthermore in diagnostic, therapeutic and prog- nostic approaches in AITP. affected by acute AITP, while adults usually suffer from Introduction chronic courses of thrombocytopenia [1, 6]. The onset of Autoimmune thrombocytopenic purpura (AITP) is an acute AITP in children is often 2–3 weeks following an acquired autoimmune bleeding disorder, characterized by immunological event like immunization or a viral infec- isolated thrombocytopenia because of destruction of tion, and the disease is often self-limiting. In contrast, autoantibody-coated platelets by Fc-receptor-mediated adult AITP has usually no preceding event, shows more phagocytosis in the reticuloendothelial system [1] and often an insidious onset and has a chronic course [6].T suppression of platelet production [2, 3]. Patients usually cell-related and cytokine abnormalities could be shown in develop mucocutaneous bleeding signs. In rare cases, life- chronic AITP [7, 8]. Acute and chronic AITP, although threatening events like intracranial haemorrhage may both are immune mediated, might be different disease occur [4]. The annual ITP incidence in adults has been entities with different underlying pathophysiologic mech- estimated at 3.2 per 10.000 [5]. With respect to the clin- anisms [9]. ical onset and the duration of symptoms, AITP is classi- The diagnosis of AITP remains one of exclusion [1]. fied in an acute (<6-month duration) and a chronic (more Usually, secondary forms (e.g. in association with sys- than 6 months) disorder. Children are more frequently temic lupus erythematosus or lymphoproliferative Ó 2011 The Authors Scandinavian Journal of Immunology Ó 2011 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 74, 489–495 489 490 Isotype of Autoantibody Influences Phagocytosis in AITP M. Hoemberg et al. .................................................................................................................................................................. disorders) should be excluded. No clinical signs or labo- Little is known about the pathophysiological differ- ratory findings that may lead to a different diagnosis like ences of AITP-mediating autoantibody isotype and the marked splenomegaly, altered bone marrow aspiration corresponding Fc receptor classes for phagocytosis of anti- samples, blood count or blood smear apart from throm- body-coated platelets, but they might be of high rele- bocytopenia are found. Several assays were designed to vance for a better understanding of the disease. It is detect anti-platelet antibodies and show a sensitivity of surprising that although much work has been carried out 49–66% and an estimated specificity of 78–92% [1, 10, to identify the IgG receptor-subclass and the AITP-medi- 11]. Though, a negative test result cannot rule out the ating autoantibody IgG subclasses, little work has been diagnosis of AITP [12]. addressed to clarify the role of the disease-mediating Current diagnostic procedures usually focus on detec- autoantibody isotype. tion of IgG antibodies and the platelet-specific glycopro- Therefore, we used the model of AITP to investigate tein target of the disease-mediating antibody. Concerning the impact of disease-mediating antibody isotype on diagnostic and pathophysiological aspects of AITP, immune phagocytosis of autologous platelets. autoantibodies of the IgM class and other isotypes of antiplatelet antibodies are often neglected. Material and methods The principal aim in the treatment of AITP is to raise platelet count to prevent the patient from severe and life- Patients and controls. Plasma samples from 11 adult threatening bleeding. Because bleeding in AITP patients is patients with AITP were obtained. Additionally, immu- rare and platelet function is preserved, many patients do noglobulin from the platelet membrane of eight AITP not need treatment. The risk of bleeding should face the patients could be eluted by acid elution technique as side effects and toxicity of treatment. Corticosteroids and used in standard procedures [20]. Material obtained by IVIg remain the most common first-line treatment for the latter procedure is referred to as eluates. All patients AITP. Anti-rhesus antibody (anti-D) is also effective in presented with clinical bleeding signs because of throm- Rh-D-positive individuals. Splenectomy might be consid- bocytopenia at the time point of investigation, and the ered for adult patients with chronic AITP. Rituximab, aza- diagnosis of AITP was made on the basis of verification thioprine, cyclosporin, cyclophosphamide, mycophenolate of platelet-reactive autoantibodies. None of the patients mofetil and etanercept are also used in AITP treatment, had received treatment prior to the collection of blood usually as second-line treatment [4]. New treatment samples. No information was available on other clinical options as thrombopoietin receptor agonists, monoclonal diagnoses. Healthy adults were enrolled as controls. Stud- anti-Fcc antibodies and inhibitors of syk kinase are cur- ies were approved by the ethics commission at the Uni- rently evaluated [13, 14]. versity of Muenster, Germany. Many concepts for the immune pathophysiology of Immunohaematological and biochemical investiga- AITP put the destruction of autoantibody-sensitized tions. Autoantibody isotype in plasma and eluate samples platelets by FccR-bearing phagocytic cells in the centre of AITP patients was determined by the platelet immu- of interest [1, 6, 15]. At least three classes of FccRs are nofluorescence test [20]. Results were confirmed using expressed on phagocytic effector cells in the reticuloendo- the monoclonal antibody-specific immobilization of plate- thelial system. High-affinity activating FccRI binds let antigens method in case of IgG antibodies [21] and monomeric and immune-complexed IgG, while low-affin- by commercially available enzyme-linked immunosorbent ity activating receptors FccRIIA and FccRIIIA bind assay (GTI, Brookfield, WI, USA) in the case of IgM only immune-complexed IgG. Additionally, inhibitory antibodies. The ELISA was performed according to the FccRIIB has recently been found [15]. In 1986, Clarkson manufacturer’s specifications with the exception that we et al. [16] showed that blocking of the receptors leads to used anti-IgM as secondary antibody instead of a mixture reduced platelet destruction in AITP patients. Further- of anti-IgM, anti-IgG and anti-IgA. Anti-human leuko- more, FccR polymorphisms that can significantly alter cyte antigen (HLA) antibodies in plasma were excluded antibody binding are significantly overexpressed in a pop- by the lymphocyte cytotoxicity assay [20]. The concentra- ulation with childhood