Human neutrophil antigen-3a antibodies induce neutrophil stiffening and conformational activation of CD11b without shedding of L-selectin
Tom Berthold
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
These authors contributed equally to this work.
Search for more papers by this authorMichael Glaubitz
Nanostructure Group, ZIK HIKE–Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
These authors contributed equally to this work.
Search for more papers by this authorStefan Muschter
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
Search for more papers by this authorStefan Groß
Department of Cardiology, Universitätsmedizin Greifswald, Greifswald, Germany
DZHK–German Centre for Cardiovascular Research, Greifswald, Germany
Search for more papers by this authorRaghavendra Palankar
Nanostructure Group, ZIK HIKE–Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorAngelika Reil
German Red Cross Blood Service West, Hagen, Germany
Search for more papers by this authorChristiane A. Helm
Institute of Physics, Ernst-Moritz-Arndt-University Greifswald
Search for more papers by this authorTamam Bakchoul
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
Search for more papers by this authorHansjörg Schwertz
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
Lichtenberg-Professor for Experimental Hemostasis, Universitätsmedizin Greifswald
Program in Molecular Medicine, University of Utah, Salt Lake City, Utah
Department of Surgery, University of Utah, Salt Lake City, Utah
Search for more papers by this authorCorresponding Author
Andreas Greinacher
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
Address reprint requests to: Prof. Dr. med. Andreas Greinacher, Institut für Immunologie und Transfusionsmedizin, Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany; e-mail: [email protected].Search for more papers by this authorMihaela Delcea
Nanostructure Group, ZIK HIKE–Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorTom Berthold
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
These authors contributed equally to this work.
Search for more papers by this authorMichael Glaubitz
Nanostructure Group, ZIK HIKE–Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
These authors contributed equally to this work.
Search for more papers by this authorStefan Muschter
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
Search for more papers by this authorStefan Groß
Department of Cardiology, Universitätsmedizin Greifswald, Greifswald, Germany
DZHK–German Centre for Cardiovascular Research, Greifswald, Germany
Search for more papers by this authorRaghavendra Palankar
Nanostructure Group, ZIK HIKE–Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorAngelika Reil
German Red Cross Blood Service West, Hagen, Germany
Search for more papers by this authorChristiane A. Helm
Institute of Physics, Ernst-Moritz-Arndt-University Greifswald
Search for more papers by this authorTamam Bakchoul
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
Search for more papers by this authorHansjörg Schwertz
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
Lichtenberg-Professor for Experimental Hemostasis, Universitätsmedizin Greifswald
Program in Molecular Medicine, University of Utah, Salt Lake City, Utah
Department of Surgery, University of Utah, Salt Lake City, Utah
Search for more papers by this authorCorresponding Author
Andreas Greinacher
Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany
Address reprint requests to: Prof. Dr. med. Andreas Greinacher, Institut für Immunologie und Transfusionsmedizin, Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany; e-mail: [email protected].Search for more papers by this authorMihaela Delcea
Nanostructure Group, ZIK HIKE–Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany
Search for more papers by this authorThis work was supported by an unrestricted grant from the Red Cross Blood Donation Service West, Hagen, Germany; by the Land Mecklenburg-Vorpommern, Exzellenzinitiative UG 07-064, and by the Germany's Ministry of Education and Research (BMBF), Innovation Center–Humoral Immune Reactions in Cardiovascular Diseases (ZIK-HIKE) FKZ 03Z2CN11 and FKZ 03Z2CN12; by the Forschungsverbund Molekulare Medizin of the University Greifswald (FOMM 2012-01); by the Deutsche Forschungsgemeinschaft (Grant GR 2232/7-1); and by the Volkswagen Stiftung (Lichtenberg Professorship to HS).
Abstract
BACKGROUND
HNA-3a antibodies induce severe transfusion-related acute lung injury (TRALI) in which neutrophils play a major role. As neutrophil passage through the pulmonary microvasculature is a critical step in the pathogenesis of TRALI, we investigated the impact of HNA-3a antibodies on two important factors that could impair granulocyte passage through lung capillaries: the elasticity of neutrophils and the expression and activation of adhesion molecules.
STUDY DESIGN AND METHODS
The impact of HNA-3a antibodies on the elasticity of neutrophils was investigated using atomic force microscopy (AFM). Neutrophils were settled on poly-2-hydroxyethyl-methacrylate–coated glass slides before treatment with anti-HNA-3a plasma samples, control plasma, or control plasma containing formyl-methionyl-leucyl-phenylalanine (fMLP). Elasticity measurements were carried out in a temperature-controlled perfusion chamber using an atomic force microscopy (AFM) device. The impact of HNA-3a antibodies on the surface expression of total CD11b, activation of CD11b, and L-selectin (CD62L) shedding was investigated by flow cytometry. The functional impact of HNA-3a antibodies on neutrophil adhesion was assessed using fibrinogen-coated plates.
RESULTS
HNA-3a antibodies induced stiffening of neutrophils (+24%-40%; p < 0.05) to a similar extent as fMLP. This effect was blocked by treatment of neutrophils with cytochalasin D. While total surface expression of CD11b and L-selectin on neutrophils was largely unaffected, HNA-3a antibodies induced alloantigen-specific activation of CD11b (+72%-107%; p < 0.05) and increased adhesion of neutrophils to fibrinogen.
CONCLUSION
Accumulation of neutrophils in the pulmonary microvasculature during severe TRALI is likely mediated by increased rigidity and CD11b-mediated adhesion of neutrophils leading to retention of neutrophils.
Supporting Information
Additional Supporting Information may be found in the online version of this article at the publisher's website:
| Filename | Description |
|---|---|
| trf13299-sup-0001-suppinfofs1.pdf34.2 KB | Fig. S1. Detection of adhesion molecules by flow cytometry. Characteristic histogram showing the detection of total CD11b (A), L-selectin (B), activated CD11b (C) and the respective isotype controls. Total CD11b and L-selectin staining was conducted using monoclonal anti-CD11b-PE-Cy5 (ICRF44, BD Bioscience) and CD62L-PE-Cy5 (BD Bioscience), respectively. Activated CD11b on the surface of neutrophils was detected using the monoclonal antibody CBRM1/5 (Biolegend) and FITC-conjugated anti-mouse IgG (aMo-IgG-FITC, Dako). Isotype staining was performed using matched mouse-IgG (Nordic-MUbio). |
| trf13299-sup-0002-suppinfotbl.doc30 KB |
Table S1. Averaged Young's Moduli E of the control groups. Mean Young's moduli E with standard error range (SElower bound - SEupper bound) of neutrophils homozygous for HNA-3a or HNA-3b corresponding to different control groups used for normalization of the AFM data (Fig. 4). The Young's moduli E of PBS-treated neutrophils (HNA-3a/a) were used for normalization of fMLP-treated neutrophils during the validation stage. Young's moduli obtained after treatment of neutrophils (HNA-3a/a) with P1 (n = 4), P2 (n = 5) and P3 (n = 5) were normalized to E values of neutrophils treated with control plasma (C) only. E values obtained from neutrophils in the presence of CD (P1 and C+fMLP treatment) were normalized to Young's moduli of plasma C-treated cells that were previously exposed to CD. Measurement of CD treated cells expectably resulted in lower values of E compared to measurements under normal conditions. Young's moduli of HNA-3b homozygous neutrophils treated with P1 and C+fMLP were normalized to E values of same neutrophils treated with control plasma C only. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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