title: Human neutrophil antigen-3a antibodies induce neutrophil stiffening and conformational activation of CD11b without shedding of L-selectin.
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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: Tom
authors.initials: T
authors.lastname: Berthold
authors.affiliation: Nanostructure Group, ZIK HIKE-Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Glaubitz
authors.affiliation: Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Muschter
authors.affiliation: Department of Cardiology, Universitätsmedizin Greifswald, Greifswald, Germany.|DZHK-German Centre for Cardiovascular Research, Greifswald, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Groß
authors.affiliation: Nanostructure Group, ZIK HIKE-Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany.
authors.forename: Raghavendra
authors.initials: R
authors.lastname: Palankar
authors.affiliation: German Red Cross Blood Service West, Hagen, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Reil
authors.affiliation: Institute of Physics, Ernst-Moritz-Arndt-University Greifswald.
authors.forename: Christiane A
authors.initials: CA
authors.lastname: Helm
authors.affiliation: Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: Tamam
authors.initials: T
authors.lastname: Bakchoul
authors.affiliation: 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.
authors.forename: Hansjörg
authors.initials: H
authors.lastname: Schwertz
authors.affiliation: Ruhr University Bochum, Bochum, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bux
authors.affiliation: Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Greinacher
authors.affiliation: Nanostructure Group, ZIK HIKE-Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Delcea
mesh_terms: D055371:Acute Lung Injury / Q000209:etiology; D039481:CD11b Antigen / Q000737:chemistry / Q000502:physiology*; D002448:Cell Adhesion; D006801:Humans; D007518:Isoantibodies / Q000502:physiology*; D007519:Isoantigens / Q000276:immunology*; D019041:L-Selectin / Q000502:physiology*; D018625:Microscopy, Atomic Force; D009504:Neutrophils / Q000502:physiology*; D011487:Protein Conformation; D065227:Transfusion Reaction
chemical_list: D039481:CD11b Antigen; C518134:HNA-3a antigen, human; C509295:ITGAM protein, human; D007518:Isoantibodies; D007519:Isoantigens; D019041:L-Selectin