title: HNA antibody-mediated neutrophil aggregation is dependent on serine protease activity.
abstract: OBJECTIVE
  Transfusion-related acute lung injury (TRALI) is often caused by antibodies against human neutrophil alloantigen-2 (HNA-2) and HNA-3a. Neutrophil aggregation is considered as a major cause of TRALI, but little is known about how HNA antibodies initiate this process. We explored mechanisms involved in neutrophil aggregation induced by HNA-2 and HNA-3a antibodies.
  
  
  METHODS
  Isolated neutrophils were pretreated with broad-spectrum or specific inhibitors against different cell functions or proteases. Granulocyte agglutination test (GAT) was performed with serially diluted anti-HNA-2 and anti-HNA-3a plasmas or control plasma, and reactivity was evaluated microscopically. Reactive oxygen species (ROS) production in neutrophils was investigated using a lucigenin-based chemiluminescence assay.
  
  
  RESULTS
  HNA-2 and HNA-3a antibody-mediated neutrophil aggregation was inhibited by pretreatment with formaldehyde, iodoacetamide and the serine protease inhibitors Pefabloc-SC, N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Nα-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK). In contrast, inhibition of actin polymerization, respiratory burst, cysteine proteases, metalloproteases or aspartic proteases did not affect neutrophil aggregation. Furthermore, HNA-3a antibodies did not directly cause ROS production in neutrophils.
  
  
  CONCLUSIONS
  Aggregation of neutrophils induced by HNA-2 and HNA-3a antibodies is an active process and depends on trypsin- or chymotrypsin-like serine proteases but is not dependent on the production of ROS. These findings may open new prospects for the pharmacologic prevention of neutrophil-associated acute lung injury.
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Berthold
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Schubert
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Muschter
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Rohr
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Wesche
authors.affiliation: Deutsches Rotes Kreuz - Blutspendedienst West, Hagen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Reil
authors.affiliation: Ruhr Universität Bochum, Bochum, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Bux
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Bakchoul
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Greinacher
mesh_terms: D000371:Agglutination; D058851:GPI-Linked Proteins / Q000276:immunology; D006801:Humans; D007519:Isoantigens / Q000276:immunology*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000276:immunology*; D011956:Receptors, Cell Surface / Q000276:immunology*; D057057:Serine Proteases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology
chemical_list: C407242:CD177 protein, human; D058851:GPI-Linked Proteins; C518134:HNA-3a antigen, human; D007519:Isoantigens; D011956:Receptors, Cell Surface; D015842:Serine Proteinase Inhibitors; D057057:Serine Proteases
keywords: HNA-2; HNA-3a; TRALI; aggregation; inhibitors; neutrophil