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HNA antibody‐mediated neutrophil aggregation is dependent on serine protease activity
Author(s) -
Berthold T.,
Schubert N.,
Muschter S.,
Rohr M.,
Wesche J.,
Reil A.,
Bux J.,
Bakchoul T.,
Greinacher A.
Publication year - 2015
Publication title -
vox sanguinis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.68
H-Index - 83
eISSN - 1423-0410
pISSN - 0042-9007
DOI - 10.1111/vox.12292
Subject(s) - proteases , chemistry , serine , antibody , biochemistry , serine protease , neutrophil extracellular traps , iodoacetamide , elastase , tosyl , chymotrypsin , trypsin , protease , enzyme , cysteine , immunology , inflammation , biology , organic chemistry
Background and Objectives 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. Materials and 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 N α ‐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. Conclusion 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.