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HNA Antibody-Mediated Neutrophil Aggregation Is Dependent on Serine Protease Activity

HNA Antibody-Mediated Neutrophil Aggregation Is Dependent on Serine Protease Activity

T Berthold, N Schubert, S Muschter, M Rohr, J Wesche, A Reil, J Bux, T Bakchoul, A Greinacher

Vox Sang. 2015 Nov;109(4):366-74.

PMID: 26084778

Abstract:

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 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.
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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP4272746	Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride		4272-74-6	Price

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