Human neutrophil antigen-3a antibodies induce neutrophil stiffening and conformational activation of CD11b without shedding of L-selectin

• Published in: Transfusion (Philadelphia, Pa.) Vol. 55; no. 12; pp. 2939 - 2948
• Main Authors: Berthold, Tom, Glaubitz, Michael, Muschter, Stefan, Groß, Stefan, Palankar, Raghavendra, Reil, Angelika, Helm, Christiane A., Bakchoul, Tamam, Schwertz, Hansjörg, Bux, Jürgen, Greinacher, Andreas, Delcea, Mihaela
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.12.2015; Wiley Subscription Services, Inc
• Subjects: Acute Lung Injury - etiology; CD11b Antigen - chemistry; CD11b Antigen - physiology; Cell Adhesion; Humans; Immunoglobulins; Isoantibodies - physiology; Isoantigens - immunology; L-Selectin - physiology; Microscopy; Microscopy, Atomic Force; Neutrophils; Neutrophils - physiology; Protein Conformation; Rodents; Transfusion Reaction
• ISSN: 0041-1132; 1537-2995
• DOI: 10.1111/trf.13299

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.