Mass spectrometric phosphoproteome analysis of small-sized samples of human neutrophils.

Background: Global analysis of stimulus-dependent changes in the neutrophil phosphoproteome will improve the understanding of neutrophil signal transduction and function in diverse disease settings. However, gel-free phosphoproteomics of neutrophils in clinical studies is hampered by limited sample amounts and requires protein extract stability, efficient tryptic digestion and sensitive phosphopeptide enrichment in a protease-rich environment. For development of an appropriate workflow, we assessed neutrophil protein stability in urea-based lysis buffers and determined feasibility of gel-free phosphoproteomic analyses using polymer-based metal ion affinity capture (PolyMAC).

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

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).

Impact of priming on the response of neutrophils to human neutrophil alloantigen-3a antibodies.

Background: Human neutrophil alloantigen-3a (HNA-3a) antibodies can induce transfusion-related acute lung injury (TRALI). The severity of TRALI varies largely among the affected patients. Severe comorbidity seems to increase the susceptibility for TRALI, potentially by priming of neutrophils.

Human neutrophil antigen-3a antibodies induce neutrophil aggregation in a plasma-free medium.

Background: Antibodies against the human neutrophil alloantigen-3a (HNA-3a) are involved in severe cases of transfusion-related acute lung injury (TRALI), but the susceptibility of patients towards HNA-3a antibody differs largely. HNA-3a antibodies induce granulocyte aggregation. However, it is unresolved whether plasma proteins are required for granulocyte aggregation.

Developments in the definition and clinical impact of human neutrophil antigens.

Purpose Of Review: This review summarizes the current genetic, molecular and functional information on human neutrophil alloantigens (HNAs), which are implicated in autoimmune and alloimmune neutropenia and in transfusion-related acute lung injury. Identification and functional characterization of these antigens improve the understanding of HNA-antibody-induced diseases and may lead to the development of antibody detection assays and new therapeutic concepts.

Recent Findings: HNA-3 is localized on choline transporter-like protein 2 (CTL2) and originates from an Arg154Gln amino acid (aa) substitution.

Epitope mapping of antibodies directed against the human neutrophil alloantigen 3a.

Background: Severe transfusion-related acute lung injury is often caused by antibodies directed against the human neutrophil alloantigen (HNA)-3a. HNA-3a results from an amino acid exchange (Arg154Gln) in the first extracellular loop of the choline transporter-like protein 2 (CTL2). The characteristics of the binding domain(s) of HNA-3a antibodies are unknown.