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). METHODS: Western blotting, phosphopeptide enrichment and mass spectrometric analyses of samples of neutrophils were performed. RESULTS: Degradation of proteins in neutrophil extracts was observed after preparation with a urea-containing lysis buffer and could be prevented by addition of highly concentrated protease inhibitors. Subsequent tryptic digestion and PolyMAC-based phosphopeptide enrichment proved efficient with accordingly prepared neutrophil samples. Applying the new workflow, formyl­methionyl­leucyl­phenylalanine-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was detected after gel-free and gel-based phosphoproteomic analyses as proof of principle from 20 ml of whole blood. Furthermore, phosphorylation of other ERK1/2 pathway-associated proteins was monitored. CONCLUSION: We provide a workflow for efficient, gel-free phosphoproteome analyses with small-sized neutrophil samples, suitable for application in clinical studies.

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). 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.

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. Thus, the impact of neutrophil priming on HNA-3a antibody-mediated neutrophil aggregation and CD11b surface expression was investigated. STUDY DESIGN AND METHODS: Neutrophils were primed using formyl-methionyl-leucyl-phenylalanine (fMLP) or bacterial lipopolysaccharide (LPS). Granulocyte aggregation and CD11b surface expression were evaluated by the granulocyte agglutination test and by flow cytometry (FC), respectively. Priming-induced changes in the surface expression of choline transporter-like protein 2 (CTL2) and the CTL2 mRNA expression were assessed by FC and quantitative real-time polymerase chain reaction, respectively. RESULTS: Priming of neutrophils lowered the amount of HNA-3a antibodies required for inducing granulocyte aggregation in a dose-dependent manner by 50% to 75%. The priming agent concentration necessary for this response differed between donors. Priming slightly enhanced binding of HNA-3a antibodies to neutrophils. However, CTL2 de novo synthesis was not induced after priming with LPS, indicating that increased HNA-3a antibody binding was likely caused by translocation of intracellular CTL2 to the surface or by increased affinity of HNA-3a antibodies to CTL2. HNA-3a antibodies influenced CD11b surface expression on neutrophils only marginally, which was also not potentiated by priming with fMLP or LPS. CONCLUSION: This study provides experimental evidence supporting the "threshold model" of TRALI. Priming of neutrophils with fMLP or LPS increases their aggregation response to HNA-3a antibodies by lowering the required antibody amount.

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. MATERIALS AND METHODS: We investigated whether HNA-3a-antibody-induced aggregation of polymorphonuclear cells is dependent on plasma factors by using and modifying the granulocyte agglutination test (GAT). RESULTS: Polymorphonuclear cells homozygous for HNA-3a did not aggregate when incubated with HNA-3a antibodies in a plasma-protein-free GAT setup. When the GAT was performed using polymorphonuclear cells re-suspended in phosphate-buffered saline containing proteins, HNA-3-mediated aggregation was observed. Moreover, using Tween® 20 for blocking the plates, reconstituted the granulocyte aggregation in a protein-free medium. This indicates that granulocyte aggregation probably occurs by direct granulocyte-granulocyte interaction(s) or is mediated by substances released by neutrophils after activation. DISCUSSION: Granulocyte aggregation induced by HNA-3a antibodies does not require human plasma proteins. Interindividual variability in the response to HNA-3a antibodies does not depend on differences in patient's plasma proteins.

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. The HNA-3a epitope is conformation sensitive. The additional single-nucleotide polymorphism (SNP) at aa 153 impairs genotyping and lowers the reactivity with some HNA-3a-antibodies. CD177 (HNA-2) interacts with PECAM-1, mediating neutrophil evasion. The percentage of the CD177-positive neutrophil subpopulation and the occurrence of two neutrophil subsets, differing in their CD177 expression, are associated with five SNPs. Glycosylphosphatidylinositol-linked CD177 anchors proteinase 3 on the cell membrane forming a potential signaling complex together with CD11b/CD18 (HNA-4a) in lipid rafts. SUMMARY: Characterization of the HNA-3 system allows identification of blood donors at risk to develop HNA-3-antibodies. FcγRIIIb (HNA-1) and CD177 (HNA-2) seem to be important in bacterial host defense. Activation of neutrophils by HNA-1 and HNA-2-antibodies potentially occurs via mPR3 and CD11b/CD18 (HNA-4a).

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. STUDY DESIGN AND METHODS: For epitope mapping, a library of 23 different HNA-3a (R(154)) and three HNA-3b (Q(154)) peptides covering different parts of the first extracellular loop of CTL2 (aa(55-231)) was synthesized in Escherichia coli and tested by Western blot with two HNA-3a alloantibody-containing plasma samples and by enzyme immunoassay (EIA) with different HNA-3a- (n = 21) and HNA-3b- (n = 1) positive plasma samples. RESULTS: Despite promising Western blot results using highly reactive plasma samples, we found widely varying reactivities of different HNA-3a plasmas in the EIA, with only 11 of 21 HNA-3a antibodies binding to any of the tested HNA-3a peptides and with no peptide recognized by more than nine of the 21 antibodies. The HNA-3b plasma did not react with R(154) peptides in the EIA nor with R(154) or Q(154) peptides in Western blot experiments. Plasma reactivity profiles with the peptides did not correlate with those observed using granulocyte agglutination and granulocyte immunofluorescence tests. CONCLUSION: Binding of HNA-3a alloantibodies depends on the conformation of the intact CTL2 protein and their binding sites may differ substantially. Peptide-based assays for detection of HNA-3a antibodies bear the risk to be insensitive and require systematic validation with a large panel of antibodies.