Combined analysis of intracellular signalling and immunophenotype of human peripheral blood basophils by flow cytometry: a proof of concept

• Published in: Clinical and experimental allergy Vol. 37; no. 11; pp. 1668 - 1675
• Main Authors: Ebo, D. G., Dombrecht, E. J., Bridts, C. H., Aerts, N. E., De Clerck, L. S., Stevens, W. J.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.11.2007; Blackwell
• Subjects: Allergic diseases; allergy; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - pharmacology; Antigens, CD - immunology; Antigens, CD - metabolism; basophil activation; Basophils - chemistry; Basophils - drug effects; Basophils - metabolism; Biological and medical sciences; CD63; Cell Membrane Permeability - drug effects; Dose-Response Relationship, Drug; flow cytometry; Flow Cytometry - methods; Fluorescent Antibody Technique, Direct - methods; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; IgE; IL-3; Imidazoles - pharmacology; Immunopathology; Immunophenotyping - methods; Interleukin-3 - pharmacology; intracellular signalling; Kinetics; Latex - pharmacology; Medical sciences; p38 MAPK; p38 Mitogen-Activated Protein Kinases - antagonists & inhibitors; p38 Mitogen-Activated Protein Kinases - immunology; p38 Mitogen-Activated Protein Kinases - metabolism; Platelet Membrane Glycoproteins - immunology; Platelet Membrane Glycoproteins - metabolism; Protein Kinase Inhibitors - pharmacology; Pyridines - pharmacology; Receptor, Epidermal Growth Factor - agonists; Receptor, Epidermal Growth Factor - immunology; Reproducibility of Results; Saponins - pharmacology; Sarcoidosis. Granulomatous diseases of unproved etiology. Connective tissue diseases. Elastic tissue diseases. Vasculitis; Signal Transduction - physiology; Temperature; Tetraspanin 30; Up-Regulation - drug effects; Human; Allergy; Flow cytometry; Immunopathology; CD63; Immunophenotype; IgE; Cytokine; Granulocyte; intracellular signalling; Activation; IL-3; p38 MAPK; Basophil; Blood flow; Signal transduction; Immunology; Interleukin 3; basophil activation; p38 Mitogen activated protein kinase; Hemodynamics; Intracellular
• ISSN: 0954-7894; 1365-2222
• DOI: 10.1111/j.1365-2222.2007.02819.x

Summary Background The signal transduction pathways and control mechanisms involved in IgE‐mediated basophil activation remain incompletely understood. Objectives To investigate whether basophilic intracellular signal transduction and immunophenotype can be analysed simultaneously by flow cytometry. Methods Basophils in whole blood were stimulated with anti‐IgE and latex antigen at various concentrations and during different time courses. Phosphorylation of p38 mitogen‐activated protein kinase (MAPK) as a representative of the intracellular signal transduction pathway and surface expression of CD63 was assessed simultaneously flow cytometrically. The effect of pre‐incubation with IL‐3 was assessed. Results Stimulation of the basophils with anti‐IgE and allergen induces a rapid phosphorylation of p38 MAPK that peaks between 1 and 5 min and returns to baseline levels after 60 min. In contrast, CD63 up‐regulation demonstrates a maximal but more continuous expression that peaks approximately 5 min later than phosphorylation of p38 MAPK. Specific inhibition of p38 MAPK reduced or almost completely abrogated up‐regulation of CD63. Pre‐incubation of the basophils with IL‐3 produces a rapid p38 MAPK phosphorylation over basal levels, but this was weaker and shorter than for anti‐IgE stimulation. Pre‐incubation of the basophils with IL‐3 did not potentiate anti‐IgE‐induced phosphorylation of p38 MAPK and did affect spontaneous or IgE‐mediated CD63 up‐regulation. Conclusions This study provides the proof that the flow cytometer allows an integrated analysis of basophilic intracellular signalling and immunophenotyping. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy. Capsule summary This study is the first to provide evidence for a combined analysis of basophilic intracellular signalling and immunophenotyping by flow cytometry. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy.