BACKGROUND: Stimulated human basophils exhibit different degranulation patterns with release of mediators and appearance of activation markers such as CD63 and CD203c. Traditionally, released mediators are quantified in the supernatant of activated cells, whereas the expression of activation markers by individual cells is analyzed by flow cytometry. Alternatively, intracellular histamine and its release by basophils and mast cells have been repeatedly studied applying an enzyme-affinity-gold method based on the affinity of the histaminase diamine oxidase for its substrate histamine. OBJECTIVE: To develop a flow cytometric technique enabling to study histamine release by individual basophils in combination with the expression of activation markers. To elucidate the principles of basophil degranulation on a single cell level. METHODS: Intracellular histamine and its release is analyzed flow cytometrically by an enzyme-affinity method using diamine oxidase conjugated to laser-excitable fluorochromes. Phenotyping of cells implied flow cytometric quantification of CD63 and CD203c. Stimuli such as allergen, anti-IgE, N-formyl-met-leu-phe (fMLP), phorbol 12-myristate 13-acetate (PMA), ionomycin and interleukin (IL-)3 are applied to obtain different degranulation profiles. RESULTS: Stimulation with anti-IgE, allergen, fMLP and PMA±ionomycin induces a rapid release of histamine that can be analyzed flow cytometrically. Analyses on a single cell level reveal that histamine release is not restricted to cells showing significant up-regulation of CD63. Alternatively, up-regulation of CD203c does not per se indicate histamine release. In some patients, priming of cells with IL-3 not only facilitates basophil responsiveness but also implies an increased ability of DAO to label the cells. CONCLUSION: This study provides the proof-of-concept that histamine and its release can be studied by multicolor flow cytometry on a single cell level (HistaFlow). Coupling the data to simultaneous phenotyping of activated basophils confirms that histamine release principally results from anaphylactic degranulation and in a lesser extent from piecemeal degranulation.
BACKGROUND: Stimulated human basophils exhibit different degranulation patterns with release of mediators and appearance of activation markers such as CD63 and CD203c. Traditionally, released mediators are quantified in the supernatant of activated cells, whereas the expression of activation markers by individual cells is analyzed by flow cytometry. Alternatively, intracellular histamine and its release by basophils and mast cells have been repeatedly studied applying an enzyme-affinity-gold method based on the affinity of the histaminase diamine oxidase for its substrate histamine. OBJECTIVE: To develop a flow cytometric technique enabling to study histamine release by individual basophils in combination with the expression of activation markers. To elucidate the principles of basophil degranulation on a single cell level. METHODS: Intracellular histamine and its release is analyzed flow cytometrically by an enzyme-affinity method using diamine oxidase conjugated to laser-excitable fluorochromes. Phenotyping of cells implied flow cytometric quantification of CD63 and CD203c. Stimuli such as allergen, anti-IgE, N-formyl-met-leu-phe (fMLP), phorbol 12-myristate 13-acetate (PMA), ionomycin and interleukin (IL-)3 are applied to obtain different degranulation profiles. RESULTS: Stimulation with anti-IgE, allergen, fMLP and PMA±ionomycin induces a rapid release of histamine that can be analyzed flow cytometrically. Analyses on a single cell level reveal that histamine release is not restricted to cells showing significant up-regulation of CD63. Alternatively, up-regulation of CD203c does not per se indicate histamine release. In some patients, priming of cells with IL-3 not only facilitates basophil responsiveness but also implies an increased ability of DAO to label the cells. CONCLUSION: This study provides the proof-of-concept that histamine and its release can be studied by multicolor flow cytometry on a single cell level (HistaFlow). Coupling the data to simultaneous phenotyping of activated basophils confirms that histamine release principally results from anaphylactic degranulation and in a lesser extent from piecemeal degranulation.
Authors: Hans Jürgen Hoffmann; Edward F Knol; Martha Ferrer; Lina Mayorga; Vito Sabato; Alexandra F Santos; Bernadette Eberlein; Anna Nopp; Donald MacGlashan Journal: Curr Allergy Asthma Rep Date: 2016-07 Impact factor: 4.806
Authors: Ignacio J Ansotegui; Giovanni Melioli; Giorgio Walter Canonica; Luis Caraballo; Elisa Villa; Motohiro Ebisawa; Giovanni Passalacqua; Eleonora Savi; Didier Ebo; R Maximiliano Gómez; Olga Luengo Sánchez; John J Oppenheimer; Erika Jensen-Jarolim; David A Fischer; Tari Haahtela; Martti Antila; Jean J Bousquet; Victoria Cardona; Wen Chin Chiang; Pascal M Demoly; Lawrence M DuBuske; Marta Ferrer Puga; Roy Gerth van Wijk; Sandra Nora González Díaz; Alexei Gonzalez-Estrada; Edgardo Jares; Ayse Füsun Kalpaklioğlu; Luciana Kase Tanno; Marek L Kowalski; Dennis K Ledford; Olga Patricia Monge Ortega; Mário Morais Almeida; Oliver Pfaar; Lars K Poulsen; Ruby Pawankar; Harald E Renz; Antonino G Romano; Nelson A Rosário Filho; Lanny Rosenwasser; Mario A Sánchez Borges; Enrico Scala; Gian-Enrico Senna; Juan Carlos Sisul; Mimi L K Tang; Bernard Yu-Hor Thong; Rudolf Valenta; Robert A Wood; Torsten Zuberbier Journal: World Allergy Organ J Date: 2020-02-25 Impact factor: 4.084
Authors: Knut Brockow; Bernhard Przybilla; Werner Aberer; Andreas J Bircher; Randolf Brehler; Heinrich Dickel; Thomas Fuchs; Thilo Jakob; Lars Lange; Wolfgang Pfützner; Maja Mockenhaupt; Hagen Ott; Oliver Pfaar; Johannes Ring; Bernhardt Sachs; Helmut Sitter; Axel Trautmann; Regina Treudler; Bettina Wedi; Margitta Worm; Gerda Wurpts; Torsten Zuberbier; Hans F Merk Journal: Allergo J Int Date: 2015