sq-23377 and methionyl-leucyl-phenylalanine

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.. 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.. 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.. 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.. 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.

Topics: Adolescent; Adult; Allergens; Amine Oxidase (Copper-Containing); Antibodies, Anti-Idiotypic; Basophil Degranulation Test; Basophils; Child; Female; Flow Cytometry; Histamine; Histamine Release; Humans; Interleukin-3; Ionomycin; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Phorbol Esters; Phosphoric Diester Hydrolases; Pyrophosphatases; Tetraspanin 30; Young Adult

The generation of superoxide anion and release of granule contents are essential to the bactericidal function of neutrophils, but may also contribute to host tissue damage during inflammation. In previous studies (J. Immunol. 146:2388), we have demonstrated that the acute phase reactant alpha-1-antichymotrypsin (ACT), a potent inhibitor of the serine protease cathepsin G, also suppresses superoxide anion generation. The inhibitory effect of ACT was not directly linked to its antiproteolytic activity and may reflect interaction at a site other than its reactive loop. To further characterize the mechanism of inhibition, we investigated the direct effects of ACT on the NADPH oxidase enzyme complex and the signaling pathways that regulate motivation of the respiratory burst. We present evidence that ACT does not intefer with agonist-stimulated calcium mobilization or translocation and activity of protein kinase C. ACT was an effective inhibitor of superoxide anion generation in membrane preparations isolated from PMA-activated cells. These results support the notion that ACT is acting on a component of the active assembled NADPH oxidase complex. Thus, ACT may have an important role in regulation of specific aspects of the inflammatory processes and the modulation of toxic oxygen-based host tissue damage.

Topics: alpha 1-Antichymotrypsin; Arachidonic Acid; Calcium; Cell Membrane; Concanavalin A; Humans; In Vitro Techniques; Ionomycin; N-Formylmethionine Leucyl-Phenylalanine; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Protein Kinase C; Respiratory Burst; Signal Transduction; Superoxides; Tetradecanoylphorbol Acetate; Time Factors