Dataset Information

Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes.

ABSTRACT:

Background

Activation of human basophils results in the release of many different mediators and the expression of new cell surface proteins. The markers CD63 and CD203c have been used in recent years to assess basophil activation but there have been many studies that demonstrate that expression of these markers can be dissociated from histamine release.

Objective

To determine the signal transduction requirements for CD203c and CD63 expression.

Methods

The current study began by exploring the dependency of CD203c and CD63 expression on protein kinase C (PKC) using known selective inhibitors of PKC.

Results

Between 30 and 300 nm, Ro-31-8220 and bisindoylmaleimide II (Bis II) had no effect on formyl-met-leu-phe- or anti-IgE-induced CD63 or CD203c but enhanced IgE-mediated expression of CD63 by an average of 15-fold at concentrations >1 microm. These results led to the suggestion that these inhibitors altered the normal pathways of degranulation (by a non-PKC dependent mechanism), shifting the normal presence of piecemeal degranulation to the process termed anaphylactic degranulation (AND). Morphological studies demonstrated that concentrations of Ro-31-8220 and Bis II>1 mum dramatically increased the presence of degranulation sacs, a morphological feature of AND.

Conclusion

It is proposed that CD63 expression results from only the AND form of histamine release.

SUBMITTER: MacGlashan D

PROVIDER: S-EPMC2927965 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Publications

Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes.

MacGlashan D D

Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 20100713 9

<h4>Background</h4>Activation of human basophils results in the release of many different mediators and the expression of new cell surface proteins. The markers CD63 and CD203c have been used in recent years to assess basophil activation but there have been many studies that demonstrate that expression of these markers can be dissociated from histamine release.<h4>Objective</h4>To determine the signal transduction requirements for CD203c and CD63 expression.<h4>Methods</h4>The current study bega ...[more]

PMID: 20633031

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Project description:Eosinophil activation leads to secretion of presynthesized, granule-stored mediators that determine the course of allergic, inflammatory, and immunoregulatory responses. CD63, a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) and present on the limiting membranes of eosinophil-specific (secretory) granules, is considered a potential surface marker for eosinophil degranulation. However, the intracellular secretory trafficking of CD63 in eosinophils and other leukocytes is not understood. Here, we provide a comprehensive investigation of CD63 trafficking at high resolution within human eosinophils stimulated with inflammatory stimuli, CCL11 and tumor necrosis factor α, which induce distinctly differing secretory processes in eosinophils: piecemeal degranulation and compound exocytosis, respectively. By using different transmission electron microscopy approaches, including an immunonanogold technique, for enhanced detection of CD63 at subcellular compartments, we identified a major intracellular pool of CD63 that is directly linked to eosinophil degranulation events. Transmission electron microscopy quantitative analyses demonstrated that, in response to stimulation, CD63 is concentrated within granules undergoing secretion by piecemeal degranulation or compound exocytosis and that CD63 tracks with the movements of vesicles and granules in the cytoplasm. Although CD63 was observed at the cell surface after stimulation, immunonanogold electron microscopy revealed that a strong CD63 pool remains in the cytoplasm. It is remarkable that CCL11 and tumor necrosis factor α triggered increased formation of CD63(+) large vesiculotubular carriers (eosinophil sombrero vesicles), which fused with granules in the process of secretion, likely acting in the intracellular translocation of CD63. Altogether, we identified active, intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways. This is important for understanding the complex secretory activities of eosinophils underlying immune responses.

Project description:BackgroundEffector cells assays provide an overall measure of responsiveness to allergen, but the lack of reliable and high-throughput assays limits the clinical utility. We aimed to develop a high-throughput basophil activation test based on human progenitor cell-derived basophils (PCB) and investigate the role of PCB activation test (PCBAT) in allergic diseases.MethodsProgenitor cell-derived basophils were differentiated from CD34+ progenitor cells and sensitized with sera from subjects sensitized to cat, peanut or atopic controls. Sensitized PCBs were stimulated with increasing concentrations of the corresponding allergens in vitro. Degranulation was assessed by measuring CD63 expression using flow cytometry. The correlations between PCBAT and clinical allergy were assessed.ResultsFollowing passive sensitization of the mature PCBs with serum and allergen stimulation, an allergen specific dose-dependent increase in CD63 expression was observed. Sera from subjects sensitized to cat (n = 35, of which 17 subjects had clinical reactivity quantified using inhaled allergen challenge), peanut allergic (n = 30, of which 15 subjects had clinical reactivity validated using double blind, placebo controlled food challenges [DBPCFC]), peanut-sensitized but tolerant subjects (n = 5) were used to sensitize PCBs. PCBAT area under the curve (AUC) correlated with sIgE (r2 = .49, p = .001) in subjects sensitized to cat (sIgE ≥ 0.35KU/L). The provocation concentration of inhaled cat allergen (PC20 ) correlated with PCBAT AUC (r2 = .33, p = .016). In subjects sensitized to peanut, PCBAT AUC was highly correlated with sIgE to Ara h 2 (r2 = .59, p < .0001). Peanut threshold cumulative dose during DBPCFC was negatively correlated with PCBAT AUC (r2 = .57, p = .001) and IgE to Ara h1 (r2 = .55, p = .007), but not with sIgE to whole peanut or Ara h2. All peanut-sensitized but tolerant subjects showed no reaction to peanut on PCBAT.ConclusionProgenitor cell-derived basophils activation test is a high-throughput assay, which correlates with clinical allergy and may confer a powerful alternative tool in allergy testing.

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Dataset Information

Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes.

Background

Objective

Methods

Results

Conclusion

Publications

Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes.

Similar Datasets

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