Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes
D. MacGlashan Jr
Johns Hopkins Asthma and Allergy Center, Baltimore, MD, USA
Search for more papers by this authorD. MacGlashan Jr
Johns Hopkins Asthma and Allergy Center, Baltimore, MD, USA
Search for more papers by this authorSummary
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 μm. 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 μm 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.
Cite this as: D. MacGlashan Jr, Clinical & Experimental Allergy, 2010 (40) 1365–1377.
Supporting Information
Figure S1. Panel A (n=3); kinetics of PMA-stimulated CD63 and CD203c expression. Basophils were stimulated with 50 ng/mL PMA and expression of CD63 and CD203c assessed by flow cytometry, (▪) CD63, (○) CD203c.
Figure S2. Example of images of degranulation sac formation during stimulation of basophils with FMLP. The image series follows a small group of cells labelled with fura-2. The sequence flows from left to right and top to bottom with intervals of approximately 10 s, the upper left representing 10 s after FMLP addition.
Figure S3. Relationship between the cytosolic calcium response and degranulation sac formation at the single cell level. The plot shows results from one preparation of basophils, labelled with fura-2 and stimulated with anti-IgE Ab @ 0.5 μg/mL. Degranulation sacs and the cytosolic calcium response for individual cells were measured as described in the methods. Each symbol represents the results for a single cell. For this particular plot, the Spearman rank correlation was 0.550.
Figure S4. Cytosolic calcium response in basophils with and without PKC inhibitor Ro-31-8220. Basophils were pre-incubated with vehicle control (DMSO) or Ro-31-8220 at 3 μm for 10 min prior to stimulation with anti-IgE Ab at 0.5 μg/mL. The traces represent the average of 3 experiments; for the control response (○), the gray region surrounding the primary trace represents the ±SEM for the averaged kinetic curve. For the response in the presence of Ro-31-8220 (•), the error bars represent ±SEM.
Table S1. Survey of the characteristics of the human basophil response under various conditions of activation. For the purposes of comparison, the three dominant methods of assessing basophil activation, histamine release, CD63 and CD203c expression, are examined as three groups, group I = histamine release, group II = CD63 expression, group III = CD203c expression. In other studies, group II and group III may contain other expression markers such as CD107a, CD107b, CD13, CD69, and CD164. In this table, there are 4 major categories of the response that differentiate the three compartments from each other. These categories are highlighted with different levels of gray background. * = determined in this study, § = normally IL-3 does not induce histamine release but it may in selected patients (see reference (8)), °‡ = supra-optimal polyclonal anti-IgE has unique characteristics, see text.
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