Unstimulated basophils in atopic and nonatopic subjects express intracellular interleukin-4: Detection by flow cytometry

Background: IgE-stimulated cultured basophils from atopic subjects are capable of secreting interleukin-4 (IL-4). We describe a flow-cytometric technique which identified intracellular IL-4 in unstimulated basophils unseparated from peripheral blood mononuclear cells (PBMC) in both atopic (AT) and nonatopic (NC) volunteers. Methods: Freshly isolated PBMC were fixed in 4% paraformaldehyde (PFA). Surface staining with 22E7, a noncompetitive anti-Fc(ε)RI-α antibody, allowed identification of basophils. Permeabilization by 0.1% saponin allowed staining of intracellular cytokines with specific monoclonal antibodies (mAbs). Two series of experiments utilizing different protocols and anticytokine mAbs were performed. The first protocol required a two-stage fluorochrome staining technique. The availability of fluorochrome- conjugated mAbs allowed a simpler, one-stage labelling ProCedUre for the second protocol. Results: With the first protocol, IL-4 (but not 1FN-γ), immunoreactivity was detectable in a majority (median 77%) of peripheral blood basophils from both AT and NC subjects (n=8). Basophil IL-4 immunoreactivity was again evident in experiment 2 but did not differ significantly between AT and NC subjects - either evaluated as percentage of IL-4+ basophils (AT median=66%, NC median=38.4%, P=0.41) or IL-4-specific mean fluorescence (AT median=0.85, NC median=0.3, P=0.07). Conclusions: This simple technique allowed the study of intracellular cytokine expression in unstimulated blood basophils. It demonstrated constitutive basophil expression of IL-4 (but not IFN-γ) in all subjects, with no significant increases in atopics.