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-cytometr ic technique which identified intracellular IL-4 in unstimulated basop hils unseparated from peripheral blood mononuclear cells (PBMC) in bot h atopic (AT) and nonatopic (NC) volunteers. Methods Freshly Isolated PBMC were fixed in 3% paraformaldehyde (PFA). Surface staining with 22 E7, a noncompetitive anti-Fc epsilon RI-alpha antibody, allowed identi fication of basophils. Permeabilization by 0.1% saponin allowed staini ng of intracellular cytokines with specific monoclonal antibodies (mAb s). Two series of experiments utilizing different protocols and anticy tokine mAbs were performed. The first protocol required a two-stage fl uorochrome staining technique. The availability of fluorochrome-conjug ated mAbs allowed a simpler, one-stage labelling procedure for the sec ond protocol. Results With the first protocol, IL-4 (but not IFN-gamma ), immunoreactivity was detectable in a majority (median 77%) of perip heral blood basophils from both AT and NC subjects (n=8). Basophil IL- 4 immunoreactivity was again evident in experiment 2 but did not diffe r significantly between AT and NC subjects - either evaluated as perce ntage of IL-4+ basophils (AT median=66%, NC median=38.4%, P=0.41) or I L-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-gamma) in all su bjects, with no significant increases in atopics.