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

Authors


O. M. Kon Allergy and Clinical Immunology Imperial College School of Medicine at the National Heart and Lung Institute Dovehouse Street London SW3 6LY UK

Abstract

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-a 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 IFN-y), 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 lL-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 lL-4 (but not IFN-y) in all subjects, with no significant increases in atopies.

Ancillary