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-FcepsilonRI-alpha 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-gamma), 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-gamma) in all subjects, with no significant increases in atopics.
Kon
O M
OM
Imperial College School of Medicine at the National Heart and Lung Institute, London, UK.
Sihra
B S
BS
Till
S J
SJ
Corrigan
C J
CJ
Kay
A B
AB
Grant
J A
JA
D000328:Adult; D000485:Allergens / Q000009:adverse effects; D000911:Antibodies, Monoclonal; D001249:Asthma / Q000209:etiology / Q000276:immunology*; D001491:Basophils / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis; D007371:Interferon-gamma / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D012220:Rhinitis / Q000209:etiology / Q000276:immunology*
D000485:Allergens; D000911:Antibodies, Monoclonal; D015847:Interleukin-4; D007073:Immunoglobulin E; D007371:Interferon-gamma