Unstimulated basophils in atopic and nonatopic subjects express intracellular interleukin-4

Detection by flow cytometry

O. M. Kon, B. S. Sihra, S. J. Till, C. J. Corrigan, A. B. Kay, J. A. Grant

Research output: Contribution to journalArticle

14 Citations (Scopus)

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(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.

Original languageEnglish (US)
Pages (from-to)891-896
Number of pages6
JournalAllergy: European Journal of Allergy and Clinical Immunology
Volume53
Issue number9
StatePublished - 1998

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Basophils
Interleukin-4
Flow Cytometry
Monoclonal Antibodies
Staining and Labeling
Fluorescent Dyes
Blood Cells
Cytokines
Saponins
Immunoglobulin E
Volunteers
Fluorescence
Antibodies

Keywords

  • Basophils
  • Cytokines
  • Flow cytometry
  • Interleukin-4

ASJC Scopus subject areas

  • Immunology

Cite this

Unstimulated basophils in atopic and nonatopic subjects express intracellular interleukin-4 : Detection by flow cytometry. / Kon, O. M.; Sihra, B. S.; Till, S. J.; Corrigan, C. J.; Kay, A. B.; Grant, J. A.

In: Allergy: European Journal of Allergy and Clinical Immunology, Vol. 53, No. 9, 1998, p. 891-896.

Research output: Contribution to journalArticle

Kon, O. M. ; Sihra, B. S. ; Till, S. J. ; Corrigan, C. J. ; Kay, A. B. ; Grant, J. A. / Unstimulated basophils in atopic and nonatopic subjects express intracellular interleukin-4 : Detection by flow cytometry. In: Allergy: European Journal of Allergy and Clinical Immunology. 1998 ; Vol. 53, No. 9. pp. 891-896.
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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(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.",
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AU - Sihra, B. S.

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AU - Corrigan, C. J.

AU - Kay, A. B.

AU - Grant, J. A.

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N2 - 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.

AB - 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.

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