A rapid activation assay for human eosinophils based on adhesion to immobilized ICAM-1, VCAM-1 and IgG.
Interleukin 5 (IL-5) is a T-cell derived cytokine that induces eosinophil growth and differentiation in both mouse and human bone marrow cultures. Elevated levels of IL-5 as well as eosinophils have been detected in the sputum and Bronchoalveolar lavage (BAL) fluids of asthmatics. Since the recruitment of inflammatory cells to tissues requires the participation of adhesion molecules, we have developed a rapid and sensitive assay to examine the effect of IL-5 and other activation stimuli on eosinophil adhesion to recombinant intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Human recombinant IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), tumour necrosis factor alpha (TNF-alpha), RANTES, MCP-3, C5a, PAF, fMLP, PMA and ConA all induced adhesion of purified eosinophils obtained from normal donors to ICAM-1 and VCAM-1 in a dose and time dependent manner. Adhesion was rapid, within 15 minutes of culture at 37 degrees C, and plateaued within 30 minutes. Activated eosinophils also adhered rapidly to immobilized IgG via the type II Fc gamma receptor (CD32). Analysis of the effect of IL-5 on surface molecule expression by FACS analysis revealed increased expression of CD11b molecules and decreased expression of L-selectin, but no change in the expression of CD11a, CD18, CD29, CD49d and CD32. We also show that Mac-i plays an important role in the regulation of eosinophil activation, since antibodies to CD11b can block IL-5 induced adhesion to IgG and IL-5 induced degranulation.
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Glaxo Wellcome, Cell Biology Unit, Medicines Research Centre, Stevenage, Herts.
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D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis; D002448:Cell Adhesion; D002478:Cells, Cultured; D018914:Cells, Immobilized; D018925:Chemokines / Q000494:pharmacology*; D016207:Cytokines / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000502:physiology; D004804:Eosinophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G; D018799:Intercellular Adhesion Molecule-1; D015848:Interleukin-5 / Q000494:pharmacology; D007700:Kinetics; D019041:L-Selectin / Q000032:analysis; D051379:Mice; D011994:Recombinant Proteins / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018925:Chemokines; D016207:Cytokines; D007074:Immunoglobulin G; D015848:Interleukin-5; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D019041:L-Selectin