A novel assay to measure the calcium flux in human basophils: effects of chemokines and nerve growth factor.
The calcium flux in human basophils was measured by flow cytometry. Peripheral blood mononuclear cells were labeled with anti-CD123 and anti-HLA-DR antibodies, loaded with fluo-3 acetoxymethyl ester (2 micromol/l) in the presence of probenecid (2.5 micromol/l) and Pluronic F-127 (0.02%) for 20 min, and equilibrated with Ca(2+) (1.8 mmol/l) and Mg(2+) (1 mmol/l) for 5 min. The levels of intracellular free calcium were monitored as changes in fluorescence. Cross-linking of surface IgE on basophils with anti-IgE antibodies caused effective calcium flux in atopic, but not in healthy, donors. Concentration-dependent responses to monocyte chemoattractant protein 1 (MCP-1), eotaxin, macrophage inflammatory protein 1 alpha (MIP-1alpha), and C5a (0.3-10 nmol/l) were observed in all subjects, with a rank order of potency of C5a = MCP-1 > eotaxin > MIP-1alpha. In contrast, the rank order of potency in causing basophil shape change (i.e., increase in forward scatter) was eotaxin > C5a > MCP-1 > MIP-1alpha. Nerve growth factor (NGF; 15 nmol/l) did not induce calcium flux in basophils, and pretreatment of cells with a low concentration of NGF (0.3 nmol/l), which has previously been shown to prime basophils for mediator release, had no effect on the calcium response to subsequent stimulation with C5a. We conclude that calcium mobilization is differentially involved in signaling to chemoattractants in basophils and that it is correlated with the agonist's efficacy to induce mediator release. The data also suggest that priming of basophil responses by NGF does not rely on enhanced calcium mobilization.
Heinemann
Akos
A
Department of Experimental and Clinical Pharmacology, Karl Franzens University, Graz, Austria. akos.heinemann@uni-graz.at
Ofner
Martina
M
Amann
Rainer
R
Peskar
Bernhard A
BA
D001491:Basophils / Q000166:cytology* / Q000276:immunology; D002118:Calcium / Q000032:analysis* / Q000276:immunology; D048429:Cell Size / Q000276:immunology / Q000502:physiology; D018925:Chemokines / Q000276:immunology / Q000502:physiology*; D006801:Humans; D006967:Hypersensitivity / Q000097:blood / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D020932:Nerve Growth Factor / Q000276:immunology / Q000502:physiology*
D018925:Chemokines; D020932:Nerve Growth Factor; D002118:Calcium