Flow cytometry reveals different lag times in rapid cytoplasmic calcium elevations in human neutrophils in response to N-formyl peptide.
Flow cytometric analyses were performed to study intracellular single-cell calcium transients ([Ca2+]i) in suspended human neutrophils during the initial phase of N-formyl peptide stimulation. Thereby, two neutrophil populations became apparent. Early maximally Ca(2+)-responding (high fluorescence) neutrophils and not-yet Ca(2+)-responding (low fluorescence) neutrophils, but no neutrophils with intermediate levels of [Ca2+]i were detected. Within 7 s the number of low fluorescence neutrophils decreased and the number of high fluorescence neutrophils increased maximally. This suggests that [Ca2+]i transients occurred abruptly in individual neutrophils within a time interval below 1 s. At lower N-formyl peptide concentrations the lag times of individual neutrophils and the interval time of maximal activation of the [Ca2+]i-responding neutrophil population increased, however the percentage of [Ca2+]i-responding cells decreased. Surprisingly, no influence of the N-formyl peptide concentration on the [Ca2+]i-induced fluorescence signal of the individual cell was observed: it was always in an almost maximal range or not responding. In parallel, binding studies performed with fluorescein-labeled N-formyl peptide revealed that the heterogeneity of [Ca2+]i-responding cells cannot be explained by different receptor occupancy. In summary, this study demonstrates that [Ca2+]i transients induced by N-formyl peptides in suspended individual human neutrophils occur very rapidly in an almost "all-or-none manner" and that the mean increasing fluorescence signal of a calcium indicator within a whole neutrophil population results from varying lag times of the individual cells, rather than from the mean simultaneous progress of many cells.
Elsner
J
J
Department of Dermatology, Albert Ludwigs University of Freiburg, Germany.
Norgauer
J
J
Dobos
G J
GJ
Emmendörffer
A
A
Schöpf
E
E
Kapp
A
A
Roesler
J
J
D002118:Calcium / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors
D009240:N-Formylmethionine Leucyl-Phenylalanine; D002118:Calcium