Cytosolic free Ca2+ signals in single adherent human neutrophils: Generation and functional role

Abstract

To study the role of cytosolic free calcium, [Ca2+]i, in cell activation, in particular during adhesion and movement on a surface in response to chemotactic peptide stimulation and during phagocytosis, we monitored [Ca2+]i in single human neutrophils. The neutrophils were loaded with fura-2 and allowed to adhere to albumin-coated glass coverslips. [Ca2+]i was monitored with a dual excitation microfluorimeter. Half of the cells showed spontaneous [Ca2+]i transients that lasted up to 15 min with an amplitude averaging 77 +/- 10 nM above basal levels (mean basal value of 110 +/- 20 nM) and a mean duration of 28 +/- 5 s. These repetitive [Ca2+]i elevations depended on the continuous presence of extracellular Ca2+ and could be dissociated from those triggered by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Cell morphology was monitored in parallel by recording fluorescent images with a high sensitivity charge coupled device (CCD) camera. The majority of the cells studied showed visible changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. Removal of extracellular Ca2+ abolished [Ca2+]i transients without impairing cell movement and spreading. Blockade of adherence and cell movement with cytochalasin B markedly inhibited [Ca2+]i transients. Monoclonal antibodies directed against the leucocyte integrin CR3 (CD11b/CD18 alpha m beta 2) blocked adherence, spreading and most of the [Ca2+]i activity. Total [Ca2+]i activity was assessed during phagocytosis of C3bi-opsonized yeast particles and correlated with fusion of secondary granules with the phagosomal membrane (P-L fusion).(ABSTRACT TRUNCATED AT 250 WORDS)