CYTOSOLIC FREE CA2- GENERATION AND FUNCTIONAL-ROLE( SIGNALS IN SINGLEADHERENT HUMAN NEUTROPHILS )

To study the role of cytosolic free calcium, [Ca2+]i, in cell activati on, in particular during adhesion and movement on a surface in respons e to chemotactic peptide stimulation and during phagocytosis, we monit ored [Ca2+]i in single human neutrophils. The neutrophils were loaded with fura-2 and allowed to adhere to albumin-coated glass coverslips. [Ca2+], 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 th e chemotactic peptide N-formyl-methionyl-leucylphenylalanine (fMLP). C ell morphology was monitored in parallel by recording fluorescent imag es with a high sensitivity charge coupled device (CCD) camera. The maj ority of the cells studied showed visible changes in shape which start ed either before or at the same time as the onset of the [Ca2+]i trans ients. Removal of extracellular Ca2+ abolished [Ca2+]i transients with out impairing cell movement and spreading. Blockade of adherence and c ell movement with cytochalasin B markedly inhibited [Ca2+]i transients . Monoclonal antibodies directed against the leucocyte integrin CR3 (C D11b/CD18 alpha(m)beta2) blocked adherence, spreading and most of the [Ca2+]i activity. Total [Ca2+]i activity was assessed during phagocyto sis of C3bi-opsonized yeast particles and correlated with fusion of se condary granules with the phagosomal membrane (P-L fusion). In Ca2+-co ntaining medium, upon contact with a yeast particle, a rapid rise in [ Ca2+]i was observed, followed by one or more Ca2+ peaks. P-L fusion wa s detected in 80% of the cells after 5-10 min. Increasing the cytosoli c Ca2+ buffering capacity by loading the cells with MAPT/AM led to a d ose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Un der conditions where basal [Ca2+]i was reduced to < 20 nM and intracel lular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. Taken together thes e results indicate that: 1. The action of leucocyte integrins is neces sary for the generation of the multiple [Ca2+]i transient observed in surface adherent human neutrophils. These [Ca2+]i transients do not pr eclude and can be dissociated from the response to fMLP and they prime the cell to subsequent stimulation. 2. Although the ingestion step of phagocytosis is a Ca2+-independent event, [Ca2+]i transients triggere d upon contact with opsonized particles are necessary to control the s ubsequent fusion of granules with the phagosomal membrane. 3. Studies at a single cell level will be necessary to detect subtle deficiencies in intracellular mediators during human diseases.