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-leucylphenylalanine (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 α_mβ₂) 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). In Ca²⁺-containing 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 was detected in 80% of the cells after 5–10 min. Increasing the cytosolic Ca²⁺ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca²⁺]_i elevations and P-L fusion. Under conditions where basal [Ca²⁺]_i was reduced to <20 nM and intracellular Ca²⁺ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. Taken together these results indicate that: 1. The action of leucocyte integrins is necessary for the generation of the multiple [Ca²⁺]_i transient observed in surface adherent human neutrophils. These [Ca²⁺]_i transients do not preclude 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 triggered upon contact with opsonized particles are necessary to control the subsequent 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.