Chemokine stimulation of human neutrophil [Ca2+]i signaling in biologic environments

Clinical neutrophil (PMN) priming is the net result of multiple stimuli, with intracellular calcium ([Ca2+](i)) being a key second messenger for PMN agonists such as the chemokines, Thus, [Ca2+](i) measurement may be a robust tool for the assessment of global PMN activation. [Ca2+](i) is difficult to measure in complex biologic environments, however, so data in this area are limited. We therefore developed an in vitro system to measure the effects of chemokines on PMN [Ca2+](i). PMN were isolated from volunteer blood. PMN [Ca2+](i) responses to interleukin (IL)-8 and Growth-Related Oncogene (GRO)-alpha were studied by fura-2-acetoxymethyl ester fluorescence with or without reincubation in autologous plasma just prior to study. The effects of IL-8 and GRO-alpha on PMN [Ca2+](i) at ascending doses, with or without plasma reincubation, given sequentially and in the presence or absence of extracellular calcium, were studied. PMN basal [Ca2+](i) was increased by plasma, as were low-dose priming and higher-dose spike responses to IL-8. GRO-alpha caused a more pronounced priming of PMN [Ca2+](i) than IL-8 at low doses, although significantly lower peak responses were observed with GRO-alpha than IL-8 at higher doses. Plasma suppressed both priming and spike responses to GRO-alpha. When given serially at clinically relevant agonist doses, GRO-a was permissive of IL-8 signaling, whereas IL-8 blocked GRO-alpha signaling. IL-8 generates high [Ca2+], spikes using intracellular calcium stores only. GRO-alpha produces lower [Ca2+](i) spikes despite using both intra- and extracellular stores. Plasma preincubation has profound effects on PMN [Ca2+](i) responses to chemokines. These can be measured accurately, as described. In clinically relevant environments, IL-8 and GRO-alpha interact in a regulatory fashion. GRO-alpha may act as a priming agent, with IL-8 activating PMN functions requiring high [Ca2+](i). This cross-cooperation is probably terminated by IL-8 regulation of GRO-alpha activity at the C-X-C chemokine receptor 2.