CHEMOKINE STIMULATION OF HUMAN NEUTROPHIL [CA2+](I) SIGNALING IN BIOLOGIC ENVIRONMENTS

Clinical neutrophil (PMN) priming is the net result of multiple stimul i, 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, howeve r, so data in this area are limited. We therefore developed an in vitr o system to measure the effects of chemokines on PMN [Ca2+](i). PMN we re isolated from volunteer blood. PMN [Ca2+](i) responses to interleuk in (IL)-8 and Growth-Related Oncogene (GRO)-alpha were studied by fura -2-acetoxymethyl ester fluorescence with or without reincubation in au tologous plasma just prior to study. The effects of IL-8 and GRO-alpha on PMN [Ca2+](i) at ascending doses, with or without plasma reincubat ion, given sequentially and in the presence or absence of extracellula r 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 l ow doses, although significantly lower peak responses were observed wi th GRO-alpha than IL-8 at higher doses. Plasma suppressed both priming and spike responses to GRO-alpha. When given serially at clinically r elevant 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. Plasm a preincubation has profound effects on PMN [Ca2+](i) responses to che mokines. These can be measured accurately, as described. In clinically relevant environments, IL-8 and GRO-alpha interact in a regulatory fa shion. 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 chem okine receptor 2.