CHEMOKINE STIMULATION OF HUMAN NEUTROPHIL [Ca2+]i SIGNALING IN BIOLOGIC ENVIRONMENTS

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Abstract
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)-α 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-a 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-α caused a more pronounced priming of PMN [Ca2+]i than IL-8 at low doses, although significantly lower peak responses were observed with GRO-α than IL-8 at higher doses. Plasma suppressed both priming and spike responses to GRO-α. When given serially at clinically relevant agonist doses, GRO-α was permissive of IL-8 signaling, whereas IL-8 blocked GRO-α signaling. IL-8 generates high [Ca2+]i spikes using intracellular calcium stores only. GRO-α 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-α interact in a regulatory fashion. GRO-α 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-α activity at the C-X-C chemokine receptor 2.

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