Membrane potential and intracellular cyclic AMP as regulators of calcium homeostasis in formyl peptide-activated human neutrophils : lessons from chronic granulomatous disease

Abstract

Neutrophils playa key role in the systemic inflammatory response which may lead to serious tissue injury and multiple organ dysfunction. In this setting, activated neutrophils, largely in response to tumour necrosis factor-alpha (TNF-α), secrete reactive oxidants, granule proteases and bioactive lipids, as well as pro-inflammatory cytokines, emphasising the importance of these cells as targets for anti-inflammatory therapies. There are, however, only a few currently available agents that directly modulate neutrophil pro-inflammatory responses in clinical practice, with corticosteroids being relatively ineffective against these cells. Although, the anti-inflammatory potential of cAMP-elevating agents has been recognised, the exact molecular/biochemical mechanisms which underlie the anti-inflammatory actions of epinephrine and related β-agonists with neutrophils, have not been established. Epinephrine treatment of neutrophils resulted in increased intracellular cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, further supporting a cAMP-mediated effect. Although epinephrine did not affect the release of Ca2+ from neutrophil intracellular stores, the rate of clearance of cytosolic Ca2+ was accelerated by this agent. In the setting of decreased efflux and a reduction in store-operated influx of Ca2+, these effects of epinephrine are compatible with enhancement of the cAMP-dependent Ca2+ sequestering/resequestering endo-membrane Ca2+-ATPase. Epinephrine therefore down-regulates the pro-inflammatory activation of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca2+. Comparison of the effects of 4 selective (fenoterol, formoterol, salbutamol and salmeterol) and 3 non-selective (epinephrine, norepinephrine and isoproterenol) β-adrenoreceptor agonists, on the pro-inflammatory activities of human neutrophils, demonstrated that the agents tested clearly differ with respect to anti-inflammatory potential. Epinephrine, isoproterenol, fenoterol and formoterol significantly increased intracellular concentrations of cAMP in neutrophils, an activity which was paralleled by inhibition of the production of reactive oxidants and release of elastase from FMLP-activated cells. Salbutamol and salmeterol on the other hand, did not cause significant suppression of the pro-inflammatory activities of these cells. The effect of norepinephrine was intermediate between these two groups. The inhibitory effects of βagonists are mediated via β2-adrenergic receptors on the neutrophil membrane. The relationship between activation of NAOPH oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from 4 subjects with chronic granulomatous disease (CGO). Activation of CGO neutrophils was accompanied by a prolonged increase in cytosolic Ca2+, occurring in the setting of trivial membrane depolarisation and accelerated influx of Ca2+. This was associated with hyperactivity of the cells with excessive elastase release, which was attenuated by the type 4 phosphodiesterase inhibitor, rolipram. These findings support the involvement of NAOPH oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses, and granuloma formation, which are characteristic of CGO. Store-operated influx of Ca2+ into activated neutrophils is stringently regulated, presumably to prevent hyperactivation of the cells. The major contributors to this physiologic, anti-inflammatory process are NAOPH oxidase which, by its membrane depolarising actions excludes extracellular Ca2+, and the plasma membrane and endomembrane Ca2+-ATPases, which mediate clearance of store-derived cation. Subsequent influx of the cation, through store-operated Ca2+ channels is controlled by the relatively slow, restraining, membrane repolarising action of the Na+/Ca2+ exchanger, enabling efficient diversion of incoming cation into stores.