Elsevier

Cellular Signalling

Volume 18, Issue 12, December 2006, Pages 2302-2313
Cellular Signalling

Inhibition of K+ efflux prevents mitochondrial dysfunction, and suppresses caspase-3-, apoptosis-inducing factor-, and endonuclease G-mediated constitutive apoptosis in human neutrophils

https://doi.org/10.1016/j.cellsig.2006.05.013Get rights and content

Abstract

Neutrophils die rapidly via apoptosis and their survival is contingent upon rescue from constitutive programmed cell death by signals from the microenvironment. In these experiments, we investigated whether prevention of K+ efflux could affect the apoptotic machinery in human neutrophils. Disruption of the natural K+ electrochemical gradient suppressed neutrophil apoptosis (assessed by annexin V binding, nuclear DNA content and nucleosomal DNA fragmentation) and prolonged cell survival within 24–48 h of culture. High extracellular K+ (10–100 mM) did not activate extracellular signal-regulated kinase (ERK) and Akt, nor affected phosphorylation of p38 MAPK associated with constitutive apoptosis. Consistently, pharmacological blockade of ERK kinase or phosphatidylinositol 3-kinase (PI 3-kinase) did not affect the anti-apoptotic action of KCl. Inhibition of K+ efflux effectively reduced, though never completely inhibited, decreases in mitochondrial transmembrane potential (ΔΨm) that preceded development of apoptotic morphology. Changes in ΔΨm resulted in attenuation of cytochrome c release from mitochondria into the cytosol and decreases in caspase-3 activity. Culture of neutrophils in medium containing 80 mM KCl with the pan-caspase inhibitor Z-VAD-FMK resulted in slightly greater suppression of apoptosis than KCl alone. High extracellular KCl also attenuated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) from mitochondria to nuclei. The DNase inhibitor, aurintricarboxylic acid (ATA) partially inhibited nucleosomal DNA fragmentation, and the effects of ATA and 80 mM KCl were not additive. These results show that prevention of K+ efflux promotes neutrophil survival by suppressing apoptosis through preventing mitochondrial dysfunction and release of the pro-apoptotic proteins cytochrome c, AIF and EndoG independent of ERK, PI 3-kinase and p38 MAPK. Thus, K+ released locally from damaged cells may function as a survival signal for neutrophils.

Introduction

Neutrophil granulocytes play a pivotal role in innate immunity. Their excessive accumulation, activation and prolonged survival also contribute to tissue damage. Recruitment of neutrophils into tissues during inflammation is intimately linked to their activation and survival. Mature neutrophils have the shortest life span among leukocytes, and die rapidly via apoptosis in vitro, and apparently, in vivo [1], [2], [3], [4]. This constitutively expressed program may promote removal of neutrophils from inflamed areas by scavenger macrophages [5] with minimal damage to the surrounding tissue, thereby facilitating the resolution of inflammation [1], [6]. The fate of neutrophils can, however, be influenced within the inflammatory microenvironment by bacterial products [2], [4], [7] and inflammatory mediators [2], [4], [8], [9]. Markedly suppressed neutrophil apoptosis has been detected in patients with inflammatory diseases, including acute respiratory distress syndrome [10] and sepsis [11].

Loss of intracellular potassium ions and the accompanying cell shrinkage are characteristic features of apoptosis [12], [13], [14]. Potassium ion loss is necessary for maximal caspase activation [15] and may be critical for the progression of the cell death program [16]. Conversely, inhibition of K+ efflux from Jurkat T lymphocytes or neurons triggered to undergo apoptosis (e.g. by engagement of the Fas receptor or exposure to amyloid) is sufficient to delay apoptosis [12], [15], [17], [18], [19], [20] presumably through inhibition cytochrome c-dependent formation of the apoptosome [18], [21]. In other apoptotic models, high extracellular K+ had no effect in preventing cell death [22], [23], suggesting cell-specific differences in K+ sensitivity. Alterations in intracellular potassium regulate cell volume, activation, migration and bactericidal activity of human neutrophils [24], [25], [26], [27]. However, the role of potassium in the regulation of neutrophil apoptosis has not been defined, yet it may be critical for the optimal expression and resolution of inflammation. Here we report that inhibition of K+ efflux rescues human neutrophils from the constitutively expressed apoptotic program, resulting in prolonged survival. We also show that maintenance of normal intracellular K+ concentration prevents collapse of mitochondrial transmembrane potential, and inhibits both caspase-dependent and independent death pathways. Indeed, K+ effectively reduced cytochrome c release and subsequent activation of caspase-3 as well as translocation of endonuclease G (EndoG) and apoptosis-inducing factor (AIF) from mitochondria to nuclei, thereby inhibiting nucleosomal DNA fragmentation. These actions were independent of activation of the MAPK/ERK kinase and phosphatidylinositol 3-kinase/Akt signaling pathways.

Section snippets

Isolation and culture of neutrophils

Neutrophils were isolated from venous blood of healthy volunteers (male and female, 23–54 years), who had not taken any drugs for at least 14 days before the experiments [9]. The Clinical Research Committee of the Maisonneuve-Rosemont Hospital approved the protocol. Neutrophils (5 × 106 cells/ml, purity > 96%, viability > 98%) were resuspended in HBSS (140 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, 0.6 mM MgCl2, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 4.2 mM NaHCO3 and 5 mM glucose, pH 7.4) supplemented with 10%

Prevention of K+ efflux prolongs neutrophil survival by delaying apoptosis

As expected, control (untreated) isolated neutrophils developed prominent features of apoptosis, including loss of membrane asymmetry (assessed by annexin V binding), diminution in cell volume, chromatin condensation, and internucleosomal cleavage of DNA, resulting in hypoploid nuclei, within 24–48 h of culture. When KCl was included in the incubation buffer, development of apoptotic morphology was markedly delayed (Fig. 1A). However, at 72 h or 96 h of culture, almost all neutrophils stained

Discussion

Circulating neutrophils are constitutively committed to apoptosis and do not undergo further differentiation or cellular division. Signals from the inflammatory microenvironment can rescue neutrophils from apoptosis, thereby prolonging their survival, a prerequisite for trafficking into injured tissues and amplification of the inflammatory response. The results presented here suggest that inhibition of K+ influx effectively suppressed the apoptotic machinery by preventing mitochondrial

Acknowledgements

This study was supported by grants MOP-12573 and MOP-64283 (to J.G.F.) and Doctoral Research Awards (to L.J. and T.K.) from the Canadian Institutes of Health Research.

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