Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro.

Whyte, Moira K. B.; Hardwick, Simon J.; Meagher, Laura C.; Savill, John; Haslett, Christopher

doi:10.1172/jci116587

Cited by 139 publications

(87 citation statements)

References 37 publications

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“…Furthermore, intracellular calcium does not appear to be elevated in cells undergoing apoptosis in a number of models (Table I and see Ref. 28) and may appear only as a late consequence of the apoptotic process (18). Indeed, elevated intracellular calcium can inhibit apoptosis in neutrophils (24). Here, we show that the appearance of PS in the outer leaflet is totally inhibited in the absence of extracellular calcium and can be dissociated from the other events of apoptosis, namely DNA degradation and loss of the aminophospholipid translocase activity.…”

Section: Discussionmentioning

confidence: 59%

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Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase

Bratton

Fadok

Richter

et al. 1997

Journal of Biological Chemistry

422

294

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Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet during apoptosis, where it triggers non-inflammatory phagocytic recognition of the apoptotic cell. The mechanism of PS appearance during apoptosis is not well understood but has been associated with loss of aminophospholipid translocase activity and nonspecific flip-flop of phospholipids of various classes. The human leukemic cell line HL-60, the T cell line Jurkat, and peripheral blood neutrophils, undergoing apoptosis induced either with UV irradiation or anti-Fas antibody, were probed in the cytofluorograph for (i) surface PS using fluorescein isothiocyanate-labeled annexin V, (ii) PS uptake by the aminophospholipid translocase using [6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl] (NBD)-labeled PS, (iii) nonspecific uptake of phospholipids (as a measure of transbilayer flip-flop) using NBDlabeled phosphatidylcholine, and (iv) the appearance of hypodiploid DNA. In all three types of cells undergoing apoptosis, the appearance of PS followed loss of aminophospholipid translocase and was accompanied by nonspecific phospholipid flip-flop. Importantly, however, in the absence of extracellular calcium, the appearance of PS was completely inhibited despite DNA fragmentation and loss of aminophospholipid translocase activity, the latter demonstrating that loss of the translocase is insufficient for PS appearance during apoptosis. Furthermore, while both the appearance of PS and nonspecific phospholipid uptake demonstrated identical extracellular calcium requirements with an ED 50 of nearly 100 M, the magnitude of PS appearance depended on the level of aminophospholipid translocase activity. Taken together, the data strongly suggest that while nonspecific flip-flop is the driving event for PS appearance in the plasma membrane outer leaflet, aminophospholipid translocase activity ultimately modulates its appearance.The appearance of phosphatidylserine in the outer leaflet of the plasma membrane appears to be a universal phenomenon in cells undergoing apoptosis, or programmed cell death (1). Importantly, outer leaflet PS likely serves as a signal in tissues for the noninflammatory engulfment of apoptotic cells (Ref. 2 and see "Discussion"), a distinctly different response than the pro-inflammatory events following tissue necrosis. While phosphatidylserine (PS) 1 is actively transported from the outer to the inner leaflet by the aminophospholipid translocase (3-5), the mechanism(s) by which PS appears in apoptosis is not well understood. Verhoven et al. (3) reported that PS appearance was accompanied by both loss of aminophospholipid translocase activity and enhanced nonspecific transbilayer movement of phospholipids, perhaps due to activation of a "scramblase." Hence, they proposed that PS may become detectable in the outer leaflet due to the combination of these two events. However, to date, the relative contribution of these two events has not been clarified. Additionally, it is not known what role...

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Section: Discussionmentioning

confidence: 59%

“…Although elevation of intracellular calcium has been shown to cause apoptosis in HL-60s (22,23), it has been shown to inhibit apoptosis of neutrophils (24). Thus it was necessary to determine intracellular calcium concentration during apoptosis.…”

Section: Resultsmentioning

confidence: 99%

Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase

Bratton

Fadok

Richter

et al. 1997

Journal of Biological Chemistry

422

294

View full text Add to dashboard Cite

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“…As noted above, A23187 treatment delays apoptosis in PMN [5]. However, PMN exposed to Ca 2ϩ ionophores increase their [free Ca] by both influx of Ca 2ϩ and release of Ca 2ϩ from internal stores, and it is unclear whether one, the other, or both of these mechanisms are necessary for the apoptosis-sparing effect.…”

Section: Discussionmentioning

confidence: 97%

“…PMN spontaneously undergo apoptosis when stored in vitro or exposure to CHX or anti-Fas antibody can be used to induce apoptosis rapidly. Previous studies have suggested that intracellular Ca 2ϩ balance can modulate this process, as a rise in [free Ca] caused by the Ca 2ϩ ionophore A23187 early in storage delays development of apoptosis [5]. Based on this report, we sought to better understand the ionic events associated with apoptosis by determining whether PMN apoptosis induced by CHX resulted in changes in [free Ca] and/or resting .…”

Section: Discussionmentioning

confidence: 99%

Membrane depolarization and depletion of intracellular calcium stores are associated with delay of apoptosis in human neutrophils

Chacón-Cruz

Oelberg

Davis

et al. 1998

Journal of Leukocyte Biology

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Apoptosis occurs rapidly in human polymorphonuclear leukocytes (PMN) after exposure to 1 mM cycloheximide (CHX). We examined whether this form of stimulated apoptosis altered either resting cytosolic free Ca 2؉ concentrations ([free Ca]) or membrane potential ( ) in PMN and found no significant effects. However, manipulation of either PMN intracellular Ca 2؉ stores or was found to delay CHX-induced apoptosis. Depletion of PMN intracellular Ca 2؉ stores with thapsigargin caused membrane depolarization and significantly delayed CHX-induced apoptosis based on both morphological and annexin-V-fluorescein isothiocyanate binding criteria. Short-term suspension (4 h) of PMN in Ca 2؉ -free buffer depleted internal Ca 2؉ stores, induced membrane depolarization at 2.5 h, and delayed spontaneous (24 h) apoptosis but had no effect on CHX-induced apoptosis. Rapid membrane depolarization with 150 mM KCl buffer significantly delayed CHX-induced apoptosis, suggesting that depolarization rather than Ca 2؉ stores depletion was the crucial event. Timing experiments revealed that depolarization within 12 min of CHX exposure significantly delayed apoptosis. Collectively, these observations suggest an early -sensitive step in the apoptosis pathway initiated by CHX. CHX exposure alone does not alter either resting PMN [free Ca] or ; accompanying depolarization of plasma membrane (either electrochemically or via depletion of internal Ca 2؉ stores) delays CHX-induced apoptosis in a time-dependent manner. J. Leukoc. Biol. 64: 759-766; 1998.

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“…When neutrophils die by apoptosis they retain their granule contents but lose chemotactic and secretory responsiveness [8] and thereafter are recognised and phagocytosed intact by macrophages [9] which fail to release pro-inflammatory mediators in response to this interaction [10]. Although neutrophils un-*Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

Hypoxia prolongs neutrophil survival in vitro

et al. 1995

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~,bstract Neutrophil apoptosis represents a major mechanism ~nvolved in the resolution of inflammation. Since hypoxia induces apoptosis in several cell lines and is of particular relevance in many disease states, we studied the effect of oxygen concentration on neutrophil survival in vitro. Hypoxia caused a dramatic decrease in neutrophil apoptosis (% apoptosis 20 h: 78.7 + 2.2% in 21% 0 2, 61.4 _+ 6.5% in 2.5% 02, 23.1 + 3.2% in 0% 02, = 5). This was additive to the effect of GM-CSF (50 Ulml), not associated with induction of bcl-2 expression, and was not mimicked by methionine (5 raM), superoxide dismutase (200 pg/ml) or Trolox (10 mM) but was mimicked by catalase (250/zg/ml).Hence, hypoxia has a bcl-2-independent effect on neutrophil apoptosis that may adversely affect the clearance of these cells from an inflammatory focus.

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Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro.

Cited by 139 publications

References 37 publications

Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase

Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase

Membrane depolarization and depletion of intracellular calcium stores are associated with delay of apoptosis in human neutrophils

Hypoxia prolongs neutrophil survival in vitro

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