Abstract
Human tissue inflammation is terminated, at least in part, by the death of inflammatory neutrophils by apoptosis. The regulation of this process is therefore key to understanding and manipulating inflammation resolution. Previous data have suggested that the short-lived pro-survival Bcl-2 family protein, Mcl-1, is instrumental in determining neutrophil lifespan. However, Mcl-1 can be cleaved following caspase activity, and the possibility therefore remains that the observed fall in Mcl-1 levels is due to caspase activity downstream of caspase activation, rather than being a key event initiating apoptosis in human neutrophils.We demonstrate that apoptosis in highly purified neutrophils can be almost completely abrogated by caspase inhibition with the highly effective di-peptide caspase inhibitor, Q-VD.OPh, confirming the caspase dependence of neutrophil apoptosis. Effective caspase inhibition does not prevent the observed fall in Mcl-1 levels early in ultrapure neutrophil culture, suggesting that this fall in Mcl-1 levels is not a consequence of neutrophil apoptosis. However, at later timepoints, declines in Mcl-1 can be reversed with effective caspase inhibition, suggesting that Mcl-1 is both an upstream regulator and a downstream target of caspase activity in human neutrophils.
Highlights
We are protected against infectious disease by a range of mechanisms, including innate immune cells such as neutrophils and macrophages
Survival signals received by the neutrophils, such as cytokines (e.g. GM-CSF [3]), bacterial products [4] and hypoxia [5] act to profoundly delay neutrophil lifespan in in vitro cultures, which are thought to mimic the in vivo behaviour of neutrophils
We show that the irreversible dipeptide caspase inhibitor QVD.OPh profoundly inhibits neutrophil apoptosis, and that Mcl-1 levels drop in advance of apoptosis, even in the presence of caspase inhibition
Summary
We are protected against infectious disease by a range of mechanisms, including innate immune cells such as neutrophils and macrophages. Neutrophils have the shortest lifespan of any healthy cell, and this brief lifespan limits pro-inflammatory functions of the neutrophil [1]. Survival signals received by the neutrophils, such as cytokines (e.g. GM-CSF [3]), bacterial products [4] and hypoxia [5] act to profoundly delay neutrophil lifespan in in vitro cultures, which are thought to mimic the in vivo behaviour of neutrophils. Mcl-1 [7] and, to a lesser extent, A1 [8] have been shown to be important for maintaining neutrophil survival, and have been implicated in signalling extended neutrophil lifespan in response to a variety of stimuli including cytokines [9], elevated cAMP [10] and hypoxia [11,12]. Therapeutic strategies using CDK inihibitors or Lipoxins to target neutrophils appear to act via modulation of Mcl-1 [16,17,18]
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