Proteinase 3–dependent caspase-3 cleavage modulates neutrophil death and inflammation
Fabien Loison, … , Yuanfu Xu, Hongbo R. Luo
Fabien Loison, … , Yuanfu Xu, Hongbo R. Luo
Published September 2, 2014
Citation Information: J Clin Invest. 2014;124(10):4445-4458. https://doi.org/10.1172/JCI76246.
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Proteinase 3–dependent caspase-3 cleavage modulates neutrophil death and inflammation

Abstract

Caspase-3–mediated spontaneous death in neutrophils is a prototype of programmed cell death and is critical for modulating physiopathological inflammatory responses; however, the underlying regulatory pathways remain ill defined. Here we determined that in aging neutrophils, the cleavage and activation of caspase-3 is independent of the canonical caspase-8– or caspase-9–mediated pathway. Instead, caspase-3 activation was mediated by serine protease proteinase 3 (PR3), which is present in the cytosol of aging neutrophils. Specifically, PR3 cleaved procaspase-3 at a site upstream of the canonical caspase-9 cleavage site. In mature neutrophils, PR3 was sequestered in granules and released during aging via lysosomal membrane permeabilization (LMP), leading to procaspase-3 cleavage and apoptosis. Pharmacological inhibition or knockdown of PR3 delayed neutrophil death in vitro and consistently delayed neutrophil death and augmented neutrophil accumulation at sites of inflammation in a murine model of peritonitis. Adoptive transfer of both WT and PR3-deficient neutrophils revealed that the delayed death of neutrophils lacking PR3 is due to an altered intrinsic apoptosis/survival pathway, rather than the inflammatory microenvironment. The presence of the suicide protease inhibitor SERPINB1 counterbalanced the protease activity of PR3 in aging neutrophils, and deletion of Serpinb1 accelerated neutrophil death. Taken together, our results reveal that PR3-mediated caspase-3 activation controls neutrophil spontaneous death.

Authors

Fabien Loison, Haiyan Zhu, Kutay Karatepe, Anongnard Kasorn, Peng Liu, Keqiang Ye, Jiaxi Zhou, Shannan Cao, Haiyan Gong, Dieter E. Jenne, Eileen Remold-O’Donnell, Yuanfu Xu, Hongbo R. Luo

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Figure 2

Serine protease activity is responsible for procaspase-3 cleavage during neutrophil spontaneous death.

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Serine protease activity is responsible for procaspase-3 cleavage during...
(A) Procaspase-3 cleavage in the cytosol of aging neutrophils was suppressed by a serine protease inhibitor. His-tagged procaspase-3 was incubated with the cytosolic fraction of aging human neutrophils (24 hours) at 37°C for 45 minutes in the presence of caspase inhibitor VI (20 mM), DFP (serine protease inhibitor, 20 mM), E-64d (calpain and cathepsins B, H, and L inhibitor, 20 mM), calpeptin (calpain-2 inhibitor, 20 mM), calpain inhibitor II (N-acetyl-L-leucyl-L-leucyl-L-methional; calpain-1 and cathepsin inhibitor, 20 mM), and pepstatin A (cathepsin D inhibitor, 1 mM). Procaspase-3 cleavage was assayed as described in Figure 1. (B) DFP did not inhibit procaspase-3 cleavage mediated by apoptosome (cytochrome c). Recombinant procaspase-3 was incubated with the cytosolic fraction of fresh human neutrophils in the presence or absence of cytochrome c (10 mM), z-VAD-fmk (20 mM), and/or DFP (20 mM). (C) Cleavage of procaspase-3 by the cytosolic fraction of aging neutrophils (24 hours) was independent of cytochrome c. Results are representative of at least 3 independent experiments.