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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Research Article Immunology

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 1

Caspase-3 activation during neutrophil spontaneous death is independent of caspase-8 or caspase-9.

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Caspase-3 activation during neutrophil spontaneous death is independent ...
(A) Neutrophil spontaneous death was caspase dependent. Human primary neutrophils were cultured in the presence of the pan-caspase inhibitor z-VAD-fmk, and the percentage of apoptotic cells was measured, normalized to a no-inhibitor control. Results are mean ± SEM of 3 independent experiments. ***P < 0.001, 2-way ANOVA. (B) Neutrophil spontaneous death was mediated by caspase-3, but was independent of caspase-8 or caspase-9. Human neutrophils were cultured with the indicated specific caspase inhibitors for 36 hours; G-CSF (50 ng/ml) was used as a positive control. Results are mean ± SD of 3 independent experiments. *P < 0.001, Student’s t test. (C) The cytosolic fraction of aging neutrophils (24 hours in culture) cleaved procaspase-3, independent of caspase-8 or caspase-9. Procaspase-3 cleavage was assayed using recombinant N-terminal His-tagged procaspase-3, and unprocessed procaspase-3 (32 kDa) and cleaved caspase-3 (N-terminal fragment; 20 kDa) were detected by Western blotting using anti-His tag antibodies, with actin as a loading control. Results are representative of 3 independent experiments.