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 5

Inhibition of serine protease activity delays neutrophil spontaneous apoptosis in vitro.

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Inhibition of serine protease activity delays neutrophil spontaneous apo...
(A) Spontaneous death of human neutrophils, analyzed by annexin V and PI staining. Human neutrophils were cultured in the presence or absence of the indicated growth factor and protease inhibitor (G-CSF, 50 ng/ml; z-VAD-fmk, 50 mM; DFP, 100 mM) for 16 hours. The percentage of viable cells (region R1), early apoptotic cells (region R2); and late apoptotic and necrotic cells (regions R3 and R4) is indicated. (B) Quantification of neutrophil spontaneous death. Results are mean ± SEM of 3 independent experiments. *P < 0.01, Student’s t test. (C) siRNA-mediated PR3 knockdown (KD) in murine neutrophils. Results are representative of 3 independent experiments. (D) Quantification of siRNA-mediated PR3 knockdown. Results are mean ± SEM of 3 independent experiments. **P < 0.01, Student’s t test. (E) siRNA-mediated PR3 knockdown prolonged survival of murine neutrophils. Neutrophil death was analyzed 24 hours after siRNA nucleofection. Results are representative of 5 independent experiments. (F) Quantitative analysis of neutrophil spontaneous death. The percentage of viable cells at each time point was normalized to the sample transfected with control siRNA. Results are mean ± SD of 5 independent experiments. *P < 0.01, Student’s t test. (G) PR3 expression was abolished in PR3-deficient murine neutrophils isolated from the femurs and tibias of 10-week-old mice. (H) Neutrophil morphology, examined by Wright-Giemsa staining. Scale bars: 10 μm. (I) Spontaneous death of WT and PR3-deficient murine neutrophils isolated from BM. Results are mean ± SD of 3 independent experiments. **P < 0.005.