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 9

Disruption of PR3 delays neutrophil spontaneous death during the course of inflammation in vivo.

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Disruption of PR3 delays neutrophil spontaneous death during the course ...
(A) Enhanced neutrophil accumulation at sites of inflammation in Prtn3–/– mice. WT and Prtn3–/– mice were injected intraperitoneally with E. coli. Total neutrophil content in the lavage fluid was calculated by microscopic examination of Wright-Giemsa–stained cytospins. All values are mean ± SD of 3 separate experiments. *P < 0.01 vs. WT. (B) Reduced neutrophil spontaneous death at sites of inflammation in Prtn3–/– mice. Apoptotic cells in peritoneal lavage fluid were detected as described in Figure 5. Neutrophils were recognized by Mac-1 and Ly6G staining. The annexin V+ cells included both PI–annexin V+ (early apoptotic) and PI+annexin V+ (late apoptotic and necrotic) cells. Since PI+annexin V– cells were rarely detected, we used percentage of annexin V+ cells as the primary outcome measure. Data are mean ± SD (n = 3 mice). *P < 0.01 vs. WT. (C) Strategy for analyzing apoptosis in adoptively transferred neutrophils in the TG-induced peritonitis model. Since the purity of the neutrophils used for adoptive transfer was >90%, the majority of SNARF-1– or CFSE-labeled cells were neutrophils. (D) Apoptosis of adoptively transferred neutrophils. Results are representative of 3 independent experiments. (E) Relative neutrophil survival, calculated as the ratio of the indicated populations in the peritoneal cavity. The total number of viable cells (PI–annexin V–) was used for calculation. All values represent mean ± SD of 3 separate experiments. *P < 0.01 versus WT (SNARF1)/WT (CFSE) control.