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.
View: Text | PDF
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

×

Figure 8

SERPINB1 negatively regulates neutrophil spontaneous death.

Options: View larger image (or click on image) Download as PowerPoint
SERPINB1 negatively regulates neutrophil spontaneous death. (A) The seri...
(A) The serine protease responsible for procaspase-3 processing was inhibited by a factor present in the cytosol of nonapoptotic neutrophils. Proteins were extracted from freshly isolated neutrophils (processed as shown at left) and cultured in the presence or absence of cytochrome c, z-VAD-fmk, or DFP. (B) SERPINB1 expression in total cell lysates and in the cytosolic fraction was measured in freshly isolated and aging (24 hours) neutrophils by Western blotting. (C) SERPINB1 in the cytosolic fraction, quantified using NIH Image and expressed relative to actin. Data are mean ± SD of 3 independent experiments. (D) SERPINB1 protein levels decreased during neutrophil spontaneous death by complexing with proteases. The cytosolic fractions were prepared from freshly isolated and aging (24 hours) neutrophils, and levels of PR3 and SERPINB1 were assessed by Western blotting. PR3 and SERPINB1 were detected both as uncomplexed proteins (~27 and ~42 kDa, respectively) and in a complex (~65 kDa; dark arrowheads). (E) Spontaneous death of WT and SERPINB1-deficient neutrophils was analyzed by FACS. Mouse neutrophils were isolated from BM and cultured for the indicated times. Neutrophil spontaneous death was detected by annexin V and PI staining. Results are representative of 3 independent experiments. (F) Quantitative analysis of spontaneous death of WT and SERPINB1-deficient neutrophils. All results were normalized to the amount of viable WT neutrophils at the 4-hour time point. Results are mean ± SEM of 3 independent experiments. ***P < 0.001, Student’s t test.