Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization

Abstract

Lysosomal membrane permeabilization (LMP) is emerging as an important regulator of cell apoptosis. Human neutrophils are highly granulated phagocytes, which respond to pathogens by exhibiting increased production of reactive oxygen species (ROS) and lysosomal degranulation. In a previous study, we observed that intracellular, nonphagosomal generation of ROS triggered by adherent bacteria induced ROS-dependent neutrophil apoptosis, whereas intraphagosomal production of ROS during phagocytosis had no effect. In the present study, we measured lysosomal membrane stability and leakage in human neutrophils and found that adherent, noningested, Type 1-fimbriated Escherichia coli bacteria induced LMP rapidly in neutrophils. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium markedly blocked the early LMP and apoptosis in neutrophils stimulated with Type 1-fimbriated bacteria but had no effect on the late LMP seen in spontaneously apoptotic neutrophils. The induced lysosomal destabilization triggered cleavage of the proapoptotic Bcl-2 protein Bid, followed by a decrease in the antiapoptotic protein Mcl-1. Involvement of LMP in initiation of apoptosis is supported by the following observations: Bid cleavage and the concomitant drop in mitochondrial membrane potential required activation of cysteine-cathepsins but not caspases, and the differential effects of inhibitors of cysteine-cathepsins and cathepsin D on apoptosis coincided with their ability to inhibit Bid cleavage in activated neutrophils. Together, these results indicate that in microbe-induced apoptosis in neutrophils, ROS-dependent LMP represents an early event in initiation of the intrinsic apoptotic pathway, which is followed by Bid cleavage, mitochondrial damage, and caspase activation.

Reduced tumour cell proliferation and delayed development of high-grade mammary carcinomas in cathepsin B-deficient mice

“…In this model of apoptosis induction, PyMT;Ctsb À/À tumour cells were found to be significantly more resistant to Leu-LeuOMe than the control PyMT tumour cells. This could be (Stoka et al, 2001;Cirman et al, 2004;Blomgran et al, 2007;Nagaraj et al, 2007). Taken together, our results suggest that absence of Ctsb in primary PyMT tumour cells does not alter their susceptibility to an inducer of tumour cell apoptosis, that is TNF-a.…”

Induction of apoptosis in human neutrophils by Mycobacterium tuberculosis is dependent on mature bacterial lipoproteins

“…As shown in Fig. 6B, the respiratory burst induced by Mtb was weaker (<15%) than that elicited by an E. coli strain (FimH+, expressing type 1 fimbriae), previously reported to cause strictly ROS-dependent apoptosis [12].…”

“…It has been shown that ROS produced during cell activation is a mediator of apoptosis in inflammatory cells [12,21]. Using luminol-amplified chemiluminescence, we measured formation of ROS in PMNs and found that both opsonized and unopsonized Mtb induced similar amount of ROS (Fig.…”

“…Oxidative damage of these membrane leads to permeabilization of the granules and leakage of lysosomal cathepsins to the cytosol [12,22,23], events closely linked to amplification of apoptotic signals. Therefore, we measured the integrity of these granules in PMNs subjected to Mtb-induced apoptosis, by monitoring cells with intact acridine orange-accumulating lysosomes (Fig.…”

“…coli-induced production of reactive oxygen species (ROS) can oxidize the membranes of azurophilic granules resulting in leakage of cathepsins to the cytosol and propagation of the mitochondrial apoptosis pathway [12]. Virulent mycobacteria interfere with the fusion between granules and the phagosome [13,14] but it is not known whether azurophilic granules are also involved in Mtb-induced PMN apoptosis.…”