Apoptosis: Killing neutrophils the cathepsin way

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Cathepsin D directly cleaves and activates caspase-8 at the beginning of a pro-apoptotic pathway in neutrophils, showing how apoptosis can be activated in the absence of death-receptor ligation.

Neutrophil apoptosis is a hallmark of the resolution of inflammatory responses. Although apoptotic pathways involving caspases have been studied extensively, the initial events that trigger caspase activation in these cells have not been clear. Now, Simon and colleagues describe a pro-apoptotic pathway in neutrophils in which caspase-8 is directly activated by cathepsin D.

Although caspases can be activated by death receptors of the tumour-necrosis factor (TNF) family, previously published work indicated that these receptors are unlikely to have a role in the initiation of neutrophil apoptosis, particularly under conditions of survival-factor withdrawal. The authors therefore focused on cathepsins, which have been suggested to act in concert with caspases in the regulation of apoptosis. Indeed, their study showed that inactivation of cathepsin D, but surprisingly not of cathepsin B, by both genetic and pharmacological means delayed neutrophil apoptosis. Moreover, cathepsin D deficiency in mice resulted in a stronger and more prolonged innate immune response as a result of defective neutrophil apoptosis following injection of low-dose lipopolysaccharide.

Immunofluorescence assays revealed that cathepsin D co-localized specifically with myeloperoxidase in the azurophilic granules of neutrophils. However, within 3 hours of culture a more diffuse pattern of cathepsin D localization was observed within the cytoplasm, suggesting granule permeabilization and release of its contents into the cytosol. Cathepsin D secretion into the cytosol was inhibited by survival cytokines but not by pan-caspase inhibitors. By contrast, mitochondrial release of the pro-apoptotic factor cytochrome c occurred after release of cathepsin D and was blocked by both survival cytokines and pan-caspase inhibitors, indicating that caspase-independent permeabilization of azurophilic granules occurs at the early stages of neutrophil apoptosis prior to the caspase-dependent release of mitochondrial pro-apoptotic factors.

Prompted by the fact that activation of mitochondrial pro-apoptotic factors was both caspase and cathepsin D dependent, the authors tested whether cathepsin D could activate caspases. They found that cathepsin D directly cleaved caspase-8, but not caspase-3, to produce enzymatically active fragments, although cleavage of caspase-3 was dependent on that of caspase-8.

This study provides an important new role for azurophilic granules — not only are they involved in the digestion of phagocytosed microorganisms but they also have a role in the induction of apoptosis in mature neutrophils — and resolves the question of how apoptosis can be activated in the absence of death-receptor ligation. Future studies will have to address questions such as how the pH-dependent activity of cathepsin D is maintained once the protease is released in the cytosol.

Marta Tufet

References

Conus, S. et al. Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation. J. Exp. Med. 205, 685–698 (2008) | Article | PubMed |