Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis

Granzyme B (GrB), acting similar to an apical caspase, efficiently activates a proteolytic cascade after intracellular delivery by perforin. Studies here were designed to learn whether the physiologic effector, GrB–serglycin, initiates apoptosis primarily through caspase-3 or through BH3-only proteins with subsequent mitochondrial permeabilization and apoptosis. Using four separate cell lines that were either genetically lacking the zymogen or rendered deficient in active caspase-3, we measured apoptotic indices within whole cells (active caspase-3, mitochondrial depolarization [ΔΨm] and TUNEL). Adhering to these conditions, the following were observed in targets after GrB delivery: (a) procaspase-3–deficient cells fail to display a reduced ΔΨm and DNA fragmentation; (b) Bax/Bak is required for optimal ΔΨm reduction, caspase-3 activation, and DNA fragmentation, whereas BID cleavage is undetected by immunoblot; (c) Bcl-2 inhibits GrB-mediated apoptosis (reduced ΔΨm and TUNEL reactivity) by blocking oligomerization of caspase-3; and (d) in procaspase-3–deficient cells a mitochondrial-independent pathway was identified which involved procaspase-7 activation, PARP cleavage, and nuclear condensation. The data therefore support the existence of a fully implemented apoptotic pathway initiated by GrB, propagated by caspase-3, and perpetuated by a mitochondrial amplification loop but also emphasize the presence of an ancillary caspase-dependent, mitochondria-independent pathway

Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop

The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells

Granzyme A Produced by g9d2 T Cells Induces Human Macrophages to Inhibit Growth of an Intracellular Pathogen

A small subset of human T cells express γ9δ2 T cell receptors and recognize unique non-peptide phosphoantigens expressed by microbes and damaged cells, such as cancer. These cells are important because: 1) they reside within skin and mucosal surfaces at critical points of initial pathogen invasion, and 2) they are not restricted by polymorphic HLA types and thus can be activated by the same cognate antigens in highly diverse populations. Many important human pathogens such as the causes of AIDS, malaria, tuberculosis and others induce potent responses in γ9δ2 T cells that can be protective. However, the key mechanisms involved in γ9δ2 T cell-mediated protective immunity are not well defined. We have found that γ9δ2 T cells produce soluble granzyme A which correlates with their ability to protect against intracellular mycobacterial growth. We show directly that highly purified granzyme A alone can trigger human monocytes to control intracellular mycobacteria. We further show that the granzyme A-induced mycobacterial inhibition required production of TNF-α by infected monocytes. These studies may have important implications for future vaccine development and novel therapeutic strategies

NF-κB protects from the lysosomal pathway of cell death

The programme of gene expression induced by RelA/NF-κB transcription factors is critical to the control of cell survival. Ligation of ‘death receptors’ such as tumor necrosis factor receptor 1 (TNF-R1) triggers apoptosis, as well as NF-κB, which counteracts this process by activating the transcription of anti-apoptotic genes. In addition to activating caspases, TNF-R1 stimulation causes the release of cathepsins, most notably cathepsin B, from the lysosome into the cytoplasm where they induce apoptosis. Here we report a mechanism by which NF-κB protects cells against TNF-α-induced apoptosis: inhibition of the lysosomal pathway of apoptosis. NF-κB can protect cells from death after TNF-R1 stimulation, by extinguishing cathepsin B activity in the cytosol. This activity of NF-κB is mediated, at least in part, by the upregulation of Serine protease inhibitor 2A (Spi2A), a potent inhibitor of cathepsin B. Indeed, Spi2A can substitute for NF-κB in suppressing the induction of cathepsin B activity in the cytosol. Thus, inhibition of cathepsin B by Spi2A is a mechanism by which NF-κB protects cells from lysosome-mediated apoptosis

Proposed model of granzyme A-mediated suppression of intracellular mycobacterial growth by γ₉δ₂ T cells.

<p>Mycobacteria-infected macrophages present antigens to γ₉δ₂ T cells (1) which secrete granzyme A upon activation (2). Granzyme A in turn induces TNF-α production by infected and/or bystander macrophages (3) apparently independent of perforin. TNF-α activates intracellular mechanisms which alone or in concert with other unknown granzyme A-induced intracellular mechanisms suppress mycobacterial growth (4).</p

Granzyme A secretion by γ₉δ₂ T cells mediates inhibition of intracellular mycobacteria by induction of inflammatory responses in mycobacteria-infected macrophages.

<p><i>A</i>, The levels of granzyme A produced by γ₉δ₂ T cells are directly and highly correlated with inhibition of intracellular mycobacterial growth (r = 0.7564; p<0.0001). Supernatant levels of granzyme A, measured by CBA, were correlated with the level of mycobacterial growth inhibition observed in γ₉δ₂ T cell co-cultures with BCG-infected macrophages. <i>B</i>, Purified granzyme A induces the production of pro-inflammatory cytokines TNF-α and IL-1β by BCG-infected macrophages (*p<0.05, n = 4; statistical comparisons performed using Friedman's test). The indicated concentrations of purified granzyme A were added to BCG-infected monocytes for 3 days and the levels of TNF-α and IL-1β in the culture supernatants determined by CBA. <i>C-D</i>, Purified granzyme A alone can induce inhibition of intracellular mycobacterial growth in the absence of perforin. The indicated concentrations of granzyme A were added to BCG-infected (<i>C</i>) or <i>M. tuberculosis</i> H37Rv-infected (<i>D</i>) co-cultures for 3 days and the surviving bacteria quantified by H³-uridine incorporation and CFU counting. (*p<0.03; n = 6–12 by Wilcoxon matched pairs test comparing levels of mycobacterial intracellular growth in cultures with and without added purified granzyme A). Purified granzyme A had no direct effects on extracellular mycobacterial growth (data not shown). <i>E</i>, siRNA knockdown of granzyme A in γ₉δ₂ T cells reduces the inhibitory activity of γ₉δ₂ T cells for mycobacterial growth (*p<0.05; n = 3 by one-way ANOVA comparing levels of intracellular mycobacterial growth inhibition in cultures with or without γ₉δ₂ T cells producing granzyme A). γ₉δ₂ T cells transduced with the indicated siRNA-lentivirus targeting GzmA or a negative control (NC) were co-cultured with BCG-infected macrophages for 3 days and surviving bacteria quantified by H³-uridine incorporation.</p