Granzyme A produced by γ(9)δ(2) T cells induces human macrophages to inhibit growth of an intracellular pathogen.

Human γ(9)δ(2) T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ(9)δ(2) T cells produced soluble factors that could pass through 0.

Interleukin-1R signaling is essential for induction of proapoptotic CD8 T cells, viral clearance, and pathology during lymphocytic choriomeningitis virus infection in mice.

The T cell granule exocytosis pathway is essential to control hepatotropic lymphocytic choriomeningitis virus strain WE (LCMV-WE) but also contributes to the observed pathology in mice. Although effective antiviral T cell immunity and development of viral hepatitis are strictly dependent on perforin and granzymes, the molecular basis underlying induction of functionally competent virus-immune T cells, including participation of the innate immune system, is far from being resolved. We demonstrate here that LCMV-immune T cells of interleukin-1 receptor (IL-1R)-deficient mice readily express transcripts for perforin and granzymes but only translate perforin, resulting in the lack of proapoptotic potential in vitro.

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.

Human perforin permeabilizing activity, but not binding to lipid membranes, is affected by pH.

The various steps that perforin (PFN), a critical mediator of innate immune response, undertakes to form a transmembrane pore remains poorly understood. We have used surface plasmon resonance (SPR) to dissect mechanism of pore formation. The membrane association of PFN was calcium dependent irrespective of pH.

Human and mouse granzyme A induce a proinflammatory cytokine response.

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis.

Detection of functional cell surface perforin by flow cytometry.

How perforin (PFN) delivers the granzymes during cytotoxic granule mediated apoptosis remains a mystery. A major obstacle has been the inability to visualize PFN in either monomeric or polymeric form after interaction with the target cell surface. An antibody based technique is described which detects cell surface PFN on intact cells by flow cytometry.

A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells.

The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme B-serglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans.

NF-kappaB protects from the lysosomal pathway of cell death.

The programme of gene expression induced by RelA/NF-kappaB 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-kappaB, 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.

Cytotoxic granule-mediated apoptosis: unraveling the complex mechanism.

The molecular details of cytotoxic granule-mediated apoptosis have been gleaned from the study of the effects of isolated granzymes and perforin on target cells. Recent evidence indicates that the physiological apoptosis-inducing form is a multi-component macro-complex consisting of cationic granule proteins non-covalently linked to the chondroitin-sulfate proteoglycan, serglycin.

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 [DeltaPsim] and TUNEL).

Cytotoxic cell granule-mediated apoptosis. Characterization of the macromolecular complex of granzyme B with serglycin.

We have recently shown that the physiological mediator of granule-mediated apoptosis is a macromolecular complex of granzymes and perforin complexed with the chondroitin-sulfate proteoglycan, serglycin (Metkar, S. S., Wang, B.

Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation.

The mechanism underlying perforin (PFN)-dependent delivery of apoptotic granzymes during cytotoxic cell granule-mediated death remains speculative. Granzyme B (GrB) and perforin were found to coexist as multimeric complexes with the proteoglycan serglycin (SG) in cytotoxic granules, and cytotoxic cells were observed to secrete exclusively macromolecular GrB-SG. Contrary to the view that PFN acts as a gateway for granzymes through the plasma membrane, monomeric PFN and, strikingly, PFN-SG complexes were shown to mediate cytosolic delivery of macromolecular GrB-SG without producing detectable plasma membrane pores.