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Summary: 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. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1β [IL-1β], TNFα, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA+ cytotoxic T lymphocytes (CTLs) induce IL-1β from primary mouse macrophages, and GzmA −/− mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator. [Copyright &y& Elsevier]

The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme Bserglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans. Using a combination of cell binding and internalization assays, granzyme B was found to exchange to sulfated glycosaminoglycans and, depending on the cell type, to higher affinity sites. Apoptosis induced by purified granzyme B and cytotoxic T-cells was diminished in targets with reduced cell surface glycosaminoglycan content. A mechanism of delivery is proposed entailing electrostatic transfer of granzyme B from serglycin to cell surface proteins. [ABSTRACT FROM AUTHOR]

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. LCMV is not cleared in IL-1R-deficient mice, and yet the infected mice develop neither splenomegaly nor hepatitis. These results demonstrate that IL-1R signaling is central to the induction of proapoptotic CD8 T cell immunity, including viral clearance and associated tissue injuries in LCMV infection. [ABSTRACT FROM AUTHOR]

Abstract: 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. However, PFN does not permeabilize large or giant unilamellar vesicles (GUV) at pH 5.5 even though the monomers bind to the membranes in the presence of calcium. It was possible to activate adsorbed PFN and to induce membrane permeabilization by simply raising pH to a physiological level (pH 7.4). These results were independently confirmed on GUV and Jurkat cells. The conformational state of PFN at either pH was further assessed with monoclonal antibodies Pf-80 and Pf-344. Pf-344 maps to a linear epitope within region 373–388 of epidermal growth factor (EGF)-like domain while the Pf-80 appears to recognize a conformational epitope. Pf-344 interacts with the EGF-like domain after PFN monomers undergo pore formation, the site recognized by Pf-80 is only accessible at acidic but not neutral pH. Thus, the Pf-80 mAb likely interacts with a region of the monomer that participates in oligomerization prior to insertion of the monomer into the lipid bilayer and thus may have therapeutic utility against PFN-mediated immunopathology. [ABSTRACT FROM AUTHOR]