Detection and Significance of Cytotoxic Cell Subsets in Biopsies of HCV-Infected Human Livers
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Granzyme B is the prototypic member of a family of serine proteases localized to the cytolytic granules of cytotoxic lymphocytes. Together with another granule protein, perforin, granzyme B is capable of inducing all aspects of apoptotic death in target cells. A number of granzyme B substrates have been identified and it has been demonstrated that granzyme B is responsible, directly or indirectly, for the morphological nuclear changes observed in target cell apoptosis, including DNA fragmentation. In an earlier study, we showed that granzyme B binds to a nuclear protein in a manner dependent on its enzymatic activity. Here, we demonstrate that granzyme B is translocated rapidly to the nucleus in cells that have been induced to undergo apoptosis by a granzyme-dependent process, and that translocation is dependent on caspase activity. Appearance of granzyme B in the nucleus of target cells precedes the detection of DNA fragmentation. Although not directly responsible for DNA fragmentation, these data suggest a nuclear role for granzyme B in target cell apoptosis. c-Abl nuclear functions.
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ABSTRACTIntroduction Granzyme B is a serine protease extensively studied for its implication in cytotoxic lymphocyte-mediated apoptosis. In recent years, the paradigm that the role of granzyme B is restricted to immune cell-mediated killing has been challenged as extracellular roles for the protease have emerged. While mostly absent from healthy tissues, granzyme B levels are elevated in several autoimmune and/or chronic inflammatory conditions. In the skin, its accumulation significantly impairs proper wound healing.Areas covered After an overview of the current knowledge on granzyme B, a description of newly identified functions will be presented, focussing on granzyme B ability to promote cell–cell and dermal-epidermal junction disruption, extracellular matrix degradation, vascular permeabilization, and epithelial barrier dysfunction. Progress in granzyme B inhibition, as well as the use of granzyme B inhibitors for the treatment of tissue damage, will be discussed.Expert opinion The absence of endogenous extracellular inhibitors renders extracellular granzyme B accumulation deleterious for the proper healing of chronic wounds due to sustained proteolytic activity. Consequently, specific granzyme B inhibitors have been developed as new therapeutic approaches. Beyond applications in wound healing, other autoimmune and/or chronic inflammatory conditions related to exacerbated granzyme B activity may also benefit from the development of these inhibitors.KEYWORDS: Granzyme Bwound healinginflammationsmall molecule inhibitortissue remodelingskin integrity Article highlights Granzyme B is a pro-apoptotic serine protease with diverse emerging extracellular functions in disease.Granzyme B levels are minimal to absent in healthy individuals but are increased in the extracellular space and biological fluids of patients suffering from autoimmune disorders, chronic inflammatory conditions, or chronic wounds.Within the extracellular space, granzyme B cleaves numerous substrates involved in cell–cell junction, basement membrane integrity, and extracellular matrix organization.Due to the absence of endogenous inhibitor in human, proteolytic activity of extracellular granzyme B is believed to be dysregulated.Genetic deletion of granzyme B in mice reduces contact dermatitis and pemphigoid disorders severity, and improves thermal-, diabetic- and age-dependent impaired wound healing.Topical application of VTI-1002, a specific granzyme B inhibitor, showed high potential to treat inflammatory dermatological conditions and wound healing in mice.EOTT – List of abbreviationAAD = allergic airway diseaseACT = α-1-antichymotrypsinAD = atopic dermatitisAMD = age-related macular degenerationApoE = apolipoprotein EBM = basement membraneBP = bullous pemphigoidCC3 = cleaved Caspase-3CD = Crohn’s diseaseCTL = cytotoxic T lymphocyteDEJ = dermal-epidermal junctionDEP = diesel exhaust particlesDH = dermatitis herpetiformisEBA = epidermolysis bullosa acquisitaECM = extracellular matrixFGFR1 = fibroblast growth factor receptor 1GzmA = granzyme AGzmB = granzyme BHFD = high-fat dietHMD = house dust miteIBD = inflammatory bowel diseases(s)ICAM = (soluble) intracellular adhesion molecule-1IEL = intraepithelial lymphocytesIL = interleukinJAM-A = junctional adhesion molecule-ALPS = lipopolysaccharideLTBP = latent TGF-β binding proteinMIP-2 = macrophage inflammatory protein-2MMP = matrix metalloproteinaseNE = neutrophil elastaseNK = natural killerOVA = ovalbuminOXA = oxazolonePAR2 = protease-activator receptor 2PD = pemphigoid diseasesPI = pressure injuriesRPE = retinal pigment epitheliumSerpina3N = serine peptidase inhibitor, clade 3, member 3NSJS = Stevens-Johnson syndromeTEN = toxic epidermal necrolysisTGF-β1 = transforming growth factor-β1TIMP = tissue inhibitor of metalloproteinaseTJ = tight junctionTNF = tumor necrosis factorTREM-1 = triggering receptor expressed by myeloid cells-1UV = ultravioletVEGF = vascular endothelial growth factorVEGFR = vascular endothelial growth factor receptorWT = wild typeZO-1 = zonula occludens protein-1Declaration of interestDavid J. Granville is the Co-Founder and Chief Scientific Officer of viDA Therapeutics, which owns proprietary granzyme B inhibitor VTI-1002. The other authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.Reviewer disclosuresPeer reviewers on this manuscript have no relevant financial or other relationships to disclose.Supplementary materialSupplemental data for this article can be accessed online at https://doi.org/10.1080/14728222.2022.2161890Additional informationFundingThis manuscript was funded by the Canadian Institutes for Health Research, Michael Smith Foundation for Health Research, National Sciences and Engineering Research Council (NSERC) of Canada, Leo Foundation, Cancer Research Society, Eczema Society of Canada, Mitacs Canada, and the Rick Hansen Institute to DJ Granville.
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Cytotoxic lymphocytes use the granule exocytosis pathway to kill pathogen-infected cells and tumor cells. Although many genes in this pathway have been extensively characterized (e.g., perforin, granzymes A and B), the role of granzyme C is less clear. We therefore developed a granzyme C-specific mAb and used flow cytometry to examine the expression of granzyme B and C in the lymphocyte compartments of wild-type and mutant GzmB(-/-) cre mice, which have a small deletion in the granzyme B gene. We detected granzyme B and C expression in CD4(+) and CD8(+) T cells activated with CD3/CD28 beads or MLRs. Stimulation of NK cells in vitro with IL-15 also induced expression of both granzymes. Granzyme C up-regulation was delayed relative to granzyme B in wild-type lymphocytes, whereas GzmB(-/-) cre cells expressed granzyme C earlier and more abundantly on a per-cell basis, suggesting that the deleted 350-bp region in the granzyme B gene is important for the regulation of both granzymes B and C. Quantitative RT-PCR revealed that granzyme C protein levels were regulated by mRNA abundance. In vivo, a population of wild-type CD8alphaalpha(+) intraepithelial lymphocytes constitutively expressed granzyme B and GzmB(-/-) cre intraepithelial lymphocytes likewise expressed granzyme C. Using a model of a persistent murine CMV infection, we detected delayed expression of granzyme C in NK cells from infected hosts. Taken together, these findings suggest that granzyme C is activated with persistent antigenic stimulation, providing nonredundant backup protection for the host when granzyme B fails.
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The serine protease granzyme B, which is secreted by cytotoxic cells, is one of the major effectors of apoptosis in susceptible targets. To examine the apoptotic mechanism of granzyme B, we have analyzed its effect on purified proteins that are thought to be components of death pathways inherent to cells. We demonstrate that granzyme B processes interleukin 1beta-converting enzyme (ICE) and the ICE-related protease Yama (also known as CPP32 or apopain) by limited proteolysis. Processing of ICE does not lead to activation. However, processing by granzyme B leads directly to the activation of Yama, which is now able to bind inhibitors and cleave the substrate poly(ADP-ribose) polymerase whose proteolysis is a marker of apoptosis initiated by several other stimuli. Thus ICE-related proteases can be activated by serine proteases that possess the correct specificity. Activation of pro-Yama by granzyme B is within the physiologic range. Thus the cytotoxic effect of granzyme B can be explained by its activation of an endogenous protease component of a programmed cell death pathway.
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Lichen planus is an inflammatory dermatosis involving either skin and/or mucosal epithelial surfaces. A cell-mediated cytotoxicity response is the main suspected mechanism of this dermatosis. Granzyme B and granulysin are components of the cytoplasmic granules of cytotoxic T lymphocytes and natural killers. They are involved in cell-mediated apoptosis. This work studies the possible implication of granzyme B and granulysin in the cell-mediated cytotoxicity response in lichen planus.In situ expression of granzyme B and granulysin was studied by real-time reverse transcriptase polymerase chain reaction in 15 biopsies of lichen planus. The distribution and the phenotype of the inflammatory infiltrate and the expression of granzyme B were studied by immunohistochemistry in seven other biopsies of lichen planus.Granzyme B and granulysin mRNA expression was one to two hundred times greater than in biopsies of normal skin. Immunohistochemical study revealed that the lymphohistiocytic infiltrate consisted mainly of CD4+ and CD8+ lymphocytes. Granzyme B+ cells were observed close to apoptotic keratinocytes.Our results suggest a central role for cell-mediated cytotoxicity by the granule exocytosis pathway probably because of auto-cytotoxic T-cell clones in the pathogenesis of lichen planus.
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Granzyme B is an important effector in the immune response induced by cytotoxic lymphocytes.Within the target cells, granzyme B rapidly activates apoptosis and induces cytotoxicity. It also has other im-portant extracellular functions. The self-adjusting of granzyme B is achieved mainly through interaction withits inhibitors belonging to the serine protease inhibitor (serpin) superfamily. These endogenous serpins are significant dominators of homeostatic regulation in cytotoxic lymphocytes and some antigen presenting cells.In clinical medicine, they also mitigate autoimmune diseases and graft rejection pertinent to granzyme B death pathways. A lot of malignantly transformed cells have been reported to encode inhibitors of granzyme Bfor escaping apoptosis. Viruses have also co-evolved with the immune system to resist anti-virus infection,and several different mechanisms of granzyme B inhibitors have been implicated in these viral processes. Re-searches on natural and synthetic inhibitors of granzyme B should enable novel therapies based on selectiveregulation of the effector response in conditions as diverse as autoimmunity and cancer. They are also in-creasingly attracting attentions for their potential application value on anti-virus therapies.
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Cytotoxic lymphocytes (CL) induce death of their targets by granule exocytosis. During this process, enzymes contained within cytotoxic granules (granzymes) are delivered to the target cell where the enzymes trigger the cell death by cleaving specific substrates. Granzyme B is the only granzyme that has been shown to induce cell death by apoptosis, but the exact pathway by which this is achieved has been the subject of hot debate. Furthermore, several other death-inducing granzymes have been identified; therefore, the exact contribution of granzyme B to CL-induced death is unclear. In this study, we discuss our recent findings on granzyme B-induced cell death and discuss the potential relevance of this pathway to CL-induced death of viral-infected and transformed cells.
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