Granzyme B in skin inflammation and disease

Granzyme B (GzmB) is a serine protease emerging as an important mediator of skin injury, inflammation and repair. Found at low levels in healthy skin, GzmB is dramatically elevated in chronic disease and inflammatory skin disorders, including diabetic ulcers, hypertrophic scarring, autoimmune skin disorders, cutaneous leishmaniasis and aging skin. Traditionally known for its pro-apoptotic function, the role of GzmB in disease has been redefined due to the discovery of additional activities involving the cleavage of extracellular matrix proteins, epithelial barrier disruption, fibrosis, vascular permeability, anoikis, inflammation and autoimmunity. In addition to the accumulation of GzmB⁺ cells in diseased tissue, and critical to the mechanistic redefinition, is the realization that GzmB often accumulates in the extracellular milieu, retains its activity in plasma, and is expressed by both immune and non-immune cells that may or may not express perforin, the pore-forming protein required for GzmB internalization into target cells. As GzmB is not normally found in the extracellular milieu, and does not appear to be regulated, GzmB-mediated proteolysis can impact processes such as tissue remodelling, barrier function, autoantigen generation and angiogenesis. The present review will summarize and critically examine the current knowledge regarding GzmB in inflammatory skin disease, providing an overview of both apoptotic and extracellular mechanisms, but with a focus on the extracellular roles of GzmB in skin health and disease.     

粒酶B与细胞凋亡

粒酶B（GrB)是杀伤性T淋巴细胞（CTL）和自然杀伤细胞（NK）颗粒（granule)中最重要的丝氨酸蛋白酶,通过caspases依赖途径。直接入核途径及不依赖caspases的胞浆途径。启动CTL介导的靶细胞凋亡。     

Annexin A1 regulates intestinal mucosal injury, inflammation, and repair

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (-/-) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (-/-) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.     

Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

Background

Genotoxic stress, such as by exposure to bromodeoxyuridine (BrdU) and cigarette smoke, induces premature cell senescence. Recent evidence indicates that cellular senescence of various types of cells is accelerated in COPD patients. However, whether the senescence of airway epithelial cells contributes to the development of airway diseases is unknown. The present study was designed to test the hypothesis that premature senescence of airway epithelial cells (Clara cells) impairs repair processes and exacerbates inflammation after airway injury.

Methods

C57/BL6J mice were injected with the Clara-cell-specific toxicant naphthalene (NA) on days 0, 7, and 14, and each NA injection was followed by a daily dose of BrdU on each of the following 3 days, during which regenerating cells were allowed to incorporate BrdU into their DNA and to senesce. The p38 MAPK inhibitor SB202190 was injected 30 minutes before each BrdU dose. Mice were sacrificed at different times until day 28 and lungs of mice were obtained to investigate whether Clara cell senescence impairs airway epithelial regeneration and exacerbates airway inflammation. NCI-H441 cells were induced to senesce by exposure to BrdU or the telomerase inhibitor MST-312. Human lung tissue samples were obtained from COPD patients, asymptomatic smokers, and nonsmokers to investigate whether Clara cell senescence is accelerated in the airways of COPD patients, and if so, whether it is accompanied by p38 MAPK activation.

Results

BrdU did not alter the intensity of the airway epithelial injury or inflammation after a single NA exposure. However, after repeated NA exposure, BrdU induced epithelial cell (Clara cell) senescence, as demonstrated by a DNA damage response, p21 overexpression, increased senescence-associated β-galactosidase activity, and growth arrest, which resulted in impaired epithelial regeneration. The epithelial senescence was accompanied by p38 MAPK-dependent airway inflammation. Senescent NCI-H441 cells impaired epithelial wound repair and secreted increased amounts of pro-inflammatory cytokines in a p38 MAPK-dependent manner. Clara cell senescence in COPD patients was accelerated and accompanied by p38 MAPK activation.

Conclusions

Senescence of airway epithelial cells impairs repair processes and exacerbates p38 MAPK-dependent inflammation after airway injury, and it may contribute to the pathogenesis of COPD.     

Oxygen radicals, inflammation, and tissue injury

Inflammatory reactions often result in the activation and recruitment of phagocytic cells (e.g., neutrophils and/or tissue macrophages) whose products result in injury to the tissue. In killing of endothelial cells by activated neutrophils as well as in lung injury produced by either activated neutrophils or activated macrophages there is evidence that H2O2 and iron play a role. HO. may be a key oxygen product related to the process of injury. Endothelial cells in some vascular compartments may be susceptible to neutrophil mediated injury in a manner that is independent of oxygen radicals. On the basis of in vitro observations, a synergy exits between platelets and neutrophils, resulting in enhanced oxygen radical formation by the latter. Finally, the cytokines, interleukin 1 and tumor necrosis factor, released from macrophages have both direct stimulatory effects on oxygen radical formation in neutrophils and can "prime" macrophages for enhanced oxygen radical responses to other agonists. Cytokines may also alter endothelial cells rendering them more susceptible to oxygen radical mediated injury by neutrophils. This suggests a complex network of interactions between phagocytic cells and peptide mediators, the result of which is acute, oxygen radical mediated tissue injury.     

Systemic biomarkers of neutrophilic inflammation, tissue injury and repair in COPD patients with differing levels of disease severity

The identification and validation of biomarkers to support the assessment of novel therapeutics for COPD continues to be an important area of research. The aim of the current study was to identify systemic protein biomarkers correlated with measures of COPD severity, as well as specific protein signatures associated with comorbidities such as metabolic syndrome. 142 protein analytes were measured in serum of 140 patients with stable COPD, 15 smokers without COPD and 30 non-smoking controls. Seven analytes (sRAGE, EN-RAGE, NGAL, Fibrinogen, MPO, TGF-α and HB-EGF) showed significant differences between severe/very severe COPD, mild/moderate COPD, smoking and non-smoking control groups. Within the COPD subjects, univariate and multivariate analyses identified analytes significantly associated with FEV(1), FEV(1)/FVC and DLCO. Most notably, a set of 5 analytes (HB-EGF, Fibrinogen, MCP-4, sRAGE and Sortilin) predicted 21% of the variability in DLCO values. To determine common functions/pathways, analytes were clustered in a correlation network by similarity of expression profile. While analytes related to neutrophil function (EN-RAGE, NGAL, MPO) grouped together to form a cluster associated with FEV(1) related parameters, analytes related to the EGFR pathway (HB-EGF, TGF-α) formed another cluster associated with both DLCO and FEV(1) related parameters. Associations of Fibrinogen with DLCO and MPO with FEV(1)/FVC were stronger in patients without metabolic syndrome (r = -0.52, p = 0.005 and r = -0.61, p = 0.023, respectively) compared to patients with coexisting metabolic syndrome (r = -0.25, p = 0.47 and r = -0.15, p = 0.96, respectively), and may be driving overall associations in the general cohort. In summary, our study has identified known and novel serum protein biomarkers and has demonstrated specific associations with COPD disease severity, FEV(1), FEV(1)/FVC and DLCO. These data highlight systemic inflammatory pathways, neutrophil activation and epithelial tissue injury/repair processes as key pathways associated with COPD.     

颗粒酶B和胱天蛋白酶-3对肌动蛋白水解作用的体外研究

已知凋亡过程的基本变化之一是细胞骨架的异常,后者在某种程度上决定凋亡细胞的形态学特征.为揭示凋亡相关蛋白酶--颗粒酶B和胱天蛋白酶-3对胞浆型肌动蛋白的水解作用,采用成年猕猴脑组织粗提物作为无细胞体系,以外源性颗粒酶B触发凋亡途径的终末反应.经一系列免疫印迹分析发现:孵育12 h方见β-肌动蛋白被剪切,产生41 ku和15 ku水解片段,并证明该水解反应为颗粒酶B依赖;颗粒酶B活化的内源性胱天蛋白酶-3和重组胱天蛋白酶-3均不能水解脑提取物中的β-肌动蛋白,尽管胱天蛋白酶-3可作用于纯化的肌动蛋白,产生15 ku片段.以上结果提示,内源性β-肌动蛋白对凋亡相关蛋白酶,尤其胱天蛋白酶-3不敏感,这可能与该蛋白质的空间结构特征或脑组织中存在的某种蛋白酶抑制因子有关.     

Granzyme A, which causes single-stranded DNA damage, targets the double-strand break repair protein Ku70

Granzyme A (GzmA) induces caspase-independent cell death with morphological features of apoptosis. Here, we show that GzmA at nanomolar concentrations cleaves Ku70, a key double-strand break repair (DSBR) protein, in target cells. Ku70 is cut after Arg(301), disrupting Ku complex binding to DNA. Cleaving Ku70 facilitates GzmA-mediated cell death, as silencing Ku70 by RNA interference increases DNA damage and cell death by GzmB cluster-deficient cytotoxic T lymphocytes or by GzmA and perforin, whereas Ku70 overexpression has the opposite effect. Ku70 has two known antiapoptotic effects-facilitating DSBR and sequestering bax to prevent its translocation to mitochondria. However, GzmA triggers single-stranded, not double-stranded, DNA damage, and GzmA-induced cell death does not involve bax. Therefore, Ku70 has other antiapoptotic functions in GzmA-induced cell death, which are blocked when GzmA proteolyses Ku70.     

Airway epithelium controls lung inflammation and injury through the NF-kappa B pathway

Although airway epithelial cells provide important barrier and host defense functions, a crucial role for these cells in development of acute lung inflammation and injury has not been elucidated. We investigated whether NF-kappaB pathway signaling in airway epithelium could decisively impact inflammatory phenotypes in the lungs by using a tetracycline-inducible system to achieve selective NF-kappaB activation or inhibition in vivo. In transgenic mice that express a constitutively active form of IkappaB kinase 2 under control of the epithelial-specific CC10 promoter, treatment with doxycycline induced NF-kappaB activation with consequent production of a variety of proinflammatory cytokines, high-protein pulmonary edema, and neutrophilic lung inflammation. Continued treatment with doxycycline caused progressive lung injury and hypoxemia with a high mortality rate. In contrast, inducible expression of a dominant inhibitor of NF-kappaB in airway epithelium prevented lung inflammation and injury resulting from expression of constitutively active form of IkappaB kinase 2 or Escherichia coli LPS delivered directly to the airways or systemically via an osmotic pump implanted in the peritoneal cavity. Our findings indicate that the NF-kappaB pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli; therefore, targeting inflammatory pathways in airway epithelium could prove to be an effective therapeutic strategy for inflammatory lung diseases.     

Granzyme B activity in target cells detects attack by cytotoxic lymphocytes

Lymphocyte-mediated cytotoxicity via granule exocytosis operates by the perforin-mediated transfer of granzymes from CTLs and NK cells into target cells where caspase activation and other death pathways are triggered. Granzyme B (GzB) is a major cytotoxic effector in this pathway, and its fate in target cells has been studied by several groups using immunodetection. In this study, we have used a newly developed cell-permeable fluorogenic GzB substrate to measure this protease activity in three different living targets following contact with cytotoxic effectors. Although no GzB activity is measurable in CTL or NK92 effector cells, this activity rapidly becomes detectable throughout the target cytoplasm after effector-target engagement. We have combined the GzB substrate with a second fluorogenic substrate selective for caspase 3 to allow both flow cytometry and fluorescence confocal microscopy studies of cytotoxicity. With both effectors, caspase 3 activity appears subsequent to that of GzB inside all three targets. Overexpression of Bcl-2 in target cells has minimal effects on lysis, NK- or CTL-delivered GzB activity, or activation of target caspase 3. Detection of target GzB activity followed by caspase 3 activation provides a unique readout of a potentially lethal injury delivered by cytotoxic lymphocytes.     

Granzyme B proteolyzes receptors important to proliferation and survival, tipping the balance toward apoptosis

Granzyme B is critical to the ability of natural killer cells and cytotoxic T lymphocytes to induce efficient cell death of virally infected or tumor cell targets. Although granzyme B can cleave and activate caspases to induce apoptosis, granzyme B can also cause caspase-independent cell death. Thirteen prospective granzyme B substrates were identified from a cDNA expression-cleavage screen, including Hsp70, Notch1, fibroblast growth factor receptor-1 (FGFR1), poly-A-binding protein, cAbl, heterogeneous nuclear ribonucleoprotein H', Br140, and intersectin-1. Validation revealed that Notch1 is a substrate of both granzyme B and caspases, whereas FGFR1 is a caspase-independent substrate of granzyme B. Proteolysis of FGFR1 in prostate cancer cells has functionally relevant consequences that indicate its cleavage may be advantageous for granzyme B to kill prostate cancer cells. Therefore, granzyme B not only activates pro-death functions within a target, but also has a previously unidentified role in inactivating pro-growth signals to cause cell death.     

Granzyme B induces BID-mediated cytochrome c release and mitochondrial permeability transition

Many cell death pathways converge at the mitochondria to induce release of apoptogenic proteins and permeability transition, resulting in the activation of effector caspases responsible for the biochemical and morphological alterations of apoptosis. The death receptor pathway has been described as a triphasic process initiated by the activation of apical caspases, a mitochondrial phase, and then the final phase of effector caspase activation. Granzyme B (GrB) activates apical and effector caspases as well as promotes cytochrome c (cyt c) release and loss of mitochondrial membrane potential. We investigated how GrB affects mitochondria utilizing an in vitro cell-free system and determined that cyt c release and permeability transition are initiated by distinct mechanisms. The cleavage of cytosolic BID by GrB results in truncated BID, initiating mitochondrial cyt c release. BID is the sole cytosolic protein responsible for this phenomenon in vitro, yet caspases were found to participate in cyt c release in some cells. On the other hand, GrB acts directly on mitochondria in the absence of cytosolic S100 proteins to open the permeability transition pore and to disrupt the proton electrochemical gradient. We suggest that GrB acts by two distinct mechanisms on mitochondria that ultimately lead to mitochondrial dysfunction and cellular demise.     

Granzyme B cleavage of fibronectin disrupts endothelial cell adhesion,migration and capillary tube formation

Dysregulated angiogenesis contributes to the pathogenesis of chronic inflammatory diseases. Modulation of the extracellular matrix by immune-derived proteases can alter endothelial cell–matrix interactions as well as endothelial cell sprouting, migration and capillary formation. Granzyme B is a serine protease that is expressed by a variety of immune cells, and accumulates in the extracellular milieu in many chronic inflammatory disorders that are associated with dysregulated angiogenesis. Although granzyme B is known to cleave fibronectin, an essential glycoprotein in vascular morphogenesis, the role of granzyme B in modulating angiogenesis is unknown. In the present study, granzyme B cleaved both plasma fibronectin and cell-derived fibronectin, resulting in the release of multiple fibronectin fragments. Granzyme B cleavage of fibronectin resulted in a dose-dependent reduction in endothelial cell adhesion to fibronectin as well as reduced endothelial cell migration and tubular formation. These events were prevented when granzyme B activity was inhibited by a small molecule inhibitor. In summary, granzyme B-mediated cleavage of fibronectin contributes to attenuated angiogenesis through the disruption of endothelial cell — fibronectin interaction resulting in impaired endothelial cell migration and tubular formation.     

Matrix metalloproteinases in liver injury,repair and fibrosis

The liver is a large highly vascularized organ with a central function in metabolic homeostasis, detoxification, and immunity. Due to its roles, the liver is frequently exposed to various insults which can cause cell death and hepatic dysfunction. Alternatively, the liver has a remarkable ability to self-repair and regenerate after injury. Liver injury and regeneration have both been linked to complex extracellular matrix (ECM) related pathways. While normal degradation of ECM components is an important feature of tissue repair and remodeling, irregular ECM turnover contributes to a variety of liver diseases. Matrix metalloproteinases (MMPs) are the main enzymes implicated in ECM degradation. MMPs not only remodel the ECM, but also regulate immune responses. In this review, we highlight some of the MMP-attributed roles in acute and chronic liver injury and emphasize the need for further experimentation to better understand their functions during hepatic physiological conditions and disease progression.     

Direct targeting of sEH with alisol B alleviated the apoptosis,inflammation, and oxidative stress in cisplatin-induced acute kidney injury

Acute kidney injury (AKI) is a pathological condition characterized by a rapid decrease in glomerular filtration rate and nitrogenous waste accumulation during hemodynamic regulation. Alisol B, from Alisma orientale, displays anti-tumor, anti-complement, and anti-inflammatory effects. However, its effect and action mechanism on AKI is still unclear. Herein, alisol B significantly attenuated cisplatin (Cis)-induced renal tubular apoptosis through decreasing expressions levels of cleaved-caspase 3 and cleaved-PARP and the ratio of Bax/Bcl-2 depended on the p53 pathway. Alisol B also alleviated Cis-induced inflammatory response (e.g. the increase of ICAM-1, MCP-1, COX-2, iNOS, IL-6, and TNF-α) and oxidative stress (e.g. the decrease of SOD and GSH, the decrease of HO-1, GCLC, GCLM, and NQO-1) through the NF-κB and Nrf2 pathways. In a target fishing experiment, alisol B bound to soluble epoxide hydrolase (sEH) as a direct cellular target through the hydrogen bond with Gln384, which was further supported by inhibition kinetics and surface plasmon resonance (equilibrium dissociation constant, K_D = 1.32 μM). Notably, alisol B enhanced levels of epoxyeicosatrienoic acids and decreased levels of dihydroxyeicosatrienoic acids, indicating that alisol B reduced the sEH activity in vivo. In addition, sEH genetic deletion alleviated Cis-induced AKI and abolished the protective effect of alisol B in Cis-induced AKI as well. These findings indicated that alisol B targeted sEH to alleviate Cis-induced AKI via GSK3β-mediated p53, NF-κB, and Nrf2 signaling pathways and could be used as a potential therapeutic agent in the treatment of AKI.     

Granzyme B regulates antiviral CD8+ T cell responses

CTLs and NK cells use the perforin/granzyme cytotoxic pathway to kill virally infected cells and tumors. Human regulatory T cells also express functional granzymes and perforin and can induce autologous target cell death in vitro. Perforin-deficient mice die of excessive immune responses after viral challenges, implicating a potential role for this pathway in immune regulation. To further investigate the role of granzyme B in immune regulation in response to viral infections, we characterized the immune response in wild-type, granzyme B-deficient, and perforin-deficient mice infected with Sendai virus. Interestingly, granzyme B-deficient mice, and to a lesser extent perforin-deficient mice, exhibited a significant increase in the number of Ag-specific CD8(+) T cells in the lungs and draining lymph nodes of virally infected animals. This increase was not the result of failure in viral clearance because viral titers in granzyme B-deficient mice were similar to wild-type mice and significantly less than perforin-deficient mice. Regulatory T cells from WT mice expressed high levels of granzyme B in response to infection, and depletion of regulatory T cells from these mice resulted in an increase in the number of Ag-specific CD8(+) T cells, similar to that observed in granzyme B-deficient mice. Furthermore, granzyme B-deficient regulatory T cells displayed defective suppression of CD8(+) T cell proliferation in vitro. Taken together, these results suggest a role for granzyme B in the regulatory T cell compartment in immune regulation to viral infections.     

Gap junctional communication in tissue inflammation and repair

Local injury induces a complex orchestrated response to stimulate healing of injured tissues, cellular regeneration and phagocytosis. Practically, inflammation is defined as a defense process whereby fluid and white blood cells accumulate at a site of injury. The balance of cytokines, chemokines, and growth factors is likely to play a key role in regulating important cell functions such as migration, proliferation, and matrix synthesis during the process of inflammation. Hence, the initiation, maintenance, and resolution of innate responses depend upon cellular communication. A process similar to tissue repair and subsequent scarring is found in a variety of fibrotic diseases. This may occur in a single organ such as liver, kidneys, pancreas, lung, skin, and heart, but fibrosis may also have a more generalized distribution such as in atherosclerosis. The purpose of this review is to summarize recent advances on the contribution of gap junction-mediated intercellular communication in the modulation of the inflammatory response and tissue repair.     

Gap junctional communication in tissue inflammation and repair

Local injury induces a complex orchestrated response to stimulate healing of injured tissues, cellular regeneration and phagocytosis. Practically, inflammation is defined as a defense process whereby fluid and white blood cells accumulate at a site of injury. The balance of cytokines, chemokines, and growth factors is likely to play a key role in regulating important cell functions such as migration, proliferation, and matrix synthesis during the process of inflammation. Hence, the initiation, maintenance, and resolution of innate responses depend upon cellular communication. A process similar to tissue repair and subsequent scarring is found in a variety of fibrotic diseases. This may occur in a single organ such as liver, kidneys, pancreas, lung, skin, and heart, but fibrosis may also have a more generalized distribution such as in atherosclerosis. The purpose of this review is to summarize recent advances on the contribution of gap junction-mediated intercellular communication in the modulation of the inflammatory response and tissue repair.