Cathepsin G-mediated proteolytic degradation of MHC class I molecules to facilitate immune detection of human glioblastoma cells.

To mount an adaptive immune response, MHC I molecules present antigenic peptides to CTLs. Transcriptional reduction of MHC I molecules is a strategy of immune evasion, which impairs the detection of infected or tumorous cells by CTLs. Natural killer (NK) cells, on the other hand, eliminate target cells specifically in the absence of MHC I. Consequently, infected or tumorous cells partly retain their MHC I at the cell surface to avoid NK recognition. However, it remains unclear which protease degrades MHC I molecules and how these cells maintain a limited set of MHC I at the cell surface. Here, we demonstrate that cathepsin G (CatG), a serine protease, found in the endocytic compartment of APCs and, to a lesser extent, CatD and CatS proteolytically degrade MHC I molecules. Inhibition of CatG boosted MHC I expression at the cell surface of primary human immune cells. In contrast, human glioblastoma cells do not harbor active CatG and might have lost the ability to proteolytically degrade MHC I during tumorigenesis to avoid NK-mediated killing. Overexpression of CatG in glioblastoma cells resulted in a rapid and efficient MHC I degradation. In conclusion, CatG is an essential protease for regulating MHC I molecules and thus modulation of CatG activity might present a new avenue for therapeutic intervention.

Cathepsin G deficiency reduces periaortic calcium chloride injury-induced abdominal aortic aneurysms in mice.

OBJECTIVE: Cathepsin G (CatG) is a serine protease that mediates angiotensin I to angiotensin II (Ang-II) conversion and is highly expressed in human abdominal aortic aneurysms (AAAs). However, it remains untested whether this protease participates in the pathogenesis of AAA. METHODS AND RESULTS: Immunofluorescent double staining demonstrated the expression of CatG in smooth muscle cells (SMCs), macrophages, and endothelial cells in human AAA lesions (n = 12) but not in AAA-free aortas (n = 10). Whereas inflammatory cytokines induced CatG expression, high glucose concentration increased CatG activity in producing Ang-II and angiotensin-converting enzyme in SMCs, which could be fully blocked by a CatG-selective inhibitor or its small interfering RNA. To test whether CatG contributes to AAA development, we generated CatG and low-density lipoprotein receptor double deficient (Ldlr(-/-)Ctsg(-/-)) mice and their littermate controls (Ldlr(-/-)Ctsg(+/+)). Absence of CatG did not affect Ang-II infusion-induced AAAs. In contrast, in Ang-II-independent AAAs induced by periaortic CaCl2 injury (n = 12 per group), CatG deficiency significantly reduced aortic diameter increase (58.33% ± 6.83% vs 31.67% ± 5.75%; P = .007), aortic lesion area (0.35 ± 0.04 mm(2) vs 0.21 ± 0.02 mm(2); P = .005), and aortic wall elastin fragmentation grade (2.75 ± 0.18 vs 1.58 ± 0.17; P = .002) along with reduced lesion collagen content grade (2.80 ± 0.17 vs 2.12 ± 0.17; P = .009) without affecting indices of lesion inflammation, angiogenesis, cell proliferation, or apoptosis. In vitro elastin degradation assays demonstrated that CaCl2-induced AAA lesions from Ldlr(-/-)Ctsg(-/-) mice contained much lower elastinolytic activity than in those from littermate control mice. Gelatin gel zymogram assay suggested that absence of CatG in CaCl2-induced AAA lesions also reduced the activity of elastinolytic matrix metalloproteinases 2 and 9. CONCLUSIONS: CatG may contribute to CaCl2-induced experimental AAAs directly through its elastinolytic activity and indirectly by regulating lesion matrix metalloproteinases 2 and 9 activities. Increased expression of CatG in vascular and inflammatory cells of human AAAs and its increased activity in producing Ang-II and angiotensin-converting enzyme by SMCs suggest an additional mechanism by which CatG contributes to AAA lesion progression.

The Role of Serine Proteases and Antiproteases in the Cystic Fibrosis Lung.

Cystic fibrosis (CF) lung disease is an inherited condition with an incidence rate of approximately 1 in 2500 new born babies. CF is characterized as chronic infection of the lung which leads to inflammation of the airway. Sputum from CF patients contains elevated levels of neutrophils and subsequently elevated levels of neutrophil serine proteases. In a healthy individual these proteases aid in the phagocytic process by degrading microbial peptides and are kept in homeostatic balance by cognate antiproteases. Due to the heavy neutrophil burden associated with CF the high concentration of neutrophil derived proteases overwhelms cognate antiproteases. The general effects of this protease/antiprotease imbalance are impaired mucus clearance, increased and self-perpetuating inflammation, and impaired immune responses and tissue. To restore this balance antiproteases have been suggested as potential therapeutics or therapeutic targets. As such a number of both endogenous and synthetic antiproteases have been trialed with mixed success as therapeutics for CF lung disease.

Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases.

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.

[Unintended Observations Leading to Macrophage Growth and Neutrophil Factor Research].

My research area in the pharmaceutical industry is innate immunity, especially in phagocytic cells. First, I studied the heat-stable growth factor of peripheral macrophages in tumorous ascitic fluid and found that lipoproteins are an influencing factor. Later, my colleagues and I found that lipid-containing substances, namely, oxidized low-density lipoprotein, dead neutrophils, or purified lipids that could be scavenged by macrophages, induce their growth. From the series of this study, I concluded that phagocytic substances induce macrophage growth by autocrine stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF). During the study, we found that neutrophils have growth-inhibitory effects against a variety of cells. Then, I elucidated that the primary factor is a zinc-binding protein, calprotectin, an abundant protein complex in the neutrophil cytosol. I found that calprotectin induces apoptosis in many cell types, including tumor cells and normal fibroblasts, and that the zinc-binding capacity is essential for its activity. Microscopic observations revealed that neutrophil extract contains factor-inducing three-dimensional cell aggregation of human mammary carcinoma, MCF-7. I elucidated that cathepsin G is responsible for this activity and that its effect is dependent on the activation of insulin-like growth factor-1. I believe that this modest, albeit novel, observation was crucial to my thirty-nine-year-long career researching phagocytic cells.

Cathepsin G-dependent modulation of platelet thrombus formation in vivo by blood neutrophils.

Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

Intracolonic infusion of fecal supernatants from ulcerative colitis patients triggers altered permeability and inflammation in mice: role of cathepsin G and protease-activated receptor-4.

BACKGROUND: Cathepsin G (Cat-G) is a neutrophil serine-protease found in the colonic lumen of ulcerative colitis (UC) patients. Cat-G is able to activate protease-activated receptor-4 (PAR(4) ) located at the apical side of enterocytes, leading to epithelial barrier disruption. However, the mechanisms through which Cat-G triggers inflammation are not fully elucidated. The aims of our study were to evaluate in vivo the effects of UC fecal supernatants and Cat-G on epithelial barrier function and inflammation, and the connection between these two parameters. METHODS: Male balb/c mice were used in this study. We evaluated the effect of a 2-hour intracolonic infusion of 1) fecal supernatants from UC patients pretreated or not with specific Cat-G inhibitor (SCGI); 2) PAR(4) -activating peptide (PAR(4) -AP); and 3) Cat-G on colonic myeloperoxidase (MPO) activity and paracellular permeability (CPP). The involvement of PAR(4) was assessed by pretreating animals with pepducin P4pal-10, which blocks PAR(4) signaling. We investigated the role of myosin light chain (MLC) kinase by using its inhibitor, ML-7, and we determined phosphorylated MLC (pMLC) levels in mice colonic mucosa. RESULTS: UC fecal supernatants, Cat-G, and PAR(4) agonist increased both CPP and MPO activity in comparison with healthy subjects fecal supernatants. ML-7 inhibited the CPP increase triggered by Cat-G by 92.3%, and the enhanced MPO activity by 43.8%. Intracolonic infusion of UC fecal supernatant determined an increased phosphorylation level of MLC. CONCLUSIONS: These observations support that luminal factors such as Cat-G play an important proinflammatory role in the pathogenesis of colitis, mainly depending on CPP increase by MLC phosphorylation.

Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.

Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases neutrophil elastase and cathepsin G, together with externalized nucleosomes, promote coagulation and intravascular thrombus growth in vivo. The serine proteases and extracellular nucleosomes enhance tissue factor- and factor XII-dependent coagulation in a process involving local proteolysis of the coagulation suppressor tissue factor pathway inhibitor. During systemic infection, activation of coagulation fosters compartmentalization of bacteria in liver microvessels and reduces bacterial invasion into tissue. In the absence of a pathogen challenge, neutrophil-derived serine proteases and nucleosomes can contribute to large-vessel thrombosis, the main trigger of myocardial infarction and stroke. The ability of coagulation to suppress pathogen dissemination indicates that microvessel thrombosis represents a physiological tool of host defense.