cathepsin-g and Disease-Models--Animal

The discovery of the association of emphysema and homozygous alpha-1-proteinase inhibitor deficiency gave rise to the hypothesis that emphysema was caused by an imbalance between endogenous proteases and antiproteases. Experimental studies with enzymes have confirmed that emphysema is induced only by enzymes with elastolytic properties. The lesion produced is similar anatomically and physiologically to human emphysema. Genetic and nutritional models of emphysema that appear to be caused by elastic fiber degradation are also known. Alpha-1-protease inhibitor is normally present in the lung; alpha-2-macroglobulin which can loosely complex with elastases, may enter the injured lung and participate in its defense. Alpha-1-protease inhibitor may be inactivated by oxidants derived from cigarette smoke or endogenous phagocytes. The elastase/antielastase hypothesis of emphysema is also believed to be relevant in emphysema caused by cigarette smoking because cigarette smoke gives rise to increased numbers of neutrophils and macrophages in the lung thus increasing the elastase burden of the lung. The precise role of oxidation of alpha-1-protease inhibitor in-vivo by cigarette smoke is not yet clear. In-vitro and experimental data suggest that oxidants can also interfere with repair of lung matrix. A variety of injuries, including proteases, can give rise to secretory cell metaplasia in the central airways. Peripheral airways injury is produced by gases with oxidant properties such as ozone and NO2. The presence of a low molecular weight protease inhibitor in airway epithelial secretory cells when considered with enzyme-induced secretory cell metaplasia raises the possibility of a protease/antiprotease hypothesis of chronic bronchial injury. Although information on the pathogenesis of emphysema is still incomplete, efforts are being made to develop antiproteases which might be used in the prevention of emphysema and chronic bronchial injury.

Topics: alpha 1-Antitrypsin; Animals; Blood Proteins; Bronchitis; Cathepsin G; Cathepsins; Disease Models, Animal; Humans; Neutrophils; Pancreatic Elastase; Pulmonary Emphysema; Serine Endopeptidases

Plasma levels of antithrombin III, alpha 2-macroglobulin and inter-alpha-trypsin inhibitor, as well as those of various clotting, complement and other plasma factors, were significantly decreased in 18 patients suffering from hyperdynamic septic shock. A similar statistically significant reduction of the concentrations of several plasma factors (prothrombin and antithrombin III, plasminogen and alpha 2-plasmin inhibitor, complement factor C3 and clotting factor XIII) was observed in experimental endotoxaemia. In this model the reduction in the plasma levels of these factors was considerably diminished by the intravenous injection of a granulocytic elastase--cathepsin G inhibitor of lower molecular weight from soybeans. The results of both studies indicate that consumption of plasma factors in the course of Gram-negative sepsis proceeds not only via the classical routes (by activation of the clotting, fibrinolytic and complement cascades by system-specific proteinases such as thrombokinase or the plasminogen activator) but also to an appreciable degree of unspecific degradation of plasma factors by neutral proteinases such as elastase and cathepsin G. The endotoxin-induced release of both sorts of proteinases, the system-specific ones and the unspecific lysosomal proteinases from leucocytes and other cells, is likely to be mainly responsible for the consumption of antithrombin III and alpha-2-macroglobulin via complex formation (followed by elimination of the complexes) and the increased turnover of the inter-alpha-trypsin inhibitor as observed in the clinical study. The therapeutic use of an exogenous elastase--cathepsin G inhibitor in the experimental model was stimulated by the observation that human mucous secretions contain and acid-stable inhibitor of the neutral granulocytic proteinases, called HUSI-I or antileucoproteinase. This inhibitor protects mucous membranes and soluble proteins against proteolytic attack by leucocytic proteinases released in the course of a local inflammatory response. Preliminary results indicate that HUSI-I, which is produced by the epithelial cells of mucous membranes, does not belong to any known structural type of acid-stable proteinase inhibitor. The search for other candidates suitable for medication in humans led to the discovery of a potent elastase--cathepsin G inhibitor, called eglin, in the leech Hirudo medicinalis. This acid-stable inhibitor with a molecular weight close to 8100 has an unusual structural propert

Topics: Adolescent; Adult; Aged; Animals; Cathepsin G; Cathepsins; Cattle; Disease Models, Animal; Dogs; Endotoxins; Escherichia coli; Humans; Middle Aged; Pancreatic Elastase; Protease Inhibitors; Sepsis; Serine Endopeptidases; Shock, Septic

Type 1 diabetes (T1D) is a chronic autoimmune disease accompanied by the immune-mediated destruction of pancreatic β-cells. In this study, we aimed to explore the regulatory effects of vitamin D (VD) supplementation on pancreatic β-cell function by altering the expression of bioinformatically identified cathepsin G (CatG) in T1D mice. A T1D mouse model was established in nonobese diabetic (NOD) mice, and their islets were isolated and purified. Pancreatic mononuclear cells (MNCs) were collected, from which CD4

Topics: Animals; Cathepsin G; CD4-Positive T-Lymphocytes; Cytokines; Diabetes Mellitus, Type 1; Dietary Supplements; Disease Models, Animal; Down-Regulation; Gene Knockdown Techniques; Immunosuppressive Agents; Insulin-Secreting Cells; Lymphocyte Activation; Mice; Mice, Inbred NOD; RNA, Small Interfering; Signal Transduction; Vitamin D

Topics: Animals; Blood Platelets; Cathepsin G; Disease Models, Animal; Gene Expression; Lipocalin-2; Megakaryocytes; Mice; Neoplasms

Inflammation has emerged as a key component of the pathophysiology of intracranial aneurysms. Mast cells have been detected in human intracranial aneurysm tissues, and their presence was associated with intramural microhemorrhage and wall degeneration. We hypothesized that mast cells play a critical role in the development of aneurysmal rupture, and that mast cells can be used as a therapeutic target for the prevention of aneurysm rupture.. Intracranial aneurysms were induced in adult mice using a combination of induced systemic hypertension and a single injection of elastase into the cerebrospinal fluid. Aneurysm formation and rupture were assessed over 3 weeks. Roles of mast cells were assessed using a mast cell stabilizer (cromolyn), a mast cell activator (C48/80), and mice that are genetically lacking mature mast cells (Kit. Pharmacological stabilization of mast cells with cromolyn markedly decreased the rupture rate of aneurysms (80% versus 19%, n=10 versus n =16) without affecting the aneurysm formation. The activation of mast cells with C48/80 significantly increased the rupture rate of aneurysms (25% versus 100%, n=4 versus n=5) without affecting the overall rate of aneurysm formation. Furthermore, the genetic deficiency of mast cells significantly prevented aneurysm rupture (80% versus 25%, n=10 versus n=8, wild-type versus Kit. These results suggest that mast cells play a key role in promoting aneurysm rupture but not formation. Stabilizers of mast cells may have a potential therapeutic value in preventing intracranial aneurysm rupture in patients.

Topics: Aneurysm, Ruptured; Animals; Cathepsin G; Chymases; Cromolyn Sodium; Disease Models, Animal; Interleukin-6; Intracranial Aneurysm; Male; Mast Cell Stabilizers; Mast Cells; Matrix Metalloproteinase 9; Mice; Mice, Transgenic; Mutation; p-Methoxy-N-methylphenethylamine; Proto-Oncogene Proteins c-kit; Receptor, Angiotensin, Type 1; RNA, Messenger; Subarachnoid Hemorrhage; Tryptases; Tumor Necrosis Factor-alpha

Acute pancreatitis is characterized by an early intracellular protease activation and invasion of leukocytes into the pancreas. Cathepsins constitute a large group of lysosomal enzymes, that have been shown to modulate trypsinogen activation and neutrophil infiltration. Cathepsin G (CTSG) is a neutrophil serine protease of the chymotrypsin C family known to degrade extracellular matrix components and to have regulatory functions in inflammatory disorders. The aim of this study was to investigate the role of CTSG in pancreatitis. Isolated acinar cells were exposed to recombinant CTSG and supramaximal cholezystokinin stimulation. In CTSG

Topics: Acinar Cells; Animals; Cathepsin G; Cells, Cultured; Ceruletide; Disease Models, Animal; Gene Knockout Techniques; Granulocytes; Male; Mice; Neutrophil Infiltration; Neutrophils; Pancreatitis; Trypsinogen

This study aims to evaluate the role of chymostatin in paraquat-induced acute lung injury. Institute of Cancer Research mice were randomly distributed into the NS, DMSO, chymostatin, paraquat or chymostatin treatment groups. Six mice from each group were intraperitoneally injected with chloral hydrate at 0, 1, 2, 4, 8, 12, 24 and 48 h after treatment administration. Blood samples were collected through cardiac puncture. Lung tissues were stained with haematoxylin and eosin for the observation of lung histology. The degree of pulmonary oedema was determined on the basis of lung wet-to-dry ratio (W/D). The serum activity of cathepsin G was determined through substrate fluorescence assay. The serum levels of endothelial cell-specific molecule-1 (endocan), tumour necrosis factor-a (TNF-a), interleukin-1β (IL-1β), IL-6 and high-mobility group box protein 1 (HMGB1) were determined through enzyme-linked immunosorbent assay. The expression levels of endocan and nuclear NF-κBp65 in the lung were quantified through Western blot. Chymostatin alleviated the pathological changes associated with acute alveolitis in mice; decreased the lung W/D ratio, the activity of cathepsin G and the serum concentrations of TNF-a, IL-1β, IL-6 and HMGB1; and increased the serum concentration of endocan. Western blot results revealed that chymostatin up-regulated endocan expression and down-regulated nuclear NF-κBp65 expression in the lung. Chymostatin reversed the inflammatory effects of paraquat-induced lung injury by inhibiting cathepsin G activity to up-regulate endocan expression and indirectly inhibit NF-κBp65 activity.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Cathepsin G; Cytokines; Cytoprotection; Disease Models, Animal; Female; HMGB1 Protein; Inflammation Mediators; Lung; Mice, Inbred ICR; Oligopeptides; Paraquat; Proteoglycans; Pulmonary Edema; Time Factors; Transcription Factor RelA

Despite the availability of vaccines,

Topics: Administration, Oral; Adult; Age Factors; Aged; alpha-Tocopherol; Animals; Anti-Infective Agents; Cathepsin G; Cell Movement; Disease Models, Animal; Epithelium; Humans; Inflammation; Leukocyte Elastase; Lung; Mice; Neutrophils; Phagocytosis; Pneumonia, Pneumococcal; Serine Proteases; Streptococcus pneumoniae; Vitamin E

Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the context of arterial myeloid cell recruitment.. Intravital microscopy of the carotid artery, the jugular vein, and cremasteric arterioles and venules in Apoe. Our observations elucidated a crucial role for CatG during arterial leukocyte adhesion, an effect not found during venular adhesion. Consequently, CatG deficiency attenuates atherosclerosis but not acute lung inflammation. Mechanistically, CatG is immobilized on arterial endothelium where it activates leukocytes to firmly adhere engaging integrin clustering, a process of crucial importance to achieve effective adherence under high-shear flow. Therapeutic neutralization of CatG specifically abrogated arterial leukocyte adhesion without affecting myeloid cell adhesion in the microcirculation. Repetitive application of CatG-neutralizing antibodies permitted inhibition of atherogenesis in mice.. Taken together, these findings present evidence of an arterial-specific recruitment pattern centered on CatG-instructed adhesion strengthening. The inhibition of this process could provide a novel strategy for treatment of arterial inflammation with limited side effects.

Topics: Animals; Arteries; Atherosclerosis; Biomarkers; Cathepsin G; Cell Adhesion; Chemokine CCL5; Chemotaxis; Disease Models, Animal; Endothelium, Vascular; Humans; Integrins; Leukocyte Rolling; Mice; Mice, Knockout; Microcirculation; Myeloid Cells; Protein Binding; Shear Strength; Venules

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to cytotoxic T cells. During an adaptive immune response, MHC molecules are regulated by several mechanisms including lipopolysaccharide (LPS) and interferon gamma (IFN-g). However, it is unclear whether the serine protease cathepsin G (CatG), which is generally secreted by neutrophils at the site of inflammation, might regulate MHC I molecules. We identified CatG, and to a higher extend CatG and lactoferrin (LF), as an exogenous regulator of cell surface MHC I expression of immune cells and glioblastoma stem cells. In addition, levels of MHC I molecules are reduced on dendritic cells from CatG deficient mice compared to their wild type counterparts. Furthermore, cell surface CatG on immune cells, including T cells, B cells, and NK cells triggers MHC I on THP-1 monocytes suggesting a novel mechanism for CatG to facilitate intercellular communication between infiltrating cells and the respective target cell. Subsequently, our findings highlight the pivotal role of CatG as a checkpoint protease which might force target cells to display their intracellular MHC I:antigen repertoire.

Topics: Animals; Cathepsin G; Cell Line; Cell Line, Tumor; Cell Membrane; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Histocompatibility Antigens Class I; Humans; Immune System; Lactoferrin; Male; Mice; Mice, Knockout; Monocytes; Neoplastic Stem Cells; Proteolysis

Inflammation is an essential process in many pulmonary diseases in which kinins are generated by protease action on kininogen, a phenomenon that is blocked by protease inhibitors. We evaluated kinin release in an

Topics: Animals; Caesalpinia; Cathepsin G; Disease Models, Animal; Kininogens; Models, Biological; Neutrophils; Phytochemicals; Plasma Kallikrein; Pneumonia; Protease Inhibitors; Rats; Seeds

Current treatments for inflammation associated with bronchopulmonary dysplasia (BPD) fail to show clinical efficacy. Foxm1, a transcription factor of the Forkhead box family, is a critical mediator of lung development and carcinogenesis, but its role in BPD-associated pulmonary inflammation is unknown. Immunohistochemistry and RNA analysis were used to assess Foxm1 in lung tissue from hyperoxia-treated mice and patients with BPD. LysM-Cre/Foxm1(-/-) mice, in which Foxm1 was deleted from myeloid-derived inflammatory cells, including macrophages, monocytes, and neutrophils, were exposed to neonatal hyperoxia, causing lung injury and remodeling. Measurements of lung function and flow cytometry were used to evaluate the effects of Foxm1 deletion on pulmonary inflammation and repair. Increased Foxm1 expression was observed in pulmonary macrophages of hyperoxia-exposed mice and in lung tissue from patients with BPD. After hyperoxia, deletion of Foxm1 from the myeloid cell lineage decreased numbers of interstitial macrophages (CD45(+)CD11b(+)Ly6C(-)Ly6G(-)F4/80(+)CD68(-)) and impaired alveologenesis and lung function. The exaggerated BPD-like phenotype observed in hyperoxia-exposed LysM-Cre/Foxm1(-/-) mice was associated with increased expression of neutrophil-derived myeloperoxidase, proteinase 3, and cathepsin g, all of which are critical for lung remodeling and inflammation. Our data demonstrate that Foxm1 influences pulmonary inflammatory responses to hyperoxia, inhibiting neutrophil-derived enzymes and enhancing monocytic responses that limit alveolar injury and remodeling in neonatal lungs.

Topics: Airway Remodeling; Alveolar Epithelial Cells; Animals; Bronchopulmonary Dysplasia; Case-Control Studies; Cathepsin G; Disease Models, Animal; Forkhead Box Protein M1; Forkhead Transcription Factors; Humans; Hyperoxia; Infant, Newborn; Lung; Lung Injury; Macrophages; Mice, Knockout; Myeloblastin; Neutrophils; Peroxidase; Pneumonia

Neutrophil elastase (NE) and cathepsin G (CG) contribute to intracellular microbial killing but, if left unchecked and released extracellularly, promote tissue damage. Conversely, mechanisms that constrain neutrophil serine protease activity protect against tissue damage but may have the untoward effect of disabling the microbial killing arsenal. The host elaborates thrombospondin-1 (TSP-1), a matricellular protein released during inflammation, but its role during neutrophil activation following microbial pathogen challenge remains uncertain. Mice deficient in TSP-1 (thbs1(-/-)) showed enhanced lung bacterial clearance, reduced splenic dissemination, and increased survival compared with wild-type (WT) controls during intrapulmonary Klebsiella pneumoniae infection. More effective pathogen containment was associated with reduced burden of inflammation in thbs1(-/-) mouse lungs compared with WT controls. Lung NE activity was increased in thbs1(-/-) mice following K. pneumoniae challenge, and thbs1(-/-) neutrophils showed enhanced intracellular microbial killing that was abrogated with recombinant TSP-1 administration or WT serum. Thbs1(-/-) neutrophils exhibited enhanced NE and CG enzymatic activity, and a peptide corresponding to amino-acid residues 793-801 within the type-III repeat domain of TSP-1 bridled neutrophil proteolytic function and microbial killing in vitro. Thus, TSP-1 restrains proteolytic action during neutrophilic inflammation elicited by K. pneumoniae, providing a mechanism that may regulate the microbial killing arsenal.

Topics: Animals; Cathepsin G; Cytotoxicity, Immunologic; Disease Models, Animal; Immunity, Innate; Klebsiella Infections; Klebsiella pneumoniae; Leukocyte Elastase; Lung; Mice; Mice, Knockout; Neutrophils; Peptides; Recombinant Proteins; Respiratory Burst; Serine Proteases; Spleen; Thrombospondin 1

Cigarette smoking is a major factor for the development of pulmonary emphysema because it induces abnormal inflammation and a protease-rich local milieu that causes connective tissue breakdown of the lungs. As a result of its capacity to degrade lung tissue and the high risk of patients lacking α1-antitrypsin to develop emphysema, much interest has focused on neutrophil elastase (NE). Two similar neutrophil serine proteases (NSPs), cathepsin G and proteinase 3, coexist with NE in humans and mice, but their potential tissue-destructive role(s) remains unclear. Using a gene-targeting approach, we observed that in contrast to their wild-type littermates, mice deficient in all three NSPs were substantially protected against lung tissue destruction after long-term exposure to cigarette smoke. In exploring the underlying basis for disrupted wild-type lung air spaces, we found that active NSPs collectively caused more severe lung damage than did NE alone. Furthermore, NSP activities unleashed increased activity of the tissue-destructive proteases macrophage elastase (matrix metalloproteinase-12) and gelatinase B (matrix metalloproteinase-9). These in vivo data provide, for the first time, compelling evidence of the collateral involvement of cathepsin G, NE, and proteinase 3 in cigarette smoke-induced tissue damage and emphysema. They also reveal a complex positive feed-forward loop whereby these NSPs induce the destructive potential of other proteases, thereby generating a chronic and pathogenic protease-rich milieu.

Topics: Animals; Blotting, Western; Cathepsin G; Disease Models, Animal; Leukocyte Elastase; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloblastin; Pulmonary Emphysema; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Smoking

Acute promyelocytic leukemia (APL) is initiated by the PML-RARA (PR) fusion oncogene and has a characteristic expression profile that includes high levels of the Notch ligand Jagged-1 (JAG1). In this study, we used a series of bioinformatic, in vitro, and in vivo assays to assess the role of Notch signaling in human APL samples, and in a PML-RARA knock-in mouse model of APL (Ctsg-PML-RARA). We identified a Notch expression signature in both human primary APL cells and in Kit+Lin-Sca1+ cells from pre-leukemic Ctsg-PML-RARA mice. Both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells from pre-leukemic Ctsg-PML-RARA mice, but had no influence on cells from age-matched wild-type mice. In addition, six of nine murine APL tumors tested displayed diminished growth in vitro when Notch signaling was inhibited pharmacologically. Finally, we found that genetic inhibition of Notch signaling with a dominant-negative Mastermind-like protein reduced APL growth in vivo in a subset of tumors. These findings expand the role of Notch signaling in hematopoietic diseases, and further define the mechanistic events important for PML-RARA-mediated leukemogenesis.

Topics: Animals; Bone Marrow Cells; Calcium-Binding Proteins; Cathepsin G; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Leukemia, Promyelocytic, Acute; Male; Membrane Proteins; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Oncogene Proteins, Fusion; Receptor, Notch1; Serrate-Jagged Proteins; Signal Transduction

Neutrophil serine proteases in cystic fibrosis (CF) lung secretions partially resist inhibition by natural and exogenous inhibitors, mostly because DNA impairs their control. Cationic polypeptides display the property of condensing DNA and retain antimicrobial properties. We hypothesized that DNA condensation by cationic polypeptides in CF sputum would result in a better control of CF inflammation and infection.. We examined whether poly-L-lysine would compact DNA in CF lung secretions and liquefy CF sputum, improve the control of extracellular proteases by exogenous inhibitors, and whether it displays antibacterial properties toward CF-associated bacteria.. We used fluorogenic methods to measure proteolytic activities and inhibition by protease inhibitors in whole sputum homogenates from patients with CF before and after treatment with poly-L-lysine. Antibacterial properties of poly-L-lysine were measured in bacterial cultures and in whole CF sputum. Poly-L-lysine toxicity was evaluated after aerosolization by histologic analysis, flow cytometry, and quantification of proinflammatory cytokines.. Poly-L-lysine compacts CF sputum DNA, generating a liquid phase that improves ciliary beating frequency at the lung epithelial surface, and allows the control of neutrophil elastase and cathepsin G by their natural inhibitors. It retains antimicrobial properties against Pseudomonas aeruginosa and Staphylococcus aureus at doses that induce no inflammation in the mouse lung after aerosol administration.. Poly-L-lysine may be an alternative to dornase-α to liquefy sputum with added benefits because it helps natural inhibitors to better control the deleterious effects of extracellularly released neutrophil serine proteases and has the ability to kill bacteria in CF sputum.

Topics: Adult; Aged; Animals; Anti-Bacterial Agents; Cathepsin G; Cystic Fibrosis; Disease Models, Animal; DNA; Female; Flow Cytometry; Humans; Leukocyte Elastase; Lung; Lysine; Male; Mice; Middle Aged; Neutrophils; Peptide Hydrolases; Proteolysis; Pseudomonas aeruginosa; Sputum; Staphylococcus aureus

Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 Å. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications.

Topics: Amino Acid Sequence; Animals; Cathepsin G; Chymases; Crystallization; Disease Models, Animal; Female; Gene Expression; Humans; Inflammation; Insect Proteins; Ixodes; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Platelet Aggregation; Protein Structure, Quaternary; Salivary Proteins and Peptides; Sequence Analysis, Protein; Serpins

Trousseau syndrome is classically defined as migratory, heparin-sensitive but warfarin-resistant microthrombi in patients with occult, mucinous adenocarcinomas. Injecting carcinoma mucins into mice generates platelet-rich microthrombi dependent on P- and L-selectin but not thrombin. Heparin prevents mucin binding to P- and L-selectin and mucin-induced microthrombi. This model of Trousseau syndrome explains resistance to warfarin, which inhibits fluid-phase coagulation but not selectins. Here we found that carcinoma mucins do not generate microthrombi in mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte ligand for P- and L-selectin. Furthermore, mucins did not activate platelets in blood from PSGL-1-deficient mice. Mucins induced microthrombi in radiation chimeras lacking endothelial P-selectin but not in chimeras lacking platelet P-selectin. Mucins caused leukocytes to release cathepsin G, but only if platelets were present. Mucins failed to generate microthrombi in cathepsin G-deficient mice. Mucins did not activate platelets in blood from mice lacking cathepsin G or protease-activated receptor-4 (PAR4), indicating that cathepsin G activates platelets through PAR4. Using knockout mice and blocking antibodies, we found that mucin-triggered cathepsin G release requires L-selectin and PSGL-1 on neutrophils, P-selectin on platelets, and Src family kinases in both cell types. Thus, carcinoma mucins promote thrombosis through adhesion-dependent, bidirectional signaling in neutrophils and platelets.

Topics: Adenocarcinoma, Mucinous; Animals; Antibodies, Neoplasm; Antibodies, Neutralizing; Blood Platelets; Cathepsin G; Cell Line, Tumor; Colonic Neoplasms; Disease Models, Animal; Humans; L-Selectin; Membrane Glycoproteins; Mice; Mice, Knockout; Mucins; Neoplasm Proteins; Neutrophil Activation; Neutrophils; P-Selectin; Platelet Activation; Receptors, Thrombin; Syndrome; Thrombosis

Mast cells and neutrophils are key contributors to the pathophysiological inflammatory processes that underpin asthma and chronic obstructive pulmonary disease, partly through the release of noxious serine proteases, including cathepsin G (Cat G) and chymase. From this standpoint, a dual inhibitor of neutrophil Cat G and mast cell chymase could protect against these disease-related inflammatory responses.. We examined the antiinflammatory pharmacology of RWJ-355871, a dual inhibitor of Cat G and chymase, in animal models of inflammation that evince pathophysiological pathways relevant to asthma and chronic obstructive pulmonary disease to determine the therapeutic potential of this compound.. In an ovalbumin (OVA)-sensitized rat model, RWJ-355871 was administered to block the mast-cell-mediated increase in paw volume caused by OVA injection. In a sheep asthma model, antigen-induced airway responses were assessed with and without aerosol treatment with RWJ-355871. In a murine tobacco-smoke model of airway inflammation, the effect of RWJ-355871 on smoke-induced neutrophilia was determined.. Intravenous treatment of OVA-sensitized rats with RWJ-355871 provided dose-dependent reduction in the increase in rat paw volume. In allergic sheep, aerosol pretreatment with RWJ-355871 showed dose-dependent inhibition of the antigen-induced early response, late response, and post-antigen-induced airway hyperreponsiveness. In tobacco-smoke-exposed mice, nebulized RWJ-355871 significantly reduced the smoke-induced neutrophilia from the levels observed in untreated mice.. The preclinical antiinflammatory effects of RWJ-355871 in these animal models of inflammation indicate that this dual inhibitor may have therapeutic utility for treating airway inflammatory diseases involving mechanisms that depend on Cat G and/or chymase.

Topics: Animals; Biomarkers; Bronchoalveolar Lavage Fluid; Cathepsin G; Chymases; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Injections, Intravenous; Lung Diseases; Mice; Organophosphonates; Piperidines; Pulmonary Disease, Chronic Obstructive; Rats; Sheep; Treatment Outcome

The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process.. We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa.. Pretreatment of the animals with BbCI (2.5 mg·kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1β or tumour necrosis factor-α, however, did not change.. Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Topics: Animals; Anti-Inflammatory Agents; Bauhinia; Carrageenan; Cathepsin G; Cathepsin L; Cell Adhesion; Cell Movement; Cytokines; Disease Models, Animal; Edema; Enzyme-Linked Immunosorbent Assay; Humans; Inflammation; Leukocyte Elastase; Leukocytes; Male; Microscopy; Plant Proteins; Rats; Rats, Wistar; Seeds

The cystic fibrosis airway is susceptible to Pseudomonas aeruginosa infection, which stimulates an intense inflammatory response leading to airway obstruction and bronchiectasis. Neutrophils migrate into the airway, and once there, release high concentrations of neutral serine proteases during phagocytosis and in death. In particular, neutrophil elastase is central to progression of bronchiectasis by interfering with bacterial clearance and directly perpetuating the inflammatory response in the airway. Using a murine model of endobronchial inflammation, we found that a different neutrophil-derived serine protease, cathepsin G, inhibited the host's ability to clear Pseudomonas from the lung, based on a 1-log reduction in bacteria recovered from cathepsin G-deficient mice. Higher antibody concentrations were found in respiratory epithelial lining fluid from mice lacking cathepsin G, but there was no difference in other opsonins, such as surfactant proteins A and D. Chemokine levels measured in the lung correlated with bacterial burden and not the animal's genotype, indicating that airway inflammation was not affected by the presence (or absence) of specific serine proteases. These findings suggest that cathepsin G interferes with airway defenses, showing that proteases other than neutrophil elastase have roles in the pathogenesis of suppurative airway diseases.

Topics: Animals; Bronchitis; Cathepsin G; Cathepsins; Cystic Fibrosis; Disease Models, Animal; Leukocyte Elastase; Lung; Mice; Mice, Knockout; Neutrophils; Pseudomonas aeruginosa; Serine Endopeptidases

Bronchopulmonary dysplasia (BPD) continues to be a major cause of morbidity in premature infants. An imbalance between neutrophil elastase and its inhibitors has been implicated in BPD. Serine protease inhibitor (SERPIN)B1 is an inhibitor of neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (cat G). Recent studies suggest that SERPINB1 could provide protection in the airways by regulating excess protease activity associated with inflammatory lung disorders. In this study, we determined the distribution and ontogeny of SERPINB1 in the baboon lung and characterized the expression of SERPINB1 in baboon models of BPD. SERPINB1 expression was detected in the conducting airway and glandular epithelial cells in addition to neutrophils, macrophages, and mast cells. SERPINB1 mRNA and protein expression increased with advancing gestational age and in the new BPD model. In contrast, SERPINB1 expression levels were decreased in the old BPD model. Furthermore, SERPINB1 was detected as a high-molecular-mass (HMM) complex in lung tissue and bronchoalveolar lavage fluid samples from the BPD group. Analysis of the HMM complex by coimmunoprecipitation showed that these complexes were formed between SERPINB1 and NE or cat G. High-performance liquid chromatography (HPLC) ion trap mass spectrometry verified the presence of SERPINB1 in HMM complexes. Finally, NE activity level was compared between new and old baboon models of BPD and was found to be significantly lower in new BPD. Thus SERPINB1 upregulation in new BPD may be protective by contributing to the regulation of neutrophil proteases NE and cat G.

Topics: Animals; Animals, Newborn; Bronchoalveolar Lavage Fluid; Bronchopulmonary Dysplasia; Cathepsin G; Cathepsins; Disease Models, Animal; Embryo, Mammalian; Embryonic Development; Gestational Age; Humans; Immunologic Techniques; Infant, Newborn; Leukocyte Elastase; Lung; Papio; RNA, Messenger; Serine Endopeptidases; Serpins; Up-Regulation

Transgenic mice expressing PML-RARalpha in early myeloid cells under control of human cathepsin G regulatory sequences all develop a myeloproliferative syndrome, but only 15% to 20% develop acute promyelocytic leukemia (APL) after a latent period of 6 to 14 months. However, this transgene is expressed at very low levels in the bone marrow cells of transgenic mice. Because the transgene includes only 6 kb of regulatory sequences from the human cathepsin G locus, we hypothesized that sequences required for high-level expression of the transgene might be located elsewhere in the cathepsin G locus and that a knock-in model might yield much higher expression levels and higher penetrance of disease. We, therefore, targeted a human PML-RARalpha cDNA to the 5' untranslated region of the murine cathepsin G gene, using homologous recombination in embryonic stem cells. This model produced a high-penetrance APL phenotype, with more than 90% of knock-in mice developing APL between 6 and 16 months of age. The latent period and phenotype of APL (including a low frequency of an interstitial deletion of chromosome 2) was similar to that of the previous transgenic model. Remarkably, however, the expression level of PML-RARalpha in bone marrow cells or APL cells was less than 3% of that measured in the low-penetrance transgenic model. Although the explanation for this result is not yet clear, one hypothesis suggests that very low levels of PML-RARalpha expression in early myeloid cells may be optimal for the development of APL in mice.

Topics: Animals; Antigens, CD34; Antineoplastic Agents; Biomarkers; Cathepsin G; Cathepsins; Cell Differentiation; Chromosomes, Mammalian; Disease Models, Animal; Female; Gene Deletion; Gene Dosage; Humans; Leukemia, Promyelocytic, Acute; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Proteins; Oncogene Proteins, Fusion; Penetrance; Recombination, Genetic; RNA, Messenger; Serine Endopeptidases; Tretinoin

Among other phenotypic defects, the beige mouse is susceptible to infection and has large neutrophil granules that apparently secrete a decreased amount of elastolytic activity. We have shown using in vitro methods that cytosolic inhibitors in beige neutrophils are normal. Although cathepsin G is tightly bound to lysosomal membranes, normal amounts of activity are released in response to degranulating agents. Decreased elastolytic activity is secreted by beige neutrophils because elastase is present in the granules as a 46 kDa proenzyme, which can be activated extracellularly by a protease-dependent mechanism. The current experiments were undertaken to explore the in vivo functions of neutrophils from C57 BI/6J (bg/bg) beige mice using the model of casein-induced acute peritonitis; normal C57 BI/6J (+/+) mice served as controls. The kinetics of neutrophil accumulation in the peritoneum were normal, suggesting normal neutrophil migration. Cathepsin G activity in the cell-free supernatant of peritoneal lavage fluid was normal; elastolytic activity was initially very low but increased to about twice baseline level after 4 h at 25 degrees C and to about 20-fold at 36 h. The appearance of this activity was inhibited to varying degree (54 to 83%) by different protease inhibitors (pepstatin, antipain, aprotinin, leupeptin and chymostatin). We conclude that the decreased amount of elastolytic activity secreted by beige neutrophils into an inflammatory exudate is due to a genetic defect that results in production of a 46 kDa proelastase rather than the normal 29 kDa active elastase; the proelastase can be spontaneously activated by a protease-dependent mechanism. In light of these data, the use of the beige mouse as a model for the Chediak-Higashi syndrome, and as a model in which neutrophils do not produce elastase, must be reconsidered.

Topics: Analysis of Variance; Animals; Ascitic Fluid; Blotting, Western; Caseins; Cathepsin G; Cathepsins; Cell Count; Chediak-Higashi Syndrome; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Leukocyte Elastase; Lysosomes; Male; Mice; Mice, Inbred C57BL; Molecular Weight; Neutrophils; Peritonitis; Protease Inhibitors; Serine Endopeptidases; Species Specificity

To determine whether peptic activity in bronchoalveolar fluid, due to the presence of the gastric proteolytic enzyme pepsin, could serve as a biochemical marker for pulmonary aspiration of gastric contents.. Prospective, experimental trial.. A university animal research laboratory.. Thirty-six New Zealand rabbits, weighing 2 to 4 kg.. New Zealand rabbits were anesthetized, intubated via tracheostomy, and mechanically ventilated. Pulmonary aspiration was induced by the intratracheal instillation of 2 mL/kg human gastric juice (pH 1.2 +/- 0.2; pepsin activity 0.02 +/- 0.006 microgram/mL; human gastric juice group, n = 24) or normal saline solution (pH 5.2 +/- 0.2; normal saline solution group; n = 12). Mechanical ventilation was continued. Bronchoalveolar lavage was performed at 15 mins (human gastric juice group, n = 8; normal saline solution group, n = 4), 30 mins (human gastric juice group, n = 8; normal saline solution group, n = 4), or 60 mins (human gastric juice group, n = 8; normal saline solution group, n = 4) postaspiration.. Peak airway pressure and PaO2 values were measured at baseline and 15 and 30 mins after aspiration. The pH of retrieved bronchoalveolar lavage fluid was measured and pepsin activity in sample fluid was determined. Changes from baseline in peak airway pressure and PaO2 were significant in human gastric juice animals at 15 and 30 mins when compared with normal saline solution animals (PaO2 -4% vs. -44%, peak airway pressure 20% vs. 36% at 15 mins; PaO2 -16% vs. -79%, peak airway pressure 28% vs. 69% at 30 mins; normal saline solution group vs. human gastric juice group, p < .02). Bronchoalveolar lavage fluid pH was not significantly different between groups at any time postaspiration (6.6 +/- 0.7 vs. 6.0 +/- 0.4 at 15 mins; 7.4 +/- 0.9 vs. 6.5 +/- 0.4 at 30 mins; 7.2 +/- 0.5 vs. 6.4 +/- 0.4 at 60 mins, normal saline solution group vs. human gastric juice group, p = NS). No peptic activity was present in bronchoalveolar lavage fluid from normal saline solution animals at any time. In the human gastric juice group, peptic activity was detected in postaspiration bronchoalveolar lavage fluid in eight of eight animals at 15 mins, six of eight animals at 30 mins, and five of eight animals at 60 mins (normal saline solution group vs. human gastric juice group; p < .001 at 15 mins, p < .01 at 30 mins, p = NS at 60 mins). Peptic activity of bronchoalveolar lavage fluid varied; mean values were greater at 15 mins than at 30 or 60 mins (pepsin activity: 0.004 +/- 0.002 microgram/mL vs. 0.002 +/- 0.001 microgram/mL vs. 0.0006 +/- 0.0001 microgram/mL, respectively, p < .05).. The results of this study suggest that peptic activity in bronchoalveolar lavage fluid can be detected up to 60 mins after induced, experimental gastric juice aspiration and may prove a clinically useful biochemical marker for episodes of occult pulmonary aspiration of gastric contents.

Topics: Animals; Biomarkers; Bronchoalveolar Lavage Fluid; Cathepsin G; Cathepsins; Disease Models, Animal; Humans; Hydrogen-Ion Concentration; Pepsin A; Pneumonia, Aspiration; Rabbits; Serine Endopeptidases

Pneumocystis carinii pneumonia (PCP) is characterized by the formation of leaky alveoli and a foamy alveolar exudate. To induce PCP, male Wistar rats were immunosuppressed by oral dexamethasone treatment for 12 weeks, during which time all rats developed PCP. Bronchoalveolar lavage fluid (BALF) was analyzed at that time and at 1, 2, and 4 weeks after the cessation of dexamethasone treatment, during which time the rats were recovering from PCP and immunosuppression (and was compared with the BALF obtained from healthy control rats), for type IV collagenase, elastase, cathepsin G, and collagenase activities. Scores for 72-kDa (matrix metalloproteinase type [MMP-2]) and 92-kDa (MMP-9) type IV collagenase-gelatinase activities correlated with those for BALF macrophages (r = 0.58; P < 0.001) and neutrophils (r = 0.66; P < 0.001), respectively, suggesting that they may, in part, be derived from these cells. However, MMP-2 was constitutively expressed and may play a role in normal tissue remodeling. MMP-9 activity was highest in the group with PCP (1.8 +/- 0.37; P > 0.05), with a gradual decline (1.0 +/- 0.48 by week 4; P > 0.05) toward normal (0.67 +/- 0.42) during recovery, which suggests a role for it in tissue-destructive inflammatory events. In rats with PCP the endogenously active collagenase was present at high levels compared with those in healthy controls (2.6 +/- 0.69 versus 0.17 +/- 0.17, respectively; P < 0.01), but they returned to normal by week 4 of recovery (0.42 +/- 0.30; P > 0.05). Collagenase activity showed a correlation with cyst number (r = 0.57; P < 0.001). The BALF of rats with PCP also contained the serine proteinases, which may act as pro-MMP activators. Ultramorphology disclosed increased pinocytotic activities, subepithelial bleb formation, and degeneration and denudation of the basal lamina. These findings suggest that the increased activities of collagenases in BALF caused by the host response against P. carinii might contribute to leaky alveoli.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cathepsin G; Cathepsins; Collagenases; Dexamethasone; Disease Models, Animal; Immunosuppressive Agents; Lung; Male; Microscopy, Electron; Pancreatic Elastase; Pinocytosis; Pneumonia, Pneumocystis; Pulmonary Alveoli; Rats; Rats, Wistar; Serine Endopeptidases

Passive anti-glomerular basement membrane (GBM) nephritis in the mouse is accompanied by acute massive albuminuria in the early heterologous phase. As we have previously shown, this albuminuria does not occur in the beige mutant of the C57BL/6J strain which is deficient for the leukocytic neutral proteinases elastase and cathepsin G. To address the question whether an intrinsic defect in the polymorphonuclear granulocyte (PMN) or local factors in the beige kidney are responsible for the lack of albuminuria in the beige mouse strain, we conducted reciprocal bone marrow transplantations (BMT) in beige and congenic control mice. Injection of anti-GBM antibody resulted in only slight albuminuria (89 +/- 47 micrograms/18 hours; N = 6) in normal (that is, non-irradiated, non-reconstituted) beige mice. By contrast, in beige mice, reconstituted with bone marrow (BM) from control mice, acute albuminuria developed (3032 +/- 1408 micrograms/18 hours; N = 8), to a degree comparable to that in non-irradiated control mice (4411 +/- 3405 micrograms/18 hours; N = 6, P < 0.01). Albuminuria in control mice, reconstituted with beige BM, was in the range of the normal beige mice (112 +/- 55 micrograms/18 hours; N = 9). Reconstitution with syngeneic bone marrow demonstrated that BMT by itself did not influence the level of albuminuria. All mice showed similar morphological lesions, with comparable influx of PMN in the glomeruli two hours after antibody injection. Elastase activities of PMN extracts in BMT groups were not different from those in donor mice. We conclude that the absence of albuminuria in beige mice is caused by an intrinsic defect in leukocytic neutral proteinase activity.

Topics: Albuminuria; Animals; Antibodies; Basement Membrane; Bone Marrow Transplantation; Cathepsin G; Cathepsins; Disease Models, Animal; Kidney Glomerulus; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nephritis; Neutrophils; Pancreatic Elastase; Serine Endopeptidases

The collagenolytic activity associated with insoluble collagen fibers separated from homogenates of inflamed paws from rats with adjuvant arthritis was quantitated using EDTA-sensitive solubilization of hydroxyproline as a measure of activity. Approximately 60% of the solubilized hydroxyproline was associated with dialyzable products. The level of collagenolytic activity in the paws increased with time after the induction of adjuvant arthritis and paralleled to a large extent the development of inflammation in both the adjuvant injected (right) hind paw and in the non-injected, contralateral paw. By day 26, the level of free collagenolytic activity in the injected paw had increased to a level 30 times normal while that in the contralateral paw had increased to a level 10 times normal. Treatment of the residues from the injected paws with trypsin resulted in the activation of a latent collagenolytic activity which, on day 26, accounted for approximately 50% of the total activity. The elevated level of collagen prolyl hydroxylase in the inflamed paw suggested that the rate of collagen synthesis was also increased. The activity of beta-glucuronidase increased in the inflamed paw with time after the induction of adjuvant arthritis while that of cathepsin G was elevated as compared to normal in paws removed, 5 but not 22 days after the induction of adjuvant arthritis. The inflamed paw of the adjuvant rat may represent a useful system in which to study the role of collagenolytic enzymes in the destruction of connective tissue by inflammatory lesions.

Topics: Animals; Arthritis; Arthritis, Experimental; Cathepsin G; Cathepsins; Collagen; Disease Models, Animal; Glucuronidase; Male; Microbial Collagenase; Peptide Hydrolases; Procollagen-Proline Dioxygenase; Rats; Serine Endopeptidases; Time Factors