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1.  HOW IMMUNE PEPTIDASES CHANGE SPECIFICITY 
Cathepsin G is a major secreted serine peptidase of neutrophils and mast cells. Studies in Ctsg-null mice suggest that cathepsin G supports antimicrobial defenses but can injure host tissues. The human enzyme has unusual “Janus-faced” ability to cleave peptides at basic (tryptic) as well as aromatic (chymotryptic) sites. Tryptic activity has been attributed to acidic Glu226 in the primary specificity pocket and underlies proposed important functions such as activation of pro-urokinase. However, most mammals, including mice, substitute Ala for Glu226, suggesting that human tryptic activity may be anomalous. To test this hypothesis, human cathepsin G was compared with mouse wild type and humanized active site mutants, revealing that mouse primary specificity is markedly narrower than that of human cathepsin G, with much greater Tyr activity and selectivity and near absence of tryptic activity. It also differs from human in resisting tryptic peptidase inhibitors (e.g., aprotinin), while favoring angiotensin destruction at Tyr4 over activation at Phe8. Ala226Glu mutants of mouse cathepsin G acquire tryptic activity and human ability to activate pro-urokinase. Phylogenetic analysis reveals that the Ala226Glu missense mutation appearing in primates 31–43 million years ago represented an apparently unprecedented way to create tryptic activity in a serine peptidase. We propose that tryptic activity is not an attribute of ancestral mammalian cathepsin G, which was primarily chymotryptic, and that primate-selective broadening of specificity opposed the general trend of increased specialization by immune peptidases and allowed acquisition of new functions.
doi:10.4049/jimmunol.1002292
PMCID: PMC3954857  PMID: 20889553
2.  Malaria-Associated l-Arginine Deficiency Induces Mast Cell-Associated Disruption to Intestinal Barrier Defenses against Nontyphoidal Salmonella Bacteremia 
Infection and Immunity  2013;81(10):3515-3526.
Coinfection with malaria and nontyphoidal Salmonella serotypes (NTS) can cause life-threatening bacteremia in humans. Coinfection with malaria is a recognized risk factor for invasive NTS, suggesting that malaria impairs intestinal barrier function. Here, we investigated mechanisms and strategies for prevention of coinfection pathology in a mouse model. Our findings reveal that malarial-parasite-infected mice, like humans, develop l-arginine deficiency, which is associated with intestinal mastocytosis, elevated levels of histamine, and enhanced intestinal permeability. Prevention or reversal of l-arginine deficiency blunts mastocytosis in ileal villi as well as bacterial translocation, measured as numbers of mesenteric lymph node CFU of noninvasive Escherichia coli Nissle and Salmonella enterica serotype Typhimurium, the latter of which is naturally invasive in mice. Dietary supplementation of malarial-parasite-infected mice with l-arginine or l-citrulline reduced levels of ileal transcripts encoding interleukin-4 (IL-4), a key mediator of intestinal mastocytosis and macromolecular permeability. Supplementation with l-citrulline also enhanced epithelial adherens and tight junctions in the ilea of coinfected mice. These data suggest that increasing l-arginine bioavailability via oral supplementation can ameliorate malaria-induced intestinal pathology, providing a basis for testing nutritional interventions to reduce malaria-associated mortality in humans.
doi:10.1128/IAI.00380-13
PMCID: PMC3811760  PMID: 23690397
3.  Mast Cell Proteases as Protective and Inflammatory Mediators 
Proteases are the most abundant class of proteins produced by mast cells. Many of these are stored in membrane-enclosed intracellular granules until liberated by degranulating stimuli, which include cross-linking of high affinity IgE receptor FcεRI by IgE bound to multivalent allergen. Understanding and separating the functions of the proteases is important because expression differs among mast cells in different tissue locations. Differences between laboratory animals and humans in protease expression also influence the degree of confidence with which results obtained in animal models of mast cell function can be extrapolated to humans. The inflammatory potential of mast cell proteases was the first aspect of their biology to be explored and has received the most attention, in part because some of them—notably tryptases and chymases—are biomarkers of local and systemic mast cell degranulation and anaphylaxis. Although some of the proteases indeed augment allergic inflammation and are potential targets for inhibition to treat asthma and related allergic disorders, they are protective and even anti-inflammatory in some settings. For example, mast cell tryptases may protect from serious bacterial lung infections and may limit the “rubor” component of inflammation caused by vasodilating neuropeptides in the skin. Chymases help to maintain intestinal barrier function and to expel parasitic worms, and may support blood pressure during anaphylaxis by generating angiotensin II. In other life-or-death examples, carboxypeptidase A3 and other mast cell peptidases limit systemic toxicity of endogenous peptides like endothelin and neurotensin during septic peritonitis, and inactivate venom-associated peptides. On the other hand, mast cell peptidase-mediated destruction of protective cytokines, like IL-6, can enhance mortality from sepsis. Peptidases released from mast cells also influence non-mast cell proteases, such as by activating matrix metalloproteinase cascades, which are important in responses to infection and resolution of tissue injury. Overall, mast cell proteases have a variety of roles—inflammatory and anti-inflammatory, protective and deleterious—in keeping with the increasingly well-appreciated contributions of mast cells in allergy, tissue homeostasis, and innate immunity.
doi:10.1007/978-1-4419-9533-9_12
PMCID: PMC3954859  PMID: 21713659
Tryptase; chymase; cathepsin G; carboxypeptidase A3; dipeptidyl peptidase I; cathepsin C; mastin; neurolysin; matrix metalloproteinase; tissue-type plasminogen activator; angiotensin; renin; endothelin; neurotensin; proteinase-activated receptor 2; calcitonin gene-related peptide; vasoactive intestinal peptide
4.  Association of Large-Airway Lymphocytic Bronchitis with Bronchiolitis Obliterans Syndrome 
Rationale: Lung transplantation offers great promise for otherwise terminal lung diseases, but the development of bronchiolitis obliterans syndrome (BOS) continues to limit survival. Although acute rejection and lymphocytic bronchiolitis have been identified as risk factors for the development of BOS, it is unclear whether large-airway lymphocytic inflammation conveys the same risk.
Objectives: We evaluated lymphocytic bronchitis on endobronchial biopsies as a risk factor for BOS and mortality.
Methods: Endobronchial biopsies were collected and graded during surveillance after lung transplantation. We assessed samples with negative cultures collected in the first 90 days from 298 subjects and compared large-airway lymphocytic bronchitis assessed by a 0–2 “E-score” and with standard A and BR pathology scores for acute rejection and small-airway lymphocytic bronchiolitis, respectively.
Measurements and Main Results: We found surprisingly little association between large- and small-airway lymphocytic inflammation scores from a given bronchoscopy. Endobronchial lymphocytic bronchitis was more prevalent in subjects in BOS stage 0p and BOS stages 1–3 at the time of biopsy. Within 90 days after transplantation, increasing maximum E-score was associated with greater risk of BOS (adjusted hazard ratio, 1.76; 95% confidence interval, 1.11–2.78; P = 0.02) and in this analysis 90-day maximum E-scores were the only score type predictive of BOS (P < 0.01).
Conclusions: These results support a multicenter study to evaluate endoscopic biopsies for the identification of patients at increased risk for BOS. The association of endobronchial lymphocytic inflammation and BOS may have mechanistic implications.
doi:10.1164/rccm.201206-1025OC
PMCID: PMC3603592  PMID: 23239157
bronchiolitis obliterans; graft rejection; bronchoscopy; lung transplantation
5.  Mast Cell Peptidases 
Mast cells make and secrete an abundance of peptidases, which are stored in such large amounts in granules that they comprise a high fraction of all cellular protein. Perhaps no other immune cell is so generously endowed with peptidases. For many years after the main peptidases were first described, they were best known as markers of degranulation, for they are released locally in response to mast cell stimulation and can be distributed systemically and detected in blood. The principal peptidases are tryptases, chymases, carboxypeptidase A3, and dipeptidylpeptidase I (cathepsin C). Numerous studies suggest that these enzymes are important and even critical for host defense and homeostasis. Endogenous and allergen or pathogen-associated targets have been identified. Belying the narrow notion of peptidases as proinflammatory, several of the peptidases limit inflammation and toxicity of endogenous peptides and venoms. The peptidases are interdependent, so that absence or inactivity of one enzyme can alter levels and activity of others. Mammalian mast cell peptidases—chymases and tryptases especially—vary remarkably in number, expression, biophysical properties, and specificity, perhaps because they hyper-evolved under pressure from the very pathogens they help to repel. Tryptase and chymase involvement in some pathologies stimulated development of therapeutic inhibitors for use in asthma, lung fibrosis, pulmonary hypertension, ulcerative colitis, and cardiovascular diseases. While animal studies support the potential for mast cell peptidase inhibitors to mitigate certain diseases, other studies, as in mice lacking selected peptidases, predict roles in defense against bacteria and parasites and that systemic inactivation may impair host defense.
doi:10.1165/rcmb.2009-0324RT
PMCID: PMC2830402  PMID: 19933375
mast cell; tryptase; chymase; dipeptidylpeptidase I; carboxypeptidase A3
6.  Mast Cell α and β Tryptases Changed Rapidly during Primate Speciation and Evolved from γ-Like Transmembrane Peptidases in Ancestral Vertebrates1 
Human mast cell tryptases vary strikingly in secretion, catalytic competence, and inheritance. To explore the basis of variation, we compared genes from a range of primates, including humans, great apes (chimpanzee, gorilla, orangutan), Old- and New-World monkeys (macaque and marmoset), and a prosimian (galago), tracking key changes. Our analysis reveals that extant soluble tryptase-like proteins, includingα- andβ-like tryptases, mastins, and implantation serine proteases, likely evolved from membrane-anchored ancestors because their more deeply rooted relatives (γtryptases, pancreasins, prostasins) are type I transmembrane peptidases. Function-altering mutations appeared at widely separated times during primate speciation, with tryptases evolving by duplication, gene conversion, and point mutation. Theα-tryptase Gly216Asp catalytic domain mutation, which diminishes activity, is present in macaque tryptases, and thus arose before great apes and Old World monkeys shared an ancestor, and before theαβsplit. However, the Arg–3Gln processing mutation appeared recently, affecting only humanα. By comparison, the transmembraneγ-tryptase gene, which anchors the telomeric end of the multigene tryptase locus, changed little during primate evolution. Related transmembrane peptidase genes were found in reptiles, amphibians, and fish. We identified soluble tryptase-like genes in the full spectrum of mammals, including marsupial (opossum) and monotreme (platypus), but not in nonmammalian vertebrates. Overall, our analysis suggests that soluble tryptases evolved rapidly from membrane-anchored, two-chain peptidases in ancestral vertebrates into soluble, single-chain, self-compartmentalizing, inhibitor-resistant oligomers expressed primarily by mast cells, and that much of present numerical, behavioral, and genetic diversity ofα- andβ-like tryptases was acquired during primate evolution.
PMCID: PMC2366170  PMID: 17947681
7.  Mast cell tryptases and chymases in inflammation and host defense 
Immunological reviews  2007;217:141-154.
Summary
Tryptases and chymases are the major proteins stored and secreted by mast cells. The types, amounts and properties of these serine peptidases vary by mast cell subtype, tissue, and mammal of origin. Membrane-anchored γ-tryptases are tryptic, prostasin-like, type I peptidases that remain membrane-attached upon release and act locally. Soluble tryptases, including their close relatives, mastins, form inhibitor-resistant oligomers that act more remotely. Befitting their greater destructive potential, chymases are quickly inhibited after release, although some gain protection by associating with proteoglycans. Most chymase-like enzymes, including mast cell cathepsin G, hydrolyze chymotryptic substrates, an uncommon capability in the proteome. Some rodent chymases, however, have mutations resulting in elastolytic activity. Secreted tryptases and chymases promote inflammation, matrix destruction, and tissue remodeling by several mechanisms, including destroying pro-coagulant, matrix, growth and differentiation factors, and activating proteinase-activated receptors, urokinase, metalloproteinases, and angiotensin. They also modulate immune responses by hydrolyzing chemokines and cytokines. At least one chymase protects mice from intestinal worms. Tryptases and chymases also can oppose inflammation by inactivating allergens and neuropeptides causing inflammation and bronchoconstriction. Thus, like mast cells themselves, mast cell serine peptidases play multiple roles in host defense and any accounting of benefit versus harm is necessarily context-specific.
doi:10.1111/j.1600-065X.2007.00509.x
PMCID: PMC2275918  PMID: 17498057
Tryptase; chymase; cathepsin G; mast cell
8.  Tryptase genetics and anaphylaxis 
Tryptases secreted by tissue mast cells and basophils can enter the bloodstream. In human subjects tryptases are encoded by several genes and alleles, including α, β, γ, and δ. Common variations include complete absence of α genes. Until recently, α tryptase was considered to be the major tryptase secreted at baseline and in mastocytosis. However, lack of α tryptase genes has little effect on circulating tryptase levels, which are now thought mainly to consist of inactive pro-β tryptase secreted constitutively rather than stored in granules with mature tryptases. Pro-β tryptase levels thus might reflect total body mast cell content. In contrast, mature β tryptase can increase transiently in severe systemic anaphylaxis and confirm the diagnosis. However, it might fail to increase in food anaphylaxis or might increase nonspecifically in samples acquired after death. Thus pro- and mature β tryptase measurements are useful but associated with false-negative and false-positive results, which need to be considered in drawing clinical conclusions in cases of suspected anaphylaxis.
doi:10.1016/j.jaci.2006.02.026
PMCID: PMC2271076  PMID: 16751005
Anaphylaxis; tryptase; mastocytosis; mast cell; basophil
9.  Mast Cells Protect Mice from Mycoplasma Pneumonia 
Rationale: As the smallest free-living bacteria and a frequent cause of respiratory infections, mycoplasmas are unique pathogens. Mice infected with Mycoplasma pulmonis can develop localized, life-long airway infection accompanied by persistent inflammation and remodeling.
Objective: Because mast cells protect mice from acute septic peritonitis and gram-negative pneumonia, we hypothesized that they defend against mycoplasma infection. This study tests this hypothesis using mast cell–deficient mice.
Methods: Responses to airway infection with M. pulmonis were compared in wild-type and mast cell–deficient KitW-sh/KitW-sh mice and sham-infected control mice.
Measurements and Main Results: Endpoints include mortality, body and lymph node weight, mycoplasma antibody titer, and lung mycoplasma burden and histopathology at intervals after infection. The results reveal that infected KitW-sh/KitW-sh mice, compared with other groups, lose more weight and are more likely to die. Live mycoplasma burden is greater in KitW-sh/KitW-sh than in wild-type mice at early time points. Four days after infection, the difference is 162-fold. Titers of mycoplasma-specific IgM and IgA appear earlier and rise higher in KitW-sh/KitW-sh mice, but antibody responses to heat-killed mycoplasma are not different compared with wild-type mice. Infected KitW-sh/KitW-sh mice develop larger bronchial lymph nodes and progressive pneumonia and airway occlusion with neutrophil-rich exudates, accompanied by angiogenesis and lymphangiogenesis. In wild-type mice, pneumonia and exudates are less severe, quicker to resolve, and are not associated with increased angiogenesis.
Conclusions: These findings suggest that mast cells are important for innate immune containment of and recovery from respiratory mycoplasma infection.
doi:10.1164/rccm.200507-1034OC
PMCID: PMC2662990  PMID: 16210667
angiogenesis; bronchitis; innate immunity; lymphangiogenesis; pneumonia
10.  The αvβ6 integrin modulates airway hyperresponsiveness in mice by regulating intraepithelial mast cells 
Allergic asthma is the most common form of asthma, affecting more than 10 million Americans. Although it is clear that mast cells have a key role in the pathogenesis of allergic asthma, the mechanisms by which they regulate airway narrowing in vivo remain to be elucidated. Here we report that mice lacking αvβ6 integrin are protected from exaggerated airway narrowing in a model of allergic asthma. Expression microarrays of the airway epithelium revealed mast cell proteases among the most prominent differentially expressed genes, with expression of mouse mast cell protease 1 (mMCP-1) induced by allergen challenge in WT mice and expression of mMCP-4, -5, and -6 increased at baseline in β6-deficient mice. These findings were most likely explained by loss of TGF-β activation, since the epithelial integrin αvβ6 is a critical activator of latent TGF-β, and in vitro–differentiated mast cells showed TGF-β–dependent expression of mMCP-1 and suppression of mMCP-4 and -6. In vitro, mMCP-1 increased contractility of murine tracheal rings, an effect that depended on intact airway epithelium, whereas mMCP-4 inhibited IL-13–induced epithelial-independent enhancement of contractility. These results suggest that intraepithelial activation of TGF-β by the αvβ6 integrin regulates airway responsiveness by modulating mast cell protease expression and that these proteases and their proteolytic substrates could be novel targets for improved treatment of allergic asthma.
doi:10.1172/JCI58815
PMCID: PMC3266785  PMID: 22232213
11.  Parasitic Infection Improves Survival from Septic Peritonitis by Enhancing Mast Cell Responses to Bacteria in Mice 
PLoS ONE  2011;6(11):e27564.
Mammals are serially infected with a variety of microorganisms, including bacteria and parasites. Each infection reprograms the immune system's responses to re-exposure and potentially alters responses to first-time infection by different microorganisms. To examine whether infection with a metazoan parasite modulates host responses to subsequent bacterial infection, mice were infected with the hookworm-like intestinal nematode Nippostrongylus brasiliensis, followed in 2–4 weeks by peritoneal injection of the pathogenic bacterium Klebsiella pneumoniae. Survival from Klebsiella peritonitis two weeks after parasite infection was better in Nippostrongylus-infected animals than in unparasitized mice, with Nippostrongylus-infected mice having fewer peritoneal bacteria, more neutrophils, and higher levels of protective interleukin 6. The improved survival of Nippostrongylus-infected mice depends on IL-4 because the survival benefit is lost in mice lacking IL-4. Because mast cells protect mice from Klebsiella peritonitis, we examined responses in mast cell-deficient KitW-sh/KitW-sh mice, in which parasitosis failed to improve survival from Klebsiella peritonitis. However, adoptive transfer of cultured mast cells to KitW-sh/KitW-sh mice restored survival benefits of parasitosis. These results show that recent infection with Nippostrongylus brasiliensis protects mice from Klebsiella peritonitis by modulating mast cell contributions to host defense, and suggest more generally that parasitosis can yield survival advantages to a bacterially infected host.
doi:10.1371/journal.pone.0027564
PMCID: PMC3217977  PMID: 22110673
12.  Accumulation of intraepithelial mast cells with a unique protease phenotype in TH2-high asthma 
Background
Previously, we found that mast cell tryptases and carboxypeptidase A3 (CPA3) are differentially expressed in the airway epithelium in asthmatic subjects. We also found that asthmatic subjects can be divided into 2 subgroups (“TH2 high” and “TH2 low” asthma) based on epithelial cell gene signatures for the activity of TH2 cytokines.
Objectives
We sought to characterize intraepithelial mast cells (IEMCs) in asthma.
Methods
We performed gene expression profiling in epithelial brushings and stereology-based quantification of mast cell numbers in endobronchial biopsy specimens from healthy control and asthmatic subjects before and after treatment with inhaled corticosteroids (ICSs). We also performed gene expression and protein quantification studies in cultured airway epithelial cells and mast cells.
Results
By means of unsupervised clustering, mast cell gene expression in the airway epithelium related closely to the expression of IL-13 signature genes. The levels of expression of mast cell genes correlate positively with lung function improvements with ICSs. IEMC density was 2-fold higher than normal in subjects with TH2-high asthma compared with that seen in subjects with TH2-low asthma or healthy control subjects (P = .015 for both comparisons), and these cells were characterized by expression of tryptases and CPA3 but not chymase. IL-13 induced expression of stem cell factor in cultured airway epithelial cells, and mast cells exposed to conditioned media from IL-13–activated epithelial cells showed downregulation of chymase but no change in tryptase or CPA3 expression.
Conclusion
IEMC numbers are increased in subjects with TH2-high asthma, have an unusual protease phenotype (tryptase and CPA3 high and chymase low), and predict responsiveness to ICSs. IL-13–stimulated production of stem cell factor by epithelial cells potentially explains mast cell accumulation in TH2-high asthmatic epithelium.
doi:10.1016/j.jaci.2010.03.003
PMCID: PMC2918406  PMID: 20451039
Asthma; mast cells; tryptase; chymase; carboxypeptidase A3; stem cell factor
13.  Humans are protected from mast cell tryptase deficiency despite frequent inheritance of loss-of-function mutations 
Background
Mast cell tryptases have proposed roles in allergic inflammation and host defense against infection. Tryptase gene loci TPSAB1 and TPSB2 are known to be polymorphic but the nature and extent of diversity at these loci have not been fully explored.
Objective
To compare functional and nonfunctional tryptase allele frequencies and establish haplotypes in human populations.
Methods
Tryptase allele frequencies were determined by direct sequencing in 270 individuals from HapMap populations of European, African, Chinese and Japanese ancestry. Haplotypes were predicted, validated in parent-child trios, and compared between populations.
Results
We identify a new frame-shifted tryptase allele (βIIIFS) carried by 23% and 19% of individuals of European and African ancestry, but 0% of Asians. Homology models predict that βIIIFS is functionless. Our genotyping assay shows that allele and haplotype distributions in each population are unique. Strong linkage disequilibrium between TPSAB1 and TPSB2 (r2 = 0.83, D’ = 0.85) yields two major and five minor tryptase haplotypes.
Conclusions
Tryptase deficiency alleles (α and newly discovered βIIIFS) are common, causing the number of inherited active genes to range from a minimum of two to a maximum of four, with major differences between populations in proportion of individuals inheriting two versus four active alleles. African and Asian populations are especially enriched in genes encoding functional and non-functional tryptases, respectively. Strong linkage of TPSAB1 and TPSB2, and pairing of deficiency alleles with functional alleles in observed haplotypes, protect humans from “knockout” genomes, and indeed from inheritance of fewer than two active alleles.
Clinical implications
Disparities in the inherited frequency in functional versus nonfunctional tryptases between populations may contribute to ethnicity-specific differences in disease phenotypes influenced by mast cell tryptase.
doi:10.1016/j.jaci.2009.07.026
PMCID: PMC2783561  PMID: 19748655
tryptase; mast cells; human genetics
14.  α2-Macroglobulin Capture Allows Detection of Mast Cell Chymase in Serum and Creates a Circulating Reservoir of Angiotensin II-generating Activity1 
Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by the MCTC subset of mast cells. When secreted from degranulating cells, it can interact with a variety of circulating anti-peptidases, but is mostly captured by α2-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that α2-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used α2-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of ~1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. α2-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin converting enzyme. These findings suggest that chymase bound to α2-macroglobulin is active, that the circulating complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that α2-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.
doi:10.4049/jimmunol.0900127
PMCID: PMC2756746  PMID: 19380825
15.  Ex Vivo Sputum Analysis Reveals Impairment of Protease-dependent Mucus Degradation by Plasma Proteins in Acute Asthma 
Rationale: Airway mucus plugs, composed of mucin glycoproteins mixed with plasma proteins, are an important cause of airway obstruction in acute severe asthma, and they are poorly treated with current therapies.
Objectives: To investigate mechanisms of airway mucus clearance in health and in acute severe asthma.
Methods: We collected airway mucus from patients with asthma and nonasthmatic control subjects, using sputum induction or tracheal aspiration. We used rheological methods complemented by centrifugation-based mucin size profiling and immunoblotting to characterize the physical properties of the mucus gel, the size profiles of mucins, and the degradation products of albumin in airway mucus.
Measurements and Main Results: Repeated ex vivo measures of size and entanglement of mucin polymers in airway mucus from nonasthmatic control subjects showed that the mucus gel is normally degraded by proteases and that albumin inhibits this degradation. In airway mucus collected from patients with asthma at various time points during acute asthma exacerbation, protease-driven mucus degradation was inhibited at the height of exacerbation but was restored during recovery. In immunoblots of human serum albumin digested by neutrophil elastase and in immunoblots of airway mucus, we found that albumin was a substrate of neutrophil elastase and that products of albumin degradation were abundant in airway mucus during acute asthma exacerbation.
Conclusions: Rheological methods complemented by centrifugation-based mucin size profiling of airway mucins in health and acute asthma reveal that mucin degradation is inhibited in acute asthma, and that an excess of plasma proteins present in acute asthma inhibits the degradation of mucins in a protease-dependent manner. These findings identify a novel mechanism whereby plasma exudation may impair airway mucus clearance.
doi:10.1164/rccm.200807-1056OC
PMCID: PMC2724713  PMID: 19423716
airway mucus; rheology; neutrophil elastase; plasma; asthma exacerbation
16.  Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis 
Gastroenterology  2007;132(7):2422-2437.
Background & Aims
We studied the role of protease-activated receptor 2 (PAR2) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis.
Methods
We injected TxA into ileal loops in PAR2 or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR2 and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333.
Results
TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR2 deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%−28% and tissue and fluid myeloperoxidase by 31%−71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR2 and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca2+ responses to PAR2 AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis.
Conclusions
PAR2 and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR2 and up-regulates PAR2 and activating proteases, and PAR2 causes inflammation by neurogenic mechanisms.
doi:10.1053/j.gastro.2007.03.101
PMCID: PMC2366898  PMID: 17570216
17.  A Pulmonary Perspective on GASPIDs: Granule-Associated Serine Peptidases of Immune Defense 
Airways are protected from pathogens by forces allied with innate and adaptive immunity. Recent investigations establish critical defensive roles for leukocyte and mast cell serine-class peptidases garrisoned in membrane-bound organelles-here termed Granule-Associated Serine Peptidases of Immune Defense, or GASPIDs. Some better characterized GASPIDs include neutrophil elastase and cathepsin G (which defend against bacteria), proteinase-3 (targeted by antineutrophil antibodies in Wegener’s vasculitis), mast cell β-tryptase and chymase (which promote allergic inflammation), granzymes A and B (which launch apoptosis pathways in infected host cells), and factor D (which activates complement’s alternative pathway). GASPIDs can defend against pathogens but can harm host cells in the process, and therefore become targets for pharmaceutical inhibition. They vary widely in specificity, yet are phylogenetically similar. Mammalian speciation supported a remarkable flowering of these enzymes as they co-evolved with specialized immune cells, including mast cells, basophils, eosinophils, cytolytic T-cells, natural killer cells, neutrophils, macrophages and dendritic cells. Many GASPIDs continue to evolve rapidly, providing some of the most conspicuous examples of divergent protein evolution. Consequently, students of GASPIDs are rewarded not only with insights into their roles in lung immune defense but also with clues to the origins of cellular specialization in vertebrate immunity.
PMCID: PMC2405921  PMID: 18516248
Neutrophil elastase; cathepsin G; tryptase; chymase; granzyme; factor D
18.  Regulation of the Epithelial Na+ Channel by Peptidases 
Recent investigations point to an important role for peptidases in regulating transcellular ion transport by the epithelial Na+ channel, ENaC. Several peptidases, including furins and proteasomal hydrolases, modulate ENaC maturation and disposal. More idiosyncratically, apical Na+ transport by ENaC in polarized epithelia of kidney, airway, and gut is stimulated constitutively by one or more trypsin-family serine peptidases, as revealed by inhibition of amiloride-sensitive Na+ transport by broad-spectrum antipeptidases, including aprotinin and bikunin/SPINT2. In vitro, the transporting activity of aprotinin-suppressed ENaC can be restored by exposure to trypsin. The prototypical channel-activating peptidase (CAP) is a type 1 membrane-anchored tryptic peptidase first identified in Xenopus kidney cells. Frog CAP1 strongly upregulates Na+ transport when coexpressed with ENaC in oocytes. The amphibian enzyme's apparent mammalian orthologue is prostasin, otherwise known as CAP1, which is coexpressed with ENaC in a variety of epithelia. In airway cells, prostasin is the major basal regulator of ENaC activity, as suggested by inhibition and knockdown experiments. Other candidate regulators of mature ENaC include CAP2/TMPRSS4 and CAP3/matriptase (also known as membrane-type serine protease 1/ST14). Mammalian CAPs are potential targets for treatment of ENaC-mediated Na+ hyperabsorption by the airway in cystic fibrosis (CF) and by the kidney in hypertension. CAPs can be important for mammalian development, as indicated by embryonic lethality in mice with null mutations of CAP1/prostasin. Mice with selectively knocked out expression of CAP1/prostasin in the epidermis and mice with globally knocked out expression of CAP3/matriptase exhibit phenotypically similar defects in skin barrier function and neonatal death from dehydration. In rats, transgenic overexpression of human prostasin disturbs salt balance and causes hypertension. Thus, several converging lines of evidence indicate that ENaC function is regulated by peptidases, and that such regulation is critical for embryonic development and adult function of organs such as skin, kidney, and lung.
doi:10.1016/S0070-2153(06)78002-4
PMCID: PMC2276519  PMID: 17338914
19.  Mast cell tryptase may modulate endothelial cell phenotype in healing myocardial infarcts 
The Journal of pathology  2005;205(1):102-111.
Mast cells and macrophages infiltrate healing myocardial infarcts and may play an important role in regulating fibrous tissue deposition and extracellular matrix remodelling. This study examined the time-course of macrophage and mast cell accumulation in healing infarcts and studied the histological characteristics and protease expression profile of mast cells in a canine model of experimental infarction. Although macrophages were more numerous than mast cells in infarct granulation tissue, macrophage density decreased during maturation of the scar, whereas mast cell numbers remained persistently elevated. During the inflammatory phase of infarction, newly recruited leucocytes infiltrated the injured myocardium and appeared to be clustered in close proximity to degranulating cardiac mast cells. During the proliferative phase of healing, mast cells had decreased granular content and were localized close to infarct neovessels. In contrast, macrophages showed no selective localization. Mast cells in healing canine infarcts were alcian blue/safranin-positive cells that expressed both tryptase and chymase. In order to explain the pro-inflammatory and angiogenic actions of tryptase — the major secretory protein of mast cells — its effects on endothelial chemokine expression were examined. Chemokines are chemotactic cytokines that play an important role in leucocyte trafficking and angiogenesis and are highly induced in infarcts. Tryptase, a proteinase-activated receptor (PAR)-2 agonist, induced endothelial expression of the angiogenic chemokines CCL2/MCP-1 and CXCL8/IL-8, but not the angiostatic chemokine CXCL10/IP-10. Endothelial PAR-2 stimulation with the agonist peptide SLIGKV induced a similar chemokine expression profile. Mast cell tryptase may exert its angiogenic effects in part through selective stimulation of angiogenic chemokines.
doi:10.1002/path.1690
PMCID: PMC2275298  PMID: 15586361
mast cell; infarction; myocardial ischaemia/reperfusion; macrophage; chemokine; tryptase; chymase; inflammation
20.  Transcript Signatures of Lymphocytic Bronchitis in Lung Allograft Biopsy Specimens 
Background
Rejection and obliterative bronchiolitis are barriers to sustained graft function in recipients of transplanted lungs. Early detection is hindered by inadequate tests and an incomplete understanding of the molecular events preceding or accompanying graft deterioration.
Methods
Hypothesizing that genes involved in immune responses and tissue remodeling produce biomarkers of rejection, we measured the expression of 192 selected genes in 72 sets of biopsy specimens from human lung allografts. Gene transcripts were quantified using a 2-step, multiplex, real-time polymerase chain reaction approach in endobronchial and transbronchial biopsy specimens from transplant recipients without acute infections undergoing routine surveillance bronchoscopy.
Results
Comparisons of histopathology in parallel biopsy specimens identified 6 genes correlating with rejection as manifested by lymphocytic bronchitis, a suspected harbinger of obliterative bronchiolitis. For example, β2-defensin and collagenase transcripts in inflamed bronchi increased 37-fold and 163-fold, respectively. By contrast, these transcripts did not correlate with acute rejection in transbronchial specimens. Further, no correspondence was noted between histopathologic bronchitis and parenchymal rejection when endobronchial and transbronchial samples were obtained from the same patient.
Conclusions
Our highly sensitive method permits quantitation of many gene transcripts simultaneously in small, bronchoscopically acquired biopsy specimens of allografts. Transcript signatures obtained by this approach suggest that airway and alveolar responses to rejection differ and that endobronchial biopsy specimens assess lymphocytic bronchitis and chronic rejection but are not proxies for transbronchial biopsy specimens. Further, they reveal changes in airway expression of the specific genes involved in host defense and remodeling and suggest that the measurement of transcripts correlating with lymphocytic bronchitis may be diagnostic adjuncts to histopathology.
doi:10.1016/j.healun.2004.06.016
PMCID: PMC2271113  PMID: 16102441
21.  Mast Cell and Neutrophil Peptidases Attack an Inactivation Segment in Hepatocyte Growth Factor to Generate NK4-like Antagonists* 
The Journal of biological chemistry  2005;281(3):1489-1494.
Hepatocyte growth factor (HGF) is a plasminogen-like protein with an α chain linked to a trypsin-like β chain without peptidase activity. The interaction of HGF with c-met, a receptor tyrosine kinase expressed by many cells, is important in cell growth, migration, and formation of endothelial and epithelial tubes. Stimulation of c-met requires two-chain, disulfide-linked HGF. Portions of an α chain containing an N-terminal segment and four kringle domains (NK4) antagonize HGF activity. Until now, no physiological pathway for generating NK4 was known. Here we show that chymases, which are chymotryptic peptidases secreted by mast cells, hydrolyze HGF, thereby abolishing scatter factor activity while generating an NK4-like antagonist of HGF scatter factor activity. Thus, chymase interferes with HGF directly by destroying active protein and indirectly by generating an antagonist. The site of hydrolysis, Leu480, lies in the α chain on the N-terminal side of the cysteine linking the α and β chains. This site appears to be specific for HGF because chymase does not hydrolyze other plasminogen-like proteins, such as macrophage-stimulating protein and plasminogen itself. Mast cell/neutrophil cathepsin G and neutrophil elastase generate similar fragments of HGF by cleaving near the chymase site. Mast cell and neutrophil peptidases are secreted during tissue injury, infection, ischemia, and allergic inflammation, where they may oppose HGF effects on epithelial repair. Thus, HGF possesses an “inactivation segment” that serves as an Achilles' heel attacked by inflammatory proteases. This work reveals a potential physiological pathway for inactivation of HGF and generation of NK4-like antagonists.
doi:10.1074/jbc.M511154200
PMCID: PMC2271111  PMID: 16303761
22.  Cathepsins L and S are not required for activation of dipeptidyl peptidase I (cathepsin C) in mice 
Biological chemistry  2006;387(8):1143-1146.
The cysteine protease dipeptidyl peptidase I (DPPI) activates granule-associated immune-cell serine proteases. The in vivo activator of DPPI itself is unknown; however, cathepsins L and S are candidates because they activate pro-DPPI in vitro. In this study, we tested whether cathepsins L and S activate pro-DPPI in vivo by characterizing DPPI activity and processing in cells lacking cathepsins L and S. DPPI activity, and the relative size and amounts of DPPI heavy and light chains, were identical in mast cells from wild-type and cathepsin L/S double-null mice. Furthermore, the activity of DPPI-dependent chymase was preserved in tissues of cathepsin L/S double-null mice. These results show that neither cathepsin L nor S is required for activation of DPPI and suggest that one or more additional proteases is responsible.
doi:10.1515/BC.2006.141
PMCID: PMC2271110  PMID: 16895486
chymase; cysteine protease; mast cell; papain
23.  Prostasin regulates epithelial monolayer function: cell-specific Gpld1-mediated secretion and functional role for GPI anchor 
Prostasin, a trypsinlike serine peptidase, is highly expressed in prostate, kidney, and lung epithelia, where it is bound to the cell surface, secreted, or both. Prostasin activates the epithelial sodium channel (ENaC) and suppresses invasion of prostate and breast cancer cells. The studies reported here establish mechanisms of membrane anchoring and secretion in kidney and lung epithelial cells and demonstrate a critical role for prostasin in regulating epithelial monolayer function. We report that endogenous mouse prostasin is glycosylphosphatidylinositol (GPI) anchored to the cell surface and is constitutively secreted from the apical surface of kidney cortical collecting duct cells. Using site-directed mutagenesis, detergent phase separation, and RNA interference approaches, we show that prostasin secretion depends on GPI anchor cleavage by endogenous GPI-specific phospholipase D1 (Gpld1). Secretion of prostasin by kidney and lung epithelial cells, in contrast to prostate epithelium, does not depend on COOH-terminal processing at conserved Arg322. Using stably transfected M-1 cells expressing wild-type, catalytically inactive, or chimeric transmembrane (not GPI)-anchored prostasins we establish that prostasin regulates transepithelial resistance, current, and paracellular permeability by GPI anchor- and protease activity-dependent mechanisms. These studies demonstrate a novel role for prostasin in regulating epithelial monolayer resistance and permeability in kidney epithelial cells and, furthermore, show specifically that prostasin is a critical regulator of transepithelial ion transport in M-1 cells. These functions depend on the GPI anchor as well as the peptidase activity of prostasin. These studies suggest that cell-specific Gpld1- or peptidase-dependent pathways for prostasin secretion may control prostasin functions in a tissue-specific manner.
doi:10.1152/ajpcell.00637.2005
PMCID: PMC2271112  PMID: 16822939
serine protease; epithelial sodium channel; glycosylphosphatidylinositol anchor; transepithelial resistance; tight junction
24.  Tryptase haplotype in mastocytosis: Relationship to disease variant and diagnostic utility of total tryptase levels 
Clinical immunology (Orlando, Fla.)  2007;123(3):268-271.
Serum mast cell tryptase levels are used as a diagnostic criterion and surrogate marker of disease severity in mastocytosis. Approximately 29% of the healthy population lacks α tryptase genes; however, it is not known whether lack of α tryptase genes leads to variability in tryptase levels or impacts on disease severity in mastocytosis. We have thus analyzed tryptase haplotype in patients with mastocytosis, computing correlations between haplotype and plasma total and mature tryptase levels; and disease category. We found: (1) the distribution of tryptase haplotype in patients with mastocytosis appeared consistent with Harvey-Weinberg equilibrium and the distribution in the general population;(2) the disease severity and plasma tryptase levels were not affected by the number of α or β tryptase alleles in this study; and (3) information about the tryptase haplotype did not provide any prognostic value about the severity of disease. Total and mature tryptase levels positively correlated with disease severity, as well as prothrombin time and partial thromboplastin time, and negatively correlated with the hemoglobin concentration.
doi:10.1016/j.clim.2007.02.007
PMCID: PMC1949411  PMID: 17449330
Mastocytosis; tryptase; haplotype; genotype; allele; prothrombin time; partial thromboplastin time
25.  Neutrophil histamine contributes to inflammation in mycoplasma pneumonia 
The Journal of Experimental Medicine  2006;203(13):2907-2917.
Mycoplasmas cause chronic inflammation and are implicated in asthma. Mast cells defend against mycoplasma infection and worsen allergic inflammation, which is mediated partly by histamine. To address the hypothesis that mycoplasma provokes histamine release, we exposed mice to Mycoplasma pulmonis, comparing responses in wild-type and mast cell–deficient KitW-sh/KitW-sh (W-sh) mice. Low histamine levels in uninfected W-sh mice confirmed the conventional wisdom that mast cells are principal sources of airway and serum histamine. Although mycoplasma did not release histamine acutely in wild-type airways, levels rose up to 50-fold above baseline 1 week after infection in mice heavily burdened with neutrophils. Surprisingly, histamine levels also rose profoundly in infected W-sh lungs, increasing in parallel with neutrophils and declining with neutrophil depletion. Furthermore, neutrophils from infected airway were highly enriched in histamine compared with naive neutrophils. In vitro, mycoplasma directly stimulated histamine production by naive neutrophils and strongly upregulated mRNA encoding histidine decarboxylase, the rate-limiting enzyme in histamine synthesis. In vivo, treatment with antihistamines pyrilamine or cimetidine decreased lung weight and severity of pneumonia and tracheobronchitis in infected W-sh mice. These findings suggest that neutrophils, provoked by mycoplasma, greatly expand their capacity to synthesize histamine, thereby contributing to lung and airway inflammation.
doi:10.1084/jem.20061232
PMCID: PMC2118183  PMID: 17158962

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