An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

Clinical relevance of inherited genetic differences in human tryptases: Hereditary alpha-tryptasemia and beyond.

OBJECTIVE: To describe our current understanding of hereditary α-tryptasemia (HαT), how HαT fits into the evolutionary context of tryptases and contemporary framework of mast cell-associated disorders, and to discuss the future clinical and therapeutic landscape for symptomatic individuals with HαT. DATA SOURCES: Primary peer-reviewed literature. STUDY SELECTIONS: Basic, clinical, and translational studies describing tryptase gene composition, generation, secretion, and elevation and the associated clinical impacts of HαT and treatment of such individuals were reviewed. RESULTS: HαT is a common autosomal dominant genetic trait caused by increased TPSAB1 copy number encoding α-tryptase. Approximately 1 in 20 White individuals have HαT, making it by far the most common cause for elevated basal serum tryptase levels. Although many individuals with HαT may not manifest associated symptoms, the prevalence of HαT is increased in patients with clonal and nonclonal mast cell-associated disorders wherein it is linked to more prevalent and/or severe anaphylaxis and increased mast cell mediator-associated symptoms. Increased generation of mature α/ß-tryptase heterotetramers, and their unique physiochemical properties, may be responsible for some of these clinical findings. CONCLUSION: HαT is a common modifier of mast cell-associated disorders and reactions. Nevertheless, whether HαT may be an independent cause of clinical phenotypes with which it has been associated remains unproven. Correct identification of HαT is critical to accurate interpretation of serum tryptase levels in the clinical evaluation of patients. Beyond HαT, we foresee tryptase genotyping as an important parameter in the standard workup of patients with mast cell-associated disorders and development of therapeutic modalities targeting these patients and associated clinical phenotypes.

Mutational tail loss is an evolutionary mechanism for liberating marapsins and other type I serine proteases from transmembrane anchors.

Human and mouse marapsins (Prss27) are serine proteases preferentially expressed by stratified squamous epithelia. However, mouse marapsin contains a transmembrane anchor absent from the human enzyme. To gain insights into physical forms, activities, inhibition, and roles in epithelial differentiation, we traced tail loss in human marapsin to a nonsense mutation in an ancestral ape, compared substrate preferences of mouse and human marapsins with those of the epithelial peptidase prostasin, designed a selective substrate and inhibitor, and generated Prss27-null mice. Phylogenetic analysis predicts that most marapsins are transmembrane proteins. However, nonsense mutations caused membrane anchor loss in three clades: human/bonobo/chimpanzee, guinea pig/degu/tuco-tuco/mole rat, and cattle/yak. Most marapsin-related proteases, including prostasins, are type I transmembrane proteins, but the closest relatives (prosemins) are not. Soluble mouse and human marapsins are tryptic with subsite preferences distinct from those of prostasin, lack general proteinase activity, and unlike prostasins resist antiproteases, including leupeptin, aprotinin, serpins, and α2-macroglobulin, suggesting the presence of non-canonical active sites. Prss27-null mice develop normally in barrier conditions and are fertile without overt epithelial defects, indicating that marapsin does not play critical, non-redundant roles in development, reproduction, or epithelial differentiation. In conclusion, marapsins are conserved, inhibitor-resistant, tryptic peptidases. Although marapsins are type I transmembrane proteins in their typical form, they mutated independently into anchorless forms in several mammalian clades, including one involving humans. Similar pathways appear to have been traversed by prosemins and tryptases, suggesting that mutational tail loss is an important means of evolving new functions of tryptic serine proteases from transmembrane ancestors.

Dipeptidyl peptidase I controls survival from Klebsiella pneumoniae lung infection by processing surfactant protein D.

Prior work established that a deficiency in the cysteine protease dipeptidyl peptidase I (DPPI) improves survival following polymicrobial septic peritonitis. To test whether DPPI regulates survival from severe lung infections, DPPI(-/-) mice were studied in a Klebsiella pneumoniae lung infection model, finding that survival in DPPI(-/-) mice is significantly better than in DPPI(+/+) mice 8d after infection. DPPI(-/-) mice have significantly fewer bacteria in the lung than infected DPPI(+/+) mice, but no difference in lung histopathology, lung injury, or cytokine levels. To explore mechanisms of enhanced bacterial clearance in DPPI(-/-) mice, we examined the status of pulmonary collectins, finding that levels of surfactant protein D, but not of surfactant protein A, are higher in DPPI(-/-) than in DPPI(+/+) BAL fluid, and that DPPI(-/-) BAL fluid aggregate bacteria more effectively than control BAL fluid. Sequencing of the amino terminus of surfactant protein D revealed two or eight additional amino acids in surfactant protein D isolated from DPPI(-/-) mice, suggesting processing by DPPI. These results establish that DPPI is a major determinant of survival following Klebsiella pneumoniae lung infection and suggest that the survival disadvantage in DPPI(+/+) mice is in part due to processing of surfactant protein D by DPPI.

Mast cells in a murine lung ischemia-reperfusion model of primary graft dysfunction.

Primary graft dysfunction (PGD), as characterized by pulmonary infiltrates and high oxygen requirements shortly after reperfusion, is the major cause of early morbidity and mortality after lung transplantation. Donor, recipient and allograft-handling factors are thought to contribute, although new insights regarding pathogenesis are needed to guide approaches to prevention and therapy. Mast cells have been implicated in ischemic tissue injury in other model systems and in allograft rejection, leading to the hypothesis that mast cell degranulation contributes to lung injury following reperfusion injury.We tested this hypothesis in a mouse model of PGD involving reversible disruption of blood flow to one lung. Metrics of injury included albumin permeability, plasma extravasation, lung histopathology, and mast cell degranulation. Responses were assessed in wild-type (Kit+/+) and mast cell-deficient (KitW-sh/W-sh) mice. Because mouse lungs have few mast cells compared with human lungs, we also tested responses in mice with lung mastocytosis generated by injecting bone marrow-derived cultured mast cells (BMCMC).We found that ischemic lung responses of mast cell-deficient KitW-sh/W-sh mice did not differ from those of Kit+/+ mice, even after priming for injury using LPS. Degranulated mast cells were more abundant in ischemic than in non-ischemic BMCMC-injected KitW-sh/W-sh lungs. However, lung injury in BMCMC-injected KitW-sh/W-sh and Kit+/+ mice did not differ in globally mast cell-deficient, uninjected KitW-sh/W-sh mice or in wild-type Kit+/+ mice relatively deficient in lung mast cells.These findings predict that mast cells, although activated in lungs injured by ischemia and reperfusion, are not necessary for the development of PGD.

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.

Cathepsin L protects mice from mycoplasmal infection and is essential for airway lymphangiogenesis.

Cathepsin L (Ctsl) is a proposed therapeutic target to control inflammatory responses in a number of disease states. However, Ctsl is thought to support host defense via its involvement in antigen presentation pathways. Hypothesizing that Ctsl helps combat bacterial infection, we investigated its role in Mycoplasma pulmonis-infected mice as a model of acute and chronic infectious airway inflammation. Responses to the airway inoculation of mycoplasma were compared in Ctsl(-/-) and Ctsl(+/+) mice. After infection, Ctsl(-/-) mice demonstrated more body weight loss, greater mortality (22% versus 0%, respectively), and heavier lungs than Ctsl(+/+) mice, but had smaller bronchial lymph nodes. The burden of live mycoplasma in lungs was 247-fold greater in Ctsl(-/-) mice than in Ctsl(+/+) mice after infection for 3 days. Ctsl(-/-) mice exhibited more severe pneumonia and neutrophil-rich, airway-occlusive exudates, which developed more rapidly than in Ctsl(+/+) mice. Compared with the conspicuous remodeling of lymphatics after infection in Ctsl(+/+) mice, little lymphangiogenesis occurred in Ctsl(-/-) mice, but blood vessel remodeling and tissue inflammation were similarly severe. Titers of mycoplasma-reactive IgM, IgA, and IgG in blood in response to live and heat-killed organisms were similar to those in Ctsl(+/+) mice. However, enzyme-linked immunosorbent spot assays revealed profound reductions in the cellular IFN-γ response to mycoplasma antigen. These findings suggest that Ctsl helps contain mycoplasma infection by supporting lymphangiogenesis and cellular immune responses to infection, and our findings predict that the therapeutic inhibition of Ctsl could increase the severity of mycoplasmal infections.

Malaria-associated L-arginine deficiency induces mast cell-associated disruption to intestinal barrier defenses against nontyphoidal Salmonella bacteremia.

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.

Update on Mast Cell Proteases as Drug Targets.

Mast cell granules are packed with proteases, which are released with other mediators by degranulating stimuli. Several of these proteases are targets of potentially therapeutic inhibitors based on hypothesized contributions to diseases, notably asthma and ulcerative colitis for ß-tryptases, heart and kidney scarring for chymases, and airway infection for dipeptidyl peptidase-I. Small-molecule and antibody-based ß-tryptase inhibitors showing preclinical promise were tested in early-phase human trials with some evidence of benefit. Chymase inhibitors were given safely in Phase II trials without demonstrating benefits, whereas dipeptidyl peptidase-I inhibitor improved bronchiectasis, in effects likely related to inactivation of the enzyme in neutrophils.

Genetically defined individual reference ranges for tryptase limit unnecessary procedures and unmask myeloid neoplasms.

Serum tryptase is a biomarker used to aid in the identification of certain myeloid neoplasms, most notably systemic mastocytosis, where basal serum tryptase (BST) levels >20 ng/mL are a minor criterion for diagnosis. Although clonal myeloid neoplasms are rare, the common cause for elevated BST levels is the genetic trait hereditary α-tryptasemia (HαT) caused by increased germline TPSAB1 copy number. To date, the precise structural variation and mechanism(s) underlying elevated BST in HαT and the general clinical utility of tryptase genotyping, remain undefined. Through cloning, long-read sequencing, and assembling of the human tryptase locus from an individual with HαT, and validating our findings in vitro and in silico, we demonstrate that BST elevations arise from overexpression of replicated TPSAB1 loci encoding canonical α-tryptase protein owing to coinheritance of a linked overactive promoter element. Modeling BST levels based on TPSAB1 replication number, we generate new individualized clinical reference values for the upper limit of normal. Using this personalized laboratory medicine approach, we demonstrate the clinical utility of tryptase genotyping, finding that in the absence of HαT, BST levels >11.4 ng/mL frequently identify indolent clonal mast cell disease. Moreover, substantial BST elevations (eg, >100 ng/mL), which would ordinarily prompt bone marrow biopsy, can result from TPSAB1 replications alone and thus be within normal limits for certain individuals with HαT.

Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells.

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na(+) channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o-) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o- epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.

Neutrophil histamine contributes to inflammation in mycoplasma pneumonia.

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.

How immune peptidases change specificity: cathepsin G gained tryptic function but lost efficiency during primate evolution.

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 an unusual "Janus-faced" ability to cleave peptides at basic (tryptic) as well as aromatic (chymotryptic) sites. Tryptic activity has been attributed to acidic Glu(226) in the primary specificity pocket and underlies proposed important functions, such as activation of prourokinase. However, most mammals, including mice, substitute Ala(226) for Glu(226), 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 Tyr(4) over activation at Phe(8). Ala(226)Glu mutants of mouse cathepsin G acquire tryptic activity and human ability to activate prourokinase. Phylogenetic analysis reveals that the Ala(226)Glu 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.

Alpha 2-macroglobulin capture allows detection of mast cell chymase in serum and creates a reservoir of angiotensin II-generating activity.

Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by alpha(2)-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 alpha(2)-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used alpha(2)-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 approximately 1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. alpha(2)-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it 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 alpha(2)-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that alpha(2)-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.

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 F(c)εRI 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 peptideslike 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 nonmast 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.

Accumulation of intraepithelial mast cells with a unique protease phenotype in T(H)2-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 ("T(H)2 high" and "T(H)2 low" asthma) based on epithelial cell gene signatures for the activity of T(H)2 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 T(H)2-high asthma compared with that seen in subjects with T(H)2-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 T(H)2-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 T(H)2-high asthmatic epithelium.

Mast cell peptidases: chameleons of innate immunity and host defense.

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.

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.

Human subjects 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: We sought to compare functional and nonfunctional tryptase allele frequencies and establish haplotypes in human populations. METHODS: Tryptase allele frequencies were determined by means of 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 (betaIII(FS)) carried by 23% and 19% of individuals of European and African ancestry but 0% of Asian subjects. Homology models predict that betaIII(FS) is functionless. Our genotyping assay shows that allele and haplotype distributions in each population are unique. Strong linkage disequilibrium between TPSAB1 and TPSB2 (r(2)=0.83, D'=0.85) yields 2 major and 5 minor tryptase haplotypes. CONCLUSIONS: Tryptase deficiency alleles (alpha and the newly discovered betaIII(FS)) are common, causing the number of inherited active genes to range from a minimum of 2 to a maximum of 4, with major differences between populations in the proportion of individuals inheriting 2 versus 4 active alleles. African and Asian populations are especially enriched in genes encoding functional and nonfunctional tryptases, respectively. Strong linkage of TPSAB1 and TPSB2 and pairing of deficiency alleles with functional alleles in observed haplotypes protect human subjects from "knockout" genomes and indeed from inheritance of fewer than 2 active alleles.