Mast Cell Peptidases - ATS Journals

Red in Translation Mast Cell Peptidases Chameleons of Innate Immunity and Host Defense Neil N. Trivedi1,2 and George H. Caughey1,2 1

Section of Pulmonary and Critical Care Medicine, Medicine Service, Veterans Affairs Medical Center, San Francisco, California; and Department of Medicine, University of California at San Francisco, San Francisco, California

2

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. Keywords: mast cell; tryptase; chymase; dipeptidylpeptidase I; carboxypeptidase A3

Peptidases are far and away the most conspicuous protein products of mast cells (MC) and are deposited in large amounts outside of degranulating cells. Therefore, a substantial portion of the past two decades of effort to understand what MC contribute to host defense—and more generally to determine what MC are for—has been devoted to peptidases. These and related basic studies led to refinements and major upgrades of concepts of the place of MC in vertebrate biology. At one time MC were considered to be primitive cells, perhaps throwbacks or vestiges of a more primitive immune system, currently

(Received in original form September 1, 2009 and in final form November 11, 2009) This work was supported by National Institutes of Health grant HL024136 (to C.H.G.), by the Northern California Institute for Research and Education, by the San Francisco Veterans Affairs Medical Center, and by a Veterans Affairs Career Development Award (to N.N.T.). Correspondence and requests for reprints should be addressed to George H. Caughey, M.D., Veterans Affairs Medical Center, Mailstop 111-D, 4150 Clement Street, San Francisco, CA 94121. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 42. pp 257–267, 2010 Originally Published in Press as DOI: 10.1165/rcmb.2009-0324RT on November 20, 2009 Internet address: www.atsjournals.org

CLINICAL RELEVANCE This work briefly reviews several decades of work by laboratories around the world, building an understanding of the nature and roles of peptidases made and secreted by mast cells. This work suggests that these peptidases contribute to defense of lung and airway from certain infections and tissue homeostasis, while in some cases augmenting deleterious pathological responses. More generally, this review article suggests ways in which basic biochemical, pharmacological, physiological, and genetic investigations conducted at the laboratory bench can lead to insights of potential importance to human health.

contributing very little in relation to the trouble they cause by overreacting to otherwise nonthreatening inhaled, ingested, or injected antigens. Based largely on studies in rodents, we now know that MC contribute to the innate as well as adaptive arms of mammalian host defense. Although they do play oftenmentioned roles as ‘‘sentinels’’ or ‘‘effectors,’’ they also produce a variety of cytokines and other mediators with immunoregulatory functions, with resulting effects on phenomena as diverse as tumor growth, immune tolerance, and control of bacterial infection. These rodent studies also established that the contribution of MC and their products, including peptidases, can be pro- or anti-inflammatory, depending on timing and context. Finally, it has become clear that MC, although their precursors originate in bone marrow, mature in different tissue microenvironments into a variety of phenotypes, which vary in migration behavior, responses to activating stimuli, sensitivity to corticosteroids, and content of stored mediators, especially peptidases.

PEPTIDASES: THE MAJOR PROTEINS OF MAST CELLS Mast cells (‘‘mastzellen’’) were first described over 130 years ago by then medical student Paul Ehrlich, who later made other seminal discoveries and indeed became a towering figure in hematology and immunology (see review by Beaven in Ref. 1). Ehrlich recognized MC as distinct from other cells based on color changes—‘‘metachromasia’’—of intracellular granules in the presence of certain aniline dyes. We now know that metachromasia is due to contact with dense accumulations of highly charged heparin and chrondroitin sulfate polyanions, which function in part to allow close packing in granules of highly cationic histamine and peptidases. So Ehrlich can be credited with the first sighting of MC granule peptidases, although he knew neither what the granules contain nor what MC do. He speculated that the cells, being so stuffed with granules, are involved in nutrition. Although right about many things, he is

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likely to be mistaken on this point, although it should be noted that observations in MC-deficient mice recently suggested roles for MC in control of diet-induced obesity (2). The first direct evidence that MC are generously endowed with peptidases came half a century later from pioneering studies of Gomori, who developed elegant enzyme histochemical techniques for detecting esterase activity inside of cells in sections of fixed tissues (3). MC stain intensely by such approaches, with the esterase activity being a manifestation of general hydrolytic activity of enzymes whose physiological targets are thought to be amides (i.e., peptide bonds) rather than esters. Although puzzling at first, the intense MC esterase activity (which continues to be used to detect MC in tissues) later was linked to serine peptidases by several lines of evidence (4, 5), including disappearance of esterase activity in mice genetically engineered to lack MC peptidase activity (6). MC are uniquely associated with high levels of granule-associated trypsin and chymotrypsin-like enzyme activity, which is notable for being readily detected within intracellular granules. In this way the MC enzymes differ from pancreatic trypsins and chymotrypsins, which are present in acinar cell granules largely as inactive zymogens (trypsinogens and chymotrypsinogens) and are activated only after release from the cell. The proof that MC tryptic and chymotryptic peptidases (tryptases and chymases—a rubric proposed by Lagunoff and Benditt 46 years ago [7]) are unique in structure, activation, and function came later from biochemical and cell biological studies. A timeline containing selected events in the discovery and characterization of tryptases and chymases is shown in Figure 1. The first MC peptidase to be purified and subjected to detailed structural and functional characterization was a rat chymase, which only belatedly was recognized to originate from MC (8). Tryptases followed a few years later (9–12). MC chymases were the first immune serine peptidases to have structures determined—and this was by classical protein sequencing, crystallization, and X-ray diffraction (8). More recently, widespread availability and application of cDNA cloning, automated genome sequencing, and recombinant protein expression has revealed far more diversity and redundancy in MC peptidases—and in immune peptidases generally—than once appreciated (13). Such studies in the molecular biology era also reveal striking species differences, and variations in peptidase expression among MC in different tissue locations. For example, rats and mice contain many chymases and chymase-like peptidases that are absent in dogs and humans. Dogs contain tryptase-like enzymes that are not present in humans, and vice versa (14–16). Even monkeys and humans, despite relatively recent shared ancestors, differ in types of expressed tryptases (17, 18). This suggests that chymases and tryptases are evolving rapidly. The ‘‘big picture’’ view afforded by genomic surveys now allows us to view MC peptidases as part of large, related families of immune peptidases (19–22), which have proliferated and evolved over time to play specialized roles. In this regard, some MC and immune peptidases, like MC themselves, may predate the development of adaptive (antibody-based) immunity (18, 21–23). Although the amounts and combinations of peptidases in MC are unique among immune cells and are major means by which subpopulations of MC differ (24), ‘‘MC’’ peptidases are not necessarily MC-selective. This is especially evident now that we have more sensitive means of detecting gene and protein expression in other immune cell populations. For example, some tryptases are expressed by basophils as well as by MC (25, 26), and chymaselike cathepsin G is expressed by neutrophils and monocytes as well as by a subset of MC (27, 28). Although these peptidases originally may have supported innate defenses, in MC they are now also connected to adaptive responses because they are

released in response to engagement of surface-bound IgE by polyvalent antigen or allergen. Thus, the phylogeny of MC and related immune peptidases may afford glimpses of the origins of vertebrate immunity.

TRYPTASES Tryptases are well known and widely used clinically as markers of mastocytosis (29) and systemic MC activation (30), that is, anaphylaxis, a condition in which tryptases also may be pathogenic (31). Notwithstanding their exceptionally high levels of expression in human MC, tryptases have been challenging to link to specific functions. Because tryptases are present in an activated form inside of MC granules, they have potential intracellular roles, including self-activation (32). On the other hand, the soluble tryptases and mastins as a class are secreted and protect themselves from circulating inhibitors by forming proteasome-like oligomers (15), with active sites facing into a central cavity in which large inhibitors do not fit (33). Thus, tryptases have the opportunity to hydrolyze extracellular peptides and proteins. Of numerous potential targets identified (reviewed in Refs. 34 and 35), among the best substrates are peptides like calcitonin gene-related peptide and vasoactive intestinal peptide, which fit readily into the central cavity of the tryptase tetramer (36). On the other hand, the most important targets of tryptases may not be endogenous, but rather pathogen-associated. Although this is largely speculative, the existence of apparently specific MC tryptase inhibitors, such as one used by medicinal leeches, is taken as indirect evidence that tryptases target peptides in pathogens (37). Because of the multiplicity of tryptase genes and allelic variants in many mammals, it is also possible that different tryptases serve host defense in fundamentally different ways, although this remains to be clarified. One way to divine function of a mystery peptidase is to examine its resemblance to peptidases of known function. In this regard, genetic studies have been less than fully revelatory, although they have been extremely helpful in revealing that tryptases are products of many more genes and are more variable in form and catalytic function than previously imagined. Genes encoding soluble MC tryptases and their close siblings (mastins and implantation serine proteases) cluster closely with a gene encoding a curious, membrane-anchored form, g-tryptase. Phylogenetic studies suggest that these tryptases are mammal-specific; however, their more distant cousins, the pancreasins (38) and prostasins (16), trace back to reptiles and amphibians, respectively, and are transmembrane peptidases expressed primarily by non-MC (39). A major function of prostasin, which is embryonically lethal when inactivated in the mouse genome, is to regulate the epithelial sodium channel (39, 40), which does not appear to connect to a current role of soluble tryptases in MC. These considerations suggest that soluble tryptases evolved from prostasin-like transmembrane ancestors and that loss of the membrane anchor allowed selection and evolution of new functions as ancestral tryptases were liberated from the membrane (16–18). Human tryptases achieve further diversity via alternative mRNA splicing (41). In humans, expressed MC tryptases originate from four tandem loci: TPSG1 (g), TPSB2 (mainly bII and bIII), TPSAB1 (mainly a and bI), and TPSD1 (d) (16, 31, 42, 43). Defective alleles are common, including a, d, and a recently described bIII frame shift mutant (42, 44). Despite this, no humans have been identified with fewer than two fully active tryptase alleles, with the majority of individuals in most populations having three active alleles, and a smaller fraction having four active alleles (42). These findings suggest that inheritance

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Figure 1. Timeline of selected milestones in chymase and tryptase research.

of fewer than two active soluble tryptase genes may be incompatible with life, and that there may be a cost (possibly excessive inflammation) to inheriting four active genes. If this is the case, then human tryptase genes are subject to stabilizing (ambidirectional) selection. Alleles at TPSB2 and TPSAB1 are in close physical association and are in linkage disequilibrium. Haplotypes are highly restricted in specific populations. Overall, the two most common haplotypes at these loci are bII-a and bIII-bI (42). Remarkably, deficiency alleles, if present, are always paired with an active allele on a given chromosome, which is why at least two active tryptases are inherited. Comparing the tryptase gene cluster in dogs, rodents, and humans suggests that at least five gene duplication events, starting from an ancestral gene (18, 45)—plus one or more

gene conversion events (17, 43)—explain the present status of the multi-gene human cluster on chromosome 16p13.3, which is a genetic hotspot. In some mammals, chymase-related genes also hyperevolved, albeit with key differences (see CHYMASES below). Despite diversification of tryptases in mammals, the ‘‘specificity triad’’ residues in the catalytic domain most critical for determining substrate amino acid preference at the site of hydrolysis are typical of tryptic peptidases (Asp188Gly215Gly225) and are highly conserved. Human a-tryptases and monkey ab tryptases possess a Gly215Asp mutation, which dramatically reduces catalytic activity (18, 46). Other notable exceptions are d-tryptases in great apes. Orangutans have the specificityaltering Gly215Asp mutation; gorillas, chimpanzees, and humans have a nonsense mutation at Trp206 causing severe truncation

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and loss of specificity triad residues 215 and 225 (17, 43). The recently described bIII frame-shifted mutant is even more severely truncated (42). Chymase-related peptidases have a greater variety of specificity triad residues, which over time yielded a collection of peptidases with specificity ranging widely from tryptic to chymotryptic, elastolytic, Leu-ase, and Asp-ase (22, 34). The major genetic mutations in tryptases resulted in loss of membrane anchor (18), defective zymogen activation (32), or loss of catalytic function (17, 47, 48), rather than major change in specificity. In tryptases, the presence of the Cys191Cys220 disulfide bridge, which narrows the primary substrate pocket and is conspicuously absent in chymase-related peptidases, may create a more rigid active site that is less tolerant of mutations in specificity triad residues than in chymase-like peptidases (see below). It is also likely that preservation of tryptic specificity is essential for MC tryptase function, which may be helpful or harmful depending on pathological context. The following paragraphs review examples of conditions in which tryptases are thought to contribute. MC are known for their contributions to IgE-dependent allergic reactions. However, as might be expected of a cell type that appears to be highly conserved in vertebrates, MC also contribute to animal well being. Among these contributions is protection from certain bacterial infections of lung and peritoneum, as revealed initially by seminal papers appearing in 1996 (49, 50), which suggested that the MC contribution related to recruitment of neutrophils to sites of infection by releasing TNF-a and other factors. Given the very high levels of tryptase expression and storage in human MC (51), several groups have examined the possibility that tryptases support MC contributions to host defense. Human b tryptase injected into guinea pig skin and mouse peritoneum stimulates influx of neutrophils and eosinophils (52), and produces neutrophilic inflammation when instilled intratracheally (53, 54). Furthermore, MC-deficient mice pretreated with human tryptase defend themselves more effectively against intratracheally delivered Klebsiella pneumoniae (54). However, not all tryptases are created equal. For example, human bI but not a tryptase induces neutrophilic pneumonitis in mice (54). Similarly, injection of mouse tryptase mast cell peptidase (MCP)-6, but not MCP-7, induces neutrophilic peritonitis (55). The mechanism of tryptase-induced neutrophil recruitment may relate to stimulation of endothelial cells to secrete neutrophil chemokines. The importance of MC tryptases in innate host defense received further support from tryptase knockout (Mcpt62/2) mice. Thakurdas and colleagues disrupted MCP-6 in C57BL/6 mice, which are naturally deficient in the other soluble MC tryptase, MCP-7, resulting in a mouse largely bereft of classical soluble tryptases (56), although there is likely to be some expression of a tryptase-related mouse mastin, also known as MCP-11 (26, 57), which is not expressed in humans (15). Mcpt62/2 MC retain other secretory granuleassociated mediators, such as chymases MCP-4 and MCP-5 and histamine, and they degranulate in response to FceRI aggregation. However, survival in Mcpt62/2 mice is dramatically lower than in wild-type mice after peritoneal challenge with K. pneumoniae, and heterozygote survival is intermediate, suggesting dependence on gene dose. On the other hand, survival in mice with peritonitis from less virulent bacteria released by cecal puncture is actually improved in Dppi2/2 mice, which have reduced levels of MCP-6, which is proposed to reduce levels of protective cytokines by inactivating MC-derived IL-6 (58). This improvement in survival is despite higher counts of bacteria in the peritoneum. Thus tryptases have the potential to help as well as to harm. Although further studies are needed to explain the mechanism of protection by tryptases from bacterial peritonitis, these studies highlight the potential contribution of tryptases to innate immunity. An

alternative model is an Mcpt62/2 mouse with retained MCP-7 expression challenged with the helminth Trichinella spiralis (59). Although intestinal expulsion of worms is delayed in mice lacking the chymase MCP-1 (60), Mcpt62/2 mice do not differ from wild type in the acute phase of infection. However, during chronic stages, Mcpt62/2 mice have fewer necrotic larvae, more live cysts, and markedly reduced eosinophils in skeletal muscle, the primary site of chronic infection. These studies emphasize the significance of tryptases in innate responses to at least two categories of pathogens (namely, bacteria and metazoan parasites). MC, which are approximately 3% of cells in normal synovial membranes, were linked to arthritic inflammation almost four decades ago in a rat model of adjuvant-induced arthritis (61), in which MC were among the first inflammatory cells to appear in synovial and articular tissues. The subsequent appearance of neutrophils and lymphocytes in inflamed joints correlated with MC adopting a degranulated phenotype. Also, biopsies from humans with rheumatoid arthritis demonstrated synovial mastocytosis (62). Correlations between synovial MC numbers and arthritis severity were reported in multiple later studies, and MC-deficient mice exhibited less articular inflammation in antigen-induced and serum transfer models of arthritis (63, 64). Testing a long-standing hypothesis that tryptase contributes to development of rheumatoid arthritis (65), human b-tryptase was injected into mouse joints, where it produced dose-dependent joint inflammation and hyperemia (66). These effects appear to be mediated through protease-activated receptor-2 (which mediates a number of effects of tryptase in lung, gut, and skin [67–70]) because receptor-deficient mice are protected from joint inflammation. The potential importance of tryptase received further support in tryptase-deficient (Mcpt62/2/Mcpt72/2) mice in a methylated BSA/IL-1b–induced arthritis model (71). Tryptasedeficient mice were protected from joint inflammation compared with wild-type mice. Both MCP-6 and MCP-7 required ablation to generate the protected phenotype, in contrast to the finding that MCP-6 alone is required to maintain defense against Klebsiella peritonitis. This may indicate that MCP-6 and MCP-7 play different roles or that their roles are expression level– dependent and partially redundant. Potential involvement of MC and tryptases in the pulmonary manifestations of arthritis and autoimmune disease warrants exploration. An early suggestion that tryptases contribute to asthma arose from demonstration that dog tryptase destroys the activity of vasoactive intestinal peptide (72, 73), a bronchodilating neuropeptide of nonadrenergic, noncholinergic airway neurons. This was subsequently confirmed by Tam and Caughey with human lung tryptase, in the work whose high citation rate prompted this review (36). The same work identified additional human tryptase substrates, including calcitonin gene-related peptide, which is the most avidly cleaved natural substrate for a tryptase identified to date. In the same year, after demonstration by Sekizawa and colleagues that dog tryptase directly increases histamine-induced contraction of isolated dog bronchi (74), Tam and coworkers showed that tryptase inhibitors potentiate VIP-induced relaxation in human bronchi (75), providing possibly the first hints that human tryptases should be targeted for pharmaceutical inhibition. Subsequently, human tryptase was shown to augment histamine-induced bronchial smooth muscle contraction in sensitized human bronchus (76). During this period additional airway effects were identified, such as that tryptases are mitogens for fibroblasts, airway smooth muscle cells, and epithelium (77–79). With regard to the airway phenotype in chronic asthma, these discoveries predicted a role for tryptase in generating subepithelial fibrosis and smooth muscle hyperplasia. The smooth muscle effects have attracted particular attention, given evidence that airway smooth

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muscle mastocytosis is a hallmark of asthma (80). In other work, human tryptase introduced into sheep airway (81) or incubated with cultured human mast cells (82) released histamine, suggesting the possibility that tryptases released from an activated mast cell could spread and amplify the stimulus by provoking degranulation of nearby mast cells. The first generation of high-potency inhibitors of human tryptase to be identified were aromatic bis-amidines (83), which were used, along with peptidic inhibitors, to reduce antigeninduced early and late airway responses in a sheep model of allergic asthma (84). Similar results were seen after tryptase inhibition in sheep, guinea pig, and mouse models of asthma using later-generation inhibitors (85–89). In the first clinical trial of a tryptase inhibitor, APC366 reduced antigen-induced late bronchoconstrictor response in individuals with atopic asthma (90). A tryptase inhibitor also appeared efficacious in an openlabel, phase 2 trial in subjects with ulcerative colitis (91). Human tryptase inhibitors remain under development for a variety of conditions. Regardless of whether a tryptase inhibitor finds its way into clinical practice for the treatment of asthma or other diseases, the early work by Tam and others exploring mechanisms by which tryptases augment bronchial narrowing helped to justify and, indeed, seed a much larger body of physiological, pathological, genetic, and pharmaceutical work in the tryptase field.

CHYMASES Chymases and tryptases inhabit the same granules, but how are they related? There is no established evolutionary connection or common ancestor of chymases and tryptases (18, 20, 21) other than that both belong to the extremely ancient subclan of serine peptidases found in bacteria as well as mammals and other eukaryotes. Rather, human chymase and cathepsin G belong to a family of immune serine peptidases whose human members include granzymes A, B, H, and M of cytolytic lymphocytes and natural killer cells, elastase and proteinase 3 of neutrophils and monocytes, and factor D of the complement system. Origins of the chymase/cathepsin G/granzyme/elastase family as a whole date back at least 420 million years to before the time at which tetrapods separated from ray-finned fishes (92). However, chymases, cathepsin G, and close relatives developed and differentiated mainly in mammals, and lack specific orthologs in fish, frogs, and other nonmammalian vertebrates. However, chymases, cathepsin G, and close relatives proliferated on a grand scale in mammals. Their relatives include closely related to mammalian immune peptidases that are prominent in some mammals but are not expressed in humans, including duodenases (cows, sheep, and some other mammals), mouse granzymes C through G and b chymases MCP-1, -2, and -4, and rat MCP-1 and -2 and vascular chymase. The rat genome famously contains 64 clustered chymase-related genes (93) compared with just 4 in humans (94). The reasons that some mammals express many more of these enzymes than others are unknown; possibly they are hyperevolving in response to positive selection by species-selective pathogens. In this regard it may be significant that in addition to wide variation in the number of expressed genes, the family is notable for a range of target specificities. Classical chymases (including the ones first described) are chymotryptic, meaning that they prefer to cleave targets after aromatic residues, especially Phe and Tyr. This gave rise to the ‘‘chymase’’ rubric and led to the original division of MC peptidases into (1) tryptic tryptases and (2) chymotryptic chymases (7, 95), a division now seen as simplistic. In recent years it has become evident that chymases exhibit a range of substrate preferences, with some

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possessing little or no chymotryptic activity. For example, mouse and rat MC peptidase (MCP)-5 (produced by the Cma1 gene, the ortholog of CMA1 encoding human chymase) is elastolytic rather than chymotryptic (96–98), even though the human enzyme is classically chymotryptic. On the other hand, guinea pig chymase is primarily a Leu-ase and Met-ase, with little chymotryptic or elastolytic activity (13, 98). Opossum chymase is chymotryptic, but prefers to cleave after the bulky aromatic amino acid Trp rather than after Phe or Tyr (99). Other chymase-like peptidases, like mouse MCP-2, are proteins with very little enzymatic activity of any specificity (100). Thus, mammalian ‘‘chymase’’ activity can range from chymotryptic (Phe, Tyr versus Trp), to elastolytic (Ala, Val), to Leu-ase or Met-ase activity—or no peptidolytic activity. Moreover, some close relatives, like human cathepsin G and bovine duodenase, are partly tryptic (Lys, Arg). Even among classic chymotryptic chymases, like dog and human, there are potentially important differences in activity, as in the tendency to create or destroy angiotensin II by cleaving at one or the other of two potential aromatic residues in angiotensin I (101, 102). Via site-directed mutagenesis and related approaches, striking differences in specificity have been attributed to a small number of amino acid changes in the vicinity of the active site. Thus it appears that the chymase active site has unusual capacity to change specificity in response to mutations. In chymotrypsin, for example, mutations are more likely to reduce activity than change specificity, and wholesale changes in the active site are required to change specificity, say, to that of trypsin. The chameleon-like ability of chymases to change specificity may relate in part to lack of a disulfide bond (Cys191-Cys220) that constricts and rigidifies the active site of most other serine peptidases of the trypsin-chymotrypsin family (13, 21, 23, 103). The tight embrace of human chymase with its best known endogenous substrate, angiotensin I, is shown in Figure 2, which conveys a sense of how small changes in enzyme topography in the vicinity of the active site can alter binding affinity. All of this begs the question of what chymases and closely related peptidases are doing if they differ so much in substrate preferences. Are they targeting endogenous or exogenous targets? Clearly they are capable of doing both, including cleaving allergens, like profilin (104), endogenous peptides like angiotensin I (105, 106), blood proteins like albumin (107) and hepatocyte growth factor (108), and endogenous immune proteins, like cytokines IL-6 and IL-13 (109). With regard to targets originating from pathogens, is co-evolution at work? Have pathogen proteins evolved to resist hydrolysis, in turn driving changes in chymase specificity? And for that matter, have basic functions of chymases evolved to be speciesspecific? These are unresolved questions in MC biology. Nonetheless, important clues concerning roles of chymases emerged from studies in genetically modified animals and from pharmacological studies using chymase inhibitors. For example, a mouse chymase MCP-1 ‘‘knockout’’ (60) supports long-suspected roles for MC products in expulsion of intestinal worms, a knockout of MCP-4 (110) supports in vitro evidence that chymases are major activators of pro–matrix metalloproteinase 9 and help to limit accumulation of extracellular matrix (111–113), a knockout of MCP-5 (114) suggests a role in promoting ischemia reperfusion injury, and transgenic expression of an angiotensin-generating rat chymase in mouse vascular smooth muscle supports potential roles for chymase in hypertension and arteriopathy (115). Other valuable insights arise from pharmacologic studies in a variety of animals using chymase-selective inhibitors, which suggest, for example, roles for chymases in promoting atherosclerosis, postinjury vascular stenosis, ventricular hypertrophy, myocardial infarction

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Figure 2. Angiotensin docked to human chymase. The decapeptide angiotensin I, which is the best known natural substrate of human chymase, was fitted to the extended substrate binding and active site of human chymase and rendered using AutoDock Vina (http://vina.scripps. edu/). The starting point for the chymase catalytic domain structure was crystal-based Protein Data Bank–deposited structure 1pjp (130), minus the bound inhibitor. The angiotensin amino terminus is at the top of the stick figure. The site of hydrolysis is at Phe8, whose phenyl side chain is seen buried in a hydrophobic slot in the catalytic domain.

and fibrosis, and peritoneal adhesions (reviewed by Miyazaki and colleagues [116]). The major challenge in translating mouse knockout phenotypes to humans is that several chymases that support host defense (as revealed by immune deficits in mice lacking specific peptidases) lack strict orthologs in humans (23, 34). Conversely, the mouse enzyme (MCP-5) phylogenetically closest to human chymase is elastolytic rather than chymotryptic, and thus is not functionally orthologous (96, 97). In regard to biophysical properties, target specificity, and expression, the mouse chymase most similar to human is MCP-4, which, like the human enzyme, is a highly cationic, heparin-binding, angiotensin II– generating (101, 117) chymotryptic peptidase strongly expressed in ‘‘connective tissue’’ MC of ear skin, peritoneum (and likely pleura), and in a subset of MC in other locations (118, 119). The parts played by human chymase in lung disease remain to be clearly established and are under active investigation. Chymase has not received as much attention as tryptases in this regard, in part because the vast majority of MC in alveolar interstitium contain little chymase (24). However, chymaseexpressing MC are prominent constituencies of MC populations of the pleura and of airway and vessel walls (120, 121). Although it has long been assumed that chymases are proinflammatory and therefore will worsen the pathology of allergic and infectious inflammation, this is not necessarily so. In humans with asthma, for example, higher numbers of chymase-expressing MC in small airways correlate with better airflow (122). On the other hand, a major increase in chymase-positive adventitial MC of small pulmonary arteries was noted in patients who died of asthma (123). Chymase-expressing MC also are prominent in scarring lung diseases, as in interstitial lung disease (124), even in the absence of inflamma-

tion, and animal studies of bleomycin-induced fibrosis suggest that chymase inhibition reduces scarring (125). Chymase could influence lung and airway function, even if not originating in the lung in light of recent evidence that chymase can circulate in an active, angiotensin II–generating form bound to a2-macroglobulin (106). In humans, clarification of suggested roles of chymase in asthma and other allergic conditions, scarring lung diseases, and pathologies affecting pleura and pulmonary vasculature must await testing of selective inhibitors, which have been developed using rational design strategies on several drug platforms, aided by the availability of high-quality, crystal-derived models of the human chymase active site (126–132).

CATHEPSIN G Although human cathepsin G is expressed in the same subset of cells (MCTC) that express chymase—and in similar amounts (27)—less is known of its MC-specific roles because it is expressed in a variety of leukocytes, including neutrophils, monocytes, and dendritic cells. Studies in mice lacking cathepsin G suggest roles in defense against injected bacteria and fungi (133, 134) and in reperfusion injury (135); however, the extent to which these roles are due to the MC enzyme is not clear, and in fact they are usually assumed (but not formally established) to be performed by neutrophil cathepsin G, which aids killing of bacteria in phagolysosomes. Human cathepsin G has a capacity that known chymases do not possess, which is to cleave tryptic as well as chymotryptic substrates, although cathepsin G has generally weaker chymotryptic activity than chymase (106, 136, 137). The crystal-derived structure of human cathepsin G reveals an acidic residue (Glu) in the primary specificity pocket of the active site that appears to be responsible for tryptic

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activity by attracting basic side chains of Lys or Arg residues in substrates (138). In contrast, mouse cathepsin G lacks this Glu residue and may be purely chymotryptic, like human chymase, raising questions concerning how accurately the phenotype of Ctsg2/2 mice models the role of cathepsin G in humans and related primates. Despite the differences between human chymase and cathepsin G in tryptic activity, their chymotryptic activity is sufficiently similar that it has been possible to develop dual-specificity inactivators (132), which recently were shown to reduce allergen-induced bronchoconstriction in a sheep model of asthma and to decrease cigarette smoke–induced neutrophilic inflammation in a mouse model of bronchitis (139). Human cathepsin G also shares some activities with chymase, such as the ability to convert angiotensin I to II (105) and to stimulate secretion by cultured serous cell gland cells (140).

MC-derived IL-6 in gram-negative peritonitis and pneumonia (155). Dppi2/2 mice have been useful in establishing a mechanism of activation shared by many innate immune serine peptidases and in revealing the potential for these peptidases to harm as well as help the host. There is a potential human counterpart (Papillon-Lefevre syndrome) to the Dppi-null mouse. Many humans with this rare, autosomal recessive disorder, which is characterized by periodontitis and palmoplantar hyperkeratosis, have full or partial defects in dipeptidylpeptidase I (156). The phenotype of lymphocytes and neutrophils in these individuals suggests that dipeptidylpeptidase I may not be responsible for activating as wide a spectrum of peptidases as in mice (157), especially of granzymes. The MC phenotype of humans with dipeptidylpeptidase I deficiency remains to be established.

MAST CELL CARBOXYPEPTIDASE

FUTURE DIRECTIONS

Carboxypeptidase A3 is a specialized metallo-exopeptidase that appears to be largely specific for MC (141, 142), where it is expressed in the same granules that contain tryptase and chymase (143). It prefers targets with C-terminal aromatic residues, and thus is in a position to act in tandem with chymase and cathepsin G when they cleave targets at aromatic sites (117). In humans, carboxypeptidase A3 not only is expressed in the same MCTC that express tryptases and chymase, but also appears to co-segregate with chymase within granules and to be released as a proteoglycan-bound complex (144). In mice, the connection between carboxypeptidase and chymase MCP-5 expression is overt because genetic deletion of either enzyme results in absence or near-absence of the other in MC granules (145). Despite the close linkage of these enzymes, studies in mice do suggest important independent MC carboxypeptidase functions, such as hydrolytic inactivation of potentially lethal endogenous peptides (e.g., neurotensin and endothelin) (146, 147), and detoxification of sarafotoxin-class snake venoms (145, 148). Toxic doses of these peptidases are more likely to be lethal in the absence of MC carboxypeptidase. The roles played by this enzyme in lung disease remain to be determined. There is recent interest in this regard, for it is among the most overexpressed transcripts in the genome in asthmatic versus nonasthmatic epithelium (149).

Future studies in this area should include exploring roles of MC peptidases in nonallergic diseases not classically associated with MC, including (in the chest) bacterial pneumonias, sepsis, acute lung injury, interstitial lung diseases, tumor metastasis, pulmonary hypertension, and pleural scarring. Such studies have the potential to be especially meaningful given numerous pharmaceutical efforts to develop selective, potent inhibitors of MC peptidases for clinical use. Investigators need to continue to examine implications of human genetic variation in MC peptidases, including mutations that may affect response to therapeutic antipeptidases. This is especially important for tryptases, which are extraordinarily diverse and exhibit a high prevalence of loss-of-function mutations. It is a particular priority to correlate genotype with phenotype in diseases like asthma, ulcerative colitis, and arthritis in which tryptases are implicated in animal models. Such human genetic studies are increasingly tenable with modern genomic tools, improved understanding of statistical pitfalls, and access to collections of genomic DNA linked with clinical databases and specific conditions. Finally, it is critical to explore roles of MC peptidases in tissue homeostasis, including lessening of inflammation, because studies increasingly support homeostatic and anti-inflammatory roles for MC as a whole. This includes anticipating the possibility that MC peptidases are important enough for innate immune defense and control of inflammatory responses that systemic pharmaceutical inhibition may be deleterious.

DIPEPTIDYLPEPTIDASE I A detailed review of the functions of dipeptidylpeptidase I (also known as cathepsin C), which is expressed in a variety of granulated immune cells, is beyond the scope of this review. However, this peptidase deserves notice here because it is important for activating MC serine peptidases from inactive zymogen forms (pro-enzymes) and for controlling levels of MC carboxypeptidase (6, 150, 151). Dipeptidylpeptidase I is a thiolclass, granule-associated peptidase that removes amino-terminal dipeptides from peptides and has scant but demonstrable endoproteolytic activity (152, 153). In uninflamed airway, it is expressed predominantly by MC (152). It can be secreted but is thought to act intracellularly for principal functions (154). In mice, dipeptidylpeptidase I is essential for activating chymases and cathepsin G and for full activation of tryptase (6, 150). Intriguingly, despite dipeptidylpeptidase I’s role in activating MC, neutrophil, and lymphocyte peptidases with roles in host defense against bacteria, Dppi2/2 mice are partially protected from death from septic peritonitis (58). This is due to lack of dipeptidylpeptidase I in MC specifically, and may relate to reduced proteolytic destruction of protective cytokines, especially IL-6 (58), as reinforced by studies of the importance of

CONCLUSIONS This review emphasizes key developments in a field that expanded in multiple directions in the two decades since Elizabeth Tam first-authored her Red Journal study on the properties of human tryptase as a peptidase (36). More than 2,500 papers with ‘‘tryptase’’ in the title or abstract appeared since that article was published. The observations in this highly cited paper themselves were possible only because of prior contributions by multiple investigators. Collectively, these observations and associated ideas fertilized the subsequent flowering of the field. Perhaps the major contribution of the Red Journal article to the surge in interest in tryptases—and the reason it is often cited—is that it hypothesized a role for tryptase in asthmatic bronchoconstriction and proposed a mechanism for how this could happen. Certainly, some of our original ideas about tryptases were inescapably naı¨ve given that they preceded a tsunami of structural, functional, and genetic knowledge that followed. In particular, we had almost no inkling of the multiplicity and markedly polymorphic nature of the human tryptase gene locus, and of the differences that

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would loom large in later work. We also harbored only primitive notions of what MC tryptases—and for that matter MC in general—might contribute to host defense. The old notion that tryptases and MC cause nothing but trouble is now replaced by a more balanced picture of a family of enzymes, including activators, regulators, and fellow travelers like chymases, cathepsin G, and carboxypeptidase, whose contributions are positive or negative, depending on biological context. More generally, evidence of MC involvement in a broader range of pathophysiology is gradually effacing the limited concept of MC solely as ‘‘effectors’’ of allergic inflammation. Conflict of Interest Statement: Neither author has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgments: The authors thank Bill Raymond for helping to prepare the chymase model.

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