Update The Structure and Airway Biology of Mast Cell Proteinases George H. Caughey Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, California

Recent studies have led to a rapid expansion of knowledge concerning the structure and biology of the two major mast cell proteinases, tryptase and chymase. Tryptase is an abundant, trypsin-like enzyme found in the secretory granules of all human lung mast cells. The subunits of the heparin-associated tryptase tetramer appear to be the products of a multigene family whose intron-exon organization is unique and is not closely related to that of other mast cell or leukocyte serine proteinases. In vitro studies suggest that tryptases may participate in lung and airway responses by regulating airway neuropeptide activity, bronchomotor tone, and fibroblast mitogenesis. Mast cell chymases are chymotrypsin-like proteinases related closely to neutrophil cathepsin G and lymphocyte granzymes. The eDNA-derived structures of tryptase and chymase suggest that the two enzymes may differ in modes of activation from proenzyme forms, although the mature enzymes are packaged and released together. Chymase expression appears to be limited to a subset of human lung mast cells most prevalent in the airway submucosa. Possible roles for chymase include inactivation of sensory neuropeptides, regulation of submucosal gland secretion, and potentiation of histamine-induced vascular permeability.

Mast cells are distributed throughout most extravascular tissues. It is estimated that if all of the mast cells in the body were assembled in one organ, their mass would equal that of the spleen. In the lungs, mast cells are particularly abundant in the airway subepithelium and in the alveolar interstitium. Occasionally, they are found free in the tracheobronchial or alveolar lumen, but generally they are confined to the tissues. The role of mast cells in normal tissue homeostasis has long been a mystery. Mast cells are found in most vertebrates, in which they may form part of a defense against parasitic invasion. However, mast cells have a widely recognized pathologic role in disorders of immediate hypersensitivity, including allergic rhinitis and asthma, which involve IgE-mediated release of stored and newly formed inflammatory mediators into respiratory tissues. Mast cells also release mediators by pathways independent of IgE, which raises the possibility that they participate in events apart from those associated with classical immediate hypersensitivity. Some of these events are considered in more detail below. (Received in originalform January 3, 1991and in revisedform January16, 1991) Addresscorrespondence to: George H. Caughey, M.D., Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0911.

Abbreviations: calcitonin gene-related peptide, CGRP; rat chymase II, RMCPII; vasoactive intestinal peptide, VIP. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 387-394, 1991

Of the substances stored in, and released from, mast cell secretory granules, histamine is the best known and most extensively studied. However, mast cells also store and release neutral serine-class proteinases (tryptase and chymase), which are the major protein constituents of secretory granules, and, indeed, of the entire cell. Many of us who work with these proteinases have hypothesized that they have biologically significant physiologic and pathologic roles to play in the lung and in other tissues. My major research effort in the past several years has been to address this hypothesis by characterizing the structure and activities of mast cell tryptases and chymases. As outlined below in this Update, major strides have been made recently by a number of investigators in this area, but many questions remain to be answered.

Mast Cell Proteinase Genes The placement and phase of introns are often conserved among genes encoding proteins that are closely related in protein evolution and in function. In particular, the genes for mammalian serine proteinases fall naturally into a few groups based on intron-exon organization. To date, the organization and nucleotide sequence of a human mast cell tryptase and a rat mast cell chymase have been determined. Tryptase A gene whose exons match the nucleotide sequence of a human tryptase cDNA reveals a unique pattern of protein cod-

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991

MAST CELL TRYPTASE

3'

5'. H

i

0

S

i' I ,1

J

mRNA

identification of multiple different expressed tryptase sequences in lung (2, 3) and skin (1) eDNA libraries suggests the existence of several human tryptases that are the products of a multigene family, a conclusion reinforced by the results of genomic Southern blotting (1). Although the region of the 5' flank of the human tryptase gene reveals a sequence similar to that of the "core enhancer" that regulates pancreasspecific expression of pancreatic serine proteinases, little specific is known of the transcriptional regulation of tryptase gene expression. Nonetheless, immunohistochemical studies suggest that tryptases are produced exclusively in mast cells, with the exception, perhaps, of small amounts in human

basophils (4, 5). Chymase

Processed MONOMER

Heparinassociated

Unlike the tryptase gene, the chymase gene (Figure 2) is closely related to that of a number of other well-described granule-associated serine proteinases, including neutrophil cathepsin G and elastase, and the cytotoxic T-Iymphocyte proteinases ("granzymes"). The gene encoding rat chymase II (RMCPII), the principal serine proteinase of rat mucosal mast cells, has been sequenced and characterized (6). Like the tryptase gene, the chymase gene has regions similar to the pancreatic core enhancer, one of which, in the chymase gene, appears to be part of a mast cell-specific enhancer that directs preferential expression of a linked reporter gene and binds specifically to mast cell DNA-binding proteins (7). The distinguishing feature of the intron/exon structure of

'l'ETRAMER

Figure 1. Expression of mast cell tryptase. Exons in the gene are represented by boxes. Nonprotein coding exons are shaded with diagonal lines. Prepropeptides and catalytic domains are designated by black and grey boxes, respectively. Phase (0, I, or II) of introns is indicated below the strand. Codons or amino acids representing essential catalytic triad amino acids (His [H], Asp [D], Ser [S]) are positioned in DNA, mRNA, and protein as indicated. In the diagram of the processed monomer, N-linked glycosylation sites are indicated by "CHO". Positions of disulfide linkages are shown below the strand. Small arrows show presumed sites of proteolytic processing of preprotryptase. This figure is based on data of Vanderslice and associates (1).

ing and noncoding regions (1). The tryptase gene contains six exons separated by five introns. The idiosyncratic first intron separates the initiator Met codon from the 5' region of the gene containing promotor and other regulatory elements (see Figure 1). Although the tryptase gene structure is unique, it more closely resembles that of trypsin and related proteinases than those of mast cell chymase, neutrophil cathepsin G and elastase, lymphocyte granzymes, or any of several serine proteinases of hemostasis or fibrinolysis whose genes have been examined. Because the tryptase gene structure differs from that of any known protease, it fails to provide helpful clues to function that one might hope to find as a result of comparisons with other serine proteinase genes of known function. However, the comparison does suggest that tryptase shared an ancestor with trypsin more recently than with chymase or most other serine proteinases. Hamster-human somatic hybrid mapping suggests that human tryptase genes are found on chromosome 16 (2). Although only one tryptase gene has been fully sequenced, the

MAST CELL CHYMASE

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Figure 2. Expression of mast cell chymase. See legend of Figure 1 for explanation of labels and symbols. This figure is based on data of Benfey and colleagues (6).

Update

chymase and related leukocyte enzymes is the loss of a phase I intron (homologous to the intron separating exon 4 from exon 5 in tryptase) and the appearance of a new phase 0 intron inserted a few nucleotides downstream from the codon for the Asp of the catalytic triad (His 57 , Asp102 , Ser 195 using standard chymotrypsinogen numbering) essential for the function of all serine proteinases. Chymase-like gene structure is also shared by the gene for adipsin, an adipocytespecific serine proteinase, which has narrow trypsin-like specificity. Thus, the group of enzymes that are similar to chymase in gene structure includes proteinases with tryptic and elastolytic activity as well as chymotrypsin-like activity. Phylogenetic analysis indicates a close evolutionary relationship among most of the group's members, some of which remain closely linked on human chromosome 14 (8). It is reasonable to speculate that many of these related mast cell and leukocyte serine proteinases subserve similar functions, although much more needs to be learned of the biology of the enzymes before such speculation can be substantiated or refuted. The large number of closely related genes makes estimation of the total number of mast cell-specific chymases difficult. Two different chymases have been identified in mouse mucosal mast cells (9, 10). In rats, there are at least two distinct chymase genes expressed in different mast cell subsets (6, 11),and all rat mast cells seem to contain one type of chymase or another. Southern blotting of nit genomic DNA suggests that there may be 10 to 15 members of the gene family that includes RMCPII (6). In humans, the distribution of chymase-containing mast cells appears to be more limited than in rodents. There is no convincing evidence of more than one type of chymase. However, an enzyme indistinguishable from neutrophil cathepsin G is found in the same subset of human mast cells that express chymase (12). Indeed, cathepsin G is so closely related to mast cell chymases in structure and activity (see below) that, if it had been discovered in mast cells first, it would be called a chymase.

Mast Cell Proteinase Structure Tryptase Mature human tryptase is a glycosylated, heparin-associated tetramer of heterogeneous, catalytically active subunits. The tryptase monomer's amino acid sequence, like its gene structure, has no close counterpart among the numerous other serine proteinases that have been characterized. The complete primary structure of dog and human tryptases has been deduced from cloned cDNAs (1-3, 13). All of these predicted sequences contain the above-mentioned catalytic triad residues common to all serine proteinases, as well as an Asp whose homologue in other trypsin-like proteinases is thought to confer specificity for substrates containing Arg or Lys residues on the C-terminal side of the scissile bond. Overall, however, the predicted catalytic domain of tryptases reveals no more than rv40 % identity in alignment with the catalytic domains of other trypsin-like proteinases. The catalytic domain of tryptases is among the largest of any serine proteinase (21 to 22 residues longer than trypsin), with the extra length attributable principally to two internal hydrophobic insertions, which are predicted to create novel surface loops

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(1, 13). At present, an understanding of the contribution of these and other unique structural features of heparin binding, oligomerization, and substrate preferences is a goal of molecular modeling efforts but lies largely in the realm of speculation. All of the tryptases described to date are subject to posttranslational modification by Asn-linked glycosylation, as indicated by the sensitivity of the purified proteins to glycosidase treatment (13-5) and by the identification of consensus glycosylation sites in the protein sequences deduced from cloned cDNAs (1-3, 13). However, the human tryptase cDNAs vary in the number of predicted glycosylation sites, a finding that may explain some of the observed heterogeneity in tryptase preparations. It is possible that this type of modification plays a role in tetramer formation, especially in the light of evidence that unglycosylated human tryptase expressed in Escherichia coli is active as a monomer (16). Other basic questions regarding the tryptase tetramer concern the nature of its subunits. It is not yet known if tetramer formation requires interaction between the protein products of more than one tryptase gene or if the products of one tryptase gene can form a homotetramer. Whether tryptase gene expression or subunit composition varies among mast cells found in different tissues is likewise unresolved. Another intriguing mystery is the mechanism of formation and maintenance of the active tetramer, which, in the absence of heparin at physiologic ionic strength, dissociates rapidly and irreversibly into inactive monomers (17). The heparin-associated tetramer appears to be maintained entirely by noncovalent interactions because high ionic strength buffers dissociate the tetramer from heparin while stabilizing the configuration of the active tetramer (17, 18). Thus, the interaction of the tetramer with heparin is mainly ionic in nature, whereas the interactions of the subunits with each other are mainly due to hydrophobic interactions. One can postulate that the tetramer-to-monomer conversion provides a mechanism for the physiologic control of tryptase activity following release of the heparin -associated complex from the mast cell. However, in the presence of heparin, the activity of tryptase in undiluted serum is preserved for hours, and there is no convincing evidence that conversion to the inactive monomer is an important means of regulating tryptase activity. The cDNA and gene sequences of tryptases indicate that the nascent protein is synthesized with a hydrophobic signal peptide and a novel propeptide, which are removed during maturation into the active tetramer (see Figure 1). The precise length of the signal peptide, which directs tryptase into the endoplasmic reticulum and is removed by signal peptidase, is not known. However, comparison of the 30-residue preprosequence deduced from dog and human tryptase cDNAs shows a high level of sequence identity among the last 11 amino acids, which probably include the propeptide (1, 13). Unlike signal sequences, propeptides typically are highly conserved among functionally related groups of serine proteinases. The prosequence of tryptases, by analogy to other eukaryotic serine proteinases, should function as an activation peptide. However, it is unique because it ends in Gly (1-3, 13). The chymases and related enzymes have a short acidic pro sequence (see below), and the great majority of other serine proteinases are activated by tryptic hydrolysis of

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activation sequences, which end in a basic residue, i.e., Lys or Arg. The presence of a unique tryptase indicates that tryptases may not share a mechanism of activation with other serine proteinases. It is also possible that the propeptide serves functions other than zymogen activation, perhaps in granule targeting or tetramer assembly. The finding of a dog cDNA encoding a protein related to tryptases in its prosequence (13) suggests that tryptases may be members of a new group of serine proteinases sharing a novel means of activation, and that other members may remain to be discovered. Chymase Chymase from human skin is a glycosylated, chymotrypsinlike, cationic monomer with a high affinity for heparin (19). A chymase-like enzyme purified from human left ventricle has similar properties (20) but may be less highly glycosylated and more susceptible to autolytic hydrolysis at an internal site. Immunohistochemical studies suggest that the skin enzyme is an exclusive product of mast cells (5, 19); however, the cell of origin of the heart chymase remains to be established. A chymase from human lung seems similar to the skin enzyme but has been less thoroughly characterized (21). Only partial amino acid sequence information is available for chymases of human origin, and it is not known whether chymases in different human tissues are the products of the same or different genes. The most detailed structural insights concerning mast cell chymases arise from studies of rat (6,22-24), mouse (9, 10), and dog (25, 26) enzymes. From these studies, it is apparent that chymases from different species and from various mast cell subsets share numerous structural features. In particular, all are likely to possess patterns of folding, domain organization, and secondary structure that mimic very closely those of RMCPII, the mast cell chymase whose crystal structure has been solved to 1.9-A resolution (23). A model based on the crystallographic coordinates of this chymase is displayed on the cover of this issue of the journal. However, the level of sequence similarity is surprisingly low for a group of proteinases whose members are considered to have diverged only recently in protein evolution (8) and are presumed to share biologic functions. Thus, to know one chymase is not to know them all. One property in which chymases differ greatly is surface charge. Rat chymase I, for example, is predicted to be highly cationic (net charge, +18) at the pH of extracellular fluids and is released from connective tissue mast cells as an insoluble complex with macromolecular heparin proteoglycan (27). RMCPII, however, is nearly neutral (net charge, +4) and appears to be released from mucosal mast cells in a freely soluble form, which can be detected in the bloodstream (28). Asn-linked glycans, which are found in dog and human chymases, are not a universal feature of chymases, for neither of the rat chymases are glycosylated. Because the C-terminal amino acid sequence deduced from cloned RMCPII cDNA is three residues longer than the sequence determined by direct amino acid sequencing (6,22), RMCPII appears to be subject to C-terminal processing (see Figure 2). However, the functional significance of such processing, and its presence or absence in other chymases, remains to be explored. Other features that distinguish mast cell chymases from tryptases, including the presence of only

three disulfide bonds and of a "signature" octapeptide (residues 9 through 16) (25), are also shared by chymaserelated enzymes, such as cathepsin G and lymphocyte granzymes. As noted above, chymase prepropeptides differ from those of tryptases. The cloned rat, mouse, and dog cDNAs reveal a conventional hydrophobic signal peptide followed by an acidic two-residue propeptide terminating in Glu. The three major neutrophil granule serine proteinases (cathepsin G, elastase, and proteinase-3), as well as most of the lymphocyte granzymes, contain similar acidic dipeptides, implying that all share a similar pathway of activation from precursors. The evidence supporting a role as an activation peptide for this two-residue sequence stems from studies of biosynthetically labeled neutrophil elastase and cathepsin G, which are synthesized as proenzymes containing the acidic dipeptide and are processed to the mature N- and Cterminally processed enzymes within minutes of translation (29). Chymase presumably follows the same paradigm, resulting in the accumulation of active enzyme in secretory granules. The subcellular site of propeptide removal and the identity of the enzyme or enzymes required for the processing event are not known.

Mast Cell Proteinase Expression, Packaging, and Release As demonstrated by immunogold electron microscopy, mast cell tryptase and chymase are packaged in the same mast cell granules (30). However, human mast cells differ in their patterns of expression of tryptase and chymase. Although tryptase is a constituent of all human mast cells examined so far, chymase is limited to a particular subset. Thus, mast cells are divided into two groups based on proteinase expression: the "MC T" subset containing tryptase without chymase and the "MC Tc " subset containing both enzymes (5). The levels of neutral proteinases stored in human mast cells are remarkably high, ranging from 11 to 35 pg/cell of tryptase in MC T and MC Tc mast cells and t'\J4.5 pg/cell of chymase in MC Tc mast cells (31). Indeed, tryptase and chymase are the most abundant proteins in mast cells. By comparison, elastase and cathepsin G are found in neutrophils at a level of t'\J 1 pg/cell. In human lung, the chymase-containing subset of mast cells is poorly represented in alveolar tissues, where they comprise only rv7% of the total (5). However, the percentage of chymase-containing mast cells is considerably higher in bronchial and bronchiolar subepithelium (5). Stimulation of mast cells provokes the release of tryptase and chymase along with other preformed mediators, such as histamine (32). Intriguingly, noncytotoxic degranulation of mast cells also releases classical lysosomal acid hydrolases (33), such as aryl sulfatase and hexosaminidase, in parallel with histamine and the neutral proteinases that are the focus of this Update. This suggests that there may be no physical or functional difference between lysosomal and secretory granule compartments in mast cells. Given this observation, one can speculate that tryptase and chymase are lysosomal hydrolases primarily serving an intracellular function, and that they are released outside of the cell with mediator molecules like histamine only because they happen to reside in the same intracellular organelle. Certainly, it can be argued that an extracellular role for the classical lysosomal hydro-

391

Update

lases, whose activity is usually confined to low-pH environments, is unlikely. However, as discussed below, tryptase and chymase readily hydrolyze targets outside of the cell because of their neutral to alkaline pH optimum. Indeed, although tryptase and chymase appear to be stored in the granule in an activated form rather than as precursor zymogens (32), their actions probably are restricted to the extracellular milieu, because, like most serine proteinases, they have negligible activity at the acidic pH of the secretory granule. Tryptase and chymase are stored in mast cell secretory granules with a heterogeneous assortment of sulfated proteoglycans, principally heparin- and chondoitin sulfate-containing species varying in extent of sulfation. The fate of human chymase following mast cell stimulation and release is not known. However, human tryptase can be measured readily in a variety of biologic fluids following mast cell activation. Indeed, tryptase has emerged recently as a marker of mast cell involvement in human pathology. After anaphylaxis, tryptase appears in the bloodstream, where, in contrast to histamine, it remains detectable for several hours (34). Its appearance has been detected in samples of nasal and lung lavage fluid from atopic subjects challenged with specific antigen (35, 36). Tryptase levels in lung lavage fluid obtained from atopic asthmatics increase after endobronchial allergen challenge, but are also elevated at baseline, implicating persistent mast cell activation in the pathogenesis of atopic asthma (36). In contrast, lung lavage tryptase levels do not increase after exercise in subjects with exercise-induced asthma (37), suggesting that mast cell activation is not involved in this type of nonatopic asthma. Some smokers of cigarettes have striking elevations of bronchoalveolar lavage fluid tryptase levels compared to nonsmoking controls (38), a finding that provides some support for the hypothesis that release of proteinases from activated mast cells could contribute to lung destruction in smoker's emphysema.

TABLE 1

Summary of biologic activities of tryptase and chymase Activity Tryptase Cleaves fibrinogen a-chains; cleaves high-molecular-weight kininogen with possible release of kinins

Significance

With heparin, may act as a local anticoagulant

Activates prostromelysin (proMMP-3); activates procollagenase (pro-MMP-I) via MMP-3

Possible role in tissue inflammation and remodeling; possible role in joint destruction in rheumatoid arthritis

Cleaves and inactivates vasoactive intestinal peptide (VIP); cleaves peptide histidine-methionine (PHM)

Possibly increases bronchoconstriction in asthma by destroying bronchodilating peptides

Cleaves calcitonin gene-related peptide (CGRP)

May regulate flare reaction in cutaneous neurogenic inflammation

Acts as a mitogen and co-mitogen for fibroblasts

Possible pathogenic role in fibrotic disorders of lung and skin

Chymase Augments histamine-induced wheal formation in skin

May regulate vascular permeability changes in immediate hypersensitivity

Cleaves substance P

Possibly plays feedback role in limiting substance P-induced mast cell degranulation in cutaneous neurogenic inflammation

Converts angiotensin I to II

Possible role in extravascular generation of vasoactive peptides

Stimulates degranulation of airway submucosal gland serous cells

Possible role as secretagogue in asthma and bronchitis

Cleaves extracellular matrix and glycocalyx

Possible role in tissue inflammation

Biologic Activities of Mast Cell Proteinases Tryptase In vivo explorations of the biologic functions of tryptase in the lungs and other tissues have been hindered by a lack of neutralizing antibodies or selective, noncytotoxic inhibitors of catalytic activity. Nonetheless, studies in vitro provide evidence of several possible roles for tryptase in the airways and pulmonary parenchyma. In isolated dog bronchi, purified dog tryptase has the striking and unexpected effect of enhancing smooth muscle contraction in response to histamine (39). Tryptase enhances both the sensitivity and magnitude of histamine-induced bronchoconstriction, and yet, in the absence of histamine, has no effect on smooth muscle tone. Tryptase-augmented bronchoconstriction is prevented by active-site inhibition of tryptase and by histamine HI-receptor and voltage-dependent Ca2+-channel antagonists. Furthermore, tryptase potentiates the effects of serotonin and KCI, whose effects on muscle contraction are thought to involve voltage-gated Ca 2+ channels. These observations, which need to be confirmed in human tissues, raise the intriguing possibility that tryptase could play a role in bronchial hyperresponsiveness to histamine, a nearly universal feature of asthma. A mechanism consistent with the physiologic observations is a hydrolytic activation of Ca2+-chan-

nel protein or of a surface-expressed protein regulating the channel. Tryptase also may influence bronchomotor tone by hydrolyzing and inactivating bronchoactive neuropeptides. Purified dog and human lung tryptase hydrolyze and inactivate vasoactive intestinal peptide (VIP) and peptide histidine-methionine (PHM) (40-42), neuropeptide transmitters of the nonadrenergic inhibitory nervous system, which, in human airways, provide the principal relaxant influence (43). VIP appears to be deficient in asthmatic airways (44), where its diminished influence has been proposed to underlie the enhanced bronchoconstriction seen in asthma. Thus, it is attractive to hypothesize that tryptase promotes bronchial hyperresponsiveness by destroying bronchodilating peptides. The most favorable kinetics of hydrolysis of a natural peptide by tryptase are found using calcitonin gene-related peptide (CORP) (42), which is a potent vasodilator coreleased with substance P from sensory neurons. Its release in the skin is probably responsible for the flare component of the "triple response" to skin injury, a process that also involves substance P-mediated mast cell degranulation. Thus, tryptase may be responsible in whole or part for the marked

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decrease in duration of the CGRP-induced flare response in vivo in the presence of mast cell degranulation (42,45). Substance P and related tachykinins are not inactivated by tryptase, but are cleaved readily by chymase (40, 42), which may play a role in limiting the actions of substance P released from cutaneous sensory nerves. Although substance P and CGRP-containing nerves have been identified in the lung and airways, the nature and importance of their physiologic roles and mast cell interactions in pulmonary tissues are less well established. However, it seems reasonable to assume that some of the regulatory principles identified in the skin apply also to the lung and airways. The recent demonstration of the potent fibroblast growthstimulating activity of tryptase (46) has provided a possible molecular link between mast cell activation and fibrosis. In the lungs, this observation has particular significance in regard to the pathogenesis of fibrotic lung diseases, which are associated with mast cell proliferation and activation, and the pathologic changes in asthmatic airways, which are characterized by subepithelial fibrosis. Incubation of tryptase with cultured fibroblasts, such as the CCL39 line of hamster lung cells, elicits increases in thymidine uptake equivalent to those of thrombin or fetal calf serum. Just as significantly, perhaps, threshold concentrations of tryptase synergize dramatically with other classical growth factors, including epidermal growth factor, fibroblast growth factor, and insulin, to produce large increases in fibroblast DNA synthesis. Several other identified actions of tryptase are potentially relevant to pulmonary physiology. Like a number of proteinases, tryptase promotes collagen digestion indirectly by activating stromelysin (matrix metalloproteinase 3), which, in turn, activates collagenase (matrix metalloproteinase 1) (47). This observation gives rise to the suggestion that tryptase, released in synovial fluid, participates in destruction of rheumatoid joints. However, it could also give tryptase a role in tissue remodeling in lung injury and fibrosis. In contrast to chymase, tryptase has little or no direct effect on digestion of collagen (types I through V), nor does it cleave other connective tissue components, such as proteoglycan, fibronectin, or laminin (48). Indeed, compared to trypsin and many other serine proteinases, tryptase is highly selective. Many proteins potentially susceptible to tryptic activation are not affected by tryptase. These include zymogens of the complement system, coagulation, and fibrinolysis, as well as plasma and tissue kallikreins (48, 49). Tryptase does, however, destroy fibrinogen and high-molecular-weight kininogen (50, 51), which may give the tryptase-heparin complex a role as a tissue anticoagulant. Chymase As is true of tryptase, there are no genetic disorders involving selective deficiency of chymase, nor are highly selective inhibitors available for probing the in vivo actions of the enzyme. Nonetheless, a number of observations strongly suggest the possibility of important roles for chymase. Recent studies in atopic dogs, involving cutaneous injections of purified chymase and histamine and endogenous release of both mediators using ragweed antigen, suggest that chymase modulates vascular permeability changes leading to wheal

formation in vivo (52). The mechanism of this effect remains to be determined. However, the observation invites speculation that similar events take place in nasal and tracheobronchial tissues of atopic individuals. The peptidase activity of chymase is its most extensively characterized feature. When their activity is profiled against a battery of peptidyl-4-nitroanilides, chymases are selective but highly active in hydrolyzing certain substrates and thus exhibit pronounced subsite preferences, especially compared to chymotrypsin (53). Dog chymase cleaves the Tyr 22-Leu23 bond of VIP and the Phe'-Phe'' bond of substance P (40, 41). The human heart chymase appears to have limited ability to hydrolyze VIP. However, human chymases have a rather striking ability to activate angiotensin I to angiotensin II, hydrolyzing the Phe 8-His9 bond (20, 54). The kinetic parameters (i.e., kca.lKm) of this converting enzyme activity are more favorable than that of angiotensin-converting enzyme itself. Chymase may account for more than 75 % of converting enzyme activity in human heart tissues (20). Thus, chymase has the potential of being a major factor in extravascular generation of angiotensin II, which is a cardiac inotrope as well as vasoconstrictor. However, it remains to be shown that chymase is released in sufficient amounts and that it remains active following release for a sufficient time to playa physiologic role in the generation of angiotensin II in the heart and other tissues. Recent observations suggest that chymase stimulates secretion from cultured airway submucosal gland cells (55). This secretagogue activity takes the form of noncytotoxic degranulation. The extent of degranulation is dramatically greater than that caused by more familiar secretagogues such as histamine and isoproterenol, and results in exocytosis of f\.J80% of sulfated granule proteoglycan. This observation suggests a potential role for chymase in airway hypersecretion, as in asthma or bronchitis. This type of activity is not an exclusive property of chymase, for neutrophil cathepsin G and elastase possess similar activity (56). However, recent morphometric analysis of human airway subepithelium (unpublished results) suggests that more than 70% of the mast cells within 20 J.'m of bronchial submucosal glands contain chymase (the highest percentage of any airway location), supporting the possibility of a role for chymase in the physiologic regulation of gland secretion. Chymase and the neutrophil enzymes also possess proteoglycanase activity, for they partially digest the chondroitin sulfate proteoglycans whose release they stimulate. In addition, other studies have established that chymase can remove components on the surface glycocalyx of cultured airway epithelial cells (57) and can degrade constituents of extracellular matrix (58). Thus, the consequences of chymase release in the immediate milieu of a mast cell degranulating in the airway could range from generation of vasoactive peptides to stimulation of gland secretion and solubilization of extracellular matrix. All of these effects require in vivo validation.

Conclusions and Future Directions In conclusion, tryptases and chymases are abundant secretory proteinases of mast cells, which are widely distributed in vertebrate tissues, including the airways and lung parenchyma. Much is known of their structure and of their activity

Update

in vitro, and a large body of morphologic and biochemical information suggests that they could be important in pulmonary pathophysiology. However, more needs to be learned of the properties and tissue-specific expression of different molecular forms of tryptase and chymase, of the rates of diffusion and half-life of active enzyme following release, and of the nature and fate of the extracellular targets of the enzymes in vivo. To evaluate the hypotheses generated by the in vitro data and to establish specific roles for the enzymes in vivo, it is especially important to develop noncytotoxic inhibitors selective for tryptases and chymases to use as pharmacologic tools. To the extent that the enzymes are important in the pathogenesis of human diseases, the development of selective inhibitors also may lead to the identification of new therapeutic agents. Acknowledgments: The writer is a recipient of Clinical Investigator Award HL01736 from the National Institutes of Health and of an RJR-Nabisco Research Scholar Award in Pulmonary.

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18.

19.

20. 21. 22. 23. 24. 25.

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