Mast Cell Proteases as Protective and Inflammatory Mediators

Abstract

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_cεRI by IgE bound to multivalent allergen. Understanding and separating the functions of the proteases is important because expression differs among mast cells in different tissue locations. Differences between laboratory animals and humans in protease expression also influence the degree of confidence with which results obtained in animal models of mast cell function can be extrapolated to humans. The inflammatory potential of mast cell proteases was the first aspect of their biology to be explored and has received the most attention, in part because some of them—notably tryptases and chymases—are biomarkers of local and systemic mast cell degranulation and anaphylaxis. Although some of the proteases indeed augment allergic inflammation and are potential targets for inhibition to treat asthma and related allergic disorders, they are protective and even anti-inflammatory in some settings. For example, mast cell tryptases may protect from serious bacterial lung infections and may limit the “rubor” component of inflammation caused by vasodilating neuropeptides in the skin. Chymases help to maintain intestinal barrier function and to expel parasitic worms, and may support blood pressure during anaphylaxis by generating angiotensin II. In other life-or-death examples, carboxypeptidase A3 and other mast cell peptidases limit systemic toxicity of endogenous peptides like endothelin and neurotensin during septic peritonitis, and inactivate venom-associated peptides. On the other hand, mast cell peptidase-mediated destruction of protective cytokines, like IL-6, can enhance mortality from sepsis. Peptidases released from mast cells also influence non-mast cell proteases, such as by activating matrix metalloproteinase cascades, which are important in responses to infection and resolution of tissue injury. Overall, mast cell proteases have a variety of roles—inflammatory and anti-inflammatory, protective and deleterious—in keeping with the increasingly well-appreciated contributions of mast cells in allergy, tissue homeostasis, and innate immunity.

INTRODUCTION

Several recent reviews provide in-depth coverage of particulars of mast cell protease biochemistry, genetics and biological function ^1–4. The present chapter emphasizes mast cell protease function as it relates to host defense and its frequent by-product, inflammation. The link between mast cell proteases and inflammation is not (as some might assume) automatic; rather, the overall effect can be anti-inflammatory and homeostatic. This chapter cannot do justice to all of the work being done in this field, which has expanded rapidly in the past five years, but it will summarize major recent findings shaping current notions of protease contributions to mast cell function and pathobiology.

PROTECTIVE and ANTI-INFLAMMATORY EFFECTS

Control of neurogenic inflammation: Tryptases, calcitonin gene-related peptide and the triple response of Lewis

A classical response to injury (such as a that created by stroking skin with a blunt instrument) is a red line appearing at the site of injury, followed by transient flare or redness surrounding the region of injury, and a wheal due to edema at and near the site injury. This is the so-called triple response of Lewis. The redness or “rubor” aspect is a cardinal sign of inflammation, along with “calor” (heat) and tumor (swelling). The red line is partly due to the release of histamine from mast cells under the influence of the neuropeptide substance P discharged from sensory nerves stimulated by the injury. The flare is attributed mainly to release of calcitonin gene-related peptide (CGRP) from sensory nerves stimulated by antidromic propagation of sensory nerve signals originating at the site of injury. The transience of CGRP-induced vasodilation is thought to be due to extracellular release of peptidases from mast cells activated by substance P ⁵. CGRP is hydrolyzed by tryptases, and, kinetically speaking, may be the best natural substrate yet identified for human β-tryptase ⁶, which inactivates CGRP’s vasodilating actions ⁷. More recent evidence suggests that primary spinal afferent neurons containing CGRP and substance P also contain protease-activated receptor (PAR)-2 ⁸, which can be activated by tryptases ^9–14. Although tryptase is weak compared to trypsin as a PAR-2 activator, mast cells are often near neurons ¹⁵, which may be exposed to high concentrations of tryptase during mast cell degranulation. Thus nerves, substance P, CGRP and tryptases may be involved in feedback loops that limit neurogenic inflammation. In effect, tryptases detoxify CGRP, which is perhaps the first-described example of a more general function for tryptases discussed below in connection with venoms and toxic non-neural peptides.

Thresholds for protective nociception and bronchoconstriction: roles of tryptases and PAR-2

Tryptase-activated neural PAR-2 is implicated in the component of itching in atopic dermatitis that is unresponsive to anti-histamines ^{16, 17}. Furthermore, a recently appreciated phenotype of PAR-2-null mice is lowered nociceptive thresholds, such as those involving sensitivity to dermal and visceral pain ¹⁸, although a role for tryptases in setting pain thresholds in vivo is partly speculative at this point. Itch and pain are both essentially protective, because they alert the host to the presence of pathogens or impending tissue damage. PAR-2 also is expressed in non-neural tissues and cells, such as airway smooth muscle ¹¹; indeed, isolated bronchi constrict in response to PAR-2 agonists, including tryptases ^19–22. Nonetheless, it is not yet clear that the bronchoconstricting activities of tryptases are mediated fully via interactions with PAR-2, especially since most studies find that tryptase, rather than constricting smooth muscle on its own, potentiates the actions of known constrictors, such as histamine, so that they act at lower concentrations and to greater maximum effect ^{19, 20, 22, 23}. Furthermore, PAR-2 agonists acting on epithelium rather than smooth muscle can cause bronchodilation by stimulating epithelial release of PGE2 ²⁴. Alternative mechanisms by which tryptase may promote bronchoconstriction include degrading bronchodilating peptides (leaving bronchoconstrictors unopposed) ^{6, 25–27}, spreading degranulation signals ²³, untethering muscle from the bronchial wall by cleaving extracellular matrix or activating matrix-cleaving proteases ²⁸, and desensitizing smooth muscle cells to bronchorelaxing β-adrenergic agonists ²⁹. Tryptase’s overall effect is to promote bronchial hyperresponsiveness, a hallmark of asthma. In essence, this is protective, if one accepts that a major purpose of airway smooth muscle is to guard airways from entry of unwanted flora, fauna and other noxious substances, for which mast cells can be sentinels. Surprisingly, the actual function and true benefits of airway smooth muscle are unknown. Clearly, the purpose is not to cause asthma.

Feedback detoxification of neurotensin by neurolysin and carboxypeptidase

Neurotensin is a 13-residue fragment of a much larger polypeptide that includes the neuromedin N. Neurotensin gains partial protection from degradation by post-translational cyclization of its N-terminal amino acid to pyro-glutamate, thereby eliminating its free amino terminus and reducing its sensitivity to shortening by amino-peptidases. Neurotensin is usually classified as a neuropeptide, since it can originate from neurons. It causes hypotension when injected into mice; moreover, endogenous neurotensin can influence mortality in septic shock, because mice lacking neurotensin are less likely to survive septic peritonitis. Serum levels of neurotensin are elevated in humans with shock and sepsis. The origin of serum and peritoneal neurotensin is suspected to be neural; however, the actual sources in these conditions are not known ³⁰. Intriguing experiments in mice subjected to cecal ligation and puncture, which models septic peritonitis from a ruptured appendix, suggest that mast cells respond to neurotensin in the peritoneum and play a detoxifying role significant enough to affect survival. Although neurotensin is not a strong mast cell degranulator, the detoxifying effect of mast cells depends at least partially on mast cell expression of neurotensin receptor 1, which may signal these cells to express an inactivating metallopeptidase, neurolysin—possibly on the cell surface (see Figure 1). Secreted carboxypeptidase A3 also appears to play a role, presumably by removing residues from neurotensin’s unprotected C-terminus. These findings suggest one explanation for the known benefit of peritoneal and mesenteric mast cells in recovering from cecal ligation and puncture. Whether mast cell-mediated detoxification of neurotensin is important in other types of sepsis and shock—and in humans—remains to be determined.

Figure 1. Mast cell-mediated protection from toxic endogenous peptides in sepsis.

Bacterial sepsis is associated with release of toxic peptides like neurotensin and endothelin-1 from nerves and vascular endothelium, respectively. These peptides are recognized by receptors on mast cells, stimulating release, activation or upregulation of detoxifying peptidases, such as carboxypeptidase A3 (CPA3), chymases, and neurolysin. This is an example of a homeostatic function of mast cells.

Detoxification of endogenous non-neural peptides and proteins

Endothelin

Many endogenous peptides have the potential to harm as well as to help when produced in response to microbial invasion or tissue injury. One particularly closely examined example, intriguing both in regard to its complexity and physiological importance, is provided by endothelins, which are family of peptides produced by endothelial cells in response to injurious stimuli. Mature endothelins act by engaging receptors on the surface a variety of cells, including vascular smooth muscle and mast cells. They can have acute and longer-term effects, including vasoconstriction, mast cell activation, and vascular remodeling. Drugs antagonizing interactions of endothelins with receptors have found a place, for example, in treating idiopathic pulmonary hypertension. Maurer and colleagues ³¹ described an intriguing nexus between endothelin and mast cells in the peritoneum (see Figure 1). Endothelin injected into the peritoneal cavity of mice is toxic—and, in sufficient doses, lethal—and also has toxic potential when produced endogenously in the context of septic peritonitis. Mouse peritoneal mast cells possess type A endothelin receptors and respond to endothelin by releasing destructive peptidases, which may include chymases and mast cell carboxypeptidase A3. Schneider and colleagues established more recently that removal of a single tryptophan residue from endothelin’s C-terminus is the principal inactivating event ³² in mice. Although other mast cell peptidases, including mouse chymases, can nick endothelin internally, this is not necessarily inactivating, because of stabilizing disulfide bonds. Indeed, the overall contribution of chymases to endothelin homeostasis is uncertain, and may be species-specific, because some chymases can process and activate endothelins from larger, precursor forms. For example, human chymase processes precursor “big” endothelins into a novel, bronchoconstricting form (endothelin [1–31]) ³³, whereas rat chymases are less selective and degrade big endothelins as well as endothelin [1–31], which possesses bronchoconstricting, vasoconstricting, and vascular smooth muscle-proliferating activity ^{33, 34}, and thus has potential to contribute to pathological bronchoconstriction in human asthma and vascular remodeling in pulmonary hypertension, systemic atherosclerosis, and re-stenosis after angioplasty. In reference to mice specifically, it should be noted that the overall role of endothelin-1 in asthma-like bronchoconstriction is not clear, since endothelin-1-null mice exhibit airway hyper-responsiveness ³⁵. In humans, then, it appears that mast cells plausibly are involved in activating or inactivating endothelin (or both in succession, since activator chymase and inactivator carboxypeptidase A3 are usually but not inevitably found in the same subsets of mast cells and released together ^36–38). Thus, it is reasonable to hypothesize that the role of human mast cells activated by already-mature endothelin is to inactivate the peptide—i.e., to limit toxicity by preventing accumulation and shortening duration of action—but that the role of human mast cells activated by other stimuli, such as allergen, first may be to activate via chymase-mediated processing of precursor big endothelins to smaller active forms, followed perhaps by mast cell carboxypeptidase A3-mediated inactivation.

Cathelicidin

In studies of human lung mast cells, Schiemann and colleagues showed that antibacterial peptide cathelicidin LL-37 (production of which is induced during inflammation) stimulates mast cells to degranulate and secrete β-tryptase, which in turn inactivates cathelicidin ³⁹. This is an example of immunomodulatory negative feedback similar to that described for endothelin. On the other hand, human chymase and cathepsin G in skin mast cells can activate platelet-derived connective tissue activating peptide III to generate neutrophil-activating peptide II, a neutrophil chemokine ⁴⁰. Therefore, downstream effects of human mast cell degranulation on inflammation will depend on the nature of mast cell stimulus, the protease phenotype of the stimulated mast cell, and the local availability of targets cleavable by the secreted proteases.

Cytokines and interleukins

Mast cell ability to degrade cytokines can be striking. In studies of activated human skin mast cells, exocytosed proteases markedly diminish immunodetection of mast cell-secreted cytokines like IL-6, IL-13 and TNFα, presumably by destroying epitopes recognized by antibodies ⁴¹. Inhibitor screens suggest that chymase and cathepsin G are more responsible than are tryptases. These effects are likely to be anti-inflammatory, to the extent that they reduce biological activity as well as immunoreactivity of inflammatory cytokines. Unless countered by inhibitors, this peptidase activity causes considerable underestimation of mast cell production of several cytokines. The in vivo importance of these effects is less clear, because chymases and cathepsin G are released into environments rich in inhibitors, such as α₂-macroglobulin and α₁-antichymotrypsin ^42–44. There are, however, hints from mice that modulation of cytokine activity can be biologically significant in vivo. In septic peritonitis, hydrolytic inactivation of mast cell-derived IL-6 by mMCP-6 tryptase (and perhaps other peptidases) appears to lead to increased mortality ⁴⁵. This suggests that cytokine-cleaving activities of mast cell proteases can be counterproductive, even while reducing inflammation.

Detoxification of snake and bee venoms

Mast cells have a well-deserved reputation for contributing to severe anaphylactic reactions to bee and wasp stings. Numerous deaths result from systemic release of mast cell mediators of shock and inflammation. Although hymenoptera venoms are complex mixtures of peptides and proteins, some of which directly degranulate mast cells, severe reactions are mediated by mast cell-bound IgE recognizing venom components based on prior sensitization. Because of this association between mast cells and fatal reactions to envenomation, recent evidence ⁴⁶ that mast cells protect mice from lethality of some venoms is at first glance counter-intuitive. One example was provided by sarafotoxins, a class of venoms produced by snakes related to mole vipers. Sarafotoxins are homologous to endothelins, which, as discussed earlier, activate mast cells to release peptidases. Metz and colleagues ⁴⁶ showed that mast cell detoxification of sarafotoxins involves similar mechanisms and affords some protection from lethal outcomes of envenomation. As is true for endothelin, carboxypeptidase A3 plays a prominent role in disarming the toxin. It is not yet clear whether this effect applies broadly to other venom peptides and proteins. However, mast cells protect mice from hypothermia and death caused by the venom of two snakes (rattlesnake and copperhead) not containing sarafotoxins. Carboxypeptidase A3 involvement in these cases is not as prominent. Thus, for some venoms, other detoxifying peptidases, like chymases and tryptases, may be important. By unclear mechanisms, mast cells also partially protect mice from effects of honeybee venom ⁴⁶, which contains mast cell-degranulating apitoxins.

Secretion, activation and destruction of matrix metalloproteinases (MMPs)

MMPs in remodeling and resolution of inflammation

The classic gelatinolytic MMPs produced by inflammatory cells are MMP-2 (gelatinase A) and MMP-9 (gelatinase B). Although mast cells and mast cell lines can secrete MMP-2 and MMP-9 ^47–51, these enzymes also are produced by a variety of other inflammatory cells, which are likely to be the dominant source in inflamed tissues. MMP-9, for example, is especially abundant in neutrophils, and its levels in inflamed tissues and fluids tend to track with the number of neutrophils. It is not clear whether the well-known and often-assessed capacity of these enzymes to hydrolyze denatured collagen (gelatin) in polyacrylamide gels mirrors or predicts their roles in vivo, for these enzymes cleave a variety of proteins ^{52, 53} and there is perhaps no equivalent of gelatin in vivo. MMP-9-null mice have phenotypic abnormalities involving long bone angiogenesis ⁵⁴, but there is no apparent deficit in airway inflammatory angiogenesis and lymphangiogenesis stimulated by mycoplasma infection—and these mice do develop neutrophilic pneumonia ⁵⁵. Although MMP-2 (which increases in lungs of infected MMP-9-null mice) potentially could compensate for lack of MMP-9, MMP-2/MMP-9 double knockout mice have a neutrophilic pneumonia and airway angiogenesis phenotype similar to that of MMP-9-null mice ⁵⁵. Data from mice suggest that allergic pulmonary inflammation may differ in this regard ^{53, 56, 57} because lack of MMP-2 and (especially) of MMP-9 hinders egress of recruited eosinophils and other leukocytes from pulmonary interstitium into the airspaces, from which they would otherwise be cleared via the mucociliary and/or macrophage-mediated apoptotic pathways. This clogs interstitial spaces with leukocytes, impairs gas transfer, and “asphyxiates” the mice. These findings are intriguing because they suggest that MMP-2 and MMP-9 promote resolution of allergic inflammation. Indeed, studies of other types of inflammation also suggest that control of inflammation and associated remodeling may be an important function of these MMPs, as in a model of bronchopulmonary dysplasia, in which transgenic Mmp9 −/− mice have more pulmonary macrophages and alveolar hypoplasia ⁵⁸.

Mast cells as a source of gelatinolytic MMPs

The most detailed evidence that mast cells produce gelatinolytic MMPs stems from studies of canine, mouse and human mast cells ^{47, 49, 50, 59}. In canine lines, MMP-9 production is regulated by kit ligand and TGFβ ⁴⁸. MMP-9 appears to be released in a constitutive and regulated manner as a pro-form bound to MMP inhibitor tissue inhibitor of metalloproteinases (TIMP)-1. Thus, MMP-9 is secreted as an inactive, pro-MMP-9/TIMP-1 complex ^{47, 60}. Activation of the mast cell-secreted complex occurs outside of the cell, especially in cells that are stimulated to release contents of serine protease-rich secretory granules. Because mast cell MMP-9 seems to be secreted independently of its serine protease activators, it is likely that the pro-MMP/TIMP-1 complex originates in a compartment separate from the classic secretory granule. Human mast cells also exhibit the interesting property of secreting MMP-9 upon direct contact with T lymphocytes ⁵⁰.

Activation, disinhibition and destruction of MMPs by chymases

The interactions of mast cell chymases with MMP-9 are biochemically intriguing and possibly unique. Studies of canine mast cell chymase and pro-MMP-9 reveal that chymase activates pro-MMP-9 by selectively hydrolyzing residues in the MMP-9 pro-peptide ^{47, 60}. This yields a large gain in solution-phase proteolytic activity. Beyond this, chymase activates MMP-9 even while bound to TIMP-1 ⁶¹ and is the only MMP-9 activator shown to possess this capability. Because much extracellular MMP-9 is imbedded in matrix as a TIMP-1-bound pro-enzyme, chymase released from mast cells can bring MMP-9 to life from latency, and launch cascades of MMP activation initiated by MMP-9, which can activate other MMPs. Chymase accomplishes activating TIMP-1-bound MMP-9 by cleaving TIMP-1 itself. Although interactions between chymase and MMP-2 have been studied less intensively, chymase inhibitors are reported to decrease intimal hyperplasia in balloon-injured canine carotid arteries, with an accompanying decrease in activated MMP-2 ⁶². Although the biochemistry of MMP-9 has been explored in greatest detail using canine enzymes and mast cell lines, the phenomenon occurs in other mammals. In mice, for example, the principal pro-mMMP-9-activating chymase is mMCP-4 ^{49, 51}, whose actions are hypothesized to be important in tumor growth and invasion in a model of skin cancer ⁴⁹ as well as in collagenous ear thickening and lung fibronectin accumulation, which are attributed to reduced matrix turnover in mMCP-4-null mice ⁵¹, which also suggest that mMCP-4 regulates levels of MMP-2 ⁵¹. More recently, a study of chymase inhibitors in a colitis model reveals markedly reduced colonic MMP-9 levels in inhibitor-treated mice ⁶³. Similar effects occur in angiotensin II-induced aortic aneurysms ⁶⁴. Chymase can destroy MMP-9 activity with prolonged incubations, although its preference appears to be for activating cleavages. It should be stressed that the net effect of MMP-9 activation by chymases could be anti-inflammatory, given MMP-9’s involvement in resolution of inflammation.

Mast cell protease-facilitated activation of the renin-angiotensin system

Updating paradigms

Research over the past two decades built a very solid body of evidence supporting physiologically important mast cell peptidase activation of the renin-angiotensin system, whose end product is the vasoactive octapeptide angiotensin II. Several aspects of involvement of mast cell peptidases in generating angiotensin II are at odds with the partly outmoded concept that the key steps in generating angiotensin II occur within vessels, starting with regulated release of the aspartyl peptidase renin into the bloodstream by kidney cells responding to drops in renal perfusion pressure. According to the classical paradigm, renin then cleaves off a portion of the N-terminus of a circulating protein, angiotensinogen to generate the decapeptide angiotensin I. This inactive peptide is then hydrolyzed by angiotensin converting enzyme (ACE), an ecto-metallopeptidase on the surface of pulmonary vascular endothelial cells, to generate the angiotensin II, which binds to receptors in arteriolar smooth muscle to constrict vessels and acts on the adrenal gland and kidney to promote retention of sodium and water. This process is homeostatic, being designed to preserve blood pressure. Additional essentially protective roles may include vasoconstriction to reduce bleeding and edema during tissue injury. In this regard it is interesting to note the association between use of ACE inhibitors and more severe systemic reactions to insect stings ⁶⁵. ACE and angiotensin II are unquestionably important in human health, as shown by the success of therapeutic agents directed against ACE or receptors of its product, angiotensin II. As discussed in more detail below, mast cells complicate the classical paradigm by providing the means of generating both angiotensin I and II outside of vessels, using pathways that neither involve ACE nor are blocked by ACE inhibitors. And because mast cells outside of the kidney can be a source of renin, they can initiate angiotensin production in a variety of tissues in response to events unconnected to regulation of blood pressure and volume. Recent research on angiotensins also suggests that angiotensin II is a trophic factor with chronic affects on growth and remodeling of many tissues, that angiotensin II-inactivating peptidases may be important in regulating angiotensin activity, and that there may be “good” and “bad” receptors for angiotensin II in the context, for example, of responses to lung injury ^{66, 67}.

Generation of angiotensin II by chymases and cathepsin G

The idea that some mast peptidases generate bioactive angiotensin II is not new. Travis, Wintroub and others showed that human mast cell chymase and cathepsin G process angiotensin I into angiotensin II in vitro more than a quarter of a century ago ^{68, 69}, not long after the enzymes were first purified. Later, convergence of work in different laboratories culminated in realization that a potent, non-ACE, angiotensin II-generating peptidase extractable from human heart tissue is, in fact, indistinguishable from human mast cell chymase ^70–73. Although some evidence supports the possibility that this chymase is expressed in endothelial, vascular or cardiac muscle cells, it is clear that mast cells have far greater capacity to store chymase than any other cell type. Thus, it is probable that most chymase in extracts of human heart muscle, as in other tissues, arises from MC_TC mast cells, which are easily detected in heart tissues. History was repeating itself, in the sense that a similar convergence occurred twice in the 1970’s in connection with the major mast cell chymotryptic protease of rat connective tissue, mast cell protease I, which was initially thought to be intrinsic to skeletal muscle ^{74, 75}, and also in connection with an enzyme extracted from rat intestine thought to function to degrade intracellular pyridoxal phosphate-dependent enzymes—but found subsequently to be made and stored by an intestinal mast cell sub-population, then called “atypical” ⁷⁶. These events in the history of mast cell peptidases are worth recounting if only because they remind us that the capacity of mast cells to store peptidases is prodigious—so much so that a minor cell in a tissue like skeletal muscle can produce the lion’s share of certain peptidase activities in tissue extracts.

Although human cathepsin G was shown long ago to be capable of selectively hydrolyzing angiotensin I to generate angiotensin II ⁶⁸—and shown later to be an abundant product of MC_TC mast cells ^{77, 78}—rather little is known of its importance in generating angiotensin II in vivo, and even less known of its role in other animals. A preliminary comparison of the properties of mouse and human enzyme suggests that the former is more active and narrower in specificity, being more purely chymotryptic. In humans, cathepsin G is a major product of neutrophils (and to a lesser extent, macrophages), in addition to being a product of MC_TC mast cells. Thus, it may contribute to the generation of angiotensin II in neutrophilic as well as mast cell-mediated pathologies. In this connection, it may be helpful that potent, dual inhibitors of human chymase and cathepsin G have been generated ^{79, 80}. In humans, chymase can generate angiotensin II while bound to macroglobulin, which prolongs chymase’s during of action and escorts chymase away from site of generation, where it can be detected in blood ⁴⁴. Chymase released into interstitial fluid or serum is much more likely to be trapped in this macroglobulin-bound but active form than to be inactivated irreversibly by serpin-class inhibitors, for which chymase has little affinity. Cathepsin G, on the other hand, is susceptible to α₁-antichymotrypsin.

In mice, notwithstanding the apparent redundancy of chymase-like peptidases, it appears that mMCP-4 makes the major contribution, as suggested by studies in mMCP-4-null mice ⁸¹. However, mMCP-4 is not as selective as human chymase for the Phe₈-His₉ bond of angiotensin I, and is both activator and inactivator of angiotensin ⁸². Nonetheless, as established by the work of Husain and Dell’Italia and others, mMCP-4 and/or similar chymases appear to be responsible for ACE inhibitor-resistant generation of angiotensin II in mice—and for most extravascular generation of angiotensin II in heart muscle ⁸³, as previously shown in dogs ⁸⁴. Although, there is potential for production of mMCP-4 and other chymases by non-mast cells, the marked reduction of interstitial angiotensin II generation in mast cell-deficient Kit^W/Kit^W-v mice suggests that most of extravascular angiotensin II-generating capacity originates from mast cells ⁸³. Presumably, the existence of this extravascular pathway serves a homeostatic purpose, like blood pressure control. However, many studies in this active area of research focus on potentially deleterious effects such as hypertension (as shown in transgenic mouse expressing rat vascular chymase ⁸⁵), re-stenosis, fibrosis, worsening lung injury in ARDS, and cardiac arrhythmias ^{83, 86–88}.

Mast cell renin

Studies by Mackins and colleagues ^{89, 90} establish that mast cells can store renin, which is released by mast cell activators like compound 48/80 and allergen and boosts local generation of angiotensin I, which is then processed to angiotensin II by ACE or chymase-like peptidases. Presumably, mast cell renin could serve homeostatic functions, although existing studies focus on potential pathological contributions. In perfused mouse hearts, mast cell deficiency is associated with less “spillover” of renin and fewer malignant ventricular arrhythmias after ischemia-reperfusion. Intriguingly, much of this work on cardiovascular implications of mast cell renin release has been conducted in guinea pigs ^{89, 90}, which appear to lack angiotensin II-generating chymase ⁸⁹. Whether mast cell-derived renin is important in myocardial ischemia and arrhythmias in humans needs to be established.

Mast cell proteases, de-worming and gut homeostasis

Perhaps the earliest in vivo evidence that a mast cell peptidase serves host defense was provided by Miller and colleagues, who developed a mouse deficient in the mucosal/intraepithelial mast cell chymase mMCP-1 ⁹¹. This was the first mammal engineered to lack a mast cell serine peptidase. These Mcpt1-null mice have, difficulty expelling Trichinella spiralis ⁹¹, which is a parasitic roundworm that infects a variety of mammalian hosts and causes trichinellosis, a disease characterized most dramatically in humans by muscle inflammation from tissue deposition of larvae. MMCP-1-expressing mast cells increase dramatically in mice after worm infestation, and expulsion of Trichinella spiralis accompanies or precedes mMCP-1 release into intestinal mucosa and lumen. This presumably increases intestinal inflammation over the short term, but by expelling worms more quickly, restores gut health sooner. Curiously, mMCP-1’s de-worming function does not extend to all nematodes. For example, although mice infected with Nippostrongylus brasiliensis develop impressive jejunal mastocytosis and dramatic increases in gut content of mMCP-1, worm burden is unaffected by lack of mMCP-1 in Mcpt1-null mice ^91–94. However, other mast cell proteases may be important. Indeed, recent studies suggest that the related β-chymase mMCP-4 plays a more general role in regulating gut barrier function, including permeability and epithelial migration, as indicated by the small intestine phenotype in Mcpt4-null mice and in mast cell-deficient mice with mast cell populations re-established by adoptive transfer of BMCMC originating from wild-type and Mcpt4 −/− mice ⁹⁵. By activating PAR-2, mast cell tryptases may play similar roles in large intestine ⁹⁶.

Chymases and control of allergic airway inflammation

It is not yet clear whether the overall effect of mast cell chymases released during airway allergen challenge is to promote or oppose allergic inflammation (or both: e.g., pro-inflammatory in the early phase and anti-inflammatory in late or chronic phases). Although it is often assumed that release of chymase will augment inflammation, several experiments suggest mechanisms by which the actions of chymases could reduce or terminate inflammation. For example, chymases could disarm allergens by cleaving them, as occurs with canine chymase degrading birch profilin and destroying epitopes recognized by IgE ⁹⁷ (see Figure 2). Furthermore, human mast cell chymase (and cathepsin G) destroy several cytokines associated with perpetuation of allergic inflammation ⁴¹. Indeed, the idea that human chymase opposes inflammation received further support from the observation that chymase-containing mast cells in the outer wall of small airways correlate with better lung function in asthmatics ⁹⁸. In a model of asthma, anti-inflammatory activity was attributed to a specific chymase, mMCP4, based on the finding of increased airway responsiveness, inflammation, and smooth muscle volume in Mcpt4 −/− mice ⁹⁹. A potential mechanism for increased smooth muscle volume is degradation of perimyocyte matrix and reduced proliferative responses to growth factors ¹⁰⁰. Such actions may be related to pro-apoptotic effects of chymases on vascular smooth muscle ^{101, 102}, which may increase susceptibility to aneurysm formation ¹⁰³. On the other hand, inhibitors of human chymase and cathepsin G oppose development of airway hyper-responsiveness and early- and late-phase rises in airway resistance in Ascaris suum-sensitized sheep challenged with allergen, and, in mice exposed to tobacco smoke, reduce lung neutrophilia and production of neutrophil chemoattractant ^{79, 80}. Explanations for observed differences in conclusions drawn from genetic versus pharmacological models likely relate in part to differences between mammals in the degree of chymase (and cathepsin G) redundancy—along with variations in enzymological properties, inhibitor susceptibilities, and cell-selective expression within and between mammals (reviewed in ⁴).

Figure 2. Self-termination of mast cell activation by allergen.

Multivalent protein allergens like birch profilin recognized by allergen-specific IgE bound to high-affinity IgE receptor on the surface of mast cells stimulates degranulation and release of epitope-destroying proteases, such as chymases. This is a negative feedback loop that reduces allergic inflammation by destroying allergen.

Tryptases and control of bacterial infection

This topic has been reviewed recently ¹⁰⁴ and is briefly summarized and updated here. Among the most persuasive early evidence that mast cells control bacterial infection in vivo came from studies of responses of mast cell-deficient mice to enterobacteria ^{105, 106}. In peritoneal infections, stimulation of mast cell release of TNFα early in infection is needed for timely recruitment of neutrophils to contain infection. Subsequent studies revealed that mast cell-derived IL-6 helps to contain peritoneal and lung infections with Klebsiella pneumoniae and other gut organisms ^{45, 107}. The possibility that secreted mast cell proteases could also help to control gut infections was suggested by studies in mice showing that the mouse tryptase mMCP6 (but not the related protease mMCP7) attracts neutrophils when injected into the peritoneal cavity ¹⁰⁸ and established that Mcpt6 −/− mice clear Klebsiella pneumoniae inefficiently and are more likely than +/+ mice to die following injection of Klebsiella pneumoniae into the peritoneal cavity ¹⁰⁹. Other studies provide glimpses of some of the subtleties of mast cell protease contributions by establishing that reduced levels of active mast cell tryptase in Dppi −/− mice correlate with increased gut bacterial load following cecal ligation and puncture but with improved short-term survival, possibly related to higher levels of immunoprotective IL-6, which can be degraded by mMCP-6 ⁴⁵. Thus, although mouse studies clearly establish that some mast cell tryptases help to control and contain some types of bacteria, the impact on survival may depend on the nature and virulence of the organism—and in some cases tryptases may lower survival. In any case, several other mast cell factors, including TNFα and IL-6, are also important. Studies with human mast cell tryptases are more limited, although it has been shown that active human βI tryptase (but not α tryptase) attracts neutrophils when introduced to mouse airways ¹¹⁰. Whether mast cell tryptases are in fact major weapons in human anti-bacterial defenses is an unanswered question, the answer to which may determine whether pharmaceutical strategies involving systemic blockade of tryptase activity will block mast cell-mediated inflammation at the expense of creating serious immune deficits. In this regard, the failure to identify humans that are entirely bereft of active mast cell tryptases, despite the high frequency of deficiency alleles in most populations, is consistent with (although not proof of) key roles in host defense ^111–113.

PRO-INFLAMMATORY ROLES of MAST CELL PEPTIDASES

Tryptases

Allergic inflammation of airway and skin

Tryptases are released with histamine from human skin mast cells in vivo in acute and chronic responses to allergen ^{114, 115}. Levels of immunoreactive tryptases also increase in the airway in asthma ¹¹⁶, and transcripts encoding tryptases are among the most abundant and upregulated gene products in brush biopsies of asthmatic bronchial epithelium, consistent with intraepithelial mast cell migration or local proliferation ^{38, 117}. It is not known if tryptases detected by immunoassays in airway fluids (or in blood, for that matter) is active. Overall, evidence from animal models as well as humans is compelling that tryptases released during allergic inflammation are not only markers of mast cell activation but contribute to resulting pathology. For example, small molecules designed to inhibit human tryptases markedly reduce airway eosinophilia and goblet cell hyperplasia in a mouse model of asthma ¹¹⁸. Similar findings are reported with nafamostat ¹¹⁹, which is a highly potent although not entirely selective inhibitor of tryptases ¹²⁰. Several lines of evidence suggest that tryptase is a bona fide in vivo target of nafamostat. For example, nafamostat reduces scratching in mice induced by skin injection of tryptase or by mast cell-degranulator compound 48/80 ¹⁷. Nafamostat’s effects on scratching are not seen in mast cell-deficient mice, and involve PAR-2, since the effects of tryptase and compound 48/80 on scratching are inhibited by PAR-2 antagonists. The simplest explanation of these findings is that tryptase from mast cells activates PAR-2 on nerves involved in itch pathways. Note that leeches make a potent and selective tryptase inhibitor ¹²¹, which is indirect evidence of a role for tryptase in signaling the presence of the leech, perhaps by activating neural itch and pain pathways or by spreading the degranulation signal.

Published studies of the effect of tryptase inhibition in humans are few. However, a topical (aerosolized) tryptase inhibitor reduced late-phase bronchoconstriction in a small study involving mild human atopic asthmatics ¹²² and decreased nasal symptoms and eosinophilia in humans with allergic rhinitis ¹²³. Among several proposed pathways by which tryptases may promote asthmatic bronchoconstriction, it is not yet clear which are the most important. One potential mechanism proposed early on is tryptase-mediated destruction of vasoactive intestinal peptide ^{6, 25–27}. A possibly distinct pathway involves augmentation of bronchoconstriction by histamine and other airway smooth muscle agonists, which is a phenomenon manifest in muscle bath preparations of bronchi from dogs and humans ^{19, 20, 22}. Tryptase released from one mast cell under the influence of allergen also may promote degranulation of nearby mast cells, as suggested by mast cell-stabilizing actions of some tryptase inhibitors ^{124, 125} and by provocation of histamine release in sheep or guinea pig skin or airway by injected or inhaled human tryptase ^{23, 126–129}. The early history of pharmaceutical interest in and development of tryptase inhibitors was thoroughly reviewed by Cairns ¹³⁰ and will not be re-reviewed here. Notwithstanding the pharmaceutical interest in blocking more acute affects of tryptases on inflammation and smooth muscle constriction, chronic affects on airway remodeling (including growth of airway fibroblasts ^131–134, smooth muscle ¹³⁵ and vessels ¹³⁶)—which may be responsible for bronchodilator-resistant airway obstruction—also provide rationales for therapeutic inhibition. The extent to which tryptase activation of PAR-2 is involved in allergic inflammatory, bronchoconstrictor and remodeling responses is not yet clear.

Arthritis and inflammatory bowel disease

A role for mast cell tryptases in arthritis is suggested by reduced inflammation in tryptase-deficient mice in two models or arthritis ^{137, 138}. One of these models (methylated bovine serum albumin/IL-1β-induced) seems to require two tryptase gene products (namely, mMCP-6 and -7) for full expression of the inflammatory phenotype ¹³⁷. These recent findings support prior speculation about the importance of mast cell products in arthritis based on studies in mast cell-deficient mice and the finding of tryptase-expressing mast cells in arthritic joints ^139–142. The mechanisms by which mast cell tryptases contribute to various forms of experimental arthritis remain to be established—and of course tryptases and mast cells are not the sole factors contributing to the phenotype ^{141, 143}. The importance of mast cells and tryptases to the pathogenesis of related human afflictions like rheumatoid arthritis also remains to be clarified. The picture is in some respects clearer in regard to inflammatory bowel disease, especially ulcerative colitis, which has been long associated with increases in mast cell numbers and activation in affected tissues ^{144, 145}. At least two pharmacological lines of evidence suggest that tryptases released from mast cells contribute to the pathology of ulcerative colitis: 1) a human trial of a β-tryptase-selective inhibitor given systemically by subcutaneous injection appeared to reduce gastrointestinal symptoms in subjects with ulcerative colitis ¹⁴⁶ and 2) treatment with the tryptase inhibitor nafamostat decreased the severity of pathological findings in a rat model of colitis caused by trinitrobenzene sulfonic acid ¹⁴⁷.

Chymases and cathepsin G

Allergic inflammation in airway and skin

The ability of human chymase and cathepsin G to cleave angiotensin I selectively at Phe₈ to generate angiotensin II, which can be a homeostatic process assisting support of blood pressure—but can also be pro-inflammatory—was summarized earlier. However, both enzymes have effects that are more classically inflammatory, especially by promoting tissue swelling. For example, dog chymase, injected into dog skin, increases the size of histamine-induced wheals without inducing wheals by itself ¹⁴⁸. Furthermore, inhibition of chymase activity in vivo reduces size of wheals generated by mast cell-degranulating agents. The mechanism of these effects are not known, but could include breakdown and untethering of extracellular matrix so that fluid extravasated under the influence of histamine travels farther than it would do otherwise. Both chymase and cathepsin G are fairly omnivorous, and can separate the dermal-epidermal junction by degrading a variety of matrix proteins ¹⁴⁹, as well as by activating MMPs ⁴⁹. Possibly, these enzymes also destroy extracellular histaminases so that histamine levels are higher and more sustained. Chymase and cathepsin G also can stimulate gland secretion ^{150, 151}. Indeed, in human airway, chymase-positive mast cells are a high fraction of mast cells lying within 20 μm of submucosal glands ¹⁵². The proteolytic activity of human chymase extends to albumin ¹⁵³; however, this cleavage would not increase oncotic pressure because the nicked fragments remain joined by disulfide linkages. Acting subacutely or chronically, chymase-like peptidases also may promote inflammatory angiogenesis, as suggested by sponge granulomas in hamsters ¹⁵⁴ and by mast cell-dependent angiogenesis in a model of skin carcinogenesis ⁴⁹. The recently reported inhibition of several animal models of allergic and non-allergic inflammation by inhibitors of chymase and cathepsin G ⁸⁰ further suggests that these enzymes are broadly capable of promoting inflammation. Possibly, they act synergistically when released from human mast cells, where they are usually found in the same granules, because their substrate preferences only partially overlap.

Ischemia-reperfusion injury, aneurysms and vascular stenosis

A mouse model of irreversible ischemia-reperfusion injury suggests that a specific mouse mast cell protease, mMCP-5, is responsible for irreversibly injuring skeletal muscle ¹⁵⁵. This work establishes the principle that a mast cell protease can be cytotoxic in the context of ischemic inflammation, which is associated with mast cell activation. The mechanism by which muscle is damaged by mMCP-5, which is an elastolytic peptidase with no known functional equivalent in human mast cells, is not clear. However, the potential for chymase-related mast cell peptidases to damage and alter vessels themselves has gathered increased experimental support over the past few years. After initial studies suggested a role for mast cells in promoting arterial enlargement in a neutrophil elastase-induced mouse model of aortic aneurysm ¹⁵⁶, subsequent studies showed at least partial dependence on mast cell expression of a particular chymase, mMCP-4 ¹⁰³. The mechanism by which this chymase promotes aneurysm formation in this model is hypothesized to include inflammatory activation of vessel wall-weakening MMPs and cathepsins and direct stimulation of aortic smooth muscle cell apoptosis. On the other hand, there is strong in vivo pharmacological evidence from studies in a variety of models of vascular injury that chymase-like enzymes promote cardiovascular remodeling, fibrosis and stenosis in response to injury ^{62, 88, 157–160}.

Dipeptidyl peptidase I and other thiol cathepsins

Membrane-bound mast cell secretory granules harboring biogenic amines, proteases and proteoglycans are related to lysosomes and partly may serve lysosomal functions. Indeed they contain some lysosome-associated proteases. Cathepsin G is not a classic cathepsin because it is a serine (not thiol) peptidase with expression restricted to specialized granules of mast and myelomonocytic cells, especially neutrophils. Another granule peptidase, dipeptidyl peptidase I (DPPI; cathepsin C) also is atypical. Although it is expressed in many cells, it is much more abundant in mast cells, myelomonocytes and other specialized granulated cells, like cytotoxic T and natural killer cells. In uninflamed dog airway, mast cells are the dominant cell type staining positively for DPPI ¹⁶¹. Like other proteins of secretory granules, DPPI can be secreted ¹⁶² and it may cleave extracellular targets ¹⁶¹. However, it is not highly destructive because its activities are restricted compared to other thiol cathepsins by an “exclusion domain” ensuring preference for cleaving N-terminal dipeptides ¹⁶³. These attributes suggest that DPPI is likely to serve an intragranular function not related to general protein degradation or typical lysosomal activity. At present, DPPI’s major identified role is to activate granule-associated immune cell peptidases related to chymases, cathepsin G, lymphocyte granzymes, and neutrophil elastase. It accomplishes this by removing the N-terminal pro-dipeptide that is a shared attribute of these enzymes ^164–167. Given the effects of genetic inactivation of DPPI on activation of an impressive range of conserved immune serine peptidases, the phenotype might be expected to be more severe than it is. In fact, genetic deletion or inactivation of DPPI is not lethal to mice protected from infections, but DPPI-deficient mice have a variety of immune deficits and altered responses to sepsis ^{14, 168}, including improved short term survival following cecal ligation and puncture ⁴⁵. Humans with defects in DPPI have chronic periodontal infections ¹⁶⁹. Mice lacking DPPI have little if any mast cell chymase activity, although at least one chymase (mMCP-4) is present in mast cell granules as an inactive pro-enzyme ¹⁶⁶. The effect on mouse tryptase mMCP-6 is less dramatic, with activity being reduced but present ¹⁶⁶. This is perhaps not surprising given that tryptases possess a much longer pro-peptide than chymases, granzymes and neutrophil elastase-like hydrolases ^{170, 171}. Schwartz and colleagues suggest that the pro-peptide is removed from human β-tryptases by tandem cleavages initiated by autocatalysis to generate a remnant pro-dipeptide removed by DPPI ¹⁷². The extent to which human chymases and tryptases depend on DPPI for activation in vivo remains to be determined. Mast cells do express classical lysosomal thiol cathepsins, including cathepsin S, which can influence levels of chymase and/or carboxypeptidase independently of DPPI in mouse mast cells ^{173, 174}. Thus, although DPPI has received more attention in studies of mast cell biology to date, other thiol cathepsins may be important to mast cell function.

Table I.

Comparison of some mast cell proteases in mice and humans.

Protease class	Human	Mouse
SERINE
Tryptase-like
Active:	βI, βII, βIII, γ	mMCP-6, -7*, γ; mastin/mMCP-11**
Inactive:	α, δ, βIIIFS; mastin	mMCP-7*
Chymase-like
Active:	CMA-1/α; Cathepsin G	mMCP-1, -4, -5***; Cathepsin G
Inactive:		mMCP-2
Plasminogen activator	t-PA	?
CYSTEINE/THIOL
Cathepsins	DPPI/C, ?others	DPPI/C, B, L, S
METALLO
Carboxypeptidase	CPA3	CPA3
Matrix metallo	MMP-9	MMP-9
ADAM, other	?	ADAM17, neurolysin
ASPARTYL	Renin	Renin

^*not expressed in some strains of laboratory mice

^**expressed primarily in basophils

^***elastolytic, not chymotryptic