Mast cell chymase. A potent secretagogue for airway gland serous cells

Submucosal glands are the major sources of airway secretions in most mammals. Mast cells are abundant in the environment of airway submucosal glands and are rich sources of secreted proteases. To investigate the hypothesis that mast cell proteases stimulate airway gland secretion, we studied the ability of the two major mast cell granule proteases, chymase and tryptase, to cause secretion of 35S-labeled macromolecules from a line of cultured bovine airway gland serous cells. Mast cell chymase and tryptase were purified from dog mastocytoma cells. Chymase markedly stimulated serous cell secretion in a concentration-dependent fashion with a threshold of 10(-10) M, whereas tryptase had no effect. The response to 10(-8) M chymase (1530 +/- 80% over base line) was approximately 10-fold higher than that evoked by other agonists such as histamine and isoproterenol. The predominant 35S-labeled macromolecule released by chymase was chondroitin sulfate proteoglycan, the glycoconjugate present in serous cell secretory granules. The response to chymase was non-cytotoxic and was blocked by active site inhibitors of chymase (soybean trypsin inhibitor and chymostatin) and by inhibitors of cellular energy metabolism (azide,2,4-dinitrophenol, dicumarol). Supernatant obtained by degranulation of mastocytoma cells caused a secretory response of comparable magnitude to that caused by chymase. These findings demonstrate that chymase, but not tryptase, is a potent secretagogue for airway gland serous cells, and they suggest a possible role for chymase-containing mast cells in the pathogenesis of airway hypersecretion.     

Mast cell chymase regulates dermal mast cell number in mice

Chymase inhibitor reduced the increase in the number of dermal mast cells in 2,4-dinitrofluorobenzene-induced dermatitis in a dose-dependent manner. Intradermal injection of human chymase to mouse ear significantly increased histamine content, the marker for mast cell number in the skin. These results suggest that chymase released by mast cells may participate in local mast cell accumulation in a positive feedback fashion. Immunohistochemical analysis revealed that the intradermal injection of chymase reduces expression of stem cell factor (SCF) on surface of the skin keratinocytes. In addition, incubation of human keratinocytes with chymase in vitro resulted in release of SCF into the culture medium. Since soluble SCF is thought to regulate mast cell number, the chymase-induced mast cell accumulation may occur via the ability of chymase to process membrane-bound SCF on the epidermal keratinocytes.     

Distribution of chymase-containing mast cells in human bronchi.

Mast cell chymase stimulates secretion from cultured airway gland serous cells and hydrolyzes bronchoactive peptides in vitro. To explore the likelihood of these interactions occurring in situ, we examined the distribution and concentration of chymase-containing mast cells near glands and smooth muscle of major human bronchi from eight individuals without known airway disease. Total airway mast cells and the subset of mast cells containing chymase were detected by staining for methylene blue metachromasia and chloroacetate esterase activity, respectively. The percentage of chymase-containing mast cells was found to differ strikingly among bronchial tissue compartments. Near glands, for example, the concentration of chymase-positive mast cells (640 +/- 120 cells/mm3) was 73 +/- 9% that of total mast cells (910 +/- 130 cells/mm3), whereas in smooth muscle the concentration of chymase-positive mast cells (450 +/- 200 cells/mm3) was only 14 +/- 4% that of total mast cells (2920 +/- 620 cells/mm3). Of all chymase-containing mast cells in the airway subepithelium, 30 +/- 4% were located within 20 microns of submucosal glands. Although the percentage of chymase-containing cells varied, the absolute concentration of chymase-containing mast cells was similar in all compartments. These results reveal a differential distribution of mast cell subpopulations in human airway and suggest that mast cells containing chymase are near gland and smooth muscle targets.     

Mast cell chymase induces smooth muscle cell apoptosis by disrupting NF-kappaB-mediated survival signaling

Chymase released from activated mast cells induces apoptosis of vascular smooth muscle cells (SMCs) in vitro by degrading the pericellular matrix component fibronectin, so causing disruption of focal adhesion complexes and Akt dephosphorylation, which are necessary for cell adhesion and survival. However, the molecular mechanisms of chymase-mediated apoptosis downstream of Akt have remained elusive. Here, we show by means of RT-PCR, Western blotting, EMSA, immunocytochemistry and confocal microscopy, that chymase induces SMC apoptosis by disrupting NF-kappaB-mediated survival signaling. Following chymase treatment, the translocation of active NF-kappaB/p65 to the nucleus was partly abolished and the amount of nuclear p65 was reduced. Pretreatment of SMCs with chymase also inhibited LPS- and IL-1beta-induced nuclear translocation of p65. The chymase-induced degradation of p65 was mediated by active caspases. Loss of NF-kappaB-mediated transactivation resulted in downregulation of bcl-2 mRNA and protein expression, leading to mitochondrial swelling and release of cytochrome c. The apoptotic process involved activation of both caspase 9 and caspase 8. The results reveal that, by disrupting the NF-kappaB-mediated survival-signaling pathway, activated chymase-secreting mast cells can mediate apoptosis of cultured arterial SMCs. Since activated mast cells colocalize with apoptotic SMCs in vulnerable areas of human atherosclerotic plaques, they may participate in the weakening and rupture of atherosclerotic plaques.     

Airway secretion: a cell-specific analysis

Respiratory mucus is a complex secretion elaborated by several cell types in the airway wall: serous and mucous gland cells in the submucosa, and ciliated and goblet cells in the epithelium. The cell types are under independent regulatory mechanisms, most of which are poorly understood. Two new approaches are described which permit analysis of these mechanisms and the role of each cell type in mucus production. Using cell-specific monoclonal antibodies, we have obtained biochemical information about mucus components contributed by the various cell types. Antibodies are also being used in enzyme immunoassays (ELISA) to detect cell-specific secretion from mixed cell biopsies. In other studies, analysis of secretory products from pure cultures of serous gland cells has revealed that the major glycoconjugates released by this cell type are chondroitin sulfate proteoglycans, hyaluronic acid and N-linked glycoproteins of complex type.     

Mechanical induction of an epithelial cell chymase associated with wound edge migration

Chymase is a chymotrypsin-like serine protease predominantly produced by mast cells. In this study, human cutaneous and gingival keratinocytes, ovary surface epithelia, and a porcine epithelial cell line were assayed by homology-based cloning, and the amplified DNA fragment was identified as a chymase. In vitro, chymase could not be induced by serum or cytokine treatment alone. Chymase was activated 3-fold within 60 min in basal media by scratch wounding cultured monolayers and further potentiated over 10-fold at 18 h by additional serum and cytokine treatment. Chymase activity was cell-associated and found to peak within 24 h of wounding and then steadily decreased as cultures healed, reaching baseline levels before confluence was reestablished. Affinity column purified enzyme effectively degraded fibronectin and was found by Western blot analysis using a human chymase antibody to be of about 30 kDa. Immunostaining revealed chymase activation at the wound edge colocalizing with reactive oxygen species generation. Specifically, chymase activation was attenuated by inhibition of nitric oxide, superoxide, and peroxynitrite. Exogenous peroxynitrite but not hydrogen peroxide also resulted in chymase activation in unwounded monolayers. Disruption of cytoskeletal stress fibers by cytochalasin D attenuated both wound-activated chymase and reactive oxygen species generation. Chymase inhibitor chymostatin reduced the loss of cell-cell contacts and the onset of porcine and human skin epithelial cell migration at the wound edge. This shows that an epithelial chymase is rapidly activated by a ligand-independent mechanism following mechanical stress via cytoskeletal and reactive oxygen species signaling and is associated with the onset of epithelial cell migration.     

Chymotrypsin- and trypsin-type serine proteases in rat mast cells: properties and functions

Two of the major enzymes present in and released from rat mast cells are chymotrypsin-type serine protease (chymase) and trypsin-type serine protease (tryptase), and these have been postulated to be important in the inflammatory reactions. There have been no clear data regarding the trypsin-type protease in rat mast cells. Tryptase was recently purified from rat peritoneal mast cells with an associated protein (trypstatin) that inhibited the protease activity above pH 7.5. Chymase was also purified from rat peritoneal cells by employing a one-step method involving hydrophobic chromatography on octyl-Sepharose 4B or arginine-Sepharose 4B. The properties of chymase and tryptase were described in relation to substrate specificity and their relative sensitivity to inhibitors. It was found that proteolytic activities of these enzymes were modulated by naturally occurring substances, such as phosphoglycerides, long-chain fatty acids, and trypstatin. There is as yet little evidence for the physiological roles of these enzymes in the inflammatory reaction. It has been found that the specific, low-molecular-weight inhibitor of chymase, chymostatin, and that of tryptase, leupeptin, inhibit histamine release induced by addition of anti-rat IgE to mast cells. However, the inhibitors with molecular weights of more than 6000 were found to have no effect in this process. The data suggest that chymase and tryptase in mast cell granules play a crucial or significant role in the process of degranulation.     

Non-peptidic inhibitors of human chymase. Synthesis, structure-activity relationships, and pharmacokinetic profiles of a series of 5-amino-6-oxo-1,6-dihydropyrimidine-containing trifluoromethyl ketones

Chymase possesses a wide variety of actions, including promotion of angiotensin II production and histamine release from mast cells. However, due to a lack of effective inhibitors featuring both high inhibitory activity and high metabolic stability, the pathophysiological role of chymase has not been fully elucidated. We designed non-peptidic inhibitors based on the predicted binding mode of the peptidic chymase inhibitor Val-Pro-Phe-CF3 and demonstrated that the Val-Pro unit is replaceable with a (5-amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)acetyl moiety. Structure-activity relationship studies revealed that phenyl substitution at the 2-position of the pyrimidinone ring is indispensable for high activity. The most potent compound 1h (Ki = 0.0506 microM) is superior in potency to the parent peptidic inhibitor Val-Pro-Phe-CF3 and has good selectivity for chymase over other proteases. The related analogue 1e was orally absorbed and maintained high plasma levels for at least 2h. These results suggest that the derivatives reported here could be developed as agents for treatment of chymase-induced disease.     

Regulation of the inflammatory response in asthma by mast cell products

In airways, mast cells lie adjacent to nerves, blood vessels and lymphatics, which highlights their pivotal importance in regulating allergic inflammatory processes. In asthma, mast cells are predominantly activated by IgE receptor cross linking. In response to activation, preformed mediators that are stored bound to proteoglycans, for example, TNF-alpha, IL-4, IL-13, histamine, tryptase and chymase, are released. New synthesis of arachidonic acid metabolites (leukotriene C4 (LTC4), leukotriene B4 (LTB4) and prostaglandin D2 (PGD2)) and further cytokines is stimulated. Mediators from degranulating mast cells are critical to the pathology of the asthmatic lung. Mast cell proteases stimulate tissue remodelling, neuropeptide inactivation and enhanced mucus secretion. Histamine stimulates smooth muscle cell contraction, vasodilatation and increased venular permeability and further mucus secretion. Histamine induces IL-16 production by CD8+ cells and airway epithelial cells; IL-16 is an important early chemotactic factor for CD4+ lymphocytes. LTC4, LTB4 and PGD2 affect venular permeability and can regulate the activation of immune cells. The best characterized mast cell cytokine in asthmatic inflammation is TNF-alpha, which induces adhesion molecules on endothelial cells and subsequent transmigration of inflammatory leucocytes. IL-13 is critical to development of allergic asthma, although its mode of action is less clear.     

Rat serosal mast cell degranulation mediated by chymase, an endogenous secretory granule protease: active site-dependent initiation at 1 degree C

Exposure at 37 degrees C of rat serosal mast cells (RSMC) to chymase, an endogenous secretory granule serine protease, results in exocytosis as determined by the release of another secretory granule enzyme, beta-hexosaminidase. Chymase-mediated RSMC degranulation does not occur at 1 degree C; however, exposure of RSMC to chymase at 1 degree C followed by the removal of buffer and the resuspension of the cells in buffer alone at 37 degrees C results in exocytosis equivalent to that obtained by direct exposure of RSMC to chymase at 37 degrees C. Maximal chymase-mediated RSMC degranulation at 37 degrees C is Ca2+-dependent and Mg2+-independent. The dose-dependent degranulation-inducing interaction of chymase and alpha-chymotrypsin with RSMC at 1 degree C is Ca2+-independent, whereas subsequent exocytosis at 37 degrees C in new buffer without added enzyme still requires Ca2+. Specific binding of 125I-labeled alpha-chymotrypsin to RSMC does not occur at 1 degree C, implying that the inducing action of chymase is not a simple ligand-receptor binding. The enzyme inhibitors diisopropyl fluorophosphate and lima bean trypsin inhibitor inhibit subsequent exocytosis at 37 degrees C only if they are added within the first 10 min of the interaction of RSMC and chymase at 1 degree C, implying that an active site-dependent inducing event occurs between RSMC and chymase at 1 degree C. Thus, chymase-induced coupled activation-secretion can be divided into a cation- and temperature-independent initiation phase, which is dependent on the active site of exogenously added chymase and a subsequent temperature-dependent and calcium-augmented cellular secretion phase.     

Rat mast cell tryptase

Rat mast cell tryptase is located largely if not totally in the cell's secretory granules. When the active site reagent [3H]diisopropyl fluorophosphate was used to label tryptase and chymase simultaneously, the ratio of tryptase:chymase active sites was determined to be 0.05. In comparison to chymase and tryptase in other species and chymase in the rat, rat tryptase is poorly bound to the granule matrix as evidenced by (1) its release parallel to histamine on induction of secretion and (2) its appearance in the supernatant when isolated granules were stripped of their membranes with hypotonic medium. Tryptase on release from the granule is moderately stable at a pH of 5.0 but unstable at pH 7.5, the pH that the enzyme encounters on secretion from the cell. These several properties indicate that the role of rat mast cell tryptase extracellularly is likely to differ greatly from that of chymase.     

Detection and partial characterization of a human mast cell carboxypeptidase

Using a high performance liquid chromatography assay that detects the cleavage of the C-terminal leucine from angiotensin I, we have identified a carboxypeptidase activity in mast cells from human lung and in dispersed mast cell preparations from human skin. The enzyme activity was detected in a preparation of dispersed human mast cells from lung of greater than 99% purity and was released with histamine after stimulation with goat anti-human IgE. In nine preparations of dispersed human mast cells from lung of 10 to 99% purity, net percentage of release of carboxypeptidase correlated with the release of histamine, localizing carboxypeptidase to mast cell secretory granules. The enzyme activity was also detected in preparations of dispersed human mast cells from skin and in extracts of whole skin. The inhibitor profile and m.w. of carboxypeptidase activity from preparations of dispersed mast cells from skin was similar to that from dispersed mast cells from lung. Mast cell carboxypeptidase had a m.w. on gel filtration of 30,000 to 35,000. The enzyme in crude lysates of dispersed mast cell preparations had optimal activity between pH 8.5 and 9.5 and was inhibited by potato inhibitor, which distinguished it from carboxypeptidase in cultured human foreskin keratinocytes and adult fibroblasts, and from other proteolytic mast cell enzymes. The enzyme activity was also inhibited by EDTA, o-phenanthroline, and, to a small extent, by 8-OH quinoline, but not by Captopril, soybean trypsin inhibitor, or pepstatin. These findings demonstrate that human mast cell secretory granules contain carboxypeptidase in addition to tryptase and chymase. It appears that mast cells from skin may have a higher content of carboxypeptidase than do mast cells from lung.     

Human skin chymotrypsin-like proteinase chymase. Subcellular localization to mast cell granules and interaction with heparin and other glycosaminoglycans

The subcellular localization of human skin chymase to mast cell granules was established by immunoelectron microscopy, and binding of chymase to the area of the dermo-epidermal junction, a basement membrane, was demonstrated immunocytochemically in cryosections incubated with purified proteinase prior to immunolabeling. Because heparin and heparan sulfate proteoglycans are major constituents of mast cell granules and basement membranes, respectively, the ability of chymase to bind to glycosaminoglycans (GAG) was investigated. Among a variety of GAGs, only binding of chymase to heparin and heparan sulfate appears physiologically significant. Binding was ionic strength-dependent, involved amino groups on the proteinase, and correlated with increasing GAG sulfate content, indicating a predominantly electrostatic association. Interaction with heparin was observed in solutions containing up to 0.5 M NaCl, and interaction with heparan sulfate was observed in solutions containing up to 0.3 M NaCl. Binding of heparin did not detectably affect catalysis of peptide substrates, but may reduce accessibility of proteinase to protein substrates. Measurements among a series of serine class proteinases indicated that heparin binding was a more common property of mast cell proteinases than proteinases stored in other secretory granules. Binding of chymase to heparin is likely to have a storage as well as a structural role within the mast cell granule, whereas binding of chymase to heparan sulfate may have physiological significance after degranulation.     

A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components

In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of secretory granules as well as the fate of the secretory granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles.     

Expression of airway secretory epithelial functions by lung carcinoma cells

We examined 12 non-small cell lung carcinoma cell lines for expression of airway goblet, serous, and mucous cell characteristics. The cells expressed some ultrastructural traits of secretory epithelial cells but none contained secretory granules typical of the airway secretory cells. Using immunocytochemistry and cell-specific monoclonal antibodies, we identified heterogeneous expression of goblet, mucous, and serous cell markers among the cell lines. After metabolic radiolabeling, cells incorporated isotope into high molecular weight material. Incubation of pulse-radiolabeled cells with a number of known mucus secretogogues revealed that 5 of the 12 cell lines released radiolabeled material in response to the agonists. However, in each cell line only one of the receptor-activated pathways tested was intact. Although we did not identify a single cell line expressing a phenotype similar to normal airway secretory cells, particular functions retained by some of these cell lines may make them useful for specific studies of mucus production or secretion.     

Mast cell heterogeneity: effects of neuroenteric peptides on histamine release

Recent reports suggesting that the actions of certain neuroenteric peptides may be mediated in part by the secretion of histamine and other mast cell contents could have important implications for gastrointestinal motility and secretion. However, evidence for a mast cell-hormonal interaction is based on studies using peritoneal or cutaneous mast cells. Because intestinal mucosal mast cells (MMC) differ functionally from peritoneal mast cells (PMC), we compared the effects of several neurotransmitters and intestinal hormones on histamine secretion from two mast cell types in the rat. MMC hyperplasia was induced in rats by infection with the nematode Nippostrongylus brasiliensis, and MMC were isolated from the small intestine by collagenase digestion. Substance P, somatostatin, vasoactive intestinal polypeptide (VIP), neurotensin, and bradykinin had a potent secretagogue effect on (10(-7) to 10(-4)M) PMC which was temperature-, energy-, and calcium-dependent. In contrast to PMC, MMC released significant amounts of histamine only when challenged with substance P. Acetylcholine, bombesin, motilin, and pentagastrin had no secretory effect on either PMC or MMC. The differences between PMC and MMC in responsiveness to peptides could not be attributed to the MMC isolation procedure because PMC treated similarly or mixed with MMC suspensions retained their responsiveness to these stimuli. Our results extend the concept of neurocrine control of mast cell function, but indicate that mast cells from different sites have distinct profiles of responsiveness to regulatory peptides.     

Eosinophil migration induced by mast cell chymase is mediated by extracellular signal-regulated kinase pathway

Mast cell chymase is known to induce eosinophil migration in vivo and in vitro. In the present study, we investigated possible involvement of mitogen-activated protein (MAP) kinases; extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, in the chymase-induced eosinophil migration. Human chymase induced a rapid phosphorylation of ERK1/2 and p38 in human eosinophilic leukemia EoL-1 cells, while no phosphorylation was detected in JNK. The chymase-induced phosphorylation of ERK and p38 was inhibited by pertussis toxin. Similar results were obtained in the experiments using mouse chymase and eosinophils. U0126 (the inhibitor for MAP/ERK kinase) suppressed chymase-induced migration of EoL-1 cells and mouse eosinophils. However, SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) showed little effect on the migration. It is suggested therefore that chymase activates ERK and p38 probably through G-protein-coupled receptor, and that ERK but not p38 cascade may have a crucial role in chymase-induced migration of eosinophils.     

Proteoglycans in haemopoietic cells

Proteoglycans are produced by all types of haemopoietic cells including mature cells and the undifferentiated stem cells. The proteinase-resistant secretory granule proteoglycan (serglycin; Ref. 14), is the most prevalent and best characterised of these proteoglycans. Although its complete pattern of distribution in the haemopoietic system is unknown, serglycin has been identified in the mast cells, basophils and NK cells, in which secretion is regulated, and in HL-60 cells and a monocytoid cell line (Kolset, S.O., unpublished data) in which secretion is constitutive. Proteinase-resistant proteoglycans have been detected in human T-lymphocytes and murine stem cells (FDCP-mix) and the core proteins may be closely related to serglycin. A variety of glycosaminoglycan chains are assembled on the serglycin protein and it is likely that this class of proteoglycan can carry out a wide variety of functions in haemopoietic cells including the regulation of immune responses, inflammatory reactions and blood coagulation. There is strong evidence that in mast cells, NK cells and platelets, the proteoglycans are complexed to basic proteins (including enzymes and cytolytic agents) and amines in secretory granules and such complexes may dissociate following secretion from the cell. The stability of the complexes may be regulated by the ambient pH which may be acidic in the granules and neutral or above in the external medium. However, proteinase-proteoglycan complexes in mast cell granules seem to remain stable after secretion and it has been proposed that the proteoglycan regulates activity of proteinases released into the pericellular domain. The functions of proteoglycans which are constitutively secreted from cells are less clear. If cells have no requirement for storage of basic proteins why do they utilise the same design of proteoglycan as cells which accumulate secretory material prior to regulated release? We should stress that the so-called constitutive secretory pathway has been identified in haemopoietic cells in culture, which are usually maintained and grown in the presence of mitogenic factors (e.g., IL-2, IL-3). the cells are therefore activated and it has not been established that continuous proteoglycan secretion occurs in quiescent cells circulating in the peripheral blood. It is possible that lymphocytes, monocytes and macrophages, in which the constitutive secretion pathway operates in vitro, may store proteoglycan in vivo unless stimulated by mitogens or other activating agents.(ABSTRACT TRUNCATED AT 400 WORDS)