George H. Caughey

Mast cell tryptase is a mitogen for cultured fibroblasts.

Mast cells appear to promote fibroblast proliferation, presumably through secretion of growth factors, although the molecular mechanisms underlying this mitogenic potential have not been explained fully by known mast cell-derived mediators. We report here that tryptase, a trypsin-like serine proteinase of mast cell secretory granules, is a potent mitogen for fibroblasts in vitro. Nanomolar concentrations of dog tryptase strongly stimulate thymidine incorporation in Chinese hamster lung and Rat-1 fibroblasts and increase cell density in both subconfluent and confluent cultures of these cell lines. Tryptase-induced cell proliferation appears proteinase-specific, as this response is not mimicked by pancreatic trypsin or mast cell chymase. In addition, low levels of tryptase markedly potentiate DNA synthesis stimulated by epidermal growth factor, basic fibroblast growth factor, or insulin. Inhibitors of catalytic activity decrease the mitogenic capacity of tryptase, suggesting, though not proving, the participation of the catalytic site in cell activation by tryptase. Differences in Ca++ mobilization and sensitivity to pertussis toxin suggest that tryptase and thrombin activate distinct signal transduction pathways in fibroblasts. These data implicate mast cell tryptase as a potent, previously unrecognized fibroblast growth factor, and may provide a molecular link between mast cell activation and fibrosis.

Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.

To investigate the hypothesis that neutrophil proteases stimulate airway gland secretion, we studied the effect of human cathepsin G and elastase on secretion of 35S-labeled macromolecules from cultured bovine airway gland serous cells. Both proteases stimulated secretion in a concentration-dependent fashion with a threshold of greater than or equal to 10(-10) M. Elastase was more potent than cathepsin G, causing a maximal secretory response of 1,810 +/- 60% over baseline at 10(-8) M. The maximal response to cathepsin G (1,810 +/- 70% over baseline at 10(-7) M) was similar to the maximal response to elastase. These responses were greater than 10-fold larger than the response to other agonists such as histamine. Protease-induced secretion was noncytotoxic and required catalytically active enzymes. The predominant sulfated macromolecule released by proteases was chondroitin sulfate proteoglycan. Immunocytochemical staining demonstrated chondroitin sulfate in cytoplasmic granules and decreased granular staining after stimulation of cells with elastase. The neutrophil proteases also degraded the proteoglycan released from serous cells. Cathepsin G and elastase in supernatant obtained by degranulation of human peripheral neutrophils also caused a secretory response. Thus, neutrophil proteases stimulate airway gland serous cell secretion of chondroitin sulfate proteoglycan and degrade the secreted product. These findings suggest a potential role for neutrophil proteases in the pathogenesis of increased and abnormal submucosal gland secretions in diseases associated with inflammation and neutrophil infiltration of the airways.

Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2.

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin-like enzymes. PAR-2 is highly expressed by small intestinal enterocytes where it is activated by luminal trypsin. The location, mechanism of activation, and biological functions of PAR-2 in the colon, however, are unknown. We localized PAR-2 to the muscularis externa of the rat colon by immunofluorescence. Myocytes in primary culture also expressed PAR-2, assessed by immunofluorescence and RT-PCR. Trypsin, SLIGRL-NH2 (corresponding to the PAR-2 tethered ligand), mast cell tryptase, and a filtrate of degranulated mast cells stimulated a prompt increase in [Ca2+]i in myocytes. The response to tryptase and the mast cell filtrate was inhibited by the tryptase inhibitor BABIM, and abolished by desensitization of PAR-2 with trypsin. PAR-2 activation inhibited the amplitude of rhythmic contractions of strips of rat colon. This response was unaffected by indomethacin, l-NG-nitroarginine methyl ester, a bradykinin B2 receptor antagonist and tetrodotoxin. Thus, PAR-2 is highly expressed by colonic myocytes where it may be cleaved and activated by mast cell tryptase. This may contribute to motility disturbances of the colon during conditions associated with mast cell degranulation.

Mast cell tryptases and chymases in inflammation and host defense

Summary:  Tryptases and chymases are the major proteins stored and secreted by mast cells. The types, amounts, and properties of these serine peptidases vary by mast cell subtype, tissue, and mammal of origin. Membrane‐anchored γ‐tryptases are tryptic, prostasin‐like, type I peptidases that remain membrane attached on release and act locally. Soluble tryptases, including their close relatives, mastins, form inhibitor‐resistant oligomers that act more remotely. Befitting their greater destructive potential, chymases are quickly inhibited after release, although some gain protection by associating with proteoglycans. Most chymase‐like enzymes, including mast cell cathepsin G, hydrolyze chymotryptic substrates, an uncommon capability in the proteome. Some rodent chymases, however, have mutations resulting in elastolytic activity. Secreted tryptases and chymases promote inflammation, matrix destruction, and tissue remodeling by several mechanisms, including destroying procoagulant, matrix, growth, and differentiation factors and activating proteinase‐activated receptors, urokinase, metalloproteinases, and angiotensin. They also modulate immune responses by hydrolyzing chemokines and cytokines. At least one chymase protects mice from intestinal worms. Tryptases and chymases can also oppose inflammation by inactivating allergens and neuropeptides causing inflammation and bronchoconstriction. Thus, like mast cells themselves, mast cell serine peptidases play multiple roles in host defense, and any accounting of benefit versus harm is necessarily context specific.

Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase.

Abstract: Proteinase‐activated receptor‐2 (PAR‐2) is a G‐protein coupled receptor. Tryptic proteases cleave PAR‐2 exposing a tethered ligand (SLIGKV), which binds and activates the receptor. Although PAR‐2 is highy expressed by cultured keratinocytes and is an inflammatory mediator, its precise localization in the normal and inflamed human skin in unknown, and the proteases that activate PAR‐2 in the skin have not been identified. We localized PAR‐2 in human skin by immunohistochemistry, examined PAR‐2 expression by RT‐PCR and RNA blotting, and investigated PAR‐2 activation by mast cell tryptase. PAR‐2 was localized to keratinocytes, especially in the granular layer, to endothelial cells, hair follicles, myoepithelial cells of sweat glands, and dermal dendritic‐like cells. PAR‐2 was also highly expressed in keratinocytes and endothelial cells of inflamed skin. PAR‐2 mRNA was detected in normal human skin by RT‐PCR, and in cultured human keratinocytes and dermal microvascular endothelial cells by Northern hybridization. Trypsin, tryptase and a peptide corresponding to the tethered ligand (SLIGKVNG2) increased [Ca2+]i in keratinocytes, measured using Fura‐2/AM. Although tryptase‐containing mast cells were sparsely scattered in the normal dermis, they were numerous in the dermis in atopic dermatitis, and in the dermis, dermal‐epidermal border, and occasionally within the lower epidermis in psoriasis. Tryptase may activate PAR‐2 on keratinocytes and endothelial cells during inflammation.

Thrombin and mast cell tryptase regulate guinea‐pig myenteric neurons through proteinase‐activated receptors‐1 and −2

1 Proteases regulate cells by cleaving proteinase‐activated receptors (PARs). Thrombin and trypsin cleave PAR‐1 and PAR‐2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR‐1 and PAR‐2. 2 We detected immunoreactive PAR‐1 and PAR‐2 in > 60 % of neurons from the myenteric plexus of guinea‐pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR‐1 and PAR‐2. We confirmed expression of PAR‐1 and PAR‐2 in the myenteric plexus by RT‐PCR using primers based on sequences of cloned guinea‐pig receptors. 3 Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR‐1 and PAR‐2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR‐1 agonists responded to PAR‐2 agonists, and > 90 % of PAR‐1 and PAR‐2 responsive neurons responded to ATP. 4 These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR‐1 and PAR‐2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.

Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs.

Supernatants obtained by degranulation of dog mastocytoma cells greatly increased the sensitivity and the magnitude of the contractile response of isolated dog bronchial smooth muscle to histamine. The enhanced contractile response was reversed completely by H1-receptor antagonists and was prevented by an inhibitor of tryptase (a major protease released with histamine from secretory granules of mast cells). The potentiation of histamine-induced contractions was reproduced by active tryptase in pure form. The contractions due to the combination of histamine and purified tryptase were abolished by the Ca2+ channel blockers nifedipine and verapamil. The bronchoconstricting effects of KCl and serotonin, which, like histamine, contract airway smooth muscle by a mechanism predominantly involving membrane potential-dependent Ca2+ transport, were also potentiated by tryptase. However, the contractile effects of acetylcholine, which contracts dog airway smooth muscle by a mechanism independent of Ca2+ channels, were unaffected by tryptase. These findings show a striking promotion of agonist-induced bronchial smooth muscle contraction by mast cell tryptase, via direct or indirect effects on Ca2+ channels, and the findings therefore suggest a novel potential mechanism of hyperresponsiveness in dog bronchi.

Dog mast cell alpha-chymase activates progelatinase B by cleaving the Phe88-Gln89 and Phe91-Glu92 bonds of the catalytic domain.

In prior work we showed that a metallogelatinase is secreted from dog mastocytoma cells and directly activated by exocytosed mast cell α-chymase. The current work identifies the protease as a canine homologue of progelatinase B (92-kDa gelatinase, MMP-9), determines the sites cleaved by α-chymase, and explores the regulation of gelatinase expression in mastocytoma cells. To obtain a cDNA encoding the complete sequence of mastocytoma gelatinase B, a 2.3-kilobase clone encoding progelatinase was isolated from a BR mastocytoma library. The sequenced cDNA predicts a 704-amino acid protein 80% identical to human progelatinase B. Regions thought to be critical for active site latency, such as the Cys-containing propeptide sequence, PRCGVPD, and the catalytic domain sequence, HEFGHALGLDHSS, are entirely conserved. Cleavage of progelatinase B by purified dog α-chymase yielded an ∼84-kDa product that contained two NH2-terminal amino acid sequences, QTFEGDLKXH and EGDLKXHHND, which correspond to residues 89–98 and 92–101 of the cDNA predicted sequence, respectively. Thus, α-chymase cleaves the catalytic domain of gelatinase B at the Phe88-Gln89 and Phe91-Glu92 bonds. Like BR cells, the C2 line of dog mastocytoma cells constitutively secrete progelatinase B which is activated by α-chymase. By contrast, non-chymase-producing C1 cells secrete a gelatinase B (which remains in its proform) only in response to 12-O-tetradecanoylphorbol-13-acetate. Whereas 12-O-tetradecanoylphorbol-13-acetate stimulation of BR cells produced a ∼15-fold increase in gelatinase B mRNA expression, dexamethasone down-regulated its expression by ∼5-fold. Thus, extracellular stimuli may regulate the amount of mast cell progelatinase B expressed by mast cells. These data further support a role for mast cell α-chymase in tissue remodeling involving gelatinase B-mediated degradation of matrix proteins.

Angiotensin II generation by mast cell α- and β-chymases

Mast cells secrete α- and β-chymases. Primate α-chymases generate angiotensin (AT) II by selectively hydrolyzing AT I’s Phe8–His9 bond. This is distinct from the AT converting enzyme (ACE) pathway. In humans, α-chymase is the major non-ACE AT II-generator. In rats, β-chymases destroy AT II by cleaving at Tyr4–Ile5. Past studies predicted that AT II production versus destruction discriminates α- from β-chymases and that Lys40 in the substrate-binding pocket determines α-chymase Phe8 specificity. This study examines these hypotheses by comparing AT II generation by human α-chymase (containing Lys40), dog α-chymase (lacking Lys40), and mouse mMCP-4 (a β-chymase lacking Lys40; orthologous to AT II-destroying rat chymase rMCP-1). The results suggest that human and dog α-chymase generate AT II exclusively and with comparable efficiency, although dog chymase contains Ala40 rather than Lys40. Furthermore, AT II is the major product generated by degranulation supernatants from cultured dog mast cells, which release tryptases and dipeptidylpeptidase as well as α-chymase. In contrast to rMCP-1, mMCP-4 β-chymase readily generates AT II. Although there is competing AT I hydrolysis at Tyr4, mMCP-4 does not destroy AT II quickly once it is formed. We conclude (1) that chymases are the dominant AT I-hydrolyzing mast cell peptidases, (2) that residues other than Lys40 are key determinants of α-chymase AT I Phe8 specificity, (3) that β-chymases can generate AT II, and (4) that α- and β-chymases are not strictly dichotomous regarding AT I cleavage specificity.

Tissue-selective mast cell reconstitution and differential lung gene expression in mast cell-deficient KitW-sh/KitW-sh sash mice

Background Mast cell‐deficient KitW/KitW‐v mice are an important resource for studying mast cell functions in vivo. However, because they are compound heterozygotes in a mixed genetic background and are infertile, they cannot be crossed easily with other mice.