Dipeptidyl peptidase I is essential for activation of mast cell chymases, but not tryptases, in mice

Dipeptidyl peptidase I (DPPI) is the sole activator in vivo of several granule-associated serine proteases of cytotoxic lymphocytes. In vitro, DPPI also activates mast cell chymases and tryptases. To determine whether DPPI is essential for their activation in vivo, we used enzyme histochemical and immunohistochemical approaches and solution-based activity assays to study these enzymes in tissues and bone marrow-derived mast cells (BMMCs) from DPPI +/+ and DPPI -/- mice. We find that DPPI -/- mast cells contain normal amounts of immunoreactive chymases but no chymase activity, indicating that DPPI is essential for chymase activation and suggesting that DPPI -/- mice are functional chymase knockouts. The absence of DPPI and chymase activity does not affect the growth, granularity, or staining characteristics of BMMCs and, despite prior predictions, does not alter IgE-mediated exocytosis of histamine. In contrast, the level of active tryptase (mMCP-6) in DPPI -/- BMMCs is 25% that of DPPI +/- BMMCs. These findings indicate that DPPI is not essential for mMCP-6 activation but does influence the total amount of active mMCP-6 in mast cells and therefore may be an important, but not exclusive mechanism for tryptase activation.     

Molecular cloning of the mouse mast cell protease-5 gene. A novel secretory granule protease expressed early in the differentiation of serosal mast cells

cDNAs were isolated that encode mouse mast cell protease-5 (MMCP-5), an approximately 30,000 Mr serine protease stored in the secretory granules of serosal mast cells (SMC) and Kirsten sarcoma virus-immortalized mast cells. Based on the deduced amino acid sequences of these cDNAs, MMCP-5 is synthesized as a 247-amino acid preproenzyme composed of a novel 19-residue hydrophobic signal peptide, a Gly-Glu activation peptide not present in other mast cell chymases, and a 226-amino acid protein that represents the mature enzyme. MMCP-5 possesses a unique Asn residue in the substrate binding cleft at residue 176 and is highly basically charged. The MMCP-5 gene was isolated, sequenced, and found to belong to a distinct subset of chymase genes. Allelic variations of the MMCP-5 gene were also detected. MMCP-5 is expressed in bone marrow-derived mast cells (BMMC), Kirsten sarcoma virus-immortalized mast cells, and SMC, but not in gastrointestinal mucosal mast cells of helminth-infected mice. The abundant levels of MMCP-5 mRNA in immature BMMC indicate that this chymase is expressed relatively early during the differentiation of mast cells. MMCP-5 is the first chymase to be molecularly cloned from progenitor mast cells and is also the first chymase shown to be expressed preferentially in the SMC subclass.     

Altered processing of fibronectin in mice lacking heparin. a role for heparin-dependent mast cell chymase in fibronectin degradation

We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2(-/-) mice show reduced levels of various mast cell mediators such as histamine and various heparin-binding mast cell proteases, including chymases, tryptases, and carboxypeptidase A. In this work we have addressed the possible functional consequences of the lack of sulfated heparin. Peritoneal cells were harvested from normal and NDST-2(-/-) mice. After culturing the cells, conditioned media were collected and were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Several differences in the protein patterns were observed, including the presence of large amounts of a approximately 250-kDa protein in medium from NDST-2(-/-) mice that was absent in normal controls. Peptide microsequencing revealed identity of this protein with fibronectin. Western blot analysis showed the presence of fibronectin degradation products in cell cultures from normal mice, which were absent in cultures from NDST-2(-/-) animals. Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2(+/+) mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner. This report thus indicates a biological function for chymase/heparin proteoglycan complexes in fibronectin turnover.     

Cloning of the gene and cDNA for human heart chymase

We have recently identified and characterized a chymotrypsin-like serine proteinase in human heart (human heart chymase) that is the most catalytically efficient enzyme described, thus far, for the cleavage of angiotensin I to yield angiotensin II and the dipeptide His-Leu. Compared to other chymases, this enzyme also has an unusually high degree of specificity for the substrate angiotensin I. We report here the molecular cloning and nucleotide sequence of the gene and cDNA encoding human heart chymase, and determination of its entire deduced amino acid sequence. These data indicate that human heart chymase is highly homologous to other members of the chymase subfamily of chymotrypsin-like proteinases and, most likely, all evolved from a common ancestral gene. Potential regulatory elements found in the 5'-untranslated region of other chymases are also found in the human heart chymase gene. However, this gene lacks mast cell-specific sequences found in the 5'- and 3'-untranslated regions of the rat chymase II gene. In addition, human heart chymase contains clusters of unique amino acid sequences located at key positions likely involved in substrate binding, which may contribute to its high substrate specificity. These contrasting features of the human heart chymase gene and cDNA, and the potential determinants of its primary structure that underlie its unique functional characteristics are considered.     

Mast Cell Chymase Degrades the Alarmins Heat Shock Protein 70, Biglycan,HMGB1, and Interleukin-33 (IL-33) and Limits Danger-induced Inflammation

During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(W^sash)-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation.     

Species differences in angiotensin II generation and degradation by mast cell chymases

Although chymases are known to exhibit species differences in regard to angiotensin (Ang) II generation and degradation, their properties have never been compared under the same experimental conditions. We analyzed the processing of Ang I by chymases of a variety of species (human chymase, dog chymase, hamster chymase-1, rat mast cell protease-1 [rMCP-1], mouse mast cell protease-4 [mMCP-4]) at physiological ionic strength and under neutral pH conditions. Human chymase generated Ang II from Ang I without further degradation, whereas the chymases of other species generated Ang II, followed by degradation at the Tyr4-Ile5 site in a time-dependent manner. Kinetic analysis showed that in terms of Ang II generating activity (analyzed by cleavage of the Phe8-His9 bond using the model peptide Ang(5-10), Ile5-His6-Pro7-Phe8-His9-Leu10), the chymases ranked as follows: dog > human > hamster > mouse > rat (kcat/Km: 18, 11, 0.69, 0.059, 0.030 microM-1min-1), and that in terms of Ang II degrading activity (i.e., cleavage of the Tyr4-Ile5 bond of Ang II), the order was hamster > rat > mouse > dog (kcat/Km: 5.4, 4.8, 0.39, 0.29 microM-lmin-1). These results suggest species differences in the contribution of chymases to local Ang II generation and degradation.     

Purification and characterization of dog mastocytoma chymase: identification of an octapeptide conserved in chymotryptic leukocyte proteinases

We isolated and characterized a chymotryptic serine proteinase from dog mastocytomas. Chymotryptic activity extracted at high ionic strength from mastocytomas propagated in nude mice was separated from tryptic activity by gel filtration and rapidly purified by sequential high-performance hydrophobic interaction and cation-exchange chromatography. The purified enzyme had an Mr of 27,000-30,000 by both analytical gel filtration and SDS-polyacrylamide gel electrophoresis, and a single amino-terminal sequence by automated Edman degradation. Like chymases from rat and human mast cells, the mastocytoma enzyme exhibited a high kcat/Km (1.1.10(5) M-1.s-1) employing succinyl-L-Val-Pro-Phe-p-nitroanilide, the best of several p-nitroanilide substrates screened. It was inhibited by diisopropyl fluorophosphate and soybean trypsin inhibitor, but not by aprotinin, distinguishing it from the otherwise closely related neutrophil enzyme, cathepsin G. The amino-terminal 25 residues of mastocytoma chymase were found to be 72 and 68% identical to the corresponding sequences of chymases from rat peritoneal and mucosal mast cells, respectively; they were also closely related to human cathepsin G and to proteinase sequences from mouse cytotoxic T-lymphocytes. The mastocytoma chymotryptic enzyme contained an octapeptide sequence which is common to all chymotryptic leukocyte proteinases sequenced to date from four mammalian species; this feature distinguishes chymases and other chymotryptic leukocyte proteinases from serine proteinases of coagulation and digestion.     

Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.     

Mechanism by which heparin proteoglycan modulates mast cell chymase activity.

Chymases are highly basic chymotrypsin-like serine proteases expressed exclusively by mast cells. Large amounts of chymases complexed with heparin proteoglycan (PG) are released in vivo during mast cell activation. The tight binding of chymase to heparin PG results in increased activity of the protease toward certain substrates, e.g., thrombin and MeO-Suc-Arg-Pro-Tyr-pNA (S-2586). In this study, the mechanism by which heparin PG modulates chymase activity was investigated, using thrombin and various chromogenic peptide substrates as model substrates. Incubation of thrombin with oligonucleotides that block the heparin-binding site of thrombin abolished the stimulatory effect of heparin PG on thrombin inactivation. Further, thrombin mutants with defects in their heparin-binding regions were less efficiently inactivated by chymase-heparin PG than wild type thrombin. These findings suggest a model for chymase stimulation where heparin PG may promote the chymase-catalyzed cleavage of heparin-binding substrates by simultaneously binding to both chymase and substrate. Experiments in which various chromogenic peptide substrates were utilized showed that heparin PG enhanced the activity of chymase toward positively charged peptide substrates such as S-2586, whereas the cleavage of uncharged substrates was not affected by the presence of heparin PG. On the basis of the latter findings, an alternative stimulation mechanism is discussed where heparin PG may stimulate chymase activity by blocking positively charged regions in chymase, thereby reducing the level of electrostatic repulsion between chymase and positively charged substrates.     

Guinea pig chymase is leucine-specific: a novel example of functional plasticity in the chymase/granzyme family of serine peptidases

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the alpha clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig alpha-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by alpha1-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster alpha-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.     

A key role for mast cell chymase in the activation of pro-matrix metalloprotease-9 and pro-matrix metalloprotease-2

Chymases, serine proteases exclusively expressed by mast cells, have been implicated in various pathological conditions. However, the basis for these activities is not known, i.e. the in vivo substrate(s) for mast cell chymase has not been identified. In this study we show that mice lacking the chymase mouse mast cell protease 4 (mMCP-4) fail to process pro-matrix metalloprotease 9 (pro-MMP-9) to its active form in vivo, whereas both the pro and active form of MMP-9 was found in tissues of wild type mice. Moreover, the processing of pro-MMP-2 into active enzyme was markedly defective in mMCP-4 null animals. Histological analysis revealed an increase in collagen in the ear tissue of mMCP-4-deficient animals accompanied by increased ear thickness and a higher content of hydroxyproline. Furthermore, both lung and ear tissue from the knock-out animals showed a markedly increased staining for fibronectin. MMP-9 and MMP-2 are known to have a range of important activities, but the mechanisms for their activation in vivo have not been clarified previously. The present study thus indicates a key role for mast cell chymase in the regulation of pro-MMP-2 and -9 activities. Moreover, the results suggest an important role for mast cell chymase in regulating connective tissue homeostasis.     

Relationship between Numerous Mast Cells and Early Follicular Development in Neonatal MRL/MpJ Mouse Ovaries

In the neonatal mouse ovary, clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. During this period, we found numerous mast cells in the ovary of MRL/MpJ mice and investigated their appearance and morphology with follicular development. The ovarian mast cells, which were already present at postnatal day 0, tended to localize adjacent to the surface epithelium. Among 11 different mouse strains, MRL/MpJ mice possessed the greatest number of ovarian mast cells. Ovarian mast cells were also found in DBA/1, BALB/c, NZW, and DBA/2 mice but rarely in C57BL/6, NZB, AKR, C3H/He, CBA, and ICR mice. The ovarian mast cells expressed connective tissue mast cell markers, although mast cells around the surface epithelium also expressed a mucosal mast cell marker in MRL/MpJ mice. Some ovarian mast cells migrated into the oocyte nests and directly contacted the compressed and degenerated oocytes. In MRL/MpJ mice, the number of oocytes in the nest was significantly lower than in the other strains, and the number of oocytes showed a positive correlation with the number of ovarian mast cells. The gene expression of a mast cell marker also correlated with the expression of an oocyte nest marker, suggesting a link between the appearance of ovarian ? 4mast cells and early follicular development. Furthermore, the expression of follicle developmental markers was significantly higher in MRL/MpJ mice than in C57BL/6 mice. These results indicate that the appearance of ovarian mast cells is a unique phenotype of neonatal MRL/MpJ mice, and that ovarian mast cells participate in early follicular development, especially nest breakdown.     

Cytokine production by skin-derived mast cells: endogenous proteases are responsible for degradation of cytokines

The current study characterizes the cytokine protein (ELISA) and mRNA (gene array and RT-PCR) profiles of skin-derived mast cells cultured under serum-free conditions when activated by cross-linking of Fc epsilonRI. Prior to mast cell activation, mRNA only for TNF-alpha was detected, while after activation mRNA for IL-5, IL-6, IL-13, TNF-alpha, and GM-CSF substantially increased, and for IL-4 it minimally increased. However, at the protein level certain recombinant cytokines, as measured by ELISAs, were degraded by proteases released by these skin-derived mast cells. IL-6 and IL-13 were most susceptible, followed by IL-5 and TNF-alpha; GM-CSF was completely resistant. These observations also held for the endogenous cytokines produced by activated mast cells. By using protease inhibitors, chymase and cathepsin G, not tryptase, were identified in the mast cell releasates as the likely culprits that digest these cytokines. Their cytokine-degrading capabilities were confirmed with purified chymase and cathepsin G. Soy bean trypsin inhibitor, when added to mast cell releasates, prevented the degradation of exogenously added cytokines and, when added to mast cells prior to their activation, prevented degradation of susceptible endogenous cytokines without affecting either degranulation or GM-CSF production. Consequently, substantial levels of IL-5, IL-6, IL-13, TNF-alpha, and GM-CSF were detected 24-48 h after mast cells had been activated, while none were detected 15 min after activation, by which time preformed granule mediators had been released. IL-4 was not detected at any time point. Thus, unless cytokines are protected from degradation by endogenous proteases, cytokine production by human mast cells with chymase and cathepsin G cells may be grossly underestimated.     

Expansion of the mast cell chymase locus over the past 200 million years of mammalian evolution

The acidic granules of natural killer (NK) cells, T cells, mast cells, and neutrophils store large amounts of serine proteases. Functionally, these proteases are involved, e.g., in the induction of apoptosis, the recruitment of inflammatory cells, and the remodeling of extra-cellular matrix. Among the granule proteases are the phylogenetically related mast cell chymases, neutrophil cathepsin G, and T-cell granzymes (Gzm B to H and Gzm N), which share the characteristic absence of a Cys₁₉₁–Cys₂₂₀ bridge. The genes of these proteases are clustered in one locus, the mast cell chymase locus, in all previously investigated mammals. In this paper, we present a detailed analysis of the chymase locus in cattle (Bos taurus) and opossum (Monodelphis domestica). The gained information delineates the evolution of the chymase locus over more than 200 million years. Surprisingly, the cattle chymase locus contains two α-chymase and two cathepsin G genes where all other studied chymase loci have single genes. Moreover, the cattle locus holds at least four genes for duodenases, which are not found in other chymase loci. Interestingly, duodenases seem to have digestive rather than immune functions. In opossum, on the other hand, only two chymase locus-related genes have been identified. These two genes are not arranged in one locus, but appear to have been separated by a marsupial-specific chromosomal rearrangement. Phylogenetic analyses place one of the opossum genes firmly with mast cell α-chymases, which indicates that the α-chymase had already evolved as a separate, clearly identifiable gene before the separation of marsupials and placental mammals. In contrast, the second gene in opossum is positioned phylogenetically between granzymes, cathepsin G, and the duodenases. These genes, therefore, probably evolved as separate subfamilies after the separation of placental mammals from marsupials. In platypus, only one chymase locus-like sequence could be identified. This previously published “granzyme” does not cluster clearly with any of the chymase locus gene families, but shares the absence of the Cys₁₉₁–Cys₂₂₀ bridge with the other chymase locus proteases. These findings indicate that all chymase locus genes are derived from a single ancestor that was present more than 200 million years ago.     

Mast cell heterogeneity in the human uvea

To identify chymase- and tryptase-positive mast cells in the human uvea, and to study their associations with different types of resident uveal cells, uveal specimens from 24 human donor eyes were cryosectioned in sagittal and tangential planes. Enzyme histochemical staining of chymase was combined with immunohistochemical staining for tryptase, detected with the APAAP method. Fluorescence immunohistochemistry was performed with antibodies against c-kit, alpha smooth muscle actin, protein gene product (PGP) 9.5, CD45, and HLA-DR. In different uveal compartments, the total amounts of mast cells were calculated and the distributions of chymase and tryptase were quantified. All uveal mast cells were c-kit and CD45 positive and HLA-DR negative. No association existed between mast cells and actin-containing cells. Only a few mast cells were in close association with PGP 9.5-labeled nerve fibers. In the choroid, most mast cells were located in the inner central part (mean density = 48.9/mm²), and contained both chymase and tryptase (96%). The ciliary muscle contained numerous mast cells (mean density = 33.7/mm²), many of them tryptase positive but chymase negative (63%). In the pars plana, a high number of chymase-positive, tryptase-negative mast cells were found (20%). In the iris only a few mast cells were present. Although the choroid contains the most common subtype of mast cells, a unique situation concerning the distribution of chymase and tryptase is present in the anterior uveal tissues. A possible role for these cells in the special immunological situation of the anterior eye chamber merits further investigation. Accepted: 16 September 1999     

Human mast cell chymase cleaves pro-IL-18 and generates a novel and biologically active IL-18 fragment

Increased release of IL-18 in the skin causes atopic dermatitis (AD)-like skin lesions, suggesting a role of IL-18 in the pathogenesis of AD. Caspase-1 is a well-known activator of IL-18, but caspase-1 knockout mice still have biologically active IL-18. Normal human keratinocyte constitutively produces pro-IL-18, but it is unable to activate it, suggesting the existence of an alternative pathway for IL-18 in the skin. Dermal accumulation of mast cells is commonly observed in AD patients and in experimental mouse models of AD. Connective tissue mast cells contain high amounts of chymase and tryptase in their cytoplasmic granules. In the present study, we demonstrated that activation of IL-18 is a novel function of human mast cell chymase. Human mast cell chymase rapidly cleaves recombinant pro-IL-18 at 56-phenylalanine and produces a biologically active IL-18 fragment that is smaller than any other reported IL-18-derived species. The human mast cell chymase and the novel IL-18-derived active peptide may be novel therapeutic targets in AD- and IL-18-associated diseases.     

IgE crosslinkage of Fcepsilon receptor I induces both production and activation of matrix metalloproteinase-9 in mast cells

Since mast cells play pivotal roles in allergic inflammations, we investigated how IgE-mediated stimulation modulated mast cell matrix metalloproteinase (MMP)-9 production, and its enzymatic activation. In this study, we clearly demonstrated that proMMP-9 released from murine bone marrow-derived cultured mast cells (BMCMC) was activated to its valid form after crosslinking of surface immunoglobulin (Ig)E. Serine protease inhibitors sensitive to chymases inhibited the phenomenon, indicating that certain chymases may be responsible for activation of proMMP-9. Although binding of IgE to its specific receptors did not alter MMP-9 production, the IgE crosslinkage increased both expression of mRNA, and production of MMP-9 in mast cells. Glucocorticoid declined extra cellular processing of proMMP-9 without affecting mRNA expression. These findings give rise to the possibility that production and activation of mast cell MMP-9 may be increased in the affected sites, thereby resulting in an exacerbation of tissue degradation in inflammatory conditions.