Abstract. Whether or not a nontransformed, mature mouse mast cell (MC) or its committed progenitor can change its granule protease phenotype during inflammatory responses, has not been determined. To address this issue, the granule morphology and protease content of the MC in the jejunum of BALB/c mice exposed to Trichinella spiralis were assessed during the course of the infection. Within 1 wk after helminth infection of the mice, increased numbers of MC appeared in the crypts at the base of the villi, and by wk 2 the number of MC throughout the villi increased by ~25-fold. Shortly after the peak of the mastocytosis, the intraepithelial population of MC disappeared, followed by a progressive loss of lamina propria MC. The presence of stellate-shaped granules containing crystalline structures in intraepithelial MC at the height of infection and the retention of such granules with fragmented crystals in lamina propria MC during resolution of the mastocytosis suggest that MC migrate during the various phases of the inflammation. As assessed by immunohistochemical analyses of serial sections, predominant chymase phenotypes were observed at the height of the infection in the muscle that expressed mouse MC protease (mMCP) 5 without mMCP-1 or mMCP-2 and in the epithelium that expressed mMCP-1 and mMCP-2 without mMCP-5. Accompanying these two MC populations were transitional forms in the submucosa that expressed mMCP-2 and mMCP-5 without mMCP-1 and in the lamina propria that expressed rnMCP-2 alone. These data suggest that jejunal MC sequentially express mMCP-2, cease expressing mMCP-5, and finally express mMCP-1 as the cells progressively appear in the submucosa, lamina propria, and epithelium, respectively. In the recovery phase of the disease, MC sequentially cease expressing mMCP-1, express mMCP-5, and finally cease expressing mMCP-2 as they present at the tips of the villi, the base of the villi, and the submucosa, respectively. That MC can reversibly alter their protease phenotypes suggests that a static nomenclature with fixed functional implications is inadequate to describe MC populations during an inflammatory process within a particular tissue.

A gene that encodes mouse mast cell protease (mMCP) 7 (also known as mouse mast cell tryptase 2) was isolated by genomic cloning with a cDNA that encodes mMCP-6, a tryptase in serosal mast cells. cDNAs encoding mMCP-7 were isolated from a bone-marrow-derived mast cell cDNA library. The mMCP-7 gene spans 2.3 kilos and contains five exons rather than six, as found in the mMCP-6 and human mast cell yptase I genes. Comparison ofthe 5' end of the transcript with the genomic sequence ted that the region corresponding to the first intron in the mMCP-6 and human tryptase I genes Is not spliced during trnscription of mMCP-7 mRNA because of a point mutation at the intron 1 acceptor splice site; this results in a 5' untransated region of 195 nucleotides, which is longer than that of any other known mast cell-specific transcpt. mMCP-7 is 71-76% homologous with mMCP-6 and with dog and human mast cell tryptases, and it is the most acidic mast cell protease, with an overall net charge of -10. RNA blot analyses revealed that the mMCP-7 gene is transcribed in bone-marrow-derived mast cells but is not transcribed in mature serosal mast cells or in mucosal mast cell-enriched intestinal tissue of Trihinefla spinl-infected mice. Transcription of the mMCP-7 gene by differentiating bone-marrow-derived mast cells occurred within 1 week of bone-marrow culture but decreased dramatically after 3 weeks. Thus, the mMCP-7 gene displays a number of unusual structurl characteristics and is distinctive in its transient and selective expression in Immature mast cells maintained in interleukin 3-enriched medium.The secretory granules of mouse mast cells contain a family of serine proteases termed mouse mast cell protease (mMCP) 1 through mMCP-6 (1). mMCP-1 (2-4), mMCP-2 (5), mMCP4 (6), and mMCP-5 (7) have been designated as chymases because of the predicted chymotryptic-like substrate specificities of their translated gene products. mMCP-6 (8) has been designated a tryptase because of its predicted trypticlike substrate specificity. The mouse mast cell chymases have overall net charges at pH 7 from +2 to + 15, whereas mMCP-6 has an overall net charge of -5. Serosal mast cells selectively express mMCP-4, mMCP-5, and mMCP-6, whereas mucosal mast cells that proliferate in the intestines of helminth-infected mice selectively express mMCP-1 and mMCP-2. The mMCP-5 and mMCP-6 genes are transcribed in bone-marrow-derived mast cells (BMMCs) obtained by culturing bone-marrow cells in medium containing interleukin (IL) 3. Because the BMMCs reconstitute all populations of mast cells in vivo when injected into mast cell-deficient

Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used this in vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34 -55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the ␣-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptasespecific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the ␣-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions.

SummaryIt is now established that the subdasses of mast cells (MC) that reside in mucosal and serosal environments can be distinguished from one another in terms of their expression of specific secretory granule-localized proteases and proteoglycans. Further, the hematopoietic-and connective tissue-derived cytokines that regulate expression of the genes that encode these constituents of the granule can now be identified using recently developed gene-specific probes and recombinant cytokines. When bone marrow-derived MC (BMMC) were developed with recombinant interleukin 3 (rlL-3) and maintained with this cytokine in the absence or presence of recombinant c-kit ligand (rKL), they remained saffanin-, produced almost no 3sS-labeled heparin proteoglycans, and contained greater levels of mouse MC protease (MMCP) -5 mKNA and mast cell carboxypeptidase A (MC-CPA) mKNA than MMCP-6 mKNA. They did not contain MMCP-4 or -2 mKNA, genes expressed late in the differentiation of progenitor cells into serosal and mucosal MCs, respectively. In contrast, BMMC developed with rKL alone or by sequential culture in medium containing rlL-3 followed by rKL expressed high levels of MMCP-4 and -6 mRNA, as well as the transcripts that encode MMCP-5 and MC-CPA. Although rKL-developed BMMC were saffanin + and produced substantial amounts of 3sS-labeled heparin proteoglycans, they contained only minimal amounts of histamine and MC-CPA enzymatic activity relative to serosal MC. These are the first studies to characterize the transcriptional granule phenotype of a population of BMMC derived using any recombinant eytokine, to demonstrate a dissociation between histochemical staining and granule maturation, and to demonstrate antagonistic regulation of late expressed protease genes by a eytokine.M ast cells (MC) 1 that reside in different tissue sites are heterogeneous in terms of their secretory granule proteoglycans and proteases. Safranin + granules of mouse serosal MC, considered to represent the connective tissue MC subclass, contain abundant amounts of heparin proteoglycans, MC carboxypeptidase A (MC-CPA), mouse MC protease (MMCP) -3, -4, -5, -6, but no MMCP-1 or -2 (1-8). In contrast, the safranin-mucosal MC subclass found in the intestines of helminth-infected mice express MMCP-1 (9) and -2 (5), but little MC-CPA (4), and no MMCP-5 (7) or -6 (8).The factors that regulate the diversity of MC proteases are unknown, but tissue-related MC heterogeneity may be a consequence of the particular panel of cytokines provided in varied t Abbreviations used in this paper: BMMC, bone marrow-derived MC; KL, c-kit ligand; MC, mast cell; MC-CPA, MC carboxypeptidase A; MMCP, mouse MC protease; SG-PG, secretory granule proteoglycan peptide core; and TSG, 0.1 M Tr/s-HCI, 0,1 M sodium sulfate, and 4 M GnHC1. microenvironments (10-14). Mouse bone marrow cells cultured in medium containing IL-3 differentiate into morphologically immature MC that express the high affinity IgE receptor . When this receptor is crosslinked with antigen, lipid mediators are generate...

As assessed by RNA blot analyses with genespecific probes, we report that the perivascular connective tissue mast ceOls (CTMCs) in the ear and skin of BALB/cJ mice contain abundant levels of the mouse mast cell protease (mMCP) 7 transcript, in addition to those protease transcripts present in their serosal mast cells (SMCs). High levels of the mMCP-7 transcript also were detected in the ears of WBB6F1/J-+/+, WCB6F1/J-+/+, WB/ReJ-+/+, and WC/ReJ-+/+ mice. However, the ears of these four strains and the SMCs from the WCB6F1/J-+/+ strain but not the BALB/cJ strain also contained high steady-state levels of the mMCP-2 transcript. The mMCP-2, mMCP-4, mMCP-5, mMCP-6, and mMCP-7 transcripts were not detected in the ears of mast-cell-deficient WBB6F1/J-W/Wv and WCB6F1/JSli/SId mice, indicating that mast cells were the source of these protease transcripts in the +/+ animals. When immunohistochemic*l analyses of serial sections of ear and skin from WBB6F1/J-+/+ mice were performed with anti-mMCP-2IgG and anti-mMCP-5 IgG, the perivascular CTMCs in these tissues were found to express both mMCP-2 and mMCP-5 in their granules. The prominent expression of mMCP-7 in constitutive perivascular CTMCs indicates that this mast cell has an extended protease phenotype relative to the SMCs of the same strains. Further, the perivascular CTMCs and SMCs of the +/+ strains differ from those in BALB/cJ mice in their prominent expression of mMCP-2.As assessed by protein sequencing and/or by cDNA cloning, mouse mast cells differentiated in vivo and in vitro express varied combinations of carboxypeptidase A (mMC-CPA) (1) and at least seven serine proteases [designated mouse mast cell protease (mMCP) 1 to mMCP-7] in their granules (2-11). The safranin+ staining serosal mast cells (SMCs) of the BALB/cJ mouse contain high steady-state levels of the transcripts that encode mMCP-4, mMCP-5, and mMCP-6 but not mMCP-1, mMCP-2, or mMCP-7. In contrast, the safranin-mucosal mast cells that proliferate in the intestine of helminth-infected BALB/cJ mice contain high steady-state levels of the mMCP-1 and mMCP-2 transcripts but not the mMCP-4, mMCP-5, mMCP-6, or mMCP-7 transcripts. Although these two histochemically distinct populations of tissue mast cells apparently express their granule proteases in a strict subclass-specific manner, nontransformed but immature populations of BALB/cJ mouse mast cells derived in vitro can express varied combinations of the eight mMCPs in response to different recombinant cytokines (12)(13)(14)(15)(16).In addition to those protease transcripts expressed by SMCs. We also present RNA and immunohistochemical data that indicate that the WBB6F1/J-+/+, WCB6F1/J-+/+, WB/ ReJ-+/+, and WC/ReJ-+/+ strains of mice differ from the BALB/cJ mouse strain in their prominent expression of mMCP-2 in both their SMCs and perivascular CTMCs. MATERIALS AND METHODSMouse Mast-Cell-Specific Protease mRNA Levels in Ear and Skin CTMCs and Isolated SMCs. Female mice of the WB/ ReJ-+/+, WC/ReJ-+/+, WBB6F1/J-+/+, WCB6F1/J-+/+, WBB6F,/J-W/Wv, WCB...

The C57BL/6 mouse differs from the BALB/c mouse in that its ear and skin mast cells and its progenitor bone marrow-derived mast cells (mBMMCs) do not express mouse mast cell protease (mMCP) 7. We now report that, as detected by nuclear run-on analysis, the mMCP-7 gene is transcribed in C57BL/6 mBMMCs at a rate comparable to that in BALB/c mBMMCs. Reverse transcriptase-polymerase chain reaction analysis and sequencing of the product revealed that the ears of C57BL/6 mice contain small amounts of a mMCP-7 transcript that possesses a 98-base pair deletion. The deletion begins at a normally quiescent cryptic splice site (G 416 TGAG), 98 base pairs upstream of the normal exon 2/intron 2 splice site (G 514 TGAG), and introduces a premature stop codon in the alternatively spliced transcript. Thus, even if translated, the mature protein would consist of only 18 amino acids as compared to 245 amino acids in normal mMCP-7. Sequence analysis of the mMCP-7 gene in the C57BL/6 mouse revealed that the cryptic splice site is activated due to a G 514 3 A point mutation at the first nucleotide of the normal exon 2/intron 2 splice site. This is the first report of a mutation of a gene that encodes a mast cell secretory granule constituent that leads to its loss of expression. Moreover, the mMCP-7 gene is the first found in any species that sequentially has undergone a splice site mutation to cause retention of an intron and then a second splice site mutation to cause activation of a cryptic splice site.

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Mouse mast cell protease 7 (mMCP-7) is a tryptase stored in the secretory granules of mast cells. At the granule pH of 5.5, mMCP-7 is fully active and is bound to heparin-containing serglycin proteoglycans. to understand the interaction of mMCP-7 with heparin inside and outside the mast cell, this trytase was first studied by comparative protein modeling. The "pro" form of mMCP-7 was then expressed in insect cells and studied by site-directed mutagenesis. Although mMCP-7 lacks known linear sequences of amino acis that interact with heparin, the three-dimensional model of mMCP-7 revealed an area on the surface of the folded protein away from the substrate-binding site that exhibits a strong positive electrostatic potential at the acidic pH of the granule. In agreement with this calculation, recombinant pro-mMCP-7 bound to a heparin-affinity column at pH 5.5 and readily dissociated from the column at pH > 6.5. Site-directed mutagenesis confirmed the prediction that the conversion of His residues 8,68, and 70 in the positively charged region into Glu prevents the binding of pro-mMCP-7 to heparin. Because the binding requires positively charged His residues, native mMCP-7 is able to dissociate from the protease/proteoglycan macromolecular complex when the complex is exocytosed from bone marrow-derived mast cells into a neutral pH environment. Many hematopoietic effector cells store positively charged proteins in granules that contain serglycin proteoglycans. The heparin/mMCP-7 interaction, which depends on the tertiary structure of the tryptase, may be representative of a general control mechanism by which hematopoietic cells maximize storage of properly folded, enzymatically active proteins in their granules.