Uptake of colloidal thorium dioxide by the mouse connective tissue mast cell.

The ability of the mouse mast cell to phagocytize colloidal thorium dioxide, Thorotrast, was investigated employing the mouse connective tissue air pouch. The connective tissue mast cell of mouse was found to have limited capability to ingest the particulate Thorotrast in comparison to the rat peritoneal mast cell which ingested this material readily. Fibroblasts and macrophages in the connective tissue removed injected Thorotrast very rapidly in contrast to mast cells that demonstrated limited phagocytic capabilities. The tissue mast cell of the mouse, therefore, should not be considered a part of the reticuloendothelial system.     

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.     

Dual targets for mouse mast cell protease-4 in mediating tissue damage in experimental bullous pemphigoid

Mouse mast cell protease-4 (mMCP-4) has been linked to autoimmune and inflammatory diseases, although the exact mechanisms underlying its role in these pathological conditions remain unclear. Here, we have found that mMCP-4 is critical in a mouse model of the autoimmune skin blistering disease bullous pemphigoid (BP). Mice lacking mMCP-4 were resistant to experimental BP. Complement activation, mast cell (MC) degranulation, and the early phase of neutrophil (PMN) recruitment occurred comparably in mMCP-4(-/-) and WT mice. However, without mMCP-4, activation of matrix metalloproteinase (MMP)-9 was impaired in cultured mMCP-4(-/-) MCs and in the skin of pathogenic IgG-injected mMCP-4(-/-) mice. MMP-9 activation was not fully restored by local reconstitution with WT or mMCP-4(-/-) PMNs. Local reconstitution with mMCP-4(+/+) MCs, but not with mMCP-4(-/-) MCs, restored blistering, MMP-9 activation, and PMN recruitment in mMCP-4(-/-) mice. mMCP-4 also degraded the hemidesmosomal transmembrane protein BP180 both in the skin and in vitro. These results demonstrate that mMCP-4 plays two different roles in the pathogenesis of experimental BP, by both activating MMP-9 and by cleaving BP180, leading to injury of the hemidesmosomes and extracellular matrix of the basement membrane zone.     

Translation and granule localization of mouse mast cell protease-5. Immunodetection with specific antipeptide Ig.

An antibody to mouse mast cell protease-5 (MMCP-5) was obtained by immunizing a rabbit with a 17-residue synthetic peptide corresponding to the unique amino acid sequence at residues 146 to 162 in this serine protease. After affinity purification, anti-MMCP-5(146-162) Ig reacted in SDS-PAGE immunoblots to recombinant MMCP-5 and to the native MMCP-5 protein present in the lysates of mouse serosal mast cells and the MC5 line of Kirsten sarcoma virus-immortalized mouse mast cells. Immunocytochemical staining localized MMCP-5 to the cytoplasmic granules of serosal mast cells and Kirsten sarcoma virus-immortalized mouse mast cells. Because mouse bone marrow-derived mast cells express abundant amounts of MMCP-5 mRNA, anti-MMCP-5(146-162) Ig was used to study the translation and granule accumulation of this protease when progenitor cells differentiate into these immature mouse mast cells. Maximal expression of MMCP-5 mRNA occurred after bone marrow cells had been cultured for 2 wk in IL-3-rich WEHI-3 cell conditioned medium, and MMCP-5 protein was detected in these cells. However, electron-microscopic analysis with gold-labeled antibody revealed that the amount of MMCP-5 in the individual granules of bone marrow-derived mast cells varied. The highest concentration of MMCP-5 was found in the most electron-dense secretory granules of the cells. These studies demonstrate the ultrastructural localization of the earliest transcribed mouse mast cell chymase, MMCP-5, and its granule accumulation during the differentiation of mouse bone marrow progenitor cells into immature mouse mast cells.     

Cloning of the cDNA and gene for mouse mast cell protease 4. Demonstration of its late transcription in mast cell subclasses and analysis of its homology to subclass-specific neutral proteases of the mouse and rat

Based on the amino-terminal amino acid sequence of the mature form of mouse mast cell protease 4 (MMCP-4), previously identified in peritoneal connective tissue mast cells (CTMC) and Kirsten sarcoma virus-immortalized mast cells (KiSV-MC), a 26-mer oligonucleotide probe was constructed and used to clone cDNAs for MMCP-4 from a KiSV-MC1 cDNA library. MMCP-4 is the first secretory granule serine protease of CTMC to be molecularly cloned. Using a cDNA probe derived from the 3'-untranslated portion of the MMCP-4 cDNA, the gene for MMCP-4 and a second highly related gene (mouse mast cell protease-like, MMCP-L) were cloned from a BALB/c mouse genomic DNA library and sequenced entirely, including approximately 2 kilobases of the 5'-flanking region. MMCP-4 and MMCP-L have five exons of identical length, four introns of nearly identical length, and approximately 900 base pairs of 5'-flanking DNA with sequence similarity by dot matrix analysis. By RNA blot analysis with gene-specific probes for MMCP-4 (bases 497-633 of the cDNA) and MMCP-L (bases 502-638 of the cDNA), mRNA for MMCP-4 was present in KiSV-MC5, CTMC, and the intestine of a mouse infected with the parasite Nippostrongylus brasiliensis markedly enriched for mucosal mast cells (MMC); MMCP-L mRNA was detected only in the intestine of the N. brasiliensis-infected mouse. MMCP-4 mRNA was not expressed in normal mouse intestine or in interleukin 3-dependent bone marrow-derived mast cells, which can serve as precursors to both MMC and CTMC. This finding suggests that MMCP-4 is transcribed relatively late in the development of both the CTMC and MMC subclasses and underscores the fact that mouse bone-marrow-derived mast cells are immature mast cells, rather than tissue culture equivalents of the MMC subclass.     

The role of SHIP in the development and activation of mouse mucosal and connective tissue mast cells

Although SHIP is a well-established suppressor of IgE plus Ag-induced degranulation and cytokine production in bone marrow-derived mast cells (BMMCs), little is known about its role in connective tissue (CTMCs) or mucosal (MMCs) mast cells. In this study, we compared SHIP's role in the development as well as the IgE plus Ag and TLR-induced activation of CTMCs, MMCs, and BMMCs and found that SHIP delays the maturation of all three mast cell subsets and, surprisingly, that it is a positive regulator of IgE-induced BMMC survival. We also found that SHIP represses IgE plus Ag-induced degranulation of all three mast cell subsets and that TLR agonists do not trigger their degranulation, whether SHIP is present or not, nor do they enhance IgE plus Ag-induced degranulation. In terms of cytokine production, we found that in MMCs and BMMCs, which are poor producers of TLR-induced cytokines, SHIP is a potent negative regulator of IgE plus Ag-induced IL-6 and TNF-α production. Surprisingly, however, in splenic or peritoneal derived CTMCs, which are poor producers of IgE plus Ag-induced cytokines, SHIP is a potent positive regulator of TLR-induced cytokine production. Lastly, cell signaling and cytokine production studies with and without LY294002, wortmannin, and PI3Kα inhibitor-2, as well as with PI3K p85α(-/-) BMMCs and CTMCs, are consistent with SHIP positively regulating TLR-induced cytokine production via an adaptor-mediated pathway while negatively regulating IgE plus Ag-induced cytokine production by repressing the PI3K pathway.     

Stimulation of mouse connective tissue-type mast cells by hemopoietic stem cell factor, a ligand for the c-kit receptor.

The proliferative capacity of mouse connective tissue-type mast cells (CTMC) was analyzed by using a newly discovered c-kit ligand, termed stem cell factor (SCF). More than 90% of CTMC in the peritoneal cavity responded to recombinant rat SCF (rrSCF) and were able to give rise to pure mast cell colonies in methylcellulose culture. Serial observation (mapping) of growth of individual CTMC in culture containing rrSCF confirmed their striking proliferative ability. No serum but accessory cells (non-CTMC cells) in the peritoneal population were required for the clonal growth of CTMC induced by rrSCF in our methylcellulose culture of whole peritoneal cells. The rrSCF-induced mast cell colony formation from peritoneal CTMC was completely inhibited by the addition of anti-c-kit antibody, which can block the binding of SCF to c-kit, to the culture. When IL-3 was combined with rrSCF, mast cell colonies dramatically increased in size. Mapping studies revealed that the combination of the two factors augmented the proliferative rate of CTMC. Approximately 60% of the constituent cells of the mast cell colonies which were formed from peritoneal CTMC in the culture containing rrSCF alone were stained with berberine sulfate, which is a characteristic of CTMC. However, most mast cells which were induced by rrSCF+IL-3 from peritoneal CTMC contained berberine(-)-safranin(-)-Alcian blue(+) granules. Although IL-4 exhibited little synergism with rrSCF in the induction of CTMC proliferation, the addition of IL-4 to the culture containing rrSCF+IL-3 resulted in an increase in mast cells which retained CTMC characteristics.     

Cloning and expression of a cDNA for the human homolog of mouse T cell and mast cell growth factor P40

A cDNA encoding the human homolog of mouse T-cell and mast cell growth factor P40 was derived from peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin and phorbol myristate acetate. Sequence analysis of the cDNA predicted a precursor protein of 144 amino acids including a signal peptide of 18 residues, a structure identical with that of mouse P40. The homology between the mouse and human proteins is 55% with a perfect conservation of the 10 cysteine residues present in the mature polypeptide. Expression of the cDNA for human P40 in a baculovirus vector yielded a protein capable of enhancing in vitro survival of human T cell lines.     

Structural requirements and mechanism for heparin-induced activation of a recombinant mouse mast cell tryptase, mouse mast cell protease-6: formation of active tryptase monomers in the presence of low molecular weight heparin

Mast cell tryptase is stored as an active tetramer in complex with heparin in mast cell secretory granules. Previously, we demonstrated the dependence on heparin for the activation/tetramer formation of a recombinant tryptase. Here we have investigated the structural requirements for this activation process. The ability of heparin-related saccharides to activate a recombinant murine tryptase, mouse mast cell protease-6 (mMCP-6), was strongly dependent on anionic charge density and size. The dose-response curve for heparin-induced mMCP-6 activation displayed a bell-shaped appearance, indicating that heparin acts by binding to more than one tryptase monomer simultaneously. The minimal heparin oligosaccharide required for binding to mMCP-6 was 8-10 saccharide units. Gel filtration analyses showed that such short oligosaccharides were unable to generate tryptase tetramers, but instead gave rise to active mMCP-6 monomers. The active monomers were inhibited by bovine pancreatic trypsin inhibitor, whereas the tetramers were resistant. Furthermore, monomeric (but not tetrameric) mMCP-6 degraded fibronectin. Our results suggest a model for tryptase tetramer formation that involves bridging of tryptase monomers by heparin or other highly sulfated polysaccharides of sufficient chain length. Moreover, our results raise the possibility that some of the reported activities of tryptase may be related to active tryptase monomers that may be formed according to the mechanism described here.