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A cDNA encoding a new type II transmembrane protein has been isolated from human mast cells by subtraction cloning. This cDNA contains an open reading frame of 186 amino acids. RT-PCR analysis showed that this gene is differentially expressed in mast cells. Therefore, the peptide encoded by this gene was termed mast cell-expressed membrane protein 1 (MCEMP1). The MCEMP1 gene contains seven exons and was mapped to human chromosome 19p13.3. The epitope-tagged MCEMP1 has been expressed in mammalian cells and found to be localized to the cellular membrane with its C-terminus extending to the outside of the membrane and N-terminus into the cytoplasmic compartment. Monoclonal antibodies against MCEMP1 were generated and characterized by immunoprecipitation and FACS. The results showed that the native MCEMP1 is expressed in cord blood-derived mast cells and HMC-1 and THP-1 cell lines, but not in other cell types that we have tested. Immunochemical staining of human lung sections showed that MCEMP1 staining is specifically associated with lung mast cells.

Modulation by balancing activating and inhibitory receptors constitutes an important mechanism for regulating immune responses. Cells that are activated following ligation of receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs) can be negatively regulated by other receptors bearing immuno-receptor tyrosine-based inhibition motifs (ITIMs). Human mast cells (MCs) are the major effector cells of type I hypersensitivity and important participants in a number of disease processes. Antigen-mediated aggregation of IgE bound to its high-affinity receptor on MCs initiates a complex series of biochemical events leading to MC activation. With great detailed description and analysis of several inhibitory receptors on human MCs, a central paradigm of negative regulation of human MC activation by these receptors has emerged.

The granules of mast cells and other inflammatory cells are known to be rich in zinc (Zn), a potent caspase inhibitor. The functions of granular Zn, its mechanism of uptake, and its relationship to caspase activation in apoptosis are unclear. The granules of a variety of mast cell types fluoresced intensely with the Zn-specific fluorophore Zinquin, and fluorescence was quenched by functional depletion of Zn using a membrane-permeable Zn chelator N, N, N', N'-tetrakis (2-pyridyl-methyl)ethylenediamine (TPEN). Zn levels were also depleted by various mast cell activators, including IgE/anti-IgE, and Zn was rapidly replenished during subsequent culture, suggesting an active uptake mechanism. In support of the latter, mast cells contained high levels of the vesicular Zn transporter ZnT(4), especially in the more apical granules. Immunofluorescence and immunogold labeling studies revealed significant pools of procaspase-3 and -4 in mast cell granules and their release during degranulation. Functional depletion of Zn by chelation with TPEN, but not by degranulation, resulted in greatly increased susceptibility of mast cells to toxin-induced caspase activation, as detected using a fluorogenic substrate assay. Release of caspases during degranulation was accompanied by a decreased susceptibility to toxins. Zn depletion by chelation, but not by degranulation, also resulted in nuclear translocation of the antiapoptotic, proinflammatory transcription factor NF-kappaB. These findings implicate a role for ZnT(4) in mast cell Zn homeostasis and suggest that granule pools of Zn may be distinct from those regulating activation of procaspase-3 and NF-kappaB.

cDNAs were recently isolated from BALB/c mouse mast cells (MCs) that encode the new signaling protein mouse Ras guanine nucleotide releasing protein 4 (mRasGRP4). The present study evaluates the expression pattern and biological activity of mRasGRP4 in a variety of mouse strains. As assessed immunohistochemically and by RNA analysis, mRasGRP4 is not coordinately expressed with any of its family members. Normally, mRasGRP4 is an MC-restricted protein in tissues, and kinetic studies revealed that mRasGRP4 is expressed relatively early in developing MCs. The expression of mRasGRP4 in the fetus before granulated MCs become abundant supports the conclusion that RasGRP4 participates in MC-specific differentiation pathways. Functional studies conducted with recombinant material revealed that mRasGRP4 is a cation-dependent, diacylglycerol (DAG)-regulated, guanine nucleotide exchange factor. Immunoelectron microscopic studies revealed that mRasGRP4 resides in either the cytosol or inner leaflet of the plasma membrane of the MC, implying that DAG controls the intracellular movement of this signaling protein in c-kit-stimulated MCs. The mRasGRP4 gene resides on chromosome 7B1 within a site that is prominently linked to baseline airway reactivity in backcrossed C3H/HeJ and A/J mice. A truncated isoform of mRasGRP4 that lacks its DAG-regulatory domain was isolated from C3H/HeJ mouse MCs. Sequence analysis showed that this isoform is the result of defective splicing of the precursor transcript. MCs play a central role in allergic inflammation. The discovery of a novel isoform of mRasGRP4 in hyporesponsive mice suggests that airway reactivity is influenced by RasGRP4-dependent signaling events in pulmonary MCs.

Selective markers for human mast cells are of paramount importance for understanding their role in physiological and pathological processes. A mouse monoclonal antibody (MAb) designated 2C7, raised against in vitro-derived human mast cells, was used in immunoenzymatic analysis of sections from a variety of human organs. Double immunolabeling with 2C7 and tryptase, chymase, Fc epsilon RIalpha, and c-kit was performed on cryostat tissue sections from skin, colon, uterus, breast, stomach, bladder, and lung. MAb 2C7 stained greater than 93% of the tryptase(+) or chymase(+) mast cells in all tissues examined. In addition, the majority of cells stained with the tryptase or chymase also stained for Fc epsilon RIalpha. However, there were a significant number of Fc epsilon RIalpha(1) cells in all tissues studied that were tryptase(-) and/or chymase(-). In contrast, MAb 2C7 in double immunoenzymatic staining co-localized with 93-96% of the Fc epsilon RIalpha(1) cells in all tissues. Analysis for c-kit expression on the different tissues revealed that the majority of tryptase(+) or chymase(+) cells in skin, uterus, bladder, and lung stained with c-kit. However, only approximately 70-78% of tryptase(+) cells in colon and stomach were c-kit(+). These data suggest that MAb 2C7 appears to identify mature mast cells and a population of Fc epsilon RIalpha(1), chymase(-), and tryptase(-) cells in a variety of human tissues.

The only tryptic enzymes identified so far in mouse mast cells (MCs) are three members of the chromosome 17A3.3 family of neutral proteases. Sequence analysis of a cDNA library revealed that BALB/c mouse bone marrow-derived MCs express neuropsin, a member of the chromosome 7B2 family of tryptic kallikreins. Kinetic studies revealed that neuropsin is expressed relatively early in MC development. As assessed immunohistochemically, the MCs residing in numerous connective tissues store neuropsin in their secretory granules. The finding that the neuropsin transcript is maximally expressed in the intestine at the height of a helminth infection indicates that MC-committed progenitors selectively increase their expression of neuropsin as they develop into mature mucosal MCs. This is the first report documenting the expression of neuropsin in an immune cell. Thus, it is now apparent that mouse MCs store at least two distinct families of tryptic-like proteases in their secretory granules.

Mast cells (MCs) are a major source of prostaglandin (PG) D(2) in connective tissues, and the expression of this eicosanoid has been linked to asthma and other inflammatory disorders. While it is known that the surface receptor c-kit controls PGD(2) expression in MCs by regulating the levels of a synthase that converts PGH(2) to PGD(2), the intracellular signaling proteins that act downstream of c-kit in this cyclooxygenase pathway have not been identified. We recently cloned a new cation-dependent, guanine nucleotide exchange factor/phorbol ester receptor (designated RasGRP4) that is required for the efficient expression of granule proteases in the human MC line HMC-1. GeneChip analysis of approximately 12,600 transcripts in RasGRP4(-) and RasGRP4(+) HMC-1 cells revealed a >100-fold difference in the levels of hematopoietic PGD(2) synthase mRNA. No other transcript in the eicosanoid pathway was influenced by RasGRP4 in a comparable manner. As assessed by SDS-PAGE immunoblot analysis, RasGRP4(+) HMC-1 cells contained substantial amounts of PGD(2) synthase protein. RasGRP4(+) MCs also produced approximately 15-fold more PGD(2) than did RasGRP4(-) MCs when both cell populations were activated by calcium ionophore. The induced transcript is therefore translated, and substantial amounts of functional PGD(2) synthase accumulate in RasGRP4(+) MCs. In support of the conclusion that RasGRP4 controls PGD(2) expression in MCs, inhibition of RasGRP4 expression in the rat MC line RBL-2H3 using a siRNA approach resulted in low levels of PGD(2) synthase protein.

We recently cloned a new mast cell (MC) restricted, Ras guanine nucleotide releasing protein (designated mRasGRP4) from IL-3-developed, mouse bone marrow-derived MCs that can activate varied members of the Ras superfamily of small GTP-binding proteins. We now describe the rat ortholog of this MC-specific guanine exchange factor. Using the mRasGRP4 gene and transcript in a homology-based cloning approach, the relevant transcript was isolated and sequenced from the spleen and lungs of Sprague-Dawley rats. Evidence for differential splicing of the rRasGRP4 transcript was obtained in the spleen. The rat basophilic leukemia 1 MC line was found to express rRasGRP4, as well as the MC-committed progenitors residing in the bone marrow and the mature MCs residing in varied tissues of Sprague-Dawley rats. Based on its deduced amino acid sequence, rRasGRP4 is 93% identical to mRasGRP4. rRasGRP4 contains all of the functional domains present in the RasGRP family of guanine nucleotide exchange factors. Like its mouse ortholog, rRasGRP4 is a MC-restricted guanine exchange factor that contains Ca(2+) and phorbol ester/diacylglycerol-binding domains C-terminal of its CDC25-like catalytic domain.

A cDNA was isolated from interleukin 3-developed, mouse bone marrow-derived mast cells (MCs) that contained an insert (designated mRasGRP4) that had not been identified in any species at the gene, mRNA, or protein level. By using a homology-based cloning approach, the approximately 2.6-kb hRasGRP4 transcript was also isolated from the mononuclear progenitors residing in the peripheral blood of normal individuals. This transcript information was then used to locate the RasGRP4 gene in the mouse and human genomes, to deduce its exon/intron organization, and then to identify 10 single nucleotide polymorphisms in the human gene that result in 5 amino acid differences. The >15-kb hRasGRP4 gene consists of 18 exons and resides on a region of chromosome 19q13.1 that had not been sequenced by the Human Genome Project. Human and mouse MCs and their progenitors selectively express RasGRP4, and this new intracellular protein contains all of the domains present in the RasGRP family of guanine nucleotide exchange factors even though it is <50% identical to its closest homolog. Recombinant RasGRP4 can activate H-Ras in a cation-dependent manner. Transfection experiments also suggest that RasGRP4 is a diacylglycerol/phorbol ester receptor. Transcript analysis of an asthma patient, a mastocytosis patient, and the HMC-1 cell line derived from a MC leukemia patient revealed the presence of substantial amounts of non-functional forms of hRasGRP4 due to an inability to remove intron 5 in the precursor transcript. Because only abnormal forms of hRasGRP4 were identified in the HMC-1 cell line, this immature MC progenitor was used to address the function of RasGRP4 in MCs. HMC-1 leukemia cells differentiated and underwent granule maturation when induced to express a normal form of RasGRP4. Thus, RasGRP4 plays an important role in the final stages of MC development.