Development of mucosal mast cells after injection of a single connective tissue-type mast cell in the stomach mucosa of genetically mast cell-deficient W/Wv mice

Mast cells may be classified into at least two phenotypically distinct populations: connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC). Mast cells in the peritoneal cavity of mice are typical CTMC, whereas mast cells in the mucosa of the stomach show morphologic characteristics of MMC. We investigated whether CTMC may change to MMC. A single peritoneal mast cell of WBB6F1-+/+ mice was identified under the phase-contrast microscope, picked up with the micromanipulator, and injected into the stomach wall of genetically mast cell-deficient WBB6F1-W/Wv mice. The cells with histochemical and electron microscopical features of MMC developed in the mucosa, and those with histochemical features of CTMC in the muscularis propria. This directly demonstrates that a certain proportion of CTMC may function as a bipotent precursor for both MMC and CTMC.     

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.     

Stem cell factor has histamine releasing activity in rat connective tissue-type mast cells.

Stem cell factor (SCF) was documented to be involved in the growth of mast cells controlled by fibroblasts. We tested the effect of recombinant rat SCF on degranulation from rat peritoneal mast cells (connective tissue-type mast cells: CTMC). SCF induced histamine release (approximately 20% of total histamine content) in a dose-dependent fashion. The release response was relatively rapid and reached a maximum within 5 min. The release showed total dependence on the presence of extracellular phosphatidylserine (PTS). These results reveal that SCF has histamine releasing activity in CTMC.     

Role of gastric mast cells in the regulation of central TRH analog-induced hyperemia in rats

RX 77368 (RX) increases gastric mucosal blood flow by a vagal cholinergic mechanism. The relative roles of mucosal and connective tissue mast cells (MMC and CTMC) were investigated in RX-injected rats. Blood flow and mast cell degranulation were measured after intracisternal RX. RX significantly increased gastric mucosal blood flow, and sequentially degranulated CTMC and MMC. Ketotifen or doxantrazole inhibited the hyperemic response. Ondansetron, RS-039604-90, or famotidine, but not ketanserin or pyrilamine, reduced hyperemia. Mast cells mediate RX-induced gastric hyperemia via 5-HT3, 5-HT4, and H2 receptors; initial increase depends upon CTMC whereas MMC contributes to the later response.     

Nerve growth factor modifies the expression of inflammatory cytokines by mast cells via a prostanoid-dependent mechanism

Nerve growth factor (NGF) is well recognized to have a number of potent effects on mast cells, including increasing mast cell numbers in vivo and inducing mast cell degranulation in vitro. More recently, NGF has been demonstrated to induce PGD2 production by mast cells through the induction of mast cell cyclooxygenase expression. We have observed that NGF at doses as low as 10 ng/ml will induce IL-6 production and inhibit TNF-alpha release from rat peritoneal mast cells in the presence of lysophosphatidylserine as a cofactor. NGF synergizes with LPS treatment of peritoneal mast cells (PMC) for the induction of IL-6. Examination of the mechanism of this phenomenon has revealed that NGF can induce both rat PMC and mouse bone marrow-derived cultured mast cells to produce substantial levels of PGE2. This response is maximal at later time points 18-24 h after NGF activation. The ability of NGF to induce PGE2 is not dependent on mast cell degranulation. Other stimuli capable of inducing IL-6, such as LPS, do not induce production of this prostanoid. Inhibition of cyclooxygenase activity by PMC using either flurbiprofen or indomethacin inhibited both the NGF-induced PGE2 synthesis and the NGF-induced alterations in TNF-alpha and IL-6 production. These results suggest a role for mast cell-derived prostanoids in the regulation of local inflammatory responses and neuronal degeneration after tissue injury involving induction of NGF production.     

Establishment and characterization of hybrid rat mast cells

Rat peritoneal mast cells (RPMC) and rat basophilic leukemia (RBL) cells are representative of connective tissue-type (CTMC) and mucosal-type (MMC) mast cells, respectively. Using polyethylene glycol, we have fused RPMC with 6-thioguanine resistant, HAT (hypoxanthine, aminopterin, thymidine) sensitive RBL-CA10.7 or RBL-CK2 cells, yielding several hybrid rat mast cell lines (HRMC). The hybridomas exhibited different size and cytoplasmic granularity when compared with parental cell lines. Analysis of both high (Fc epsilon RI) and low affinity (Fc epsilon RL) receptors for IgE revealed that the hybrid lines had more variable receptor patterns than the parent lines. Three hybridoma lines were chosen for further study. Differential histochemical staining with alcian blue and safranin O dyes indicated the hybrids to be predominantly of the MMC type: however, a few cells of one of these uncloned hybridomas were found to be of the CTMC type. Attempts to isolate the CTMC hybridomas yielded one culture which was predominantly of the CTMC phenotype and in a number of other cultures, cells were found expressing simultaneously both the CTMC and the MMC phenotype. After 3 weeks in culture, however, all hybridomas, including those which were cloned further, expressed only the MMC histochemical phenotype. This was found to correlate with the presence of rat mast cell protease II (RMCPII) and the absence of RMCPI in all hybridomas, as detected by Western blot analysis. In addition, the histamine content of all cells was significantly lower than that of the parent RPMC. Most hybrid mast cells expressed both Fc epsilon RI and Fc epsilon RL which in some cases exhibited significant variations in the Mr. These results indicate that somatic cell hybrids expressing the MMC and CTMC phenotype can be produced by the fusion of RBL and RPMC. The CTMC phenotype, however, is unstable, and possible reasons for this are discussed.     

Genetically mast cell-deficient W/Wv mice as a tool for studies of differentiation and function of mast cells

Genetically mast cell-deficient W/Wv mice are useful for the analysis of mast cell biology, especially as recipients of bone marrow cells and skin pieces. Inasmuch as suspension and clonal cultures of mast cells have been developed, we combined these in vivo and in vitro systems. Suspension-cultured mast cells had morphological and biochemical characteristics similar to those of mucosal mast cells (MMC). However, i.p. injection of such cultured mast cells gave rise to development of cells with characteristics similar to those of connective tissue mast cells (CTMC). When peritoneal cells of normal +/+ mice were cultured in methylcellulose, pure mast cell colonies appeared. Cells from individual mast cell colonies were divided and injected into the skin and stomach wall of W/Wv mice; CTMC developed in the skin and MMC in the stomach mucosa. This indicates the presence of a common precursor for CTMC and MMC. Morphology of such bipotent mast cell precursors was studied by using micromanipulation. About 4% of morphologically identifiable peritoneal mast cells may function as the bipotent precursors. Although W/Wv mice showed a defect in resistance against ixodid ticks, injection of suspension-cultured mast cells normalized the defect. The four examples mentioned above indicate that combinations of in vivo and in vitro systems increase the usefulness of W/Wv mice.     

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.     

Stochastic model for mast cell proliferation in culture of murine peritoneal cells

We recently identified two types of mast cell colonies derived from murine peritoneal cells: type 1 and type 2. Type 1 mast cell colonies consisted of berberine sulfate(+)- safranin(+) connective tissue-type mast cells (CTMC) and were derived from mature CTMC in the heaviest fraction obtained by Percoll density gradient centrifugation. In contrast, type 2 mast cell colonies consisted of alcian blue(+)- berberine sulfate(-)- safranin(-) mucosal mast cells (MMC) and were derived from immature progenitors in low density fractions. We replated a total of 60 type 1 and 60 type 2 mast cell colonies and examined their capability for producing secondary colonies. Although all of the primary colonies yielded secondary colonies, the replating efficiencies of individual colonies varied over a wide range. Cumulative distributions of secondary colonies from both type 1 and type 2 primary colonies could be fitted well by gamma distributions obtained by computer simulation. These findings are in agreement with the stochastic model for CTMC- and MMC proliferation. Cytological analyses of secondary colonies from primary type 1 colonies revealed heterogeneous distributions of alcian blue(+)- safranin(-)- berberine sulfate(-) mast cells, suggesting that transdifferentiation from mature CTMC to safranin(-)- berberine sulfate(-) mast cells is also governed by stochastic mechanisms.     

Histochemical heterogeneity of dermal mast cells in athymic and normal rats

Summary Mucosal mast cells (MMC) and connective tissue mast cells (CTMC) of the rat contain different proteoglycans, which can be distinguished using histochemical methods. The chondroitin sulphate proteoglycan of the MMC, unlike the heparin of the CTMC, does not show fluorescent berberine binding, is susceptible to aldehyde fixatives and stains preferentially with Alcian Blue in a staining sequence with Safranin. The majority of the dermal mast cells are typical CTMC and are located in the deep part of the dermis. Subepidermal mast cells are comparatively few in normal rats but numerous in athymic rats and mice. These cells differ from other dermal mast cells in that they stain preferentially with Alcian Blue and they appear to contain little histamine. We examined some of the histochemical properties of the skin mast cells of female PVG-rnu/rnu rats and their heterozygous littermates aged from 5 to 29 weeks. The thiazine dye-binding of the subepidermal mast cells was partially blocked by formaldehyde fixation and only about half of them showed a weakly fluorescent berberine binding. The critical electrolyte concentration of the Alcian Blue staining of the subepidermal mast cells was between that of CTMC and MMC. Deaminative cleavage with nitrous acid abolished the staining of all skin mast cells, while that of the MMC was unaffected. There were no statistically significant differences in the staining patterns of the dermal mast cells between different ages or groups of rat. These results indicate that the subepidermal mast cells contain a heparin proteoglycan which is, however, different from that of the typical CTMC of other sites. They thus appear to represent a second example of a mast cell within a defined anatomical location exhibiting a distinct proteoglycan expression.     

Somatostatin inhibits intestinal mucosal mast cell degranulation in normal conditions and during mast cell hyperplasia

Several studies demonstrate that intestinal mucosal mast cells (IMMC) are modulated by nervous reflexes as well as by intraluminal content. We recently demonstrated that peptones, such as ovalbumin hydrolysate (OVH), induce the release of rat mast cell protease II (RMCP II), indicating IMMC degranulation. The response is due to complex neuroendocrine reflexes. Somatostatin (SS) and its analogues have been used as potential treatments for inflammation in other body systems with contradictory results. The aim of this study was to evaluate if somatostatin could contribute to the reduction of intestinal mucosal mast cell degranulation. Anesthetized rats were prepared for duodenal perfusion and mast cell activation was measured by analysis of RMCP II concentration in the duodenal perfusate. Somatostatin significantly decreased RMCP II concentration in both nonstimulated conditions and after ovalbumin hydrolysate perfusion. However, when somatostatin was given previously to OVH, the peptone still induced a slight increase of RMCP II. Similar effects were observed in animals previously treated with capsaicin. These protocols were repeated in animals infected with Trichinella spiralis, which induces mucosal mast cell hyperplasia. In these cases, somatostatin blocked the effect of OVH, thus, preventing an increase in RMCP II concentration. Fresh frozen tissue sections from the duodenum were processed in an attempt to demonstrate the presence of SS receptors in mast cells using immunofluorescence and Fluo-peptide labeling techniques. Confocal images from duodenum specimens demonstrate the existence of SS receptors in positive cells for RMCP II. Taken together, these results indicate that somatostatin diminishes mast cell activity and in consequence could prevent the intestinal responses to mast cell hyperplasia.     

Rapid separation of rat peritoneal mast cells with Percoll

Rat peritoneal mast cells were separated by using density gradients of PVP-coated silica particles (Percoll). Mast cells were either isolated on preformed Percoll gradients or cell separation was made simultaneously with the gradient formation. Both procedures resulted in mast cell suspensions of 95 to 99 per cent purity. As tested by Ruthenium red staining and electron microscopy, the isolated mast cells showed a very good preservation of cell structure and reacted easily to the degranulating agent Compound 48/80. Practically all mast cells could be recovered from the peritoneal cell suspension. Percoll was found to be superior to earlier isolation procedures by giving a practically pure and intact mast cell suspension and by avoiding cell aggregation.     

Generation of leukotriene C4, leukotriene B4, and prostaglandin D2 by immunologically activated rat intestinal mucosa mast cells

Mucosal mast cells (MMC) were isolated from the intestine of Nippostrongylus brasiliensis-infected rats and then activated with Ag or with anti-IgE in order to assess their metabolism of arachidonic acid to leukotriene (LT) C4, LTB4, and prostaglandin D2 (PGD2). After challenge of MMC preparations of 19 +/- 1% purity with five worm equivalents of N. brasiliensis Ag, the net formation of immunoreactive equivalents of LTC4, LTB4, and PGD2 was 58 +/- 8.3, 22 +/- 4.5, and 22 +/- 3.4 ng/10(6) mast cells, respectively (mean +/- SE, n = 7). When MMC preparations of 56 +/- 9% purity were activated by Ag, the net generation of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) MMC was 107 +/- 15, 17 +/- 5.4, and 35 +/- 18 ng, respectively. These data indicate that the three eicosanoids originated from the MMC rather than from a contaminating cell. Analysis by reverse phase HPLC of the C-6 sulfidopeptide leukotrienes present in the supernatants of the activated MMC preparations of lower purity revealed LTC4, LTD4, and LTE4. In a higher purity MMC preparation only LTC4 was present, suggesting that other cell types in the mucosa are able to metabolize LTC4 to LTD4 and LTE4. The release of histamine and the generation of eicosanoids from intestinal MMC and from peritoneal cavity-derived connective tissue-type mast cells (CTMC) isolated from the same N. brasiliensis-infected rats were compared. When challenged with anti-IgE, these MMC released 165 +/- 41 ng of histamine/10(6) mast cells, and generated 29 +/- 3.6, 12 +/- 4.2, and 4.7 +/- 1.0 ng (mean +/- SE, n = 3) of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) mast cells, respectively. In contrast, CTMC isolated from the same animals and activated with the same dose of anti-IgE released approximately 35 times more histamine (5700 +/- 650 ng/10(6) CTMC), generated 7.5 +/- 2.3 ng of PGD2/10(6) mast cells, and failed to release LTC4 or LTB4. These studies establish, that upon immunologic activation, rat MMC and CTMC differ in their quantitative release of histamine and in their metabolism of arachidonic acid to LTC4 and LTB4.     

A comparison of reactive oxygen species generation by rat peritoneal macrophages and mast cells using the highly sensitive real-time chemiluminescent probe pholasin: inhibition of antigen-induced mast cell degranulation by macrophage-derived hydrogen peroxide

Mast cells and macrophages live in close proximity in vivo and reciprocally regulate one another's function in various ways. Although activated macrophages possess a powerful reactive oxygen species (ROS) generating system, there is conflicting evidence regarding whether mast cells can produce ROS. We used the highly sensitive real-time chemiluminescent probe Pholasin to examine ROS release by peritoneal macrophages and mast cells isolated from OVA-sensitized rats. Macrophages stimulated with PMA (0.8 microM) or ionomycin (1 microM), but not OVA (1 microg/ml), released high-level ROS, levels of which peaked after 3-7 min and declined to baseline levels within 1 h. Superoxide was identified as the major ROS species induced by PMA but not by ionomycin. In contrast, purified mast cells stimulated with PMA released low-level ROS, which was entirely due to the contaminating (2%) macrophages, and did not release any detectable ROS in response to ionomycin or OVA at concentrations that induced degranulation. Stimulation of mixed cell populations with PMA to induce macrophage ROS release led to 50% inhibition of serotonin release from mast cells stimulated 5 min later with OVA. The PMA-induced inhibitory factor was identified as hydrogen peroxide. In conclusion, activated rat peritoneal macrophages but not mast cells produce ROS, and macrophage-derived hydrogen peroxide inhibits mast cell degranulation. The latter could be an important mechanism whereby phagocytic cells regulate mast cell activation and promote resolution of IgE-mediated inflammation.     

Ileal mucosal mast cell, eosinophil, and goblet cell populations during Hymenolepis diminuta infection of the rat.

The population dynamics in the enteric connective tissues of eosinophils, mucosal mast cells (MMC), and in the mucosal epithelium of goblet cells were examined morphometrically in fixed ileal tissue of outbred Sprague Dawley rats during the first 32 days of infection with the tapeworm Hymenolepis diminuta. MMC and eosinophils were present in the lamina propria and submucosa; however, only eosinophils were also present in the muscularis externa. Eosinophilic infiltrate was first observed in the lamina propria at 15 days postinfection (dpi) and the numbers of eosinophils remained elevated through 32 dpi. Initial mucosal mastocytosis was detected on 6 dpi and MMC numbers continued to rise over the study period without reaching a plateau. Goblet cell hyperplasia occurred only at 32 dpi. In contrast to some intestinal nematode infections where these same 3 cell types are associated with the host's expulsion responses, H. diminuta is not lost by a rapid host response in the outbred Sprague Dawley rat strain used in these experiments. We suggest that either the induction of hyperplasia of these host effector cells in ileum tissue during H. diminuta infection is not capable of triggering parasite rejection mechanisms, or the function of the induced hyperplasia is necessary for some as yet unassociated physiological or tissue architecture change in the host's intestine.     

Mast cell heterogeneity: effects of neuroenteric peptides on histamine release

Recent reports suggesting that the actions of certain neuroenteric peptides may be mediated in part by the secretion of histamine and other mast cell contents could have important implications for gastrointestinal motility and secretion. However, evidence for a mast cell-hormonal interaction is based on studies using peritoneal or cutaneous mast cells. Because intestinal mucosal mast cells (MMC) differ functionally from peritoneal mast cells (PMC), we compared the effects of several neurotransmitters and intestinal hormones on histamine secretion from two mast cell types in the rat. MMC hyperplasia was induced in rats by infection with the nematode Nippostrongylus brasiliensis, and MMC were isolated from the small intestine by collagenase digestion. Substance P, somatostatin, vasoactive intestinal polypeptide (VIP), neurotensin, and bradykinin had a potent secretagogue effect on (10(-7) to 10(-4)M) PMC which was temperature-, energy-, and calcium-dependent. In contrast to PMC, MMC released significant amounts of histamine only when challenged with substance P. Acetylcholine, bombesin, motilin, and pentagastrin had no secretory effect on either PMC or MMC. The differences between PMC and MMC in responsiveness to peptides could not be attributed to the MMC isolation procedure because PMC treated similarly or mixed with MMC suspensions retained their responsiveness to these stimuli. Our results extend the concept of neurocrine control of mast cell function, but indicate that mast cells from different sites have distinct profiles of responsiveness to regulatory peptides.     

Identification of NDRG1 as an early inducible gene during in vitro maturation of cultured mast cells

Coculture of mouse bone marrow-derived mast cells (BMMC) with fibroblasts in the presence of stem cell factor (SCF) facilitates morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype. By means of cDNA subtraction, we identified several inducible genes during this mast cell maturation process. Of approximately 100 sequenced clones induced, nearly 50% were chromosome 14-associated serine proteases. Approximately 14% encoded NDRG1, a 43-kDa cytosolic protein that has been implicated in cell differentiation. NDRG1 was distributed in the cytosol of cultured mast cells and CTMC in rat skin. Overexpression of NDRG1 in RBL-2H3 cells resulted in enhanced degranulation in response to various stimuli. Thus, NDRG1 may be a mast cell maturation-associated inducible protein that allows the cells to be susceptible to extracellular stimuli leading to degranulation. Additionally, several unique maturation-associated inducible genes were identified, molecular and functional characterization of which will provide new insights into mast cell biology.     

Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family

Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and from a mouse Kirsten sarcoma virus-immortalized mast cell line (KiSV-MC), and a cDNA that encodes this exopeptidase has been cloned from a KiSV-MC-derived cDNA library. KiSV-MC-derived mast cell carboxypeptidase A was purified with a potato-derived carboxypeptidase-inhibitor affinity column and was found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a Mr 36,000 protein. Secretory granule proteins from KiSV-MC and from mouse peritoneal CTMC were then resolved by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences were obtained for the prominent Mr 36,000 protein present in the granules of both cell types. Based on the amino-terminal sequence, an oligonucleotide probe was synthesized and used to isolate a 1,470-base pair cDNA that encodes this mouse exopeptidase. The deduced amino acid sequence revealed that, after cleavage of a 15-amino acid hydrophobic signal peptide and a 94-amino acid activation peptide from a 417-amino acid preproenzyme, the mature mast cell carboxypeptidase A protein core has a predicted Mr of 35,780 and a high positive charge [Lys + Arg) - (Asp + Glu) = 17) at neutral pH. Although critical zinc-binding amino acids (His67, Glu70, His195), substrate-binding amino acids (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), and cysteine residues that participate in intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) of pancreatic carboxypeptidases were also present in mast cell carboxypeptidase A, the overall amino acid sequence identities for mouse mast cell carboxypeptidase A relative to rat pancreatic carboxypeptidases A1, A2, and B were only 43, 41, and 53%, respectively. RNA and DNA blot analyses revealed that mouse peritoneal CTMC, KiSV-MC, and bone marrow-derived mast cells all express a prominent 1.5-kilobase mast cell carboxypeptidase A mRNA which is transcribed from a single gene. We conclude that mouse mast cell carboxypeptidase A is a prominent secretory granule enzyme of mast cells of the CTMC subclass and represents a novel addition to the carboxypeptidase gene family.     

Hepatic mast cells in rats infected with Taenia taeniaeformis

Mast cells appeared in the liver around metacestodes of Taenia taeniaeformis by 13 days after infection (DAI) of rats. The cells often occurred in clusters. The population increased until 28 DAI, then gradually declined. These hepatic mast cells (HMC) were compared to intestinal mucosal mast cells (MMC) and connective tissue mast cells (CTMC) histochemically, morphologically and in their response in vivo to Compound (built|48/80) and dexamethasone. Hepatic mast cells were similar to MMC in that they stained strongly blue with Astra blue at pH 1·0, could not be demonstrated with 0·005% toluidine blue, disappeared after treatment with dexamethasone, and were unaffected by 48/80. Immunoglobulin-containing cells in the liver were characterized by immunofluorescence. Immunoglobulin E-positive, and to a lesser extent IgG_2a-, and IgG_2c-positive cells surround the parasites in increasing numbers until 28 DAI, then declined. Many IgE-positive cells were HMC, and the IgE was frequently located intracytoplasmically. These cells were clearly distinguishable from eosinophils which stained characteristically with Giemsa and did not react with the anti-IgE probe. The results suggest that mast cell progenitors may be induced to localize and proliferate at the host-parasite interface in the Taenia-infected livers, giving rise to a cell population comparable to the MMC often seen at parasitized mucosal surfaces.     

Impaired mast cell maturation and degranulation and attenuated allergic responses in Ndrg1-deficient mice

We have previously reported that N-myc downstream regulated gene-1 (NDRG1) is an early inducible protein during the maturation of mouse bone marrow-derived mast cells (BMMCs) toward a connective tissue mast cell-like phenotype. To clarify the function of NDRG1 in mast cells and allergic responses, we herein analyzed mast cell-associated phenotypes of mice lacking the Ndrg1 gene. Allergic responses including IgE-mediated passive systemic and cutaneous anaphylactic reactions were markedly attenuated in Ndrg1-deficient mice as compared with those in wild-type mice. In Ndrg1-deficient mice, dermal and peritoneal mast cells were decreased in number and morphologically abnormal with impaired degranulating ability. Ex vivo, Ndrg1-deficient BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of stem cell factor, a condition that facilitates the maturation of BMMCs toward a CTMC-like phenotype, displayed less exocytosis than replicate wild-type cells after the cross-linking of FcepsilonRI or stimulation with compound 48/80, even though the exocytotic response of IL-3-maintained, immature BMMCs from both genotypes was comparable. Unlike degranulation, the production of leukotriene and cytokines by cocultured BMMCs was unaffected by NDRG1 deficiency. Taken together, the altered phenotypes of Ndrg1-deficient mast cells both in vivo and ex vivo suggest that NDRG1 has roles in the terminal maturation and effector function (degranulation) of mast cells.