Requirement of Smad3 for mast cell growth

The involvement of the TGF-beta family in cell growth of bone marrow-derived mast cells (BMMC) cultured with medium containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was examined. Doubling time of BMMC from Smad3-null mice was longer than that from wild-type (WT) mice, and the differences tended to be larger with time of culture. Consistent with the results, uptake and reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was lower in Smad3-deficient BMMC. Cell cycle analyses revealed no apparent differences between WT BMMC and Smad3-deficient BMMC, suggesting that longer doubling time in Smad3-deficient BMMC resulted from increased cell death. TGF-beta and activin A were supplied by PWM-SCM rather than by self-production by BMMC. Blocking the TGF-beta pathway by anti-TGF-beta neutralizing antibody or an inhibitor for the type I receptors for ligands including TGF-beta and activin, SB431542, inhibited MTS uptake and reduction in WT BMMC, whereas anti-activin A antibody and SB431542 tended to inhibit them in Smad3-deficient BMMC. The present results suggest that TGF-beta-induced and Smad3-mediated signaling is essential for maximal cell growth in mast cells, and that the activin pathway may be required for it when mast cell context is modulated by Smad3 depletion.     

Plasma membrane and intracellular expression of globotetraosylceramide (globoside) in mouse bone marrow-derived mast cells

The cellular localization of globotetraosylceramide (globoside), one of the predominant neutral glycosphingolipids of mouse interleukin 3-dependent, bone marrow culture-derived mast cells (BMMC), has been determined by immunologic and chemical methods. Although less than 10% of BMMC expressed globoside on their surface, as assessed by cytofluorographic analysis of the binding of a mouse monoclonal IgM anti-globoside antibody, treatment of BMMC with nonactivating doses of pronase, trypsin, or neuraminidase increased the percentage of BMMC binding anti-globoside antibody by an average of six, three, or sixfold respectively. That most BMMC had globoside on their plasma membrane was confirmed by the surface radiolabeling of globoside with galactose oxidase and sodium borotritide, as detected by autoradiography of thin layer chromatograms of the extracted neutral glycosphingolipids. Thus, BMMC expressed globoside on their plasma membrane, but accessibility of a large probe such as IgM antibody to the glycosphingolipid was impeded by surrounding surface molecules. All BMMC bound anti-globoside antibody intracellularly, as assessed by indirect immunofluorescence staining and fluorescence microscopy on acetone-permeabilized cells, and the pattern of staining suggested that globoside was associated with the secretory granules of BMMC. Immunologic activation of BMMC resulted in a fivefold increase in the surface expression of globoside, as detected by cytofluorographic analysis of the binding of monoclonal anti-globoside antibody. The findings suggest that activation of BMMC causes a reorganization of the plasma membrane such that globoside is more exposed or that activation is accompanied by movement of globoside from internal membranes to the plasma membrane. The increased expression of globoside is a novel marker of the activated mouse BMMC.     

Internalization and degradation of type IIA phospholipase A(2) in mast cells

Whereas exogenous types IB and X secretory phospholipase A(2) (sPLA(2)) elicited prostaglandin D(2) (PGD(2)) production in mouse bone marrow-derived mast cells (BMMC), sPLA(2)-IIA was unable to do so. In search of a mechanism underlying this cellular refractoriness to exogenous sPLA(2)-IIA, we now report that this isozyme is promptly associated with cell surfaces, internalized, and then degraded in BMMC. Adsorption of sPLA(2)-IIA to BMMC was prevented by addition of heparin to the medium. Moreover, a heparin-nonbinding sPLA(2)-IIA mutant did not bind to BMMC. These results indicate that this sPLA(2)-IIA inactivation process depends on its rapid binding to heparan sulfate proteoglycan (HSPG) on BMMC surfaces. Thus, the present observations represent a particular situation in which cell surface HSPG exhibit a negative regulatory effect on cellular function of sPLA(2)-IIA, and argue that HSPG does not always act as a functional adapter for heparin-binding sPLA(2)s in mammalian cells as has been demonstrated before.     

Carboxypeptidase A in mouse mast cells. Identification, characterization, and use as a differentiation marker

By using a conventional spectrophotometric assay with hippuryl-L-phenylalanine as the substrate, 10(6) BALB/c mouse serosal mast cells possessed 1.5 +/- 0.43 U (mean +/- SE, n = 5, range = 0.48 to 2.5) of carboxypeptidase A activity, while T cell factor-dependent, mouse bone marrow-derived mast cells (BMMC) had barely detectable levels of 0.01 +/- 0.001 U/10(6) cells (mean +/- SE, n = 3). In order to characterize the carboxypeptidase A present in the BMMC, a sensitive assay was developed that used angiotensin I as the substrate and reverse phase-high performance liquid chromatography to separate and quantify production of the cleavage product des-leu-angiotensin I. Using this assay, mouse BMMC carboxypeptidase A had a neutral to basic pH optimum and hydrolyzed angiotensin I with a Km of 0.78 mM. The antigen-induced net percent release of carboxypeptidase A from IgE-sensitized BMMC was proportional to that of the secretory granule component beta-hexosaminidase which indicates a secretory granule location for the exopeptidase. As defined by exclusion during Sepharose CL-2B chromatography, carboxypeptidase A was exocytosed as a greater than 1 X 10(7) m.w. complex bound to proteoglycans. Because BMMC cocultured with mouse skin-derived 3T3 fibroblasts are known to undergo an increase in histamine content and biosynthesis of 35S-labeled heparin proteoglycans, carboxypeptidase A activity was measured during BMMC/fibroblast coculture for 0 to 28 days. The carboxypeptidase A activity increased progressively during 28 days of co-culture from 0.004 +/- 0.002 U/10(6) starting BMMC (mean +/- SE, n = 3) to 0.36 +/- 0.10 U/10(6) co-cultured mast cells. These findings indicate that carboxypeptidase A, a neutral protease, is exocytosed from the secretory granules of mouse mast cells bound to proteoglycan and is increased during the in vitro differentiation of mouse BMMC from mucosal-like mast cells to serosal-like mast cells.     

Maturation-related changes in the expression of Fc gamma RII and Fc gamma RIII on mouse mast cells derived in vitro and in vivo.

The expression and function of Fc gamma RII and Fc gamma RIII on three mouse mast cell populations that differ in maturity as assessed by secretory granule constituents were analyzed by cellular and immunochemical approaches. As quantified by flow cytometric analysis of the binding of the rat 2.4G2 anti-Fc gamma RII/III mAb, mouse serosal mast cells (SMC) purified from the peritoneal cavity expressed more receptors per cell than did mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC), which are progenitors of SMC. Coculture of BMMC with mouse 3T3 fibroblasts for 2 wk, which alters the secretory granule composition toward that of SMC, also increased receptor epitope expression to a level equivalent to that of SMC. As assessed by rosette assays with mouse mAb to SRBC, all three mast cell populations bound IgG1, IgG2a, and IgG2b, essentially all binding was inhibited by 2.4G2 antibody, and greater quantities of the antibody were required to block immune adherence by cocultured mast cells and SMC as compared with BMMC. Immunoprecipitation and SDS-PAGE analysis of Fc gamma RII and Fc gamma RIII from BMMC, cocultured mast cells, and SMC that were surface radiolabeled with Na125I revealed predominant native forms of 62, 57, and 56 kDa, respectively, and an additional surface form of 43 kDa in SMC. Removal of N-linked carbohydrate from immunoprecipitates demonstrated that BMMC expressed peptide cores of 38 kDa (Fc gamma RII-1 gene product) and 31 kDa (Fc gamma RII-2 gene product), and barely detectable amounts of a 28-kDa (Fc gamma RIII gene product) core. The expression of all three was increased by coculture with 3T3 fibroblasts, consistent with the increased expression of their common epitope by cytofluorographic analysis. SMC expressed primarily the Fc gamma RII-1 and some Fc gamma RIII gene product. Thus, the three populations of mast cells express different amounts and ratios of the Fc gamma RII and Fc gamma RIII gene products, and maturation of BMMC during coculture with fibroblasts in vitro and in the peritoneal cavity in vivo augments cell-surface expression of the receptors and immune adherence function.     

FES kinase promotes mast cell recruitment to mammary tumors via the stem cell factor/KIT receptor signaling axis

KIT receptor is required for mast cell development, survival, and migration toward its ligand stem cell factor (SCF). Many solid tumors express SCF and this leads to mast cell recruitment to tumors and release of mediators linked to tumor angiogenesis, growth, and metastasis. Here, we investigate whether FES protein-tyrosine kinase, a downstream effector of KIT signaling in mast cells, is required for migration of mast cells toward SCF-expressing mammary tumors. Using a novel agarose drop assay for chemotaxis of bone marrow-derived mast cells (BMMC) toward SCF, we found that defects in chemotaxis of fes-null BMMCs correlated with disorganized microtubule networks in polarized cells. FES displayed partial colocalization with microtubules in polarized BMMCs and has at least two direct microtubule binding sites within its N-terminal F-BAR and SH2 domains. An oligomerization-disrupting mutation within the Fer/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain had no effect on microtubule binding, whereas microtubule binding to the SH2 domain was dependent on the phosphotyrosine-binding pocket. FES involvement in mast cell recruitment to tumors was tested using the AC2M2 mouse mammary carcinoma model. These tumor cells expressed SCF and promoted BMMC recruitment in a KIT- and FES-dependent manner. Engraftment of AC2M2 orthotopic and subcutaneous tumors in control or fes-null mice, revealed a key role for FES in recruitment of mast cells to the tumor periphery. This may contribute to the reduced tumor growth and metastases observed in fes-null mice compared with control mice. Taken together, FES is a potential therapeutic target to limit the progression of tumors with stromal mast cell involvement.     

Tyrosine phosphorylation is required for mast cell activation by Fc epsilon RI cross-linking.

We investigated the possible role of tyrosine phosphorylation in the activation process of mast cells by cross-linking of cell-bound IgE antibodies. Bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE antiDNP mAb and then challenged with multivalent Ag DNP conjugates of human serum albumin. Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that cross-linking of cell-bound IgE antibodies induced a marked increase in tyrosine phosphorylation of several proteins. To obtain direct evidence for activation of protein-tyrosine kinases (PTK), phosphotyrosine-containing proteins in lysates of mast cells were affinity purified, and kinase activity of the immunoprecipitates was assessed by an in vitro kinase assay. The results clearly showed activation of PTK upon cross-linking of Fc epsilon RI. Activation of PTK was not detected by the same assay when the sensitized BMMC were challenged with monovalent DNP-lysine. Treatment of sensitized BMMC with either Ca2+ ionophore or PMA failed to induce the activation of PTK. A representative IgE-independent secretagogue, thrombin, induced histamine release from BMMC but failed to induce activation of PTK. The results excluded the possibility that PTK activation is the consequence of an increase in intracellular Ca2+ or activation of protein kinase C. Addition of genistein, a PTK inhibitor, to sensitized BMMC before Ag challenge inhibited not only Ag-induced PTK activation, but also inositol 1,4,5-trisphosphate production, and histamine release in a similar dose-response relationship. Other PTK inhibitors, such as lavendustin A and tyrphostin RG50864, also inhibited the Ag-induced activation of PTK and histamine release. The results collectively suggest that activation of PTK is an early event upstream of the activation of phospholipase C, and is involved in transduction of IgE-dependent triggering signals to mediator release.     

Rapid remodeling of arachidonate from phosphatidylcholine to phosphatidylethanolamine pools during mast cell activation.

The objective of the present study was to better understand the remodeling of arachidonic acid (AA) in phospholipids of the mouse bone marrow-derived mast cell (BMMC) during Ag and ionophore A23187 activation. Initial studies were designed to understand the movement of AA in phospholipid classes under resting conditions. BMMC pulse labeled with AA incorporated greater than 95% of the label into the major phospholipid classes. Phosphatidylcholine (PC) subclasses, 1-acyl-2-arachidonoyl-(sn-glycero-3-phosphocholine (GPC)) in particular, initially accounted for most of the label incorporated into the cells with phosphatidylinositol/phosphatidylserine (PI/PS) and phosphatidylethanolamine (PE) subclasses containing much smaller quantities. Prolonged incubation of labeled BMMC resulted in a decrease in the radioactivity in PC with a concomitant increase in PE such that 1-alk-1-enyl-2-arachidonoyl-(sn-glycero-3-phosphoethanolamine (GPE)) became the single largest labeled AA pool by 24 h. Further experiments indicated that 24 h was the time required to reach isotopic equilibrium among AA-containing phospholipids of the BMMC. In the next series of experiments, BMMC phospholipids were labeled to different specific activities by either labeling the cells for 0.5 h or for 24 h followed by stimulation. Under isotopic equilibrium conditions (24 h), stimulation resulted in AA release from PE greater than PC much greater than PI/PS with 1-alk-1-enyl-2-arachidonoyl-GPE providing the bulk of AA released from the BMMC. By contrast, cells labeled for 0.5 h released AA from PC much greater than PI/PS, with 1-acyl-2-arachidonoyl-GPC accounting for most of the AA released from BMMC phospholipids. Label associated with PE subclasses under nonequilibrium conditions remained unchanged or slightly increased throughout a 10-min stimulation period. Finally, BMMC were double labeled with [14C]-AA for 24 h and then with [3H]-AA for 0.5 h. Cell stimulation resulted in a decrease in the [3H]/[14C] ratio in PC and PI and an increase in the ratio in PE. The decrease in [3H]/[14C] ratio in PC was mainly in 1-acyl-2-arachidonoyl-GPC, whereas the increase in PE subclasses was primarily in 1-alk-1-enyl-2-arachidonoyl-GPE. The [3H]/[14C] ratio in cellular neutral lipids and in supernatant fluid products were at values between PC and PE subclasses. Taken together, these data suggest that during Ag activation, the release of free arachidonic acid is from predominantly PE subclasses. Concomitant with the release of AA, there is a rapid remodeling of AA from PC subclasses into PE subclasses (1-alk-1-enyl-2-acyl-GPE).     

Chemotaxis of rat mast cells toward adenine nucleotides.

Rat mucosal mast cells express P2 purinoceptors, occupation of which mobilizes cytosolic Ca2+ and activates a potassium conductance. The primary function of this P2 system in mast cell biology remains unknown. Here, we show that extracellular ADP causes morphological changes in rat bone marrow-cultured mast cells (BMMC) typical of those occurring in cells stimulated by chemotaxins, and that the nucleotides ADP, ATP, and UTP are effective chemoattractants for rat BMMC. ADP was also a chemotaxin for murine J774 monocytes. The nucleotide selectivity and pertussis toxin sensitivity of the rat BMMC migratory response suggest the involvement of P2U receptors. Poorly hydrolyzable derivatives of ADP and ATP were effective chemotaxins, obviating a role for adenosine receptors. Buffering of external Ca2+ at 100 nM or reduction of the electrical gradient driving Ca2+ entry (by elevating external K+) blocked ADP-driven chemotaxis, suggesting a role for Ca2+ influx in this process. Anaphylatoxin C5a was a potent chemotaxin (EC50 approximately 0.5 nM) for J774 monocytes, but it was inactive on rat BMMC in the presence or absence of laminin. Ca2+ removal or elevated [K+] had modest effects on C5a-driven chemotaxis of J774 cells, implicating markedly different requirements for Ca2+ signaling in C5a- vs ADP-mediated chemotaxis. This is supported by the observation that depletion of Ca2+ stores with thapsigargin completely blocked migration induced by ADP but not C5a. These findings suggest that adenine nucleotides liberated from parasite-infested tissue could participate in the recruitment of mast cells by intestinal mucosa.     

Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death independent of perforin

Mast cells respond to pathogens and allergens by secreting a vast array of preformed and newly synthesized mediators, including enzymes, vasoactive amines, lipid mediators, cytokines and chemokines, thereby affecting innate and adaptive immune responses and pathogenesis. Here, we present evidence that skin-, but not lung-associated primary mast cells as well as in vitro-differentiated bone marrow-derived mast cells (BMMC) express granzyme (gzm) B, but not gzmA or perforin (perf). GzmB is associated with cytoplasmic granules of BMMC and secreted after Fcepsilon-receptor-mediated activation. BMMC from wild type but not gzmB-deficient mice cause cell death in susceptible adherent target cells, indicating that the perf-independent cytotoxicity of BMMC is executed by gzmB. Furthermore, gzmB induces a disorganization of endothelial cell-cell contacts. The data suggest that activated mast cells contribute, via secreted gzmB, to cell death, increased vascular permeability, leukocyte extravasation and subsequent inflammatory processes in affected tissues.     

Effect of nonhydrolyzable guanosine phosphate on IgE-mediated activation of phospholipase C and histamine release from rodent mast cells

Rat mast cells and bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE mAb, and permeabilized by ATP to introduce guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) and/or guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) into the cells. After ATP-induced lesions were resealed with Mg2+, the cells were challenged by Ag to determine the effect of the nonhydrolyzable guanosine phosphate on Ag-induced hydrolysis of phosphoinositides and histamine release. Introduction of GTP gamma S into permeabilized rat mast cells or BMMC, followed by exposure of the cells to extracellular Ca2+, resulted in histamine release, but failed to induce hydrolysis of phosphoinositides. It was also found that introduction of GTP gamma S into the cells did not synergistically enhance Ag-induced histamine release. Introduction of GDP beta S into sensitized BMMC inhibited the GTP gamma S-dependent, Ca2+-induced histamine release but failed to inhibit Ag-induced histamine release. The results suggest that GTP gamma S-dependent, Ca2+-induced histamine release and Ag-induced histamine release go through independent biochemical pathways. It was also found that introduction of GTP gamma S or GDP beta S into sensitized BMMC neither enhanced nor inhibited Ag-induced formation of inositol phosphates. These results together with previous findings that pretreatment of BMMC with either pertussis toxin or cholera toxin does not affect Ag-induced hydrolysis of phosphoinositides, indicate that a G protein is not involved in the transduction of IgE-mediated triggering signals to phospholipase C in rodent mast cells.     

曲古菌素A对FcεRI介导的肥大细胞活化的影响

曲古菌素A（TSA）是一种组蛋白去乙酰化酶抑制剂,在变态反应等免疫性疾病和抗肿瘤方面显示了潜在的治疗效应。主要观察了TSA对变态反应的效应细胞—肥大细胞活化的影响。TSA可诱导小鼠骨髓来源的肥大细胞（BMMC）凋亡,并抑制表面FcεRI的表达。TSA处理的BMMC经anti-IgE/IgE刺激之后,脱颗粒及分泌TNF-α、IL-6和IL-13生物活性介质的能力显著受到抑制。由此提示,TSA可能通过诱导BMMC凋亡和/或下调FcεRI表达,进而抑制FcεRI介导的BMMC活化,这为TSA进一步在肥大细胞相关的变态反应性疾病中的应用提供了实验数据。     

Pleiotropic role of Rac in mast cell activation revealed by a cell permeable Bordetella dermonecrotic fusion toxin

To activate the GTPase Rac in rat basophilic leukemia (RBL) cells and mouse bone marrow-derived mast cells (BMMC) a TAT fusion toxin of Bordetella dermonecrotic toxin (DNT-TAT) was constructed. The fusion toxin activated Rac1 and RhoA in vitro but only Rac in RBL cells and BMMC. DNT-TAT caused an increase in inositol phosphate formation, calcium mobilization, ERK activation and degranulation of mast cells. All these effects were inhibited by the Rho GTPase-inactivating Clostridium difficile toxin B and Clostridium sordellii lethal toxin. Also the calcium ionophore A23187 caused mast cell activation, including ERK phosphorylation, by processes involving an activation of Rac. The data indicate pleiotropic functions of Rac in mast cell 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.     

Granule-associated serine neutral proteases of the mouse bone marrow-derived mast cell that degrade fibronectin: their increase after sodium butyrate treatment of the cells

Mouse bone marrow-derived mast cells, differentiated in vitro with concanavalin A splenocyte-conditioned medium and sensitized with monoclonal IgE, release neutral serine proteases after activation with specific antigen. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the supernatants from immunologically activated mast cells revealed the presence of four prominent proteins of 27,000, 29,000, 30,000 and 31,000 m.w. When the supernatants and sonicated residual cells from antigen-challenged or nonactivated IgE-sensitized mast cells were incubated with [3H]diisopropylfluorophosphate ([3H]DFP) and the proteins were subjected to SDS-PAGE followed by autoradiography, proteins of 27,000 to 31,000 m.w. were labeled with [3H]DFP. The antigen-dependent release of labeled proteins was accompanied by a corresponding depletion of similarly sized [3H]DFP-labeled proteins from these cell pellets relative to unactivated cells. The SDS gels were also stained with Coomassie Blue and were sectioned to separate the individual proteins for measurement of their incorporated radioactivity; the net percent antigen-dependent release of all four [3H]DFP-labeled proteins ranged from 64 to 68% and was comparable to that of the secretory granule markers, beta-hexosaminidase and histamine. That the [3H]DFP-labeled proteins were derived from the secretory granules of the cells was supported by studies in which mast cells were cultured for 4 days in the presence of 1 mM sodium butyrate. This treatment produced a differential increase in their cellular content of histamine (10-fold), [3H]DFP binding proteins (two- to fourfold), and beta-hexosaminidase (minimally), while the net percent antigen-dependent release of each of these constituents was unchanged. After sensitization and antigen activation, the net percent release of histamine, beta-hexosaminidase, and the four [3H]DFP-labeled proteins was 51, 59, and 53 to 61%, respectively, for sodium butyrate-treated cells, and 53, 60, and 64 to 68%, respectively, for cells not exposed to sodium butyrate. Human plasma fibronectin was used as a substrate to demonstrate that the exocytosed proteins possessed proteolytic activity. As assessed by optical density scanning of stained SDS-PAGE gels of the substrate, the proteases present in the supernatants of antigen-activated cells, but not of sensitized unchallenged cells, rapidly degraded native fibronectin at pH 7.0. This degradation was prevented by pretreatment of the exocytosed proteins from immunologically activated cells for 90 min at 37 degrees C with 2 mM DFP.(ABSTRACT TRUNCATED AT 400 WORDS)