Conservation of signal transduction mechanisms via the human Fc epsilon RI alpha after transfection into a rat mast cell line, RBL 2H3.

The high affinity receptor for IgE (Fc epsilon RI) is present on mast cells and basophils, and the aggregation of IgE-occupied receptors by Ag is responsible for the release of allergic mediators. The Fc epsilon RI is composed of at least three different subunits, alpha, beta, and gamma, with the alpha subunit binding IgE. The series of biochemical events linking receptor aggregation to the release of mediators has not been fully delineated. As a step towards understanding these processes, and for the development of functional cell lines, we have transfected the human Fc epsilon RI alpha subunit into the rat mast cell line RBL 2H3. These human Fc epsilon RI alpha-transfected cell lines have been characterized with respect to the association of the human alpha subunit with endogenous rat beta and gamma subunits and the ability of aggregated Fc epsilon RI alpha subunits to mediate a variety of biochemical events. The signal transduction events monitored include phosphoinositide hydrolysis, Ca2+ mobilization, tyrosine phosphorylation, histamine release, and arachidonic acid metabolism. In all cases, the events mediated by aggregating human Fc epsilon RI alpha subunits were indistinguishable from those produced via the rat Fc epsilon RI alpha. These results demonstrate that the human Fc epsilon RI alpha subunit can functionally substitute for the rat Fc epsilon RI alpha subunit during signal transduction. The availability of this cell line will provide a means of evaluating potential Fc epsilon RI antagonists.     

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.     

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.     

The effectiveness of different rat IgG subclasses as IgE-blocking antibodies in the rat basophil leukaemia cell model

The degranulation of mast cells in an allergic response is initiated by the aggregation of high-affinity IgE receptors (Fc epsilon RI) by IgE and antigen. Recently it has been shown that such degranulation can be inhibited by cross-linking Fc epsilon RI and low-affinity IgG receptors (Fc gamma RII) which are also expressed by mast cells. The ability of various monoclonal antibodies to block the degranulation of rat basophil leukaemia (RBL) cells sensitized with IgE antidinitrophenyl (DNP) antibodies has been investigated. Sensitized cells were challenged with immune complexes formed using varying concentrations of antigen, and of both high- and low-valency antigen. It is reported here that rat IgG1 antibodies, which are associated in the rat with a Th1-type response, act as highly effective blocking antibodies over a wide concentration range. Rat IgG2a antibodies, which are associated with a Th2-type response, were able only to inhibit degranulation when immune complexes were formed with very low concentrations of high-valency antigen (DNP32-HSA). Under these conditions, some inhibitory activity was seen with high-affinity murine IgA anti-DNP but not with low-affinity rat IgG2b anti-DNP antibody-containing immune complexes. In addition to this inhibitory activity, IgG2a antibodies were shown to be capable of inducing degranulation of cells via unoccupied Fc epsilon RI. These results demonstrate that blocking activity may arise via both inhibitory receptors and by masking of antigen.     

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen-induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied.     

The murine lymphocyte receptor for IgE. III. Use of chemical cross-linking reagents to further characterize the B lymphocyte Fc epsilon receptor

Cross-linking reagents were used to further characterize the murine B cell receptor for the Fc portion of IgE (Fc epsilon R) and compare this receptor to the well-characterized high-affinity Fc epsilon R on rat basophilic leukemia (RBL) cells. The disulfide cleavable and noncleavable reagents 3,3'-dithiobis(sulfosuccinimidyl) propionate (DTSSP) and bis(sulfosuccinimidyl) suberate (BS3) were used. With these reagents, efficient cross-linking of the alpha component of the high-affinity RBL Fc epsilon R to the membrane-buried beta and gamma components occurred only if the membrane was solubilized before the cross-linking reaction. In studies with purified murine B cells, IgE could be cross-linked to the Fc epsilon R on intact cells with either DTSSP or BS3. Under the same conditions, up to 10% of the B cell surface immunoglobulin (sIg) (both IgM and IgD) was also found to cross-link to a portion of the IgE/Fc epsilon R complex, suggesting that on the intact murine B cell the Fc epsilon R is frequently in close association with sIg. The B cell Fc epsilon R was also examined for the presence of receptor-associated proteins. Under conditions where the high-affinity RBL Fc epsilon R was substantially cross-linked to the alpha, beta, gamma complex, no evidence was seen for similar cross-linking of the B cell Fc epsilon R. Cross-linking experiments on affinity-purified Fc epsilon R preparations also gave no evidence for receptor-associated proteins with the B cell Fc epsilon R, although evidence for receptor-receptor association was seen. Thus, these data further support the concept that there may be little relationship between the high-affinity mast cell/basophil Fc epsilon R and the low-affinity lymphocyte Fc epsilon R.     

The role of mast cell degranulation products in mast cell hyperplasia. I. Mechanism of action of nerve growth factor

A variety of mast cell degranulating agents have previously been shown to induce mast cell hyperplasia in adult rats. In neonates 2.5 S nerve growth factor (NGF) induces a hyperplasia of both mucosal and connective tissue mast cells (MMC and CTMC). We have examined the role of the potent mast cell degranulating properties of NGF on its ability to induce mast cell hyperplasia. Administration of NGF in combination with the mast cell stabilizing agent disodium cromoglycate was found to abrogate the CTMC hyperplasia induced by NGF alone. Treatment of neonatal rats with the alternate degranulating agent compound 48/80 was found to induce a limited CTMC but not a MMC hyperplasia. A supernatant obtained by degranulating purified adult rat peritoneal mast cells with anti-IgE was found to induce hyperplasia of the CTMC population similar to that observed with NGF administration. However, this degranulation product supernatant only induced a limited MMC hyperplasia as judged by RMCP II content of the tissues. These results suggest that NGF has dual action inducing mast cell hyperplasia; CTMC hyperplasia being dependent on the ability of NGF to degranulate mast cells. MMC hyperplasia induced by NGF is independent of CTMC degranulation. Degranulation products from peritoneal mast cells act to increase both MMC and CTMC populations in the neonate. These data suggest that the CTMC population may be regulated by an autocrine positive feedback mechanism in vivo.     

Molecular perspective of antigen-mediated mast cell signaling

Antigen-mediated cross-linking of the high affinity receptor for IgE (Fc epsilon RI), in the plasma membrane of mast cells, is the first step in the allergic immune response. This event triggers the phosphorylation of specific tyrosines in the cytoplasmic segments of the beta and gamma subunits of Fc epsilon RI by the Src tyrosine kinase Lyn, which is anchored to the inner leaflet of the plasma membrane. Lyn-induced phosphorylation of Fc epsilon RI occurs in a cholesterol-dependent manner, leading to the hypothesis that cholesterol-rich domains, or "lipid rafts," may act as functional platforms for IgE receptor signaling. Testing this hypothesis under physiological conditions remains challenging because of the notion that these functional domains are likely transient and much smaller than the diffraction limit of optical microscopy. Here we use ultrafast fluorescence dynamics to investigate the correlation between nanostructural changes in the plasma membrane (labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (diI-C18)) and IgE-Fc epsilon RI cross-linking in adherent RBL mast cells stimulated with multivalent antigen. Time-dependent two-photon fluorescence lifetime imaging microscopy of diI-C18 shows changes in lifetime that agree with the kinetics of stimulated tyrosine phosphorylation of Fc epsilon RI, the first identifiable biochemical step of the allergic response, under the same conditions. In addition, two-photon fluorescence lifetime imaging microscopy of Alexa Fluor 488-labeled IgE indicates that F?rster resonance energy transfer occurs with diI-C18 in the plasma membrane. Our live cell studies provide direct evidence for the association of IgE-Fc epsilon RI with specialized cholesterol-rich domains within approximately 4-nm proximity and with an energy transfer efficiency of 0.22 +/- 0.01 at maximal association during IgE receptor signaling.     

A comparison of mediators released or generated by IFN-gamma-treated human mast cells following aggregation of Fc gamma RI or Fc epsilon RI

The high affinity receptor for IgG (Fc gamma RI, CD64) is expressed on human mast cells, where it is up-regulated by IFN-gamma and, thus, may allow mast cells to be recruited through IgG-dependent mechanisms in IFN-gamma-rich tissue inflammation. However, the mediators produced by human mast cells after aggregation of Fc gamma RI are incompletely described, and it is unknown whether these mediators are distinct from those produced after activation of human mast cells via Fc epsilon RI. Thus, we investigated the release of histamine and arachidonic acid metabolites and examined the chemokine and cytokine mRNA profiles of IFN-gamma-treated cultured human mast cells after Fc gamma RI or Fc epsilon RI aggregation. Aggregation of Fc gamma RI resulted in histamine release and PGD(2) and LTC(4) generation. These responses were qualitatively indistinguishable from responses stimulated via Fc epsilon RI. Aggregation of Fc epsilon RI or Fc gamma RI led to an induction or accumulation of 22 cytokine and chemokine mRNAs. Among them, seven cytokines (TNF-alpha, IL-1beta, IL-5, IL-6, IL-13, IL-1R antagonist, and GM-CSF) were significantly up-regulated via aggregation of Fc gamma RI compared with Fc epsilon RI. TNF-alpha mRNA data were confirmed by quantitative RT-PCR and ELISA. Furthermore, we confirmed histamine and TNF-alpha data using IFN-gamma-treated purified human lung mast cells. Thus, aggregation of Fc gamma RI on mast cells led to up-regulation and/or release of three important classes of mediators: biogenic amines, lipid mediators, and cytokines. Some cytokines, such as TNF-alpha, were released and generated to a greater degree after Fc gamma RI aggregation, suggesting that selected biologic responses of mast cells may be preferentially generated through Fc gamma RI in an IFN-gamma-rich environment.     

新生大鼠雌激素注射后睾丸肥大细胞的变化

新生大鼠注射雌二醇后,睾丸肥大细胞于第30天可见到,细胞数量随年龄增长而增多,生后4－6个月,睾丸网附近仍可见大量肥大细胞。睾丸内的肥大细胞比皮肤内的结缔组织肥大细胞（CTMC）小而与小肠粘膜的粘膜肥大细胞（MMC）相近,AB－S染色后基本着蓝色,硫酸小檗碱荧光染色后呈现中等强度黄色荧光,结果提示,新生大鼠雌激素注射后睾丸内肥大细胞的增多可能与免疫过程有关,睾丸内肥大细胞与CTMC和MMC皆有所不同。     

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response.     

Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.     

Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases

Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.     

A novel cell-permeable cromoglycate derivative inhibits type I Fc epsilon receptor mediated Ca2+ influx and mediator secretion in rat mucosal mast cells.

Type I Fc epsilon receptor (Fc epsilon RI) mediated Ca2+ uptake and secretion of rat serosal mast cells have been shown to be inhibited by disodium 1,3-bis [(2'-carboxylatochromon-5'-yl) oxy]-2-hydroxypropane (disodium cromoglycate, DSCG), which is widely employed in the treatment of allergic asthma [Foreman et al. (1977) Br. J. Pharmacol. 59, 473P-474P; Cox (1967) Nature (London) 216, 1328-1329]. This drug was also found to modify the protein phosphorylation pattern of these mast cells. [Theoharides et al. (1980) Science 207, 80-82]. We have isolated by affinity chromatography on a water-insoluble cromoglycate-carrying matrix a cytosolic enzyme recently identified as a nucleoside 5'-diphosphate kinase. In order to examine a possible intracellular activity of the drug, a cell-permeant cromoglycate derivative, 1,3-bis [[2'-[[(acetoxymethyl)oxy]carbonyl]chromon-5'- yl]oxy]-2-hydroxypropane [bis(acetoxymethyl) cromoglycate, CG/AM], has been synthesized, and its uptake and effect on the Fc epsilon RI-mediated exocytosis of mast cells was investigated. A tritium-labeled CG/AM derivative, used as radioactive tracer, was found to permeate mucosal mast cells of the rat line RBL-2H3 and accumulate intracellularly up to 40-fold its extracellular concentration following hydrolysis by cytoplasmic hydrolases. A CG/AM dose dependent inhibition of the Fc epsilon RI-induced mediator secretion was observed in RBL-2H3 cells loaded with this compound (I50 approximately 40 microM extracellular CG/AM). A similar dose-dependent inhibition was observed for both the Fc epsilon RI-mediated transient rise in the concentration of cytosolic free Ca2+ ions [( Ca2+]i) and the net Ca2+ influx, as monitored by the fluorescent indicator Quin2 and the radioactive tracer 45Ca2+, respectively. These results clearly show that cell-permeant cromoglycate inhibits the Fc epsilon RI-mediated Ca2+ influx into the cell and further underscore the dominant role of this process in the coupling of stimulus to secretion in RBL cells. Furthermore, with the identification of nucleoside 5'-diphosphate kinase as a potential intracellular target for CG activity, distinct mechanisms of action may be inferred for cell-permeant and nonpermeant forms of CG.     

Compartmentalization of the Type I Fc epsilon receptor and MAFA on mast cell membranes

The Mast cell Function-associated Antigen (MAFA) is a membrane glycoprotein on rat mast cells (RBL-2H3) expressed at a ratio of approximately 1:30 with respect to the Type I Fc epsilon receptor (Fc epsilon RI). Despite this stoichiometry, clustering MAFA by its specific mAb G63 substantially inhibits secretion of both granular and de novo synthesized mediators induced upon Fc epsilon RI aggregation. Since the Fc epsilon RIs apparently signal from within raft micro-environments, we investigated possible co-localization of MAFA within these membrane compartments containing aggregated Fc epsilon RI. We used cholera toxin B subunit (CTB) to cluster the raft component ganglioside GM1 and studied the effects of this perturbation on rotation of Fc epsilon RI and MAFA by time-resolved phosphorescence anisotropy of erythrosin-conjugated probes. CTB treatment would be expected to substantially inhibit rotation of raft-associated molecules. Experimentally, CTB has no effect on rotational parameters such as the long-time anisotropy (r(infinity)) of unperturbed Fc epsilon RI or MAFA. However, on cells where Fc epsilon RI-IgE has previously been clustered by antigen (DNP(14)-BSA), CTB treatment increases the Fc epsilon RI-IgE's r(infinity) by 0.010 and MAFA's by 0.014. Similarly, CTB treatment of cells where MAFA had been clustered by mAb G63 increases MAFA's r(infinity) by 0.010 but leaves Fc epsilon RI's unaffected. Evaluation of raft localization of Fc epsilon RI and MAFA using sucrose gradient ultracentrifugation of Triton X-100 treated membrane fragments demonstrates that a significant fraction of MAFA molecules sediments with rafts when Fc epsilon RI is clustered by antigen or when MAFA itself is clustered by mAb G63. The large excess of Fc epsilon RI over MAFA explains why clustering MAFA does not substantively affect Fc epsilon RI dynamics. Moreover, in single-particle tracking studies of individual Fc epsilon RI-IgE or MAFA molecules, these proteins, upon clustering by antigen, move into small membrane compartments of reduced, but similar, dimensions. This provides additional indication of constitutive interactions between Fc epsilon RI and MAFA. Taken together, these results of distinct methodologies suggest that MAFA functions within raft microdomains of the RBL-2H3 cell membrane and thus in close proximity to the Fc epsilon RI which themselves signal from within the raft environment.     

Downstream of kinase, p62(dok), is a mediator of Fc gamma IIB inhibition of Fc epsilon RI signaling

The low-affinity receptor for IgG, Fc gamma RIIB, is expressed widely in the immune system and functions to attenuate Ag-induced immune responses. In mast cells, coaggregation of Fc gamma RIIB with the high-affinity IgE receptor, Fc epsilon RI, leads to inhibition of Ag-induced degranulation and cytokine production. Fc gamma RIIB inhibitory activity requires a conserved motif within the Fc gamma RIIB cytoplasmic domain termed the immunoreceptor tyrosine-based inhibition motif. When coaggregated with an activating receptor (e.g., Fc epsilon RI, B cell Ag receptor), Fc gamma RIIB is rapidly phosphorylated on tyrosine and recruits the SH2 domain-containing inositol 5-phosphatase (SHIP). However, the mechanisms by which SHIP mediates Fc gamma RIIB inhibitory function in mast cells remain poorly defined. In this report we demonstrate that Fc gamma RIIB coaggregation with Fc epsilon RI stimulates enhanced SHIP tyrosine phosphorylation and association with Shc and p62(dok). Concurrently, enhanced p62(dok) tyrosine phosphorylation and association with RasGAP are observed, suggesting that SHIP may mediate Fc gamma RIIB inhibitory function in mast cells via recruitment of p62(dok) and RasGAP. Supporting this hypothesis, recruitment of p62(dok) to Fc epsilon RI is sufficient to inhibit Fc epsilon RI-induced calcium mobilization and extracellular signal-regulated kinase 1/2 activation. Interestingly, both the amino-terminal pleckstrin homology and phosphotyrosine binding domains and the carboxyl-terminal proline/tyrosine-rich region of p62(dok) can mediate inhibition, suggesting activation of parallel downstream signaling pathways that converge at extracellular signal-regulated kinase 1/2 activation. Finally, studies using gene-ablated mice indicate that p62(dok) is dispensable for Fc gamma RIIB inhibitory signaling in mast cells. Taken together, these data suggest a role for p62(dok) as a mediator of Fc gamma RIIB inhibition of Fc epsilon RI signal transduction in mast cells.     

Peroxisome proliferator-activated receptor ligands negatively regulate the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells

The high-affinity IgE receptor Fc epsilon RI is expressed on the cell surface of mast cells and basophils, and plays a central role in IgE-mediated inflammatory reactions. Recently, peroxisome proliferator-activated receptors (PPARs) have been implicated in the anti-inflammatory response. To investigate a possible role for PPAR in human basophils, the effect of PPAR ligands on Fc epsilon RI expression in human basophilic KU812 cells was studied. The PPARalpha ligand, leukotriene B(4), did not affect the cell surface expression of Fc epsilon RI. However, prostaglandin (PG) A(1) and 15-deoxy-Delta(12,14) PGJ(2) (15d-PGJ(2)), which are PPARbeta and gamma ligands, respectively, were both able to decrease Fc epsilon RI expression. Treatment with PGA(1) or 15d-PGJ(2) separately also reduced histamine release from KU812 cells in response to cross-linkage of Fc epsilon RI. In addition, RT-PCR analysis showed that KU812 cells expressed the mRNA for PPARalpha, beta, and gamma, indicating that PPARbeta or gamma may negatively regulate the cell activation via Fc epsilon RI. Cells treated with 15d-PGJ(2) expressed lower levels of Fc epsilon RI alpha and gamma mRNA, and PGA(1) treatment decreased the level of Fc epsilon RI gamma mRNA. These results suggest that the suppression of Fc epsilon RI expression by PPARs may be due to the down-regulation of Fc epsilon RI alpha or gamma mRNA.