RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells.

Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.

FcεRI expression and dynamics on mast cells.

Mast cells are key effector and immunoregulatory cells in IgE-associated immune responses, including allergic disorders. IgE antibodies bind to the high-affinity IgE receptor, FcεRI, expressed on the surface of mast cells; antigen-induced cross-linking of FcεRI-bound IgE molecules activates the mast cell to release an array of proinflammatory and immunomodulatory mediators. Because mast cells often respond to very low levels of antigen in vivo, the level of FcεRI expressed on the surface of these cells is an important factor in determining the responsiveness of these cells to antigen. FcεRI surface expression is regulated by a number of processes, including FcεRI stabilization, FcεRI recycling, and antigen-induced internalization. Although members of the Rab family of small GTPases and the ubiquitin ligase, Cbl, have recently emerged as major regulators of many of the membrane trafficking events that govern FcεRI expression levels, the mechanisms and intracellular pathways that regulate FcεRI trafficking remain poorly defined. This chapter outlines a number of flow cytometry-based assays that can be used to investigate cell surface FcεRI expression and dynamics (stabilization, recycling, and internalization) on bone marrow-derived mast cells (BMCMCs), the most commonly used model system for studying mast cells in vitro. Given the importance of FcεRI levels to mast cell responsiveness and function, the characterization of FcεRI expression and dynamics on different mast cell populations is critical when trying to compare IgE-dependent processes between different mast cell populations.

A beneficial role for immunoglobulin E in host defense against honeybee venom.

Allergies are widely considered to be misdirected type 2 immune responses, in which immunoglobulin E (IgE) antibodies are produced against any of a broad range of seemingly harmless antigens. However, components of insect venoms also can sensitize individuals to develop severe IgE-associated allergic reactions, including fatal anaphylaxis, upon subsequent venom exposure. We found that mice injected with amounts of honeybee venom similar to that which could be delivered in one or two stings developed a specific type 2 immune response that increased their resistance to subsequent challenge with potentially lethal amounts of the venom. Our data indicate that IgE antibodies and the high affinity IgE receptor, FcεRI, were essential for such acquired resistance to honeybee venom. The evidence that IgE-dependent immune responses against venom can enhance survival in mice supports the hypothesis that IgE, which also contributes to allergic disorders, has an important function in protection of the host against noxious substances.

Mast cells suppress murine GVHD in a mechanism independent of CD4+CD25+ regulatory T cells.

To investigate the role of mast cells in hematopoietic cell transplantation, we assessed graft-versus-host disease (GVHD) in C57BL/6-Kit(W-sh/W-sh) recipients, which virtually lack mast cells, compared with C57BL/6 WT recipients. GVHD was severely exacerbated in C57BL/6-Kit(W-sh/W-sh) mice (median survival time = 13 vs 60 days in wild-type [WT] mice; P < .0001). The increased mortality risk in C57BL/6-Kit(W-sh/W-sh) hosts correlated with increased T-cell numbers in lymph nodes, liver, and gastrointestinal tract sites, as indicated by bioluminescence imaging (P < .001). We did not detect any deficit in the number or function of CD4(+)CD25(+) regulatory T cells (Tregs) in C57BL/6-Kit(W-sh/W-sh) mice. Furthermore, Tregs were equally effective at reducing GVHD in C57BL/6-Kit(W-sh/W-sh) recipients compared with WT recipients containing mast cells. Furthermore, we found that survival of C57BL/6-Kit(W-sh/W-sh) mice during GVHD was significantly improved if the mice were engrafted with bone marrow-derived cultured mast cells from WT C57BL/6 mice but not from interleukin (IL)-10-deficient C57BL/6 mice. These data indicate that the presence of mast cells can significantly reduce GVHD independently of Tregs, by decreasing conventional T-cell proliferation in a mechanism involving IL-10. These experiments support the conclusion that mast cells can mediate a novel immunoregulatory role during hematopoietic cell transplantation.

Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells.

BACKGROUND: Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. OBJECTIVES: We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. METHODS: C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. RESULTS: Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. CONCLUSIONS: Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro.

Mast cell anaphylatoxin receptor expression can enhance IgE-dependent skin inflammation in mice.

BACKGROUND: Mast cells express receptors for complement anaphylatoxins C3a and C5a (ie, C3a receptor [C3aR] and C5a receptor [C5aR]), and C3a and C5a are generated during various IgE-dependent immediate hypersensitivity reactions in vivo. However, it is not clear to what extent mast cell expression of C3aR or C5aR influences C3a- or C5a-induced cutaneous responses or IgE-dependent mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo. OBJECTIVE: We sought to assess whether mouse skin mast cell expression of C3aR or C5aR influences (1) the cells' responsiveness to intradermal injections of C3a or C5a or (2) the extent of IgE-dependent mast cell degranulation and PCA in vivo. METHODS: We measured the magnitude of cutaneous responses to intradermal injections of C3a or C5a and the extent of IgE-dependent mast cell degranulation and PCA responses in mice containing mast cells that did or did not express C3aR or C5aR. RESULTS: The majority of the skin swelling induced by means of intradermal injection of C3a or C5a required that mast cells at the site expressed C3aR or C5aR, respectively, and the extent of IgE-dependent degranulation of skin mast cells and IgE-dependent PCA was significantly reduced when mast cells lacked either C3aR or C5aR. IgE-dependent PCA responses associated with local increases in C3a levels occurred in antibody-deficient mice but not in mice deficient in FcɛRIγ. CONCLUSION: Expression of C3aR and C5aR by skin mast cells contributes importantly to the ability of C3a and C5a to induce skin swelling and can enhance mast cell degranulation and inflammation during IgE-dependent PCA in vivo.

Evidence questioning cromolyn's effectiveness and selectivity as a 'mast cell stabilizer' in mice.

Cromolyn, widely characterized as a 'mast cell stabilizer', has been used in mice to investigate the biological roles of mast cells in vivo. However, it is not clear to what extent cromolyn can either limit the function of mouse mast cells or influence biological processes in mice independently of effects on mast cells. We confirmed that cromolyn (at 10 mg/kg in vivo or 10-100 µM in vitro) can inhibit IgE-dependent mast cell activation in rats in vivo (measuring Evans blue extravasation in passive cutaneous anaphylaxis (PCA) and increases in plasma histamine in passive systemic anaphylaxis (PSA)) and in vitro (measuring peritoneal mast cell (PMC) ß-hexosaminidase release and prostaglandin D(2) synthesis). However, under the conditions tested, cromolyn did not inhibit those mast cell-dependent responses in mice. In mice, cromolyn also failed to inhibit the ear swelling or leukocyte infiltration at sites of PCA. Nor did cromolyn inhibit IgE-independent degranulation of mouse PMCs induced by various stimulators in vitro. At 100 mg/kg, a concentration 10 times higher than that which inhibited PSA in rats, cromolyn significantly inhibited the increases in plasma concentrations of mouse mast cell protease-1 (but not of histamine) during PSA, but had no effect on the reduction in body temperature in this setting. Moreover, this concentration of cromolyn (100 mg/kg) also inhibited LPS-induced TNF production in genetically mast cell-deficient C57BL/6-Kit(W-sh/W-sh) mice in vivo. These results question cromolyn's effectiveness and selectivity as an inhibitor of mast cell activation and mediator release in the mouse.

Reduced mast cell and basophil numbers and function in Cpa3-Cre; Mcl-1fl/fl mice.

It has been reported that the intracellular antiapoptotic factor myeloid cell leukemia sequence 1 (Mcl-1) is required for mast cell survival in vitro, and that genetic manipulation of Mcl-1 can be used to delete individual hematopoietic cell populations in vivo. In the present study, we report the generation of C57BL/6 mice in which Cre recombinase is expressed under the control of a segment of the carboxypeptidase A3 (Cpa3) promoter. C57BL/6-Cpa3-Cre; Mcl-1(fl/fl) mice are severely deficient in mast cells (92%-100% reduced in various tissues analyzed) and also have a marked deficiency in basophils (58%-78% reduced in the compartments analyzed), whereas the numbers of other hematopoietic cell populations exhibit little or no changes. Moreover, Cpa3-Cre; Mcl-1(fl/fl) mice exhibited marked reductions in the tissue swelling and leukocyte infiltration that are associated with both mast cell- and IgE-dependent passive cutaneous anaphylaxis (except at sites engrafted with in vitro-derived mast cells) and a basophil- and IgE-dependent model of chronic allergic inflammation, and do not develop IgE-dependent passive systemic anaphylaxis. Our findings support the conclusion that Mcl-1 is required for normal mast cell and basophil development/survival in vivo in mice, and also suggest that Cpa3-Cre; Mcl-1(fl/fl) mice may be useful in analyzing the roles of mast cells and basophils in health and disease.

Antiinflammatory and immunosuppressive functions of mast cells.

Through the release of biologically active products, mast cells function as important effector and immunoregulatory cells in diverse immunological reactions and other biological responses; for example, mast cells promote inflammation and other tissue changes in immunoglobulin E (IgE)-associated allergic disorders, as well as in certain innate and adaptive immune responses that are thought to be independent of IgE. Despite the mast cell's well-deserved reputation as a promoter of inflammation, others and we have used bone marrow-derived cultured mast cell (BMCMC) engrafted mast cell-deficient c-kit-mutant mice (so-called "mast cell knock-in" mice) to show that mast cells can also have important antiinflammatory and immunosuppressive functions in vivo. An early study showed that mast cells can contribute to susceptibility to ultraviolet B (UVB)-induced immunosuppression in one model of contact hypersensitivity (CHS), through effects mediated at least in part by histamine. Subsequently, it was reported that mast cells can mediate negative immunomodulatory effects following Anopheles mosquito bites, and in peripheral tolerance to skin allografts; however, the mechanism(s) by which mast cells mediate immunosuppressive functions in these two studies remains to be elucidated. Finally, we showed that mast cells and mast cell-derived IL-10 can limit the magnitude of and promote the resolution of certain CHS responses, and suppress the inflammation and skin injury associated with innate cutaneous responses to chronic low-dose UVB irradiation. This chapter outlines the generation of BMCMCs, a powerful model system commonly used to: (1) identify potential mast cell mediators in vitro; (2) study the mechanisms of mast cell activation and mediator release in response to specific stimuli in vitro; and (3) engraft mast cell-deficient mice to study the effector and immunoregulatory roles of mast cells or specific mast cell mediators in diverse immunological responses in vivo.

Anaphylaxis: mechanisms of mast cell activation.

Anaphylaxis is a severe systemic allergic response that is rapid in onset and potentially lethal, and that typically is induced by an otherwise innocuous substance. In IgE-dependent and other examples of anaphylaxis, tissue mast cells and circulating basophilic granulocytes (basophils) are thought to represent major (if not the major) sources of the biologically active mediators that contribute to the pathology and, in unfortunate individuals, fatal outcome, of anaphylaxis. In this chapter, we will describe the mechanisms of mast cell (and basophil) activation in anaphylaxis, with a focus on IgE-dependent activation, which is thought to be responsible for most examples of antigen-induced anaphylaxis in humans. We will also discuss the use of mouse models to investigate the mechanisms that can contribute to anaphylaxis in that species in vivo, and the relevance of such mouse studies to human anaphylaxis.

Mast cell-derived TNF can exacerbate mortality during severe bacterial infections in C57BL/6-KitW-sh/W-sh mice.

We used mast cell-engrafted genetically mast cell-deficient C57BL/6-Kit(W-sh/W-sh) mice to investigate the roles of mast cells and mast cell-derived tumor necrosis factor in two models of severe bacterial infection. In these mice, we confirmed findings derived from studies of mast cell-deficient WBB6F(1)-Kit(W/W-v) mice indicating that mast cells can promote survival in cecal ligation and puncture (CLP) of moderate severity. However, we found that the beneficial role of mast cells in this setting can occur independently of mast cell-derived tumor necrosis factor. By contrast, using mast cell-engrafted C57BL/6-Kit(W-sh/W-sh) mice, we found that mast cell-derived tumor necrosis factor can increase mortality during severe CLP and can also enhance bacterial growth and hasten death after intraperitoneal inoculation of Salmonella typhimurium. In WBB6F(1)-Kit(W-sh/W-sh) mice, mast cells enhanced survival during moderately severe CLP but did not significantly change the survival observed in severe CLP. Our findings in three types of genetically mast cell-deficient mice thus support the hypothesis that, depending on the circumstances (including mouse strain background, the nature of the mutation resulting in a mast cell deficiency, and type and severity of infection), mast cells can have either no detectable effect or opposite effects on survival during bacterial infections, eg, promoting survival during moderately severe CLP associated with low mortality but, in C57BL/6-Kit(W-sh/W-sh) mice, increasing mortality during severe CLP or infection with S. typhimurium.

New developments in mast cell biology.

Mast cells can function as effector and immunoregulatory cells in immunoglobulin E-associated allergic disorders, as well as in certain innate and adaptive immune responses. This review focuses on exciting new developments in the field of mast cell biology published in the past year. We highlight advances in the understanding of FcvarepsilonRI-mediated signaling and mast cell-activation events, as well as in the use of genetic models to study mast cell function in vivo. Finally, we discuss newly identified functions for mast cells or individual mast cell products, such as proteases and interleukin 10, in host defense, cardiovascular disease and tumor biology and in settings in which mast cells have anti-inflammatory or immunosuppressive functions.

IgE-induced mast cell survival requires the prolonged generation of reactive oxygen species.

We show in this study that the ability of five different monomeric IgEs to enhance murine bone marrow-derived mast cell (BMMC) survival correlates with their ability to stimulate extracellular calcium (Ca(2+)) entry. However, whereas IgE+Ag more potently stimulates Ca(2+) entry, it does not enhance survival under our conditions. Exploring this further, we found that whereas all five monomeric IgEs stimulate a less robust Ca(2+) entry than IgE+Ag initially, they all trigger a more prolonged Ca(2+) influx, generation of reactive oxygen species (ROS), and ERK phosphorylation. These prolonged signaling events correlate with their survival-enhancing ability and positively feedback on each other to generate the prosurvival cytokine, IL-3. Interestingly, the prolonged ERK phosphorylation induced by IgE appears to be regulated by a MAPK phosphatase rather than MEK. IgE-induced ROS generation, unlike that triggered by IgE+Ag, is not mediated by 5-lipoxygenase. Moreover, ROS inhibitors, which block both IgE-induced ROS production and Ca(2+) influx, convert the prolonged ERK phosphorylation induced by IgE into the abbreviated phosphorylation pattern observed with IgE+Ag and prevent IL-3 generation. In support of the essential role that IgE-induced ROS plays in IgE-enhanced BMMC survival, we found the addition of H(2)O(2) to IgE+Ag-stimulated BMMCs leads to IL-3 secretion.

Rabaptin-5 regulates receptor expression and functional activation in mast cells.

Rab5 is a small GTPase that regulates early endocytic events and is activated by RabGEF1/Rabex-5. Rabaptin-5, a Rab5 interacting protein, was identified as a protein critical for potentiating RabGEF1/Rabex-5's activation of Rab5. Using Rabaptin-5 shRNA knockdown, we show that Rabaptin-5 is dispensable for Rab5-dependent processes in intact mast cells, including high affinity IgE receptor (FcepsilonRI) internalization and endosome fusion. However, Rabaptin-5 deficiency markedly diminished expression of FcepsilonRI and beta1 integrin on the mast cell surface by diminishing receptor surface stability. This in turn reduced the ability of mast cells to bind IgE and significantly diminished both mast cell sensitivity to antigen (Ag)-induced mediator release and Ag-induced mast cell adhesion and migration. These findings show that, although dispensable for canonical Rab5 processes in mast cells, Rabaptin-5 importantly contributes to mast cell IgE-dependent immunologic function by enhancing mast cell receptor surface stability.

Mast cell-derived interleukin 10 limits skin pathology in contact dermatitis and chronic irradiation with ultraviolet B.

Allergic contact dermatitis, such as in response to poison ivy or poison oak, and chronic low-dose ultraviolet B irradiation can damage the skin. Mast cells produce proinflammatory mediators that are thought to exacerbate these prevalent acquired immune or innate responses. Here we found that, unexpectedly, mast cells substantially limited the pathology associated with these responses, including infiltrates of leukocytes, epidermal hyperplasia and epidermal necrosis. Production of interleukin 10 by mast cells contributed to the anti-inflammatory or immunosuppressive effects of mast cells in these conditions. Our findings identify a previously unrecognized function for mast cells and mast cell-derived interleukin 10 in limiting leukocyte infiltration, inflammation and tissue damage associated with immunological or innate responses that can injure the skin.

Roles of RabGEF1/Rabex-5 domains in regulating Fc epsilon RI surface expression and Fc epsilon RI-dependent responses in mast cells.

RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (-/-) mast cells and examined Fc epsilon RI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in -/- mast cells, including decreased basal Fc epsilon RI expression, slowed Fc epsilon RI internalization, elevated IgE + Ag-induced degranulation and IL-6 production, and the decreased ability of -/- cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface Fc epsilon RI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen-activated -/- mast cells. By contrast, correction of these -/- phenotypes required a functional Vps9 domain. Thus, Fc epsilon RI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity.

RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells.

We recently reported that RabGEF1 is a negative regulator of high-affinity Fc receptor for IgE (Fc epsilonRI)-dependent mast cell activation and that mice lacking RabGEF1 develop severe skin inflammation and increased numbers of dermal mast cells. To better understand how RabGEF1 can regulate signaling events and biological responses in mast cells, we examined the responses of bone marrow-derived cultured mast cells (BMCMCs) from wild-type (+/+) and Rabgef1 knockout (-/-) mice after stimulation with the c-Kit ligand, stem cell factor (SCF), an important regulator of mast cell development, survival, proliferation, and activation. We found that RabGEF1-deficient mast cells exhibited enhanced and prolonged activation of Ras and extracellular regulated kinase, and significantly elevated IL-6 secretion, after stimulation with SCF. SCF-induced activation of c-Jun N-terminal kinase was increased in Rabgef1-/- BMCMCs, but without corresponding significant increases in SCF-induced migration or adhesion. SCF-mediated activation of the survival-enhancing kinase, Akt, also was increased in Rabgef1-/- BMCMCs, and these cells had a survival advantage over their +/+ counterparts in vitro. Despite enhanced Ras activation in the absence of RabGEF1, SCF-induced proliferation was lower in Rabgef1-/- BMCMCs compared with their +/+ counterparts. Finally, we found that c-Kit internalization was delayed in the absence of RabGEF1, probably reflecting a positive role for RabGEF1 in the regulation of endocytic events, and that infection of Rabgef1-/- BMCMCs with a wild-type RabGEF1 lentiviral construct normalized c-Kit internalization to the levels seen in +/+ BMCMCs. Thus, RabGEF1 plays a critical role in the regulation of SCF/c-Kit-mediated signaling events and biological responses in mast cells.

Mast cells as "tunable" effector and immunoregulatory cells: recent advances.

This review focuses on recent progress in our understanding of how mast cells can contribute to the initiation, development, expression, and regulation of acquired immune responses, both those associated with IgE and those that are apparently expressed independently of this class of Ig. We emphasize findings derived from in vivo studies in mice, particularly those employing genetic approaches to influence mast cell numbers and/or to alter or delete components of pathways that can regulate mast cell development, signaling, or function. We advance the hypothesis that mast cells not only can function as proinflammatory effector cells and drivers of tissue remodeling in established acquired immune responses, but also may contribute to the initiation and regulation of such responses. That is, we propose that mast cells can also function as immunoregulatory cells. Finally, we show that the notion that mast cells have primarily two functional configurations, off (or resting) or on (or activated for extensive mediator release), markedly oversimplifies reality. Instead, we propose that mast cells are "tunable," by both genetic and environmental factors, such that, depending on the circumstances, the cell can be positioned phenotypically to express a wide spectrum of variation in the types, kinetics, and/or magnitude of its secretory functions.

RabGEF1, a negative regulator of Ras signalling, mast cell activation and skin inflammation.

Mast cell activation induced by the aggregation of FcepsilonRI with IgE and antigen is mediated through the activation of multiple protein kinase cascades. This process induces mast cells to undergo degranulation, to synthesize and release lipid mediators, and to secrete multiple cytokines, chemokines and growth factors. We found that RabGEF1 (Rabex-5) binds to Ras and negatively regulates Ras activation and downstream effector pathways during FcepsilonRI-dependent mouse mast cell activation. Mast cells derived from RabGEF1-deficient mice exhibit significantly enhanced levels of degranulation, release of lipid mediators and secretion of cytokines in response to FcepsilonRI aggregation. RabGEF1 knockout mice have increased perinatal mortality and the mice that do survive develop severe skin inflammation and increased numbers of mast cells in the dermis, some of which exhibit morphological evidence of degranulation. These mice also show elevated concentrations of serum histamine and IgE. Thus, RabGEF1 is a negative regulator of Ras signalling and FcepsilonRI-dependent mast cell activation in vitro, and a lack of RabGEF1 results in the development of elevated numbers of mast cells in the skin and severe skin inflammation in vivo.