Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

• Keywords
• FRAP, FcεRI signaling, STIM1-ORAI1 coupling, alkanol, flow-FRET, heat shock response, membrane fluidizer, store-operated calcium entry,
• MeSH
• Cholesterol MeSH
• Cytokines MeSH
• Heptanol MeSH
• Mast Cells * MeSH
• Signal Transduction * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol MeSH
• Cytokines MeSH
• Heptanol MeSH

Pentacyclic triterpenoids, including ursolic acid (UA), are bioactive compounds with multiple biological activities involving anti-inflammatory effects. However, the mode of their action on mast cells, key players in the early stages of allergic inflammation, and underlying molecular mechanisms remain enigmatic. To better understand the effect of UA on mast cell signaling, here we examined the consequences of short-term treatment of mouse bone marrow-derived mast cells with UA. Using IgE-sensitized and antigen- or thapsigargin-activated cells, we found that 15 min exposure to UA inhibited high affinity IgE receptor (FcεRI)-mediated degranulation, calcium response, and extracellular calcium uptake. We also found that UA inhibited migration of mouse bone marrow-derived mast cells toward antigen but not toward prostaglandin E2 and stem cell factor. Compared to control antigen-activated cells, UA enhanced the production of tumor necrosis factor-α at the mRNA and protein levels. However, secretion of this cytokine was inhibited. Further analysis showed that UA enhanced tyrosine phosphorylation of the SYK kinase and several other proteins involved in the early stages of FcεRI signaling, even in the absence of antigen activation, but inhibited or reduced their further phosphorylation at later stages. In addition, we show that UA induced changes in the properties of detergent-resistant plasma membrane microdomains and reduced antibody-mediated clustering of the FcεRI and glycosylphosphatidylinositol-anchored protein Thy-1. Finally, UA inhibited mobility of the FcεRI and cholesterol. These combined data suggest that UA exerts its effects, at least in part, via lipid-centric plasma membrane perturbations, hence affecting the functions of the FcεRI signalosome.

• Keywords
• immunoglobulin E, lipid raft, mast cell, plasma membrane, signal transduction, tumor necrosis factor, tyrosine kinase,
• MeSH
• Antigens metabolism   MeSH
• Cell Degranulation MeSH
• Ursolic Acid MeSH
• Lipids pharmacology   MeSH
• Mast Cells metabolism   MeSH
• Mice MeSH
• Receptors, IgE * metabolism   MeSH
• Triterpenes * pharmacology   metabolism   MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens MeSH
• Lipids MeSH
• Receptors, IgE * MeSH
• Triterpenes * MeSH
• Calcium MeSH

A better understanding of the molecular mechanisms leading to mast cell migration and chemotaxis is the long-term goal in mast cell research and is essential for comprehension of mast cell function in health and disease. Various techniques have been developed in recent decades for in vitro and in vivo assessment of mast cell motility and chemotaxis. In this chapter, three microscopy assays facilitating real-time quantification of mast cell chemotaxis and migration are described, focusing on individual cell tracking and data analysis.

• Keywords
• Cell migration, Cell tracking, Chemoattractant, Chemokine, Chemotaxis, Mast cells,
• MeSH
• Cell Migration Assays methods   MeSH
• Biological Assay methods   MeSH
• Cell Tracking methods   MeSH
• Chemotaxis physiology   MeSH
• Fibronectins metabolism   MeSH
• Humans MeSH
• Mast Cells cytology   physiology   MeSH
• Microscopy methods   MeSH
• Mice MeSH
• Computer Systems MeSH
• Cell Movement physiology   MeSH
• Environment, Controlled MeSH
• Sepharose MeSH
• Software MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fibronectins MeSH
• Sepharose MeSH

Protein 4.1R, a member of the 4.1 family, functions as a bridge between cytoskeletal and plasma membrane proteins. It is expressed in T cells, where it binds to a linker for activation of T cell (LAT) family member 1 and inhibits its phosphorylation and downstream signaling events after T cell receptor triggering. The role of the 4.1R protein in cell activation through other immunoreceptors is not known. In this study, we used 4.1R-deficient (4.1R-KO) and 4.1R wild-type (WT) mice and explored the role of the 4.1R protein in the high-affinity IgE receptor (FcεRI) signaling in mast cells. We found that bone marrow mast cells (BMMCs) derived from 4.1R-KO mice showed normal growth in vitro and expressed FcεRI and c-KIT at levels comparable to WT cells. However, 4.1R-KO cells exhibited reduced antigen-induced degranulation, calcium response, and secretion of tumor necrosis factor-α. Chemotaxis toward antigen and stem cell factor (SCF) and spreading on fibronectin were also reduced in 4.1R-KO BMMCs, whereas prostaglandin E2-mediated chemotaxis was not affected. Antibody-induced aggregation of tetraspanin CD9 inhibited chemotaxis toward antigen in WT but not 4.1R-KO BMMCs, implying a CD9-4.1R protein cross-talk. Further studies documented that in the absence of 4.1R, antigen-mediated phosphorylation of FcεRI β and γ subunits was not affected, but phosphorylation of SYK and subsequent signaling events such as phosphorylation of LAT1, phospholipase Cγ1, phosphatases (SHP1 and SHIP), MAP family kinases (p38, ERK, JNK), STAT5, CBL, and mTOR were reduced. Immunoprecipitation studies showed the presence of both LAT1 and LAT2 (LAT, family member 2) in 4.1R immunocomplexes. The positive regulatory role of 4.1R protein in FcεRI-triggered activation was supported by in vivo experiments in which 4.1R-KO mice showed the normal presence of mast cells in the ears and peritoneum, but exhibited impaired passive cutaneous anaphylaxis. The combined data indicate that the 4.1R protein functions as a positive regulator in the early activation events after FcεRI triggering in mast cells.

• Keywords
• 4.1R protein, chemotaxis, degranulation, mast cell, passive cutaneous anaphylaxis,
• MeSH
• Chemotaxis immunology   MeSH
• Cell Degranulation immunology   MeSH
• Mast Cells immunology   metabolism   MeSH
• Microfilament Proteins immunology   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Passive Cutaneous Anaphylaxis immunology   MeSH
• Receptors, IgE immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epb41 protein, mouse MeSH Browser
• Microfilament Proteins MeSH
• Receptors, IgE MeSH

C-terminal Src kinase (CSK) is a major negative regulator of Src family tyrosine kinases (SFKs) that play critical roles in immunoreceptor signaling. CSK is brought in contiguity to the plasma membrane-bound SFKs via binding to transmembrane adaptor PAG, also known as CSK-binding protein. The recent finding that PAG can function as a positive regulator of the high-affinity IgE receptor (FcεRI)-mediated mast cell signaling suggested that PAG and CSK have some non-overlapping regulatory functions in mast cell activation. To determine the regulatory roles of CSK in FcεRI signaling, we derived bone marrow-derived mast cells (BMMCs) with reduced or enhanced expression of CSK from wild-type (WT) or PAG knockout (KO) mice and analyzed their FcεRI-mediated activation events. We found that in contrast to PAG-KO cells, antigen-activated BMMCs with CSK knockdown (KD) exhibited significantly higher degranulation, calcium response, and tyrosine phosphorylation of FcεRI, SYK, and phospholipase C. Interestingly, FcεRI-mediated events in BMMCs with PAG-KO were restored upon CSK silencing. BMMCs with CSK-KD/PAG-KO resembled BMMCs with CSK-KD alone. Unexpectedly, cells with CSK-KD showed reduced kinase activity of LYN and decreased phosphorylation of transcription factor STAT5. This was accompanied by impaired production of proinflammatory cytokines and chemokines in antigen-activated cells. In line with this, BMMCs with CSK-KD exhibited enhanced phosphorylation of protein phosphatase SHP-1, which provides a negative feedback loop for regulating phosphorylation of STAT5 and LYN kinase activity. Furthermore, we found that in WT BMMCs SHP-1 forms complexes containing LYN, CSK, and STAT5. Altogether, our data demonstrate that in FcεRI-activated mast cells CSK is a negative regulator of degranulation and chemotaxis, but a positive regulator of adhesion to fibronectin and production of proinflammatory cytokines. Some of these pathways are not dependent on the presence of PAG.

• Keywords
• C-terminal Src kinase, LYN, SHP-1, STAT5, cytokines, degranulation, mast cell, phosphoprotein associated with glycosphingolipid-enriched microdomains,
• MeSH
• Analysis of Variance MeSH
• Bone Marrow Cells physiology   MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Cytokines metabolism   MeSH
• Cell Degranulation MeSH
• Fibronectins metabolism   MeSH
• Phosphoproteins metabolism   MeSH
• Phosphorylation MeSH
• Genetic Vectors MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mast Cells physiology   MeSH
• Membrane Proteins metabolism   MeSH
• Intercellular Signaling Peptides and Proteins MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Receptors, IgE metabolism   MeSH
• Signal Transduction immunology   MeSH
• src-Family Kinases metabolism   physiology   MeSH
• STAT5 Transcription Factor metabolism   MeSH
• Tyrosine metabolism   MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 metabolism   MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CSK Tyrosine-Protein Kinase MeSH
• CSK protein, human MeSH Browser
• Cytokines MeSH
• Fibronectins MeSH
• Phosphoproteins MeSH
• lyn protein-tyrosine kinase MeSH Browser
• Membrane Proteins MeSH
• Intercellular Signaling Peptides and Proteins MeSH
• Pag protein, mouse MeSH Browser
• Pag1 protein, mouse MeSH Browser
• Ptpn6 protein, mouse MeSH Browser
• Receptors, IgE MeSH
• src-Family Kinases MeSH
• STAT5 Transcription Factor MeSH
• Tyrosine MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 MeSH
• Calcium MeSH

The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

• Keywords
• CD9, IgE receptor, LAT, NTAL, membrane microdomains, plasma membrane, signal transduction,
• Publication type
• Journal Article MeSH
• Review MeSH

Mast cells play crucial roles in both innate and adaptive arms of the immune system. Along with basophils, mast cells are essential effector cells for allergic inflammation that causes asthma, allergic rhinitis, food allergy and atopic dermatitis. Mast cells are usually increased in inflammatory sites of allergy and, upon activation, release various chemical, lipid, peptide and protein mediators of allergic reactions. Since antigen/immunoglobulin E (IgE)-mediated activation of these cells is a central event to trigger allergic reactions, innumerable studies have been conducted on how these cells are activated through cross-linking of the high-affinity IgE receptor (FcεRI). Development of mature mast cells from their progenitor cells is under the influence of several growth factors, of which the stem cell factor (SCF) seems to be the most important. Therefore, how SCF induces mast cell development and activation via its receptor, KIT, has been studied extensively, including a cross-talk between KIT and FcεRI signaling pathways. Although our understanding of the signaling mechanisms of the FcεRI and KIT pathways is far from complete, pharmaceutical applications of the knowledge about these pathways are underway. This review will focus on recent progresses in FcεRI and KIT signaling and chemotaxis.

• Keywords
• Chemotaxis, IgE receptor, KIT receptor, Mast cell, Plasma membrane, Signal transduction,
• MeSH
• Chemotaxis * drug effects   MeSH
• Humans MeSH
• Mast Cells cytology   drug effects   MeSH
• Signal Transduction * drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH

Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells.

• MeSH
• Actins metabolism   MeSH
• Cell Adhesion MeSH
• Chemotaxis MeSH
• Cytokines genetics   metabolism   MeSH
• Phosphorylation MeSH
• Galectin 3 genetics   metabolism   MeSH
• Lysosomes metabolism   MeSH
• RNA, Small Interfering MeSH
• Mast Cells cytology   physiology   MeSH
• Mice, Inbred BALB C MeSH
• Prostaglandin D2 metabolism   MeSH
• Receptors, IgE genetics   metabolism   MeSH
• Signal Transduction MeSH
• Ubiquitination MeSH
• Calcium metabolism   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Actins MeSH
• Cytokines MeSH
• Galectin 3 MeSH
• Lgals3 protein, mouse MeSH Browser
• RNA, Small Interfering MeSH
• Prostaglandin D2 MeSH
• Receptors, IgE MeSH
• Calcium MeSH

Single-nucleotide polymorphism studies have linked the chromosome 17q12-q21 region, where the human orosomucoid-like (ORMDL)3 gene is localized, to the risk of asthma and several other inflammatory diseases. Although mast cells are involved in the development of these diseases, the contribution of ORMDL3 to the mast cell physiology is unknown. In this study, we examined the role of ORMDL3 in antigen-induced activation of murine mast cells with reduced or enhanced ORMDL3 expression. Our data show that in antigen-activated mast cells, reduced expression of the ORMDL3 protein had no effect on degranulation and calcium response, but significantly enhanced phosphorylation of AKT kinase at Ser 473 followed by enhanced phosphorylation and degradation of IκBα and translocation of the NF-κB p65 subunit into the nucleus. These events were associated with an increased expression of proinflammatory cytokines (TNF-α, IL-6, and IL-13), chemokines (CCL3 and CCL4), and cyclooxygenase-2 dependent synthesis of prostaglandin D2. Antigen-mediated chemotaxis was also enhanced in ORMDL3-deficient cells, whereas spreading on fibronectin was decreased. On the other hand, increased expression of ORMDL3 had no significant effect on the studied signaling events, except for reduced antigen-mediated chemotaxis. These data were corroborated by increased IgE-antigen-dependent passive cutaneous anaphylaxis in mice with locally silenced ORMDL3 using short interfering RNAs. Our data also show that antigen triggers suppression of ORMDL3 expression in the mast cells. In summary, we provide evidence that downregulation of ORMDL3 expression in mast cells enhances AKT and NF-κB-directed signaling pathways and chemotaxis and contributes to the development of mast cell-mediated local inflammation in vivo.

• Keywords
• Chemotaxis, Degranulation, Mast cell, ORMDL3 knockdown, Proinflammatory cytokines, Prostaglandin D2, RNA interference,
• MeSH
• Chemotaxis * MeSH
• Cytokines genetics   immunology   MeSH
• Cell Degranulation * MeSH
• Down-Regulation MeSH
• Cells, Cultured MeSH
• Mast Cells cytology   immunology   metabolism   MeSH
• Membrane Proteins genetics   immunology   MeSH
• RNA, Messenger genetics   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Receptors, IgE immunology   MeSH
• Up-Regulation MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• Membrane Proteins MeSH
• RNA, Messenger MeSH
• ORMDL3 protein, mouse MeSH Browser
• Receptors, IgE MeSH

Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of FcεRI-cholesterol signalosomes at the plasma membrane.

• MeSH
• Cholesterol metabolism   MeSH
• Cytokines genetics   metabolism   MeSH
• Ethanol pharmacology   MeSH
• Gene Expression drug effects   MeSH
• Phosphorylation MeSH
• Mast Cells drug effects   metabolism   MeSH
• Mice MeSH
• Receptors, IgE metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Calcium metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol MeSH
• Cytokines MeSH
• Ethanol MeSH
• Receptors, IgE MeSH
• Calcium MeSH