Regulation of the Tyrosine Phosphorylation of Phospholipid Scramblase 1 in Mast Cells That Are Stimulated through the High-Affinity IgE Receptor

Engagement of high-affinity immunoglobulin E receptors (FcεRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3-kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FcεRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRγ chain. FcεRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FcεRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FcεRI-activated mast cells and that PLSCR1 sits at a crossroads between Lyn and Fyn pathways.     

Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

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.     

Differential Effects of the Toll-Like Receptor 2 Agonists,PGN and Pam3CSK4 on Anti-IgE Induced Human Mast Cell Activation

Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.     

The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

Background

IgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcεRI in human intestinal epithelium.

Methodology/Principal Findings

FcεRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FcεRIα positive epithelial cells co-expressed FcεRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcεRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcεRI and to bind IgE, whereas confluent cells were negative for γ-chains.

Conclusions/Significance

Our data provide the first evidence that the components of a functional FcεRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FcεRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested.     

Regulation of Fc?RI Signaling in Mast Cells by G Protein-coupled Receptor Kinase 2 and Its RH Domain

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) promotes their desensitization and internalization. Here, we sought to determine the role of GRK2 on FcϵRI signaling and mediator release in mast cells. The strategies utilized included lentiviral shRNA-mediated GRK2 knockdown, GRK2 gene deletion (GRK2^flox/flox/cre recombinase) and overexpression of GRK2 and its regulator of G protein signaling homology (RH) domain (GRK2-RH). We found that silencing GRK2 expression caused ∼50% decrease in antigen-induced Ca²⁺ mobilization and degranulation but resulted in ablation of cytokine (IL-6 and IL-13) generation. The effect of GRK2 on cytokine generation does not require its catalytic activity but is mediated via the phosphorylation of p38 and Akt. Overexpression of GRK2 or its RH domain (GRK2-RH) enhanced antigen-induced mast cell degranulation and cytokine generation without affecting the expression levels of any of the FcϵRI subunits (α, β, and γ). GRK2 or GRK2-RH had no effect on antigen-induced phosphorylation of FcϵRIγ or Src but enhanced tyrosine phosphorylation of Syk. These data demonstrate that GRK2 modulates FcϵRI signaling in mast cells via at least two mechanisms. One involves GRK2-RH and modulates tyrosine phosphorylation of Syk, and the other is mediated via the phosphorylation of p38 and Akt.     

IgE by Itself Affects Mature Rat Mast Cell Preformed and De Novo-Synthesized Mediator Release and Amplifies Mast Cell Migratory Response

Background

Immunoglobulin E (IgE) binds to high affinity receptor FcεRI numerously expressed on mast cells. Recent findings have revealed that IgE by itself may regulate various aspects of mast cell biology, however, detailed data is still limited.

Methodology/Findings

Here, we have examined the influence of IgE alone, used at different concentrations, on mast cell activity and releasability. For the study we have employed in vivo differentiated mature tissue mast cells isolated from rat peritoneal cavity. Mast cells were exposed to IgE alone and then the release of preformed and de novo-synthesized mediators, surface FcεRI expression and mast cell migratory response were assessed. IgE by itself was found to up-regulate FcεRI expression and activate mast cells to degranulation, as well as de novo synthesis and release of cysteinyl leukotrienes and TNF. We have provided evidence that IgE alone also amplified spontaneous and CCL5- or TNF-induced migration of mast cells. Importantly, IgE was effective only at concentrations ≥ 3 µg/mL. A molecular basis investigation using an array of specific inhibitors showed that Src kinases, PLC/PLA₂, MAP kinases (ERK and p38) and PI3K were entirely or partially involved in IgE-induced mast cell response. Furthermore, IgE alone stimulated the phosphorylation of MAP kinases and PI3K in rat mast cells.

Conclusion

Our results clearly demonstrated that IgE by itself, at higher concentrations, influences mast cell activity and releasability. As there are different conditions when the IgE level is raised it might be supposed that in vivo IgE is one of the important factors modulating mast cell biology within tissues.     

Histone Deacetylase-3 Mediates Positive Feedback Relationship between Anaphylaxis and Tumor Metastasis

Allergic inflammation has been known to enhance the metastatic potential of tumor cells. The role of histone deacetylase-3 (HDAC3) in allergic skin inflammation was reported. We investigated HDAC3 involvement in the allergic inflammation-promotion of metastatic potential of tumor cells. Passive systemic anaphylaxis (PSA) induced HDAC3 expression and FcϵRI signaling in BALB/c mice. PSA enhanced the tumorigenic and metastatic potential of mouse melanoma cells in HDAC3- and monocyte chemoattractant protein 1-(MCP1)-dependent manner. The PSA-mediated enhancement of metastatic potential involved the induction of HDAC3, MCP1, and CD11b (a macrophage marker) expression in the lung tumor tissues. We examined an interaction between anaphylaxis and tumor growth and metastasis at the molecular level. Conditioned medium from antigen-stimulated bone marrow-derived mouse mast cell cultures induced the expression of HDAC3, MCP1, and CCR2, a receptor for MCP1, in B16F1 mouse melanoma cells and enhanced migration and invasion potential of B16F1 cells. The conditioned medium from B16F10 cultures induced the activation of FcϵRI signaling in lung mast cells in an HDAC3-dependent manner. FcϵRI signaling was observed in lung tumors derived from B16F10 cells. Target scan analysis predicted HDAC3 to be as a target of miR-384, and miR-384 and HDAC3 were found to form a feedback regulatory loop. miR-384, which is decreased by PSA, negatively regulated HDAC3 expression, allergic inflammation, and the positive feedback regulatory loop between anaphylaxis and tumor metastasis. We show the miR-384/HDAC3 feedback loop to be a novel regulator of the positive feedback relationship between anaphylaxis and tumor metastasis.     

Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 10³-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.     

Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca²⁺ response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)₂ or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcϵRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)₂ fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcϵRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.     

Transcription Factor GATA1 Is Dispensable for Mast Cell Differentiation in Adult Mice

Although previous studies have shown that GATA1 is required for mast cell differentiation, the effects of the complete ablation of GATA1 in mast cells have not been examined. Using conditional Gata1 knockout mice (Gata1^−/y), we demonstrate here that the complete ablation of GATA1 has a minimal effect on the number and distribution of peripheral tissue mast cells in adult mice. The Gata1^−/y bone marrow cells were capable of differentiating into mast cells ex vivo. Microarray analyses showed that the repression of GATA1 in bone marrow mast cells (BMMCs) has a small impact on the mast cell-specific gene expression in most cases. Interestingly, however, the expression levels of mast cell tryptases in the mouse chromosome 17A3.3 were uniformly reduced in the GATA1 knockdown cells, and GATA1 was found to bind to a 500-bp region at the 5′ end of this locus. Revealing a sharp contrast to that observed in the Gata1-null BMMCs, GATA2 deficiency resulted in a significant loss of the c-Kit⁺ FcεRIα⁺ mast cell fraction and a reduced expression of several mast cell-specific genes. Collectively, GATA2 plays a more important role than GATA1 in the regulation of most mast cell-specific genes, while GATA1 might play specific roles in mast cell functions.     

Rat Embryonic Mast Cells Originate in the AGM

Mast cells originate from pluripotent hematopoietic stem cells. Two mast cell specific antibodies, mAbsAA4 and BGD6, have previously been used to identify and study committed mast cell precursors (MCcps) in the bone marrow of adult mice and rats. However, the embryonic origin of MCcps is still not known. In the present study, we identified MCcps in rat embryos using these previously characterized mast cell specific antibodies. The MCcps were found in the AGM (aorta-gonad-mesonephros) region of rat embryos at E11.5. These cells were BGD6+, CD34⁺, c-kit⁺, CD13⁺, FcεRI⁻, AA4⁻ CD40⁻, and Thy-1⁻. By PCR the cells contained message for the α and β subunits of FcεRI and mast cell specific proteases. In vitro, the MCcps differentiated into metachromatic mast cells. With age of gestation the percent of MCcps diminished while the percent of mast cell progenitors increased. An increased knowledge of the biology and embryonic origin of mast cells may contribute to a greater understanding of allergy, asthma, and other mast cell related diseases.     

Transglutaminase II/MicroRNA-218/-181a Loop Regulates Positive Feedback Relationship between Allergic Inflammation and Tumor Metastasis

The molecular mechanism of transglutaminase II (TGaseII)-mediated allergic inflammation remains largely unknown. TGaseII, induced by antigen stimulation, showed an interaction and co-localization with FcϵRI. TGaseII was necessary for in vivo allergic inflammation, such as triphasic cutaneous reaction, passive cutaneous anaphylaxis, and passive systemic anaphylaxis. TGaseII was necessary for the enhanced metastatic potential of B16F1 melanoma cells by passive systemic anaphylaxis. TGaseII was shown to be a secreted protein. Recombinant TGaseII protein increased the histamine release and β-hexosaminidase activity, and enhanced the metastatic potential of B16F1 mouse melanoma cells. Recombinant TGaseII protein induced the activation of EGF receptor and an interaction between EGF receptor and FcϵRI. Recombinant TGaseII protein displayed angiogenic potential accompanied by allergic inflammation. R2 peptide, an inhibitor of TGaseII, exerted negative effects on in vitro and in vivo allergic inflammation by regulating the expression of TGaseII and FcϵRI signaling. MicroRNA (miR)-218 and miR-181a, decreased during allergic inflammation, were predicted as negative regulators of TGaseII by microRNA array and TargetScan analysis. miR-218 and miR-181a formed a negative feedback loop with TGaseII and regulated the in vitro and in vivo allergic inflammation. TGaseII was necessary for the interaction between mast cells and macrophages during allergic inflammation. Mast cells and macrophages, activated during allergic inflammation, were responsible for the enhanced metastatic potential of tumor cells that are accompanied by allergic inflammation. In conclusion, the TGaseII/miR-218/-181a feedback loop can be employed for the development of anti-allergy therapeutics.     

Involvement of Class II Phosphoinositide 3-Kinase α-Isoform in Antigen-Induced Degranulation in RBL-2H3 Cells

In this study, we present findings that suggest that PI3K-C2α, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of FcεRI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2α. The knockdown impaired the FcεRI-induced release of a lysosome enzyme, β-hexosaminidase, without affecting the intracellular Ca²⁺ mobilization. The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells. The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α. In wild-type cells, FcεRI stimulation induced the formation of large vesicles, which were associated with CD63, a marker protein of secretory granules. On the vesicles, the existence of PI3K-C2α and PtdIns(3,4)P₂ was observed. These results indicated that PI3K-C2α and its product PtdIns(3,4)P₂ may play roles in the secretory process.     

IgE Regulates the Expression of smMLCK in Human Airway Smooth Muscle Cells

Previous studies have shown that enhanced accumulation of contractile proteins such as smooth muscle myosin light chain kinase (smMLCK) plays a major role in human airway smooth muscle cells (HASM) cell hypercontractility and hypertrophy. Furthermore, serum IgE levels play an important role in smooth muscle hyperreactivity. However, the effect of IgE on smMLCK expression has not been investigated. In this study, we demonstrate that IgE increases the expression of smMLCK at mRNA and protein levels. This effect was inhibited significantly with neutralizing abs directed against FcεRI but not with anti-FcεRII/CD23. Furthermore, Syk knock down and pharmacological inhibition of mitogen activated protein kinases (MAPK) (ERK1/2, p38, and JNK) and phosphatidylinositol 3-kinase (PI3K) significantly diminished the IgE-mediated upregulation of smMLCK expression in HASM cells. Taken together, our data suggest a role of IgE in regulating smMLCK in HASM cells. Therefore, targeting the FcεRI activation on HASM cells may offer a novel approach in controlling the bronchomotor tone in allergic asthma.