Characterization of human Fc?RI? chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines.
ABSTRACT: Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (Fc?RIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of Fc?RIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected Fc?RIH chain in humanized RBL cell lines. We compared surface levels of Fc?RI?H by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. Fc?RI?H copy numbers were assessed by qPCR, and the sequence verified. Transfection with Fc?RI?H cDNA was assessed for its ability to increase Fc?RI?H expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower Fc?RI?H expression, respectively. This was neither related to Fc?RIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, Fc?RI?H surface expression appeared to correlate with the co-expression of Fc?RI?H. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFc?RI gamma, which constitutively expresses Fc?RI?H, increased Fc?RI?H chain expression levels. Levels of Fc?RI?H surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.
Project description:<h4>Background</h4>For the detection of allergen-specific IgE in sera, solid-phase IgE-binding assays like the CAP test are commonly used. Although such immunochemical methods are very sensitive, they frequently produce false positives. Degranulation of the human IgE receptor (Fc?RI)-transfected rat mast cell (RBL) lines seems to be a possible indicator for human IgE, but spontaneous mediator release from these cells in the presence of human sera is not negligible.<h4>Methods</h4>The nuclear factor of activated T-cells (NFAT)-responsive luciferase reporter gene was stably transfected into human Fc?RI-expressing RBL-SX38 cells. One established clone (RS-ATL8) was sensitized with 1?:?100 dilution of sera from patients with egg white allergy and then stimulated with purified or a crude extract of egg white allergen.<h4>Results</h4>Sensitization with 15?pg/ml IgE was sufficient to detect IgE crosslinking-induced luciferase expression (EXiLE) by anti-IgE stimulation. Allergen-specific EXiLE was elicited by as little as 1?fg/ml of egg white protein without cytotoxicity. There was a good correlation between results with EXiLE and oral food challenge tests on patients with egg allergy (P?=?0.001687, Fisher's exact test). The measured values of EXiLE and the CAP test also correlated well (R?=?0.9127, Spearman's test).<h4>Conclusion</h4>The EXiLE test using RS-ATL8 cells is a promising in vitro IgE test to evaluate the biological activity of the binding between IgE and allergens.
Project description:The protein-tyrosine phosphatase (PTP) Shp2 has been implicated in many immunoreceptor signaling pathways, but its role in immunoreceptor Fc?RI signaling, which leads to the activation of mast cells and blood basophils, is still largely undefined. Using Shp2 knockdown RBL-2H3 (RBL) mast cells, we here reported that Shp2 is required for the activation of RBL cells induced by Fc?RI. Fc?R?-evoked degranulation, calcium mobilization, and synthesis of cytokine transcripts (IL-1?, IL-10, and monocyte chemoattractant protein 1 (MCP-1)) were reduced in Shp2 knockdown RBL cells. Signaling regulatory mechanism investigation using immunoblotting, immunoprecipitation, and GST pull-down assay reveals that the down-regulation of Shp2 expression in RBL cells leads to decreased activities of Fyn, PLC?, JNK, p38MAPK, and Ras/Erk1/2 after Fc?R? aggregation. Further studies suggest that Paxillin phosphoryaltion was also impaired, but PAG phosphorylation was normal after Fc?R? stimulation as a consequence of the inhibition of Shp2 expression in RBL cells. Collectively, our data strongly indicate that Shp2 is essential for the activation of RBL cells in response to Fc?R? aggregation. Shp2 regulates this process through Fyn and Ras with no involvement of PAG. In addition, we identify Paxillin as an indirect substrate of Shp2 in Fc?R?-initiated signaling of RBL cells.
Project description:Hydrogen sulfide (H2 S) modulates many pathophysiological processes, including inflammation and allergic reactions, in which mast cells act as major effector cells. IgE receptor (Fc?RI) cross linking leads to an increase in intracellular calcium ([Ca+2 ]i ), a critical step in mast cell degranulation. The aim of this study was to investigate the role of H2 S in [Ca+2 ]i -dependent mast cell activation.We investigated the effects of H2 S, either endogenously produced or released by the slow H2 S donor 4-carboxy-phenyl isothiocyanate (PhNCS-COOH), on antigenic- and non-antigenic degranulation of native murine mast cells, and human and rat (RBL-2H3) mast cell lines. We measured the release of specific mast cell degranulation markers (?-hexosaminidase and renin), as well as changes in [Ca+2 ]i and phosphorylation of proteins downstream of Fc?RI activation.Endogenously produced H2 S inhibited antigen-induced degranulation in RBL-2H3. Similarly, H2 S released by PhNCS-COOH (10-300 ?M) reduced, in a concentration-dependent manner, antigenic and non-antigenic degranulation and renin release in all mast cell types. Notably, PhNCS-COOH also prevented in a concentration-dependent mode the increase in [Ca+2 ]i elicited by Ca+2 ionophore, thapsigargin and Fc?RI activation. Moreover, PhNCS-COOH attenuated the phosphorylation of Syk, cPLA-2 and PLC?1 in antigen-stimulated RBL-2H3 cells.Collectively, our results demonstrate that, by attenuating the phosphorylation of proteins downstream of Fc?RI cross-linking on mast cells, H2 S diminishes [Ca+2 ]i availability and thus mast cell degranulation and renin release. These findings suggest that PhNCS-COOH could be a strategic therapeutic tool in mast cell-mediated allergic conditions.
Project description:Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca(2+) oscillations. Although oscillations arise from regenerative Ca(2+) release, they are sustained by store-operated Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca(2+) oscillations, CRAC channel activity, and downstream Ca(2+)-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FC?RI receptors evoked Ca(2+) oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca(2+) signals and downstream responses.
Project description:In this study, we present findings that suggest that PI3K-C2?, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of Fc?RI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2?. The knockdown impaired the Fc?RI-induced release of a lysosome enzyme, ?-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-C2?-deficient cells. The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2?. In wild-type cells, Fc?RI 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-C2? and PtdIns(3,4)P2 was observed. These results indicated that PI3K-C2? and its product PtdIns(3,4)P2 may play roles in the secretory process.
Project description:Antigen-mediated cross-linking of IgE bound to its receptor, Fc?RI, initiates a transmembrane signaling cascade that results in mast cell activation in the allergic response. Using immunogold labeling of intact RBL mast cells and scanning electron microscopy (SEM), we visualize molecular reorganization of IgE-Fc?RI and early signaling proteins on both leaflets of the plasma membrane, without the need for ripped off membrane sheets. As quantified by pair correlation analysis, we observe dramatic changes in the nanoscale distribution of IgE-Fc?RI after binding of multivalent antigen to stimulate transmembrane signaling, and this is accompanied by similar clustering of Lyn and Syk tyrosine kinases, and adaptor protein LAT. We find that Lyn co-redistributes with IgE-Fc?RI into clusters that cross-correlate throughout 20 min of stimulation. Inhibition of tyrosine kinase activity reduces the numbers of both IgE-Fc?RI and Lyn in stimulated clusters. Coupling of these proteins is also decreased when membrane cholesterol is reduced either before or after antigen addition. These results provide evidence for involvement of Fc?RI phosphorylation and cholesterol-dependent membrane structure in the interactions that accompany IgE-mediated activation of RBL mast cells. More generally, this SEM view of intact cell surfaces provides new insights into the nanoscale organization of receptor-mediated signaling complexes in the plasma membrane.
Project description:C-reactive protein (CRP) is an important biomarker for inflammatory diseases. However, its role in inflammation beyond complement-mediated pathogen clearance remains poorly defined. We identified the major IgA receptor, Fc?RI, as a ligand for pentraxins. CRP recognized Fc?RI both in solution and on cells, and the pentraxin binding site on the receptor appears distinct from that recognized by IgA. Further competitive binding and mutational analysis showed that Fc?RI bound to the effector face of CRP in a region overlapping with complement C1q and Fc? receptor (Fc?R) binding sites. CRP cross-linking of Fc?RI resulted in extracellular signal-regulated kinase (ERK) phosphorylation, cytokine production, and degranulation in Fc?RI-transfected RBL cells. In neutrophils, CRP induced Fc?RI surface expression, phagocytosis, and TNF-? secretion. The ability of CRP to activate Fc?RI defines a function for pentraxins in inflammatory responses involving neutrophils and macrophages. It also highlights the innate aspect of otherwise humoral immunity-associated antibody receptors.
Project description:Antigen-mediated cross-linking of Immunoglobulin E (IgE) bound to mast cells/basophils via Fc?RI, the high affinity IgE Fc-receptor, is a well-known trigger of allergy. In humans, but not mice, dendritic cells (DCs) also express Fc?RI that is constitutively occupied with IgE. In contrast to mast cells/basophils, the consequences of IgE/Fc?RI signals for DC function remain poorly understood. We show that humanized mice that express Fc?RI on DCs carry IgE like non-allergic humans and do not develop spontaneous allergies. Antigen-specific IgE/Fc?RI cross-linking fails to induce maturation or production of inflammatory mediators in human DCs and Fc?RI-humanized DCs. Furthermore, conferring expression of Fc?RI to DCs decreases the severity of food allergy and asthma in disease-relevant models suggesting anti-inflammatory IgE/Fc?RI signals. Consistent with the improved clinical parameters in vivo, antigen-specific IgE/Fc?RI cross-linking on papain or lipopolysaccharide-stimulated DCs inhibits the production of pro-inflammatory cytokines and chemokines. Migration assays confirm that the IgE-dependent decrease in cytokine production results in diminished recruitment of mast cell progenitors; providing a mechanistic explanation for the reduced mast cell-dependent allergic phenotype observed in Fc?RI-humanized mice. Our study demonstrates a novel immune regulatory function of IgE and proposes that DC-intrinsic IgE signals serve as a feedback mechanism to restrain allergic tissue inflammation.
Project description:This study is aimed at determining whether Sesamum indicum Linn. beneficially influences Fc?RI-mediated allergic reactions in RBL-2H3 mast cells; it is also aimed at further investigating Lyn/Fyn and Syk signaling pathways. To examine the antiallergic effect of Sesamum indicum Linn. extract (SIE), we treated antigen/immunoglobulin E- (IgE-) sensitized mast cells with extracts of various concentrations. We examined the degranulation release and concentrations of inflammatory mediators. Additionally, the expressions of genes involved in the Fc?RI and arachidonate signaling pathways were examined. SIE inhibited the degranulation and secretion of inflammatory mediators in antigen/IgE-sensitized mast cells. SIE reduced the expressions of Fc?RI signaling-related genes, such as Syk, Lyn, and Fyn, and the phosphorylation of extracellular signal-regulated kinase in antigen/IgE-sensitized mast cells. Additionally, in late allergic responses, SIE reduced PGD2 release and COX-2 and cPLA2 phosphorylation expression in Fc?RI-mediated mast cell activation. Lastly, 250-500?mg/kg SIE significantly attenuated the Ag/IgE-induced passive cutaneous anaphylaxis (PCA) reaction in mice. The potent effect of SIE on RBL-2H3 mast cell activation indicates that the extract could potentially be used as a novel inhibitor against allergic reactions.
Project description:Black tiger shrimp Penaeus monodon is one of the common causes of shellfish allergy that is increasing worldwide. One of the important problems in the management of shellfish allergy is the lack of accurate diagnostic assay because the biological and immunological properties of allergens in black tiger shrimp have not been well characterized. This study aims to detect proteins with the ability to bind and cross-link immunoglobulin E (IgE) from black tiger shrimp by enzyme-linked immunosorbent assay (ELISA), Western blot, and a humanized rat basophilic leukemia reporter cell line RS-ATL8.Sera from shrimp allergic subjects were subjected to ELISA and Western blots using raw or cooked shrimp extract as antigens. Pooled sera were used to sensitize the RS-ATL8 reporter cell line and cells were activated by shrimp extract. Eluted protein extracts separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were tested on the RS-ATL8 cell line and subjected to mass spectrometry to identify potential candidate allergens.Allergic sera reacted stronger to raw shrimp extract than cooked shrimp extract (P=0.009). Western blot demonstrated that major IgE reactivity protein bands were at 32-39 kDa and 91-230 kDa in both raw and cooked shrimp extracts. The eluted protein bands at the molecular weight of 38 and 115 kDa from raw shrimp extract induced IgE cross-linking as assayed by the RS-ATL8 cell line. These protein bands were subjected to mass spectrometry for analysis. Ubiquitin-activating enzyme and crustacyanin were identified as potential candidate novel shrimp allergens.The RS-ATL8 reporter cell line can be used to identify potential new shrimp allergens that can functionally cross-link IgE and induce mast cell degranulation.