RBL cells as models for in vitro studies of mast cells and basophils.

Since their establishment in 1981, RBL-2H3 cells have been widely used as a mast cell (MC) model. Their ability to be easily grown in culture in large amounts, their responsiveness to FcεRI-mediated triggers and the fact that they can be genetically manipulated, have provided advantages over primary MCs, in particular for molecular studies relying on genetic screening. Furthermore, the ability to generate clones that stably express proteins of interest, for example, a human receptor, have marked the RBL cells as an attractive MC model for drug screening. Indeed, 3 RBL reporter cell lines (RS-ATL8, NFAT-DsRed, and NPY-mRFP) have been generated providing useful models for drug and allergen screening. Similarly, RBL cells stably expressing the human MrgprX2 receptor provide a unique paradigm for analyzing ligand interactions and signaling pathways of the unique human receptor. Finally, transient co-transfections of RBL cells allow functional genomic analyses of MC secretion by combining library screening with simultaneous expression of a reporter for exocytosis. RBL cells thus comprise powerful tools for the study of intracellular membrane trafficking and exocytosis and the detection of allergens, vaccine safety studies and diagnosis of allergic sensitization. Their recent uses as an investigative tool are reviewed here.

The Humanised NPY-mRFP RBL Reporter Cell Line Is a Fast and Inexpensive Tool for Detection of Allergen-Specific IgE in Human Sera.

Rat basophilic leukaemia (RBL) cells have been used for decades as a model of high-affinity Immunoglobulin E (IgE) receptor (FcεRI) signalling. Here, we describe the generation and use of huNPY-mRFP, a new humanised fluorescent IgE reporter cell line. Fusion of Neuropeptide Y (NPY) with monomeric red fluorescent protein (mRFP) results in targeting of fluorescence to the granules and its fast release into the supernatant upon IgE-dependent stimulation. Following overnight sensitisation with serum, optimal release of fluorescence upon dose-dependent stimulation with allergen-containing extracts could be measured after 45 min, without cell lysis or addition of any reagents. Five substitutions (D194A, K212A, K216A, K226A, and K230A) were introduced into the FcεRIα cDNA used for transfection, which resulted in the removal of known endoplasmic reticulum retention signals and high surface expression of human FcεRIα* in huNPY-mRFP cells (where * denotes the penta-substituted variant), comparable to the ~500,000 FcεRIα molecules per cell in the RS-ATL8 humanised luciferase reporter, which is a human FcεRIα/FcεRIγ double transfectant. The huNPY-mRFP reporter was used to demonstrate engagement of specific IgE in sera of Echinococcus granulosus-infected individuals by E. granulosus elongation factor EgEF-1ß and, to a lesser extent, by EgEF-1δ, which had been previously described as IgE-immunoreactive EgEF-1ß/δ.

Measurement of Exocytosis in Genetically Manipulated Mast Cells.

The hallmark of mast cell activation is secretion of immune mediators by regulated exocytosis. Measurements of mediator secretion from mast cells that are genetically manipulated by transient transfections provide a powerful tool for deciphering the underlying mechanisms of mast cell exocytosis. However, common methods to study regulated exocytosis in bulk culture of mast cells suffer from the drawback of high signal-to-noise ratio because of their failure to distinguish between the different mast cell populations, that is, genetically modified mast cells versus their non-transfected counterparts. In particular, the low transfection efficiency of mast cells poses a significant limitation on the use of conventional methodologies. To overcome this hurdle, we developed a method, which discriminates and allows detection of regulated exocytosis of transfected cells based on the secretion of a fluorescent secretory reporter. We used a plasmid encoding for Neuropeptide Y (NPY) fused to a monomeric red fluorescent protein (NPY-mRFP), yielding a fluorescent secretory granule-targeted reporter that is co-transfected with a plasmid encoding a gene of interest. Upon cell trigger, NPY-mRFP is released from the cells by regulated exocytosis, alongside the endogenous mediators. Therefore, using NPY-mRFP as a reporter for mast cell exocytosis allows either quantitative, via a fluorimeter assay, or qualitative analysis, via confocal microscopy, of the genetically manipulated mast cells. Moreover, this method may be easily modified to accommodate studies of regulated exocytosis in any other type of cell.

NPY-mRFP Rat Basophilic Leukemia (RBL) Reporter: A Novel, Fast Reporter of Basophil/Mast Cell Degranulation.

Humanized rat basophilic leukemia (RBL) reporter cell lines are increasingly used for the detection of allergen-specific IgE and other purposes, such as the detection of allergens and standardization of allergen preparations. Existing reporter systems have many strengths and advantages but can be expensive or require longer incubation times. The new NPY-mRFP reporter cell line addresses such problems, as it requires neither expensive substrates nor overnight incubation for detection of activation. The fusion of Neuropeptide Y (NPY) with monomeric Red Fluorescent Protein (mRFP) results in localization of the fluorescent protein in granules.  As NPY-mRFP is preformed in granules, the reporter system activation can be assessed using fluorescence measurements after as soon as 45-60 min, as described in this chapter, without the need to add any substrates.