5.3 Humanized RBL cells
As discussed above, RBL cells originated from rats. It was established that rat IgE can bind to human IgE receptor 95, however rat IgE receptor did not recognize human IgE 96. RBL cells expressing the high affinity IgE receptor FcεRI implied that the cell line may be suitable for the detection of allergen specific IgE and allergens59. Stable transfection of human FcεRI α chain in RBL cells was achieved in the 90s with the receptor expressed mediated antigen-induced signaling and mediator release97–100. These humanized RBL cells were then utilized to investigate the ability of pure peanut allergens and other food allergens to induce degranulation 101–104. RBL-SX38 is one of the commonly used humanized RBL cell lines that expresses α, β, and γ chains of human FcεRI 105.
The addition of a reporter gene (firefly luciferase) to humanized RBL cells paved the way for straightforward and extremely sensitive detection of cellular activity via the IgE receptor59. The first and most common system was a Nuclear Factor of Activated T-cells (NFAT)-firefly luciferase reporter that is linked to IgE dependent signal transduction named RS-ATL8 cells 106. The calcium influx from stimulation of the cells activates calcineurin, a phosphatase that dephosphorylates NFAT. This resulted in the unveiling of the nuclear localization signal in the N-terminal and leads to nuclear translocation. NFAT then attaches to specific promoter regions and initiates gene transcription of the luciferase reporter gene107. By detecting luciferase activity with suitable substrates and a luminescence detector, activation of these cells may be detected 106. RS-ATL8 assay is very sensitive and well suited for high throughput format. The assay had since been employed for the identification of food or other allergens, as well as determining the allergenicity of vaccines 108–110.
From our literature retrieval, we identified two articles using the humanized RBL cells, RBL-SX38 111,112 and one article using the reporter humanized cells, RS-ALT8 113 to investigate the release of β-hexosaminidase. RBL-SX38 was obtained from a research group in Harvard Medical School, USA while the study using RS-ALT8 did not specify the cells’ origin. Both RBL-SX38 and RS-ALT8 cells were grown in MEM supplemented with 10% FBS, L-glutamine and antibiotics. One study using RBL-SX38 did not specify the culture condition. In all three studies, cells were sensitized with allergen specific human IgE and induced by their respective allergens.