Respiratory allergic diseases are increasing worldwide with air pollutants and climate change playing an important role in the severity of asthma and allergic rhinitis. Therefore, continuous characterization of allergens and the development of diagnostic allergy tests are necessary for better diagnosis. Although the SPT is a good diagnostic test, interpretation is often difficult because sensitization and clinical history do not coincide, and there is a risk of anaphylaxis in some cases. An alternative is in vitro IgE tests that use cells such as BAT or RBL. Previously, RBL cell lines have been developed and successfully used in allergic tests. However, the problem is that these cell lines are not commercially available. Hence, the present study aims to develop a cellular model with immortal RBL-2H3 cells as an alternative and complementary method to diagnose allergies in vitro.

Since RBL cells are derived from leukemia rats, human IgE does not recognize their IgE receptor. As a result, several groups have cloned the three subunits of human IgE receptors in RBL cells with different expression levels [6]. Nevertheless, Taudou and colleagues reported that the α-chain is the minimal subunit needed in RBL cells to be sensitized to human IgE [7]. In this sense, we used a commercial plasmid that encodes the α-chain of the human FcεRI receptor with the resistance gene to G418 to humanize RBL-2H3 cells. It is crucial to assess the activation potential of these cells, and employing reporter genes offers significant benefits compared to biochemical assays. Kalli and colleagues have used an RBL cell line that uses the nuclear factor of activated T cells (NFAT) a responsive luciferase reporter gene [8]. Luciferase can be measured using appropriate chemiluminescent substrates. This process is rapid and more sensitive than measuring beta-hexosaminidase release when RBL cells are activated. Then, the plasmid pSIRV-NF-kB-eGFP was selected because it contains a GFP reporter gene under activation of the NF-kB transcription factor. The activation of NF-kB signaling in immune cells drives the expression of pro-inflammatory cytokinesassociated with allergic diseases [9].

Transfected cells were tested with the sera of allergenic patients to Ligustrum tree pollen. Interestingly, in 2015, our group identified six IgE-binding proteins in this pollen employing an immunoproteomics approach. Indeed, 2-DE immunoblotting and mass spectrometry allowed the  identification of the following allergens: profilin, enolase, β-1,3-glucanase, polygalacturonase, alanine aminotransferase, and two beta subunits of ATP-synthase [10]. A limitation is that using a pool of patients’ sera and pollen proteins makes it impossible to determine what each patient is allergic to. Nevertheless, at this time, employing these sera and the proteins, we found that the method we developed is a suitable assay for detecting allergen-specific IgE antibodies. As reported in similar experiments, we employed two sera dilutions, 1:50 and 1:100, with high protein concentrations [11]. Cells responded better with the diluted sera concentration. This could be explained because of the cytotoxicity of the sera over RBL cells, which can be due to serum components such as activated complement. Higher dilutions are recommended but can be insufficient to sensitize cells. Our results showed that cells are neither activated by themselves nor employing sera derived from healthy patients. Likewise, sera and proteins alone cannot stimulate RBL-2H3 cells. Spontaneous degranulation near 1% was observed with the negative controls; we hypothesized that this observation could be due to incubation with sera alone or by senescent cells. Cellular senescence-associated changes can lead to mast cell degranulation (IgE-independent) [12].

This model was activated at levels near positive control using sera 1:100 and 100 µg/mL of pollen proteins. Compound 48/80 has been reported as an activator of degranulation in mast cells [13]. When RBL were stimulated with the same sera concentration and 10 µg/mL of pollen proteins, cell activation was seven-fold lower than that caused by C48/80. These results show that both sera and protein concentrations mediate cellular activation, and GFP is expressed as a result of cell activation. Of particular interest was the finding that changes in cellular shape and granule appearance were observed upon RBL cell activation. These granules  indicate basophil degranulation by IgE binding to the FcεRI receptor. The cellular changes could be taken as a parameter of cell activation without using a fluorescent microscope. The advantages of working with RBL cells lie in their easy cultivation and the ability to test them with various sera and allergenic sources. Indeed, the cellular model described in the present manuscript can be established using commercially available plasmids, allowing its implementation in laboratories with limited infrastructure, as it does not require the use of a flow cytometer. Additionally, integrating  a GFP-ELISA kit could enhance the  accuracy of the results.

The major challenge in allergy diagnosis is the creation of accessible and dependable diagnostic methods that enable accurate prediction. In this study, we have shown that integrating reporter genes into humanized RBL cells could emerge as a potent tool for the clinical diagnosis of intricate allergic conditions and evaluating innovative anti-allergic medications.