5.2 RBL-2H3 cell sensitization, induction, and degranulation
Following the crosslinking of their IgE-bound FceRI by multivalent allergens, RBL-2H3 cells, like mast cells and basophils, respond with degranulation, releasing a variety of mediators that trigger a potent immunological allergic response77. The cells were demonstrated to have a bell-shaped dose-response to anti-DNP IgE as observed in primary mast cells78,79. A range of non-immunological triggers such as the C48/80 and A23187 can also cause RBL-2H3 cells to degranulate80. Calcium ionophore A23187 can induce mast cell degranulation by increasing calcium entry into the cells through pore formation or as a transporting carrier 81. RBL-2H3 cells behave similarly to basophils and mast cells when exposed to the A23187 82,83. A23187 at a concentration of 5 µg/mL caused RBL-2H3 cells to degranulate about 50% of available histamine, which is 1.65× more than that of IgE 67. On the other hand, degranulation of MCs, especially connective tissue MCs can be induced by polybasic compounds like C48/80. C48/80 can interact with G0 and Gi and their subfamilies of G-protein coupled receptors (GPCRs) to activate their downstream signaling of degranulation 84–86. However, polybasic compounds were found to be inactive on some MC subfamilies, including RBL cells which may be due to their lack of Gi-387,88. Nonetheless, depending on the culture conditions, they can develop sensitivity to C48/80. Interestingly, quercetin can cause a rise in the expression of Gi-3’s subunits, triggering RBL-2H3 response to C48/80 88. Another study also reported that RBL-2H3 cells change into a C48/80 active phenotype when co-cultured with 3T3 fibroblast89. Unfortunately, they were unable to ascertain the factors implicated with this phenomenon.
Review on the assays performed using RBL-2H3 cells (Table 6) demonstrated that most of the studies sensitize the cells with DNP-BSA and later induce the cells with DNP-HSA (91 of 131) and measured the degranulation of β-hexosaminidase (115 of 131) and histamine (61 of 131). The induction times were mainly reported to be within 1 h (91 of 131), with most of the studies reportedly induced RBL cells for a full 1 h (29 of 131). One study used RBL-2H3 cells for cytotoxicity assay but did not investigate any mediators released nor used any inducers. β-hexosaminidase is an exoglycosidase enzyme that is associated with degranulation, and it is released together with histamine80. Based on our review, β-hexosaminidase is the most common marker used to measure degranulation for RBL-2H3 cells. On the other hand, histamine had a significant role in allergy and inflammatory reactions as it mediates the interactions between inflammatory cells90. The pre-stored histamine within the granules of RBL-2H3 cells were inconclusive as wide range of histamine content had been reported per 1 × 105 cells: from 20-45 ng91, to 100 ng 92 and up to 700 ng67. MCs had been known to produce pro-inflammatory cytokines like IL-4, IL-13 and TNF-α through the induction of TLR4 and TLR2 pathways 93. RBL-2H3 has been observed to release up to 180 pg/mL of IL-13 and 60 ng/mL of TNF-α 94. Our review revealed that 48 of 105 publications had investigated and demonstrated cytokines production in RBL-2H3 cells. However, there are studies reported conflicting findings that RBL-2H3 cells did not respond to lipopolysaccharide induction 66,67. Those studies also demonstrated that RBL-2H3 cells did not expressed CD14 and MyD88 that is implicated in TLR4 signaling pathway. Additionally, they also reported that RBL-2H3 cells were also unresponsive to TLR2 ligands due to the lack of TLR2 receptors 66,67.