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.