The RBL-2H3 cell line: its provenance and suitability as a model for the mast cell (1)
RBL-2H3 cells have been commonly and successfully used, in particular, in studies on binding of IgE to FceRI receptors and subsequent downstream events. Basophils and mast cells are two functionally similar but distinct cell types that play pivotal roles in the initiation and development of type I hypersensitivity reactions. Mucosal mast cells (MMC) and basophils respond in different ways to LPS (via TLR4), and show a different expression pattern of other TLRs.
Degranulation
The release of b-hexosaminidase after stimulation by mast cell degranulating peptide (MCDP) was tested. MCDP (0.3–10 ug/ml) failed to produce a degranulation from RBL-2H3 cells. This unresponsiveness of RBL-2H3 to compound MCDP and other polybasic compounds is not the only characteristic that this cell line shares with MMC. RBL-2H3 cells respond well to calcium ionophore A23187 in a fashion similar to both mast cells and basophils. Calcium ionophore A23187 at 5 ug/ml resulted in a degranulation in RBL-2H3 cells of almost 50% of total histamine content; this represents a value 1.65 times that obtained by IgE.
Granular mediators
Histamine is certainly present in RBL-2H3 cells, but the amount of histamine in this cell line, and relative to mast cells, is unclear. For example the histamine content of RBL-2H3 cells has been reported to be between 20–45 ng histamine per 1 x 105 cells, and 100 ng per 1 x 105 cells. b-Hexosamidinase is commonly used as a marker for degranulation in both RBL-2H3 cells and RPMC since it is released in parallel with histamine. Release of serotonin from RBL-2H3 would appear to be biphasic, with release from large granules minutes after activation, and the bulk (>70%) of serotonin from small granules released over a period of 10 minutes. Serotonin release to IgE appears to follow a bell-shaped concentration/release curve in a manner similar to that of histamine.
Stabilisers
Mast cells can be triggered to release newly synthesized mediators, such as TNF-a, after TLR4 stimu- lation by lipopolysaccaharide (LPS), without concurrent degranulation. They can also degranulate through a TLR2- dependent pathway. RBL-2H3 are unresponsive to
LPS, although TLR4 receptors are expressed on the cell surface. This is inconsistent with previously published data where others have shown RBL-2H3 releasing up to 180 pg/ml of IL-13 and 60 ng/ml of TNF-a; it is not clear why there should be such a conflict of data. In support of their findings, Frankish and co-workers have since shown that RBL-2H3 cells do not express associated elements of the TLR4 signaling pathway: they are deficient in both CD14 and MyD88. In addition, RBL-2H3 cells are unresponsive to TLR-2 ligands such as peptidoglycan and, in fact, TLR-2 receptors are not expressed in this cell line.
Since RBL-2H3 cells are tumorous cells, they obviously present aberrant characteristics and abnormalities. Additionally, culture conditions can affect their phenotype, leading to different strains. Even within the same strain, experimental conditions can have a profound effect on their function. RBL-2H3 cells show many characteristics common to both MMC and basophils; they present similar responses to LPS as basophils but diverge from both cell types in their TLR expression.