3.2 Inducers and mediators release for HMC-1 cells
As the HMC-1 cell line lacks the expression of FcεRI, an external stimulus is required to induce mast cell degranulation. The combination of PMA and CI is widely used (25 of 50 and 28 of 50 respectively). Other inducers include OVA, IL-33, TSLP, protein kinase activator C, RANKL, LPS, histamine, and more. However, three of the 50 publications did not specify the type of inducers used. The induction time implemented by most studies falls within 5 to 8 hours (h) (23 of 50), followed by an hour and below (20 of 50), within 13 to 24 h (16 of 50), 2 to 4 h (13 of 50) and 9 to 12 h (2 of 50). However, four publications did not indicate the induction time. The degranulation of mast cells can be characterized by the concentration of cytokines secreted. Many of the publications studied the release of TNF-α (29 of 50), IL-6 (27 of 50), IL-1β (20 of 50), histamine (17 of 50), TSLP (14 of 50), and IL-8 (12 of 50). Table 2 summarizes our analysis of the type of inducers, induction time, and type of mediators often studied by researchers using HMC-1 cells.
Mast cell activation depends on the cross-linking of IgE antibodies on the FcεRI and the subsequent signal transduction cascade, including intracellular Ca2+ mobilization, influx, and protein kinase C (PKC) activation. As such, the combination of PMACI is a strong candidate as an inducer owing to its ability to enhance Ca2+ influx and activation of MAP kinases 35,36. PMA stimulates PKC activity, while CI raises the intracellular level of Ca2+ 37,38. Many clinical studies have shown the upregulation of cytokines released (Th1-related cytokines) upon PMACI stimulation, namely the IL-2, IL-6, IL-17, IFN-γ, and TNF-α. However, these studies did not affect the expression of IL-4 and IL-10 39–41. Another point to note in future research is that the expressions of IL-10, IL-17, IL-22, IFN-γ, and TNF-α are higher in IMDM than RPMI-1640 upon PMACI stimulation due to the concentration of Ca2+. According to Zimmermann and his colleagues (2015), IMDM consists of 1.49 mM of Ca2+, while RPMI-1640 contains 0.42mM of Ca2+. Therefore, by increasing the Ca2+ concentration in RPMI-1640 to 1.5 mM, the expression of cytokines is comparable to IMDM. Vice versa, the reduction of Ca2+ concentration in IMDM resulted in a lowered cytokine expression. Thus, implying that 1.5 mM of Ca2+ is optimal for maximal ionomycin stimulation in cells 42.
When compared between PMACI, lectin phytohaemagglutinin (PHA), LPS, Con-A, and pokeweed mitogen, PMACI expresses the most potent cytokine production in a short period without significant damage to the cells43. On the other hand, C48/80 acts as a ”selective” mast cell activator by stimulating the trimeric G-proteins and activating phospholipase C and D pathways 44. C48/80 activation can bypass Ca2+ and PKC signal transduction, thus beneficial when PMACI cannot be used35.