4.2 Inducers and mediators release for LAD2 cells
As LAD2 cells express the high affinity IgE receptor, they can be sensitized with antibody to induce degranulation. Among the 75 shortlisted articles, only 24 of them reported that they sensitized LAD2 cells with the IgE antibody. Other studies directly activate the LAD2 cells to degranulate using their chosen inducer(s). Several inducers have been used in studies involving LAD2 cells. This includes the common C48/80 which was being used by 36 of the studies. C48/80 is a known mast cell degranulator that has been used in countless allergy-related studies 48. Substance P is also commonly used as an inducer of LAD2 cells (14 of 75). Substance P is an undecapeptide found in the human body and it is secreted from the terminals of specific sensory nerves 49 and it is a known ligand of MRGPRX250. A major product of the adrenomedullin precursor, PAMP(9-20) 49 was used by three research groups to induce LAD2 cells in this analysis. Other inducers that were being used to activate LAD2 cells include Tween 20, LL-37, (R)-ZINC-3573, CST-14, Thapsigargin and complement C3a as shown in Table 4. Apart from these common inducers, several studies have analyzed the degranulation effects of antidepressants (clomipramine, paroxetine and desipramine), contrast media (gadopentetate meglumine, iodinated contrast media and iopamidol), p-Phenylenediamine (PPD), P17, morphine derivatives (thebaine and pethidine) and fluoroquinolones and many others. PPD is a component found in hair dyes implicated to induce immediate allergy, acute dermatitis and contact dermatitis51. On the other hand, P17 is a short host defense peptide from the ant Tetramonium bicarinatum venom52. LAD2 cells are commonly used to study MRGPRX2 related pseudo-allergy reactions and thus they were used to identify several novel mast cells compounds 53. Besides that, the effects following exposure to influenza A virus were also studied using LAD2 cells 54. As LAD2 cells are mast cells, some researchers chose to pre-sensitize the cells first before inducing them with the corresponding inducers. These studies used DNP-IgE as sensitizer (18 of 75) and then followed by either DNP-HSA, DNP-BSA, DNP-streptomycin, or streptavidin as inducers in their studies. Other combinations such as biotinylated IgE with streptavidin and human myeloma IgE with anti-IgE were also used by several researchers.
From the articles analyzed, histamine (36 of 75) and β-hexosaminidase (62 of 75) are the two common mediators that were often evaluated. Both mediators are pro-inflammatory mediators that play key roles as degranulation biomarkers 55. Other mediators include cytokines and chemokines. Thirty out of 75 articles evaluated the levels of MCP-1, while 28 analyzed TNF-α. Several ILs were analyzed too, and these were IL-8 (27 of 75); IL-6 (5 of 75) and others. Several other mediators evaluated include 5-hydroxytryptamine (5-HT), tryptase, MIP-1α, MIP2, prostaglandin 2 (PGD2), leukotriene B4 (LTB4), granulocyte-macrophage colony-stimulating factor (GM-CSF) and many others. The induction time differs based on the type of mediators studied. From our analysis, generally, LAD2 cells were induced between 0-1 h when β-hexosaminidase and histamine were to be evaluated. The duration of 30 minutes (mins) was the most common induction period while some groups induced the cells for 40 mins, with the longest being 2 h56. The shortest duration used to stimulate LAD2 cells to produce β-hexosaminidase was 15 mins by Park et al. (2019) and histamine for 10 mins by Sun et al. (2021) 57,58. For the evaluation of late mediators such as TNF-α, ILs, MCP-1 and others, longer induction time was needed, and it ranged from 6 to 8 h, with 6 h being the most used induction time. Two other induction times were noted which were 12 h and between 24 and 48 h. In the study involving influenza A virus, the cytokines and chemokines were analyzed at days 1, 2 and 4 (maximum) post infection 54. In another study using 12 h induction time, the researchers were evaluating the effects of substance P and PAMP(9-20) on LAD2 cells and analyzed the levels of TNF-α, MCP-1, IL-8 and IL-31 49. On the induction time of 24-48 h, the study studied the effects of P17 on LAD2 cells, analyzing MCP-1 and MIP-1α release 52. Table 4 summarizes our analysis on the type of inducers, induction time and type of mediators often used and studied by researchers using LAD2 cells.