Utility of mast cell P815 for evaluating FcεRI-dependent and FcεRI-independent allergic effects
Main Article Content
Keywords
anaphylaxis, G protein-coupled receptor, mast cell degranulation, signal transduction, mast cell
Abstract
Introduction and Objectives: The degranulation and release of inflammatory mediators mediated by the high-affinity immunoglobulin E (IgE) receptor (FcεRI) on mast cells in response to allergen contact is the driving force of anaphylaxis. This study shows that P815 cells, which were previously thought not to express FcεRI, cause a reaction similar to FcεRI-mediated degranulation in the presence of antigen and IgE.
Materials and Methods: The kinetics of degranulation were evaluated by comparing P815 cells with FcεRI-expressing a rat basophilic leukemia (RBL-2H3) (that typically indicates the specific clone or subline within that cell line) cell lines. Degranulation activity was measured using the release rate of β-hexosaminidase as an indicator. P815 cells showed significant degranulation when compound 48/80 or anti-dinitrophenyl (DNP)-IgE antibody and DNP-human serum albumin (HSA) antigen were added simultaneously. Gene expression analysis confirmed the expression of each FcεRI subunit—specifically, the γ subunit expressed markedly. Moreover, the expression of the phosphorylation enzymes Lyn, spleen tyrosine kinase (Syk), Fyn, and Bruton tyrosine kinase (Btk), which are involved in degranulation, was upregulated.
Results: FcεRI has three subunits: α, β, and γ. P815 cells do not express FcεRI because they have messenger ribonucleic acid (mRNA) for the γ subunit but not for the α and β subunits. However, P815 expressed each subunit protein (α, β, and γ), as detected in the western blotting analysis of cell extracts in the presence of DNP-HSA antigen and anti-DNP-IgE.
Conclusion: These results suggest that P815 may cause degranulation via FcεRI. Therefore, P815 is considered to be a cell model that can evaluate both FcεRI-mediated and FcεRI-independent degranulation reactions in response to allergens.
References
2 Blank U, Ra C, Miller L, White K, Metzger H, Kinet JP. Complete structure and expression in transfected cells of high affinity IgE receptor. Nature. 1989;337 (6203):187–9. 10.1038/337187a0
3 Ra C, Jouvin MH, Kinet JP. Complete structure of the mouse mast cell receptor for IgE (Fc epsilon RI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells. J Biol Chem. 1989;264(26):15323–7. 10.1016/S0021-9258(19)84829-9
4 Dunn TB, Potter M. A transplantable mast-cell neoplasm in the mouse. J Natl Cancer Inst. 1957;18(4):587–601.
5 Miller L, Alber G, Varin-Blank N, Ludowyke R, Metzger H. Transmembrane signaling in P815 mastocytoma cells by transfected IgE receptors. J Biol Chem. 1990;265(21):12444–53. 10.1016/S0021-9258(19)38366-8
6 Nagata Y, Suzuki R. FcεRI. A Master regulator of mast cell functions. Cells. 2022;11(4):622. 10.3390/cells11040622
7 Yu Y, Blokhuis BR, Garssen J, Redegeld FA. Non-IgE mediated mast cell activation. Eur J Pharmacol. 2016;778:33–43. 10.1016/j.ejphar.2015.07.017
8 Nilsson G, Johnell, Hammer CH, Tiffany HL, Nilsson K, Metcalfe DD, et al. C3a and C5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway. J Immunol. 1996;157(4):1693–8.
9 Kirshenbaum AS, Swindle E, Kulka M, Wu Y, Metcalfe DD. Effect of lipopolysaccharide (LPS) and peptidoglycan (PGN) on human mast cell numbers, cytokine production, and protease composition. BMC Immunol. 2008;9:45. 10.1186/1471-2172-9-45
10 Hültner L, Kölsch S, Stassen M, Kaspers U, Kremer J-P, Mailhammer R, et al. In activated mast cells, IL-1 upregulates the production of several Th2-related cytokines including IL-9. J Immunol. 2000;164(11):5556–63. 10.4049/jimmunol.164.11.5556
11 Irman-Florjanc T, Erjavec F. Compound 48/80 and substance P induced release of histamine and serotonin from rat peritoneal mast cells. Agents Act. 1983;13(2-3):138–41. 10.1007/BF01967317
12 Kashem SW, Subramanian H, Collington SJ, Magotti P, Lambris JD, Ali H. G protein coupled receptor specificity for C3a and compound 48/80-induced degranulation in human mast cells: Roles of mas-related genes MrgX1 and MrgX2. Eur J Pharmacol. 2011;668(1-2):299–304. 10.1016/j.ejphar.2011.06.027
13 Zylka MJ, Dong X, Southwell AL, Anderson DJ. Atypical expansion in mice of the sensory neuron-specific Mrg G protein-coupled receptor family. Proc Natl Acad Sci USA. 2003;100(17):10043–8. 10.1073/pnas.1732949100
14 Wang N, Liu R, Liu Y, Zhang R, He L. Sinomenine potentiates P815 cell degranulation via upregulation of Ca2+ mobilization through the Lyn/PLCγ/IP3R pathway. Int J Immunopathol Pharmacol. 2016;29(4):676–83. 10.1177/0394632015621768
15 Hu X, Wen Y, Liu S, Luo J, Tan X, Li Z, et al. Evaluation of the anaphylactoid potential of andrographis paniculata extracts using the popliteal lymph node assay and P815 cell degranulation in vitro. J Transl Med. 2015;13:121. 10.1186/s12967-015-0478-0
16 Péterfy H, Tóth G, Pecht I, Erdei A. C3a-derived peptide binds to the type I FcepsilonR and inhibits proximal-coupling signal processes and cytokine secretion by mast cells. Int Immunol. 2008;20(10):1239–45. 10.1093/intimm/dxn083
17 Matsuda H, Nakamura S, Yoshikawa M. Degranulation inhibitors from medicinal plants in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells. Chem Pharm Bull. 2016;64(2):96–103. 10.1248/cpb.c15-00781
18 Goldring JPD. Measuring protein concentration with absorbance, Lowry, Bradford Coomassie Blue, or the Smith bicinchoninic acid assay before electrophoresis. Methods Mol Biol. 2019;1855:31–9. 10.1007/978-1-4939-8793-1_3
19 Kalesnikoff J, Huber M, Lam V, Damen JE, Zhang J, Siraganian RP, et al. Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity. 2001;14(6):801–11. 10.1016/s1074-7613(01)00159-5
20 Wang J, Liu J, Yang Y, Sun G, Yang D, Yin S, et al. Inhibitory effect of phellodendrine on C48/80-induced allergic reaction in vitro and in vivo. Int Immunopharmacol. 2024;134:112256. 10.1016/j.intimp.2024.112256
21 Zhang X, Yao H, Qian Q, Li N, Jin W, Qian Y. Cerebral mast cells participate in postoperative cognitive dysfunction by promoting astrocyte activation. Cell Physiol Biochem. 2016;40(1-2):104–16. 10.1159/000452528
22 Bugajev V, Bambousková M, Dráberová L, Dráber P. What precedes the initial tyrosine phosphorylation of the high affinity IgE receptor in antigen-activated mast cell? FEBS Lett. 2010;584(2-4):4949–55. 10.1016/j.febslet.2010.08.045
23 Gilfillan AM, Rivera J. The tyrosine kinase network regulating mast cell activation. Immunol Rev. 2009;228(1):149–69. 10.1111/j.1600-065X.2008.00742.x
24 Kageyama-Yahara N, Wang X, Katagiri T, Wang P, Yamamoto T, Tominaga M, et al. Suppression of phospholipase Cγ1 phosphorylation by cinnamaldehyde inhibits antigen-induced extracellular calcium influx and degranulation in mucosal mast cells. Biochem Biophys Res Commun. 2011;416(3-4):283–8. 10.1016/j.bbrc.2011.11.014
25 Ito N, Yokomizo T, Sasaki T, Kurosu H, Penninger J, Kanaho Y, et al. Requirement of phosphatidylinositol 3-kinase activation and calcium influx for leukotriene B4-induced enzyme release. J Biol Chem. 2002;277(47):44898–904. 10.1074/jbc.M208051200
26 Wang Z, Franke K, Bal G, Li Z, Zuberbier T, Babina M. MRGPRX2-mediated degranulation of human skin mast cells requires the operation of Gαi, Gαq, Ca++ channels, ERK1/2 and PI3K—Interconnection between early and late signaling. Cells. 2022;11(6):953. 10.3390/cells11060953
27 Thathiah P, Sanapala S, Rodriguez AR, Yu JJ, Murthy AK, Guentzel MN, et al. Non-FcεR bearing mast cells secrete sufficient interleukin-4 to control Francisella tularensis replication within macrophages. Cytokine. 2011;55(2):211–20. 10.1016/j.cyto.2011.04.009
28 Li E, Lin N, Hao R, Fan X, Lin L, Hu G, et al. 5-HMF induces anaphylactoid reactions in vivo and in vitro. Toxicol Rep. 2020;7:1402–11. 10.1016/j.toxrep.2020.10.010
29 Tashiro M, Kawakami Y, Abe R, Han W, Hata D, Sugie K, et al. Increased secretion of TNF-alpha by costimulation of mast cells via CD28 and Fc epsilon RI. J Immunol. 1997;158(5):2382–9.
30 Fukamachi H, Yamada N, Miura T, Kato T, Ishikawa M, Gulbins E, et al. Identification of a protein, SPY75, with repetitive helix-turn-helix motifs and an SH3 domain as a major substrate for protein tyrosine kinase(s) activated by Fc epsilon RI cross-linking. J Immunol. 1994;152(2):642–52.
31 Li Y, Leung PSC, Gershwin ME, Song J. New mechanistic advances in fcεri-mast cell-mediated allergic signaling. Clin Rev Allergy Immunol. 2022;63(3):431–46. 10.1007/s12016-022-08955-9
32 Karasuyama H, Miyake K, Yoshikawa S, Kawano Y, Yamanishi Y. How do basophils contribute to Th2 cell differentiation and allergic responses? Int Immunol. 2018;30(9):391–6. 10.1093/intimm/dxy026
33 Dwyer DF, Barrett NA, Austen KF. Immunological genome project C. Expression profiling of constitutive mast cells reveals a unique identity within the immune system. Nat Immunol. 2016;17(7):878–87. 10.1038/ni.3445
34 Motakis E, Guhl S, Ishizu Y, Itoh M, Kawaji H, de Hoon M, et al. Redefinition of the human mast cell transcriptome by Deep-CAGE sequencing. Blood. 2014;123(17):e58–67. 10.1182/blood-2013-02-483792
35 Miyake K, Ito J, Karasuyama H. Role of basophils in a broad spectrum of disorders. Front Immunol. 2022;13:902494. 10.3389/fimmu.2022.902494
36 Hussain M, Borcard L, Walsh KP, Pena Rodriguez M, Mueller C, Kim BS, et al. Basophil-derived IL-4 promotes epicutaneous antigen sensitization concomitant with the development of food allergy. J Allergy Clin Immunol. 2018;141(1):223–34. 10.1016/j.jaci.2017.02.035
37 Muto T, Fukuoka A, Kabashima K, Ziegler SF, Nakanishi K, Matsushita K, et al. The role of basophils and proallergic cytokines, thymic SLP and IL-33, in cutaneously sensitized food allergy. Int Immunol. 2014;26(10):539–49. 10.1093/intimm/dxu058
38 Noti M, Kim BS, Siracusa MC, Rak GD, Kubo M, Moghaddam AE, et al. Exposure to food allergens through inflamed skin promotes intestinal food allergy through the thymic stromal lymphopoietin-basophil axis. J Allergy Clin Immunol. 2014;133(5):1390–9. 10.1016/j.jaci.2014.01.021
39 Miyake K, Shibata S, Yoshikawa S, Karasuyama H. Basophils and their effector molecules in allergic disorders. Allergy. 2021;76(6):1693–706. 10.1111/all.14662