Functional MRGPRX2 expression on peripheral blood-derived human mast cells increases at low seeding density and is suppressed by interleukin-9 and fetal bovine serum

Front Immunol. 2024 Dec 13:15:1506034. doi: 10.3389/fimmu.2024.1506034. eCollection 2024.

Abstract

Primary human mast cells (MC) obtained through culturing of blood-derived MC progenitors are the preferred model for the ex vivo study of MRGPRX2- vs. IgE-mediated MC activation. In order to assess the impact of culture conditions on functional MRGPRX2 expression, we cultured CD34+-enriched PBMC from peripheral whole blood (PB) and buffy coat (BC) samples in MethoCult medium containing stem cell factor (SCF) and interleukin (IL)-3, modified through variations in seeding density and adding or withholding IL-6, IL-9 and fetal bovine serum (FBS). Functional expression of MRGPRX2 was assessed after 4 weeks via flow cytometry. We found similar proportions of CD34+ MC-committed progenitors in BC and PB. Higher seeding densities (≥ 1x105 cells/mL) and exposure to IL-9 and FBS suppressed functional MRGPRX2 expression at 4 weeks, while leaving MC yield largely unaffected. IL-6 had no impact on MRGPRX2 expression. MRGPRX2-expressing MC upregulated CD63 upon stimulation with polyclonal anti-IgE, substance P and compound 48/80 at 4 weeks. Ketotifen and dasatinib but not cromolyn sodium inhibited both IgE- and MRGPRX2-dependent pathways. Our results confirm the feasibility of functional MC activation studies on PB-derived MC after a short 4-week culture and highlight the impact of culture conditions on functional MRGPRX2 expression.

Keywords: MRGPRX2; blood; culture; human; interleukin-9; ketotifen; mast cell; serum.

MeSH terms

  • Animals
  • Cattle
  • Cells, Cultured
  • Humans
  • Interleukin-9* / metabolism
  • Mast Cells* / immunology
  • Mast Cells* / metabolism
  • Nerve Tissue Proteins* / metabolism
  • Receptors, G-Protein-Coupled* / genetics
  • Receptors, G-Protein-Coupled* / metabolism
  • Receptors, Neuropeptide* / genetics
  • Receptors, Neuropeptide* / metabolism

Substances

  • Interleukin-9
  • Receptors, Neuropeptide
  • MRGPRX2 protein, human
  • Nerve Tissue Proteins
  • Receptors, G-Protein-Coupled
  • IL9 protein, human

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This project was supported by a UZ Leuven Clinical Research Council grant (KOOR, S65209) and a Vlaanderen National Fund for Scientific Research (FWO) TBM grant (T001417N). TI is supported by a grant from the Study Center For Allergy Projects (Haarlem, The Netherlands). A-CJ was supported by a FWO predoctoral strategic basic research fellowship (1S20418N). WR is supported by a FWO predoctoral fundamental research fellowship (11PDZ24N). DB and RS are supported by a FWO senior clinical investigator fellowship (1805518N, 1805523N, and 1801019N) and DB is currently supported by a KU Leuven Special Research Fund mandate (BOF-FKO, 3M230694). The funder(s) had no role in the design and conduct of the study, collection, management, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.