Retinoic Acid Negatively Impacts Proliferation and MCTC Specific Attributes of Human Skin Derived Mast Cells, but Reinforces Allergic Stimulability

Abstract

The Vitamin-A-metabolite retinoic acid (RA) acts as a master regulator of cellular programs. Mast cells (MCs) are primary effector cells of type-I-allergic reactions. We recently uncovered that human cutaneous MCs are enriched with RA network components over other skin cells. Yet, direct experimental evidence on the significance of the RA-MC axis is limited. Here, skin-derived cultured MCs were exposed to RA for seven days and investigated by flow-cytometry (BrdU incorporation, Annexin/PI, FcεRI), microscopy, RT-qPCR, histamine quantitation, protease activity, and degranulation assays. We found that while MC size and granularity remained unchanged, RA potently interfered with MC proliferation. Conversely, a modest survival-promoting effect from RA was noted. The granule constituents, histamine and tryptase, remained unaffected, while RA had a striking impact on MC chymase, whose expression dropped by gene and by peptidase activity. The newly uncovered MRGPRX2 performed similarly to chymase. Intriguingly, RA fostered allergic MC degranulation, in a way completely uncoupled from FcεRI expression, but it simultaneously restricted MRGPRX2-triggered histamine release in agreement with the reduced receptor expression. Vitamin-A-derived hormones thus re-shape skin-derived MCs numerically, phenotypically, and functionally. A general theme emerges, implying RA to skew MCs towards processes associated with (allergic) inflammation, while driving them away from the skin-imprinted MC_TC ("MCs containing tryptase and chymase") signature (chymase, MRGPRX2). Collectively, MCs are substantial targets of the skin retinoid network.

Keywords: IgER; cell cycle; chymase; mast cell; proliferation; retinoic acid; skin; tryptase.

Figure 1

Retinoic acid (RA) inhibits cell cycle progression of skin-derived mast cells (MCs), but also modestly interferes with cell death. Cultured skin-derived MCs (at the proliferative stage) were treated with RA as described in the Experimental section and harvested after 7 days. (A) Increase in MC numbers (relative to day 0) in the presence versus absence of RA in a variety of donors, indicated by interconnected dots (n = 17); (B) Annexin⁺ cells (in % of total cells) after culture with and without RA, mean ± SEM, n = 5; (C) Expression of apoptosis related genes in RA treated cells normalized to the “no RA” control, mean ± SEM, n = 9, dotted line drawn at 1 to highlight control expression (in the absence of RA); (D,E) BrdU incorporation (added 5 days before harvest), as determined by flow-cytometry, (D) representative example, (E) mean ± SEM of n = 7; (F) Expression of cell cycle associated genes in RA treated cells normalized to the “no RA” control, mean ± SEM, n = 9; (G) Visualization of MCs upon acidic toluidine blue staining reveals no conspicuous changes by RA; note the bright purple granules and the accumulation of cytoplasm, the latter typical of MCs exposed to SCF; (A,B,E) Paired t-test, (C,F) one-sample t-test versus control (set as 1). * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus control.

Figure 2

Granule-associated mediators: RA dampens chymase mRNA and chymase activity. (A) Relative mRNA expression of histidine decarboxylase (HDC, involved in histamine generation), tryptase and chymase normalized to β actin, respectively, in RA treated and control cells (mean ± SEM, n = 9); (B) Mediator contents. Histamine was quantified by an auto-analyzer based method, while tryptase and chymase activity were measured by cleavage of specific substrates in RA pretreated and control cells, as described in the Experimental section. Each dot corresponds to one MC preparation and dots representing the same donor are connected. n = 11 (histamine) or 9 (tryptase, chymase). ** p < 0.01, *** p < 0.001 versus control (by paired t-test).

Figure 3

RA reinforces FcεRI-mediated degranulation, but has no effect on the expression of the FcεRI receptor complex. (A,B) FcεRI cell surface expression by flow-cytometry; (A) Representative example, (B) cumulative data from n = 8 MC preparations; MFI = Mean fluorescence intensity; (C) Left, Histamine release in the absence of a specific stimulus (spontaneous) in % of the complete cellular histamine present in the respective MC preparation; right, net histamine release specifically elicited by FcεRI-aggregation (note that spontaneous release, determined for each preparation, was subtracted from the corresponding value after FcεRI-triggering) in RA pretreated and control MCs. n = 14; (B,C) Each dot corresponds to one MC preparation and dots representing the same donor are connected; (D) Dose-response of histamine release in RA pretreated and control cells triggered by different concentrations of the IgER-aggregating antibody 29C6, experiment as in (C) but using another set of MC preparations and other concentrations, n = 7. (E) Relative mRNA expression of the three chains forming the FcεRI complex normalized to β actin (mean ± SEM, n = 9). * p < 0.05, ** p < 0.01, **** p < 0.0001 significantly higher than control, + p < 0.05 significantly lower than control; ns = not significant.

Figure 4

Impact of RA on other genes and MC-restricted processes. Relative mRNA expression, normalized to β actin, for (A) the pan-hematopoietic sialoprotein CD43 (the latter used as control of an RA-upregulated gene); (B) the IL-33 receptor ST2/T1, and (C) the MC-selective receptor MRGPRX2, Mean ± SEM, n = 9; (D) net histamine release elicited by MRGPRX2 triggering (by use of its agonist compound 48/80 at 10 μg/mL) in RA pretreated and control MCs (analogous to Figure 3C,D). n = 12. Each dot corresponds to one MC preparation and dots representing the same donor are connected. ** p < 0.01, *** p < 0.001, **** p < 0.0001 significantly different from control (by paired t-test); ns = not significant.