Apoptotic resistance of human skin mast cells is mediated by Mcl-1

Abstract

Mast cells (MCs) are major effector cells of allergic reactions and contribute to multiple other pathophysiological processes. MCs are long-lived in the tissue microenvironment, in which they matured, but it remains ill-defined how longevity is established by the natural habitat, as research on human MCs chiefly employs cells generated and expanded in culture. In this study, we report that naturally differentiated skin MCs exhibit substantial resilience to cell death with considerable portions surviving up to 3 days in the complete absence of growth factors (GF). This was evidenced by kinetic resolution of membrane alterations (Annexin-V, YoPro), DNA degradation (propidium iodide), mitochondrial membrane disruption (Depsipher), and Caspase-3 activity. Because of the high basal survival, further protection by SCF was modest. Conversely, survival was severely compromised by staurosporine, implying functional caspase machinery. Contrary to the resistance of freshly purified MCs, their culture-expanded counterpart readily underwent cell death upon GF deprivation. Searching for the molecular underpinnings explaining the difference, we identified Mcl-1 as a critical protector. In fact, silencing Mcl-1 by RNAi led to impaired survival in skin MCs ex vivo, but not their cultured equivalent. Therefore, MCs matured in the skin have not only higher expression of Mcl-1 than proliferating MCs, but also greater reliance on Mcl-1 for their survival. Collectively, we report that human skin MCs display low susceptibility to cell death through vast expression of Mcl-1, which protects from mortality and may contribute to MC longevity in the tissue.

MRGPRX2 is negatively targeted by SCF and IL‐4 to diminish pseudo‐allergic stimulation of skin mast cells in culture

“…We asked whether deprivation of IL‐4, SCF or both impacts MRGPRX2 expression and function. Because cultured MCs lose viability on SCF withdrawal, deprivation was limited to 16 hours, after which time survival is robust (Figure ). We observed a profound drop in MRGPRX2 mRNA levels after re‐addition of SCF (with or without IL‐4) (Figure A).…”

“…To allow analysis at different time points, cells from 2 to 9 foreskins were pooled and utilized as one preparation. Cells were used ex vivo and upon reaching optimal proliferation with SCF+IL‐4). For deprivation experiments, cultured MCs were replated in fresh media (Iscove/10% FCS) and divided into 4 groups: no additive, IL‐4, SCF, SCF+IL‐4 and harvested 16 hours thereafter for downstream analyses.…”

“…Contrary to FcεRI, MRGPRX2 is mostly confined to MC TC type MCs (MCs in skin), and skin MCs were the only samples to express MRGPRX2 across the FANTOM5 atlas . MCs used for research purposes are typically expanded in culture, and this provides a different microenvironment compared to the cells’ natural habitat, inducing adaptations in the MCs . Cultured skin‐derived MCs also adopt stronger degranulation responses to FcεRI‐aggregation vs ex vivo counterparts …”

Yin-Yang of IL-33 in Human Skin Mast Cells: Reduced Degranulation, but Augmented Histamine Synthesis through p38 Activation

“…Accell-mediated small interfering RNA (siRNA) selfdelivery has recently been introduced as a method to perturb endogenous levels of proteins in tissue MCs (Hazzan et al, 2017a(Hazzan et al, , 2017b. Exploiting this advancement, we employed JNK1-specific and p38a-specific RNA interference to eliminate their expression after verification that these were the major isoforms expressed in skin MCs (Motakis et al, 2014).…”

“…MCs were stained by the Annexin V-FITC Apoptosis detection kit (eBioscience, San Diego, CA) and analyzed on FACSCalibur (BD Biosciences, Heidelberg, Germany) as described previously (Hazzan et al, 2017a).…”