Abstract
Mast cells are in close proximity to the vasculature and cause endothelial activation, plasma extravasation, and vascular dysfunction (Gupta & Harvima, Immunol Rev 2018). Vascular dysfunction in sickle cell disease (SCD) is accompanied by increased expression of P-selectin. Treatment with Crizanlizumab, an antibody against P-selectin, led to significantly less sickle cell-related pain crises (Ataga et al., NEJM 2017) highlighting the role of endothelial P-selectin in vasoocclusive crises (VOC). Earlier studies demonstrated that mast cell activation with morphine or ischemia/reperfusion stimulates endothelial E- and/or P-selectin expression. However, it is unknown how mast cells stimulate endothelial selectin expression in SCD. Endothelial dysfunction contributes significantly to the pathobiology of SCD including VOC and may play a critical role in increased blood-brain barrier (BBB) permeability, which may contribute to stroke, another major comorbidity of SCD. One of the known triggers of endothelial dysfunction, inflammation and oxidative stress is endoplasmic reticulum (ER) stress. We hypothesize that in a sickle microenvironment, mediators derived from activated mast cells stimulate endothelial P-selectin expression via ER stress leading to increased BBB permeability. We examined the ability of mast cells to stimulate P-selectin expression and BBB permeability via ER stress in a sickle microenvironment.
We isolated MCs from HbAA-BERK and HbSS-BERK, control and sickle mice, respectively; incubated them in vitro and collected mast cell conditioned media (MCCM) from HbAA MCs and HbSS MCs. Normal mouse brain microvascular endothelial cells (mBMECs) were treated with unconditioned MCCM, HbAA MCCM, or HbSS MCCM to examine the effect of mast cell activation on endothelium. We observed increased mast cell activity in HbSS mice evinced by significantlyhigher plasma and skin histamine levels, compared to HbAA mice (p< 0.02 for both). Mast cells from HbSS mouse skin showed significantly increased expression of histamine compared to HbAA skin mast cells (p< 0.04). mBMECs incubated with HbAA and HBSS MCCM exhibited about 3- and 6-fold fold increases in P-selectin expression, compared to unconditioned culture medium, respectively (p< 0.0001 for both). Therefore, mast cells in culture release substances that stimulate P-selectin expression which is further increased by mast cells from sickle (HbSS) microenvironment. Preincubation of mBMEC with 5 microM salubrinal, an inhibitor of dephosphorylation of elongation initiation factor-a, which reduces ER stress, significantly inhibited HbSS MCCM-induced P-selectin expression on mBMEC (p< 0.0001) to the level induced by HbAA-MCCM. In contrast, salubrinal did not inhibit HbAA-MCCM-induced P-selectin expression on mBMEC, suggesting that in a sickle microenvironment mast cells contribute to P-selectin expression via ER stress.
We next examined mast cell activity on endothelial permeability in vitro on mBMEC monolayers and in vivo in the brain of HbSS mice. mBMECs incubated with HbSS MCCM showed a significant increase in Evans blue leakage compared to unconditioned or HbAA MCCM (p<0.0001 for both), which was inhibited by preincubation of mBMEC with 5 microM salubrinal prior to incubation with HbSS MCCM (p< 0.0001). In vivo female HbSS mice showed a significantly increased leakage in the brain of FITC-dextran injected through tail vein compared to HbAA mice (p< 0.01). HbSS mice treated with 1 mg/kg salubrinal demonstrated inhibition of FITC-dextran leakage in the brain compared to vehicle (p< 0.05). Thus, ER stress contributes to increased BBB permeability in HbSS mice. We observed activated degranulating mast cells in the brain parenchyma of HbSS mice. In HbAA mice, quiescent mast cells were confined to the meninges of the brain but not seen in the parenchyma. Together, these data suggest that mast cell activation contributes to BBB permeability in a sickle microenvironment via ER stress-mediated P-selectin expression. In turn, this mast cell-initiated activity in the brain may underlie the pathobiology of stroke in SCD. Inhibitors of mast cells and P-selectin have been tested clinically leading to reduced VOC in SCD without known adverse events. Therefore, mast cell activation-induced P-selectin via ER stress may serve as a treatable target for reducing the risk of stroke in SCD.
Disclosures
Gupta: Novartis: Honoraria; Tau tona: Consultancy.
Mast cell activation contributes to sickle cell pathobiology and pain in mice
