Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

• Keywords
• FRAP, FcεRI signaling, STIM1-ORAI1 coupling, alkanol, flow-FRET, heat shock response, membrane fluidizer, store-operated calcium entry,
• MeSH
• Cholesterol MeSH
• Cytokines MeSH
• Heptanol MeSH
• Mast Cells * MeSH
• Signal Transduction * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol MeSH
• Cytokines MeSH
• Heptanol MeSH

Pentacyclic triterpenoids, including ursolic acid (UA), are bioactive compounds with multiple biological activities involving anti-inflammatory effects. However, the mode of their action on mast cells, key players in the early stages of allergic inflammation, and underlying molecular mechanisms remain enigmatic. To better understand the effect of UA on mast cell signaling, here we examined the consequences of short-term treatment of mouse bone marrow-derived mast cells with UA. Using IgE-sensitized and antigen- or thapsigargin-activated cells, we found that 15 min exposure to UA inhibited high affinity IgE receptor (FcεRI)-mediated degranulation, calcium response, and extracellular calcium uptake. We also found that UA inhibited migration of mouse bone marrow-derived mast cells toward antigen but not toward prostaglandin E2 and stem cell factor. Compared to control antigen-activated cells, UA enhanced the production of tumor necrosis factor-α at the mRNA and protein levels. However, secretion of this cytokine was inhibited. Further analysis showed that UA enhanced tyrosine phosphorylation of the SYK kinase and several other proteins involved in the early stages of FcεRI signaling, even in the absence of antigen activation, but inhibited or reduced their further phosphorylation at later stages. In addition, we show that UA induced changes in the properties of detergent-resistant plasma membrane microdomains and reduced antibody-mediated clustering of the FcεRI and glycosylphosphatidylinositol-anchored protein Thy-1. Finally, UA inhibited mobility of the FcεRI and cholesterol. These combined data suggest that UA exerts its effects, at least in part, via lipid-centric plasma membrane perturbations, hence affecting the functions of the FcεRI signalosome.

• Keywords
• immunoglobulin E, lipid raft, mast cell, plasma membrane, signal transduction, tumor necrosis factor, tyrosine kinase,
• MeSH
• Antigens metabolism   MeSH
• Cell Degranulation MeSH
• Ursolic Acid MeSH
• Lipids pharmacology   MeSH
• Mast Cells metabolism   MeSH
• Mice MeSH
• Receptors, IgE * metabolism   MeSH
• Triterpenes * pharmacology   metabolism   MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens MeSH
• Lipids MeSH
• Receptors, IgE * MeSH
• Triterpenes * MeSH
• Calcium MeSH

CD8+ T cells protect against tumors and intracellular pathogens. The inflammatory cytokines IL-2, IL-15, and IL-7 are necessary for their expansion. However, elevated serum levels of these cytokines are often associated with cancer, poorer prognosis of cancer patients, and exhaustion of antigen-expanded CD8+ T cells. The impact of acute conditioning of antigen-expanded CD8+ T cells with these cytokines is unknown. Here, we generated antigen-expanded CD8+ T cells using dendritic cells and PC-3 cells. The cells were acutely (18-24 h) conditioned with IL-2 and either the GSK3β inhibitor TWS119, the mTORC1 inhibitor rapamycin, or the mTORC1/2 inhibitor Torin1, then their immediate and post-re-expansion (distal) cytokine responses after antigen rechallenge were evaluated. We found that acute IL-2 conditioning upregulated the immediate antigen-induced cytokine response of the tested cells. Following their re-expansion, however, the cells showed a decreased cytokine response. These IL-2 conditioning-mediated impacts were counteracted with TWS119 or rapamycin but not with Torin1. Our data revealed that the acute conditioning of antigen-expanded CD8+ T cells with IL-2 modulates the GSK3β-mTORC signaling axis. This modulation differentially affected the immediate and distal cytokine responses of the cells. The acute targeting of this signaling axis could, therefore, represent a novel strategy for the modulation of antigen-expanded CD8+ T cells.

• Keywords
• CD8+ T cells, GSK-3β, TWS119, Tim-3, Torin1, antigen rechallenge, cytokine starvation, mTOR, rapamycin,
• Publication type
• Journal Article MeSH