Membrane Cholesterol Removal from Human Eosinophils Disrupts Cholesterol Rich Membrane Microdomains Resulting in Down Regulated Map Kinase Signaling but Not Jak/Stat Signaling

Eosinophils contribute to allergic asthma exacerbation and can undergo excessive recruitment to the lungs where their activation leads to tissue damage and fibrosis. Interleukin-5 (IL-5) family cytokine receptors, which are critical for eosinophil recruitment/activation, are proposed to exist in membrane microdomains. Because cholesterol-rich microdomains are linked to signal regulation in diverse receptor systems, and because hypercholesterolemia is an asthma risk-factor, we tested the hypothesis that cholesterol-rich plasma membrane microdomains are central to IL-5-family cytokine action in eosinophils. Purified human blood eosinophils were incubated (1 hr) with the cholesterol-chelating agent methyl-β-cyclodextrin (MβCD) or soluble cholesterol (MβCD pre-loaded with cholesterol), followed by cholesterol/membrane microdomain analyses via flow cytometry and confocal microscopy or stimulation with IL-5. Eosinophil activation was determined via immunoblotting for activated p38 MAPK, activated transcriptional regulator STAT5, cyclin D3 (MAPK-dependent), or Pim1 (STAT-dependent). MβCD decreases, and soluble cholesterol increases, membrane cholesterol content in a dose-dependent manner as assessed by flow cytometry. Likewise, confocal microscopy confirmed MβCD disrupts membrane microdomains. Furthermore, MβCD attenuates IL-5-induced p38 phosphorylation compared to control (p<0.001, N=10), whereas soluble cholesterol restores IL-5-induced p38 phosphorylation and significantly elevates basal phosphorylation (p<0.05, N=10). Cyclin D3 up-regulation is blocked by MβCD treatment but unaffected by soluble cholesterol addition (N=3). Neither MβCD nor soluble cholesterol appears to alter IL-5-induced STAT5 phosphorylation (N=3) or Pim1 up-regulation, suggesting a selective action of these agents (N=2). These studies reveal that disturbances in eosinophil membrane cholesterol content selectively affect eosinophil signaling, suggesting that in vivo cholesterol levels may direct eosinophilic function and inflammatory capacity.