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.

RATIONALE: Eosinophils have been proposed to play an important role in the exacerbation of allergic asthma, as these cells can undergo excessive recruitment to the lungs where unregulated activation leads to tissue damage and fibrosis. Furthermore, interleukin-5 (IL-5) family cytokine receptors, critical for eosinophil recruitment and activation, are proposed to exist in membrane microdomains. Given that cholesterol-rich microdomains are linked to signal transduction regulation in numerous receptor systems, and because hypercholesterolemia is a risk factor for asthma, our current studies test the hypothesis that cholesterol-rich plasma membrane microdomains are important for IL-5-family cytokine stimulation of eosinophils. METHODS: Purified human peripheral blood eosinophils were incubated 1 hour with the cholesterol-chelating agent methyl-β-cyclodextrin (MβCD) or soluble cholesterol (MβCD pre-loaded with cholesterol), followed by stimulation with 100 pM IL-5. Eosinophil activation was determined via immunoblotting for activated MAP Kinase family members (phospho-ERK1/2, phospho-p38) or the transcriptional regulator phospho-STAT5. RESULTS: Addition of MβCD significantly decreases IL-5-induced ERK1/2 phosphorylation as compared to media control (p<0.001, N=8). Soluble cholesterol enhances basal ERK1/2 phosphorylation and restores the IL-5-induced ERK1/2 phosphorylation that was blocked by treatment with MβCD alone (p<0.05, N=8). Neither MβCD nor soluble cholesterol alters IL-5-induced STAT5 phosphorylation (N=8) suggesting a selective action of these agents. Also, trends indicate MβCD does not affect IL-5-induced p38 phosphorylation whereas soluble cholesterol augments both basal and IL-5-induced p38 phosphorylation. CONCLUSIONS: These studies suggest that disturbances in eosinophil cellular membrane cholesterol may affect only certain aspects of eosinophilic function and inflammatory activity. Thus, in vivo cholesterol levels may influence eosinophilic function/inflammatory/capacity.