Allergy Asthma Immunol Res.  2013 Nov;5(6):402-408. 10.4168/aair.2013.5.6.402.

Effect of Cholesterol Depletion on Interleukin-8 Production in Human Respiratory Epithelial Cells

• Kim MJ
• Hong JY
• Lee KE
• Kim KW
• Sohn MH
• Kim KE

• KMID: 2260285
• DOI: http://doi.org/10.4168/aair.2013.5.6.402

Abstract

PURPOSE
The lipid entities of cell membranes are components of the immune system and important mediators of inflammation. Despite increasing interest in the function of epithelial cells in inflammation, the role of cholesterol in this process has not been described. Here, we investigated the effect of cholesterol depletion on the inflammatory process in airway epithelial cells via the expression of interleukin (IL)-8 as a marker of inflammation.
METHODS
A 549 cells were treated with 0.5% methyl-beta-cyclodextrin as a selective cholesterol extractor. The IL-8 level was assessed by enzyme-linked immunosorbent assay and reassessed after cholesterol repletion. Mitogen-activated protein kinase (MAPK) inhibitors were used to determine the upstream signaling pathway for IL-8 production in cholesterol-depleted cells.
RESULTS
We found a relationship between the amount of cholesterol in A 549 cells and inflammation of the airway. IL-8 production was increased in cholesterol-depleted A 549 cells and restored by cholesterol repletion. IL-8 production was decreased by pretreatment with the extracellular signal-regulated kinase (ERK) inhibitor U0126 but not with JNK inhibitor II or the p38 MAPK inhibitor SB202190.
CONCLUSIONS
Our findings suggest that inflammatory responses are increased in cholesterol-depleted epithelial cells via the MAPK signaling system, predominantly by the ERK pathway. We conclude that the lipid components of airwayepithelial cells may play a role in the inflammatory process.

Keyword

Cholesterol; epithelial cell; inflammation; interleukin-8; MAP kinase signaling system

MeSH Terms

beta-Cyclodextrins
Butadienes
Cell Membrane
Cholesterol
Enzyme-Linked Immunosorbent Assay
Epithelial Cells
Humans
Immune System
Inflammation
Inflammation Mediators
Interleukin-8
Interleukins
MAP Kinase Signaling System
Nitriles
p38 Mitogen-Activated Protein Kinases
Phosphotransferases
Protein Kinases
Butadienes
Cholesterol
Inflammation Mediators
Interleukin-8
Interleukins
Nitriles
Phosphotransferases
Protein Kinases
beta-Cyclodextrins
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Determination of the cholesterol level in untreated and MβCD-treated A 549 cells. The amount of cholesterol in the cell decreased after MβCD treatment. Each bar represents the mean±SEM of 3 independent experiments. Cholesterol in the cell was at its lowest level following treatment with 0.5% MβCD.

• 

  Fig. 2 Cholesterol depletion by 0.5% MβCD does not significantly change A 549 cell viability. A 549 cells preincubated with the indicated concentrations of 0.5% MβCD for 1 h did not show any significant change in viability compared to control cells. The data are expressed as the mean±SEM showing 96.8, 95.3, and 94.2% cell viability. *P<0.05 vs. the control alone.

• 

  Fig. 3 Effects of cholesterol depletion on IL-8 production in airway epithelial cells. (A) A 549 cells were treated with 0.5% MβCD and the supernatants were assessed by ELISA after 2, 5, 10, and 24 h. IL-8 production was increased in cholesterol-depleted cells and recovered after cholesterol repletion. (B) The induction of IL-8 mRNA expression in human respiratory epithelial cells. Total RNA was isolated and reverse-transcribed, and the resulting cDNA was amplified by real-time PCR. Cells stimulated with 0.5% MβCD showed enhanced IL-8 mRNA expression. *P<0.05 vs. the control.

• 

  Fig. 4 Effects of pretreatment with MAPK inhibitors on MβCD-induced IL-8 production. (A) IL-8 production was decreased by pretreatment with the ERK inhibitor U0126 (50 µM) but not by JNK inhibitor II (100 µM) or the p38 MAPK inhibitor SB202190 (50 µM). **P<0.01 vs. MβCD-treated cells. (B) Phosphorylation assays of MAPK activity. Cells treated with MβCD were harvested at the indicated time points and then lysed. Equal amounts of the cell extracts were resolved on 10% acrylamide gels and then subjected to Western blot analysis.

• 

  Fig. 5 Effect of cholesterol depletion on IL-6 and TNF-α mRNA expression. (A) A 549 cells were treated with 0.5% MβCD and/or 70 µg/mL cholesterol for the indicated time. Total RNA was isolated and reverse-transcribed, and the resulting cDNA was amplified by real-time PCR. The cholesterol-depleted cells showed enhanced proinflammatory cytokine release. The mRNA expression of IL-6 (A) and TNF-α (B) increased and peaked at 2 h following MβCD treatment. *P<0.05 vs. the control.

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