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. 2013 Sep 23;9(9):966-79.
doi: 10.7150/ijbs.6996. eCollection 2013.

Interleukin-8 regulates endothelial permeability by down-regulation of tight junction but not dependent on integrins induced focal adhesions

Hongchi Yu  1 , Xianliang Huang, Yunlong Ma, Min Gao, Ou Wang, Ting Gao, Yang Shen, Xiaoheng Liu
Affiliations

Interleukin-8 regulates endothelial permeability by down-regulation of tight junction but not dependent on integrins induced focal adhesions

Hongchi Yu et al. Int J Biol Sci. .

Abstract

Interleukin-8 (IL-8) is a common inflammatory factor, which involves in various non-specific pathological processes of inflammation. It has been found that increased endothelial permeability accompanied with high expression of IL-8 at site of injured endothelium and atherosclerotic plaque at early stages, suggesting that IL-8 participated in regulating endothelial permeability in the developing processes of vascular disease. The purpose of this study is to investigate the regulation effects of IL-8 on the vascular endothelial permeability, and the mRNA and protein expression of tight junction components (i.e., ZO-1, Claudin-5 and Occludin). Endothelial cells were stimulated by IL-8 with the dose of 50, 100 and 200 ng/mL, and duration of 2, 4, 6, 8h, respectively. The mRNA and protein expression level of tight junction components with IL-8 under different concentration and duration was examined by RT-PCR and Western blot, respectively. Meanwhile, the integrins induced focal adhesions event with IL-8 stimulation was also investigated. The results showed that IL-8 regulated the permeability of endothelium by down-regulation of tight junction in a dose- and time-dependence manner, but was not by integrins induced focal adhesions. This finding reveals the molecular mechanism in the increase of endothelial cell permeability induced by IL-8, which is expected to provide a new idea as a therapeutic target in vascular diseases.

Keywords: Claudin-5; Endothelial permeability; IL-8; Occludin; Tight junction; ZO-1..

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Fig 1
Fig 1
The changes of endothelial permeability induced by IL-8 exhibited dose- and time-dependent manners. Permeability assay was perform as depicted in 2.1, and coefficient (Pa) in groups was calculated by formula Pa = [Ca]/t ×1/S ×V/[Cl]. (A) The transwell cultured with endothelium were treated with 50, 100 and 200 ng/ml IL-8, respectively. (B) The transwell cultured with endothelium were treated with 200 ng/ml IL-8 for 2, 4, 6 and 8h, respectively. Values represented the mean±S.E (n=3). *, P<0.05 denoted statistically significant difference compares to control.
Fig 2
Fig 2
Cultured cells treated with 0 (as controls), 50, 100 and 200 ng/ml IL-8 for 4h were stained with F-actin, and examined by laser confocal microscopy. The white arrows in figures showed visible cell-cell gaps formation. Scale bar in all images = 25 μm.
Fig 3
Fig 3
TEM images showed the different concentrations (50, 100 and 200 ng/ml) and treated durations (2, 4, 6 and 8h) of IL-8 on cell-cell junctions morphology. The black arrows in figures showed the junctions and gaps between two cells. Scale bar in all images = 1 μm.
Fig 4
Fig 4
The effect of IL-8 on occludin, claudin-5 and ZO-1 mRNA expression. IL-8 induced tight junction permeability in endothelium with decreased mRNA level of occludin, claudin-5 and ZO-1 in a dose- and time-dependence manner. (A) Representative bands of tight junction mRNA expression. (B), (C) and (D) The level of β-actin in each group was used as intrinsic controls, and relative expression mRNA of occludin, claudin-5 and ZO-1 were calculated, respectively. Values represented the mean±S.D. (n=3). *, P<0.05 denoted statistically significant difference compares to control.
Fig 4
Fig 4
The effect of IL-8 on occludin, claudin-5 and ZO-1 mRNA expression. IL-8 induced tight junction permeability in endothelium with decreased mRNA level of occludin, claudin-5 and ZO-1 in a dose- and time-dependence manner. (A) Representative bands of tight junction mRNA expression. (B), (C) and (D) The level of β-actin in each group was used as intrinsic controls, and relative expression mRNA of occludin, claudin-5 and ZO-1 were calculated, respectively. Values represented the mean±S.D. (n=3). *, P<0.05 denoted statistically significant difference compares to control.
Fig 5
Fig 5
The effect of IL-8 with different concentrations and treated durations on the protein expression and distribution of tight junction contents. IL-8 induces tight junction permeability in endothelium with decreased expression level of Occludin, Claudin-5 and ZO-1. (A) Representative western blot of tight junction proteins expression. Quantification of each protein expression level by image analysis of the western blot bands in fig A. The expression level of β-actin in each group was used as intrinsic controls, and relative expression were calculated. (B-D) The expression level of Occludin, Claudin-5 and ZO-1 with different doses of IL-8 (50, 100 and 200 ng/ml) for 2h. (E-G) The expression level of Occludin, Claudin-5 and ZO-1 with 100 ng/ml IL-8 for 2, 4, 6 and 8h, respectively. Values represented the mean±S.D. (n=3). *, P<0.05 denoted statistically significant difference compared to control. (H) Double-labeled immunofluorescence analyzed the effects of increased concentration of IL-8 on distribution and expression of ZO-1 (red) and Occludin (green) proteins, enlarged images of designated regions, labeled by square frames (blue: DAPIstained nucleus). Yellow scale bar = 25 μm.
Fig 5
Fig 5
The effect of IL-8 with different concentrations and treated durations on the protein expression and distribution of tight junction contents. IL-8 induces tight junction permeability in endothelium with decreased expression level of Occludin, Claudin-5 and ZO-1. (A) Representative western blot of tight junction proteins expression. Quantification of each protein expression level by image analysis of the western blot bands in fig A. The expression level of β-actin in each group was used as intrinsic controls, and relative expression were calculated. (B-D) The expression level of Occludin, Claudin-5 and ZO-1 with different doses of IL-8 (50, 100 and 200 ng/ml) for 2h. (E-G) The expression level of Occludin, Claudin-5 and ZO-1 with 100 ng/ml IL-8 for 2, 4, 6 and 8h, respectively. Values represented the mean±S.D. (n=3). *, P<0.05 denoted statistically significant difference compared to control. (H) Double-labeled immunofluorescence analyzed the effects of increased concentration of IL-8 on distribution and expression of ZO-1 (red) and Occludin (green) proteins, enlarged images of designated regions, labeled by square frames (blue: DAPIstained nucleus). Yellow scale bar = 25 μm.
Fig 6
Fig 6
IL-8 regulated the expression and distribution of E-Cad and N-Cad. Double-labeled immunofluorescence analyzed the effects of increased concentration (from 0 to 200 ng/ml) of IL-8 on distribution and expression of E-Cad (green) and N-Cad (red) proteins. (blue: DAPI stained nucleus). Yellow scale bar = 25 μm.
Fig 7
Fig 7
The effect of IL-8 on the protein expression level of Integrin subunits, α2, α5, β1 and β3. There were not significant differences of expressions level of Integrin subunits treated by either concentration or duration of IL-8. (A) Representative Western blot bands of integrins expression. Quantification of each protein expression level by image analysis of the Western blot bands in fig A. The relative expression were calculated by expression level of β-actin in each group as intrinsic controls. (B-E) The expression level of Integrin subunits, α2, α5, β1, and β3 with different doses of IL-8 (50, 100 and 200 ng/ml) for 2h. (F-I) The expression level of Integrin subunits, α2, α5, β1, and β3 with 100 ng/ml IL-8 for 2, 4, 6 and 8h, respectively. Values represented the mean±S.D. (n=3). *, P<0.05 denoted statistically significant difference compared to control.
Fig 8
Fig 8
The effect of IL-8 on the protein expression of focal adhesion components, Paxillin, Vinculin and Talin. The expression levels of Paxillin, Vinculin and Talin were not changed by either concentration or duration of IL-8. (A) Representative Western blot bands of integrins expression. Quantification of each protein expression level by image analysis of the Western blot bands in fig A. The expression level of β-actin in each group was used as intrinsic controls, and relative expression were calculated. (B-D) The expression level of focal adhesion components, Paxillin, Vinculin and Talin with different doses of IL-8 (50, 100 and 200 ng/ml) for 2h. (E-G) The expression level of focal adhesion components with 100 ng/ml IL-8 for 2, 4, 6 and 8h, respectively. (H & I) The ratios of phosphorylated Paxillin (pPax) in total Paxillin (Pax) with different doses and treated durations of IL-8 were calculated and compared. Values represented the mean±S.D. (n=3).
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