IL-1beta causes an increase in intestinal epithelial tight junction permeability

Abstract

IL-1beta is a prototypical proinflammatory cytokine that plays a central role in the intestinal inflammation amplification cascade. Recent studies have indicated that a TNF-alpha- and IFN-gamma-induced increase in intestinal epithelial paracellular permeability may be an important mechanism contributing to intestinal inflammation. Despite its central role in promoting intestinal inflammation, the role of IL-1beta on intestinal epithelial tight junction (TJ) barrier function remains unclear. The major aims of this study were to determine the effect of IL-1beta on intestinal epithelial TJ permeability and to elucidate the mechanisms involved in this process, using a well-established in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. IL-1beta (0-100 ng/ml) produced a concentration- and time-dependent decrease in Caco-2 transepithelial resistance. Conversely, IL-1beta caused a progressive time-dependent increase in transepithelial permeability to paracellular marker inulin. IL-1beta-induced increase in Caco-2 TJ permeability was accompanied by a rapid activation of NF-kappaB. NF-kappaB inhibitors, pyrrolidine dithiocarbamate and curcumin, prevented the IL-1beta-induced increase in Caco-2 TJ permeability. To further confirm the role of NF-kappaB in the IL-1beta-induced increase in Caco-2 TJ permeability, NF-kappaB p65 expression was silenced by small interfering RNA transfection. NF-kappaB p65 depletion completely inhibited the IL-1beta-induced increase in Caco-2 TJ permeability. IL-1beta did not induce apoptosis in the Caco-2 cell. In conclusion, our findings show for the first time that IL-1beta at physiologically relevant concentrations causes an increase in intestinal epithelial TJ permeability. The IL-1beta-induced increase in Caco-2 TJ permeability was mediated in part by the activation of NF-kappaB pathways but not apoptosis.

FIGURE 1

A, Effect of increasing concentration of IL-1β (0, 1, 10, 50, and 100 ng/ml) on Caco-2 TER. Filter-grown Caco-2 monolayers were treated with IL-1β for a 48-h experimental period. IL-1β produced a concentration-dependent decrease in Caco-2 TER. Data represent means ± SE of TER (n = 6). *, p < 0.0001 vs control. B, The effect of IL-1β mAb (100 ng/ml) on an IL-1β-induced drop in Caco-2 TER. IL-1β mAb pre-treatment prevented the IL-1β-induced drop in Caco-2 TER. Data represent means ± SE of TER (n = 6).**, p < 0.0001 vs control.

FIGURE 2

Time-course effect of IL-1β on Caco-2 TER and paracellular permeability. The effect of IL-1β (10 ng/ml) on Caco-2 TER and mucosal-to-serosal flux of paracellular marker, inulin were measured over a 72-h experimental period. A, Time-course effect of IL-1β on Caco-2 TER (n = 4). B, Time-course effect of IL-1β on transepithelial inulin flux (n = 3). C, Graph of TER vs inulin flux (r = 0.97).

FIGURE 3

Time-course effect of IL-1β on NF-κB activation in Caco-2 monolayers. Filter-grown Caco-2 monolayers were treated with IL-1β (10 ng/ml) for increasing time periods (0–60 min). A, The IκB-α protein expression was determined by Western blot analysis as described in Materials and Methods. B, Filter-grown Caco-2 monolayers were treated with IL-1β (10 ng/ml) for 30 min, and changes in the NF-κB p65 expression in the cytoplasmic and nuclear fractions were assayed by Western blot analysis. The effect of IL-1β (10 ng/ml) on NF-κB p65 cytoplasmic-to-nuclear translocation as determined by immunofluorescent Ab labeling as described in Materials and Methods. C, Control or untreated Caco-2 monolayers. D, Caco-2 monolayers treated for 30 min with IL-1β (10 ng/ml). Caco-2 monolayers pretreated with (E) curcumin (5 μM) or (F) PDTC (100 μM) before the 30-min treatment with IL-1β. IL-1β caused a rapid cytoplasmic-to-nuclear translocation of NF-κB p65 subunit. Original magnification, ×400. G, The effect of NF-κB inhibitors PDTC and curcumin on IL-1β-induced drop in Caco-2 TER. PDTC (100 μM) and curcumin (5 μM) significantly prevented the IL-1β-induced drop in Caco-2 TER (n = 6). *, p < 0.01 vs control. **, p < 0.01 vs IL-1β-treated monolayers.

FIGURE 4

ELISA-binding assay of IL-1β-activated NF-κB p65 binding to the oligonucleotide probe containing the κB-binding site. The binding of p65 to binding site was expressed as a concentration of NF-κB p65 (nanograms per milliliter) bound to the binding site. A, IL-1β caused a significant increase in NF-κB binding. NF-κB inhibitors PDTC (100 μM) and curcumin (5 μM) inhibited the IL-1β-induced NF-κB binding. B, The addition of wild-type (WT) oligonucleotide containing the consensus NF-κB-binding site in excess (100:1 ratio) as a competitive inhibitor prevented the binding of NF-κB to the DNA probe. In contrast, the addition of excess oligonucleotide containing a mutated NF-κB-binding motif did not inhibit the NF-κB binding. C, Effect of NF-κB p65 depletion by siRNA on the IL-1β-induced drop on Caco-2 TER. Caco-2 monolayers were transfected with NF-κB p65 siRNA for a 96-h time period as described in Materials and Methods. D, NF-κB p65 siRNA resulted in a marked depletion in NF-κB p65 protein expression. B, NF-κB p65 siRNA transfection prevented the IL-1β-induced drop in Caco-2 TER. *, p < 0.001 vs control; **, p < 0.001vs IL-1β treatment.

FIGURE 5

The effect of IL-1β on Caco-2 cell apoptosis. The IL-1β effect on Caco-2 cell apoptosis and necrosis was assessed by Annexin V^FITC and propidium iodide labeling as described in Materials and Methods. Flow cytometric analysis of Annexin V^FITC and propidium iodide staining was performed. A, The dot plots represent a total of 20,000 events for each sample. Upper left panel, Necrotic cells; lower right panel, apoptotic cells. Caco-2 monolayers were treated with IL-1β (10 ng/ml) for 48 h. Subsequently, Caco-2 cells were trypsinized and labeled with Annexin V^FITC (apoptosis) or propidium iodide (necrosis). Camptothecin was used as a positive control for apo-ptosis. IL-1β did not induce (B) apoptosis or (C) cell necrosis in IL-1β-treated Caco-2 cells.

FIGURE 6

A, Time-course effect of IL-1β on TJ protein expression in Caco-2 monolayers. Filter-grown Caco-2 monolayers were treated with IL-1β (10 ng/ml) for increasing time periods (0–48 h). The protein expression was determined by Western blot analysis as described in Materials and Methods. IL-1β did not cause any change in the ZO-1 protein expression. IL-1β caused a progressive down-regulation in occludin protein expression and up-regulation in claudin-1 expression. B, Effect of IL-1β on occludin junctional localization. The junctional localization of occludin protein in filter-grown Caco-2 monolayers was assessed by immunofluorescent Ab labeling. IL-1β produced a significant disturbance in occludin junctional localization (arrows). PDTC prevented the IL-1β-induced alteration in junctional localization of occludin protein.

FIGURE 7

A, Effect of IL-1β on occludin mRNA levels was assessed by real-time PCR. Filter-grown Caco-2 cells were treated with 10 ng/ml IL-1β for 16 h. B, Pretreatment with PDTC (100 μM) prevented the IL-1β-induced decrease in occludin mRNA levels. Data are represented as means of four replicates ± SE; *, p < 0.001 vs control. C, Effect of NF-κB inhibitor, PDTC (100 μM), and (D) NF-κB p65 depletion by siRNA on IL-1β-induced down-regulation of occludin protein expression. Caco-2 monolayers were treated with IL-1β for the 48-h experimental period. PDTC (100 μM) and NF-κB p65 siRNA significantly prevented the IL-1β-induced down-regulation in occludin protein expression.