Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-alpha increases intestinal TJ permeability. Because TNF-alpha levels are markedly increased in CD, TNF-alpha increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-alpha modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-alpha produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-alpha-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-kappa B activation. Inhibition of TNF-alpha-induced NF-kappa B activation by selected NF-kappa B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-kappa B activation was required for the TNF-alpha-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-alpha modulation of ZO-1 protein expression and junctional localization were also prevented by NF-kappa B inhibitors. TNF-alpha did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-alpha-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-alpha-induced increase in Caco-2 TJ permeability was mediated by NF-kappa B activation. The increase in permeability was associated with NF-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.

Cytokines play a crucial role in the modulation of inflammatory response in the gastrointestinal tract. Pro-inflammatory cytokines including tumor necrosis factor-α, interferon-γ, interleukin-1β (IL-1β), and interleukin-12 are essential in mediating the inflammatory response, while anti-inflammatory cytokines including interleukin-10 and transforming growth factor-β are important in the attenuation or containment of inflammatory process. It is increasingly recognized that cytokines have an important physiological and pathological effect on intestinal tight junction (TJ) barrier. Consistent with their known pro-inflammatory activities, pro-inflammatory cytokines cause a disturbance in intestinal TJ barrier, allowing increased tissue penetration of luminal antigens. Recent studies indicate that the inhibition of cytokine induced increase in intestinal TJ permeability has an important protective effect against intestinal mucosal damage and development of intestinal inflammation. In this review, the effects of various pro-inflammatory and anti-inflammatory cytokines on intestinal TJ barrier and the progress into the mechanisms that mediate the cytokine modulation of intestinal TJ barrier are reviewed.

No abstract

plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-␣-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-␣-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-␣-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-␣ (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-␣ increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-␣-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-␣ increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-␣ increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-␣ increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-␣ increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg 2ϩ -myosin ATPase, and metabolic energy prevented the TNF-␣ increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-␣-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-␣ increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.

No abstract