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Interferon-gamma decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin
1Department of Biochemistry, Immunex Corporation, Seattle, Washington 98101, USA. ayouakim@immunex.com
Insights
Interferon-gamma (IFN-gamma) disrupts intestinal barrier function by reducing zonula occludens-1 (ZO-1) and altering actin organization in T84 cells. This leads to increased intestinal permeability.
Area of Science:
- Cell Biology
- Gastroenterology
- Immunology
Background:
- Tight junctions regulate intestinal epithelial barrier function.
- Interferon-gamma (IFN-gamma) is a cytokine involved in immune responses and inflammation.
- Disruption of the intestinal barrier is implicated in various gastrointestinal diseases.
Purpose of the Study:
- To investigate the effects of IFN-gamma on tight junction proteins and barrier function in T84 human intestinal epithelial cells.
- To elucidate the molecular mechanisms underlying IFN-gamma-induced changes in intestinal epithelial cells.
Main Methods:
- T84 human intestinal epithelial cells were treated with IFN-gamma.
- Monolayer permeability was assessed using transepithelial electrical resistance (TER).
- Expression and localization of tight junction proteins (ZO-1, ZO-2, occludin) were analyzed by immunoblotting and confocal microscopy.
- Protein and RNA levels were analyzed using pulse-chase assays and RT-PCR.
- Actin organization was examined using immunofluorescence microscopy.
Main Results:
- IFN-gamma treatment increased monolayer permeability in a dose- and time-dependent manner.
- IFN-gamma caused a significant decrease in zonula occludens-1 (ZO-1) levels and altered the localization of ZO-2 and occludin.
- ZO-1 protein turnover increased, synthesis decreased, and RNA levels were reduced, while ZO-2 and occludin showed minimal changes in these parameters.
- IFN-gamma disrupted apical actin organization, which was closely linked to ZO-1 expression changes.
- These alterations collectively led to tight junction disorganization and increased paracellular permeability.
Conclusions:
- IFN-gamma impairs intestinal epithelial barrier function by downregulating ZO-1 and disrupting apical actin organization.
- These molecular changes result in tight junction disorganization and increased paracellular permeability in T84 cells.
- The findings suggest a role for IFN-gamma in modulating intestinal barrier integrity.
Abstract:
The effects of interferon-gamma (IFN-gamma) on tight junctions in T84 human intestinal epithelial cells were investigated. Treatment of T84 cells with IFN-gamma caused a dose- and time-dependent increase in monolayer permeability as assessed by transepithelial electrical resistance measurements. Examination of specific proteins associated with tight junctions by immunoblotting and confocal microscopy revealed changes in the expression levels and localization of some of these proteins after exposure of the cells to IFN-gamma. Specifically, IFN-gamma treatment resulted in an almost total loss of zonula occludens (ZO)-1, whereas the levels of ZO-2 and occludin showed relatively modest decreases compared with untreated cells. Loss of ZO-1 was associated with the altered localization of ZO-2 and occludin. In IFN-gamma-treated cells, ZO-2 and occludin were diffusely distributed, whereas, in control cells, they, along with ZO-1, were predominantly localized to the tight junctions. Analysis of ZO-1 protein and RNA by pulse chase and RT-PCR, respectively, showed an increase in protein turnover, a decrease in protein synthesis, and a reduction in RNA levels following IFN-gamma treatment. In contrast to ZO-1, ZO-2 and occludin did not show any major changes in these parameters. In addition, the organization of actin in the apical and tight junction regions, but not in the mid- or basal regions, of the cells was also perturbed by IFN-gamma treatment of cells. Time-course analysis of IFN-gamma-induced alterations in ZO-1 expression and apical actin perturbation indicated that these two effects were intimately linked and could not be dissociated. These results suggest that IFN-gamma affects barrier function in T84 cells by decreasing the levels of ZO-1 and perturbing apical actin organization, which leads to a disorganization of the tight junction and an increase in paracellular permeability.
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