OBJECTIVETo investigate the effect of combination of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) on intestinal epithelial barrier function.

METHODSThe Caco-2 monolayers were cultured in DMEM nutrient solution, and then they were inoculated in 24-well or 6-well plate with Transwell inserts.They were divided into control group (ordinary treatment), IFN-γ group (with addition of 10 ng/mL IFN-γ), TNF-α group (with addition of 10 ng/mL TNF-α), and IFN-γ plus TNF-α group (with addition of 10 ng/mL TNF-α and 10 ng/mL IFN-γ). Monolayers inoculated in 24-well plate were collected for determination of transepithelial electrical resistance (TER) with an ohmmeter at post treatment hour (PTH) 0, 6, 12, 24, 36, and 48, the permeability of monolayers with fluorescein isothiocyanate-labeled dextran (FITC-dextran) tracer method at PTH 48, the distribution and morphological change of tight junction occludin with immunofluorescence assay at PTH 48. Monolayers inoculated in 6-well plate were collected for determination of protein expression of occludin, myosin light chain kinase (MLCK), and phosphorylated MLC (pMLC) with Western blot at PTH 24. Data were processed with one-way analysis of variance and t test.

RESULTS(1) There was no obvious difference in TER in control group at each time point (F = 0.86, P > 0.05). TER in IFN-γ group and TNF-α group were gradually decreased during PTH 6-48, but showed no statistical difference as compared with that at PTH 0 (with F value respectively 1.69, 2.47, P values all above 0.05). TER in IFN-γ plus TNF-α group was significantly decreased from PTH 24 as compared with that at PTH 0 (t = 4.97, P < 0.05) and that in each of the other three groups (F = 11.54, P < 0.05). (2) The permeability of monolayers in IFN-γ plus TNF-α group [(1197 ± 215)pmol] was significantly higher than that in control group, IFN-γ group, and TNF-α group [(303 ± 93), (328 ± 76), (797 ± 177) pmol, with t value respectively 4.8, 5.0, 6.9, P values all below 0.01]. (3) There was no statistical difference in occludin expression at PTH 24 among four groups (F = 0.26, P > 0.05). The occludin in control group at PTH 48 was regular in arrangement, while that in IFN-γ and TNF-α groups was irregular in arrangement. The arrangement of occludin in IFN-γ plus TNF-α group at PTH 48 was interrupted, with obvious redistribution in cytoplasm. (4) The protein expression of pMLC in IFN-γ plus TNF-α group (0.95 ± 0.05) was significantly higher than that in control group, IFN-γ group, or TNF-α group (0.57 ± 0.12, 0.56 ± 0.07, 0.59 ± 0.10, respectively, F = 17.97, P < 0.01). The protein expression of MLCK in IFN-γ plus TNF-α group (1.57 ± 0.36) was also significantly higher than that in control, IFN-γ, TNF-α groups (0.85 ± 0.18, 1.04 ± 0.23, 1.00 ± 0.07, respectively, F = 9.05, P < 0.05).

CONCLUSIONSCombination of IFN-γ and TNF-α can induce intestinal epithelial barrier dysfunction by up-regulating MLCK protein expression and promoting MLC phosphorylation.