IFN-γ and TNF-α synergize to reduce barrier function in Caco-2 cell monolayers. A: Caco-2 monolayers were incubated with the indicated cytokine(s) (IFN-γ = 10 ng/ml, TNF-α = 2.5 ng/ml) added to media in the basal chamber. Conditions were no cytokines (control), continuous IFN-γ (IFN-γ), continuous TNF-α (TNF-α), continuous IFN-γ and TNF-α (IFN-γ + TNF-α), TNF-α for 24 hours followed by IFN-γ (TNF-α → IFN-γ), or IFN-γ for 24 hours followed by TNF-α (IFN-γ → TNF-α). TER fell only in cells exposed to IFN-γ and TNF-α continuously or IFN-γ followed by TNF-α (n = 3 in this representative experiment). B: Caco-2 monolayers were incubated with the indicated cytokine(s) added to the basal chamber, as above. Conditions were no cytokines (control), continuous IFN-γ for 32 hours (IFN-γ), no cytokines for 24 hours followed by TNF-α for 8 hours (TNF-α), or IFN-γ for 24 hours followed by TNF-α for 8 hours (IFN-γ/TNF-α). At that time monolayers were assayed for permeability to 3kD fluorescein isothiocyanate-dextran. Paracellular permeability was increased only in cells exposed to IFN-γ followed by TNF-α (P < 0.001) (n = 6 in this representative experiment). C: Caco-2 monolayers were incubated with (filled circles) or without (open circles) IFN-γ (10 ng/ml) for 24 hours, washed, and transferred to media with TNF-α at indicated concentrations. TER shown is 48 hours after TNF-α addition. TNF-α caused dose-dependent TER decreases in IFN-γ-primed monolayers, but not in monolayers without IFN-γ pretreatment (n = 3 in this representative experiment).

IFN-γ- and TNF-α-induced barrier dysfunction is associated with apoptosis-independent tight junction disruption. A: Caco-2 monolayers were treated with IFN-γ (10 ng/ml) for 24 hours followed by TNF-α (2.5 ng/ml) for times indicated. Cell lysates were analyzed for cleavage of caspase-3, caspase-8, and PARP as markers of apoptosis. No increases in cleaved caspase-3, caspase-8, or PARP were detected in Caco-2 cells treated with the indicated cytokine(s). The positions of intact (I) and cleaved (C) proteins are shown. Data are representative of four similar experiments, each performed in triplicate. B: Caco-2 monolayers were treated with IFN-γ (10 ng/ml) for 24 hours followed by TNF-α (2.5 ng/ml) for 8 hours. Tight junction proteins (ZO-1, occludin, and claudin-1) were detected by immunofluorescence microscopy. Treatment of IFN-γ-primed monolayers with TNF-α caused obvious disruptions of the distribution of tight junction proteins. The tight junction staining was decreased in intensity and became irregular. Increased intracellular pools of occludin and claudin-1 were also apparent. Data are representative of six similar experiments, each performed in duplicate. C: Caco-2 monolayers were treated with IFN-γ (10 ng/ml) for 24 hours followed by TNF-α (2.5 ng/ml) for 8 hours, and harvested at 4°C in TBS with 1% Triton X-100. Cell lysates (adjusted to 40% sucrose) were loaded under 0 to 40% continuous sucrose gradients and centrifuged at 180,000 × g for 18 hours.40 SDS-PAGE immunoblots for occludin and claudin-1 were performed. Fractions from 1.06 to 1.16 g/ml are shown, with lipid raft and load regions designated. Treatment with IFN-γ and TNF-α caused both occludin and claudin-1 to be removed from the raft fraction. Data are representative of three similar experiments, each performed in duplicate. D: Immunoblots detecting occludin (Figure 2C) were analyzed quantitatively. The amount of occludin present in each fraction is shown as a percentage of the sum of occludin detected in all fractions. The peak occludin content within raft fractions was detected at a density of 1.082 g/ml and was 9.9% of total in control monolayers (open symbols) but only 2.9% of total in monolayers treated with IFN-γ and TNF-α (filled symbols). Data are representative of three similar experiments, each performed in duplicate. E: Immunoblots detecting claudin-1 (Figure 2C) were analyzed quantitatively. The amount of claudin-1 present in each fraction is shown as a percentage of the sum of claudin-1 detected in all fractions. The peak claudin-1 content within raft fractions was detected at a density of 1.082 g/ml and was 3.6% of total in control monolayers (open symbols) but only 1.6% of total in monolayers treated with IFN-γ and TNF-α (filled symbols). Data are representative of three similar experiments, each performed in duplicate. F: Caco-2 monolayers were treated with IFN-γ (10 ng/ml) for 24 hours or IFN-γ for 24 hours followed by TNF-α (2.5 ng/ml) for 18 hours. Cell lysates were analyzed for the expression of tight junction proteins ZO-1, occludin, and claudin-1 by SDS-PAGE immunoblot. Expression of these tight junction proteins was not significantly changed by cytokine treatment. Data are representative of four similar experiments, each performed in triplicate.

IFN-γ- and TNF-α-dependent TER decreases are accompanied by increases in MLC phosphorylation and MLCK expression. A: Caco-2 monolayers were incubated in media with or without IFN-γ (10 ng/ml) for 24 hours and then transferred to media with or without TNF-α (2.5 ng/ml), as indicated. In the designated conditions, 200 μmol/L PIK, a highly specific membrane-permeant inhibitor of MLC kinase, was added to the apical media 2 hours after TNF-α addition. As we have shown previously,11 PIK alone induced a small increase in TER. However, in monolayers treated sequentially with IFN-γ and TNF-α, PIK was able to nearly completely prevent TER loss (P < 0.02) (n = 3 in this representative experiment). B: Caco-2 monolayers were incubated in media with or without IFN-γ (10 ng/ml) for 24 hours and then transferred to media with or without TNF-α (2.5 ng/ml), as indicated. Monolayers were harvested 8 hours after TNF-α addition. MLC phosphorylation was assessed by SDS-PAGE immunoblot. TNF-α (2.5 ng/ml) caused a small increase in MLC phosphorylation in monolayers without IFN-γ (10 ng/ml) pretreatment (P > 0.05), but a significant increase in MLC phosphorylation in cells with IFN-γ pretreatment (P < 0.05) (n = 2 in this representative experiment). C: Caco-2 monolayers were incubated in media with or without IFN-γ (10 ng/ml) for 24 hours and then transferred to media with or without TNF-α (2.5 ng/ml), as indicated. Monolayers were harvested 8 hours after TNF-α addition. MLCK protein expression was analyzed by SDS-PAGE immunoblot. TNF-α (2.5 ng/ml) caused a slight increase MLCK expression in monolayers without IFN-γ (10 ng/ml) pretreatment (P > 0.05), but a significant increase in MLCK expression occurred after TNF-α treatment of monolayers primed with IFN-γ (P < 0.05) (n = 2 in this representative experiment).

Barrier defects induced by TNF-α and IFN-γ are not abrogated by NF-κB inhibition. A: Caco-2 monolayers were cultured with IFN-γ (10 ng/ml) for 24 hours and transferred to media with 10 μmol/L BAY 11-7085 (BAY), 10 μmol/L capsaicin (CAP), 0.5 mmol/L SSA, 2 mmol/L SSA, 18 μmol/L SN50, 30 μmol/L MG132, 5 μmol/L curcumin, or 1 μmol/L triptolide with or without TNF-α (2.5 ng/ml). None of the NF-κB inhibitors significantly altered TER without TNF-α treatment. TER of monolayers treated with TNF-α is normalized to identical monolayers, treated with the same NF-κB inhibitor, but without TNF-α. SSA (0.5 mmol/L) prevented TER decreases, but 2 mmol/L SSA, 18 μmol/L SN50, 30 μmol/L MG132, and 1 μmol/L triptolide exacerbated TER decreases. BAY 11-7085 (10 μmol/L), 10 μmol/L capsaicin, and 5 μmol/L curcumin had no significant effects (n = 3 in this representative experiment). B: Caco-2 monolayers were cultured with (closed circles) or without (open circles) IFN-γ (10 ng/ml) for 24 hours and transferred to TNF-α (2.5 ng/ml). SSA was also included at indicated doses (0.01 to 2 mmol/L). Maximal barrier protection occurred at 0.5 mmol/L. Higher SSA doses were not protective and, as shown in A, 2 mmol/L SSA exacerbated TER loss (n = 3 in this representative experiment). C: Caco-2 monolayers were cultured with IFN-γ (10 ng/ml) for 24 hours and transferred to TNF-α (2.5 ng/ml) with 0.5 mmol/L SSA, 0.5 mmol/L 5-aminosalicylic acid (5-ASA), 0.5 mmol/L sulfapyridine (SPD), 0.5 mmol/L 4-aminosalicylic acid (4-ASA), 10 μmol/L caffeic acid phenethyl ester, 1 mmol/L and 10 mmol/L N^G-nitro-l-arginine-methyl-ester (L-NAME), 10 μmol/L adenosine, and 10 μmol/L 5′-N-ethylcarboxamidoadenosine (NECA). TER of monolayers treated with TNF-α is normalized to identical monolayers, treated with the same drug, but without TNF-α. Only SSA prevented barrier dysfunction induced by IFN-γ and TNF-α (n = 3 in this representative experiment). D: NF-κB RelA p65 (closed bars) was detected by SDS-PAGE immunoblot of nuclear fractions isolated 10 minutes after TNF-α (2.5 ng/ml) and drug addition to monolayers with or without IFN-γ (10 ng/ml) pretreatment for 24 hours, as indicated. A representative immunoblot of nuclear fractions is shown below the graph. NF-κB (RelA p65) nuclear translocation was inhibited by 2 mmol/L SSA and 30 μmol/L MG132, but not by 0.5 mmol/L SSA [n = 2 in this experiment which is representative of nine similar experiments, each with duplicate samples (total n = 18)]. NF-κB-dependent luciferase expression (open bars) was assessed in Caco-2 cells transiently transfected with pNFκB-TA-Luc reporter construct. Monolayers were then treated identically to the translocation assay, but harvested 8 hours after TNF-α addition to allow for luciferase synthesis. NF-κB-dependent luciferase expression was inhibited by 2 mmol/L SSA and 30 μmol/L MG132, but not by 0.5 mmol/L SSA [n = 2 in this experiment which is representative of four similar experiments, each with duplicate samples (total n = 8)].

Low dose SSA prevents morphological tight junction disruption, increased MLC phosphorylation, and increased MLCK expression. A: Caco-2 monolayers were treated with IFN-γ (10 ng/ml) for 24 hours followed by TNF-α (2.5 ng/ml) and 0.5 mmol/L SSA for 8 hours. Tight junction proteins (ZO-1, occludin, and claudin-1) were detected by immunofluorescence microscopy. 0.5 mmol/L SSA completely prevented ZO-1, occludin, and claudin-1 redistribution in IFN-γ-primed TNF-α-treated monolayers. Data are representative of three similar experiments, each performed in triplicate. B: Caco-2 monolayers were incubated with IFN-γ (10 ng/ml) for 24 hours and then transferred to media with TNF-α (2.5 ng/ml) and 0.5 mmol/L SSA, 2 mmol/L SSA, or 30 μmol/L MG132, as indicated. Monolayers were harvested 8 hours after TNF-α addition. Lysates were assayed for phosphorylated MLC by SDS-PAGE immunoblot. SSA (0.5 mmol/L) prevented increases in MLC phosphorylation induced by IFN-γ and TNF-α (P < 0.01). In contrast, 2 mmol/L SSA and 30 μmol/L MG132 failed to prevent increases in MLC phosphorylation (P > 0.05 for 2 mmol/L SSA, P = 0.04 for 30 μmol/L MG132) (n = 2 in this representative experiment). C: Caco-2 monolayers were incubated with IFN-γ (10 ng/ml) for 24 hours and then transferred to media with TNF-α (2.5 ng/ml) and 0.5 mmol/L SSA, 2 mmol/L SSA, or 30 μmol/L MG132, as indicated. Monolayers were harvested 8 hours after TNF-α addition. Lysates were assayed for MLCK by SDS-PAGE immunoblot. SSA (0.5 mmol/L) prevented increases in MLCK expression induced by IFN-γ and TNF-α (P < 0.02). In contrast, 2 mmol/L SSA and 30 μmol/L MG132 failed to prevent increases in MLCK expression (P > 0.05 for 2 mmol/L SSA or 30 μmol/L MG132) (n = 2 in this representative experiment).