NF-κB inhibitors impair lung epithelial tight junctions in the absence of inflammation

Abstract

NF-κB (p50/p65) is the best characterized transcription factor known to regulate cell responses to inflammation. However, NF-κB is also constitutively expressed. We used inhibitors of the classical NF-κB signaling pathway to determine whether this transcription factor has a role in regulating alveolar epithelial tight junctions. Primary rat type II alveolar epithelial cells were isolated and cultured on Transwell permeable supports coated with collagen for 5 d to generate a model type I cell monolayer. Treatment of alveolar epithelial monolayers overnight with one of 2 different IκB kinase inhibitors (BAY 11-7082 or BMS-345541) resulted in a dose-dependent decrease in TER at concentrations that did not affect cell viability. In response to BMS-345541 treatment there was an increase in total claudin-4 and claudin-5 along with a decrease in claudin-18, as determined by immunoblot. However, there was little effect on the total amount of cell-associated claudin-7, occludin, junctional adhesion molecule A (JAM-A), zonula occludens (ZO)-1 or ZO-2. Moreover, treatment with BMS-345541 resulted in altered tight junction morphology as assessed by immunofluorescence microscopy. Cells treated with BMS-345541 had an increase in claudin-18 containing projections emanating from tight junctions ("spikes") that were less prominent in control cells. There also were several areas of cell-cell contact which lacked ZO-1 and ZO-2 localization as well as rearrangements to the actin cytoskeleton in response to BMS-345541. Consistent with an anti-inflammatory effect, BMS-345541 antagonized the deleterious effects of lipopolysaccharide (LPS) on alveolar epithelial barrier function. However, BMS-345541 also inhibited the ability of GM-CSF to increase alveolar epithelial TER. These data suggest a dual role for NF-κB in regulating alveolar barrier function and that constitutive NF-κB function is required for the integrity of alveolar epithelial tight junctions.

Keywords: ARDS, Acute Respiratory Distress Syndrome; GM-CSF, Granulocyte Macrophage Colony Stimulating Factor; IL, interleukin; IκB, Inhibitor of κB; JAM-A, junctional adhesion molecule A; LPS, lipolysaccharide; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PBS, phosphate buffered saline; TER, transepithelial resistance; TNF, Tumor Necrosis Factor; ZO, zonula occludens; alveolus; claudin; lung barrier; tight junction.

Figure 1.

NF-κB inhibitors decrease alveolar epithelial cell barrier function. Primary rat alveolar epithelial cells were isolated and cultured on Transwell permeable supports for 5 d to produce model type I cell monolayers. The cells were then further incubated overnight with varying amounts of either BAY 11–7082 (A) or BMS-345541 (C) or vehicle control (0) and transepithelial resistance (TER) was measured using an Evohm voltmeter. For both agents, there was a statistically significant decrease in TER with increasing levels of drug treatment (*P < 0.05, n = 3–8). To rule out cell death as a possible cause of compromised barrier function, we assessed parallel cultures using the Invitrogen Live/Dead assay (B, D). Although there was considerable cell death at 0.4 and 1.0 μM BAY 11–7082 and some loss of viability at 10 μM BMS-345541 (B), TER also significantly decreased at lower concentrations of BAY 11–7082 and BMS-345541 where cell viability was maintained (*P < 0.05, n = 5).

Figure 2.

Effect of BMS-345541 on alveolar tight junction protein expression. Primary rat alveolar epithelial cells were isolated and cultured on Transwell permeable supports for 5 d to produce model type I cell monolayers. The cells were then incubated overnight with varying amounts of either BMS-345541 or vehicle control (0), harvested and analyzed by immunoblot for tight junction proteins. We found that with increasing BMS-345541 concentration there was a significant increase in claudin-4 and claudin-5 and a significant decrease in claudin-18 (*p<0 .05, n = 3). The other proteins examined showed no significant changes.

Figure 3.

Effect of BAY 11–7082 on alveolar tight junction protein expression. Primary rat alveolar epithelial cells were isolated and cultured on Transwell permeable supports for 5 d to produce model type I cell monolayers. The cells were then incubated overnight with varying amounts of either BAY 11–7082 or vehicle control (0), harvested and analyzed by immunoblot for tight junction proteins. We found that BMS-345541 did not cause signficiant changes to claudin-4, claudin-5 or claudin-18.

Figure 4.

BMS-345541 destabilized tight junction associated claudin-18. Primary rat alveolar epithelial cells were isolated and cultured on Transwell permeable supports for 5 d to produce model type I cell monolayers. The cells were then incubated overnight with vehicle control (A, C) or 4 μM BMS-345541(B, D), fixed and processed to image claudin-18 by immunofluorescence microscopy. Magnified insets are shown in C, D. In contrast to control cells, where claudin-18 was strongly localized to tight junctions at the plasma membrane, cells treated with BMS-345541 showed linear trails emanating from the plasma membrane (tight junction spikes; arrowheads). Bar – 10 micron.

Figure 5.

BMS-345541 disrupts association of ZO-1 and ZO-2 with tight junctions. Model type I cell monolayers were incubated overnight with vehicle control (A, B) or 4 μM BMS-345541 (C, D), fixed and processed to image ZO-1 (A, C) or ZO-2 (B, D) by immunofluorescence microscopy. In contrast to control cells, BMS-345541 treated cells had large areas of cell-cell contact which lacked strong ZO-1 or ZO-2 labeling (arrowheads). Bar – 10 micron.

Figure 6.

BMS-345541 induces actin cytoskeletal rearrangements. Model type I cell monolayers were incubated overnight with vehicle control (A-C, G-I) or 4 μM BMS-345541 (D-F, J-L), fixed and processed to image claudin-18 (A, D, G, J) and actin (B, E, H, K) by immunofluorescence microscopy using rhodamine-phalloidin to label F-actin. Merged images are in C, F, I, L. Images in G-L represent magnifications of the regions in C, F denoted by the box. Cells treated with BMS-345541 showed decreases in cortical actin as compared with control cells (arrowheads). Actin frequently co-localized with claudin-18 localized to tight junction spikes (arrows), particularly in cells treated with BMS-345541. Bar – 10 micron (A-F), 2 micron (G-L).

Figure 7.

BMS-345541 alters the extent of β-catenin expression. Model type I cell monolayers were incubated overnight with vehicle control (A-C) or 4 μM BMS-345541 (D-F), fixed and processed to image claudin-18 (A, D) and β-catenin (B, E) by immunofluorescence microscopy. Merged images are in C, F. Cells treated with BMS-345541 showed an overall decrease in β-catenin as compared with controls although the overall appearance was comparable. Note that the majority of β-catenin was opposed to areas which have tight junction spikes (arrowheads), although spikes did have some β-catenin as well. Bar – 10 micron.

Figure 8.

Differential effects of BMS-345541 on alveolar epithelial barrier function. Primary rat alveolar epithelial cells were isolated from rats fed either a control Lieber-DeCarli diet (A) or a Lieber-DeCarli diet containing dietary alcohol (B). The cells were cultured on Transwell permeable supports for 5 d to produce model type I cell monolayers and then incubated overnight with varying amounts of either BMS-345541 or vehicle control (0) and TER was measured. There was a statistically significant decrease in TER with increasing levels of drug treatment (*P < 0.05, n = 3). At all but the highest concentration of BMS-345541 cells from alcohol fed rats had significantly lower TER than cells isolated from control rats (#P < 0.05, n = 3). C. Cells from alcohol fed rats were further incubated with either 1 μg lipopolysaccharide (LPS) or 20 μg/ml GM-CSF overnight in either the presence or absence of 4 μg/ml BMS-345541. LPS significantly decreased TER while GM-CSF significantly increased TER. BMS-345541 reversed the effects of LPS and GM-CSF. *− P < 0.05, n = 3 vs. untreated and BMS-345541 co-treated cells.