Requirement of NF-κB activation for cytokine-mediated reductions in TEER.

<p>A: Concentration-dependent inhibition of induction of ICAM-1 expression by IκK-β-inhibitor Bay11, confirming the effect of Bay11 on TNF induction of NF-κB-dependent genes. Conditions are lane 1, DMSO Control; lanes, 2, 3, 4 and 5, Bay11 at 0.375, 0.75, 1.5 and 3.0 μM, respectively all after 0.8 ng/ml TNF for 16 hours. B: Effect of Bay11 concentration on the TNF-induced TEER decrease. ECIS analysis. X-axis: Duration of 0.8 ng/ml TNF treatment. Y-axis: TEER (ohms) normalized to basal barrier level prior to addition of TNF. TEER levels were not affected by these concentrations of Bay11 in the absence of TNF (not shown). The corrected basal TEER for this experiment was 69.9 ± 1.3 Ω·cm². n = 6,6,6,6. C: Effects of SR-IκB dominant negative overexpression on HDMEC barrier responses. Upper panel: A time course of TNF treatment (1 ng/ml for 12 h) in control-transduced (black trace) and SR-IκB-transduced (red trace) HDMEC. n = 4,4. Lower panel: A time course of thrombin (1 U/ml for 12 h) in control-transduced (black trace) and SR-IκB-transduced (red trace) HDMEC. The corrected basal TEER for this experiment was 72.4 ± 0.9 Ω·cm² for SR-IκB-transduced HDMEC and 80.0 ± 0.7 Ω·cm² for vector control-transduced HDMEC. n = 3,3. Note that the phase 1 and phase 2 decreases initiated by TNF are markedly inhibited in SR-IκB-relative to control-transduced HDMEC but that thrombin-induced TEER decreases are similar in the same control- and SR-IκB-transduced HDMEC lines. D: Effects of Bay11 (used at 1 or 3 μM as labeled) on disruption of CL5 staining by 6 hours of TNF at 10 ng/ml. Morphometric measurements of TNF-induced disruption of CL5 junctional staining as described in the Methods (left). Immunofluorescence images representative of those used to assess the extent of disruption (right). Anti-CL5 (green), and anti-ICAM (red). Scale bar, 15 μm. E: Effects of SR-IκB transduction on disruption of CL5 staining by 6 hours of TNF at 10 ng/ml. Morphometric measurements (left) and representative immunofluorescence (right) images as in (D). Anti-CL5 (green), and anti-ICAM (red). Scale bar, 15 μm. Note that Bay 11 and SR-IκB each prevented the induction of ICAM-1 as well as the induction of a disrupted pattern of anti-CL5 immunofluorescence staining (arrows in D and E) by TNF. F: Effects of SR-IκB dominant negative overexpression on IL-1β-leak (ECIS). Note that IL-1β over a 12 hour time course was ineffective at decreasing TEER in SR-IκB-transduced HDMEC. The corrected basal TEER for this experiment was 64.5 ± 2.8 Ω·cm² for SR-IκB-transduced HDMEC and 59.6 ± 1.1 Ω·cm² for vector control-transduced HDMEC. n = 3,3. Representative of 3 (A, B and C) or 2 (D, E and F) independent experiments with similar results.