Strain-dependent activation of NF-kappaB in the airway epithelium and its role in allergic airway inflammation

Abstract

NF-kappaB activation in the airway epithelium has been established as a critical pathway in ovalbumin (Ova)-induced airway inflammation in BALB/c mice (Poynter ME, Cloots R, van Woerkom T, Butnor KJ, Vacek P, Taatjes DJ, Irvin CG, Janssen-Heininger YM. J Immunol 173: 7003-7009, 2004). BALB/c mice are susceptible to the development of allergic airway disease, whereas other strains of mice, such as C57BL/6, are considered more resistant. The goal of the present study was to determine the proximal signals required for NF-kappaB activation in the airway epithelium in allergic airway disease and to unravel whether these signals are strain-dependent. Our previous studies, conducted in the BALB/c mouse background, demonstrated that transgenic mice expressing a dominant-negative version of IkappaBalpha in the airway epithelium (CC10-IkappaBalpha(SR)) were protected from Ova-induced inflammation. In contrast to these earlier observations, we demonstrate here that CC10-IkappaBalpha(SR) transgenic mice on the C57BL/6 background were not protected from Ova-induced allergic airway inflammation. Consistent with this finding, Ova-induced nuclear localization of the RelA subunit of NF-kappaB was not observed in C57BL/6 mice, in contrast to the marked nuclear presence of RelA in BALB/c mice. Evaluation of cytokine profiles in bronchoalveolar lavage demonstrated elevated expression of TNF-alpha in BALB/c mice compared with C57BL/6 mice after an acute challenge with Ova. Finally, neutralization of TNF-alpha by a blocking antibody prevented nuclear localization of RelA in BALB/c mice after Ova challenge. These data suggest that the mechanism of response of the airway epithelium of immunized C57BL/6 mice to antigen challenge is fundamentally different from that of immunized BALB/c mice and highlight the potential importance of TNF-alpha in regulating epithelial NF-kappaB activation in allergic airway disease.

Fig. 1.

NF-κB activation in airway epithelium of C57BL/6 mice is not required for airway inflammation. Wild-type (WT, transgene-negative littermates) and CC10-IκBα_SR mice in the C57BL/6 background were immunized with ovalbumin (Ova) or mock-immunized [aluminum hydroxide (Alum)] and challenged with aerosolized Ova for 30 min/day for 3 consecutive days. Lungs were harvested 48 h later, and inflammation was assessed by enumeration of total cells and differentials in bronchoalveolar lavage (BAL) fluid (A) and analysis of histopathology in lung sections stained with hematoxylin and eosin (B). Data are representative of 7 mice in each of the Ova/Ova groups and 5 mice in each of the Alum/Ova groups.

Fig. 2.

CC10-IκBα_SR mice on the C57BL/6 background express elevated IκBα in lung tissue before and after sensitization and challenge with Ova. A: RT-PCR analysis of IκBα mRNA expression in homogenized lung tissue from CC10-IκBα_SR transgenic mice on the C57BL/6 and BALB/c background. B: quantitative PCR measurement of CC10-IκBα_SR transgene expression in WT and transgenic BALB/c and C57BL/6 mice. Data are expressed as number of PCR cycles to threshold (C_T) for 1 WT mouse compared with 4 transgenic mice of each strain. A decrease in C_T indicates more abundant levels of gene expression. C_T scale is expressed in log₂, indicating that a C_T change of 1 is equivalent to a 2-fold change in expression. C: RT-PCR analysis of IκBα expression in homogenized lung tissue from control (Alum/Ova) or Ova-sensitized and challenged (Ova/Ova) WT and CC10-IκBα_SR mice on the C57BL/6 background. Samples were run on the same gels, and images were cropped and reassembled for consistency of presentation. Expression of β-actin, a housekeeping gene, was assessed as a control. Data are representative of 7 mice in each of the Ova/Ova groups and 5 mice in each of the Alum/Ova groups. Different lanes represent results from individual mice. D: LPS-induced expression of CCL20 in whole lung homogenates. WT and transgenic mice were instilled with LPS for 2 h, and whole lung RNA was analyzed for CCL20 expression. Data are representative of 3 mice in each of the groups. *P < 0.05 (by ANOVA).

Fig. 3.

CC10-IκBα_SR expression moderately attenuates mucus metaplasia and enhances airway hyperresponsiveness (AHR) in C57BL/6 mice. A: mucus metaplasia in periodic acid-Schiff-stained airway epithelium from CC10-IκBα_SR-expressing and WT mice sensitized and challenged with Ova (Ova/Ova). Alum/Ova represent control (mock-sensitized) groups (Alum/Ova). *P < 0.05 (by ANOVA). B: expression of Clca3 (Gob5) is suppressed in CC10-IκBα_SR mice compared with WT controls. RNA from whole lung homogenates of Ova-sensitized and challenged mice (Ova/Ova) or mock-sensitized mice (Alum/Ova) was analyzed by quantitative RT-PCR, and results were normalized to the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase. Data are expressed as fold increases in expression compared with WT Alum/Ova controls. *P < 0.05 (by ANOVA). C: WT or CC10-IκBα_SR transgenic mice on the C57BL/6 background were mock-sensitized and challenged with Ova (Alum/Ova) or sensitized and exposed to aerosolized Ova (Ova/Ova). At 48 h after the 3rd Ova challenge, mice were ventilated and challenged with 3.125, 12.5, or 50 mg/ml methacholine (MCh). AHR was assessed by measurement of oscillatory mechanics, and data were fit to the constant-phase model. Airway resistance (a), tissue resistance (b), and tissue elastance (c) are expressed as percent increases from baseline. *P < 0.05 (by ANOVA) vs. WT Ova/Ova groups. Data were obtained from 7 mice in each of the Ova/Ova groups and 5 mice in each of the Alum/Ova groups.

Fig. 4.

C57BL/6 mice fail to activate NF-κB in airway epithelium early following Ova challenge. A: immunized BALB/c and C57BL/6 mice were challenged with aerosolized Ova once for 30 min, and lungs were harvested 2 h later. Localization of RelA was evaluated via immunofluoresence and confocal microscopy [nuclear SYTOX green (green), RelA (red), and overlap (yellow)]. Data are representative of 4 mice per group and evaluations of multiple images per mouse. Magnification ×200. B: NF-κB DNA-binding activity by EMSA in whole lung homogenates from immunized BALB/c and C57BL/6 mice challenged with aerosolized Ova for 30 min/day for 3 consecutive days and harvested 48 h later. NF-κB-labeled region depicts electrophoretic mobility shift due to RelA and p50 DNA binding. NS indicates nonspecific DNA binding. Each lane represents an independent mouse.

Fig. 5.

Primary mouse tracheal epithelial cells (MTEC) derived from BALB/c and C57BL/6 mice activate the NF-κB pathway similarly in vitro. MTEC cultures were established in parallel from BALB/c and C57BL/6 mice. Confluent MTEC on Transwell inserts were treated with TNF-α (50 ng/ml) for 0–60 min. IκB kinase (IKK) activity was determined by an in vitro kinase assay. GST-IκBα indicates phosphorylated IκBα substrate. Western blotting for β-actin was performed on the same cell lysates run on parallel gels as a loading control.

Fig. 6.

Role of TNF-α in rapid activation of NF-κB in airway epithelium of BALB/c mice after Ova sensitization and challenge. Ova-immunized BALB/c mice were treated with TNF-α-neutralizing antibody (anti-TNF-α), control nonspecific IgG (control IgG), or vehicle control (n = 4 mice/group). Mice were then challenged with aerosolized Ova for 30 min and harvested 2 h later. Frozen lung sections were assessed for nuclear localization of RelA by confocal laser scanning microscopy [nuclear SYTOX green (green), RelA (red), and overlap (yellow)]. Results are representative of 5 images collected, on average, per group. Magnification ×200.