The role of Nuclear Factor-kappa B (NF-kB) in the regulation of lung inflammation.
Everhart, Michael Brett
The NF-kappaB pathway has been shown to play a critical role in both adaptive and innate immunity and has been implicated as a focal point for induction of lung inflammation by a variety of inflammatory stimuli; however, the role of NF-kappaB in specific lung cell types remains unclear. We hypothesized that individual cell types in the lungs make important and unique contributions to the NF-kappaB dependent innate immune response. To determine the temporal and cell specific activation of NF-kappaB in vivo, an NF-kappaB reporter mouse in which expression of an enhanced green fluorescent protein (eGFP)/luciferase fusion protein cDNA driven by an NF-kappaB inducible promoter (NGL mouse) was generated. NF-kappaB activity was detected in intact, anesthetized animals by bioluminescence imaging and at the cellular level by detection of GFP on lung tissue sections. Using Eschericia coli lipopolysaccharide (LPS) and Pseudomonas aeruginosa models of lung inflammation, the timing and duration of NF-kappaB activation in alveolar macrophages, neutrophils, airway epithelium, and endothelium was determined to be dependent on the dose, duration, and route of delivery. To determine the role NF-kappaB in alveolar macrophages, bone marrow chimeras were generated by fetal liver transplantation (FLT) using the HLL transgenic NF-kappaB reporter mouse (HIV-LTR driving expression of luciferase) as the recipient and WT, IkappaB-alpha heterozygous, IkappaB-alpha knockout, and p50 knockout mice as donors. After LPS administration, lung NF-kappaB activation (bioluminescence imaging) and inflammation (neutrophil influx) were detected. IkappaB-alpha heterozygous FLT chimeras displayed enhanced and prolonged NF-kappaB activation and inflammation compared to WT FLT chimeras, while IkappaB-alpha knockout FLT and p50 knockout FLT chimeras displayed severe NF-kappaB activation and inflammation accompanied by mortality. To study macrophage/epithelium communication, co-culture studies using bone marrow derived macrophages from the above donors and epithelial cells containing an NF-kappaB reporter were performed. After LPS treatment, IkappaB-alpha knockout and p50 knockout macrophages demonstrated prolonged NF-kappaB activation, compared to WT, resulting in sustained NF-kappaB activation in epithelial cells. Taken together, these studies provide important insight into the importance of the NF-kappaB pathway in regulating lung inflammation and suggest IkappaB-alpha and p50 play a critical role in the active “turn-off” of NF-kappaB.