Esophageal Expression of Active IκB Kinase-β in Mice Up-Regulates Tumor Necrosis Factor and Granulocyte-Macrophage Colony-Stimulating Factor, Promoting Inflammation and Angiogenesis.

BACKGROUND &

aimsIκB kinase-β (IKKβ) mediates activation of the nuclear factor-κB, which regulates immune and inflammatory responses. Although nuclear factor-κB is activated in cells from patients with inflammatory diseases or cancer, little is known about its roles in the development and progression of esophageal diseases. We investigated whether mice that express an activated form of IKKβ in the esophageal epithelia develop esophageal disorders.

methodsWe generated ED-L2-Cre/Rosa26-IKK2caSFL mice, in which the ED-L2 promoter activates expression of Cre in the esophageal epithelia, leading to expression of a constitutively active form of IKKβ (IKKβca) in epithelial cells but not in inflammatory cells or the surrounding stroma (IKKβca mice). Mice lacking the Cre transgene served as controls. Some mice were given intraperitoneal injections of neutralizing antibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor (TNF), or immunoglobulin G1 (control), starting at 1 month of age. Epithelial tissues were collected and analyzed by immunofluorescence, immunohistochemical, and quantitative real-time polymerase chain reaction assays. Transgenes were overexpressed from retroviral vectors in primary human keratinocytes.

resultsIKKβca mice developed esophagitis and had increased numbers of blood vessels in the esophageal stroma, compared with controls. Esophageal tissues from IKKβca mice had increased levels of GM-CSF. Expression of IKKβca in primary human esophageal keratinocytes led to 11-fold overexpression of GM-CSF and 200-fold overexpression of TNF. Incubation of human umbilical vein endothelial cells with conditioned media from these keratinocytes increased endothelial cell migration by 42% and promoted formation of capillary tubes; these effects were blocked by a neutralizing antibody against GM-CSF. Injections of anti-GM-CSF reduced angiogenesis and numbers of CD31+ blood vessels in esophageal tissues of IKKβca mice, but did not alter the esophageal vasculature of control mice and did not alter recruitment of intraepithelial leukocytes to esophageal tissues of IKKβca mice. Injections of anti-TNF prevented the development of esophagitis in IKKβca mice.

conclusionsConstitutive activation of IKKβ in the esophageal epithelia of mice leads to inflammation and angiogenesis, mediated by TNF and GM-CSF, respectively.