Immunohistochemical analysis of the activation of NF-?B and expression of associated cytokines and adhesion molecules in human models of allergic inflammation

Abstract

To investigate the role of NF-kappaB in regulating allergic inflammation, a monoclonal antibody directed to the activated form of NF-kappaB has been developed and immunohistochemistry has been employed to study the pro-inflammatory transcriptive function of NF-kappaB and the adhesion molecules and cytokines that it regulates. Human umbilical vein endothelial cells (HUVECs) exposed to physiological levels of TNFalpha demonstrated dose- and time-dependent cytoplasmic and nuclear activation of NF-kappaB, followed by up-regulation of ICAM-1. This was suppressed by the selective inhibitors of NF-kappaB activation, calpain and gliotoxin. Using monoclonal antibodies directed to NF-kappaB and associated cytokines and adhesion molecules, immunohistochemistry was applied to bronchial explants stimulated ex vivo with TNFalpha, and to nasal polyp tissue, embedded in glycol methacrylate. Stimulation of the bronchial explants increased expression of NF-kappaB, IL-8, and GM-CSF in the epithelium and endothelium and ICAM-1 in the endothelium. In nasal polyp, expression of NF-kappaB was in the epithelium, the endothelium and in submucosal mast cells, eosinophils, T and B lymphocytes, and macrophages. Thus, immunohistochemistry can be used to determine the cellular provenance of NF-kappaB and its activation status in single cell and complex tissue systems, in parallel with appropriate inflammatory markers.