CONSTITUTIVE NF-κB LEVELS IN HUMAN ALVEOLAR MACROPHAGES FROM NORMAL VOLUNTEERS

Abstract

Alveolar macrophages regulate the inflammatory and immune responses within the lung through cytokine production. Nuclear factor kappa B (NF-κB), a transcription factor, controls the synthesis of cytokines such as interleukin 1β, tumour necrosis factor α, and interleukin 8. In quiescent cells, NF-κB is located in the cytosol as a dimer of protein components (p50, p65) bound to an inhibitor (IκB). Upon activation, NF-κB translocates to the nucleus and binds to DNA. To determine the constitutive level of NF-κB activation in non-smoking normal volunteers, immunohistochemical analysis of alveolar macrophages from 29 subjects was performed with antibody directed against the p65 component of NF-κB. These results were confirmed in four subjects by electrophoretic mobility shift assay (EMSA). A human monocytic cell line, THP-1 with and without endotoxin stimulation was used as positive and negative controls, respectively.The mean number of positive cells was 4.1%±0.8. EMSA performed on whole cell extracts from four normal volunteers demonstrated minimal constitutive binding compared to the positive control. Supershift assay revealed the presence of the p65 dimer. By both immunohistochemistry and EMSA, alveolar macrophages from healthy non-smoking individuals demonstrate minimal NF-κB activation. Immunohistochemistry is a sensitive and quantifiable technique requiring only a minimal number of cells, and this technique may be useful in monitoring small changes in NF-κB activation in inflammatory diseases of the lung.