Constitutive NF-kappa B levels in human alveolar macrophages from normal volunteers

Alveolar macrophages regulate the inflammatory and immune responses within the lung through cytokine production, Nuclear factor kappa B (NF-kappa B), a transcription factor, controls the synthesis of cytokines such as interle ukin 1 beta, tumour necrosis factor alpha, and interleukin 8, In quiescent cells, NF-kappa B is located in the cytosol as a dimer of protein component s (p50, p65) bound to an inhibitor (I kappa B), Upon activation, NF-kappa B translocates to the nucleus and binds to DNA, To determine the constitutiv e level of NF-kappa B activation in non-smoking normal volunteers, immunohi stochemical analysis of alveolar macrophages from 29 subjects was performed with antibody directed against the p65 component of NF-kappa B, These resu lts were confirmed in four subjects by eletrophoretic mobility shift assay (EMSA), A human monocytic cell line, THP-1, with and without endotoxin stim ulation was used as positive and negative controls, respectively. The mean number of positive cells was 4.1% +/- 0.8, EMSA performed on whole cell ext racts from four normal volunteers demonstrated minimal constitutive binding compared to the positive control. Supershift assay revealed the presence o f the p65 dimer, By both immunohistochemistry and EMSA, alveolar macrophage s from healthy non-smoking individuals demonstrate minimal NF-kappa B activ ation. Immunohistochemistry is a sensitive and quantifiable technique requi ring only a minimal number of cells, and this technique may be useful in mo nitoring small changes in NF-kappa B activation in inflammatory diseases of the lung. (C) 1998 Academic Press.