Functional assay of NF-κB translocation into nuclei by laser scanning cytometry: inhibitory effect by dexamethasone or theophylline

Abstract

Nuclear factor-κ B (NF-κB)/Rel transcription factors play an important role in the inducible regulation of a variety of cytokine genes in epithelial cells. We assessed accumulation of fluorescence-stained NF-κB into propi-dium iodide-stained nuclei using laser scanning cytometry. “Activity-specific” antibodies to the Rel A (p65) and NF-κB1 (p50) subunits of NF-κB were detected in the nuclei of A549 cells (an immortalized human type II alveolar epithelial cell line). Exposure to TNF-alpha caused p65 and p50 to translocate into nuclei in a dose- and time-dependent manner. Preincubation with dexamethasone for 24 h or 30 min, or with theophylline for 30 min before TNF-alpha stimulation attenuated the nuclear translocation of Rel A and p50. These findings suggest that theophylline, as well as dexamethasone, may inhibit translocation of NF-κB.