Characterization and Quantitation of NF-κB Nuclear Translocation Induced by Interleukin-1 and Tumor Necrosis Factor-α DEVELOPMENT AND USE OF A HIGH CAPACITY FLUORESCENCE CYTOMETRIC SYSTEM

Abstract A new quantitative cytometric technique, termed the ArrayScan™, is described and used to measure NF-κB nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-α (TNFα). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-κB was found to move to the nucleus with a half-time of 7–8 min for HeLa and 12–13 min for chondrocytes, a rate in each case about 4–5 min slower than that of IκBα degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNFα and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNFα stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNFα stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent IκBα degradation and subsequent NF-κB translocation could also be discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor with an IC50 of 2 μm, while clastolactacystin β-lactone was a complete inhibitor with an IC50 of 10 μm. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNFα stimulation in both cell types with an IC50 of 0.4 μm. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNFα concentration measured after 17 h of stimulation. These results suggest that the ArrayScan™ technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-κB.