Characterization and Quantitation of NF-κB Nuclear Translocation Induced by Interleukin-1 and Tumor Necrosis Factor-α

Abstract

A new quantitative cytometric technique, termed the ArrayScan™, is described and used to measure NF-B nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-␣ (TNF␣). 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-B 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 IB␣ degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNF␣ and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNF␣ 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 TNF␣ 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 IB␣ degradation and subsequent NF-B translocation could also be discriminated; LeuLeu-Leu aldehyde was only a partial inhibitor with an IC 50 of 2 M, while clastolactacystin ␤-lactone was a complete inhibitor with an IC 50 of 10 M. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNF␣ stimulation in both cell types with an IC 50 of 0.4 M. 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 TNF␣ 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-B. IL-11 and TNF␣ are two master cytokines that induce an almost identical proinflammatory response, including the production of chemotactic cytokines, adhesion molecules, and enzymes such as cyclooxygenase, nitric-oxide synthetase, and matrix metalloproteinases (1, 2). Many of these effects are a result of the activation by both IL-1 and TNF␣ of the NF-B transcription factor pathway, which is associated with the activation of many cellular defense genes (3, 4). Composed of p65 (RelA) and p50 proteins, NF-B is normally present in the cytoplasm in an inactive state in a complex with members of the IB inhibitor protein family, chiefly the 37-kDa IB␣ form. In this complexed form, a nuclear localization sequence found on NF-B is masked by the IB␣, preventing nuclear translocation of NF-B, DNA binding, and subsequent transcriptional activation (5-12). IL-1 or TNF␣ receptor activation induces within several minutes the specific phosphorylation of Ser 32 and Ser 36 on IB␣, the destruction of the phosphorylated IB␣ protein by proteasomes, and the translocation of NF-B to the nucleus (13-1...

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