CHARACTERIZATION AND QUANTITATION OF NF-KAPPA-B NUCLEAR TRANSLOCATIONINDUCED BY INTERLEUKIN-1 AND TUMOR-NECROSIS-FACTOR-ALPHA - DEVELOPMENT AND USE OF A HIGH-CAPACITY FLUORESCENCE CYTOMETRIC SYSTEM

A new quantitative cytometric technique, termed the ArrayScan(TM), is described and used to measure NF-KB nuclear translocation induced by i nterleukin (IL)-1 and tumor necrosis factor-alpha (TNF alpha). The amo unt of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a presel ected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-kappa B w as found to move to the nucleus with a half-time of 7-8 min for HeLa a nd 12-13 min for chondrocytes, a rate in each case about 4-5 min slowe r than that of I kappa B alpha degradation. IL-1 receptor antagonist a nd anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNF alp ha and monoclonal anti-TNF receptor 1 antibodies on the other hand cou ld be shown to respectively inhibit IL-1 and TNF alpha stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiser um exhibited both a 50% agonism and a 50% antagonism to a TNF alpha st imulation in a dose-dependent fashion, indicating that subtle function al responses to complex agonist and antagonist stimuli could be measur ed. The effects of different proteasome inhibitors to prevent I kappa B alpha degradation and subsequent NF-kappa B translocation could also be discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor w ith an IC50 of 2 mu M, while clastolactacystin beta-lactone was a comp lete inhibitor with an IC50 of 10 mu M. The nonselective kinase inhibi tor K252a completely inhibited both IL-1 and TNF alpha stimulation in both cell types with an IC50 of 0.4 mu M. This concentration determine d after a 20-min stimulation, was shown to be comparable with that obt ained for inhibition of IL-6 production induced by a 100-fold lower IL -1 and TNF alpha concentration measured after 17 h of stimulation. The se results suggest that the ArrayScan(TM) technology provides a rapid, sensitive, quantitative technique for measuring early events in the s ignal transduction of NF-kappa B.