Activation of nuclear factor kappa B (NF-kappaB) assayed by laser scanning cytometry (LSC).

The transcriptional activator nuclear factor kappa B (NF-κB) plays a critical role in cellular response to a variety of stimuli such as pro-inflammatory cytokines, cytotoxic agents, bacterial or viral components as well as other physiological and nonphysiological agents (1–3,9,22,25,27). The factor is involved not only in regulation of the inflammatory response but also in a wide spectrum of other physiological and pathological states, including cancer, tissue injury, sepsis and immunological diseases. With exception of B lymphocyte lineage cells where NF-κB is also localized in the nucleus, in other cell types it remains in the cytoplasm sequestered through interaction with one of the inhibitory proteins of IκB family (1–3, 22,27). The mechanism of activation of NF-κB involves formation of the reactive oxygen intermediates, which act downstream along the activation pathway, triggering phosphorylation and degradation of IκB (1,3,21). Phosphorylation, which is carried out by specific kinase complexes, occurs at critical serine residues of IκB and results in a release of NF-κB which then moves into the nucleus. Free NF-κB is a dimer consisting of p65 (product of RelA gene) and p50 (product of NFκB-1 gene). The rapid translocation of NF-κB from cytoplasm to nucleus triggered by extracellular signals is one of the most characteristic features of this ubiquitous transcription factor (1–3,9,22,25,27). Current methods of detection of NF-κB activation suffer shortcomings. Measurement of the presence of the DNA-bound factor in the nucleus (i.e. active form of the factor) requires nuclear isolation, followed by bulk analysis of NF-κB in the nuclear fraction or its binding to DNA by the DNA gel mobility shift assay (19,24). This approach does not provide any information on cellular heterogeneity, activation of different cell subpopulations, relationship of the activation to cell cycle position, etc. Microscopic observation of the translocation of NF-κBs into the nucleus (following its immunocytochemical staining) is subjective, not quantitative, and not always possible without confocal microscopy. A flow cytometric method was recently described to detect activation of NF-κB in granulocytes and mononuclear cells of human peripheral blood by endotoxin (12). The active, nuclear bound, fraction of NF-κB was detected by lysing the cells and using “washless” procedure to immunocytochemically label this factor in the isolated nuclei (12,16). Based on differences in light scatter of the isolated nuclei it was possible to distinguish activation of granulocytes from mononuclear cells (12). In the present study we describe an approach to analyze activation of NF-κB using the microscope-based laser scanning cytometer (LSC; 15). Similar to flow cytometry, LSC allows one to measure large cell populations rapidly and with high accuracy (4–6,13–15,17). Because there is no cell loss during cell preparation prior to fluorescence measurement, LSC can be used to analyze cell samples containing relatively few cells such as fine needle aspirates (13). In addition, the instrument offers a possibility to perform repeated measurements of the same of cells, a feature useful for kinetic studies or for sequential cell staining with different fluorochromes (6). Among other advantages, LSC allows one to morphologically identify the cells whose fluorescence has been measured as well as to reveal the intracellular distribution of the fluorescence, e.g. its localization in the nucleus or cytoplasm. The latter feature tempted us to test utility of LSC in assaying the activation of NF-κB. The translocation of this factor from cytoplasm to nucleus was presently studied in U-937, HL-60 and Jurkat cells as well as in normal human fibroblasts during their activation by tumor necrosis factor α (TNF-α) or phorbol myristate acetate (PMA). The data indicate that LSC offers a possibility of rapid assay of NF-κB activation, which also can be combined with morphological identification of the activated cells. Bivariate analysis of NF-κB expression in nucleus or cytoplasm makes it also possible to correlate NF-κB activation with the cell cycle position.