NF-kB Binding Activity in Mixed Bal Cells From Patients with Sirs and Ards

Abstract

NF-KB is a transcription factor required for maximal gene expression for most pro-inflammatory cytokines but only limited clinical evidence has directly linked the NF-KB activation pathway to the pathogenesis of acute lung injury. We measured NF-KB binding activity in BAL cells of 22 mechanically ventilated patients with severe sepsis, acute lung injury, or ARDS. BAL was performed with three aliquots of 50 ml of physiologic saline in the right middle lobe or lingula at the time of study entry (T0) and 72 hours later (T72). Following centrifugation, the cell fraction was cryopreserved at −70°in 10% glycerol for storage until NF-KB DNA binding activity was measured in mixed BAL cells nuclear protein extracts by the electrophoretic mobility shift assay (EMSA). Binding activity was quantitated by densitometry of NF-KB bands from patient samples compared to a standard curve of NF-KB binding activity generated from PMA-stimulated NRK cell nuclear extracts (binding activity in 1 μg NRK cell extract = 1 NRK unit). Low level NF-KB binding activity (0–1 NRK units) was detected in 11 samples, between 1 and 2 NRK units was detected in 9 samples, between 2 and 5 NRK units in 10 samples, and more than 5 NRK units in 14 samples. There was an overall 41% increase in NF-KB activation at T72 compared to T0 (p = 0.03) and the T72 value was closely correlated to the T0 value (R = 0.703, P = 0.001). No relationship was found between NF-KB binding, activity, IL-8 concentrations, percentage of BAL neutrophils, PaO2/FiO2 ratio or the diagnosis of ARDS or ALI, presence of shock, time on the ventilator or in a critical care unit, or 28-day survival. Although measurement of NF-KB binding activity does not correlate with the biological markers or outcome measures we tested, NF-KB activation in BAL cells may still give clues to the pathogenesis of neutrophilic lung inflammation; however, further studies are necessary to examine the significance of activated BAL cell NF-KB measurements.