SHORT-TIME EXPOSURE TO LIPOPOLYSACCHARIDE IS SUFFICIENT TO ACTIVATE HUMAN MONOCYTES

Very little is known about early events in LPS binding and about the d uration of LPS exposure required to activate monocytes. In the present study, we have investigated the kinetics of LPS binding to human mono cytes and the time of exposure required to trigger the synthesis of TN F-alpha. We directly followed the binding of FITC-labeled LPS to monoc ytes by flow cytometry or confocal laser microscopy. LPS was able to b ind to the cell surface within 1 min of exposure, and was internalized within 5 min. Equilibrium was reached within 15 min at all but the lo west LPS concentration tested (10 ng/ml). Binding was dependent on the presence of LPS-binding protein, supplied either as a plasma componen t or in purified form, and inhibited by an anti-CD1 4 mAb (MY4). Polym yxin B, an inhibitor of LPS-mediated activation, essentially abrogated the LPS-binding protein- and CD14-dependent binding of LPS to monocyt es. Using either the anti-CD1 4 mAb or polymyxin B to block further LP S binding, we found that 5 to 10 min of exposure was sufficient to tri gger maximal TNF-alpha release. Similarly, monocytes washed after 5 to 15 min exposure to eliminate LPS also produced high levels of TNF-alp ha transcripts without further presence of LPS in the medium. We concl ude that a few minutes of exposure to physiologically relevant concent rations of LPS are sufficient to trigger both maximal binding and acti vation of monocytes.