Lipopolysaccharide‐stimulated TNF‐α release from cultured rat Kupffer cells: sequence of intracellular signaling pathways

We recently hypothesized that lipopoly- saccharide (LPS) stimulation of rat Kupffer cells to induce tumor necrosis factor a (TNF-a) release requires internalization of LPS, acidification of endosomes, elevation of intracellular calcium, pro- tein kinase C (PKC) activation, and protein tyrosine kinase (PTK) activation. This study uses inhibitors in pulse-chase experiments to determine the se- quence of events of intracellular signals required for LPS-stimulated TNF-a release from Kupffer cells. Inhibitors of internalization (cytochalasin B, monodansylcadaverine) prevented LPS-stimulated TNF-a release when added simultaneously with LPS but when added 10 min after LPS, no signifi- cant inhibition occurred. The inhibitor of PTK, tyrphostin AG, blocked TNF-a release by only 39 6 4% (P F 0.001 compared with TNF-a release when added simultaneously with LPS) when added 10 min after LPS. Inhibitors of endosomal acidification (bafilomycin A, monensin) inhibited LPS-stimulated TNF-a release by 92 6 11% (P F 0.001 when no inhibitor was used) when added 10 min after LPS and their effect was totally abrogated when added 45 min after LPS. The PKC inhibitor, H-7, blocked TNF-a release by 94 6 9% (P F 0.001 when no inhibitor was used) when added 30 min after LPS. The calcium channel blocker, nisol- dipine, still inhibited LPS-stimulated TNF-a release when added 45 min after LPS. These data support the hypothesis that for LPS-stimulated TNF-a re- lease in Kupffer cells, LPS must first be internal- ized, which may stimulate PTK activation. An intermediate step of signaling involves endosomal acidification. Elevation of intracellular calcium and PKC activation occur as late intracellular signaling events. J. Leukoc. Biol. 64: 368-372; 1998.