Lipopolysaccharide-stimulated TNF-alpha release from cultured rat Kupffer cells: sequence of intracellular signaling pathways.

We recently hypothesized that lipopolysaccharide (LPS) stimulation of rat Kupffer cells to induce tumor necrosis factor alpha (TNF-alpha) release requires internalization of LPS, acidification of endosomes, elevation of intracellular calcium, protein kinase C (PKC) activation, and protein tyrosine kinase (PTK) activation. This study uses inhibitors in pulse-chase experiments to determine the sequence of events of intracellular signals required for LPS-stimulated TNF-alpha release from Kupffer cells. Inhibitors of internalization (cytochalasin B, monodansylcadaverine) prevented LPS-stimulated TNF-alpha release when added simultaneously with LPS but when added 10 min after LPS, no significant inhibition occurred. The inhibitor of PTK, tyrphostin AG, blocked TNF-alpha release by only 39 +/- 4% (P < 0.001 compared with TNF-alpha release when added simultaneously with LPS) when added 10 min after LPS. Inhibitors of endosomal acidification (bafilomycin A, monensin) inhibited LPS-stimulated TNF-alpha release by 92 +/- 11% (P < 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-alpha release by 94 +/- 9% (P < 0.001 when no inhibitor was used) when added 30 min after LPS. The calcium channel blocker, nisoldipine, still inhibited LPS-stimulated TNF-alpha release when added 45 min after LPS. These data support the hypothesis that for LPS-stimulated TNF-alpha release in Kupffer cells, LPS must first be internalized, 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.