title: Endocytosis and Ca2+ are required for endotoxin-stimulated TNF-alpha release by rat Kupffer cells.
abstract: Endotoxin [lipopolysaccharide (LPS)] is a cell wall polymer derived from Gram-negative bacteria that stimulates macrophages to produce a variety of inflammatory mediators. In these studies, we examined LPS-stimulated formation of tumor necrosis factor-alpha (TNF-alpha) by cultured rat Kupffer cells. Cytochalasin B and methylpalmitate, blockers of endocytosis, decreased LPS-stimulated TNF-alpha release by > 92%. Bafilomycin A, monensin, and chloroquine, which prevent endosomal acidification, also blocked LPS-stimulated release of TNF-alpha by > 90%. Cytochalasin B and bafilomycin A decreased TNF-alpha mRNA levels by > 90% after LPS stimulation. Consistent with the requirement for LPS uptake and processing was the observation that Kupffer cells required 30 min of contact with LPS for maximal TNF-alpha release. LPS-stimulated TNF-alpha release was unaltered by incubation in Ca(2+)-free ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid medium, and A-23187, a Ca2+ ionophore, failed to stimulate TNF-alpha release in the absence of LPS. However, nisoldipine, a Ca2+ channel blocker, suppressed LPS-stimulated TNF-alpha release in cells cultured both in Ca(2+)-containing and Ca(2+)-free media. Although thapsigargin did not block TNF-alpha release, this depleter of intracellular Ca2+ stores blocked LPS-stimulated TNF-alpha synthesis in Ca(2+)-free medium and decreased TNF-alpha mRNA levels by 80%. Furthermore, LPS induced a late rise in intracellular free Ca2+ demonstrated by video microscopy of fura 2-loaded Kupffer cells. De novo protein and RNA synthesis were required, since cycloheximide and actinomycin D also inhibited LPS-stimulated TNF-alpha release. We compared free TNF-alpha secreted into culture supernatants with cell-associated TNF-alpha and found that cytochalasin B, bafilomycin A, chloroquine, monensin, and nisoldipine did not increase bound, cell-associated TNF-alpha. We conclude that endocytosis and endocytic processing may be necessary for LPS-stimulated TNF-alpha release from Kupffer cells. Ca2+ release, regulated by dihydropyridine-sensitive Ca2+ channels, also appears to be necessary for LPS-induced signaling and may arise from intracellular stores associated with the endosome/lysosome compartment.
authors.affiliation: Department of Pediatrics, University of North Carolina, Chapel Hill 27599-7220, USA.
authors.forename: S N
authors.initials: SN
authors.lastname: Lichtman
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: C
authors.initials: C
authors.lastname: Zhang
authors.forename: J J
authors.initials: JJ
authors.lastname: Lemasters
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D020126:Brefeldin A; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004705:Endocytosis; D004731:Endotoxins / Q000633:toxicity*; D004926:Escherichia coli; D007700:Kinetics; D007728:Kupffer Cells / Q000187:drug effects / Q000502:physiology*; D008070:Lipopolysaccharides / Q000633:toxicity*; D018942:Macrolides; D008297:Male; D008985:Monensin / Q000494:pharmacology; D015737:Nisoldipine / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D003517:Cyclopentanes; D004731:Endotoxins; D008070:Lipopolysaccharides; D018942:Macrolides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C057620:bafilomycin A; D020126:Brefeldin A; D003571:Cytochalasin B; D015737:Nisoldipine; D008985:Monensin; D002118:Calcium