ENDOCYTOSIS AND CA2-STIMULATED TNF-ALPHA RELEASE BY RAT KUPFFER CELLS( ARE REQUIRED FOR ENDOTOXIN)

Endotoxin [lipopolysaccharide (LPS)] Is a cell wall polymer derived fr om Gram-negative bacteria that stimulates macrophages to produce a var iety of inflammatory mediators. In these studies, we examined LPS-stim ulated formation of tumor necrosis factor-alpha (TNF-alpha) by culture d rat Kupffer cells. Cytochalasin B and methylpalmitate, blockers of e ndocytosis, decreased LPS-stimulated TNF-alpha release by >92%. Bafilo mycin A, monensin, and chloroquine, which prevent endosomal acidificat ion, also blocked LPS-stimulated release of TNF-alpha by >90%. Cytocha lasin B and bafilomycin A decreased TNF-alpha mRNA levels by >90% afte r LPS stimulation. Consistent with the requirement for LPS uptake and processing was the observation that Kupffer cells required 30 min of c ontact with LPS for maximal TNF-alpha release. LPS-stimulated TNF-alph a release was unaltered by incubation in Ca2+-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 absen ce of LPS. However, nisoldipine, a Ca2+ channel blocker, suppressed LP S-stimulated TNF-alpha release in cells cultured both in Ca2+-containi ng and Ca2+-free media. Although thapsigargin did not block TNF-alpha release, this depleter of intracellular Ca2+ stores blocked LPS-stimul ated TNF-alpha synthesis in Ca2+-free medium and decreased TNF-alpha m RNA levels by 80%. Furthermore, LPS induced a late rise in intracellul ar free Ca2+ demonstrated by video microscopy of fura 2-loaded Kupffer cells. De novo protein and RNA synthesis were required, since cyclohe ximide and actinomycin D also inhibited LPS-stimulated TNF-alpha relea se. 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 proc essing may be necessary for LPS-stimulated TNF-alpha release from Kupf fer cells. Ca2+ release, regulated by dihydropyridine-sensitive Ca2+ c hannels, also appears to be necessary for LPS-induced signaling and ma y arise from intracellular stores associated with the endosome/lysosom e compartment.