Endocytosis and calcium are required for endotoxin-stimulated tnf(
release by rat kupffer cells.
Lichtman, Steven N., Jian Wang, Chuan Zhang, and John J. Lemasters.
Departments of Pediatrics and Cell Biology & Anatomy, University
of North Carolina, Chapel Hill, North Carolina
APStracts 3:0135G, 1996.
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() by
cultured rat Kupffer cells. Cytochalasin B and methylpalmitate,
blockers of endocytosis, decreased LPS-stimulated TNF( release by
greater than 92%. Bafilomycin A, monensin, and chloroquine, which
prevent endosomal acidification, also blocked LPS-stimulated release
of TNF( by more than 90%. Cytochalasin B and bafilomycin A decreased
TNF( mRNA levels by greater than 90% after LPS stimulation.
Consistent with the requirement for LPS uptake and processing was the
observation that Kupffer cells required 30 minutes of contact with
LPS for maximal TNF( release. LPS-stimulated TNF( release was
unaltered by incubation in Ca2+-free/EGTA medium, and A23187, a
calcium ionophore, failed to stimulate TNF( release in the absence of
LPS. However, nisoldipine, a Ca2+ channel blocker, suppressed LPS
-stimulated TNF( release in cells cultured both in Ca2+-containing and
Ca2+-free media. Although thapsigargin did not block TNF( release,
this depleter of intracellular Ca2+ stores blocked LPS-stimulated
TNF( synthesis in Ca2+-free medium and decreased TNF( 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( release. We compared
free TNF( secreted into culture supernatants to cell-associated TNF(
and found that cytochalasin B, bafilomycin A, chloroquine, monensin
and nisoldipine did not increase bound, cell-associated TNF(. We
conclude that endocytosis and endocytic processing may be necessary
for LPS-stimulated TNF( release from Kupffer cells. Ca2+ release,
regulated by dihydropyridine-sensitive Ca2+ channels, also appears
necessary for LPS-induced signaling and may arise from intracellular
stores associated with the endosome/lysosome compartment.
Received 29 June 1996; accepted in final form 9 February 1996.
APS Manuscript Number G53-6.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 25 July 1996