Activation of Kupffer cells and caspase-3 involved in rat hepatocyte apoptosis induced by endotoxin.
OBJECTIVE
Sepsis and lipopolysaccharides (LPS) cause mild to severe hepatic dysfunction. In this study, Kupffer cell activation, involvement of TNFalpha and caspases downstream of the TNFalpha receptor were examined in hepatocyte apoptosis induced by LPS.


METHODS
In in vivo experiments, male Sprague-Dawley rats were injected intravenously with LPS, and small amounts of the blood and liver were sampled to evaluate apoptosis. Kupffer cells were inactivated by pretreatment with gadolinium chloride for 2 days. In in vitro experiments, hepatocytes and Kupffer cells were separately isolated from rat livers using collagenase perfusion.


RESULTS
LPS induced time-dependent and dose-dependent increases in the number of TUNEL-positive cells, which coincided with the apoptotic features of hepatocytes demonstrated by electron microscopy and DNA ladder. Activation of caspase-3-like proteases was observed with an increase in the number of apoptotic hepatocytes. Immunostaining with activated caspase-3-specific antibody showed that caspase-3 was activated only in the cytoplasm of TUNEL-positive hepatocytes. Inactivation of Kupffer cells by gadolinium chloride was concomitantly accompanied by the prevention of caspase-3 activation, hepatocyte apoptosis and liver injury induced by LPS. The co-culture system of hepatocytes and Kupffer cells, but neither cell culture system, individually, showed LPS-induced hepatocyte apoptosis. Kupffer cell-conditioned medium induced hepatocyte apoptosis, whereas addition of anti-TNFalpha antibody to Kupffer cell-conditioned medium did not. Additions of acetyl-DEVD-CHO, acetyl-YVAD-CHO, and acetyl-IETD-CHO to Kupffer cell-conditioned medium decreased the number of apoptotic hepatocytes.


CONCLUSIONS
These results suggest that the activation of Kupffer cells, TNFalpha and caspases downstream of TNFR1 were involved in hepatocyte apoptosis induced by LPS.
Hamada
E
E
First Department of Surgery, Osaka University Medical School, Suita, Japan.
Nishida
T
T
Uchiyama
Y
Y
Nakamura
J
J
Isahara
K
K
Kazuo
H
H
Huang
T P
TP
Momoi
T
T
Ito
T
T
Matsuda
H
H
D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D017209:Apoptosis; D001219:Aspartate Aminotransferases / Q000097:blood; D001663:Bilirubin / Q000097:blood; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D003593:Cytoplasm / Q000201:enzymology; D004731:Endotoxins / Q000008:administration & dosage / Q000633:toxicity*; D004789:Enzyme Activation; D005682:Gadolinium / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007275:Injections, Intravenous; D007728:Kupffer Cells / Q000187:drug effects / Q000473:pathology / Q000502:physiology*; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000633:toxicity*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology / Q000648:ultrastructure; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D017077:Culture Media, Conditioned; D004731:Endotoxins; D008070:Lipopolysaccharides; D005682:Gadolinium; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C038958:gadolinium chloride; D001663:Bilirubin