Tyrosine kinase activation in LPS stimulated rat Kupffer cells.
Kupffer cells, a majority of the body's fixed macrophages, are a major site of bacterial lipopolysaccharide (LPS) metabolism and are mediators in the body's response to sepsis. Uptake of LPS is different in Kupffer cells than other macrophages. Signal transduction in other macrophages in response to LPS involves phosphorylation of proteins in the 50-60 kDa range. We hypothesized that Kupffer cells may have unique signal transduction pathways in response to LPS. Rat Kupffer cells were exposed to LPS (1 microgram/mL) for varying times ranging from 15 to 90 min. Cell lysates were Western blotted using an anti-phosphotyrosine antibody. The blots showed an increase in the amount of tyrosine phosphorylation on two proteins of 119 kDa and 83 kDa. The effects of varying LPS concentration (1 ng/mL-1 microgram/mL) showed an increasing amount of phosphorylation with increasing LPS concentration. To associate the importance of tyrosine phosphorylation in the response of Kupffer cells to LPS, the tyrosine kinase inhibitors, tyrphostin, lavendustin, and genisten were used to study the effects of inhibiting phosphorylation on TNF-alpha production. Kupffer cells were preincubated in the presence of the inhibitor and exposed to LPS (1 microgram/mL). TNF-alpha was measured in the conditioned media by ELISA. A 70% or greater decrease in TNF-alpha production was observed. When phagocytosis of latex beads by rat Kupffer cells was measured in vivo using intravital video microscopy, LPS treatment significantly increased uptake. This increase in phagocytosis was inhibited by tyrphostin. These results show what may be unique phosphorylation events in Kupffer cells that are related to LPS induced production of TNF-alpha.
Schultze
R L
RL
Department of Surgery, Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
Gangopadhyay
A
A
Cay
O
O
Lazure
D
D
Thomas
P
P
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D007728:Kupffer Cells / Q000201:enzymology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D018715:Microscopy, Video; D010587:Phagocytosis / Q000187:drug effects; D010636:Phenols / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D010636:Phenols; D014409:Tumor Necrosis Factor-alpha; D020032:Tyrphostins; C069028:AG 127; C062898:lavendustin A; D019833:Genistein; D011505:Protein-Tyrosine Kinases