Macrophage TNF secretion in endotoxin tolerance: role of SAPK, p38, and MAPK.
OBJECTIVE
Endotoxin (LPS) activation of macrophages results in phosphorylation of mitogen-activated protein kinases (MAPK), stress-activated protein kinases (SAPK), and p38 kinase. LPS pretreatment inhibits subsequent LPS-stimulated MAPK activation and TNF release and both were reversed if macrophages were treated with phorbol myristate acetate (PMA) before LPS stimulation. In this study we sought to determine if SAPK and p38 tyrosine kinases are required for TNF production and if LPS pretreatment alters their activation.


METHODS
TNF production by murine peritoneal exudate macrophages was determined 6 h after stimulation with 100 ng/mL of LPS +/- 24 h pretreatment with 10 ng/mL of LPS. The active, diphosphorylated forms of MAPK (p42, p44), SAPK (p46, p54), and p38 were assayed 30 min after LPS stimulation by Western immunoblot using specific antibodies. In some experiments a p38 kinase inhibitor (SB202190) or the protein kinase C activator (PMA) was added 1 h before LPS stimulation.


RESULTS
LPS activated MAPK, SAPK, and p38. LPS pretreatment significantly inhibited MAPK, SAPK, and p38 activation by LPS stimulation. TNF protein secretion and MAPK activation in tolerant macrophages were restored by PMA treatment, but this did not restore SAPK activation. The p38 inhibitor SB202190 blocked LPS-stimulated TNF production.


CONCLUSIONS
LPS pretreatment-induced tolerance decreased LPS-stimulated MAP, SAP, and p38 kinase activation. LPS tolerance in murine macrophages appears to be associated with specific, PMA-reversible defects in MAPK and p38 kinase activation.
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004361:Drug Tolerance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007375:Interleukin-1 / Q000096:biosynthesis; D007700:Kinetics; D008070:Lipopolysaccharides / Q000633:toxicity*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000502:physiology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D020928:Mitogen-Activated Protein Kinases; D011494:Protein Kinases / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases