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Macrophage TNF secretion in endotoxin tolerance: role of SAPK, p38, and MAPK
J Kraatz1, L Clair, J L Rodriguez
1Hennepin County Medical Center, University of Minnesota, Minneapolis, Minnesota 55415, USA.
Purpose:
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.
Conclusion:
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.
Insights
Lipopolysaccharide (LPS) tolerance in macrophages reduces kinase activation. Phorbol myristate acetate (PMA) partially reverses this tolerance by restoring mitogen-activated protein kinase (MAPK) and p38 kinase activity, but not stress-activated protein kinase (SAPK) activation.
Area of Science:
- Immunology
- Cellular Biology
- Molecular Biology
Background:
- Macrophages play a crucial role in innate immunity, responding to lipopolysaccharide (LPS) by activating signaling pathways like mitogen-activated protein kinases (MAPK), stress-activated protein kinases (SAPK), and p38 kinase.
- LPS pretreatment can induce a state of 'tolerance' in macrophages, characterized by reduced responsiveness to subsequent LPS stimulation, including diminished TNF release and MAPK activation.
- Phorbol myristate acetate (PMA) can reverse LPS-induced tolerance, suggesting a role for protein kinase C (PKC) in regulating macrophage responsiveness.
Purpose of the Study:
- To investigate whether SAPK and p38 tyrosine kinases are essential for TNF production by macrophages.
- To determine if LPS pretreatment, leading to tolerance, alters the activation status of SAPK and p38 kinases.
- To elucidate the role of PMA in reversing LPS-induced tolerance at the level of SAPK and p38 kinase activation.
Main Methods:
- Murine peritoneal exudate macrophages were stimulated with LPS, with or without prior LPS pretreatment, to assess TNF production.
- Western immunoblotting with specific antibodies was used to quantify the active, diphosphorylated forms of MAPK (p42, p44), SAPK (p46, p54), and p38 kinase.
- Inhibition of p38 kinase activity was achieved using SB202190, and PKC activation was induced with PMA prior to LPS stimulation in select experiments.
Main Results:
- LPS stimulation robustly activated MAPK, SAPK, and p38 kinases in macrophages.
- LPS pretreatment significantly attenuated the activation of MAPK, SAPK, and p38 kinases in response to subsequent LPS challenge.
- While PMA treatment restored TNF secretion and MAPK activation in tolerant macrophages, it did not rescue SAPK activation.
- The p38 kinase inhibitor SB202190 effectively blocked LPS-stimulated TNF production, highlighting the critical role of p38 in this process.
Conclusions:
- LPS pretreatment induces tolerance in macrophages, which is associated with reduced activation of MAPK, SAPK, and p38 kinases.
- LPS tolerance involves specific, PMA-reversible defects in MAPK and p38 kinase activation pathways.
- These findings suggest that p38 kinase is a key mediator of LPS-induced TNF production and that its regulation is altered during LPS tolerance.