Selective activation and functional significance of p38α mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils
Jerry A. Nick, … , Gary L. Johnson, G. Scott Worthen
Jerry A. Nick, … , Gary L. Johnson, G. Scott Worthen
Published March 15, 1999
Citation Information: J Clin Invest. 1999;103(6):851-858. https://doi.org/10.1172/JCI5257.
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Selective activation and functional significance of p38α mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils

Abstract

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38α and p38δ were detected in neutrophils. LPS stimulation selectively activated p38α. Specific inhibitors of p38α MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-κB) activation, and synthesis of tumor necrosis factor-α (TNF-α). Inhibition of p38α MAPk resulted in a transient decrease in TNF-α mRNA accumulation but persistent loss of TNF-α synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38α MAPk, ultimately regulating adhesion, NF-κB activation, enhanced gene expression of TNF-α, and regulation of TNF-α synthesis.

Authors

Jerry A. Nick, Natalie J. Avdi, Scott K. Young, Lisa A. Lehman, Patrick P. McDonald, S. Courtney Frasch, Marcella A. Billstrom, Peter M Henson, Gary L. Johnson, G. Scott Worthen

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Figure 5

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Quantification of TNF-α mRNA by RPA. (a) RPA autoradiograph of TNF-α mRN...
Quantification of TNF-α mRNA by RPA. (a) RPA autoradiograph of TNF-α mRNA at 0, 30, and 60 min after stimulation with LPS (100 ng/ml) at 37°C in the presence (+) and absence (–) of SB203580 (10 μM). An increase in TNF-α mRNA seen after 30 min of stimulation (lane 3) is substantially reduced in cells treated with SB203580 (lane 4). At 60 min, the quantity of TNF-α mRNA in the untreated and SB203580-treated cells (lanes 5 and 6) is equivalent. The blot is representative of three consecutive experiments. (b) Normalized plot of TNF-α mRNA synthesis as quantified by RPA. Amount of TNF-α mRNA present for each condition was expressed as a fraction of GAPDH present for each sample to correct for potential differences in sample loading. Each value was then normalize to the amount of TNF-α mRNA present at 30 min of LPS stimulation in the untreated neutrophils (lane 3) to correct for variability in response between donors. After 30 min of LPS stimulation, SB203580-treated neutrophils (closed bar) demonstrated 42% of the TNF-α mRNA present in the untreated cells (hatched bar). After 60 min of stimulation, no significant difference is seen. The panel represents mean values and SEM of three consecutive experiments. (c) Release of TNF-α under conditions studied. TNF-α released from the neutrophils studied in b was assayed. The quantity of TNF-α released per 106 neutrophils was plotted for each time in the presence (closed bars) or absence (hatched bars) of SB203580. The panel represents mean values and SEM of the three consecutive experiments described for b. GAPDH, glyceraldehyde phosphate dehydrogenase; RPA, RNase protection assay.