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* From the Department of Medicine (Drs. Nick, Avdi, Young, Billstrom, and Worthen), Department of Pediatrics (Drs. McDonald and Henson), Division of Basic Sciences (Dr. Johnson), and Program in Molecular Signal Transduction (Drs. Johnson and Worthen), National Jewish Medical and Research Center, Denver, CO; and the Departments of Medicine (Drs. Nick, Henson, and Worthen) and Pharmacology (Dr. Johnson), University of Colorado School of Medicine, Denver, CO.
Correspondence to: Jerry A. Nick, MD, National Jewish Medical and Research Center, 1400 Jackson St, Denver, CO 80206; e-mail: nickj{at}njc.org
Activation and accumulation of neutrophils in the lung by proinflammatory stimuli is a central event in the pathogenesis of ARDS. Following exposure of the neutrophil to a stimulus, sequential phosphorylation of kinases creates an intracellular signal that results in specific functional responses. Integral to many intracellular signaling pathways is activation of a three-part mitogen-activated protein (MAP) kinase cascade. MAP kinases (MAPk) are a highly conserved superfamily that are central to the regulation of cell growth, differentiation, and stress responses.1 At least three distinct families of MAPks exist in mammalian cells: the p42/44 extracellular signal-regulated kinase (ERK) MAPks,2 c-Jun NH2-terminal kinases,3 and the p38 MAPk.4 Stimulation of human neutrophils with lipopolysaccharide (LPS) has been shown to result in the phosphorylation and activation of the p38 MAP kinase (MAPk), but not the p42/44 MAPks or the c-Jun NH2-terminal MAPk.5 6 7 The goal of this study is to expand our knowledge of the intracellular signaling pathway utilized by the neutrophil in response to LPS by addressing two fundamental questions: what upstream kinase activates p38 MAPk, and what are the functional consequences of this activation?
Activation of a member of the MAPk family is the final step in a three-part intracellular signal transduction cascade in which a MAP/ERK kinase kinase (MEKK) or Raf activates (through phosphorylation) a MAP/ERK kinase (MEK or MAP kinase kinase [MKK]), which in turn phosphorylates a specific tyrosine and threonine residue on a MAPk.1 At least three members of the MKK superfamily are capable of activating p38 MAPk. When overexpressed in transfected cell lines MKK3, MKK4, and MKK6 can each activate p38 MAPk.8 9 To determine which MKK family member is utilized by the neutrophil to activate p38 MAPk in response to LPS stimulation, we sequentially isolated each relevant MKK from LPS-stimulated cells and combined it with recombinant human p38 MAPk. Both MKK3 and MKK4 were phosphorylated following stimulation with LPS. The MKK3 immunoprecipitated from neutrophils stimulated with LPS was shown to phosphorylate and activate recombinant human p38 MAPk. However, under identical conditions, MKK4 did not phosphorylate or activate p38 MAPk. MKK6 was not detected in neutrophils under these conditions, and we have reported previously that MKK1 and MKK2 are not activated by LPS.6
To determine if inflammatory responses by the neutrophil are dependent
on the activation of p38 MAPk, we studied the effect of p38 MAPk
inhibition on selected LPS-induced functions. Stimulation of
neutrophils with LPS evokes several "immediate" functional
responses, including actin assembly, adherence, activation of nuclear
factor-kappa B (NF-
B), and the ability to prime for an enhanced
secretory response. As a single stimulus, LPS is ineffective in evoking
chemokinesis, chemotaxis, or the release of superoxide anion or
granular enzymes. Functional responses to LPS that are dependent on
de novo protein synthesis primarily consist of the release
of cytokines and chemokines. We hypothesize that neutrophils utilize
the p38 MAPk cascade to link proinflammatory stimuli to an array of
functional responses. The bicyclic imidazoles, SK&F 86002 and SB203580,
have been demonstrated to inhibit p38 MAPk activity with insignificant
effect on other mammalian signaling kinases.10
11
12
Pretreatment of neutrophils with SK&F 86002 or SB 203580 was
shown to significantly inhibit adhesion in response to LPS, but have
little effect on actin assembly. In contrast, the release of tumor
necrosis factor-
(TNF-) was almost completely blocked by the p38
MAPk inhibitors in a concentration-dependent manner. We found that p38
MAPk inhibition at 30 min results in a significant reduction of TNF-
messenger RNA synthesis, but by 60 min, this effect is no longer seen.
Activation of NF-kB was found to be inhibited by SB 203580,
perhaps accounting for the early transient decrease in transcription.
At both early and later time points, the inhibition of p38 MAPk results
in a lack of intracellular TNF- synthesis, suggesting inhibition of
translation. These results support a proposed pathway in which LPS
binding to CD14 on the neutrophil surface results in a series of yet
undefined signaling events leading to activation of MKK3, which in turn
activates p38 MAPk, ultimately resulting in enhanced adhesion as well
as posttranscriptional and translational regulation of TNF- release
(Fig 1)
.
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References
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