A critical feature of sepsis-induced acute lung injury is the release of cytokines from endotoxin (LPS)- stimulated alveolar macrophages (AM). LPS is also known to activate various members of the mitogen- activated protein kinase (MAPK) family in other types of cells. In this study, we evaluated whether multiple members of the MAPK family regulate cytokine gene expression in LPS-stimulated AM. We found that LPS activates both the extracellular signal-regulated kinase (Erk) and p38 kinases, and that this activation is augmented when the cells are cultured in serum. Inhibition of either the Erk (with PD98059) or p38 (with SB203580) kinase pathway resulted in only a partial reduction in cytokine (interleukin-6 and tumor necrosis factor) messenger RNA accumulation and cytokine release, whereas inhibition of both pathways simultaneously resulted in a decrease in cytokine gene expression to near-control levels. Nuclear run-on assays showed that the effect of these MAPK pathways on LPS-induced expression of the cytokine genes was attributable, at least in part, to regulation of gene transcription. These findings suggest that activation of both the Erk and p38 kinase pathways is necessary for optimal cytokine gene expression in LPS-stimulated human AM, and that the MAPK pathways play a critical role in the inflammatory response that occurs in sepsis-induced acute lung injury.

A critical feature of sepsis-induced adult respiratory distress syndrome (ARDS) is the release of cytokines (such as interleukin [IL]-6, IL-8, and tumor necrosis factor [TNF]) from endotoxin (lipopolysaccharide [LPS])-activated alveolar macrophages (AM). Nuclear factor kappa B (NF-kappaB) is activated in AM from patients with ARDS, and it is essential for the transcription of many cytokine genes. In these studies, we evaluated the regulation of LPS-induced cytokine release and the activation of NF-kappaB in human AM. We found that the activation of NF-kappaB and the release of IL-6, IL-8, and TNF from AM exposed to LPS was protein kinase C-independent and tyrosine kinase- and phosphatidylcholine-specific phospholipase C-dependent. We also found that LPS-induced activation of NF-kappaB was enhanced in AM cultured in serum or in the presence of LPS-binding protein, simulating conditions in the lung that are present in ARDS. In addition, LPS triggered the activation of several different NF-kappaB complexes in AM, and different forms of NF-kappaB bound to the IL-6, IL-8, and TNF promoter sequences. These observations suggest that physiologic abnormalities present in the lungs of patients with ARDS facilitate the activation of NF-kappaB and local release of cytokines.

Ceramide is a bioactive lipid mediator that has been observed to induce apoptosis in vitro. The purpose of this study was to determine whether endogenous ceramide, generated in response to in vivo administration of tumor necrosis factor-α (TNF-α), increases apoptosis in primary rat alveolar type II epithelial cells. Intratracheal instillation of TNF-α (5 μg) produced a decrease in sphingomyelin and activation of a neutral sphingomyelinase. These changes were associated with a significant increase in lung ceramide content. TNF-α concomitantly activated the p42/44 extracellular signal-related kinases and induced nuclear factor-κB activation in the lung. Hypodiploid nuclei studies revealed that intratracheal TNF-α did not increase type II cell apoptosis compared with that in control cells after isolation. A novel observation from separate in vitro studies demonstrated that type II cells undergo a gradual increase in apoptosis after time in culture, a process that was accelerated by exposure of cells to ultraviolet light. However, culture of cells with a cell-permeable ceramide, TNF-α, or a related ligand, anti-CD95, did not increase apoptosis above the control level. The results suggest that ceramide resulting from TNF-α activation of sphingomyelin hydrolysis might activate the mitogen-activated protein kinase and nuclear factor-κB pathways without increasing programmed cell death in type II cells.

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This study uses human alveolar macrophages to determine whether activation of a phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) is linked to activation of the p42/44 (ERK) kinases by LPS. LPS-induced ERK kinase activation was inhibited by tricyclodecan-9-yl xanthogenate (D609), a relatively specific inhibitor of PC-PLC. LPS also increased amounts of diacylglycerol (DAG), and this increase in DAG was inhibited by D609. LPS induction of DAG was, at least in part, derived from PC hydrolysis. Ceramide was also increased in LPS-treated alveolar macrophages, and this increase in ceramide was inhibited by D609. Addition of exogenous C2 ceramide or bacterial-derived sphingomyelinase to alveolar macrophages increased ERK kinase activity. LPS also activated PKC ζ, and this activation was inhibited by D609. LPS-activated PKC ζ phosphorylated MAP kinase kinase, the kinase directly upstream of the ERK kinases. LPS-induced cytokine production (RNA and protein) was also inhibited by D609. As an aggregate, these studies support the hypothesis that one way by which LPS activates the ERK kinases is via activation of PC-PLC and that activation of a PC-PLC is an important component of macrophage activation by LPS.