Dawn M. Flaherty

Lipopolysaccharide Activates Akt in Human Alveolar Macrophages Resulting in Nuclear Accumulation and Transcriptional Activity of β-Catenin

Exposure of human alveolar macrophages to bacterial LPS results in activation of a number of signal transduction pathways. An early event after the alveolar macrophage comes in contact with LPS is activation of the phosphatidylinositol 3 kinase (PI 3-kinase). This study evaluates the downstream effects of that activation. We observed that LPS exposure results in phosphorylation of Akt (serine 473). We found this using both phosphorylation-specific Abs and also by in vivo phosphorylation with 32P-loaded cells. AKT activation resulted in the phosphorylation-dependent inactivation of glycogen synthase kinase (GSK-3) (serine 21/9). We found that both of these events were linked to PI 3-kinase because the PI 3-kinase inhibitors, wortmannin and LY294002, inhibited LPS-induced phosphorylation of both AKT and GSK-3. Inactivation of GSK-3 has been shown to reduce the ubiquitination of β-catenin, resulting in nuclear accumulation and transcriptional activity of β-catenin. Consistent with this, we found that LPS caused an increase in the amounts of PI 3-kinase-dependent nuclear β-catenin in human alveolar macrophages and expression of genes that require nuclear β-catenin for their activation. This is the first demonstration that LPS exposure activates AKT, inactivates GSK-3, and causes accumulation and transcriptional activity of β-catenin in the nucleus of any cell, including alveolar macrophages.

Protein Kinase C ζ Plays a Central Role in Activation of the p42/44 Mitogen-Activated Protein Kinase by Endotoxin in Alveolar Macrophages

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC ζ, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC ζ, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC ζ partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC ζ to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC ζ. PKC ζ has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC ζ/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC ζ activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC ζ.

Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA.

Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity. PI3K inhibition resulted in an increase in cyclooxygenase 2 (COX2) protein, mRNA, and mRNA stability. PI3K negatively regulated activation of the p38 pathway (p38, MKK3/6, and MAPKAP2), and an active p38 was necessary for COX2 production. The data suggest that PI3K inhibition of p38 modulates COX2 expression via destabilization of LPS-induced COX2 mRNA.

Ceramide Regulates Lipopolysaccharide-Induced Phosphatidylinositol 3-Kinase and Akt Activity in Human Alveolar Macrophages

The phosphatidylinositol (PI) 3-kinase pathway is an important regulator of cell survival. In human alveolar macrophages, we found that LPS activates PI 3-kinase and its downstream effector, Akt. LPS exposure of alveolar macrophages also results in the generation of ceramide. Because ceramide exposure induces apoptosis in other cell types and the PI 3-kinase pathway is known to inhibit apoptosis, we determined the relationship between LPS-induced ceramide and PI 3-kinase activation in alveolar macrophages. We found that ceramide exposure activated PI 3-kinase and Akt. When we blocked LPS-induced ceramide with the inhibitor D609, we blocked LPS-induced PI 3-kinase and Akt activation. Evaluating cell survival after ceramide or LPS exposure, we found that blocking PI 3-kinase induced a significant increase in cell death. Because these effects of PI 3-kinase inhibition were more pronounced in ceramide- vs LPS-treated alveolar macrophages, we also evaluated NF-κB, which has also been linked to cell survival. We found that LPS, to a greater degree than ceramide, induced NF-κB translocation to the nucleus. As a composite, these studies suggest that the effects of ceramide exposure in alveolar macrophages may be very different from the effects described for other cell types. We believe that LPS induction of ceramide results in PI 3-kinase activation and represents a novel effector mechanism that promotes survival of human alveolar macrophages in the setting of pulmonary sepsis.

Cooperative Prosurvival Activity by ERK and Akt in Human Alveolar Macrophages is Dependent on High Levels of Acid Ceramidase Activity

Human alveolar macrophages are unique in that they have an extended life span in contrast to precursor monocytes. In evaluating the role of sphingolipids in alveolar macrophage survival, we found high levels of sphingosine, but not sphingosine-1-phosphate. Sphingosine is generated by the action of ceramidase(s) on ceramide, and alveolar macrophages have high constitutive levels of acid ceramidase mRNA, protein, and activity. The high levels of acid ceramidase were specific to alveolar macrophages, because there was little ceramidase protein or activity (or sphingosine) in monocytes from matching donors. In evaluating prolonged survival of alveolar macrophages, we observed a requirement for constitutive activity of ERK MAPK and the PI3K downstream effector Akt. Blocking acid ceramidase but not sphingosine kinase activity in alveolar macrophages led to decreased ERK and Akt activity and induction of cell death. These studies suggest an important role for sphingolipids in prolonging survival of human alveolar macrophages via distinct survival pathways.

Oxidant-Mediated Increases in Redox Factor-1 Nuclear Protein and Activator Protein-1 DNA Binding in Asbestos-Treated Macrophages

Alveolar macrophages have been implicated in the pathogenesis of a number of acute and chronic lung disorders. We have previously shown that normal human alveolar macrophages exhibit decreased DNA binding activity of the transcription factor, AP-1, compared with monocytes. Furthermore, this decrease in AP-1 DNA binding appears to be due to a decrease in the redox active protein, redox factor (Ref)-1. Ref-1 is an important redox regulator of a number of transcription factors, including NF-κB and AP-1. In this study we evaluated the role of asbestos, a prototypic model of chronic fibrotic lung disease, in Ref-1 expression and activity. We found that incubation with low concentrations of crocidolite asbestos (0.5–1.25 μg/cm2) resulted in an increase in nuclear Ref-1 protein after 5 min, with a persistent elevation in protein up to 24 h. Additionally, an increase in nuclear Ref-1 could be induced by treating the cells with an oxidant-generating stimulus (iron loading plus PMA) and inhibited by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. The asbestos-induced accumulation of nuclear Ref-1 was associated with an increase in AP-1 DNA binding activity. These findings suggest that an exposure associated with fibrotic lung disease, i.e., asbestos, modulates accumulation of nuclear Ref-1 in macrophages, and that this effect is mediated by an oxidant stimulus.

Active ERK contributes to protein translation by preventing JNK-dependent inhibition of protein phosphatase 1.

Human alveolar macrophages, central to immune responses in the lung, are unique in that they have an extended life span in contrast to precursor monocytes. We have shown previously that the ERK MAPK (ERK) pathway is constitutively active in human alveolar macrophages and contributes to the prolonged survival of these cells. We hypothesized that ERK maintains survival, in part, by positively regulating protein translation. In support of this hypothesis, we have found novel links among ERK, JNK, protein phosphatase 1 (PP1), and the eukaryotic initiation factor (eIF) 2α. eIF2α is active when hypophosphorylated and is essential for initiation of protein translation (delivery of initiator tRNA charged with methionine to the ribosome). Using [35S]methionine labeling, we found that ERK inhibition significantly decreased protein translation rates in alveolar macrophages. Decreased protein translation resulted from phosphorylation (and inactivation) of eIF2α. We found that ERK inhibition increased JNK activity. JNK in turn inactivated (via phosphorylation) PP1, the phosphatase responsible for maintaining the hypophosphorylated state of eIF2α. As a composite, our data demonstrate that in human alveolar macrophages, constitutive ERK activity positively regulates protein translation via the following novel pathway: active ERK inhibits JNK, leading to activation of PP1α, eIF2α dephosphorylation, and translation initiation. This new role for ERK in alveolar macrophage homeostasis may help to explain the survival characteristic of these cells within their unique high oxygen and stress microenvironment.

First-Generation Adenovirus Vectors Shorten Survival Time in a Murine Model of Sepsis

Adverse immunological reactions to adenoviral vectors have significantly impacted the utility of this virus for treating genetic and environmentally induced diseases. In this study, we evaluate the effect of adenoviral vectors on an animal model of sepsis. Systemic delivery of first-generation adenoviral vectors to septic mice (cecal ligation and puncture) resulted in a shortened survival time. This effect was not observed with second-generation or inactivated first-generation vectors. The accelerated death was accompanied by a number of important changes in the disease. These changes included increased liver cell apoptosis (including Kupffer cells) and a marked increase in liver bacterial load. In the lung, the combination induced an increase in bacterial load, as well as greater lung injury. In the serum, the combination was associated with decreased TNF-α levels and an increase in bacterial load. Finally, a profound degree of lymphocyte apoptosis was observed in these animals. These observations suggest that prior exposure to first-generation adenovirus gene therapy vectors may worsen the outcome of some forms of sepsis.

Human Alveolar Macrophages Are Deficient in PTEN THE ROLE OF ENDOGENOUS OXIDANTS

Human alveolar macrophages play a critical role in host defense and in the development of inflammation and fibrosis in the lung. Unlike their precursor cells, blood monocytes, alveolar macrophages are long-lived and tend to be resistant to apoptotic stimuli. In this study, we examined the role of differentiation in altering baseline phosphatidylinositol (PI) 3-kinase/Akt activity. We found that differentiation increased activity of pro-survival PI 3-kinase/Akt while decreasing amounts of the negative PI 3-kinase regulator, PTEN. PTEN is a lipid phosphatase with activity against phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3), the major bioactive product of PI 3-kinase. Examining in vivo differentiation of alveolar macrophages (by comparing blood monocytes to alveolar macrophages from single donors), we found that differentiation resulted in increased baseline reactive oxygen species (ROS) in the alveolar macrophages. This led to a deficiency in PTEN, increased activity of Akt, and prolonged survival of alveolar macrophages. These data support the hypothesis that alterations in ROS levels contribute to macrophage homeostasis by altering the balance between PI 3-kinase/Akt and the phosphatase, PTEN.

Color analysis of the human airway wall

A bronchoscope can be used to examine the mucosal surface of the airways for abnormalities associated with a variety of lung diseases. The diagnosis of these abnormalities through the process of bronchoscopy is based, in part, on changes in airway wall color. Therefore it is important to characterize the normal color inside the airways. We propose a standardized method to calibrate the bronchoscopic imaging system and to tabulate the normal colors of the airway. Our imaging system consists of a Pentium PC and video frame grabber, coupled with a true color bronchoscope. The calibration procedure uses 24 standard color patches. Images of these color patches at three different distances (1, 1.5, and 2 cm) were acquired using the bronchoscope in a darkened room, to assess repeatability and sensitivity to illumination. The images from the bronchoscope are in a device-dependent Red-Green-Blue (RGB) color space, which was converted to a tri-stimulus image and then into a device-independent color space sRGB image by a fixed polynomial transformation. Images were acquired from five normal human volunteer subjects, two cystic fibrosis (CF) patients and one normal heavy smoker subject. The hue and saturation values of regions within the normal airway were tabulated and these values were compared with the values obtained from regions within the airways of the CF patients and the normal heavy smoker. Repeated measurements of the same region in the airways showed no measurable change in hue or saturation.