Activation of second messenger pathways in alveolar macrophages by endotoxin.

The alveolar macrophage plays an important role in immune surveillance of the lung. Early responses to infectious agents by macrophages can decrease tissue injury and promote recovery of the host. Macrophage responses to pathogens are the cornerstone of the innate or nonspecific immune system. In particular, the response of macrophages to endotoxin from gram negative bacteria has been the focus of many recent studies. The recent discovery of the endotoxin receptor has accelerated the study of signalling in macrophages. This review focuses on the downstream events that occur following exposure of the alveolar macrophage to endotoxin.

Serum surfactant proteins-A and -D as biomarkers in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) has a high mortality rate, and current therapies are only marginally effective. A serum biomarker that predicts clinical outcome would be useful to stage disease, indicate prognosis and the need for aggressive therapy, and help stratify patients for clinical trials. The goals of this study were to determine whether serum levels of surfactant protein-A (SP-A) or surfactant protein-D (SP-D) would distinguish between IPF and other types of interstitial lung disease and whether serum SP-A or SP-D levels predict outcome in patients with IPF. The authors found that serum SP-A and SP-D levels were significantly elevated in patients with IPF and systemic sclerosis compared to sarcoidosis, beryllium disease and normal controls, and that SP-D correlated with radiographic abnormalities in patients with IPF. In addition, the authors found that both serum SP-A and SP-D levels were highly predictive of survival in patients with IPF. This is the largest North American data set of surfactant protein measurements in idiopathic pulmonary fibrosis and the first report using multivariate analysis comparing serum surfactant proteins-A and -D to other commonly measured predictors of survival in idiopathic pulmonary fibrosis. Based on these results, the authors propose that serum surfactant proteins may prove to be useful biomarkers in patients with idiopathic pulmonary fibrosis.

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-kappaB, which has also been linked to cell survival. We found that LPS, to a greater degree than ceramide, induced NF-kappaB 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.

Lung fibroblasts inhibit activation-induced death of T cells through PGE(2)-dependent mechanisms.

Activation-induced cell death (AICD) is a regulatory mechanism eliminating excess activated T cells, mainly through a Fas/Fas ligand-dependent mechanism. The goal of this study was to determine whether mouse primary lung fibroblasts are capable of modulating AICD. Using T cell hybridoma DO11.10, we found that fibroblasts in coculture rescue T cells from AICD. Fibroblast-conditioned medium (FCM) also inhibited apoptosis of T cells activated with immobilized anti-CD3 antibody. The effects of lung fibroblasts are mediated, in part, by secreted prostaglandin E(2) (PGE(2)) because treatment of fibroblasts with indomethacin decreased antiapoptotic activity of FCM. Addition of exogenous PGE(2) to FCM from fibroblast cultures treated with indomethacin restored the inhibitory activity of FCM. Expression of Fas receptor and Fas ligand by anti-CD3-activated DO11.10 cells was not affected by PGE(2). However, the same concentrations of PGE(2) significantly downregulated activation of caspase-8 and caspase-3. These results demonstrate that lung fibroblasts inhibit the AICD of T cells by secreting PGE(2), which downregulates caspase activation and apoptosis.

Idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a rapidly progressive illness of unknown cause characterized by sequential acute lung injury with subsequent scarring and end-stage lung disease. Treatment at present remains largely supportive, with evidence that patients' satisfaction and survival may be improved by referral to centers specializing in the evaluation of interstitial lung diseases. Although no drug therapy has clearly been demonstrated to benefit patients with idiopathic pulmonary fibrosis, a number of novel investigational agents hold promise for future study. Given the poor prognosis associated with idiopathic pulmonary fibrosis, patients should be referred to regional centers of expertise for enrollment in therapeutic clinical trials or for lung transplantation.

GM-CSF increases AP-1 DNA binding and Ref-1 amounts in human alveolar macrophages.

Alveolar macrophages have been implicated in the pathogenesis of a number of acute and chronic lung disorders. A characteristic feature of many of the chronic lung diseases is that the types of macrophages in the lung change, and in most instances, the cells resemble monocyte-like cells. We have previously shown that normal human alveolar macrophages have a decreased capacity to express protein kinase C (PKC)-induced DNA binding activity of the transcription factor activator protein (AP)-1 compared with monocytes. This decrease in AP-1 DNA binding appears to be due to a defect in redox regulation of AP-1 proteins via a decrease in the redox active protein Ref-1. The hypothesis for this study is that there are factors generated during the development of chronic lung disease that increase AP-1 DNA binding activity and Ref-1 production in human alveolar macrophages. We have focused specifically on granulocyte-macrophage colony-stimulating factor (GM-CSF) as a prototype mediator that can be released by alveolar macrophages and is related to the fibrotic process in the lung. We found that after a 24-h incubation with GM-CSF, AP-1 DNA binding was significantly increased in both unstimulated, interleukin (IL)-13, and phorbol myristate acetate (PMA)-stimulated alveolar macrophages and that there was a corresponding increase in Ref-1 protein by Western blot analysis in the PMA-stimulated group. This suggests that disease-related cytokines such as GM-CSF and IL-13 may modulate AP-1 DNA binding activity in alveolar macrophages.

The absence of activator protein 1-dependent gene expression in THP-1 macrophages stimulated with phorbol esters is due to lack of p38 mitogen-activated protein kinase activation.

Activator protein 1 (AP-1) binds to the promoters of many genes involved in immune and inflammatory responses. We have previously shown that the p38 mitogen-activated protein (MAP) kinase regulates NF-kappa B-dependent gene expression by modulating the phosphorylation and subsequent activation of TATA-binding protein (TBP). In this study, we asked whether the p38 MAP kinase regulated the transcriptional activity of AP-1. We found that phorbol 12-myristate 13-acetate (PMA) was unable to drive the AP-1-dependent reporter gene in THP-1 cells. PMA activated both the extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase MAP kinases, but it did not activate the p38 MAP kinase. We found that cells expressing MAP kinase kinase 6(Glu), which is the upstream kinase that activates the p38 MAP kinase, had significantly increased AP-1-dependent gene expression alone and when stimulated with PMA. These cells also had increased phosphorylation of native c-Jun, suggesting that both c-Jun NH(2)-terminal kinase and p38 MAP kinases phosphorylate c-Jun. More importantly, expression of a constitutive active MAP kinase kinase 6(Glu) resulted in the phosphorylation of a His-TBP fusion protein and increased direct interaction of TBP with c-Jun. These findings suggest that in macrophages, the p38 MAP kinase regulates AP-1-driven transcription by modulating the activation of TBP.

Utility of a lung biopsy for the diagnosis of idiopathic pulmonary fibrosis.

It is not known if a surgical lung biopsy is necessary in all patients for the diagnosis of idiopathic pulmonary fibrosis (IPF). We conducted a blinded, prospective study at eight referring centers. Initially, cases were evaluated by clinical history and examination, transbronchial biopsy, and high-resolution lung computed tomography scans. Pulmonologists at the referring centers then assessed their certainty of the diagnosis of IPF and provided an overall diagnosis, before surgical lung biopsy. The lung biopsies were reviewed by a pathology core and 54 of 91 patients received a pathologic diagnosis of IPF. The positive predictive value of a confident (certain) clinical diagnosis of IPF by the referring centers was 80%. The positive predictive value of a confident clinical diagnosis was higher, when the cases were reviewed by a core of pulmonologists (87%) or radiologists (96%). Lung biopsy was most important for diagnosis in those patients with an uncertain diagnosis and those thought unlikely to have IPF. These studies suggest that clinical and radiologic data that result in a confident diagnosis of IPF by an experienced pulmonologist or radiologist are sufficient to obviate the need for a lung biopsy. Lung biopsy is most helpful when clinical and radiologic data result in an uncertain diagnosis or when patients are thought not to have IPF.

Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B.

Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.

Lipopolysaccharide activates Akt in human alveolar macrophages resulting in nuclear accumulation and transcriptional activity of beta-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 (32)P-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 beta-catenin, resulting in nuclear accumulation and transcriptional activity of beta-catenin. Consistent with this, we found that LPS caused an increase in the amounts of PI 3-kinase-dependent nuclear beta-catenin in human alveolar macrophages and expression of genes that require nuclear beta-catenin for their activation. This is the first demonstration that LPS exposure activates AKT, inactivates GSK-3, and causes accumulation and transcriptional activity of beta-catenin in the nucleus of any cell, including alveolar macrophages.

Rate limiting steps of AAV transduction and implications for human gene therapy.

Despite the fact that adeno-associated virus type 2 (AAV2) is an extremely attractive gene therapy vector, its application has been limited to certain tissues such as muscle and the brain. In an attempt to broaden the array of target organs for this vector, molecular studies on the mechanism(s) of AAV transduction have expanded over the past several years. These studies have led to the development of innovative strategies capable of overcoming intracellular barriers to AAV2 transduction. The basis of these technologic breakthroughs has stemmed from a better understanding of the molecular processes that control AAV entry and intracellular trafficking to the nucleus. This review will focus on the identification of molecular components important for recombinant AAV (rAAV) transduction while highlighting the techniques used to discover them and potential clinical application of research findings.

Protein kinase C zeta 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 zeta, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC zeta, 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 zeta 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 zeta to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC zeta. PKC zeta 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 zeta/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC zeta activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC zeta.

Effects of ragweed and Th-2 cytokines on the secretion of IL-8 in human airway epithelial cells.

Ragweed-induced asthma is a very common pulmonary disease. An important part of asthma is a large increase in inflammatory cells, including neutrophils and eosinophils, in ragweed-sensitilized subjects. In this study, we determined if ragweed treatment could induce the release of interleukin-8 (IL-8) by human airway epithelial cells. We found that ragweed induces a substantial increase of the secretion of IL-8 from A549 cells in a dose- and time-dependent manner. Th-2 cytokines, IL-4 and IL-13, partially inhibited the ragweed-induced secretion of IL-8. Our results suggest that airway epithelial cells may be one of the cell sources that provide IL-8 during ragweed-induced asthma. The results also indicate that IL-4 and IL-13 may exert inhibitory effects on IL-8 secretion by airway epithelium.

A constitutive active MEK --> ERK pathway negatively regulates NF-kappa B-dependent gene expression by modulating TATA-binding protein phosphorylation.

Endotoxin-induced cytokine gene expression is regulated, in part, by NF-kappaB. We have shown that both the ERK and p38 mitogen-activated protein (MAP) kinases are necessary for cytokine gene transcription and that the p38 MAP kinase is required for NF-kappaB-driven transcription, so we hypothesized that the MEK --> ERK pathway regulated NF-kappaB-driven transcription as well. We found that a constitutive active MEK --> ERK pathway inhibited NF-kappaB-driven transcription. In addition, both PD 98059 and a dominant negative ERK2 augmented NF-kappaB-driven transcription; however, neither PD 98059 nor MEK1 altered NF-kappaB activation at any level. The constitutive active MEK --> ERK pathway inhibited the phosphorylation of TBP, which is necessary for both interaction with RelA and binding to the TATA box. Due to the fact that we have shown that the p38 MAP kinase modulates TBP activation, we evaluated the effect of the constitutive active MEK --> ERK pathway on p38 MAP kinase activity. We found that the MEK --> ERK pathway negatively regulates NF-kappaB-driven transcription, in part, by inhibiting p38 MAP kinase activity. Thus, the ERK and p38 MAP kinases have differential effects on NF-kappaB-driven transcription.

Asbestos stimulation triggers differential cytokine release from human monocytes and alveolar macrophages.

Inhalation of asbestos fibers results in a variety of lung diseases, including pulmonary fibrosis. Various animal models have demonstrated the importance of cytokines in the pathogenesis of pulmonary fibrosis. Alveolar macrophages from patients exposed to asbestos spontaneously release increased amounts of cytokines. The purpose of these studies was to determine whether asbestos directly stimulates cytokine release from human alveolar macrophages after in vitro exposure. We demonstrate that, although asbestos triggers cytokine release from blood monocytes, normal alveolar macrophages do not respond to asbestos stimulation with cytokine release. However, normal alveolar macrophages are activated by asbestos particles, in vitro, as determined by the upregulation of mRNAs for cytokines, and activation of the p38 kinase, which has been shown to be important in the translation of cytokine message into protein. These studies demonstrate that asbestos stimulates both normal blood monocytes and normal alveolar macrophages, but that there is a block in translation of cytokine mRNAs in the macrophages.

Activation of ERK2 by respiratory syncytial virus in A549 cells is linked to the production of interleukin 8.

The airway inflammation that results from respiratory syncytial virus infection is associated with a marked increase in interleukin 8 and neutrophils in the infected sites of the lung. In this study, the relationship between production of interleukin 8, infection of A549 cells by the virus, and activation of mitogen-activated protein kinases (MAPKs) was investigated. Infection of A549 cells by the virus caused an increase on the activity of extracellular signal-regulated kinase 2 (ERK2) by about 10-fold compared with the noninfected cells. The increase in the activity of ERK2 during the viral infection was an immediate event and occurred prior to the viral replication process. PD98059, which blocks the activation of MAPK/ERK kinase 1 (MEK1), inhibited the increase in the activity of ERK2 by infection of respiratory syncytial virus by about 50% at 10 microM. Pretreatment of A549 cells with PD98059 before the viral infection also inhibited the increase in the production of interleukin 8 by 50%, but had little effect on the mRNA level. The viral infection had no effect on the activities of p38 and c-jun N-terminal kinase (JNK). These observations suggest that activation of ERK2 by respiratory syncytial virus infection may be one of the mechanisms that result in the increase of the production of interleukin 8.