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.

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.

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.

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.

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.

The p38 mitogen-activated protein kinase is required for NF-kappaB-dependent gene expression. The role of TATA-binding protein (TBP).

Endotoxin-induced cytokine gene transcription in monocytes and macrophages is regulated in part by NF-kappaB. We have previously shown that the p38 mitogen-activated protein (MAP) kinase is necessary for endotoxin-induced cytokine gene transcription. Due to the fact that most cytokine promoter sequences have active NF-kappaB sites, we hypothesized that the p38 MAP kinase was necessary for NF-kappaB-dependent gene expression. We found that NF-kappaB-dependent gene expression was reduced to near control levels with either SB 203580 or a dominant-negative p38 MAP kinase expression vector. Inhibition of the p38 MAP kinase did not alter NF-kappaB activation at any level, but it significantly reduced the DNA binding of TATA-binding protein (TBP) to the TATA box. The dominant-negative p38 MAP kinase expression vector interfered with the direct interaction of native TFIID (TBP) with a co-transfected p65 fusion protein. Likewise, this dominant-negative plasmid also interfered with the direct interaction of a co-transfected TBP fusion protein with the native p65 subunit. The p38 kinase also phosphorylated TFIID (TBP) in vitro, and SB 203580 inhibited phosphorylation of TFIID (TBP) in vivo. Thus, the p38 MAP kinase regulates NF-kappaB-dependent gene transcription, in part, by modulating activation of TFIID (TBP).

Viral infection modulates expression of hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is a granulomatous, inflammatory lung disease caused by inhalation of organic Ags, most commonly thermophilic actinomycetes that cause farmer's lung disease. The early response to Ag is an increase in neutrophils in the lung, whereas the late response is a typical Th1-type granulomatous disease. Many patients who develop disease report a recent viral respiratory infection. These studies were undertaken to determine whether viruses can augment the inflammatory responses in HP. C57BL/6 mice were exposed to the thermophilic bacteria Saccharopolyspora rectivirgula (SR) for 3 consecutive days per wk for 3 wk. Some mice were exposed to SR at 2 wk after infection with respiratory syncytial virus (RSV), whereas others were exposed to SR after exposure to saline alone or to heat-inactivated RSV. SR-treated mice developed a typical, early neutrophil response and a late granulomatous inflammatory response. Up-regulation of IFN-gamma and IL-2 gene expression was also found during the late response. These responses were augmented by recent RSV infection but not by heat-inactivated RSV. Mice with a previous RSV infection also had a greater early neutrophil response to SR, with increased macrophage inflammatory protein-2 (MIP-2, murine equivalent of IL-8) release in bronchoalveolar lavage fluid. These studies suggest that viral infection can augment both the early and late inflammatory responses in HP.

Human alveolar macrophages are markedly deficient in REF-1 and AP-1 DNA binding activity.

Although many functions of human alveolar macrophages are altered compared with their precursor cell, the blood monocyte (monocyte), the reason(s) for these functional changes have not been determined. We recently reported that human alveolar macrophages do not express AP-1 DNA binding activity (Monick, M. M., Carter, A. B., Gudmundsson, G., Geist, L. J., and Hunninghake, G. W. (1998) Am. J. Physiol. 275, L389-L397). To determine why alveolar macrophages do not express AP-1 DNA binding activity, we first showed that there was not a decrease in expression of the FOS and JUN proteins that make up the AP-1 complex. There was, however, a significant difference in the amounts of the nuclear protein, REF-1 (which regulates AP-1 DNA binding by altering the redox status of FOS and JUN proteins), in alveolar macrophages compared with monocytes. In addition, in vitro differentiation of monocytes to a macrophage-like cell resulted in decreased amounts of REF-1. Finally, addition of REF-1 from activated monocytes to alveolar macrophage nuclear proteins resulted in a marked increase in AP-1 DNA binding. These studies strongly suggest that the process of differentiation of monocytes into alveolar macrophages is associated with a loss of REF-1 and AP-1 activity. This observation may explain, in part, some of the functional differences observed for alveolar macrophages compared with monocytes.

Both Erk and p38 kinases are necessary for cytokine gene transcription.

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 phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide-stimulated human alveolar macrophages.

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 zeta, and this activation was inhibited by D609. LPS-activated PKC zeta 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.

Changes in PKC isoforms in human alveolar macrophages compared with blood monocytes.

Alveolar macrophages play an important role in host defense and in other types of inflammatory processes in the lung. These cells exhibit many alterations in function compared with their precursor cells, blood monocytes. To evaluate a potential mechanism for these differences in function, we evaluated expression of protein kinase C (PKC) isoforms. We found an increase in Ca2+-dependent PKC isoforms in monocytes compared with alveolar macrophages. We also found differential expression of the Ca2+-independent isoforms in alveolar macrophages compared with monocytes. One consequence of the activation of PKC can be increased expression of mitogen-activated protein (MAP) kinase pathways. Therefore, we also evaluated activation of the MAP kinase extracellular signal-regulated kinase (ERK) 2 by the phorbol ester phorbol 12-myristate 13-acetate (PMA). PMA activated ERK2 kinase in both alveolar macrophages and monocytes; however, monocytes consistently showed a significantly greater activation of ERK2 kinase by PMA compared with alveolar macrophages. Another known consequence of the activation of PKC and subsequent activation of ERK kinase is activation of the transcription factor activator protein-1 (AP-1). We evaluated the activation of AP-1 by PMA in both monocytes and macrophages. We found very little detectable activation of AP-1, as assessed in a gel shift assay, in alveolar macrophages, whereas monocytes showed a substantial activation of AP-1 by PMA. These studies show that the differential expression of PKC isoforms in alveolar macrophages and blood monocytes is associated with important functional alterations in the cells.

IL-12 modulates expression of hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is a granulomatous, inflammatory lung disease caused by inhalation of organic Ags, most commonly thermophilic actinomycetes. Only a minority of individuals exposed to these Ags develops disease, suggesting that host factors are important for the expression of HP. We compared the expression of HP in a sensitive strain of mice, C57BL/6, and in a resistant strain of mice, DBA/2. They were exposed to the thermophilic bacteria Saccharopolyspora rectivirgula (SR) or to saline alone for 3 consecutive days/week for 3 wk. After exposure to Ag, C57BL/6 mice, but not DBA/2 mice, developed granulomatous inflammation with an increase in lung index (lung weight). Both strains had similar amounts of Ag delivered to the lungs after intranasal installation, as determined with 14C-labeled Ag. Both also had similar increases in total bronchoalveolar cells after Ag exposure, but the C57BL/6 mice had more lymphocytes. Compared with the resistant strain, the sensitive strain had a significantly greater Ag-induced increase in IL-12 and IFN-gamma gene expression. DBA/2 mice resembled sensitive, C57BL/6 mice if they received IL-12 augmentation therapy at the time of Ag exposure. These findings were not limited to lung, since both unstimulated and SR-stimulated spleen cells from C57BL/6 mice released significantly more IL-12 than cells from DBA/2 mice. However, spleen cells from DBA/2 mice made more IFN-gamma when exposed to IL-12, than cells from C57BL/6 mice. These results suggest that the IL-12 response to Ag may modulate in part the expression of HP.

Low grade rhinovirus infection induces a prolonged release of IL-8 in pulmonary epithelium.

Rhinoviruses are important respiratory pathogens implicated in asthma exacerbations. The mechanisms by which rhinoviruses trigger inflammatory responses in the lower airway are poorly understood, in particular their ability to infect the lower airway. Bronchial inflammatory cell (lymphocyte and eosinophil) recruitment has been demonstrated. IL-8 is a potent proinflammatory chemokine that is chemotactic for neutrophils, lymphocytes, eosinophils, and monocytes and may be important in the pathogenesis of virus-induced asthma. Increased levels of IL-8 have been found in nasal samples in natural and experimental rhinovirus infections. In these studies we therefore examine the ability of rhinovirus to infect a transformed lower airway epithelial cell line (A549) and to induce IL-8 protein release and mRNA induction. We observed that rhinovirus type 9 is able to undergo full viral replication in A549 cells, and peak viral titers were found 24 h after inoculation. Rhinovirus infection induced a dose- and time-dependent IL-8 release up to 5 days after infection and an increase in IL-8 mRNA expression that was maximal between 3 and 24 h after infection. UV inactivation of the virus completely inhibited replication, but only reduced IL-8 protein production and mRNA induction by half, while prevention of virus-receptor binding completely inhibited virus-induced IL-8 release, suggesting that part of the observed effects was due to viral replication and part was due to virus-receptor binding. These studies demonstrate that rhinoviruses are capable of infecting a pulmonary epithelial cell line and inducing IL-8 release. These findings may be important in understanding the pathogenesis of rhinovirus-induced asthma exacerbations.

Activator protein-1 is the preferred transcription factor for cooperative interaction with nuclear factor-kappaB in respiratory syncytial virus-induced interleukin-8 gene expression in airway epithelium.

The role of "oxidant-sensitive" transcription factors activator protein (AP)-1, nuclear factor (NF)-kappaB, and NF-IL6 in respiratory syncytial virus (RSV)-induced interleukin (IL)-8 gene expression in A549 epithelial cells was evaluated. RSV infection resulted in increased binding of each of these transcription factors. Transfection of A549 cells with plasmids containing serial truncations of the 5'-flanking region of the IL-8 gene revealed a positive cooperative effect of the binding sites for AP-1 and NF-kappaB. Mutation of either region markedly diminished responsiveness of the promoter to RSV. Mutation of the NF-IL6 site had minimal effect in the presence of intact binding sites for NF-kappaB and AP-1. The antioxidants NAC (N-acetylcysteine), DMSO, and DMPO (5,5-dimethyl-1-pyrroline N-oxide) did not inhibit RSV-induced binding of NF-kappaB; however, binding of AP-1 and NF-IL6 was inhibited. These observations suggest that AP-1 may be the preferred transcription factor (over NF-IL6) for cooperative interaction with NF-kappaB in RSV-induced IL-8 production.

Lipopolysaccharide-induced NF-kappaB activation and cytokine release in human alveolar macrophages is PKC-independent and TK- and PC-PLC-dependent.

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.