Biomedical subjects

Martha M Monick

Publications and source records attributed to Martha M Monick.

32 records · Page 2Linked to original sources

PubMed2003

Respiratory syncytial virus up-regulates TLR4 and sensitizes airway epithelial cells to endotoxin.

Airway epithelial cells are unresponsive to endotoxin (lipopolysaccharide (LPS)) exposure under normal conditions. This study demonstrates that respiratory syncytial virus (RSV) infection results in increased sensitivity to this environmental exposure. Infection with RSV results in increased expression of Toll-like receptor (TLR) 4 mRNA, protein, and increased TLR4 membrane localization. This permits significantly enhanced LPS binding to the epithelial monolayer that is blocked by disruption of the Golgi. The increased TLR4 results in an LPS-induced inflammatory response as demonstrated by increased mitogen-activated protein (MAP) kinase activity, IL-8 production, and tumor necrosis factor alpha production. RSV infection also allowed for tumor necrosis factor alpha production subsequent to TLR4 cross-linking with an immobilized antibody. These data suggest that RSV infection sensitizes airway epithelium to a subsequent environmental exposure (LPS) by altered expression and membrane localization of TLR4. The increased interaction between airway epithelial cells and LPS has the potential to profoundly alter airway inflammation.

Blotting, Western↗

PubMed2003

Adenoviral infection decreases mortality from lipopolysaccharide-induced liver failure via induction of TNF-alpha tolerance.

Effects of adenoviral infection on in vivo responses to LPS mediated by TNF-alpha were evaluated in a murine model. Adenovirus-infected mice showed decreased mortality from fulminant hepatitis induced by administration of LPS or staphylococcal enterotoxin B in the presence of D-galactosamine. Importantly, TNF-alpha resistance genes within adenoviral E3 region were not required, because E1,E3-deleted vectors showed similar effects. Adenovirus-infected mice exhibited higher TNF-alpha levels after LPS stimulation, no difference in TNFR1 expression, and similar mortality from Fas-induced fulminant hepatitis. Decreased production of IL-6 and KC in response to exogenous TNF-alpha, in addition to protection from TNF-alpha, suggested that adenoviral infection results in TNF-alpha tolerance.

Adenovirus Infections, Human↗

PubMed2003

Inhibition of Rho family GTPases results in increased TNF-alpha production after lipopolysaccharide exposure.

These studies demonstrate that treatment of macrophages with lovastatin, a cholesterol-lowering drug that blocks farnesylation and geranylgeranylation of target proteins, increases LPS-induced TNF-alpha production. This is reversed by the addition of mevalonate, which bypasses the lovastatin block. Examination of membrane localization of RhoA, Cdc42, Rac1, and Ras demonstrated decreased membrane localization of the geranylgeranylated Rho family members (RhoA, Cdc42, and Rac1) with no change in the membrane localization of farnesylated Ras. LPS-induced TNF-alpha production in the presence of the Rho family-specific blocker (toxin B from Clostridium difficile) was significantly enhanced consistent with the lovastatin data. One intracellular signaling pathway that is required for TNF-alpha production by LPS is the extracellular signal-regulated kinase (ERK). Significantly, we found prolonged ERK activation after LPS stimulation of lovastatin-treated macrophages. When we inhibited ERK, we blocked the lovastatin-induced increase in TNF-alpha production. As a composite, these studies demonstrate a negative role for one or more Rho family GTPases in LPS-induced TNF-alpha production.

Animals↗

PubMed2003

Hyperoxia induces macrophage cell cycle arrest by adhesion-dependent induction of p21Cip1 and activation of the retinoblastoma protein.

Hyperoxia induces growth arrest, apoptosis, necrosis, and morphological changes (spreading and adhesion) in various types of cells. The mechanism of hyperoxia-induced cell growth arrest has not been well elucidated, especially in macrophages. One possible mechanism is a role of cell adhesion in hyperoxia-induced cell cycle arrest. To evaluate this finding, macrophages were cultured in normoxia (21% O2) or hyperoxia (95% O2) in adhesion or low adhesion conditions. Incubation of macrophages in hyperoxia induced cell cycle arrest. The hyperoxia-induced cell cycle arrest was prevented by low adhesion conditions. To evaluate pathways potentially involved in hyperoxia-induced growth arrest, we measured extracellular regulated kinase and retinoblastoma protein activation and p21Cip1 and p53 accumulation. Hyperoxia strongly induced activation of extracellular regulated kinase and retinoblastoma protein as well as up-regulation of p21Cip1. These effects of hyperoxia were attenuated under low adhesion conditions, suggesting a role for integrin-dependent signaling. The induction of p21Cip1 and activation of retinoblastoma protein occurred via a p53-independent mechanism. These results suggest that adhesion-dependent pathways are required for hyperoxia-induced cell cycle arrest in macrophages.

Animals↗

PubMed2003

Role of repeated lung injury and genetic background in bleomycin-induced fibrosis.

Current hypotheses of the pathogenesis of many forms of pulmonary fibrosis suggest that (i) a stimulus results in repeated or prolonged episodes of lung injury, and (ii) genetic factors modulate the outcome of the injury. The commonly employed single-exposure bleomycin model results in only temporary fibrosis. Therefore, we evaluated whether repeated bleomycin exposures, in the setting of a genetic background more likely to develop a T helper 2 (Th2) response, would induce prolonged fibrosis. Lung fibrosis was induced by intratracheal bleomycin injection, either as a single exposure or as three consecutive exposures. We found that bleomycin induced a Th2-like environment in both Th1-biased C57BL/6J and Th2-biased DBA/2 mice. We also found histologic changes and collagen increases consistent with lung injury/fibrosis at early time points, but prolonged fibrosis only after multiple exposures in the Th2-biased DBA/2 mice. We also determined if impaired healing of bleomycin-induced injury would prolong fibrosis in the C57BL/6J mice. Endothelial nitric oxide (which protects endothelial cells from oxidant-induced injury) synthase knockout animals on a C57BL/6J background also had prolonged fibrosis, similar to DBA/2 mice, after multiple bleomycin exposures. This was specific to eNOS, as inducible nitric oxide synthase knockout animals cleared the fibrosis as effectively as wild-type C57BL/6J mice. This data indicate that healing of injury/fibrosis after bleomycin is complex and can be determined by a number of genetic and environmental factors.

Animals↗

PubMed2003

Integrin receptors are crucial for the restimulation of activated T lymphocytes.

Stimulation via the T-cell receptor results in proliferation of naive T cells and activation-induced death of activated T cells. The expression of Fas ligand and activation-induced cell death are major mechanisms by which immune responses are modulated in the lung. Although it is known that the binding of integrin receptors to extracellular matrix proteins provides co-stimulatory signals to naive T cells, it is not clear whether these signals are critical for activated T cells. The activation and differentiation of T cells is marked by significant changes in integrin expression and affinity. To determine the role of integrin signaling in restimulation of activated T cells, we blocked integrin receptors with RGD peptides. Using murine activated CD4+ T cells and the T-cell hybridoma DO11.10, we found that RGD peptides inhibit tyrosine phosphorylation of CD3 epsilon-chain and ZAP-70, clustering of T-cell receptors, extracellular signal-regulated kinase mitogen-activated protein-kinase activation, and Fas ligand expression and prevent activation-induced cell death. We demonstrate that activated T cells are sensitive to integrin co-stimulation and that integrin receptors are required for the successful restimulation of activated T cells. This indicates that matrix proteins may play a major role in regulating T-cell-mediated immune responses in the lung.

Animals↗

PubMed2002

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-alpha 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.

Adenoviridae↗

PubMed2002

Altered IL-4 mRNA stability correlates with Th1 and Th2 bias and susceptibility to hypersensitivity pneumonitis in two inbred strains of mice.

Previously, we have shown in a model of hypersensitivity pneumonitis that Th1-biased C57BL/6 mice are susceptible and Th2-biased DBA/2 mice are resistant to disease. We also showed that this was explained in part by differential regulation of IL-12 by IL-4. For these reasons, we postulated that C57BL/6 and DBA/2 mice differentially express IL-4. In this study, we show that C57BL/6 immune cells express Th2 but not Th1 cytokines at lower levels than DBA/2 cells. We also found that C57BL/6 splenocytes exhibit decreased mRNA stability of Th2 cytokines, relative to DBA/2 splenocytes. Stability of IL-2 and IFN-gamma were similar in the two strains of mice. Differences in Th2 cytokine mRNA stability between C57BL/6 and DBA/2 cells were not due to sequence polymorphism at specific regions of the IL-4/IL-13 locus. Furthermore, expression of Th1- and Th2-specific transcription factors T-bet and GATA-3, as well as the nuclear factor of activated T cells transcription factor, NFATc, was not significantly different between the two mice. Our data suggest that decreased mRNA stability of Th2 cytokines in C57BL/6 splenocytes may underlie the differential susceptibility to hypersensitivity pneumonitis between C57BL/6 and DBA/2 mice. Moreover, our results indicate that regulation of mRNA stability may serve as an important mechanism underlying Th1/Th2 immune polarization.

Alveolitis, Extrinsic Allergic↗

PubMed2002

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.

Animals↗

PubMed2002

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-kappaB 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 microg/cm(2)) 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.

Animals↗

PubMed2002

Second messenger pathways in pulmonary host defense.

The alveolar macrophage responds to bacterial infection with the production of inflammatory mediators that include TNFalpha. Early production of TNFalpha results in increased bacterial clearance, whereas too much TNFalpha results in many of the hallmarks of bacterial sepsis. TNFalpha production is regulated at many levels, including multiple signaling pathways, that lead to transcription, translation, and release of functional TNFalpha. Interactions of mitogen-activated protein (MAP) kinases, lipid signaling pathways, and oxidant-mediated mechanisms regulate the response of alveolar macrophages to infection. Animal models of sepsis support the central role played by macrophage-derived TNFalpha in sepsis.

Animals↗

PubMed2002

Mitochondrial K(ATP) channel openers activate the ERK kinase by an oxidant-dependent mechanism.

Extracellular signal-regulated kinases (ERKs) are key regulatory proteins that mediate cell survival, proliferation, and differentiation. Reactive oxygen species (ROS) may play a role in activation of the ERK pathway. Because mitochondria are a major source of ROS, we investigated whether mitochondria-derived ROS play a role in ERK activation. Diazoxide, a potent mitochondrial ATP-sensitive K+ (K(ATP)) channel opener, is known to depolarize the mitochondrial membrane potential and cause a reversible oxidation of respiratory chain flavoproteins, thus increasing mitochondrial ROS production. Using THP-1 cells as a model, we postulated that opening mitochondrial K(ATP) channels would increase production of ROS and, thereby, regulate the activity of the ERK kinase. We found that opening mitochondrial K(ATP) channels by diazoxide induced production of ROS as determined by an increased rate of dihydroethidium and dichlorofluorescein fluorescence. This increased production of ROS was associated with increased phosphorylation of ERK kinase in a time-dependent fashion. The MEK inhibitors PD-98059 and U-0126 blocked ERK activation mediated by diazoxide. N-acetylcysteine, but not diphenyleneiodonium, attenuated ERK activation mediated by diazoxide. Adenovirus-mediated overexpression of manganese superoxide dismutase, which is expressed in mitochondria, decreased the rate of dihydroethidium oxidation as well as ERK activation. We conclude that mitochondrial K(ATP) channel openers trigger ERK activation via mitochondria-derived ROS.

Adenosine Triphosphate↗

PubMed2002

Interaction of matrix with integrin receptors is required for optimal LPS-induced MAP kinase activation.

Exposure of macrophages to endotoxin [lipopolysaccharide (LPS)] results in a cascade of events resulting in the release of multiple inflammatory and anti-inflammatory mediators. The Toll-like receptor (TLR) 4 complex is the major receptor that mediates LPS signaling. However, there is evidence that other surface molecules may play a complementary role in the TLR-induced events. Integrin receptors are one class of receptors that have been linked to LPS signaling. This study investigates the role of macrophage integrin receptors in the activation of mitogen-activated protein (MAP) kinases by LPS. In conditions where macrophages were not permitted to adhere to matrix or a tissue culture surface, we found a decrease in LPS signaling as documented by a marked reduction in tyrosine phosphorylation of whole cell proteins. This was accompanied by a significant decrease in extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase MAP kinase activation. Inhibition of integrin signaling, with EDTA or RGD peptides, decreased LPS-induced MAP kinase activity. The functional consequence of blocking integrin signaling was demonstrated by decreased LPS-induced tumor necrosis factor-alpha production. These observations demonstrate that, in addition to the TLR receptor complex, optimal LPS signaling requires complementary signals from integrin receptors.

Animals↗

PubMed2001

Respiratory syncytial virus inhibits apoptosis and induces NF-kappa B activity through a phosphatidylinositol 3-kinase-dependent pathway.

Respiratory syncytial virus (RSV) infects airway epithelial cells, resulting in cell death and severe inflammation through the induction of NF-kappaB activity and inflammatory cytokine synthesis. Both NF-kappaB activity and apoptosis regulation have been linked to phosphatidylinositol 3-kinase (PI 3-K) and its downstream effector enzymes, AKT and GSK-3. This study evaluates the role of PI 3-K and its downstream mediators in apoptosis and inflammatory gene induction during RSV infection of airway epithelial cells. Whereas RSV infection alone did not produce significant cytotoxicity until 24-48 h following infection, simultaneous RSV infection and exposure to LY294002, a blocker of PI 3-K activity, resulted in cytotoxicity within 12 h. Furthermore, we found that RSV infection during PI 3-K blockade resulted in apoptosis by examining DNA fragmentation, DNA labeling by terminal dUTP nick-end labeling assay, and poly(ADP-ribose) polymerase cleavage by Western blotting. RSV infection produced an increase in the phosphorylation state of AKT, GSK-3, and the p85 regulatory subunit of PI 3-K. The activation of PI 3-K by RSV and its inhibition by LY294002 was confirmed in direct PI 3-K activity assays. Further evidence for the central role of a pathway involving PI 3-K and AKT in preserving cell viability during RSV infection was established by the observation that constitutively active AKT transfected into A549 cells prevented the cytotoxicity and apoptosis of combined RSV and LY294002 treatment. Finally, both PI 3-K inhibition by LY294002 and AKT inhibition by transfection of a dominant negative enzyme blocked RSV-induced NF-kappaB transcriptional activity. These data demonstrate that anti-apoptotic signaling and NF-kappaB activation by RSV are mediated through activation of PI 3-K-dependent pathways. Blockade of PI 3-K activation resulted in rapid, premature apoptosis and inhibition of RSV-stimulated NF-kappaB-dependent gene transcription.

Apoptosis↗