![[logo] US EPA](https://webarchive.library.unt.edu/eot2008/20081106053117im_/http://www.epa.gov/epafiles/images/logo_epaseal.gif){kind=logo}
Final Report: Role of RSV Infection and Endotoxin in Airway Inflammation
EPA Grant Number: R826711C003Subproject: this is subproject number 003 , established and managed by the Center Director under grant R826711
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: University of Iowa Children's Environmental Airway Disease Center
Center Director: Hunninghake, Gary W.
Title: Role of RSV Infection and Endotoxin in Airway Inflammation
Investigators: Hunninghake, Gary W. , Schwartz, David
Institution: University of Iowa
EPA Project Officer: Fields, Nigel
Project Period: January 1, 1998 through January 1, 2002
Project Amount: Refer to main center abstract for funding details.
RFA: Centers for Children's Environmental Health and Disease Prevention Research (1998)
Research Category: Children's Health , Health Effects
Description:
Objective:The hypothesis of this project is that respiratory syncytical virus (RSV) infection upregulates the response of airway epithelium to endotoxin. We further hypothesize that, in the setting of RSV infection, endotoxin triggers a marked increase in activation of multiple nitrogen-activated protein kinase (MAPK) pathways that regulate IL-8 gene expression. The objective of this research project was to explore these hypotheses by asking the following questions: (1) Does RSV infection upregulate the response to endotoxin in airway epithelial cells? (2) Does the RSV-induced response to endotoxin result in upregulation of critical MAPK pathways? (3) Is the activation of these MAPK pathways linked to activation of specific transcription factors that regulate IL-8 gene expression? (4) How is the p42/44 (ERK) kinase pathway activated by RSV infection?
Summary/Accomplishments (Outputs/Outcomes):RSV is a major cause of viral bronchiolitis and pneumonia in children. The airway inflammation that results from the viral infection is associated with a marked increase in IL-8 and neutrophils in the infected sites of the lung. In this study, the relationship between release of IL-8, infection of A549 cells (a human lung epithelial carcinoma cell line) by RSV, and activation of mitogen-activated protein kinases was investigated. Infection of A549 cells by the virus caused an increase on the activity of 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, a synthetic chemical inhibitor that blocks the activation of MEK1, inhibited the increase in the activity of ERK2 by infection of RSV by about 50 percent at 10 μM. Pretreatment of A549 cells with PD98059 before the viral infection also inhibited the increase in the production of IL-8 by 50 percent. This treatment had little effect on the virus-induced increase in IL-8 mRNA. The viral infection had no effect on the activities of p38 and JNK. These observations suggest that activation of ERK2 by RSV infection may be one of the mechanisms that result in the increase of the production of IL-8.
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-sensitized subjects. In this study, we determined if ragweed treatment could induce the release of 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. Th2 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.
RSV is an important respiratory pathogen that preferentially infects epithelial cells in the airway and causes a local inflammatory response. Very little is known about the second messenger pathways involved in this response. To characterize some of the acute response pathways involved in RSV infection, we utilized cultured human epithelial cells (A549) and optimal tissue culture infective (TCID)50 doses of RSV. We previously have shown that RSV-induced IL-8 release is linked to activation of the ERK MAPK pathway. In this study we evaluated the upstream events involved in ERK activation by RSV. RSV activated ERK at two time points, an early time point consistent with viral binding and a later sustained activation consistent with viral replication. We next evaluated the role of protein kinase C (PKC) isoforms in RSV-induced ERK kinase activity. We found that A549 cells contain the Ca++ dependent isoforms α and β1, and the Ca++ independent isoforms δ, ε, η, μ, θ, and ζ. Western analysis showed that RSV caused no change in the amounts of these isoforms. Kinase activity assays, however, demonstrated activation of ζ at the early time point and sustained activation of β1, δ, ε, and μ later in the infection. A cell permeable peptide inhibitor specific for the ζ isoform decreased early ERK kinase activation by RSV. Downregulation of the other PKC isoforms with phorbol ester blocked the late sustained activation of ERK by RSV. These studies suggest that RSV activates multiple PKC isoforms, resulting in the downstream activation of ERK kinase.
RSV causes epithelial cell death and produces severe airway inflammation through the induction of NF-κB activity and inflammatory cytokines synthesis. Both NF-κB activity and apoptosis have been linked to phosphatidylinositol 3-kinase (PI 3-K) and its downstream effector enzymes AKT and GSK-3. We have undertaken these studies in A549 cells to determine the role of PI 3-K activity in apoptosis and inflammatory gene induction during RSV infection. Although RSV infection alone did not produce significant cytotoxicity until 24 to 48 hours following infection, simultaneous blockade of PI 3-K with LY294002 resulted in cytotoxicity within 12 hours as measured by lactate dehydrogenase release assay. Furthermore, by examination of DNA fragmentation, DNA labeling by terminal deoxynucleotidyl transferase biotin-DUTP nick end labeling (TUNEL) assay, and poly(ADP-ribose)polymerase-1 (PARP) cleavage by Western blotting, we have shown that RSV infection during PI 3-K blockade promotes apoptotic cell death. Therefore, PI 3-K-dependent pathways suppress proapoptotic and cytotoxic effects of RSV early in the course of RSV infection and permit continued cell survival. RSV infection produces an increase in the phosphorylation state of AKT, GSK-3, and the p85 regulatory subunit of PI 3-K. These effects are blocked by the pretreatment of cells with LY294002. LY294002 also blocked NF-κB-driven transcription assessed by luciferase reporter assays, although it did not inhibit nuclear translocation. These data demonstrate that antiapoptotic signaling and NF-κB 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 NF-κB-dependent gene transcription.
Previously, we have shown in a model of hypersensitivity pneumonitis (HP), that Th1-biased C57BL/6 mice aresusceptible 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. Here 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 interferon (IFN)-γ were similar in the two strains of mice. Differences in Th2 cytokine mRNA stability between C57BL/6 and DBA/2 cells were not because of 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 HP 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.
We found that airway epithelial cells are unresponsive to endotoxin (lipopolysaccharide [LPS]) exposure under normal conditions and that 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 MAPK activity, IL-8 production, and TNF-a production. We were also able to show increased TLR4 responsiveness after RSV by cross-linking TLR4 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.
Significant Achievement
Asthma is characterized by a Th2 phenotype. Using an animal model, we defined a unique genetic phenotype where Th2 expression is defined by Th2 mRNA stability.
Journal Articles on this Report: 6 Displayed | Download in RIS Format
| Other subproject views: | All 6 publications | 6 publications in selected types | All 6 journal articles |
| Other center views: | All 33 publications | 32 publications in selected types | All 32 journal articles |
| Type | Citation | ||
|---|---|---|---|
|
|
Butler NS, Monick MM, Yarovinsky TO, Powers LS, Hunninghake GW. Altered IL-4 mRNA stability correlates with Th1 and Th2 bias and susceptibility to hypersensitivity pneumonitis in two inbred strains of mice. Journal of Immunology 2002;169(7):3700-3709. |
R826711 (Final) R826711C003 (Final) |
not available |
|
|
Monick MM, Staber JM, Thomas KW, Hunninghake GW. Respiratory syncytial virus infection results in activation of multiple protein kinase C isoforms leading to activation of mitogen-activated protein kinase. Journal of Immunology 2001;166(4):2681-2687. |
R826711 (Final) R826711C001 (2000) R826711C003 (Final) |
not available |
|
|
Monick MM, Yarovinsky TO, Powers LS, Butler NS, Carter AB, Gudmundsson G, Hunninghake GW. Respiratory syncytial virus up-regulates TLR4 and sensitizes airway epithelial cells to endotoxin. Journal of Biological Chemistry 2003;278(52):53035-53044. |
R826711 (Final) R826711C003 (Final) |
not available |
|
|
Thomas KW, Monick MM, Staber JM, Yarovinsky TO, Carter AB, Hunninghake GW. RSV inhibits apoptosis and induces NF-κB activity through a PI-3K dependent pathway. Journal of Biological Chemistry 2002;277(1):492-501. |
R826711 (Final) R826711C003 (Final) |
not available |
|
|
Chen W, Monica MM, Carter AB, Hunninghake GW. Activation of ERK2 by respiratory syncytial virus in A549 cells is linked to the production of interleukin 8. Experimental Lung Research 2000;6(1):13-26. |
R826711 (Final) R826711C001 (2000) R826711C003 (Final) |
not available |
|
|
Chen W, Hunninghake GW. Effects of ragweed and Th-2 cytokines on the secretion of IL-8 in human airway epithelial cells. Experimental Lung Research 2000;26(4):229-239. |
R826711 (Final) R826711C001 (2000) R826711C003 (Final) |
not available |
RSV infection, agricultural community, airway disease, airway inflammation, allergic airway, assessment of exposure, asthma, biological response, childhood respiratory disease, children, endotoxin, exposure assessment, gene expression, genetic susceptibility, health effects, human exposure, inhalation, interleukin, mitogen activated protein kinase, respiratory problems, rural communities,
,
Scientific Discipline, Health, RFA, Susceptibility/Sensitive Population/Genetic Susceptibility, Biology, Risk Assessments, genetic susceptability, Health Risk Assessment, Children's Health, Atmospheric Sciences, Environmental Chemistry, Allergens/Asthma, exposure assessment, gene expression, health effects, inhalation, respiratory problems, interleukin, rural communities, assessment of exposure, childhood respiratory disease, RSV infection, allergic airway, genetic susceptibility, endotoxin, agricultural community, etiology, interluken, sensitive populations, biological response, mitogem activated protein kinase, airway disease, children, disease, children's vulnerablity, asthma, human exposure
Progress and Final Reports:
Original Abstract
Main Center Abstract and Reports:
R826711 University of Iowa Children's Environmental Airway Disease Center
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R826711C001 Mechanisms that Initiate, Promote, and Resolve Grain Dust/LPS Induced Inflammation
R826711C002 Multi-component Intervention Study of Asthma in Children from Rural Communities
R826711C003 Role of RSV Infection and Endotoxin in Airway Inflammation
R826711C004 A Model to Study the Development of Persistent Environmental Airway Disease