* Center for Environmental Medicine, Asthma, and Lung Biology; Curriculum of Toxicology;
Department of Pediatrics, Division of Infectious Diseases and Host Defense; University of North Carolina at Chapel Hill, North Carolina 275997310; and
U.S. Environmental Protection Agency, Human Studies Division; Chapel Hill, North Carolina 27599
1 To whom correspondence should be addressed at CEMALB, CB#7310, 104 Mason Farm Rd., University of North Carolina at Chapel Hill, Chapel Hill, NC 275997310. Fax: (919) 966-9863. E-mail: Ilona_Jaspers{at}med.unc.edu.
Received January 6, 2005; accepted March 3, 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: influenza; diesel exhaust; in vitro; epithelial cells; oxidative stress.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Numerous studies have demonstrated that exposure to DE has adjuvant activity and enhances allergic sensitization in laboratory animals and human volunteers (Pandya et al., 2002; reviewed by D'Amato, 2002
), by increasing the levels of allergen-specific IgE and pro-allergic cytokines (IL-4, IL-5, IL-6, IL-10, and IL-13) (Diaz-Sanchez, 1997
). Hence, DE has important effects on adaptive immune responses. Furthermore, several studies have demonstrated that exposure to DE decreases bacterial clearance and modifies pulmonary macrophage function (Castranova et al., 2001
; Saito et al., 2002a
; Steerenberg et al., 2004
; Yang et al., 2001
; Yin et al., 2002
), both potentially contributing to decreased host defense and increased susceptibility to microbial infections. Interestingly, fewer studies have examined the effects of DE on viral infections. Hahon et al. (1985)
have shown that mice chronically exposed to DE for 6 months and infected with influenza virus have decreased ability to produce interferon (by 78%) and depressed viral killing, which led to increased viral multiplication. Recently, Harrod et al. (2003)
demonstrated that exposure of mice to diesel engine emissions for 7 days enhanced the pro-inflammatory response to a subsequent infection with respiratory syncytial virus (RSV) and increased the expression of RSV genes without decreasing interferon levels. This study also showed that exposure to diesel engine emission decreased the expression of innate immune defense mediators, specifically surfactant protein A (SP-A) and Clara Cell Secretory Protein (CCSP), which are important in controlling RSV infections (LeVine et al., 1999
; Wang et al., 2003
). Taken together, these studies indicate that both chronic and subchronic exposures to DE can enhance the susceptibility to viral infections, possibly through two different mechanisms.
Despite large-scale vaccination efforts and antiviral therapies, morbidity and mortality associated with influenza infections have not significantly changed over the past several years (Thompson et al., 2003, 2004
). Factors such as age, nutritional status, and preexisting pulmonary disease can affect susceptibility to influenza (Beck and Matthews, 2000
). In addition, the studies by Hahon et al. (1985)
and Harrod et al. (2003)
suggest that exposure to DE can also affect the susceptibility to viral infections. Airway epithelial cells are the primary site for influenza virus infection and replication. Virus-infected epithelial cells respond to influenza infection by synthesizing and releasing numerous cytokines and immunoregulatory mediators, which recruit and activate inflammatory cells to aid in the defense and the clearance of the invading virus. Among the mediators released by epithelial cells upon influenza infection, RANTES, MCP-1, IL-8, IL-6, and eotaxin recruit and activate pro-inflammatory cells, while type I interferons (IFN
and IFNß) induce the synthesis and activity of mediators involved in turning off viral replication within the host cell. Both groups of mediators are essential in the successful clearance of an influenza infection. However, other host cell-derived factors also control the susceptibility to viral infections. For example, innate immune defense mediators, such as calcium-dependent collagen-like lectins (collectins) released by epithelial cells, can aggregate the viral pathogen, thus neutralizing the ability of the virus to attach and infect the host cell.
Differentiated primary human respiratory epithelial cells, when grown under defined culture condition (see Methods), retain many characteristics seen in vivo (Clark et al., 1995; Gray et al., 1996
; Ostrowski et al., 1995
), including the presence of ciliated, nonciliated, and mucus-secreting cells, as well as tight junctions (Clark et al., 1995
) and the ability to produce innate immune defense mediators (Hawgood et al., 2004
; Wu et al., 1986
). This study was designed to examine whether acute exposures to an aqueous-trapped solution of DE (DEas) can affect influenza infections in human respiratory epithelial cells, using in vitro models of differentiated human nasal and bronchial epithelial cells as well as a respiratory epithelial cell line. Our data demonstrate that acute exposure to DEas increases the number of influenza-infected cells in an oxidative stress-dependent manner, and that this may be caused by the ability of DEas to increase influenza virus attachment and possibly entry into epithelial cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primary human bronchial cells were obtained from healthy nonsmoking adult volunteers by cytologic brushing at bronchoscopy. Primary human nasal cells were obtained from healthy nonsmoking adult volunteers by gently stroking the inferior surface of the turbinate several times with a Rhino-Probe curette (Arlington Scientific, Arlington, TX), which was inserted through an otoscope with a large aperture. The protocols for the acquisition of both primary human bronchial and nasal epithelial cells were reviewed by the University of North Carolina Institutional Review Board and informed written consent was obtained from all subjects. Both primary human bronchial and nasal epithelial cells were expanded to passage 2 in bronchial epithelial growth medium (BEGM, Cambrex Bioscience Walkersville, Inc., Walkersville, MD) and then plated on collagen-coated filter supports with a 0.4 µm pore size (Trans-CLR; Costar, Cambridge, MA) and cultured in a 1:1 mixture of bronchial epithelial cell basic medium (BEBM) and DMEM-H with SingleQuot supplements (Cambrex), bovine pituitary extracts (13 mg/ml), bovine serum albumin (BSA, 1.5 µg/ml), and nystatin (20 units). Upon confluency, all-trans retinoic acid was added to the medium, and air liquid interface (ALI) culture conditions (removal of the apical medium) were created to promote differentiation. Mucociliary differentiation was achieved after 1821 days post-ALI.
Exposure to aqueous-trapped solution of diesel exhaust (DEas).
DEas was generated as described before (Madden et al., 2003). Briefly, we used emissions from a Caterpillar diesel engine, model 3304, which was used to power a 113 KW generator. This type of engine was chosen because it is used in nonroad vehicles, which are significant contributors to ambient diesel exhaust levels, and because the projected trend for emissions from nonroad diesel engines is expected to remain at the same level or even increase in the future (U. S. EPA, 2002
). The diesel exhaust emissions from this Caterpillar diesel engine were passed through a tubing system with a filter impactor and two impinger tubes (containing 100 ml PBS each) submerged in an ice bath. Impinger glassware was washed and heated to remove and destroy endotoxin. Of the two impinger tubes, the emissions (at 10 l/min) that entered and remained in the first (primary) tube, but not the secondary tube, were utilized for the cell exposure studies. Extracts were generated and collected during a one-hour period when the engine was under high load (HL). This type of preparation was chosen because it contains DE particles, as well as polar and thus water soluble DE gas-phase components. To determine the mass of the emissions retained within the PBS in an impinger tube, an aliquot was dried overnight at 56°C and corrected for the mass of the PBS contribution (which was determined in a similar manner by overnight drying) and dilution with water from the exhaust. Aliquots of the DEas were kept at 20°C until use.
For all cell types used in this study, DEas was added 2 h before infection with influenza. Specifically, for the differentiated human nasal and bronchial epithelial cells, DEas was diluted in 200 µl media to achieve 22 or 44 µg DEas per cm2 of cell layer and added to the apical side. After the 2-h incubation with DEas, the diluted DEas was removed, and influenza virus diluted in the same volume of media was added to the apical side for 2 h, after which it was removed to establish ALI culture conditions again. For the experiments using A549 cells, DEas was diluted in F12K media plus BSA plus antibiotics to achieve 6.25, 12.5, or 25 µg/cm2 and added to the cells. After 2-h incubation with DEas, influenza virus was added to the cells. The effects of exposure to DEas on cell viability were assessed by analyzing cell culture supernatants for lactate dehydrogenase (LDH) activity using a commercially available kit according to the supplier's instructions (CytoTox 96®, Promega, Madison, WI).
Infection with influenza.
Throughout this study we used influenza A/Bangkok/1/79 (H3N2 serotype) which was propagated in 10-day-old embryonated hen's eggs. The virus was collected in the allantoic fluid and titered by 50% tissue culture infectious dose in Madin-Darby canine kidney cells and hemagglutination as described before (Beck et al., 2001). Stock virus was aliquoted and stored at 80°C until use. Unless otherwise indicated, for infection of differentiated bronchial or nasal cells as well as A549 cells, approximately 3 x 105 cells were infected with 320 hemagglutination units (HAU) of influenza A Bangkok 1/79.
RT-PCR.
Total RNA was extracted using TRizol (Invitrogen) as per the supplier's instruction. First-strand cDNA synthesis and real-time RT-PCR was performed as described previously (Jaspers et al., 1999, 2001
). The sequences for the primers and probes used in this study are as following:
Hemagglutinin (HA): probe, 5'-FAM-TGATGGGAAAAACTGCACACTGATAGATGC-TAMRA-3'; sense, 5'-CGACAGTCCTCACCGAATCC-3'; antisense, 5'-TCACAATGAGGGTCTCCCAATAG-3'; IFNß: probe, 5'-FAM-AGCAGCAATTTTCAGTGTCAGAAGCTCCTG-TAMRA-3'; sense, 5'-CAACTTGCTTGGATTCCTACAAAG-3'; antisense, 5'-AGCCTCCCATTCAATTGCC-3'; MxA: probe, 5'-FAM-AGGCCAGCAAGCGCATCTCCAG-TAMRA-3'; sense, 5'-CAGCACCTGATGGCCTATCAC-3'; antisense, 5'-CATGAAGAACTGGATGATCAAAGG-3'; GAPDH: probe, 5'-JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA-3'; sense, 5'-GAAGGTGAAGGTCGGAGTC-3'; antisense, 5'-GAAGATGGTGATGGGATTTC-3'.
Virus attachment assay.
A549 or differentiated nasal epithelial cells were exposed to DEas and infected with influenza as described above. Nonattached virus was removed by rinsing the cells twice with media either immediately (t = 0) or at 15, 30, 60, or 120 min post-infection. RNA from the rinse containing the nonattached virus was isolated using a Viral RNA Isolation kit (QIAamp Viral RNA Isolation Kit, Qiagen, Valencia, CA) and analyzed for HA RNA levels using gene-specific primer anchored RT-PCR (QuantiTectTM Probe RT-PCR Kit, Qiagen). HA RNA levels at the specific time points were normalized to the HA RNA levels at t = 0 of the respective exposure group. Decreased HA RNA levels would indicate less nonattached virus and are used here as a measure of increased virus attachment.
Western blotting.
Whole cell lysates were prepared by lysing the cells in RIPA buffer containing 1% Nonidet P (NP)-40, 0.5% deoxycholate, 0.1% SDS, and protease inhibitors (Cocktail Set III; Calbiochem, San Diego, CA). Nuclear extracts were prepared as described before (Jaspers et al., 2001). Fifty micrograms of whole cell lysate or 20 µg of nuclear extract was separated by SDSPAGE as described before (Jaspers et al., 2001
). This was followed by immunoblotting using specific antibodies to tyr-701 phospho-specific STAT1 (1:1000, Cell Signaling, Beverly, MA), interferon-stimulated gene factor 3 gamma (ISGF3
; 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), or influenza A H3N2 (1:500; US Biologicals, Swampscott, MA). Antigen-antibody complexes were stained with anti-rabbit horseradish peroxidase-conjugated antibody (1:2000, Santa Cruz Biotechnology) and SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). The chemiluminescent signals were acquired using a 16-bit CCD camera (GeneGnome system; Syngene, Frederick, MD) and analyzed using the GeneSnap software (Syngene).
Promoter-reporter assays.
An interferon-stimulated response element (ISRE)-dependent promoter reporter construct (pISRE-luc; Stratagene, La Jolla, CA) was used. A constitutively active SV-40 promoter-ß-galactosidase construct (ß-gal; Promega, Madison, WI) was used to adjust for well-to-well variation in cell number and transfection efficiency. A549 cells grown to about 5080% confluence in 24-well tissue culture dishes were transfected with 500 ng of the pISRE-luc plasmid using 0.75 µl FuGENE6 Transfection Reagent (Roche Diagnostics, Mannheim, Germany). Twenty-four h post-transfection, the cells were serum-starved for 24 h and exposed to DEas and infected with influenza as described above. Luciferase and ß-galactosidase activities were determined using the Dual Light Reporter assay System (Perkin Elmer) and an AutoLumat LB953 luminometer (Berthold Analytical Instruments, Nashua, NH). Luciferase activity was normalized to ß-galactosidase activity and expressed as fold induction over infected but nonexposed cells.
Immunohistochemistry.
A549 cells were grown on chamber slides (Lab-Tek® Chamber slides, Nalge Nunc International, Naperville, IL) and exposed to DEas and infected with influenza as described above. In some experiments, cells were treated with GSH-ET (10 mM; Sigma) 30 min before exposure to DEas. At 24 h post-infection, cells were acetone fixed and stained using FITC-tagged mouse anti-influenza A/Texas antibody (10 µg/ml; ViroStat, Portland, ME), which recognizes nucleoprotein of H3N2 influenza A. Samples were washed twice with phosphate-buffered saline and photographed on a Nikon Microphot-SA fluorescence microscope using standard fluorescein excitation and emission filter sets. To determine the average number of influenza-infected cells per microscopic field, a protocol for a systematic randomization procedure was applied. Briefly, each immunohistochemically stained sample was divided into four quadrants. Influenza-infected cells were counted in one field per quadrant as well as one field in the center of the sample. Every field was checked for confluency using phase contrast microscopy. Only fields in which cells reached >90% confluency were considered appropriate for counting, and fields with less than 90% confluent cell monolayer were excluded from the analysis.
Analysis of oxidative stress.
Protein carbonyl levels were detected by immunoblotting using the OxyBlotTM Protein Oxidation Kit (Chemicon, Temcula, CA) as per the supplier's instruction. Briefly, cells were lysed in RIPA buffer containing 1% Nonidet P (NP)-40, 0.5% deoxycholate, 0.1% SDS, protease inhibitors (Cocktail Set III; Calbiochem), and 50 mM dithiothreitol (DDT). The carbonyl groups in 5 µl cell lysates were derivatized to 24-dinitrophenyl hydrazone (DNP-hydrazone) by reaction with 24-dinitrophenyl hydrazine (DNPH). The DNP-derivatized protein samples were separated by 12% SDSPAGE, followed by immunoblotting with antibodies against the DNP moiety of the proteins. Antigenantibody complexes were stained and visualized as described above. To assure equal loading, blots were stripped using Blot Restore Membrane Rejuvenation Kit (Chemicon) as per the supplier's instruction, and re-probed using antibodies against -tubulin (Sigma).
Analysis of oxidative stress using 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) in A549 cells was conducted as described before (Jaspers et al., 2000).
Statistical Analysis.
Data are expressed as means ± SEM of at least three separate experiments. The RT-PCR and promoter reporter data were expressed as percent of the infected but nonexposed control and analyzed using the Wilcoxon Signed Rank Test, assuming a theoretical mean of 100. All other data were analyzed using a two-tailed Student's t-test (Fig.1D) or one-way analysis of variance followed by the Newman-Keul's post hoc test for multigroup analysis. A value of p < 0.05 was considered to be significant.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Enhanced levels of influenza virus replication could be caused by either increased viral production in each infected cell or increasing the number of infected cells. To analyze whether the increased levels of HA RNA and viral protein levels in DEas-exposed cells stems from increasing viral replication in each infected cell or from increasing the number of influenza-infected cells, we examined the effects of DEas exposures on the number of influenza-infected cells immunohistochemically. A549 cells were grown in chamber slides, exposed to DEas, and infected with three doses of influenza. Twenty-four h post-infection, cells were acetone-fixed and stained using a FITC-tagged anti-influenza antibody. The number of influenza-infected cells was examined using an epifluorescent microscope. Figure 1C shows that, independent of the dose of influenza used for infection, exposure to DEas increases the number of influenza-infected epithelial cells. In addition, the number of fluorescently stained cells in the control and DEas-exposed cells that were infected with 256 HAU influenza virus were counted. Figure 1D shows that DEas exposure significantly increases the number of influenza-infected A549 cells. Taken together, these results demonstrate that exposure to DEas increases the susceptibility of respiratory epithelial cells to become infected with influenza virus, resulting in a greater number of infected cells.
Exposure to DEas Increases the Susceptibility to Influenza Infections in Differentiated Human Respiratory Epithelial Cells
Primary human nasal and bronchial epithelial cells obtained from healthy human volunteers grown under defined culture conditions differentiate into a mucociliary phenotype, resembling many characteristics of human epithelium found in vivo, such as large beds of beating cilia and the presence of different cell types, including ciliated, nonciliated, and mucus-producing cells (Clark et al., 1995). We used these models of differentiated human nasal and bronchial epithelial cells to confirm our findings obtained with A549 cells. Briefly, cultures of differentiated human nasal and bronchial epithelial cells were exposed to DEas for 2 h and subsequently infected with influenza A. Similar to our experiments using A549 cells, we analyzed viral RNA and protein levels in control and DEas-exposed cells 24 h post-infection. Figure 2 shows that exposure to DEas increased HA RNA levels in differentiated bronchial epithelial cells (Figure 2A) and differentiated nasal epithelial cells (Figure 2B), and that this difference was statistically significant for the nasal epithelial cells exposed to 22 µg/cm2 and was approaching statistical significance for bronchial epithelial cells exposed to 44 µg/cm2 (p = 0.07). Figures 2C (bronchial epithelial cells) and 2D (nasal epithelial cells) also show that exposure of these cells to 44 µg/cm2 DEas enhanced viral protein levels. Taken together, these results suggest that, similar to A549 cells, exposure to DEas also increases the susceptibility to influenza infection in cultures of primary differentiated human respiratory epithelial cells.
|
|
Among the IFN-inducible antiviral mediators that are important in fighting influenza infections is myxovirus resistance protein (MxA). The importance of MxA in influenza infection is demonstrated by studies showing that cells constitutively expressing MxA are resistant to influenza infections (Horisberger, 1995). The expression of MxA is ISRE dependent, and although our data indicated that exposure to DEas did not suppress activation of STAT-1 or ISGF3
or ISRE-dependent promoter reporter activity, it is conceivable that DEas could affect the expression of essential antiviral mediators through different mechanisms. Therefore, we determined the effects of exposure to DEas on influenza-induced expression of MxA in A549 cells. Figure 3D demonstrates that the effect of DEas on influenza-induced MxA expression closely reflects the effects of DEas on HA RNA and IFNß mRNA levels. Specifically, exposure of A549 cells to DEas enhanced MxA mRNA levels in these cells, indicating the DEas exposure does not compromise the ability of human respiratory epithelial cells to express antiviral mediators. While suppression of interferon-dependent antiviral defense responses would increase the susceptibility to influenza infections and reinfection of neighboring cells, increased interferon levels in the presence of increased viral replication would suggest that the interferon-dependent antiviral defense responses are working in accordance with a greater initial infection. Taken together, the data suggest that DEas enhances influenza virus replication without suppressing the production of IFNß or IFNß-inducible genes, such as MxA.
Effects of DEas Exposure on Influenza-Induced IFN Responses in Differentiated Human Respiratory Epithelial Cells
Again, to confirm our results on the effects of DEas exposure on influenza-induced IFN responses in primary epithelial cells, we exposed differentiated human nasal and bronchial epithelial cells to DEas and subsequently infected them with influenza A. Similar to experiments conducted in A549 cells, we analyzed the expression of IFNß and MxA 24 h post-infection. Figures 4A and 4B show that, analogous to the effects seen in A549 cells, exposure to DEas also increases influenza-induced IFNß mRNA levels in differentiated human bronchial (Fig. 4A) and nasal (Fig. 4B) epithelial cells. Moreover, exposure to DEas also increases influenza-induced MxA mRNA levels in differentiated human bronchial (Fig. 4C) and nasal (Fig. 4D) epithelial cells. Thus, similar to the effects of DEas on influenza-induced IFN responses seen in A549 cells, exposure to DEas also increases influenza-induced IFNß and MxA mRNA levels in differentiated human nasal and bronchial epithelial cells. Again, these data suggest that the increased susceptibility to influenza infection seen after DEas exposure is not caused by impaired production of antiviral defense mediators.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The enhanced susceptibility to influenza virus infections after DEas exposures in our in vitro models was not caused by suppression of IFNß or IFN-dependent mediator production. In fact, our data demonstrated that the levels of IFNß, IFNß-dependent signaling, and MxA expression correlated well with the levels of influenza virus replication. Enhanced IFN production in the presence of enhanced influenza virus replication would suggest that the ability to generate IFN-dependent antiviral defense responses was not impaired by DEas exposure, but that the host tissue was responding appropriately to a greater level of viral infection. Indeed, our data demonstrated that the number of influenza-infected cells was enhanced by DEas exposures, which would explain the increased levels of IFN and IFN-dependent antiviral defense response. On the other hand, suppressed IFN production in the presence of enhance influenza virus replication would suggest that the host cells are incapable of limiting the viral infection, which enhances replication and reinfection, thus increasing the spreading of the infection. Previously published reports demonstrated that chronic exposure to DE enhanced influenza virus replication and decreased lung IFN levels (Hahon et al., 1985), suggesting that the ability of the host to limit and clear the infection was impaired by chronic DE exposures. In contrast, subchronic exposure to DE enhanced the susceptibility to infection with respiratory syncytial virus (RSV) without impairing IFN production (Harrod et al., 2003
). These studies suggest that chronic and subchronic exposures to DE may increase the susceptibility to respiratory virus infections through IFN-dependent and IFN-independent mechanisms, respectively. Our studies only examined the effects of acute DEas exposures on influenza infections in epithelial cells in vitro, and since epithelial cells only produce type I interferons, we focused on the effects of DEas on influenza-induced IFNß production. However, it is conceivable that acute exposures to DEas could impair influenza-induced IFN
production, a type II interferon, by infected monocytes, macrophages, or other immune cells.
However, other non-IFN-dependent antiviral defense strategies applied by respiratory epithelial cells to limit influenza infection may have been affected by acute and subchronic exposures to DEas. For example, studies by Harrod et al. (2003) have demonstrated that subchronic exposures to DE enhances RSV titers in mice and that this effect was associated with decreased levels of other mediators with potential antiviral function. Specifically, this study demonstrated that exposure to DE decreased lung surfactant protein A (SP-A) and Clara cell secretory protein (CCSP) levels, which can both have RSV-scavenging activity (Harrod et al., 1998
, 1999
). SP-A as well as surfactant protein D (SP-D) both belong to the family of collectins, which recognize and interact with glycoconjugates on the surface of microorganisms and thus increase clearance of the microorganisms by phagocytic cells (Wright, 2004
). Both SP-A and SP-D are released by alveolar type II cells as well as nonciliated bronchial epithelial cells or Clara cells (Madsen et al., 2000
, 2003
) and are very important for innate immediate defense responses against influenza infections (Hartshorn et al., 1994
; Hawgood et al., 2004
). Specifically, SP-D and SP-A can agglutinate influenza virus, neutralize the virus, and therefore prevent attachment to the host cell (Hartshorn et al., 2000
; Hawgood et al., 2004
). Given the previous reports on the effects of DE exposures on SP-A levels in mice (Harrod et al., 2003
) and the fact that SP-A and SP-D are produced by alveolar type II cells and nonciliated bronchial epithelial cells, it is plausible that in our in vitro models of human respiratory epithelial cells acute exposures to DEas modified the ability of SP-D or SP-A to neutralize influenza virus in these cells.
Our data show that DEas-induced oxidative stress increased the susceptibility to influenza infection in human respiratory epithelial cells and that DEas exposure increased markers of oxidative stress within 2 h post-exposure. In our experimental model, respiratory epithelial cells were exposed to DEas 2 h prior to infection with influenza, suggesting that, at the time of infection, DEas-exposed cells were already oxidatively stressed. Previous studies have shown that influenza virus replication in vitro and in vivo was inhibited by the antioxidant GSH (Cai et al., 2003), suggesting that oxidative stress would favor influenza virus replication. The mechanisms by which oxidative stress could increase the susceptibility to influenza infections are not clear. Based on our observations that DEas exposure enhances influenza virus attachment and entry within 2 h post-infection, it seems likely that redox-dependent post-translational modification of components regulating susceptibility to influenza virus infections could be involved. For example, influenza virus hemagglutinin needs to be proteolytically cleaved for the virus to become infectious and enter the cells, which is mediated by trypsin-like serine proteases released by epithelial cells (Kido et al., 1992
; Sakai et al., 1993
). The activity of such proteases in turn is regulated by mucus antiproteases, such as secretory leukocyte protease inhibitor (SLPI) (Kido et al., 1999
). Interestingly, oxidative stress derived from cigarette smoke or addition of reactive oxygen intermediates can decrease antiprotease activity (Cavarra et al., 2001
; Vogelmeier et al., 1997
) and increase influenza infectivity (Hennet et al., 1992
). Thus, post-translational modification of antiproteases by DEas-induced oxidative stress could increase the ability to proteolytically activate influenza virus and therefore enhance the susceptibility to influenza infections.
In addition, the activity of collectins, specifically SP-A, is sensitive to oxidative modifications. Wang et al. (2002) have shown that oxidative modification of SP-A after ozone exposure alters the ability of SP-A to stimulate cytokine production in a monocytic cell line. As stated above, previous studies have shown that subchronic exposures to DE can decrease the transcriptional levels of SP-A in mice, which may have contributed to the increase susceptibility to RSV infections in these animals (Harrod et al., 2003
). However, the results presented here showed increased influenza virus attachment after only a brief exposure (2 h) to DEas, suggesting that the transcriptional levels of SP-A or other collectins may not have changed during this short exposure period. Alternatively, it is also possible that post-translational oxidative modification of SP-A or other collectins reduced the ability to aggregate and neutralize influenza virus and therefore increased susceptibility to infection. Indeed, post-translational nitration of SP-A through reactive nitrogen species has been demonstrated to decrease the ability of SP-A to aggregate lipids, bind mannose, and adhere P. carinii to macrophages (Haddad et al., 1996
; Zhu et al., 1996
, 1998
), all of which are important functions of SP-A in vivo. Thus, in addition to modifying the transcriptional level of SP-A and other innate immune defense mediators, oxidative stress induced upon exposure to DE could also reduce the function of SP-A and other collectins through post-translational oxidation of these proteins. Future studies will examine the effects of DEas and oxidative modification of SP-D on its ability to neutralize influenza in vitro.
All of the experiments described in this study were performed in vitro using differentiated primary human respiratory epithelial cells or a human lung carcinoma cell line (A549 cells). Interestingly, the primary cells and the cell line responded very similarly to the treatment with DEas and influenza infection, although the responses did not always reach statistical significance for the bronchial epithelial cells. Differentiated primary human respiratory epithelial cells, when grown under defined culture condition (see Methods), retain many characteristics seen in vivo (Clark et al., 1995; Gray et al., 1996
; Ostrowski et al., 1995
). Interestingly, our data indicate that differentiated human nasal epithelial cells may be more sensitive to the effects of DEas on influenza virus replication than human bronchial epithelial cells. This becomes even more important, considering the nasal epithelium as the site where a large fraction of DE particles are potentially deposited during inhalation and exhalation (Wiesmiller et al., 2003
) and as the site of initial infection with influenza virus. However, the role of the respiratory epithelium during influenza infections goes beyond viral replication and innate immune defense responses. Respiratory epithelial cells produce and release a number of pro-inflammatory cytokines and chemokines in response to influenza infections (Adachi et al., 1997
; Julkunen et al., 2000
), which are responsible for recruitment and activation of mononuclear cells, costimulation of Th lymphocytes, induction of lymphocyte/fibroblast proliferation, acting as endogenous pyrogens, and other activities. Thus, the respiratory epithelium is not only an entryway for influenza infections, but also a key element which orchestrates many of the subsequent immune and inflammatory responses. Consequently, enhanced susceptibility of respiratory epithelial cells to influenza infections after exposure to DE will determine the severity of the infection and injury to the surrounding tissue.
Taken together, the data presented here demonstrate that acute exposures to DEas significantly affect the susceptibility of human respiratory epithelial cells to influenza virus infections by enhancing virus attachment and entry. The aqueous-trapped solution of DE used throughout these studies contains both the particulate fraction and water-soluble gas-phase components of DE derived from an off-road diesel engine. In both differentiated human epithelial cells and A549 cells, the doses of DEas used throughout the study did not cause cytotoxicity (data not shown), suggesting that DEas did not significantly affect the integrity of the epithelial layer. Future studies will determine whether different preparations of DE particles as well as other ambient air pollution particles have similar effects on influenza infections in respiratory epithelial cells. Concurrent exposures to DE or other PM mixtures and influenza infections are likely in urban populations. Therefore, enhanced susceptibility to influenza infections resulting from DE or PM mixtures could have a significant impact on public health.
![]() |
NOTES |
---|
![]() |
ACKNOWLEDGMENTS |
---|
This work was funded by grants from the Environmental Protection Agency (CR829522) and the American Chemistry Council (#545974). This publication has not been formally reviewed by the American Chemistry Council. The views expressed in this document are solely those of the authors. Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through cooperative agreement CR829522 with the Center for Environmental Medicine, Asthma, and Lung Biology, it has not been subjected to the Agency's required peer and policy review and, therefore, does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
Conflict of Interest: The corresponding author, Dr. Ilona Jaspers, acknowledges that she has received a grant from the American Chemistry Council to conduct research in this area; however, the funding organization does not have control over the resulting publications.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beck, M. A., and Matthews, C. C. (2000). Micronutrients and host resistance to viral infection. Proc. Nutr. Soc. 59, 581585.[ISI][Medline]
Beck, M. A., Nelson, H. K., Shi, Q., Van Dael, P., Schiffrin, E. J., Blum, S., Barclay, D., and Levander, O. A. (2001). Selenium deficiency increases the pathology of an influenza virus infection. FASEB J. 15, 14811483.
Bonvallot, V., Baeza-Squiban, A., Baulig, A., Brulant, S., Boland, S., Muzeau, F., Barouki, R., and Marano, F. (2001). Organic compounds from diesel exhaust particles elicit a proinflammatory response in human airway epithelial cells and induce cytochrome p450 1A1 expression. Am. J. Respir. Cell Mol. Biol. 25, 515521.
Cai, J., Chen, Y., Seth, S., Furukawa, S., Compans, R. W., and Jones, D. P. (2003). Inhibition of influenza infection by glutathione. Free Radic. Biol. Med. 34, 928936.[CrossRef][ISI][Medline]
Castranova, V., Ma, J. Y., Yang, H. M., Antonini, J. M., Butterworth, L., Barger, M. W., Roberts, J., and Ma, J. K. (2001). Effect of exposure to diesel exhaust particles on the susceptibility of the lung to infection. Environ. Health Perspect. 109 (Suppl. 4), 609612.[ISI][Medline]
Cavarra, E., Lucattelli, M., Gambelli, F., Bartalesi, B., Fineschi, S., Szarka, A., Giannerini, F., Martorana, P. A., and Lungarella, G. (2001). Human SLPI inactivation after cigarette smoke exposure in a new in vivo model of pulmonary oxidative stress. Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L412L417.
Clark, A. B., Randell, S. H., Nettesheim, P., Gray, T. E., Bagnell, B., and Ostrowski, L. E. (1995). Regulation of ciliated cell differentiation in cultures of rat tracheal epithelial cells. Am. J. Respir. Cell Mol. Biol. 12, 329338.[Abstract]
D'Amato, G. (2002). Urban air pollution and respiratory allergy. Monaldi Arch. Chest Dis. 57, 136140.[Medline]
Diaz-Sanchez, D. (1997). The role of diesel exhaust particles and their associated polyaromatic hydrocarbons in the induction of allergic airway disease. Allergy 52, 5256.[ISI][Medline]
Gray, T. E., Guzman, K., Davis, C. W., Abdullah, L. H., and Nettesheim, P. (1996). Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 14, 104112.[Abstract]
Haddad, I. Y., Zhu, S., Ischiropoulos, H., and Matalon, S. (1996). Nitration of surfactant protein A results in decreased ability to aggregate lipids. Am. J. Physiol. 270, L281L288.[ISI][Medline]
Hahon, N., Booth, J. A., Green, F., and Lewis, T. R. (1985). Influenza virus infection in mice after exposure to coal dust and diesel engine emissions. Environ. Res. 37, 4460.[CrossRef][ISI][Medline]
Harrod, K. S., Jaramillo, R. J., Rosenberger, C. L., Wang, S. Z., Berger, J. A., McDonald, J. D., and Reed, M. D. (2003). Increased susceptibility to RSV infection by exposure to inhaled diesel engine emissions. Am. J. Respir. Cell Mol. Biol. 28, 451463.
Harrod, K. S., Mounday, A. D., Stripp, B. R., and Whitsett, J. A. (1998). Clara cell secretory protein decreases lung inflammation after acute virus infection. Am. J. Physiol. 275, L924L930.[ISI][Medline]
Harrod, K. S., Trapnell, B. C., Otake, K., Korfhagen, T. R., and Whitsett, J. A. (1999). SP-A enhances viral clearance and inhibits inflammation after pulmonary adenoviral infection. Am J Physiol 277, L580L588.[ISI][Medline]
Hartshorn, K. L., Crouch, E. C., White, M. R., Eggleton, P., Tauber, A. I., Chang, D., and Sastry, K. (1994). Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses. J. Clin. Invest. 94, 311319.[ISI][Medline]
Hartshorn, K. L., White, M. R., Voelker, D. R., Coburn, J., Zaner, K., and Crouch, E. C. (2000). Mechanism of binding of surfactant protein D to influenza A viruses: Importance of binding to haemagglutinin to antiviral activity. Biochem. J. 351(2), 449458.[CrossRef][ISI][Medline]
Hashimoto, S., Gon, Y., Takeshita, I., Matsumoto, K., Jibiki, I., Takizawa, H., Kudoh, S., and Horie, T. (2000). Diesel exhaust particles activate p38 MAP kinase to produce interleukin 8 and RANTES by human bronchial epithelial cells and N-acetylcysteine attenuates p38 MAP kinase activation. Am. J. Respir. Crit. Care Med. 161, 280285.
Hawgood, S., Brown, C., Edmondson, J., Stumbaugh, A., Allen, L., Goerke, J., Clark, H., and Poulain, F. (2004). Pulmonary collectins modulate strain-specific influenza a virus infection and host responses. J. Virol. 78, 85658572.
Hennet, T., Peterhans, E., and Stocker, R. (1992). Alterations in antioxidant defences in lung and liver of mice infected with influenza A virus. J. Gen. Virol. 73(1), 3946.[Abstract]
Hiura, T. S., Kaszubowski, M. P., Li, N., and Nel, A. E. (1999). Chemicals in diesel exhaust particles generate reactive oxygen radicals and induce apoptosis in macrophages. J. Immunol. 163, 55825591.
Hiura, T. S., Li, N., Kaplan, R., Horwitz, M., Seagrave, J. C., and Nel, A. E. (2000). The role of a mitochondrial pathway in the induction of apoptosis bychemicals extracted from diesel exhaust particles. J. Immunol. 165, 27032711.
Horisberger, M. A. (1995). Interferons, Mx genes, and resistance to influenza virus. Am. J. Respir. Crit. Care Med. 152, S67S71.[ISI][Medline]
Jaspers, I., Samet, J. M., Erzurum, S., and Reed, W. (2000). Vanadium-induced kB-dependent transcription depends upon peroxide-induced activation of the p38 mitogen-activated protein kinase. Am. J. Resp. Cell Mol. Biol. 23, 95102.
Jaspers, I., Samet, J. M., and Reed, W. (1999). Arsenite Exposure of Cultured Airway Epithelial Cells Activates kB-dependent IL-8 Gene Expression in the Absence of NF-kB Nuclear Translocation. J. Biol. Chem. 274(43), 3102531033.
Jaspers, I., Zhang, W., Fraser, A., Samet, J. M., and Reed, W. (2001). Hydrogen peroxide has opposing effects on IKK activity and proteasomal degradation of IkBa in airway epithelial cells. Am. J. Resp. Cell Mol. Biol. 24, 769777.
Julkunen, I., Melen, K., Nyqvist, M., Pirhonen, J., Sareneva, T., and Matikainen, S. (2000). Inflammatory responses in influenza A virus infection. Vaccine 19 (Suppl. 1), S32S37.[CrossRef][ISI][Medline]
Kido, H., Beppu, Y., Imamura, Y., Chen, Y., Murakami, M., Oba, K., and Towatari, T. (1999). The human mucus protease inhibitor and its mutants are novel defensive compounds against infection with influenza A and Sendai viruses. Biopolymers 51, 7986.[CrossRef][ISI][Medline]
Kido, H., Yokogoshi, Y., Sakai, K., Tashiro, M., Kishino, Y., Fukutomi, A., and Katunuma, N. (1992). Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein. J. Biol. Chem. 267, 1357313579.
LeVine, A. M., Gwozdz, J., Stark, J., Bruno, M., Whitsett, J., and Korfhagen, T. (1999). Surfactant protein-A enhances respiratory syncytial virus clearance in vivo. J. Clin. Invest. 103, 10151021.
Li, N., Wang, M., Oberley, T. D., Sempf, J. M., and Nel, A. E. (2002). Comparison of the pro-oxidative and proinflammatory effects of organic diesel exhaust particle chemicals in bronchial epithelial cells and macrophages. J. Immunol. 169, 45314541.
Madden, M. C., Dailey, L. A., Stonehuerner, J. G., and Harris, B. D. (2003). Responses of cultured human airway epithelial cells treated with diesel exhaust extracts will vary with the engine load. J. Toxicol. Environ. Health A 66, 22812297.[ISI][Medline]
Madsen, J., Kliem, A., Tornoe, I., Skjodt, K., Koch, C., and Holmskov, U. (2000). Localization of lung surfactant protein D on mucosal surfaces in human tissues. J. Immunol. 164, 58665870.
Madsen, J., Tornoe, I., Nielsen, O., Koch, C., Steinhilber, W., and Holmskov, U. (2003). Expression and localization of lung surfactant protein A in human tissues. Am. J. Respir. Cell Mol. Biol. 29, 591597.
Marano, F., Boland, S., Bonvallot, V., Baulig, A., and Baeza-Squiban, A. (2002). Human airway epithelial cells in culture for studying the molecular mechanisms of the inflammatory response triggered by diesel exhaust particles. Cell Biol. Toxicol. 18, 315320.[CrossRef][ISI][Medline]
Nardone, L. L., and Andrews, S. B. (1979). Cell line A549 as a model of the type II pneumocyte. Phospholipid biosynthesis from native and organometallic precursors. Biochim. Biophys. Acta 573, 276295.[ISI][Medline]
Nencioni, L., Iuvara, A., Aquilano, K., Ciriolo, M. R., Cozzolino, F., Rotilio, G., Garaci, E., and Palamara, A. T. (2003). Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. FASEB J. 17, 758760.
Ostrowski, L. E., Randell, S. H., Clark, A. B., Gray, T. E., and Nettesheim (1995). Ciliogenesis of rat tracheal epithelial cells in vitro. Methods Cell Biol. 47, 5763.[ISI][Medline]
Pandya, R. J., Solomon, G., Kinner, A., and Balmes, J. R. (2002). Diesel exhaust and asthma: Hypotheses and molecular mechanisms of action. Environ. Health Perspect. 110 (Suppl. 1), 103112.
Rabovsky, J., Judy, D. J., Rodak, D. J., and Petersen, M. (1986). Influenza virus-induced alterations of cytochrome P-450 enzyme activities following exposure of mice to coal and diesel particulates. Environ. Res. 40, 136144.[ISI][Medline]
Saito, Y., Azuma, A., Kudo, S., Takizawa, H., and Sugawara, I. (2002a). Effects of diesel exhaust on murine alveolar macrophages and a macrophage cell line. Exp. Lung Res. 28, 201217.[CrossRef][ISI][Medline]
Saito, Y., Azuma, A., Kudo, S., Takizawa, H., and Sugawara, I. (2002b). Long-term inhalation of diesel exhaust affects cytokine expression in murine lung tissues: Comparison between low- and high-dose diesel exhaust exposure. Exp. Lung Res. 28, 493506.[CrossRef][ISI][Medline]
Sakai, K., Kawaguchi, Y., Kishino, Y., and Kido, H. (1993). Electron immunohistochemical localization in rat bronchiolar epithelial cells of tryptase Clara, which determines the pneumotropism and pathogenicity of Sendai virus and influenza virus. J. Histochem. Cytochem. 41, 8993.
Samuel, C. E. (2001). Antiviral actions of interferons. Clin. Microbiol. Rev. 14, 778809.
Spackman, E., Senne, D. A., Myers, T. J., Bulaga, L. L., Garber, L. P., Perdue, M. L., Lohman, K., Daum, L. T., and Suarez, D. L. (2002). Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J. Clin. Microbiol. 40, 32563260.
Steerenberg, P., Verlaan, A., De Klerk, A., Boere, A., Loveren, H., and Cassee, F. (2004). Sensitivity to ozone, diesel exhaust particles, and standardized ambient particulate matter in rats with a listeria monocytogenes-induced respiratory infection. Inhal. Toxicol. 16, 311317.[CrossRef][ISI][Medline]
Takizawa, H., Abe, S., Okazaki, H., Kohyama, T., Sugawara, I., Saito, Y., Ohtoshi, T., Kawasaki, S., Desaki, M., Nakahara, K., et al. (2003). Diesel exhaust particles upregulate eotaxin gene expression in human bronchial epithelial cells via nuclear factor-kappa B-dependent pathway. Am. J. Physiol. Lung Cell. Mol. Physiol. 284, L1055 L1062.
Takizawa, H., Ohtoshi, T., Kawasaki, S., Abe, S., Sugawara, I., Nakahara, K., Matsushima, K., and Kudoh, S. (2000). Diesel exhaust particles activate human bronchial epithelial cells to express inflammatory mediators in the airways: A review. Respirology 5, 197203.[CrossRef][Medline]
Thompson, W. W., Shay, D. K., Weintraub, E., Brammer, L., Bridges, C. B., Cox, N. J., and Fukuda, K. (2004). Influenza-associated hospitalizations in the United States. JAMA 292, 13331340.
Thompson, W. W., Shay, D. K., Weintraub, E., Brammer, L., Cox, N., Anderson, L. J., and Fukuda, K. (2003). Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 289, 179186.
U.S. EPA (2002). Health Assessment Document for Diesel Engine Exhaust. United States Environmental Protection Agency, EPA/600/890/057F.
Vogelmeier, C., Biedermann, T., Maier, K., Mazur, G., Behr, J., Krombach, F., and Buhl, R. (1997). Comparative loss of activity of recombinant secretory leukoprotease inhibitor and alpha 1-protease inhibitor caused by different forms of oxidative stress. Eur. Respir. J. 10, 21142119.
Wang, G., Umstead, T. M., Phelps, D. S., Al-Mondhiry, H., and Floros, J. (2002). The effect of ozone exposure on the ability of human surfactant protein a variants to stimulate cytokine production. Environ. Health Perspect. 110, 7984.[ISI][Medline]
Wang, S. Z., Rosenberger, C. L., Bao, Y. X., Stark, J. M., and Harrod, K. S. (2003). Clara cell secretory protein modulates lung inflammatory and immune responses to respiratory syncytial virus infection. J. Immunol. 171, 10511060.
Wiesmiller, K., Keck, T., Leiacker, R., Sikora, T., Rettinger, G., and Lindemann, J. (2003). The impact of expiration on particle deposition within the nasal cavity. Clin Otolaryngol. 28, 304307.[CrossRef][ISI][Medline]
Wright, J. R. (2004). Host defense functions of pulmonary surfactant. Biol. Neonate 85, 326332.[CrossRef][ISI][Medline]
Wu, R., Sato, G. H., and Whitcutt, M. J. (1986). Developing differentiated epithelial cell cultures: Airway epithelial cells. [Review] [55 refs]. Fundam. Appl. Toxicol. 6, 580590.[CrossRef][ISI][Medline]
Yang, H. M., Antonini, J. M., Barger, M. W., Butterworth, L., Roberts, B. R., Ma, J. K., Castranova, V., and Ma, J. Y. (2001). Diesel exhaust particles suppress macrophage function and slow the pulmonary clearance of Listeria monocytogenes in rats. Environ. Health Perspect. 109, 515521.[ISI][Medline]
Yin, X. J., Dong, C. C., Ma, J. Y., Antonini, J. M., Roberts, J. R., Stanley, C. F., Schafer, R., and Ma, J. K. (2004). Suppression of Cell-Mediated Immune Responses to Listeria Infection by Repeated Exposure to Diesel Exhaust Particles in Brown Norway Rats. Toxicol. Sci. 77, 263271.
Yin, X. J., Schafer, R., Ma, J. Y., Antonini, J. M., Weissman, D. D., Siegel, P. D., Barger, M. W., Roberts, J. R., and Ma, J. K. (2002). Alteration of pulmonary immunity to Listeria monocytogenes by diesel exhaust particles (DEPs). I. Effects of DEPs on early pulmonary responses. Environ. Health Perspect. 110, 11051111.[ISI][Medline]
Zhu, S., Haddad, I. Y., and Matalon, S. (1996). Nitration of surfactant protein A (SP-A) tyrosine residues results in decreased mannose binding ability. Arch. Biochem. Biophys. 333, 282290.[CrossRef][ISI][Medline]
Zhu, S., Kachel, D. L., Martin, W. J., II, and Matalon, S. (1998). Nitrated SP-A does not enhance adherence of Pneumocystis carinii to alveolar macrophages. Am. J. Physiol. 275, L1031 L1039.[ISI][Medline]
|