CIIT Centers for Health Research, 6 Davis Drive, P. O. Box 12137, Research Triangle Park, North Carolina 27709-2137
Received June 21, 2002; accepted January 7, 2003
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ABSTRACT |
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Key Words: RSV; chemokine; cytokine; ultrafine particles; alveolar macrophages; lymphocytes; mouse; ribonuclease protection assay.
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INTRODUCTION |
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While the effects of PM on animal models of asthma (Gavett et al., 1999; Lambert et al., 1999
, 2000
) have been studied, there is a paucity of research on the effects of PM on animal models of human viral infections. Respiratory syncytial virus (RSV) is an important etiological agent of acute respiratory tract infections in children (Simoes, 1999
). Elderly and otherwise immunocompromised individuals are also prone to infection with this virus (Mlinaric-Galinovic et al., 1996
). RSV is a negative-sense enveloped RNA virus that is classified as a pneumovirus within the paramyxovirus family. The virus first replicates in the epithelial cells of the upper respiratory tract and then migrates to the bronchoalveolar region where it induces inflammation in terminal bronchioles and alveoli; epithelial cell necrosis and mucus plugs lead to airway obstruction and hyperinflation of the alveoli (Shay et al., 1999
; Simoes, 1999
). Severe RSV is strongly associated with wheezing, childhood asthma, and repeated episodes of bronchospastic bronchitis, which can continue into adulthood (Kimpen, 2000
; Simoes, 2001
).
Despite the lack of in vivo studies, cell culture systems have been used to address the effects of PM on RSV infection. Becker and Soukup (1999) demonstrated that the macrophage inflammatory response to RSV and viral uptake were attenuated in the presence of PM, suggesting a mechanism for increased spread of infection and viral pneumonia in PM-exposed populations.
The purpose of the present study was to develop a mouse model of RSV infection to examine the effects of ultrafine carbon black particles on the pulmonary immune response to the virus. While chemically inert, carbon black particles have inflammogenic properties in experimental models (Li et al., 1999) and are environmentally relevant because carbonaceous material comprises approximately 50% of the mass of PM and forms the core of many ambient air particles, such as diesel exhaust (Clarke et al., 1984
). Our hypothesis is that preexposure to ultrafine particles might alter viral immune responses via the attenuation of macrophage function or damage to the airway epithelium, resulting in exacerbation of both RSV-induced pulmonary inflammation and lung function parameters. Our objectives were to examine the time course of responses during RSV infection with or without particle exposure and to determine the mediators responsible for physiologic responses. We show here that RSV infection in mice mimics human viral infection by increasing lymphocyte and neutrophil recruitment to the airways and upregulating proinflammatory cytokines and chemokines in the lung. Preexposure to ultrafine CB modulated some, but not all, of these RSV-induced responses.
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MATERIALS AND METHODS |
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Carbon black and RSV.
Ultrafine carbon black (surface area = 150 m2/g) was obtained from Columbian Chemicals Company (Marietta, GA). The particles were deemed free of endotoxin by a Limulus amebocyte lysate agglutination assay (BioWhittaker, Walkersville, MD). Direct-pelleted respiratory syncytial virus (RSV, A-2 strain) was obtained from Advanced Biotechnologies Incorporated (Columbia, MD) and had a stock concentration of 5 x 106 pfu/ml. RSV was kept at 70°C in minimum essential media with Earle's salts plus 10% FBS and 50 µl/ml gentamicin until use.
Particle and virus exposures.
Preliminary dose-response studies with 20, 40, and 80 µg carbon black were conducted to determine the dose required to produce neutrophilic inflammation at 24 h post instillation. The midrange dose of 40 µg was chosen because it induced a moderate neutrophilic inflammatory response (data not shown). Thus, at eight weeks of age, mice (1820 g) were anesthetized by isofluorane inhalation and received intratracheal (i.t.) instillations of either 40 µg carbon black in 100 µl sterile saline (carbon black solutions were sonicated for 5 min prior to use), or 100 µl sterile saline using a 1-ml syringe and round-tip needle. One day later, mice were infected by i.t. instillation of 200 µl medium containing 1 x 106 pfus RSV, or were sham-infected with 200 µl of uninfected medium. The experiment was repeated twice; end points were assessed 1, 2, 4, and 7 days (experiment 1) or 2, 4, 7, and 10 days (experiment 2) following RSV or sham instillations, for a total of 10 mice per group on days 2, 4, and 7, and 5 mice per group on days 1 and 10. To verify infection in the mice, lung homogenates were subject to an ELISA assay using antibodies to RSV (ViroStat, Portland, ME). This ELISA has been shown to correlate well with the standard plaque assays typically used to determine viral titer in lung tissue (Malley et al., 2000).
Bronchoalveolar lavage and cell differentials.
Mice were euthanized by anesthesia with 50 mg/kg Nembutal (Abbott Laboratories, Chicago, IL) and lavaged with two separate 1-ml aliquots of PBS. Approximately 1.8 ml of the 2.0-ml total volume (90%) was consistently recovered. Supernatants were collected after centrifugation (300 x g, 10 min) and stored at 80°C for cytokine protein analysis. Cell pellets were resuspended in F12 media (Gibco, Gaithersburg, MD) and counted (Coulter, Hialeah, FL). Slides were made (Cytospin 3, Shandon, Pittsburgh, PA) and stained with Diff Quik (American Scientific Inc., Sewickly, PA). Approximately 200 cells were differentiated per slide. For assessment of pulmonary inflammation, left lung lobes from day 7 were removed and inflated with Tissue-Tek OCT (Sakura Finetek U.S.A., Torrance, CA), placed on a brass chuck and embedded in OCT, snap-frozen in liquid nitrogen, and stored at 80°C until sectioned. Lungs from four mice per treatment group were sectioned at 5 µm on a cryostat, and the tissues were placed on glass slides and stained with hematoxylin and eosin for pathology. Because of the observed spike in neutrophil numbers on day 7, we exposed mice in a separate experiment to CB prior to RSV instillation exactly as described previously and extracted whole, unlavaged lungs on day 7 for further pathological analyses. Briefly, lungs (n = 3 per treatment group) were perfused and fixed with 10% buffered formalin. Tissues were embedded in paraffin, and 5-µm sections were stained with hematoxylin and eosin. All tissues were analyzed by a pathologist for the degree of pulmonary inflammation due to RSV infection, and the effect of carbon black particles on viral-induced inflammation.
Cytokine protein quantitation.
Whole lungs were homogenized in PBS supplemented with protease inhibitors (Roche Diagnostics, Manheim, Germany), and were centrifuged (500 x g, 30 min). BAL and homogenate supernatants were analyzed using commercial enzyme-linked immunosorbant assay (ELISA) kits for TNF- (BALF supernatants; Biosource International, Camarillo, CA) and IL-13 (lung homogenates; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Quantitation of interferon-gamma inducible protein 10 (IP-10) mRNA.
Right lungs were snap-frozen in liquid nitrogen and stored at 80°C until total RNA was isolated by the thiocyanate-phenol chloroform method using RNA STAT-60 (Tel-Test, Friendswood, TX). A gene-specific relative RT-PCR kit (Ambion, Austin, TX) was used for IP-10 quantitation. Briefly, total RNA (2.5 µg) was reverse transcribed at 42°C for 1 h with 2.5 mM dNTPs, 50 µM random primers, 10x RT-PCR buffer, 100 units M-MuLV, and RNase inhibitor. Specific cDNA for IP-10 was amplified by adding 5 µl RT reaction to 27.5 µl 10 x PCR buffer, 2.5 mM dNTPs, 5 µM primers, 1.4 µl DNA polymerase, 4 µl 18S PCR primer pair, and 6 µl 18S competimers and heated to 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s for 35 cycles. PCR products were run on a 2% agarose gel containing ethidium bromide and visualized under UV illumination. Band intensities of IP-10 were normalized to those of the 18s rRNA subunit using Alpha Ease densitometry software (Alpha Innotech, San Leandro, CA).
Ribonuclease protection assays.
Chemokine mRNA expression was determined by ribonuclease protection assay (RPA) using the RiboQuant kit (BD-Pharmingen, San Diego, CA) according to the manufacturer's protocol. The multiprobe template mCK-5 contains DNA templates for lymphotactin, RANTES, eotaxin, MIP-1ß, MIP-1, MIP-2, MCP-1, and TCA-3 and housekeeping genes L32 and GAPDH. Other probes used in these experiments included RNA templates for interleukins 10, 13, 15, 9, 2, 6, and IFN-
(mCK-1) and interleukins 10, 1
, 1ß, 1R
, 6, and IFN-
(mCK-2) plus L32 and GAPDH genes. Briefly, after labeling the probe with 32P dUTP and hybridizing with 10 µg total RNA, samples were loaded onto a 6% Urea-PAGE gel (Invitrogen) and identified by comparing migration distances to the unprotected probe. Band intensities were determined using ImageQuant software on a Phosphorimager SI (both by Molecular Dynamics, Sunnyvale, CA). Samples were normalized to their respective housekeeping gene, L32.
Statistical analyses.
Within each experiment, treatment groups were compared by ANOVA using statistical packages from SAS Institute, Inc. (Cary, NC), or Statview (Abacus Concepts, Berkeley, CA). If the F-test was significant, Tukey's test was used to find differences among treatments. Bonferroni correction for multiple subtesting was also used throughout the statistical analyses. A p value < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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CB pretreatment tipped the immune response away from a Th1 phenotype and toward Th2, as evidenced by an increase in the Th2 cytokine, IL-13, and decreases in TNF-, and the Th1 cytokines IP-10 and IFN
in CB-exposed RSV-infected mice. Other investigators have observed similar Th2-enhancing effects of CB preexposure in models of ovalbumin sensitization (Lovik et al., 1997
; van Zijverden et al., 2000
). In accordance with other studies (Li et al., 1999
) exposure of BALB/c mice to ultrafine CB induced a significant, acute neutrophilic pulmonary inflammation. Because neutrophils are capable of generating reactive oxygen metabolites that may damage airway epithelium (Wang and Forsyth, 2000
; Wang et al., 1998
), we initially hypothesized that exposure to ultrafine CB particles prior to RSV infection would allow for enhanced viral access and replication in the epithelium. No differences in viral replication or clearance were observed in our studies, however (data not shown).
Resistance to viral infection requires cooperation between macrophages (innate immune response) and T cells (acquired immune response), as well as certain inflammatory cytokines secreted by each cell type. TNF-, produced by activated macrophages, is an early response cytokine important for its ability to signal cells to generate additional cytokines necessary to maintain the immune response (Kunkel et al., 1999
, Luster et al., 1999
). TNF-
has antiviral activity (Mestan et al., 1986
), is a chemoattractant for T lymphocytes, and may play a role in recruiting cells for specific RSV immune responses (Franke-Ullmann et al., 1995
). We show here that CB exposure attenuates RSV-induced TNF-
production in the lung, which suggests a blunting of the innate immune response, suppression of macrophage function, and subsequent reduction of immune signaling to T cells.
Another macrophage-derived inflammatory mediator, IP-10, is a CXC chemokine that plays an important role in Th1 immunity. It is a potent activator and chemoattractant for neutrophils that interact with the CXCR3 receptor expressed on Th1 cells (Neville et al., 1997). IP-10 is also a chemoattractant for T cells, monocytes, and NK cells and participates in the innate immune response by the initiation of a Th1 polarized adaptive immune response to viral infection (Wiley et al., 2001
). In our study, IP-10 mRNA was enhanced in the lung 24 h post-RSV exposure but was significantly reduced in CB + RSV-exposed mice. Additionally, CB-exposed mice had reductions in IP-10 mRNA compared to saline-exposed controls, suggesting a shift toward a Th2 phenotype due to particle exposure alone. Our findings of more severe lung pathology (secondary bacterial infection) and increased IL-13 in CB + RSV mice on day 7 of infection support the notion that the reduction of this Th1-inducing chemokine may have promoted the exacerbation of RSV infection.
The kinetics of the recruitment of RSV-induced immune cells to the lung appeared to be altered in CB-exposed mice such that later time points were adversely affected. For example, lymphocyte numbers steadily increased throughout the time course of RSV infection, but lagged behind in CB + RSV-exposed mice, and were statistically lower on day 4 of infection. This decrease in lymphocyte numbers, presumably CD4+ T-helper 1 and CD8+ cytotoxic T cell populations resulted in reductions in both IFN- and lymphotactin mRNAs in the lung tissue on day 1 and on day 4 (data not shown). Attenuation of these Th1 T lymphocyte-derived factors could compromise an individual with respect to immune defense against pulmonary viruses (Borthwick et al., 1997
; Giancarlo et al., 1996
; Müller et al., 1995
), and may have promoted RSV disease progression. Thus, by day 7 of infection, lymphocyte numbers in CB + RSV-exposed mice increased and were comparable to those of mice infected with RSV alone. There was also a trend for an increase in neutrophil numbers in CB + RSV mice on day 7, suggesting an exacerbation of infectious disease. Pulmonary inflammation in CB + RSV mice on day 7 of our study was accompanied by increased TNF-
protein in the BALF, and elevations in mRNA expression of the cytokines IL-6 (proinflammatory), IFN-
(Th1), IL-1 receptor antagonist (IL-1Ra), and IL-2 (T cell proliferative factor). IL-1Ra production is triggered by LPS (Janson et al., 1991
) as well as viral gene products (Kline et al., 1994
).
In summary, our studies show that preexposure of BALB/c mice to ultrafine particles did not enhance RSV replication, nor significantly affect viral clearance. However, a Th2 environment may have been created in the lung by the CB-induced inflammatory milieu, promoting allergic immune responses rather than Th1 responses necessary for microbial defense.
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ACKNOWLEDGMENTS |
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NOTES |
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