* School of Life Sciences, Jawaharlal Nehru University, New Delhi, India;
Analytical Services Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Center for Disease Control and Prevention, Morgantown, West Virginia 26505; and Indian Council of Medical Research, New Delhi, India
Received November 25, 2002; accepted January 23, 2003
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ABSTRACT |
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Key Words: diesel exhaust; BCG; interferon; T cells; NK cells; macrophages; nitric oxide; lung; infection.
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INTRODUCTION |
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Diesel exhaust constitutes an important component of urban air pollution and is associated with a variety of lung diseases (McClellan 1987; Sydbom et al., 2001
). Diesel exhaust particulate (DEP) contained in diesel exhaust accumulates in the lungs of people living in areas where the air is polluted with diesel exhaust. It has been reported that treatment with DEP may inhibit the macrophage function (Yang et al., 1999
, 2001
). Since M. tuberculosis resides in macrophages, and protective immunity to tuberculosis is dependent upon macrophage activation, it is important to understand if exposure to DEP may influence the course of M. tuberculosis infection. In the present study, we have examined the hypothesis that exposure to DEP may alter susceptibility to mycobacterial infections, perhaps by interfering with macrophage function and/or local immune responses in lungs.
As is the case in human M. tuberculosis infection, mice contain, but do not eliminate, pulmonary BCG infection. The BCG mouse infection model has therefore been used extensively to study the molecular and cellular basis of protective immunity to mycobacterial infection (Erb et al., 1999; Fulton et al., 2000
; Ibsen et al., 1997
; Saxena et al., 2002a
,b
; Wakeham et al., 1998
). In previous studies using this model, we found the pulmonary clearance of BCG from the mouse lung was associated with IFN
production by T cells (Saxena, 2002b
). In the present study, we have used this model to test the hypothesis of a possible relationship between DEP exposure and mycobacterial infection. In particular, we examined the effects of DEP on INF
production and the INF
-induced response of macrophages.
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MATERIALS AND METHODS |
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Reagents.
Standard DEP (reference material 1650) was obtained from the National Institute of Standards and Technology, Gaithersburg, MD. Monoclonal antibodies (Mabs) labeled with FITC or PE (anti-CD3 [Clone 17A2], anti-NK1.1 [clone PK136], anti-CD4 [clone GK1.5], anti-CD8 [(clone 536.6], anti-CD16/32 [Fc-block, clone 2.4G2], and antimouse IFN [clone XMG1.2)], and their respective isotypic controls were obtained from Pharmingen (Pharmingen/Becton Dickinson, San Diego, CA). Unless otherwise specified, all other analytical reagents and culture media were obtained from Sigma (St. Louis, MO).
BCG and IT instillation.
A seed culture of BCG (M. bovis Pasteur, TMCC # 1011) was kindly provided by Professor Ian Orme of the Microbiology Department, Colorado State University, Fort Collins, CO. BCG was grown in Middlebrook 7H9 culture medium supplemented with OADC® (oleic acid-albumin-dextrose-catalase) and 0.05% Tween 80. Viable BCG were counted by plating bacterial suspensions at different dilutions on Middlebrook 7H10 agar plates supplemented with OADC® and counting colonies after two weeks. Intrapulmonary instillation of BCG and/or DEP (50 µl bacterial suspension containing 2.5 x 104 bacteria, with or without 100 µg DEP per mouse) was carried out as previously described (Keane-Myers et al., 1998). Control mice received 50 µl of sterile 0.1-M phosphate buffered saline (pH 7.4, PBS) by the same procedure.
Processing of tissues.
Mice were sacrificed by pentobarbital overdose. Isolation of lung interstitial cells was done by enzymatic digestion of lung tissue, mechanical dispersion, and separation on a discontinuous Percoll gradient, as previously described (Saxena et al., 2002a). The BCG load in different organs was determined by plating different dilutions of tissue homogenates on Middlebrook agar 7H10 supplemented with OADC® and counting the bacterial colonies two weeks later.
Flow cytometry.
Cells derived from lungs were stained with Mabs against several membrane markers as well as against IFN, to detect cytoplasmic IFN
. Cell suspensions of 2 to 3 x 106 cells per ml were distributed 0.1 ml per well in a deep 96-well plate. Cells were washed two times with staining buffer (1% FCS and 0.1% sodium azide in PBS) and suspended in 20 µl of staining buffer containing 1 µg of anti-CD16/32 mab (Fc-Block) and incubated at room temperature for 20 min. Staining Mabs (1 µg in 20 µl staining buffer) were then added and incubation continued for an additional 30 min at room temperature in the dark. Cells were washed twice with staining buffer and fixed by adding 0.1 ml of 0.4% paraformaldehyde solution to the loosened pellets of stained cells. For cytoplasmic staining for IFN
, 0.30.5 x 106 cells in 200 µl RPMI-1640 media containing 10% fetal calf serum were cultured for 4 h with 50 ng/ml of phorbol myristic acid (PMA) and 500 ng/ml of ionomycin. Fixing/permeabalization and staining of cells for cytoplasmic IFN
and membrane markers for T or NK cells was done by using a kit and the protocol recommended by the manufacturer (Pharmingen, San Diego, CA). Cells were analyzed on a Becton Dickinson FACSCalibur flow cytometer. Cells stained with isotypic control antibodies were used to demarcate the lymphocyte window as well as to set gates for discrimination between cells, with or without specific stain.
Isolation of pure lymphoid subpopulations and real-time PCR.
Pure lung-derived lymphocyte subpopulations were isolated by negative selection following treatment of cells with appropriate monoclonal antibodies coupled with magnetic beads. Equipment, reagents, and kits for this purpose were obtained from Stem Cell Technologies, Inc., Vancouver, WA, and detailed protocols for cell separation, supplied by the manufacturer, were followed. Purity of CD4+ T cells, CD8+ T cells, and NK cells, isolated by using these kits, was above 95% in all cases as determined by flow cytometric analysis.
Purified cell preparations were stored in RNALater® (Ambion, Austin, TX) at -20°C until used for RNA isolation. Prior to preparation of total RNA, RNALater was diluted by 50% with PBS and cells (12 x 106) pelleted by centrifugation at 4000 x g for 10 min. The supernatant was removed and total RNA extracted from the cell pellet using Trizol reagent (Invitrogen, Carlsbad, CA). RNA was diluted in 20 µl DEPC-treated H2O and subjected to DNase treatment (DNA-Free kit, Ambion, Austin, TX). Reverse transcription of extracted RNA was performed using random hexamers (Taqman Reverse Transcription Reagents kit, Applied Biosystems, Foster City, CA). Conditions for the reverse transcription reaction were as follows: 10 min at 25°C, 60 min at 48°C, and 5 min at 95°C. Ten µl per reaction of the resulting cDNA was used to perform separate Taqman real-time polymerase chain reactions to amplify cDNA encoding IFN and G3PDH. Taqman primers, probes, and PCR master mix were obtained from a commercial source (Applied Biosystems, Foster City, CA). Reaction mixtures were incubated in the thermal cycler (iCycler, BioRad, Hercules, CA) for two min at 50°C and then for ten min at 95°C. Thermal cycling was conducted for 60 cycles of 15 s at 95°C and 1 min at 60°C, with measurement of fluorescence done during the 60°C step. Abundance of mRNA was determined as cycle threshold (CT), defined as the cycle at which fluorescence exceeded baseline plus 10 standard deviations. CT is inversely related to mRNA abundance; the greater the initial amount of mRNA present, the lower the CT value. Due to the exponential nature of PCR, a difference in CT value of one (1) represents a two-fold difference in mRNA abundance. Based on these principles, IFN
mRNA abundance in each sample is expressed as a percentage of G3PDH mRNA abundance according to the following formula:
Abundance of IFN message = (100) * (2-
CT), where
CT = (CTIFN) (CTG3PDH).
Nitric oxide (NO) assay.
Alveolar macrophages from normal mice were obtained by bronchoalveolar lavage and cultured in RPMI1640 culture medium supplemented with 10% fetal calf serum, 300 µg/ml glutamine, and 60 µg/ml gentamycin. NO production was measured by determining the nitrite levels in cell culture supernatants using modified Griess reagent (Sigma, catalog no. G4410). Culture supernatants were mixed 1:1 (v/v) with Griess reagent and the color generated after a 15-min incubation at room temperature was read at 540 nm.
Statistical analysis.
Two-way comparisons were performed by the Students t-test using computer software Sigmastat (SPSS, Chicago, IL). Comparisons were considered significantly different at a level of p 0.05.
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RESULTS |
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DISCUSSION |
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IFN plays a crucial role in imparting protection from mycobacterial infection (Flynn et al., 1993
). IFN
gene-knockout mice infected with M. tuberculosis have disseminated disease and succumbed to the infection (Cooper et al., 1993
). We have previously shown that in mice T cells as well as NK cells are important sources of IFN
in lung interstitium (Saxena et al., 2002b
). After initiation of BCG lung infection in mice, IFN
-producing cells accumulate in lung interstitium. Bacterial load, as well as the number of IFN
-producing cells, attain maxima five weeks post-infection and subside thereafter (Saxena et al., 2002b
). Both T cells and NK cells contribute to the IFN
response in BCG-infected lungs (Saxena et al., 2002b
). Results of the present study indicate that (1) the accumulation of T and NK cells in BCG-infected lungs was not inhibited in DEP-exposed mice, (2) expression levels of IFN
mRNA in pulmonary NK cells, CD4 T cells, and CD8 T cells isolated after BCG infection were not affected by coexposure to DEP, and (3) accumulation of IFN
-producing lymphocytes in the lungs after BCG infection was not decreased by coexposure to DEP. Taken together, these results suggest that the development of local IFN
responses in the lungs of BCG-infected mice was not reduced as a result of exposure to DEP. A defective IFN
response is therefore unlikely to be the cause of impaired BCG clearance from DEP-exposed mice.
IFN activates macrophages to kill or stop the intracellular growth of mycobacteria. NO generated in macrophages in response to IFN
signal is a crucial mediator of antibacterial action of activated macrophages (Ehrt et al., 2001
; Xing et al., 2001
). Even if IFN
response was not altered as a result of DEP exposure, an alternate cause for impaired BCG clearance could be that the pulmonary macrophages from DEP-treated mice could not properly respond to the activation signal of IFN
. This possibility was examined by assessing IFN
-induced NO release by control and DEP-treated mouse alveolar macrophages. Our results indicated that DEP-exposed alveolar macrophages released significantly lower amounts of NO in response to IFN
. This result is compatible with the hypothesis that DEP exposure impairs BCG clearance, at least in part by impairing the ability of pulmonary macrophages to mount response critical to host defense, such as NO production after stimulation with IFN
. DEP particles have a variety of adsorbed bioactive organic molecules, some of which are known to have an inhibitory effect on macrophage function (Yang et al., 1999
, 2001
). It has been proposed that a key mechanism of action for these toxicants is induction of intracellular oxidative stress (Al-Humadi et al., 2002
; Whitekus et al., 2002
). Although only NO production was evaluated in this study, a variety of other macrophage functions relevant to host defense might also be affected by DEP exposure and contribute to the enhanced in vivo load of BCG infection in the mouse model. It should also be noted that DEP from a single source was used throughout these studies, and that DEP samples from other sources may have different toxicities or potencies.
In conclusion, DEP exposure appears to impair clearance of murine experimental pulmonary BCG infection. Impaired clearance is not the result of impairment in pulmonary IFN-producing lymphocytes or T-cell responses. Rather it appears to result, at least in part, from impaired ability of pulmonary macrophages to respond to IFN
and engage in functions critical to host defense, such as production of NO.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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