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1Division of Respiratory Medicine, University of British Columbia and James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, St. Paul's Hospital, Vancouver, British Columbia, Canada; 2Division of Respiratory Disease, University of Occupational and Environmental Health, Kitakyushu, Fukuoka, Japan; and 3Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada
Submitted 1 February 2007 ; accepted in final form 2 May 2008
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ABSTRACT |
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air pollution; alveolar macrophage; systemic inflammation; interleukin-6
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METHODS |
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Animals. This study was approved by the Animal Experimentation Committee of the University of British Columbia. The acute study was based on 15 female New Zealand White rabbits with an average weight of 2.4 ± 0.2 kg (Charles River Laboratories, Montreal, QC, Canada), and the chronic study was based on 16 female New Zealand White rabbits with an average weight of 2.7 ± 0.3 kg (Covance Research Products, Denver, PA). All animals were 12 wk old at the start of the experimental protocol and were fed standard rabbit chow.
Urban air particles. The animals were exposed to urban air PM10 (EHC-93), which was collected in 1993 over Ottawa, Canada, with filters with a nominal cutoff of 0.3 µm, from a single-pass air filtration system of the Environmental Health Centre in Ottawa (100% outdoor air) (29). The particles had a mean diameter of 0.8 ± 0.4 µm (mean ± SD), with 99% (in number, not mass) of particles <3.0 µm (29).
Experimental Protocol
The rabbits were anesthetized with 7 mg/kg ketamine and 5 mg/kg xylazine by intramuscular injection assisted by 2.5% isoflurane. For the acute experiments, animals (n = 9) were exposed to PM10 (2.6 mg/kg) suspended in saline (1 ml) or saline alone (n = 6) by intrapharyngeal instillation three times (1st, 3rd, and 5th days). For the chronic experiments, animals (n = 8) were exposed to PM10 (2 mg/kg) by intrapharyngeal instillation of particles suspended in saline (1 ml), while the control animals (n = 8) were exposed only to saline (1 ml) with the same method, twice weekly for 4 wk with a model we have previously described in detail (17). The rabbits were euthanized after the final instillations with an overdose of pentobarbital sodium.
Measurement of Vascular Tone
After euthanasia, the carotid arteries were harvested and isolated from the rabbits and placed in ice-cold physiological salt solution (PSS). Without damaging the endothelium, the vessels were carefully cleaned of connective tissue, sectioned into 2-mm rings, and mounted on a wire myograph (model 610M; Danish Myo Technology, Aarhus, Denmark). Each vessel was bathed in oxygenated PSS at 37°C for an hour, during which time the resting tension was gradually increased to 10 mN with three changes of PSS at 10-min intervals followed by stabilization of the vessels at resting tension (10 mN) for 30 min. Thereafter the vessels were twice stimulated with 80 mM KCl. The relaxant responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were determined after precontraction with phenylephrine (PE, 10 µM). Once sustained contraction was evoked, cumulative concentrations of ACh (10 nM–0.1 mM) were applied to evaluate endothelium-dependent nitric oxide (NO)-mediated vasorelaxation. An identical protocol was used to study the effects of SNP (10 nM-0.1 mM) to determine endothelium-independent (NO mediated) relaxation.
Solutions and Chemicals
PSS consisted of the following (in mM): 119 NaCl, 4.7 KCl, 1.18 KH2PO4, 24 NaHCO3, 1.17 MgSO4·7H2O, 1.6 CaCl2, 5.5 glucose, and 0.026 EDTA. All reagents were purchased from Sigma (St. Louis, MO).
Histological Studies of the Lung
After euthanasia, the lungs were harvested from the rabbits. The left lung was inflated by instilling 10% phosphate-buffered formalin through the trachea at 25 cmH2O. After full inflation, the trachea was ligated and the lung was suspended in 10% phosphate-buffered formalin for 24 h. The next day, the lung was divided into four equal parts and embedded in paraffin. The right lung was inflated by instilling OCT (Tissue-Tek, Sakura Finetek, Torrance, CA) diluted 1:1 with saline through the trachea at 25 cmH2O and suspended over liquid nitrogen until frozen. These tissue samples were then stored in –80°C conditions.
Morphometric Studies of the Lung
Paraffin-embedded lung tissue was sectioned 4 µm thick, mounted on glass slides, and stained with hematoxylin and eosin (H & E). On each slide, the lung tissue was visually divided into four equal parts. Two images were then randomly taken from each section under x100 magnification. The images were captured with a spot digital camera (Microspot; Nikon, Tokyo, Japan) and were examined by a single investigator, who was blinded to the experimental condition used in the rabbit in question.
Quantitative Analysis of Lung Inflammation
Volume fractions of macrophages and neutrophils were determined with a modified stereological analysis described by Cruz-Orive and Weibel (5). The coded images were analyzed with a point-counting grid that was superimposed onto the captured images (Image Pro Plus; Media Cybernetics, Silver Spring, MD). The volume fractions of alveolar macrophages, tissue macrophages, alveolar polymorphonuclear leukocytes (PMN), and tissue PMN were calculated as the number of grid points that superimposed on the target cell divided by the total number of grid points on the screen. As for positive and activated alveolar macrophages, we counted 400 macrophages on each slide at x400 magnification and calculated the proportion of alveolar macrophages that contained particulate matter. The latter were defined as positive macrophages. Activated macrophages were defined as macrophages that stained positively with CD11b antibodies.
Immunohistochemical Staining of the Lung
Paraffin-embedded lung tissue. The tissue was sectioned 4 µm thick, and the sections were mounted on glass slides. Serial sections were cut and then stained: one section was stained with H & E, one with mouse anti-rabbit monoclonal antibody RAM11 (DAKO, Mississauga, ON, Canada) to identify macrophages, one with mouse anti-rabbit monoclonal antibody NP-5 (Abcam, Cambridge, MA) to identify PMN, and one with mouse monoclonal antibody to CD11b (clone 198; Serotec, Raleigh, NC) to identify activated macrophages. The three-step avidin-biotin complex (ABC) staining method was used for immunohistochemical labeling of the cells according to the method outlined by Hsu et al. (11). Briefly, the slides were deparaffinized and then autoclaved in Citra Antigen Retrieval Buffer, pH 6.0 (BioGenex, San Ramon, CA) for 22 min at 121°C. Sections were then treated with Protein Serum Free Block (DAKO) for 15 min. All antibodies were diluted in Tris-buffered saline (TBS), pH 7.6 with 1% bovine serum albumin, and all incubations were carried out at room temperature. Primary antibodies were used at 3, 5, and 20 µg/ml, respectively, and applied to sections for 1 h. Sections were washed with TBS and then incubated in biotinylated goat anti-mouse (Abcam) at a concentration of 20 µg/ml for 30 min. After washing, the sections were incubated in ABC-AP (Dako) for 30 min. Sections were then washed and incubated for 20 min with New Fuchsin/AS-BI Naphthol as a substrate to detect binding of the antibodies. resulting in a pink/red deposit. Sections were counterstained with Gill hematoxylin.
Frozen tissue. Frozen tissue was sectioned at 8 µm and mounted on glass slides. Sections were then fixed in a –70°C solution of methanol-acetone (1:1) for 10 min, followed by blocking with Protein Block as above. These sections were incubated with goat anti-mouse monoclonal antibody to IL-6 (AF406NA; R&D Systems, Minneapolis, MN) at a concentration of 0.1 mg/ml. Subsequent labeling was performed with ABC as previously described. For confocal microscopy, frozen sections were labeled with RAM11 (same concentration as above) and monoclonal rat CD11b (Abcam; concentration 20 µg/ml) to colocalize these two antigens. After blocking and primary antigen incubation, the sections were incubated with Alexa Fluor 594-tagged goat anti-mouse IgG and Alexa Fluor 488-tagged goat anti-rat IgG (Molecular Probes, Eugene, OR) for 30 min. Slides were then incubated in DAPI for 2 min as counterstain for the nuclei.
Blood Count and Measurements of IL-6, NO, and Endothelin
Blood samples were obtained weekly from the central ear artery before each PM10 or saline instillation. Each sample was separated into two components. One was for the blood count for white blood cells (WBC) and PMN. Another was centrifuged, and the sera were stored at –80°C for measurement of circulating IL-6, NO, and endothelin.
WBC and differential leukocyte counts were determined on an Abbott Diagnostics Cell-Dyn 3700 instrument (Abbott Park, IL) calibrated for analysis for rabbit blood cell counts.
From the serum samples, IL-6 was measured with a human IL-6 ELISA Kit (12-MKL6-1; Alpco Diagnostics, Salem, NH), NO was measured with a Total NO/Nitrite/Nitrate Assay Kit (KGE001; R&D Systems), and endothelin was measured with an Endothelin ELISA Kit (04-BI-20082; Alpco Diagnostics).
Statistical Analysis
All results are expressed as means ± SE. Data on inflammation were analyzed with SAS 9.1 and R 2.3, using t-tests, linear regression, or a mixed linear effects model as appropriate. Owing to the longitudinal nature of the serum measurements, we applied a mixed effects model for serum IL-6 analysis. Data on vascular function were analyzed with NCSS 2000 and PASS 2000 software, using analysis of variance (ANOVA) and/or repeated-measures ANOVA. Bonferroni's correction factor was applied to adjust for multiple comparisons. All results of statistical tests were considered statistically significant at P < 0.05 (2-tailed).
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RESULTS |
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Particles were observed in alveolar macrophages in both the PM10-exposed and control groups. However, there were more macrophages (P = 0.024), alveolar macrophages (P = 0.011), positive alveolar macrophages (i.e., macrophages that contained a particulate matter in the cytoplasm) (Fig. 1A; P = 0.001), and activated alveolar macrophages (i.e., macrophages that had CD11b expression) (Fig. 1, E and F; P = 0.006) in the PM10-exposed group than in the control group (see Table 1). There were fewer tissue macrophages in the PM10-exposed group than in the control group (P = 0.024). There were no significant differences in the PMN count in either the tissue or the air spaces between the two groups.
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Circulating WBC and PMN were determined over the 4 wk of the experimental protocol and are summarized in Supplemental Fig. S1.1 In the first week of the experiment relative to baseline, WBC and PMN were significantly higher in the PM10-exposed group than in the control group. However, by weeks 2, 3, and 4, no significant differences were noted. The increase in counts in the first week correlates with the number of activated alveolar macrophages (R2 = 0.254; P = 0.079) and impaired maximal vasodilatory responses in the carotid arteries to ACh (R2 = 0.259; P = 0.076).
Serum IL-6 levels over the 4 wk of the experimental protocol are summarized in Table 2. The lower limit of detection for the assay was 1.2 pg/ml. In the first 2 wk of the experiment, serum IL-6 levels were significantly higher in the PM10-exposed group than in the control group. However, by weeks 3 and 4, no significant differences were noted. There were no significant differences in those two groups in serum NO and endothelin levels across the 4 wk. The notable exception was at week 1, when serum NO levels were higher in the PM10-exposed group (see Supplemental Table S1).
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In the acute model, PM10 exposure significantly reduced ACh-stimulated relaxation (74.6 ± 16.0% vs. 48.9 ± 16.3%, control group vs. PM10 group, P < 0.05; Fig. 2A). Similarly, ACh-stimulated relaxation was also significantly reduced in the acute model (92.5 ± 3.4% vs. 69.6 ± 7.5%, control group vs. PM10 group, P < 0.05; Fig. 2C). To determine whether the impairment of ACh relaxation was due to the reduction of smooth muscle sensitivity to NO and/or to the impairment of smooth muscle function, we examined SNP-induced endothelium-independent vasorelaxation and smooth muscle contractility in response to 80 mM KCl and PE, respectively. PM10 exposure did not alter SNP-stimulated relaxation in either the acute model (81.9 ± 7.0% vs. 71.5 ± 13.8%, control vs. PM10; Fig. 2B) or the chronic model (95.2 ± 3.6% vs. 97.3 ± 2.9%, control vs. PM10; Fig. 2D) and also did not affect PE- and KCl-elicited vasocontraction (data not shown).
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Overall, there was a significant relationship between the volume fraction of positive alveolar macrophages (R2 = 0.322; P = 0.034) as well as activated alveolar macrophages (R2 = 0.318; P = 0.036) and changes in serum IL-6 levels over the 4 wk. The relationship was strongest at week 1 (Supplemental Fig. S2) and then dissipated over the ensuing weeks. There was no statistically significant correlation between lung cells and the other biomarkers (data not shown).
Association Between Circulating Biomarkers and Vascular Endothelial Dysfunction
In the acute model, there was a significant inverse relationship between IL-6 levels and maximal relaxation responses to ACh (R2 = 0.477, P = 0.0269) supported by a significant inverse relationship between changes in serum IL-6 level both at week 1 (R2 = 0.406; P = 0.014; Fig. 3) and at week 2 (R2 = 0.372; P = 0.021) to the maximal relaxation responses to ACh in the chronic model. Together these data highlight the possible importance of systemic inflammation on endothelial function persisting for a prolonged period of time. There was no statistically significant correlation between vascular endothelial dysfunction and the other biomarkers (data not shown).
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There was a significant inverse relationship between both the volume fraction of positive alveolar macrophages (R2 = 0.363, P = 0.023; Supplemental Fig. S3A) and activated alveolar macrophages (R2 = 0.389; P = 0.017; Supplemental Fig. S3B) and the maximal response to ACh.
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DISCUSSION |
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The model we have used to test our hypothesis has been established in our laboratory and is well characterized with regard to both to the local and the systemic response (17, 26). The particulate matter we used (EHC-93), ambient particles collected over Ottawa in 1993, has also been well characterized and chemically analyzed, is complex in nature, and contains >26 metals (30). Several components of particulate matter have been implicated in the adverse vascular effects of air pollution, including inorganic (e.g., sulfates and nitrates) and carbonaceous components of PM10 and metal components of PM10 (31, 32). This suggests that it is unlikely that a single component (e.g., metals such as zinc) is responsible for all its downstream biological/vascular effects. Our study was not designed to identify the key constituent(s) of PM10 responsible for the cardiopulmonary effects observed, and future studies are needed to address this critical question.
We have used an instillation method because it provides more accurate dosing, given that rabbits are nose breathers that filter most inhaled particles. With an estimated 5.9-m2 alveolar surface for a 2.5-kg rabbit, the calculated alveolar exposure was 4.3 ng/cm2 for each dose or 34.4 ng/cm2 over the entire experimental period in the chronic model and 
50% of this exposure in the acute model. This chronic exposure is similar to that of a human exposed to 150 µg/m3 for 27 days, which occurred during the Southeast Asian forest fires of 1997 (28). For the acute exposure model we elected to expose rabbits to the same dose of EHC-93 that rabbits received during the first 2 wk of the chronic model (when IL-6 levels were elevated) spread over three exposures within a week. This exposure simulates an acute episode of exposure and is similar to exposures used previously by other investigators (1, 9, 10, 27).
We have used morphometric methods to quantify lung inflammation and enumerate inflammatory cells in the lung. We showed that PM10 produced a predominant macrophage/monocytic inflammatory response in the lung (Fig. 1). We previously showed (28) that macrophages exposed to the ambient particles (EHC-93) ex vivo produced a variety of proinflammatory cytokines including tumor necrosis factor (TNF)-
, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and IL-1β in a dose-dependent fashion. We suspect that these cytokines may in turn spill into the systemic circulation, inducing a state of systemic inflammation. In the same model, we showed previously that EHC-93 exposure produces a brisk bone marrow response, causing a rapid and persistent elevation in blood leukocyte and platelet counts. The intensity of the bone marrow response correlated with the amount of particles phagocytosed by alveolar macrophages in the lung, indicating a strong link between lung and systemic inflammation. These data are consistent with findings by Fukushima and colleagues (8). Using a guinea pig model, they found that TNF levels were over two times higher in the aorta compared with the right atrium after intratracheal instillation of Escherichia coli organisms caused by a significant spillage of TNF from the lungs into the systemic circulation. An alternate explanation is that PM10 particles translocate directly from the lung compartments into the systemic circulation, inducing systemic effects. A study from Bagate and colleagues (1) reported that particulate matter (EHC-93) decreased vasorelaxation of male spontaneously hypertensive rat (SHR) aorta, with the peak effect time at 4 h after instillation, which coincides with peak blood levels of soluble components such as transition metals (zinc and vanadium). This suggests that ultrafine particulate matter or at least soluble components of particles could enter the bloodstream and influence vessel function directly. Interestingly, they also reported that pulmonary inflammation reached a maximum at 24 h, and this coincides with impairment of vasorelaxation, suggesting that different mechanisms could operate at different times after exposure. Mills and colleagues (16) demonstrated that 95.6 ± 1.7% of 99mTc-radiolabeled carbon nanoparticles inhaled by 10 healthy volunteers were detected in the lungs at 6 h after inhalation, representing <3% of aerosolized nanoparticles translocated from the lungs into the systemic circulation. There are currently no in vivo data demonstrating that translocated particles themselves impact either blood cells or vessels in vivo. We suspect that either inflammatory mediators (such as IL-6 and TNF) translocate from the lung into the bloodstream or mediators released from circulating leukocytes (such as monocytes) interacting with translocated particles impact blood vessels, resulting in vascular events.
We have elected to use IL-6 and circulating leukocytes as biomarkers of the systemic response induced by PM10 exposure. Elevated circulating levels of IL-6 have been associated with the atherosclerosis burden (14) and acute vascular events (19, 21), and IL-6 is also expressed in walls of diseased blood vessels (24, 25), implicating a role for IL-6 in the pathogenesis of atherosclerosis. We took serial blood measurements over the 4 wk of PM10 exposure and showed IL-6 and PMN response in the first 2 wk after exposure, signifying an acute systemic inflammatory response. We previously showed (28) that the alveolar macrophages are potent producers of IL-6 when exposed ex vivo to EHC-93, supported by immunohistochemical staining for IL-6 in alveolar macrophages of rabbit lungs (Fig. 1D). The local lung inflammation as well as the impaired carotid endothelial function correlated best with circulating IL-6 in the first 2 wk (both acute and chronic models). These data suggest that the vascular dysfunction persists after IL-6 levels return back to baseline, supporting the notion of a more prolonged effect of PM10 exposure on blood vessels. The mechanism(s) of this prolonged vascular effect is unclear. Circulating endothelin and NO metabolites were not elevated at the 4 wk time point (Supplemental Table S1), and we speculate that persistent lung inflammation with the production of secondary mediators could be responsible for this prolonged vascular effect. Future studies are needed to address this important and novel observation.
Abnormal ACh-induced endothelium-dependent relaxation is an early manifestation of endothelial injury (20) and signals an abnormality in NO-mediated pathways and predicts increased risk for myocardial infarction and stroke (7, 15). Salient to our findings, Saura et al. (23) found that IL-6 has direct effects on endothelial cells, inhibiting endothelial nitric oxide synthase expression by modulating signal transducer and transactivator-3. Furthermore, Schieffer and colleagues (24) also showed IL-6 sequestration in diseased coronary blood vessel walls, and we postulate that via these mechanisms PM10 exposure reduced local expression of NO in the vascular endothelium, resulting in endothelial dysfunction. The positive relationship between IL-6 levels and endothelial dysfunction supports this notion. However, further studies are needed to establish a cause-and-effect relationship between PM10-induced increases in IL-6 and endothelial dysfunction.
We previously showed (26), using a similar exposure protocol in Watanabe heritable hyperlipidemic rabbits (which develop atherosclerosis naturally), that lung inflammation (notably infiltration of positive alveolar macrophages) was associated with an increased atherosclerotic burden, increased cellular content of the atherosclerotic lesions, and thinning of atherosclerotic caps. These data in conjunction with the present findings suggest that the lung and systemic inflammatory responses to PM10 are important contributors to vascular dysfunction and atherosclerosis. Numerous epidemiologic studies have shown an increase in cardiac events within days after an increase in ambient particulate matter (12, 22), and our study extended this observation by showing a prolonged downstream vascular effect following exposure to ambient particles, supporting our data on the contribution of chronic PM10 exposure to the progression of atherosclerosis (26).
In summary, repeated exposure to PM10 caused lung and systemic inflammation as well as endothelial dysfunction. The inflammatory responses in the lung were characterized predominantly by activation and infiltration of alveolar macrophages, and the systemic inflammatory response was characterized by an increase in circulating leukocytes and IL-6. These inflammatory responses were associated with endothelial dysfunction. We conclude that exposure to ambient particulate matter has a prolonged impact on blood vessels, and we speculate that this contributes to the vascular events associated with exposure to air pollution.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The online version of this article contains supplemental material. 
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, data from control rabbits; x, data from PM10-exposed rabbits.