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Statins Reduce Ambient Particulate Matter-Induced Lung Inflammation by Promoting the Clearance of Particulate Matter < 10 μm From Lung Tissues
CHEST
Original Research OCCUPATIONAL AND ENVIRONMENTAL LUNG DISEASES
Statins Reduce Ambient Particulate Matter-Induced Lung Inflammation by Promoting the Clearance of Particulate Matter , 10 mm From Lung Tissues Ryohei Miyata, MD, PhD; Ni Bai, PhD; Renaud Vincent, PhD; Don D. Sin, MD, MPH, FCCP; and Stephan F. Van Eeden, MD, PhD
Background: The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) suppress ambient particulate matter , 10 mm (PM10)-induced inflammatory response in vitro. The aim of this study was to determine the effect of statins on PM10-induced lung inflammation in vivo. Methods: New Zealand white rabbits were exposed to either PM10 (1.0 mg/kg) or saline by direct intratracheal instillation three times a week for 4 weeks ⫾ lovastatin 5.0 mg/kg/d. BAL fluid was assessed for cell counts and proinflammatory cytokine levels. Lung inflammation was quantified using immunohistochemical techniques and morphometric methods. Ex vivo phagocytosis assay of alveolar macrophages using PM10 particles was performed. Distribution of PM10 particles in lung tissues and draining lymph nodes was quantified morphometrically to evaluate the clearance of PM10 particles. Results: PM10 exposure increased the production of IL-6 and IL-8, promoted the recruitment of macrophages and polymorphonuclear leukocytes into the lung, and activated these recruited leukocytes. Lovastatin significantly suppressed all these effects. Lovastatin increased the phagocytic activity of macrophages and promoted the migration of PM10-laden macrophages to the regional lymph nodes. Conclusions: Lovastatin attenuates the PM10-induced recruitment and activation of alveolar macrophages and polymorphonuclear leukocytes, reduces local proinflammatory cytokine production, and promotes the clearance of PM10 particles from lung tissues to regional lymph nodes. These novel pleiotropic properties of statins are most likely to contribute to the downregulation CHEST 2013; 143(2):452–460 of PM10-induced lung inflammation. Abbreviations: BALF 5 BAL fluid; NF-kB 5 nuclear factor kB; PM 5 particulate matter; PM10 5 particulate matter , 10 mm; PMN 5 polymorphonuclear leukocyte
adverse health effects of ambient particulate Thematter (PM) exposure on respiratory health have
been well documented.1 Exposure to PM increases the risk of daily exacerbations of asthma and COPD.1,2 Exposure to PM also doubles the risk of pneumonia and, thus, is responsible for . 800,000 deaths from pneumonia annually in children aged , 5 years.3 Therefore, attenuating the lung inflammatory response induced by PM could significantly help to reduce the morbidity and mortality from these deleterious respiratory conditions. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) exhibit their primary pharmacologic
action by inhibiting the conversion of 3-hydroxy3-methylglutaryl coenzyme A to l-mevalonate. In addition to reducing cholesterol levels through the sterol metabolism pathway, they also inhibit geranylgeranylpyrophosphate through the nonsterol metabolism pathway. Isoprenylation of geranylgeranyl-pyrophosphate is required for the activation of small guanosine triphosphatases, including RhoA and Rac1 (Rho-related C3 botulinum toxin substrate). Statins decrease the availability of RhoA and Rac1, which subsequently leads to the deactivation of nuclear factor kB (NF-kB) activity,4 allowing statins to possess significant antiinflammatory effects. Other pleiotropic effects of statins include
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the regulation of oxidative stress and phagocytic activity.5,6 These effects may contribute to a reduction in lung inflammatory responses induced by PM exposure. Alveolar macrophages and lung epithelial cells are primarily responsible for processing inhaled ambient particles.7 Studies from our laboratory showed that statins suppress PM-stimulated proinflammatory cytokine production by both alveolar macrophages and bronchial epithelial cells in vitro.8 In addition, we previously showed in a rabbit model that long-term exposure to PM , 10 mm (PM10), EHC-93, induces a lung inflammatory response.9-11 Therefore, the current study was designed to determine the effect of statins on the lung inflammatory response induced by long-term exposure to PM10, with the hypothesis that statins will attenuate the degree of PM10-induced lung inflammation. Specifically, we quantified the effect of statins on leukocyte recruitment into lung tissues, activation of the recruited leukocytes, and the production of proinflammatory cytokines. We also evaluated the effects of statins on the clearance of PM10 particles from the lung. Materials and Methods
diet and water ad libitum. The protocol was approved by the Animal Experimentation Committee of The University of British Columbia (A09-0071). Experimental Protocol The animals were assigned randomly to four experimental groups (each group, n 5 6) as follows: (1) saline instillation, (2) saline instillation with lovastatin treatment, (3) PM10 instillation, and (4) PM10 instillation with lovastatin treatment. Either PM10 or saline was administrated by a direct intratracheal instillation method three times a week for 4 weeks. Lovastatin (Toronto Research Chemicals) 5 mg/kg/d was given orally for 8 weeks, beginning 4 weeks before the PM10 or saline exposure. Four hours after the final (12th) exposure, the rabbits were killed with an overdose of pentobarbital sodium. A detailed description of the protocol can be found in e-Appendix 1. Histologic Studies of the Lung and Mediastinal Lymph Nodes Lungs and mediastinal lymph nodes were harvested and processed for histologic quantification. See e-Appendix 1 for more details. BAL Alveolar cells were harvested by BAL, fixed with methanol, and underwent Wright-Giemsa staining for cell differential counts as detailed in e-Appendix 1. Differential counts were determined by morphologic criteria under oil immersion light microscopy with evaluation of 200 cells/slide in randomly selected fields of view.
Urban Air Particulates
Immunohistochemistry
Urban air PM10 (EHC-93) was obtained from the Environmental Health Directorate, Health Canada. The detailed characteristics of EHC-93, including particle preparation and chemical composition, have been described elsewhere12 as well as in e-Appendix 1.
Immunohistochemical techniques were used to identify specific leukocytes in lung tissues and the distribution of macrophages in mediastinal lymph nodes. Macrophages were identified by RAM11 (monoclonal mouse antirabbit macrophage-specific primary antibody) (M0633; Dako Canada Inc), polymorphonuclear leukocytes (PMNs) by monoclonal mouse antirabbit defensin 5 (LS-C50934; LifeSpan BioSciences, Inc), and activated leukocytes by monoclonal mouse antirabbit CD11b (MCA802; AbD Serotec a Division of MorphoSys) as detailed in e-Appendix 1.
Experimental Animals Female New Zealand white rabbits (n 5 24; 12 weeks old; weight, 2.6 ⫾ 0.1 kg) (Charles River Laboratories International, Inc) were used in this study. They were housed in a clean-air and viral-free room with restricted access and given a standard rabbit Manuscript received May 16, 2012; revision accepted July 1, 2012. Affiliations: From the UBC James Hogg Research Centre (Drs Miyata, Bai, Sin, and Van Eeden), Institute for Heart 1 Lung Health, St. Paul’s Hospital, The University of British Columbia, Vancouver, BC; Department of Anesthesiology, Pharmacology and Therapeutics (Dr Bai), The University of British Columbia, Vancouver, BC; and Environmental Health, Science and Research Bureau (Dr Vincent), Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, ON, Canada. Funding/Support: This study was supported by operating grants from the Heart and Stroke Foundation of Canada, Michael Smith Foundation for Health Research, and the Canadian Institutes of Health Research. Correspondence to: Stephan F. Van Eeden, MD, PhD, UBC James Hogg Research Centre, Institute for Heart 1 Lung Health, St. Paul’s Hospital, The University of British Columbia, Room 166, 1081 Burrard St, Vancouver, BC V6Z 1Y6, Canada; e-mail: [email protected] © 2013 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.12-1237
Quantitative Analysis of Lung Inflammation The degree of lung inflammation was quantified by standard morphometric techniques. See e-Appendix 1 for more details. Cytokine Analyses IL-6 and IL-8 levels in the BAL fluid (BALF) were analyzed by enzyme-linked immunosorbent assay using commercially available kits (CSB-E06903RB and CSB-E06905RB; Cusabio Biotech Co, Ltd) according to manufacturer instructions. Each sample was measured in duplicate. Distribution of PM10 in the Lung The burden of PM10 both in lung tissues and in mediastinal lymph nodes was quantified. See e-Appendix 1 for more details. Ex Vivo Phagocytosis Assay of Alveolar Macrophages Phagocytic ability of alveolar macrophages for fluorescent microspheres or PM10 particles was evaluated either in suspension or in adherent cell culture by flow cytometry or microscopic quantification, respectively. See e-Appendix 1 for more details.
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Statistics Values are expressed as mean ⫾ SEM. Data for the four experimental groups were compared by two-way factorial analysis of variance, and those for two groups were compared by Student t test. Statistical significance was considered at P , .05 (e-Appendix 1).
Results Leukocyte Counts in BALF The total leukocyte count in BALF was increased by PM10 exposure (P , .001) (Fig 1), which was suppressed by lovastatin (P , .01). This increase in leukocytes was predominantly due to a marked influx of PMNs and band cells (P , .001) (Table 1), both of which were strongly reduced by lovastatin (P , .001), whereas lovastatin showed no suppressive effect on the macrophage count. Proinflammatory Cytokines in BALF PM10 exposure increased both IL-6 and IL-8 levels in BALF, the former to a lesser extent than the latter (IL-6, P , .05 vs saline; IL-8, P , .001 vs saline) (Fig 2). Lovastatin reduced both IL-6 and IL-8 levels. The lovastatin effect was more evident in IL-8 levels (IL-6, P , .05 vs PM10; IL-8, P , .001 vs PM10) (Fig 2). Leukocyte Recruitment Into Lung Tissue PM10 exposure increased the volume fraction of alveolar macrophages in the airspaces as well as in the lung parenchymal tissues, and lovastatin suppressed these recruitments, an effect also seen in the lovastatintreated saline-exposed groups (Fig 3A). The majority of PMNs were in the parenchymal tissues, and their recruitment was attenuated by lovastatin (Fig 3B). PM10 exposure strongly increased CD11b expression on leukocytes (alveolar macrophages and PMNs) in the airspaces and in the parenchyma (Fig 3C). In the airspaces of the control animals, alveolar macrophages were the predominant leukocytes expressing CD11b (macrophages, 75.3% ⫾ 4.0%; PMNs, 24.7% ⫾ 4.0%), and this predominance was reduced with PM10 exposure (macrophages, 62.4%⫾2.1%; PMNs, 37.6%⫾2.1%), suggesting that PM10 exposure preferentially activates PMNs in the airspaces. In the lung parenchyma, however, the ratio of macrophages to PMNs expressing CD11b was approximately one to one, and the ratio did not change with PM10 exposure. Lovastatin suppressed CD11b expressions in both the airspaces and the parenchymal tissues, but the lovastatin effect was less in the parenchyma. Reduction in the Number of PM10 Particles in the Lung by Lovastatin PM10 particles were present throughout the lung and were predominantly found in the cytoplasm of
alveolar macrophages. The proportion of alveolar macrophages with PM10 particles in their cytoplasm was significantly higher in the PM10 groups than in the saline groups (Fig 4A). Lovastatin significantly reduced this fraction of alveolar macrophages with PM10. Quantitatively, the total lung burden of PM10 particles in all lung tissues was significantly reduced by lovastatin (Fig 4B). Clearance of PM10 Particles Into Mediastinal Lymphatic Tissues Mediastinal lymph nodes were stained with RAM11 to evaluate the distribution of macrophages and with Hansel stain to quantify the number of PM10 particles that migrated from the lung (Fig 5A). PM10 exposure strongly promoted the migration of macrophages to lymphatic tissues (Fig 5A), and morphometric analysis confirmed that PM10 exposure significantly increased the volume fraction of macrophages in the mediastinal lymph nodes (Fig 5B). Although not statistically significant, lovastatin further increased this migration (Fig 5B); however, lovastatin significantly increased the number of PM10 particles in lymph nodes compared with PM10 exposure alone (Fig 5C). Effect of Lovastatin on Alveolar Macrophage Phagocytic Activity To determine the effect of lovastatin on the phagocytic activity of alveolar macrophages, rabbits were pretreated with or without lovastatin 5 mg/kg/d for 4 weeks, and alveolar macrophages were collected for ex vivo phagocytosis assay using fluorescent microspheres
Figure 1. Cellular profile of BALF. Four hours after the final (12th) instillation, rabbits were killed, and left lung lavage was performed. PM10 exposure increased the total cell count, and lovastatin reversed this effect. In contrast, lovastatin had no suppressive effect on alveolar macrophages, which were significantly increased by PM10 exposure. PM10 also strongly stimulated the recruitment of PMNs, and lovastatin inhibited this recruitment. ***P , .001 vs saline; ##P , .01 vs PM10; ###P , .001 vs PM10. BALF 5 BAL fluid; PM10 5 particulate matter , 10 mm; PMN 5 polymorphonuclear leukocyte.
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Data are presented as mean ⫾ SEM. Cell counts are expressed in 3 103/mL. PM10 5 particulate matter , 10 mm; PMN 5 polymorphonuclear leukocyte. P , .05 vs saline. bP , .001 vs saline. cP , .01 vs PM . 10 dP , .001 vs PM . 10 a
1.2 ⫾ 0.2 (0.96 ⫾ 0.17) 0.1 ⫾ 0.1a (0.06 ⫾ 0.06) 4.2 ⫾ 1.2b (0.76 ⫾ 0.21) 0.2 ⫾ 0.2d (0.07 ⫾ 0.06) 4.0 ⫾ 0.5 (3.3 ⫾ 0.4) 0.4 ⫾ 0.2 (0.3 ⫾ 0.2) 32.0 ⫾ 2.0b (5.8 ⫾ 0.4) 1.5 ⫾ 0.6d (0.5 ⫾ 0.2) 3.2 ⫾ 0.6 (2.6 ⫾ 0.5) 0.4 ⫾ 0.2 (0.3 ⫾ 0.2) 30.3 ⫾ 3.8b (5.5 ⫾ 0.7) 1.8 ⫾ 1.0d (0.6 ⫾ 0.3) 82 ⫾ 6 (68 ⫾ 5) 111 ⫾ 2 (92 ⫾ 1) 325 ⫾ 30b (59 ⫾ 5) 262 ⫾ 9 (83 ⫾ 3) 121 ⫾ 13 121 ⫾ 20 553 ⫾ 32b 315 ⫾ 36c Saline Saline 1 lovastatin PM10 PM10 1 lovastatin
34 ⫾ 6 (28 ⫾ 5) 9 ⫾ 2 (8 ⫾ 1) 191 ⫾ 30b (35 ⫾ 5) 51 ⫾ 9d (16 ⫾ 3)
31 ⫾ 5 (26 ⫾ 4) 9 ⫾ 2 (7 ⫾ 1) 161 ⫾ 28b (29 ⫾ 5) 49 ⫾ 8d (16 ⫾ 3)
Eosinophils (%) Lymphocytes (%) Segmented Cells (%) Band Cells (%) PMNs (%) Macrophages (%) Total Cells Group
Table 1—Cellular Profile of BAL Fluid
Figure 2. IL-6 and IL-8 levels in BAL fluid collected from New Zealand white rabbits exposed to PM10 ⫾ lovastatin. The BAL fluid was analyzed by enzyme-linked immunosorbent assay. PM10 exposure increased both IL-6 and IL-8 levels, the former to a lesser extent than the latter. Lovastatin suppressed both IL-6 and IL-8 levels, with a more pronounced effect on IL-8 levels. *P , .05 vs saline; ***P , .001 vs saline; #P , .05 vs PM10; ###P , .001 vs PM10. See Figure 1 for expansion of abbreviation.
assessed by flow cytometry. Lovastatin strongly enhanced ex vivo uptake of both opsonized and unopsonized microspheres (Fig 6A). To determine the effect of lovastatin on the phagocytic activity of alveolar macrophages for PM10 particles, we pretreated rabbits with lovastatin as follows: (1) 5 mg/kg/d for 4 weeks, (2) 5 mg/kg/d for 2 days, or (3) 50 mM in vitro for 1 h. We then determined the macrophage uptake of particles using microscopic quantification (Fig 6B). Lovastatin significantly enhanced the phagocytic activity of alveolar macrophages for PM10 particles (Fig 6C) with prolonged (4 weeks) lovastatin pretreatment but not with short-term (2 days) or in vitro pretreatment. Discussion In the present study, we showed that statins have potent suppressive effects against air pollution-induced lung inflammation as evidenced by a reduction in the recruitment and activation of alveolar macrophages and PMNs in the lung. Statins also reduce the levels of IL-6 and IL-8 in the lung. Moreover, statins promote the clearance of particles from the lung by enhancing the phagocytic activity of alveolar macrophages for PM10 particles and promoting the lymphatic removal of PM10-containing macrophages to the regional lymph nodes. Collectively, these data suggest that PM10induced lung inflammation can be attenuated by statins through their ability to regulate (1) the recruitment and activation of leukocytes, (2) proinflammatory cytokine production, and (3) the clearance of PM10 particles. These novel effects of statins on the regulation of lung inflammation could broaden their application for other inflammatory lung conditions.
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Figure 3. Volume fraction of specific leukocytes in the lung exposed to PM10 ⫾ lovastatin. A, Volume fraction of alveolar macrophages (RAM11 [monoclonal mouse antirabbit macrophage-specific primary antibody]-positive cells). PM10 exposure promoted the macrophage recruitment, and lovastatin suppressed the recruitment at both locations. Lovastatin also suppressed the volume fraction of alveolar macrophages in the saline-exposed group at both locations. B, Volume fraction of PMNs (defensin 5-positive cells). PMNs were preferentially located at parenchymal tissues. PM10 exposure strongly enhanced the recruitment of PMNs into the lung, whereas lovastatin inhibited this recruitment. C, Volume fraction of activated leukocytes (CD11b-positive cells). PM10 exposure significantly activated the airspace and parenchymal leukocytes. The lovastatin effect was less in the parenchyma. *P , .05 vs saline; **P , .01 vs saline; ***P , .001 vs saline; ##P , .01 vs PM ; ###P , .001 vs PM . See Figure 1 legend for expansion of abbreviations. 10 10
We have previously shown that exposure to PM10 elicits a brisk pulmonary inflammatory response characterized by an increase in the recruitment and activation of alveolar macrophages, PM10-containing alveolar macrophages, and circulating IL-6 levels,11 which is in keeping with the findings of the present study. Our group also showed that statins suppress PM10-induced cytokine production, including IL-6 and IL-8, by human alveolar macrophages and bronchial epithelial cells in in vitro settings.8 The new results further add to the understanding of the antiinflammatory nature of statins by showing that they reduce PM10-induced proinflammatory cytokines in in vivo settings. Although the growing literature indicates the antiinflammatory properties of statins,5 the antiinflammatory properties against lung inflammation, particularly PM-induced lung inflammation, is poorly studied. However, it is established that statins are capable of reducing reactive oxygen species production,6 inactivating nicotinamide
adenine dinucleotide phosphate-oxidase,13 and inhibiting NF-kB,14 all of which are likely to participate in the downregulation of PM-induced proinflammatory cytokine production as demonstrated in Figure 2. To our knowledge, no studies to date have addressed whether statins modulate the clearance of PM, including cigarette smoke. We speculate that their ability to downregulate NF-kB contributes, in part, to their well-established antiinflammatory properties. More interestingly, we showed herein that statins promote the clearance of macrophages to the regional lymph nodes. This concept provides a new insight into the underlying mechanisms for their antiinflammatory properties. The existing body of evidence is consistent and plausible enough to confirm the link between PM air pollution and cardiovascular diseases.11,15-19 There exist three proposed molecular mechanisms by which PM increases the risk of cardiovascular events: (1) direct
Figure 4. Quantification of PM10 particle distribution in the lung exposed to PM10 ⫾ lovastatin. A, Percentages of alveolar macrophages containing PM10 particles were assessed using the lung tissue specimens. Lovastatin significantly reduced the fraction of PM10-containing alveolar macrophages. B, Comparison of total lung burden of PM10. Lovastatin also significantly reduced the total lung burden of PM10. ***P , .001 vs saline; ###P , .001 vs PM10. See Figure 1 legend for expansion of abbreviation. 456
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Figure 5. A, Microphotographs of the distribution of macrophages (RAM11 [monoclonal mouse antirabbit macrophage-specific primary antibody]-positive cells) and PM10 particles in the mediastinal lymph nodes (RAM11 and Hansel stains, original magnification 3 320). Resident macrophages were stained positive in the saline-exposed animals. Most lymphatic vessels were packed with a cluster of macrophages in the PM10-exposed animals, particularly in the PM10 1 lovastatin group, indicating the migration of macrophages from the lung. The migrated macrophages were spread out throughout the lymph follicles. PM10 exposure strongly promoted the deposition of particulate matter in the mediastinal lymph nodes. Most particulate matter was found inside tingible body macrophages in germinal centers. Lovastatin further increased the deposition of particles. The bars represent 20 mm. B, Volume fraction of macrophages (RAM11-positive cells) in the mediastinal lymph nodes. PM10 exposure significantly increased the volume fraction of macrophages in the mediastinal lymph nodes, but statistically, lovastatin showed no additional effect to increase the fraction of macrophages. *P , .05 vs saline; **P , .01 vs saline. C, Volume fraction of PM10 particles in the mediastinal lymph nodes. PM10 exposure increased the volume fraction of PM10 particles deposited in the lymph nodes. The deposition was further increased by lovastatin. *P , .05 vs saline; ###P , .001 vs PM10. N.S. 5 not significant. See Figure 1 legend for expansion of other abbreviation.
penetration of PM into the blood vessels; (2) translocation of inflammatory mediators from the lung to the systemic circulation; and (3) activation of the lung autonomic nervous system.15 Although the exact mechanism is not yet fully characterized, the systemic spillover of lung inflammation hypothesis has the most experimental support.2,20 To support this notion, our group showed that PM-induced lung inflammatory mediators (eg, IL-6) translocate to the systemic circulation,11,18 directly implicating lung inflammation as a key driver for the downstream adverse vascular effects of PM exposure. The potent antiinflammatory effects of statins on PM-induced lung inflammation demonstrated in the current study are critically important in terms of counteracting the adverse vascular
effects. This finding could also have implications for other inflammatory lung conditions, such as COPD, which is known to be strongly associated with cardiovascular diseases.21 The effect of statins on leukocyte phagocytic function is controversial. Statins have been reported to inhibit phagocytosis of IgG-opsonized bacteria.22 In contrast, other researchers reported that statins enhance phagocytosis of latex beads.23 These discrepancies may be due to phagocytosis pathway differences (Fc dependent or Fc independent). We showed that lovastatin significantly enhances phagocytosis of PM10 particles if given for a long period, whereas short-term or in vitro lovastatin treatment has no positive effect on their phagocytic function (Fig 6C). These data suggest the
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Figure 6. A, Ex vivo phagocytosis assay of fluorescent microspheres. New Zealand white rabbits were pretreated identically (lovastatin 5 mg/kg/d for 4 wk). Alveolar macrophages were collected and incubated with fluorescent microspheres for 2 h. Lovastatin strongly enhanced ex vivo uptake of both opsonized and unopsonized microspheres. ***P , .001 vs control subjects. B, Microphotographs of ex vivo phagocytosis assay of PM10 particles (Diff-Quick stain, original magnification 3 600). The bars represent 10 mm. C, Ex vivo phagocytosis assay of PM10 particles. New Zealand white rabbits were pretreated with lovastatin as follows: (1) 5 mg/kg/d for 4 wk (n 5 4), (2) 5 mg/kg/d for 2 d (n 5 4), or (3) 50 mM in vitro for 1 h (n 5 4). Alveolar macrophages were collected and incubated with PM10 particles for 2 h. Long-term (4-wk) lovastatin pretreatment strongly enhanced ex vivo uptake of PM10 particles but not short-term (2-d) or in vitro lovastatin pretreatment. **P , .01 vs control subjects. See Figure 1 legend for expansion of abbreviation.
importance of structural change in lipid bilayer membrane composition of phagocytes after a long-term lovastatin treatment, which is supported by studies where a reduction of intracellular cholesterol by pravastatin diminished membrane fluidity, resulting in the dysregulation of neutrophil phagocytosis function.24 Once having ingested foreign materials, macrophages do not remain in the airspaces but emigrate to the draining lymph nodes.25 This concept was supported by the present study, which showed an increase in PM10 particles in lymphatic tissues in rabbits exposed to PM10. Lovastatin decreases the number of PM10 particles in lung tissues (Fig 4) and increases the number in lymphatic tissues (Fig 5), suggesting that it promotes the emigration of PM10-laden macrophages to the regional lymph nodes. These findings were supported by our in vitro studies showing that lovastatin promotes the phagocytosis of PM10 particles (Fig 6C). The relatively poor clearance of PM-laden macrophages in the PM10-exposed group (Fig 4B) could contribute to the brisk influx and activation of PMNs in the lung (Fig 3B), thereby amplifying the local inflammatory responses. These data suggest that the enhancement of phagocytosis by lovastatin facilitates alveolar macrophages to phagocytose PM10 particles in the airspaces
more effectively and promotes the emigration of the PM-laden macrophages to draining lymph nodes. Collectively, the enhancement of particle clearance from the lung could contribute to the antiinflammatory properties of statins in the lung. Limitations of the present study must be noted. First, we have used the direct intratracheal instillation method because it provides an accurate dosing and lung deposition of particles in rabbits, which are predominantly nose breathers capable of efficiently filtering, capturing, and clearing most inhaled particles in the nasal cavity. We have also improved on our previous instillation method10 by using a microsprayer, which enabled more homogeneous distribution of PM across the entire lung surface. The dose of PM10 that the rabbits were exposed to for the 4-week experimental period is similar to the calculated human exposure while living in a polluted city (150 mm3) for 3 weeks.26 Nevertheless, extrapolation of the data from the intratracheal instillation method should be validated in inhalation models. Second, CD11b expression was used to assess the activation of leukocytes because CD11b molecules can be upregulated in response to PM.27 We showed that the expression of CD11b on leukocytes was increased by PM10 exposure,
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and lovastatin abolished this increase (Fig 3C), suggesting that CD11b is a good marker to determine the antiinflammatory effect of statins. However, CD11b is ubiquitously expressed on many different leukocytes, including monocytes, granulocytes, macrophages, and natural killer cells, and is not specific for activation of alveolar macrophages or PMNs. In conclusion, repeated instillations of PM10 for 4 weeks increase the recruitment of PMNs and alveolar macrophages into the lung, activate these leukocytes, and induce the local production of IL-6 and IL-8. Lovastatin treatment suppresses all these proinflammatory effects. Lovastatin also promotes the clearance of particles to regional lymphatic tissues. These findings suggest that statins can attenuate not only lung inflammation induced by PM10 exposure but also, potentially, the downstream adverse cardiovascular effects. Acknowledgments Author contributions: Dr Van Eeden had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Dr Miyata: contributed to the data collection and analysis, writing of the manuscript, and approval of the final manuscript. Dr Bai: contributed to the data collection and analysis and approval of the final manuscript. Dr Vincent: contributed to the provision of the study material, data analysis, manuscript review, and approval of the final manuscript. Dr Sin: contributed to the study conception and design, provision of the study material, financial and administrative support, revision of the manuscript, and approval of the final manuscript. Dr Van Eeden: contributed to the study conception and design, provision of study material, financial and administrative support, revision of the manuscript, and approval of the final manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Dr Sin has received grant funding from AstraZeneca and has served on advisory boards of Merck Canada Inc, Novartis Pharmaceuticals Corporation, and Takeda Pharmaceuticals International GmbH. Drs Miyata, Bai, Vincent, and Van Eeden have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript. Other contributions: We thank Beth Whalen, PhD, for performing the flow cytometry analysis. Dr Miyata is a recipient of a Canadian Institutes of Health Research Integrated and Mentored Pulmonary and Cardiovascular Training Strategic training postdoctoral fellowship. Dr Van Eeden is an American Lung Association Career Investigator, the recipient of the William Thurlbeck Distinguish Research Award, Michael Smith Foundation Senior Investigator Award, and the current GlaxoSmithKline plc/Canadian Institutes of Health Research professor in COPD. Additional information: The e-Appendix can be found in the ”Supplemental Materials” area of the online article.
References 1. Perez L, Rapp R, Künzli N. The Year of the Lung: outdoor air pollution and lung health. Swiss Med Wkly. 2010;140: w13129. 2. Hogg JC, van Eeden S. Pulmonary and systemic response to atmospheric pollution. Respirology. 2009;14(3):336-346.
3. World Health Organization. Indoor Air Pollution and Health (Fact sheet no. 292). Geneva, Switzerland: World Health Organization; 2005. 4. Delerive P, Gervois P, Fruchart JC, Staels B. Induction of IkBa expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor-alpha activators. J Biol Chem. 2000;275(47):36703-36707. 5. Jasińska M, Owczarek J, Orszulak-Michalak D. Statins: a new insight into their mechanisms of action and consequent pleiotropic effects. Pharmacol Rep. 2007;59(5):483-499. 6. Adam O, Laufs U. Antioxidative effects of statins. Arch Toxicol. 2008;82(12):885-892. 7. Miyata R, van Eeden SF. The innate and adaptive immune response induced by alveolar macrophages exposed to ambient particulate matter. Toxicol Appl Pharmacol. 2011;257(2): 209-226. 8. Sakamoto N, Hayashi S, Mukae H, Vincent R, Hogg JC, van Eeden SF. Effect of atorvastatin on PM10-induced cytokine production by human alveolar macrophages and bronchial epithelial cells. Int J Toxicol. 2009;28(1):17-23. 9. Mukae H, Hogg JC, English D, Vincent R, van Eeden SF. Phagocytosis of particulate air pollutants by human alveolar macrophages stimulates the bone marrow. Am J Physiol Lung Cell Mol Physiol. 2000;279(5):L924-L931. 10. Mukae H, Vincent R, Quinlan K, et al. The effect of repeated exposure to particulate air pollution (PM10) on the bone marrow. Am J Respir Crit Care Med. 2001;163(1):201-209. 11. Tamagawa E, Bai N, Morimoto K, et al. Particulate matter exposure induces persistent lung inflammation and endothelial dysfunction. Am J Physiol Lung Cell Mol Physiol. 2008; 295(1):L79-L85. 12. Vincent R, Bjarnason SG, Adamson IY, et al. Acute pulmonary toxicity of urban particulate matter and ozone. Am J Pathol. 1997;151(6):1563-1570. 13. Wassmann S, Laufs U, Bäumer AT, et al. HMG-CoA reductase inhibitors improve endothelial dysfunction in normocholesterolemic hypertension via reduced production of reactive oxygen species. Hypertension. 2001;37(6):1450-1457. 14. Ortego M, Bustos C, Hernández-Presa MA, et al. Atorvastatin reduces NF-kappaB activation and chemokine expression in vascular smooth muscle cells and mononuclear cells. Atherosclerosis. 1999;147(2):253-261. 15. Brook RD, Franklin B, Cascio W, et al; Expert Panel on Population and Prevention Science of the American Heart Association. Air pollution and cardiovascular disease: a statement for healthcare professionals from the Expert Panel on Population and Prevention Science of the American Heart Association. Circulation. 2004;109(21):2655-2671. 16. Pope CA III, Burnett RT, Krewski D, et al. Cardiovascular mortality and exposure to airborne fine particulate matter and cigarette smoke: shape of the exposure-response relationship. Circulation. 2009;120(11):941-948. 17. Suwa T, Hogg JC, Quinlan KB, Ohgami A, Vincent R, van Eeden SF. Particulate air pollution induces progression of atherosclerosis. J Am Coll Cardiol. 2002;39(6):935-942. 18. Kido T, Tamagawa E, Bai N, et al. Particulate matter induces translocation of IL-6 from the lung to the systemic circulation. Am J Respir Cell Mol Biol. 2011;44(2):197-204. 19. Bai N, Kido T, Kavanagh TJ, et al. Exposure to diesel exhaust up-regulates iNOS expression in ApoE knockout mice. Toxicol Appl Pharmacol. 2011;255(2):184-192. 20. van Eeden SF, Hogg JC. Systemic inflammatory response induced by particulate matter air pollution: the importance of bone-marrow stimulation. J Toxicol Environ Health A. 2002;65(20):1597-1613. 21. Sin DD, Man SF. Why are patients with chronic obstructive pulmonary disease at increased risk of cardiovascular
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diseases? The potential role of systemic inflammation in chronic obstructive pulmonary disease. Circulation. 2003; 107(11):1514-1519. 22. Benati D, Ferro M, Savino MT, et al. Opposite effects of simvastatin on the bactericidal and inflammatory response of macrophages to opsonized S. aureus. J Leukoc Biol. 2010; 87(3):433-442. 23. Djaldetti M, Salman H, Bergman M, Bessler H. Effect of pravastatin, simvastatin and atorvastatin on the phagocytic activity of mouse peritoneal macrophages. Exp Mol Pathol. 2006;80(2):160-164. 24. Sipka S, Dey I, Buda C, Csongor J, Szegedi G, Farkas T. The mechanism of inhibitory effect of eicosapentaenoic acid on
phagocytic activity and chemotaxis of human neutrophil granulocytes. Clin Immunol Immunopathol. 1996;79(3):224-228. 25. Bellingan GJ, Caldwell H, Howie SE, Dransfield I, Haslett C. In vivo fate of the inflammatory macrophage during the resolution of inflammation: inflammatory macrophages do not die locally, but emigrate to the draining lymph nodes. J Immunol. 1996;157(6):2577-2585. 26. Tan WC, Qiu D, Liam BL, et al. The human bone marrow response to acute air pollution caused by forest fires. Am J Respir Crit Care Med. 2000;161(4 pt 1):1213-1217. 27. Solovjov DA, Pluskota E, Plow EF. Distinct roles for the alpha and beta subunits in the functions of integrin alphaMbeta2. J Biol Chem. 2005;280(2):1336-1345.
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Original Research OCCUPATIONAL AND ENVIRONMENTAL LUNG DISEASES
Statins Reduce Ambient Particulate Matter-Induced Lung Inflammation by Promoting the Clearance of Particulate Matter , 10 mm From Lung Tissues Ryohei Miyata, MD, PhD; Ni Bai, PhD; Renaud Vincent, PhD; Don D. Sin, MD, MPH, FCCP; and Stephan F. Van Eeden, MD, PhD
Background: The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) suppress ambient particulate matter , 10 mm (PM10)-induced inflammatory response in vitro. The aim of this study was to determine the effect of statins on PM10-induced lung inflammation in vivo. Methods: New Zealand white rabbits were exposed to either PM10 (1.0 mg/kg) or saline by direct intratracheal instillation three times a week for 4 weeks ⫾ lovastatin 5.0 mg/kg/d. BAL fluid was assessed for cell counts and proinflammatory cytokine levels. Lung inflammation was quantified using immunohistochemical techniques and morphometric methods. Ex vivo phagocytosis assay of alveolar macrophages using PM10 particles was performed. Distribution of PM10 particles in lung tissues and draining lymph nodes was quantified morphometrically to evaluate the clearance of PM10 particles. Results: PM10 exposure increased the production of IL-6 and IL-8, promoted the recruitment of macrophages and polymorphonuclear leukocytes into the lung, and activated these recruited leukocytes. Lovastatin significantly suppressed all these effects. Lovastatin increased the phagocytic activity of macrophages and promoted the migration of PM10-laden macrophages to the regional lymph nodes. Conclusions: Lovastatin attenuates the PM10-induced recruitment and activation of alveolar macrophages and polymorphonuclear leukocytes, reduces local proinflammatory cytokine production, and promotes the clearance of PM10 particles from lung tissues to regional lymph nodes. These novel pleiotropic properties of statins are most likely to contribute to the downregulation CHEST 2013; 143(2):452–460 of PM10-induced lung inflammation. Abbreviations: BALF 5 BAL fluid; NF-kB 5 nuclear factor kB; PM 5 particulate matter; PM10 5 particulate matter , 10 mm; PMN 5 polymorphonuclear leukocyte
adverse health effects of ambient particulate Thematter (PM) exposure on respiratory health have
been well documented.1 Exposure to PM increases the risk of daily exacerbations of asthma and COPD.1,2 Exposure to PM also doubles the risk of pneumonia and, thus, is responsible for . 800,000 deaths from pneumonia annually in children aged , 5 years.3 Therefore, attenuating the lung inflammatory response induced by PM could significantly help to reduce the morbidity and mortality from these deleterious respiratory conditions. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) exhibit their primary pharmacologic
action by inhibiting the conversion of 3-hydroxy3-methylglutaryl coenzyme A to l-mevalonate. In addition to reducing cholesterol levels through the sterol metabolism pathway, they also inhibit geranylgeranylpyrophosphate through the nonsterol metabolism pathway. Isoprenylation of geranylgeranyl-pyrophosphate is required for the activation of small guanosine triphosphatases, including RhoA and Rac1 (Rho-related C3 botulinum toxin substrate). Statins decrease the availability of RhoA and Rac1, which subsequently leads to the deactivation of nuclear factor kB (NF-kB) activity,4 allowing statins to possess significant antiinflammatory effects. Other pleiotropic effects of statins include
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the regulation of oxidative stress and phagocytic activity.5,6 These effects may contribute to a reduction in lung inflammatory responses induced by PM exposure. Alveolar macrophages and lung epithelial cells are primarily responsible for processing inhaled ambient particles.7 Studies from our laboratory showed that statins suppress PM-stimulated proinflammatory cytokine production by both alveolar macrophages and bronchial epithelial cells in vitro.8 In addition, we previously showed in a rabbit model that long-term exposure to PM , 10 mm (PM10), EHC-93, induces a lung inflammatory response.9-11 Therefore, the current study was designed to determine the effect of statins on the lung inflammatory response induced by long-term exposure to PM10, with the hypothesis that statins will attenuate the degree of PM10-induced lung inflammation. Specifically, we quantified the effect of statins on leukocyte recruitment into lung tissues, activation of the recruited leukocytes, and the production of proinflammatory cytokines. We also evaluated the effects of statins on the clearance of PM10 particles from the lung. Materials and Methods
diet and water ad libitum. The protocol was approved by the Animal Experimentation Committee of The University of British Columbia (A09-0071). Experimental Protocol The animals were assigned randomly to four experimental groups (each group, n 5 6) as follows: (1) saline instillation, (2) saline instillation with lovastatin treatment, (3) PM10 instillation, and (4) PM10 instillation with lovastatin treatment. Either PM10 or saline was administrated by a direct intratracheal instillation method three times a week for 4 weeks. Lovastatin (Toronto Research Chemicals) 5 mg/kg/d was given orally for 8 weeks, beginning 4 weeks before the PM10 or saline exposure. Four hours after the final (12th) exposure, the rabbits were killed with an overdose of pentobarbital sodium. A detailed description of the protocol can be found in e-Appendix 1. Histologic Studies of the Lung and Mediastinal Lymph Nodes Lungs and mediastinal lymph nodes were harvested and processed for histologic quantification. See e-Appendix 1 for more details. BAL Alveolar cells were harvested by BAL, fixed with methanol, and underwent Wright-Giemsa staining for cell differential counts as detailed in e-Appendix 1. Differential counts were determined by morphologic criteria under oil immersion light microscopy with evaluation of 200 cells/slide in randomly selected fields of view.
Urban Air Particulates
Immunohistochemistry
Urban air PM10 (EHC-93) was obtained from the Environmental Health Directorate, Health Canada. The detailed characteristics of EHC-93, including particle preparation and chemical composition, have been described elsewhere12 as well as in e-Appendix 1.
Immunohistochemical techniques were used to identify specific leukocytes in lung tissues and the distribution of macrophages in mediastinal lymph nodes. Macrophages were identified by RAM11 (monoclonal mouse antirabbit macrophage-specific primary antibody) (M0633; Dako Canada Inc), polymorphonuclear leukocytes (PMNs) by monoclonal mouse antirabbit defensin 5 (LS-C50934; LifeSpan BioSciences, Inc), and activated leukocytes by monoclonal mouse antirabbit CD11b (MCA802; AbD Serotec a Division of MorphoSys) as detailed in e-Appendix 1.
Experimental Animals Female New Zealand white rabbits (n 5 24; 12 weeks old; weight, 2.6 ⫾ 0.1 kg) (Charles River Laboratories International, Inc) were used in this study. They were housed in a clean-air and viral-free room with restricted access and given a standard rabbit Manuscript received May 16, 2012; revision accepted July 1, 2012. Affiliations: From the UBC James Hogg Research Centre (Drs Miyata, Bai, Sin, and Van Eeden), Institute for Heart 1 Lung Health, St. Paul’s Hospital, The University of British Columbia, Vancouver, BC; Department of Anesthesiology, Pharmacology and Therapeutics (Dr Bai), The University of British Columbia, Vancouver, BC; and Environmental Health, Science and Research Bureau (Dr Vincent), Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, ON, Canada. Funding/Support: This study was supported by operating grants from the Heart and Stroke Foundation of Canada, Michael Smith Foundation for Health Research, and the Canadian Institutes of Health Research. Correspondence to: Stephan F. Van Eeden, MD, PhD, UBC James Hogg Research Centre, Institute for Heart 1 Lung Health, St. Paul’s Hospital, The University of British Columbia, Room 166, 1081 Burrard St, Vancouver, BC V6Z 1Y6, Canada; e-mail: [email protected] © 2013 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.12-1237
Quantitative Analysis of Lung Inflammation The degree of lung inflammation was quantified by standard morphometric techniques. See e-Appendix 1 for more details. Cytokine Analyses IL-6 and IL-8 levels in the BAL fluid (BALF) were analyzed by enzyme-linked immunosorbent assay using commercially available kits (CSB-E06903RB and CSB-E06905RB; Cusabio Biotech Co, Ltd) according to manufacturer instructions. Each sample was measured in duplicate. Distribution of PM10 in the Lung The burden of PM10 both in lung tissues and in mediastinal lymph nodes was quantified. See e-Appendix 1 for more details. Ex Vivo Phagocytosis Assay of Alveolar Macrophages Phagocytic ability of alveolar macrophages for fluorescent microspheres or PM10 particles was evaluated either in suspension or in adherent cell culture by flow cytometry or microscopic quantification, respectively. See e-Appendix 1 for more details.
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Statistics Values are expressed as mean ⫾ SEM. Data for the four experimental groups were compared by two-way factorial analysis of variance, and those for two groups were compared by Student t test. Statistical significance was considered at P , .05 (e-Appendix 1).
Results Leukocyte Counts in BALF The total leukocyte count in BALF was increased by PM10 exposure (P , .001) (Fig 1), which was suppressed by lovastatin (P , .01). This increase in leukocytes was predominantly due to a marked influx of PMNs and band cells (P , .001) (Table 1), both of which were strongly reduced by lovastatin (P , .001), whereas lovastatin showed no suppressive effect on the macrophage count. Proinflammatory Cytokines in BALF PM10 exposure increased both IL-6 and IL-8 levels in BALF, the former to a lesser extent than the latter (IL-6, P , .05 vs saline; IL-8, P , .001 vs saline) (Fig 2). Lovastatin reduced both IL-6 and IL-8 levels. The lovastatin effect was more evident in IL-8 levels (IL-6, P , .05 vs PM10; IL-8, P , .001 vs PM10) (Fig 2). Leukocyte Recruitment Into Lung Tissue PM10 exposure increased the volume fraction of alveolar macrophages in the airspaces as well as in the lung parenchymal tissues, and lovastatin suppressed these recruitments, an effect also seen in the lovastatintreated saline-exposed groups (Fig 3A). The majority of PMNs were in the parenchymal tissues, and their recruitment was attenuated by lovastatin (Fig 3B). PM10 exposure strongly increased CD11b expression on leukocytes (alveolar macrophages and PMNs) in the airspaces and in the parenchyma (Fig 3C). In the airspaces of the control animals, alveolar macrophages were the predominant leukocytes expressing CD11b (macrophages, 75.3% ⫾ 4.0%; PMNs, 24.7% ⫾ 4.0%), and this predominance was reduced with PM10 exposure (macrophages, 62.4%⫾2.1%; PMNs, 37.6%⫾2.1%), suggesting that PM10 exposure preferentially activates PMNs in the airspaces. In the lung parenchyma, however, the ratio of macrophages to PMNs expressing CD11b was approximately one to one, and the ratio did not change with PM10 exposure. Lovastatin suppressed CD11b expressions in both the airspaces and the parenchymal tissues, but the lovastatin effect was less in the parenchyma. Reduction in the Number of PM10 Particles in the Lung by Lovastatin PM10 particles were present throughout the lung and were predominantly found in the cytoplasm of
alveolar macrophages. The proportion of alveolar macrophages with PM10 particles in their cytoplasm was significantly higher in the PM10 groups than in the saline groups (Fig 4A). Lovastatin significantly reduced this fraction of alveolar macrophages with PM10. Quantitatively, the total lung burden of PM10 particles in all lung tissues was significantly reduced by lovastatin (Fig 4B). Clearance of PM10 Particles Into Mediastinal Lymphatic Tissues Mediastinal lymph nodes were stained with RAM11 to evaluate the distribution of macrophages and with Hansel stain to quantify the number of PM10 particles that migrated from the lung (Fig 5A). PM10 exposure strongly promoted the migration of macrophages to lymphatic tissues (Fig 5A), and morphometric analysis confirmed that PM10 exposure significantly increased the volume fraction of macrophages in the mediastinal lymph nodes (Fig 5B). Although not statistically significant, lovastatin further increased this migration (Fig 5B); however, lovastatin significantly increased the number of PM10 particles in lymph nodes compared with PM10 exposure alone (Fig 5C). Effect of Lovastatin on Alveolar Macrophage Phagocytic Activity To determine the effect of lovastatin on the phagocytic activity of alveolar macrophages, rabbits were pretreated with or without lovastatin 5 mg/kg/d for 4 weeks, and alveolar macrophages were collected for ex vivo phagocytosis assay using fluorescent microspheres
Figure 1. Cellular profile of BALF. Four hours after the final (12th) instillation, rabbits were killed, and left lung lavage was performed. PM10 exposure increased the total cell count, and lovastatin reversed this effect. In contrast, lovastatin had no suppressive effect on alveolar macrophages, which were significantly increased by PM10 exposure. PM10 also strongly stimulated the recruitment of PMNs, and lovastatin inhibited this recruitment. ***P , .001 vs saline; ##P , .01 vs PM10; ###P , .001 vs PM10. BALF 5 BAL fluid; PM10 5 particulate matter , 10 mm; PMN 5 polymorphonuclear leukocyte.
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Data are presented as mean ⫾ SEM. Cell counts are expressed in 3 103/mL. PM10 5 particulate matter , 10 mm; PMN 5 polymorphonuclear leukocyte. P , .05 vs saline. bP , .001 vs saline. cP , .01 vs PM . 10 dP , .001 vs PM . 10 a
1.2 ⫾ 0.2 (0.96 ⫾ 0.17) 0.1 ⫾ 0.1a (0.06 ⫾ 0.06) 4.2 ⫾ 1.2b (0.76 ⫾ 0.21) 0.2 ⫾ 0.2d (0.07 ⫾ 0.06) 4.0 ⫾ 0.5 (3.3 ⫾ 0.4) 0.4 ⫾ 0.2 (0.3 ⫾ 0.2) 32.0 ⫾ 2.0b (5.8 ⫾ 0.4) 1.5 ⫾ 0.6d (0.5 ⫾ 0.2) 3.2 ⫾ 0.6 (2.6 ⫾ 0.5) 0.4 ⫾ 0.2 (0.3 ⫾ 0.2) 30.3 ⫾ 3.8b (5.5 ⫾ 0.7) 1.8 ⫾ 1.0d (0.6 ⫾ 0.3) 82 ⫾ 6 (68 ⫾ 5) 111 ⫾ 2 (92 ⫾ 1) 325 ⫾ 30b (59 ⫾ 5) 262 ⫾ 9 (83 ⫾ 3) 121 ⫾ 13 121 ⫾ 20 553 ⫾ 32b 315 ⫾ 36c Saline Saline 1 lovastatin PM10 PM10 1 lovastatin
34 ⫾ 6 (28 ⫾ 5) 9 ⫾ 2 (8 ⫾ 1) 191 ⫾ 30b (35 ⫾ 5) 51 ⫾ 9d (16 ⫾ 3)
31 ⫾ 5 (26 ⫾ 4) 9 ⫾ 2 (7 ⫾ 1) 161 ⫾ 28b (29 ⫾ 5) 49 ⫾ 8d (16 ⫾ 3)
Eosinophils (%) Lymphocytes (%) Segmented Cells (%) Band Cells (%) PMNs (%) Macrophages (%) Total Cells Group
Table 1—Cellular Profile of BAL Fluid
Figure 2. IL-6 and IL-8 levels in BAL fluid collected from New Zealand white rabbits exposed to PM10 ⫾ lovastatin. The BAL fluid was analyzed by enzyme-linked immunosorbent assay. PM10 exposure increased both IL-6 and IL-8 levels, the former to a lesser extent than the latter. Lovastatin suppressed both IL-6 and IL-8 levels, with a more pronounced effect on IL-8 levels. *P , .05 vs saline; ***P , .001 vs saline; #P , .05 vs PM10; ###P , .001 vs PM10. See Figure 1 for expansion of abbreviation.
assessed by flow cytometry. Lovastatin strongly enhanced ex vivo uptake of both opsonized and unopsonized microspheres (Fig 6A). To determine the effect of lovastatin on the phagocytic activity of alveolar macrophages for PM10 particles, we pretreated rabbits with lovastatin as follows: (1) 5 mg/kg/d for 4 weeks, (2) 5 mg/kg/d for 2 days, or (3) 50 mM in vitro for 1 h. We then determined the macrophage uptake of particles using microscopic quantification (Fig 6B). Lovastatin significantly enhanced the phagocytic activity of alveolar macrophages for PM10 particles (Fig 6C) with prolonged (4 weeks) lovastatin pretreatment but not with short-term (2 days) or in vitro pretreatment. Discussion In the present study, we showed that statins have potent suppressive effects against air pollution-induced lung inflammation as evidenced by a reduction in the recruitment and activation of alveolar macrophages and PMNs in the lung. Statins also reduce the levels of IL-6 and IL-8 in the lung. Moreover, statins promote the clearance of particles from the lung by enhancing the phagocytic activity of alveolar macrophages for PM10 particles and promoting the lymphatic removal of PM10-containing macrophages to the regional lymph nodes. Collectively, these data suggest that PM10induced lung inflammation can be attenuated by statins through their ability to regulate (1) the recruitment and activation of leukocytes, (2) proinflammatory cytokine production, and (3) the clearance of PM10 particles. These novel effects of statins on the regulation of lung inflammation could broaden their application for other inflammatory lung conditions.
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Figure 3. Volume fraction of specific leukocytes in the lung exposed to PM10 ⫾ lovastatin. A, Volume fraction of alveolar macrophages (RAM11 [monoclonal mouse antirabbit macrophage-specific primary antibody]-positive cells). PM10 exposure promoted the macrophage recruitment, and lovastatin suppressed the recruitment at both locations. Lovastatin also suppressed the volume fraction of alveolar macrophages in the saline-exposed group at both locations. B, Volume fraction of PMNs (defensin 5-positive cells). PMNs were preferentially located at parenchymal tissues. PM10 exposure strongly enhanced the recruitment of PMNs into the lung, whereas lovastatin inhibited this recruitment. C, Volume fraction of activated leukocytes (CD11b-positive cells). PM10 exposure significantly activated the airspace and parenchymal leukocytes. The lovastatin effect was less in the parenchyma. *P , .05 vs saline; **P , .01 vs saline; ***P , .001 vs saline; ##P , .01 vs PM ; ###P , .001 vs PM . See Figure 1 legend for expansion of abbreviations. 10 10
We have previously shown that exposure to PM10 elicits a brisk pulmonary inflammatory response characterized by an increase in the recruitment and activation of alveolar macrophages, PM10-containing alveolar macrophages, and circulating IL-6 levels,11 which is in keeping with the findings of the present study. Our group also showed that statins suppress PM10-induced cytokine production, including IL-6 and IL-8, by human alveolar macrophages and bronchial epithelial cells in in vitro settings.8 The new results further add to the understanding of the antiinflammatory nature of statins by showing that they reduce PM10-induced proinflammatory cytokines in in vivo settings. Although the growing literature indicates the antiinflammatory properties of statins,5 the antiinflammatory properties against lung inflammation, particularly PM-induced lung inflammation, is poorly studied. However, it is established that statins are capable of reducing reactive oxygen species production,6 inactivating nicotinamide
adenine dinucleotide phosphate-oxidase,13 and inhibiting NF-kB,14 all of which are likely to participate in the downregulation of PM-induced proinflammatory cytokine production as demonstrated in Figure 2. To our knowledge, no studies to date have addressed whether statins modulate the clearance of PM, including cigarette smoke. We speculate that their ability to downregulate NF-kB contributes, in part, to their well-established antiinflammatory properties. More interestingly, we showed herein that statins promote the clearance of macrophages to the regional lymph nodes. This concept provides a new insight into the underlying mechanisms for their antiinflammatory properties. The existing body of evidence is consistent and plausible enough to confirm the link between PM air pollution and cardiovascular diseases.11,15-19 There exist three proposed molecular mechanisms by which PM increases the risk of cardiovascular events: (1) direct
Figure 4. Quantification of PM10 particle distribution in the lung exposed to PM10 ⫾ lovastatin. A, Percentages of alveolar macrophages containing PM10 particles were assessed using the lung tissue specimens. Lovastatin significantly reduced the fraction of PM10-containing alveolar macrophages. B, Comparison of total lung burden of PM10. Lovastatin also significantly reduced the total lung burden of PM10. ***P , .001 vs saline; ###P , .001 vs PM10. See Figure 1 legend for expansion of abbreviation. 456
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Figure 5. A, Microphotographs of the distribution of macrophages (RAM11 [monoclonal mouse antirabbit macrophage-specific primary antibody]-positive cells) and PM10 particles in the mediastinal lymph nodes (RAM11 and Hansel stains, original magnification 3 320). Resident macrophages were stained positive in the saline-exposed animals. Most lymphatic vessels were packed with a cluster of macrophages in the PM10-exposed animals, particularly in the PM10 1 lovastatin group, indicating the migration of macrophages from the lung. The migrated macrophages were spread out throughout the lymph follicles. PM10 exposure strongly promoted the deposition of particulate matter in the mediastinal lymph nodes. Most particulate matter was found inside tingible body macrophages in germinal centers. Lovastatin further increased the deposition of particles. The bars represent 20 mm. B, Volume fraction of macrophages (RAM11-positive cells) in the mediastinal lymph nodes. PM10 exposure significantly increased the volume fraction of macrophages in the mediastinal lymph nodes, but statistically, lovastatin showed no additional effect to increase the fraction of macrophages. *P , .05 vs saline; **P , .01 vs saline. C, Volume fraction of PM10 particles in the mediastinal lymph nodes. PM10 exposure increased the volume fraction of PM10 particles deposited in the lymph nodes. The deposition was further increased by lovastatin. *P , .05 vs saline; ###P , .001 vs PM10. N.S. 5 not significant. See Figure 1 legend for expansion of other abbreviation.
penetration of PM into the blood vessels; (2) translocation of inflammatory mediators from the lung to the systemic circulation; and (3) activation of the lung autonomic nervous system.15 Although the exact mechanism is not yet fully characterized, the systemic spillover of lung inflammation hypothesis has the most experimental support.2,20 To support this notion, our group showed that PM-induced lung inflammatory mediators (eg, IL-6) translocate to the systemic circulation,11,18 directly implicating lung inflammation as a key driver for the downstream adverse vascular effects of PM exposure. The potent antiinflammatory effects of statins on PM-induced lung inflammation demonstrated in the current study are critically important in terms of counteracting the adverse vascular
effects. This finding could also have implications for other inflammatory lung conditions, such as COPD, which is known to be strongly associated with cardiovascular diseases.21 The effect of statins on leukocyte phagocytic function is controversial. Statins have been reported to inhibit phagocytosis of IgG-opsonized bacteria.22 In contrast, other researchers reported that statins enhance phagocytosis of latex beads.23 These discrepancies may be due to phagocytosis pathway differences (Fc dependent or Fc independent). We showed that lovastatin significantly enhances phagocytosis of PM10 particles if given for a long period, whereas short-term or in vitro lovastatin treatment has no positive effect on their phagocytic function (Fig 6C). These data suggest the
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Figure 6. A, Ex vivo phagocytosis assay of fluorescent microspheres. New Zealand white rabbits were pretreated identically (lovastatin 5 mg/kg/d for 4 wk). Alveolar macrophages were collected and incubated with fluorescent microspheres for 2 h. Lovastatin strongly enhanced ex vivo uptake of both opsonized and unopsonized microspheres. ***P , .001 vs control subjects. B, Microphotographs of ex vivo phagocytosis assay of PM10 particles (Diff-Quick stain, original magnification 3 600). The bars represent 10 mm. C, Ex vivo phagocytosis assay of PM10 particles. New Zealand white rabbits were pretreated with lovastatin as follows: (1) 5 mg/kg/d for 4 wk (n 5 4), (2) 5 mg/kg/d for 2 d (n 5 4), or (3) 50 mM in vitro for 1 h (n 5 4). Alveolar macrophages were collected and incubated with PM10 particles for 2 h. Long-term (4-wk) lovastatin pretreatment strongly enhanced ex vivo uptake of PM10 particles but not short-term (2-d) or in vitro lovastatin pretreatment. **P , .01 vs control subjects. See Figure 1 legend for expansion of abbreviation.
importance of structural change in lipid bilayer membrane composition of phagocytes after a long-term lovastatin treatment, which is supported by studies where a reduction of intracellular cholesterol by pravastatin diminished membrane fluidity, resulting in the dysregulation of neutrophil phagocytosis function.24 Once having ingested foreign materials, macrophages do not remain in the airspaces but emigrate to the draining lymph nodes.25 This concept was supported by the present study, which showed an increase in PM10 particles in lymphatic tissues in rabbits exposed to PM10. Lovastatin decreases the number of PM10 particles in lung tissues (Fig 4) and increases the number in lymphatic tissues (Fig 5), suggesting that it promotes the emigration of PM10-laden macrophages to the regional lymph nodes. These findings were supported by our in vitro studies showing that lovastatin promotes the phagocytosis of PM10 particles (Fig 6C). The relatively poor clearance of PM-laden macrophages in the PM10-exposed group (Fig 4B) could contribute to the brisk influx and activation of PMNs in the lung (Fig 3B), thereby amplifying the local inflammatory responses. These data suggest that the enhancement of phagocytosis by lovastatin facilitates alveolar macrophages to phagocytose PM10 particles in the airspaces
more effectively and promotes the emigration of the PM-laden macrophages to draining lymph nodes. Collectively, the enhancement of particle clearance from the lung could contribute to the antiinflammatory properties of statins in the lung. Limitations of the present study must be noted. First, we have used the direct intratracheal instillation method because it provides an accurate dosing and lung deposition of particles in rabbits, which are predominantly nose breathers capable of efficiently filtering, capturing, and clearing most inhaled particles in the nasal cavity. We have also improved on our previous instillation method10 by using a microsprayer, which enabled more homogeneous distribution of PM across the entire lung surface. The dose of PM10 that the rabbits were exposed to for the 4-week experimental period is similar to the calculated human exposure while living in a polluted city (150 mm3) for 3 weeks.26 Nevertheless, extrapolation of the data from the intratracheal instillation method should be validated in inhalation models. Second, CD11b expression was used to assess the activation of leukocytes because CD11b molecules can be upregulated in response to PM.27 We showed that the expression of CD11b on leukocytes was increased by PM10 exposure,
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and lovastatin abolished this increase (Fig 3C), suggesting that CD11b is a good marker to determine the antiinflammatory effect of statins. However, CD11b is ubiquitously expressed on many different leukocytes, including monocytes, granulocytes, macrophages, and natural killer cells, and is not specific for activation of alveolar macrophages or PMNs. In conclusion, repeated instillations of PM10 for 4 weeks increase the recruitment of PMNs and alveolar macrophages into the lung, activate these leukocytes, and induce the local production of IL-6 and IL-8. Lovastatin treatment suppresses all these proinflammatory effects. Lovastatin also promotes the clearance of particles to regional lymphatic tissues. These findings suggest that statins can attenuate not only lung inflammation induced by PM10 exposure but also, potentially, the downstream adverse cardiovascular effects. Acknowledgments Author contributions: Dr Van Eeden had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Dr Miyata: contributed to the data collection and analysis, writing of the manuscript, and approval of the final manuscript. Dr Bai: contributed to the data collection and analysis and approval of the final manuscript. Dr Vincent: contributed to the provision of the study material, data analysis, manuscript review, and approval of the final manuscript. Dr Sin: contributed to the study conception and design, provision of the study material, financial and administrative support, revision of the manuscript, and approval of the final manuscript. Dr Van Eeden: contributed to the study conception and design, provision of study material, financial and administrative support, revision of the manuscript, and approval of the final manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Dr Sin has received grant funding from AstraZeneca and has served on advisory boards of Merck Canada Inc, Novartis Pharmaceuticals Corporation, and Takeda Pharmaceuticals International GmbH. Drs Miyata, Bai, Vincent, and Van Eeden have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript. Other contributions: We thank Beth Whalen, PhD, for performing the flow cytometry analysis. Dr Miyata is a recipient of a Canadian Institutes of Health Research Integrated and Mentored Pulmonary and Cardiovascular Training Strategic training postdoctoral fellowship. Dr Van Eeden is an American Lung Association Career Investigator, the recipient of the William Thurlbeck Distinguish Research Award, Michael Smith Foundation Senior Investigator Award, and the current GlaxoSmithKline plc/Canadian Institutes of Health Research professor in COPD. Additional information: The e-Appendix can be found in the ”Supplemental Materials” area of the online article.
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