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,1
* Environmental Health Sciences Division, National Institute for Environmental Studies, Ibaraki, 305-8506 Japan; Inflammation and Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, 602-8566 Japan; Seikagaku Kogyo, Ltd, Tokyo 207-0021 Japan; and Department of Veterinary Pathology, Faculty of Agriculture, Tottori University, Tottori, 680-8553 Japan
1 To whom requests for reprints should be addressed at Environmental Health Sciences Division, National Institute for Environmental Studies, 16–2 Onogawa, Tsukuba 305-8506, Japan. E-mail: htakano{at}nies.go.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: DEP • components • lung inflammation • LPS • endothelial damage
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In urban areas, diesel engines are the major source of PM2.5 (1). Diesel engine–derived diesel exhaust particles (DEP) have been experimentally correlated with a variety of respiratory disorders, such as lung cancer, pulmonary fibrosis, chronic alveolitis (4), pulmonary edema (5), and allergic airway inflammation (6). We have previously shown that DEP (7) and their components (8) enhance lung inflammation related to bacterial endotoxin (lipopolysaccharide [LPS]). Cellular profile of bronchoalveolar lavage (BAL) fluid and lung histology have shown that the enhancing effects are stronger with carbonaceous nuclei of DEP after extraction (washed DEP) than with the organic chemicals in DEP extracted with dichloromethane (CH2Cl2) (DEP-OC), which is concomitant with the lung expression of proinflammatory proteins, including interleukin (IL)-1ß, macrophage inflammatory protein (MIP)-1
, macrophage chemoattractant protein (MCP)-1, and keratinocyte chemo-attractant (KC) (8).
DEP can reportedly induce an inflammatory response not only in the airways but also in the circulatory blood partly through enhanced prothrombotic activity in vivo (9). On the other hand, LPS causes endothelial damage, resulting in multiple organ dysfunction syndrome frequently accompanied by consumptive hemostatic changes, which often leads to disseminated intravascular coagulation, including the activation of coagulation and fibrinolysis (10, 11). These previous studies raise the possibility that the coexistence of DEP and LPS synergistically aggravates endothelial damage and consequent coagulopathy. Also, it can be imagined that severe endothelial damage allows large molecules such as LPS to pass into the circulation. Indeed, lung-to-systemic translocation of LPS has been evidenced under large tidal volume ventilation in vivo (12).
In the current study, we elucidated the effects of pulmonary exposure to DEP components on endothelial damage, coagulatory disturbance, systemic inflammatory response, and LPS translocation from the lung-to-systemic circulation after LPS exposure to the murine respiratory system.
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Materials and Methods |
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Preparation of Particle Samples.
A 4JB1-type, light-duty, four-cylinder, 2.74-L Isuzu diesel engine (Isuzu Automobile Co., Tokyo, Japan) under computer control was connected to a dynamometer (Meiden-sya, Tokyo, Japan). The details on the condition of engine and the collection of DEP were previously described (13).
Preparation of DEP-OC and Washed DEP.
DEP were extracted with CH2Cl2 (8). Briefly, DEP were suspended in CH2Cl2 and sonicated for 5 mins (UD-201; Tomy Seiko, Tokyo, Japan). The suspension was centrifuged at 200 g for 20 mins. The supernatants were transferred to another tube, and the residue was washed with CH2Cl2. This manipulation was repeated three times. The residual particles of DEP were prepared as washed DEP. The extracts were combined, evaporated, dissolved in 100% dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan), and prepared as DEP-OC.
Preparation of LPS.
LPS (Escherichia coli O55: B5) were obtained from Difco Laboratories (Detroit, MI). The LPS was extracted by the Boivin trichloracetic acid procedure.
Study Protocol.
Mice were divided into six groups. The vehicle group received phosphate-buffered saline at pH 7.4 (GIBCO BRL, Life Technology, Grand Island, NY) containing 0.025% Tween 80 (Nacalai Tesque, Kyoto, Japan) and 0.25% DMSO. The washed DEP group or the DEP-OC group received 4 mg/kg of washed DEP or DEP-OC, respectively. The LPS group received 2.5 mg/kg of LPS. The washed DEP + LPS and DEP-OC + LPS groups received respective additions of LPS to each component before experimentation. In each group, vehicle, DEP components, LPS, or DEP components + LPS were dissolved in 0.1-ml aliquots and inoculated by the intratracheal route through a polyethylene tube under anesthesia with 4% halothane (Hoechst, Japan, Tokyo, Japan). Twenty-four hours after the intratracheal administration, all mice were sacrificed and examined. The studies reported were carried out in accordance with the Guide for the Care and Use of Laboratory animals as adopted and promulgated by the National Institutes of Health. All animal studies were approved by the Institutional Review Board.
Histological Evaluation.
After exsanguination, the lungs were fixed by intratracheal instillation of 10% neutral phosphate-buffered formalin at a pressure of 20 cm H2O for at least 72 hrs. Slices 2 to 3 mm thick of all pulmonary lobes were embedded in paraffin. Sections of 3-µm thickness were stained with hematoxylin and eosin (H&E). Neutrophil infiltration was expressed as the number of neutrophils per high-power field by counting the number of over 30 fields at a magnification of x400 in each slide (n = 5 in each group). Histologic sections were evaluated in a blind fashion.
Pulmonary Vascular Permeability.
In a separate series of experiments, the bilateral lungs were weighed immediately after exsanguination and dried in an oven at 95°C for 48 hrs. Thereafter, lung water content was estimated by calculating the ratio of wet lung weight to dry lung weight (mg)/body weight (g) (Ref. 5: n = 10 in each group). In another experiment, protein concentrations in BAL fluid were estimated by previous method by Bradford (Ref. 14: n = 8 in each group).
Assays for Circulatory Fibrinogen, Activated Protein C (APC), Antithrombin (AT)-III, von Wille-brand Factor (vWF), IL-1ß, IL-6, KC, E-Selectin, and LPS Level.
After deep anesthesia, the chest and abdominal walls were opened, and blood was retrieved by cardiac puncture. Blood samples were collected from the right ventricle and moved into 3.8% sodium citrate in a ratio of 10:1 and centrifuged at 2,500 g for 10 mins. Citrate plasma fibrinogen, APC, AT-III, and vWF (n = 7–10 in each group) were determined using commercial kits (Diagnostica Stago, Roche, Tokyo, Japan) on STA Compact (Diagnostica Stago, Roche) as described previously (15). Serum samples (n = 10 in each group) were analyzed by enzyme-linked immunosorbent assays for IL-1ß (Endogen, Cambridge, MA), IL-6 (Biosource, Nivelles, Belgium), KC, and E-selectin (R&D Systems, Minneapolis, MN) according to the manufacturers’ instructions. Values obtained at 550 nm were subtracted from values obtained at 450 nm and converted to pg/ml using values obtained from standard curves generated with varying concentrations of recombinant IL-1ß, IL-6, KC, and E-selectin with limits of detection of 3, 3, 2, and 3.8 pg/ml, respectively. The LPS level was determined by automated kinetic assay using an LPS-specific limulus amebocyte lysate reagent (Endospecy ES-50M; Seikagaku Corp., Tokyo, Japan) combined with an incubator installed micro-plate reader (Wellreader SK603; Seikagaku Corp.) as described previously (16). The increase in OD by released p-nitroaniline was continuously measured at 405 nm against a reference of 492 nm. LPS level was automatically calculated from a calibration curve with standard LPS preparation. No interference with the assay has been observed with sufficient recovery of nearly 100% by the addition-recovery test.
Statistical Analysis.
LPS concentration is expressed as mean ± SD. Other data except for LPS concentrations are reported as mean ± SEM. Differences between groups in LPS concentrations were analyzed by the Kruskal-Wallis test followed by the Mann-Whitney U test using StatView version 4.0 (Abacus Concepts, Inc., Berkeley, CA). Differences in other data were examined for statistical significance using analysis of variance and Bonferroni’s test. Significance was assigned to P values smaller than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). No pathological changes were observed in lung specimens in the vehicle group. Infiltration of inflammatory cells, including neutrophils, was slightly seen in the DEP components groups and moderately seen in the LPS group. In the washed DEP + LPS group, markedly exaggerated neutrophil sequestration and pulmonary edema were observed. On the other hand, the DEP-OC + LPS group revealed moderate neutrophilic inflammation.
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). The number of neutrophils in the lung was significantly greater in the LPS group or the DEP components + LPS groups than in the vehicle group (P < 0.01). The number was further increased in the DEP components + LPS group (P < 0.01; washed DEP + LPS group vs. the LPS group).
Effects of DEP Components on Pulmonary Permeability during Lung Inflammation.
To estimate pulmonary permeability, we next evaluated the lung water content and protein level in the BAL fluid 24 hrs after intratracheal instillation (Table 1). Lung water content was significantly greater in the washed DEP, the LPS, and the DEP components +LPS groups than in the vehicle group (P < 0.01). The value was significantly greater in the washed DEP + LPS group than in the LPS group (P < 0.01). The protein level in the BAL fluid was also significantly increased in the washed DEP group (P < 0.01) and DEP-OC + LPS groups compared to the vehicle group and was further increased in the washed DEP +LPS group (P < 0.01 vs. LPS group). The value was also significantly greater in the washed DEP + LPS group than in the LPS group (P < 0.01).
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). Fibrinogen levels were significantly higher in the washed DEP, the LPS, and the DEP components+LPS groups than in the vehicle group (Table 2; P < 0.01) with values further increased in the DEP components+LPS groups (P < 0.01 vs. the LPS group). APC was significantly smaller in the washed DEP, the LPS, and the DEP components+LPS groups than in the vehicle group (P < 0.01). The level was further reduced in the washed DEP +LPS group (P < 0.05 vs. the LPS group). AT-III values were also significantly less in the LPS and DEP-OC +LPS groups compared to the vehicle group (P < 0.01) and were further reduced in the washed DEP + LPS group (P < 0.05 vs. the LPS group). Finally, the level of vWF was significantly greater in the washed DEP + LPS group than in the vehicle group (P < 0.05).
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). IL-1ß levels were greater in the LPS and the DEP components + LPS groups than in the vehicle group (P < 0.01). The LPS group showed elevation of IL-6 level as compared with the vehicle group. IL-6 levels were also increased in the washed DEP + LPS group compared to both the vehicle and the LPS groups (P < 0.01). In addition, KC levels were significantly increased in the LPS (P < 0.05), the DEP-OC + LPS (P < 0.05), and the washed DEP + LPS (P < 0.01) groups compared to the vehicle group, and the values were further increased in the washed DEP + LPS group (P < 0.01 compared to LPS alone). E-selectin levels were significantly greater in the LPS and the DEP components + LPS groups (P < 0.01 vs. the vehicle group) and were further increased in the DEP-OC + LPS group (P < 0.05 vs. the LPS group) and in the washed DEP +LPS group (P < 0.01 vs. the LPS group) (Table 3). The LPS concentration was greater in the LPS and the DEP components + LPS groups than in the vehicle group (P < 0.01). The concentration was further increased in the washed DEP + LPS group (P < 0.05 vs. LPS).
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Discussion |
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, MCP-1, and KC, in the lung compared with LPS treated alone. In the present study, we have reconfirmed that DEP components aggravate lung inflammation induced by intratracheal instillation of LPS (Fig. 1). In addition, we have demonstrated that both DEP components exacerbate vascular permeability (Table 1) and the increased fibrinogen (Table 2) and E-selectin (Table 3) levels induced by LPS. With overall trends, the exacerbation is more prominent with washed DEP rather than with DEP-OC. Finally, washed DEP + LPS significantly decreased APC and AT-III (Table 2) and elevated circulatory levels of IL-6, KC, and LPS as compared with LPS alone (Table 3).
PM exposure results in adverse health effects not only on respiratory systems but also on circulation (1–3). Indeed, inflammatory respiratory diseases and vascular diseases are highly sensitive to DEP exposure (1, 6–8). In the present study, we reconfirm that DEP components, especially washed DEP, enhance LPS-induced neutrophil inflammation (Fig. 1B). Furthermore, recent epidemiological analysis has reported that respiratory infection can act as a risk factor in the development of cardiovascular diseases, including ischemic heart diseases (17). Nonetheless, there is a shortage of molecular and/or experimental evidence to clarify the role of DEP in susceptibility to coagulatory disturbance and systemic inflammation during infectious inflammation. In the present study, we have focused on whether the effects of DEP components on lung inflammation can spread to coagulatory changes and systemic inflammation.
Lung inflammation induced by intratracheal inoculation of LPS has reportedly elevated fibrinogen level in rats (18). However, fibrinogen levels have not been examined in the other species. Furthermore, the previous report did not examine other proinflammatory mediators, such as cytokines and chemokines, in the systemic circulation (18). In the present murine model, pulmonary exposure to LPS induced significantly higher levels of circulatory fibrinogen (Table 2) and IL-1ß (Table 3) than vehicle exposure. IL-1ß is an important proinflammatory initiator in mammalian innate immunity (19). This study represents the first demonstration that lung inflammation related to LPS induces a systemic inflammation characterized by enhanced levels of circulating proinflammatory cytokines in vivo.
Intratracheal instillation of DEP leads to a rapid activation of the thrombohemostatic system, including platelet activation, suggesting that DEP can increase the risk of ischemic vascular diseases (9). On the other hand, epidemiological data have demonstrated the close correlation between high fibrinogen levels and the risk of ischemic cardiovascular diseases (20). However, the impacts of pulmonary exposure to DEP and/or their components on thrombohemostatic disturbance and circulatory inflammatory response during lung inflammation have never been experimentally examined. In the present study, the increased circulatory fibrinogen caused by LPS alone was further significantly enhanced by its combination with the DEP components (Table 2). Furthermore, anticoagulatory factors such as APC and AT-III were lower in the DEP components + LPS groups, especially in the washed DEP + LPS group, than in the LPS group (Table 2). These findings suggest that pulmonary exposure to DEP, especially to washed DEP, activates coagulatory changes during lung inflammation. Alternatively, on the basis of the present results and the previous reports, the effects of pulmonary exposure to PM or DEP on cardiovascular events might be explained, at least partly, via the activated thrombohemostatic system, including fibrinogen hyperproduction.
DEP reportedly damages vascular endothelial cells in vitro (21). However, in vivo studies to examine the effects of DEP on endothelial damage in the lungs have not been performed. APC suppresses E-selectin–mediated inflammatory cell adhesion to endothelial cells (22, 23). E-selectin is an important vascular adhesion molecule in the early phase of inflammatory reactions induced by LPS (24). On the other hand, vWF is a proper marker of endothelial damage (25). In the present study, washed DEP+LPS significantly decreased APC (Table 2) and significantly elevated the levels of E-selectin (Table 3) as compared with LPS alone. In addition, vWF was significantly higher in the washed DEP+LPS group than in the vehicle group (Table 2). Furthermore, pulmonary permeability of both water and protein was exaggerated in the DEP components + LPS groups, especially in the washed DEP+LPS group, as compared with the vehicle group or the LPS group (Table 1). These findings support the adverse effects of DEP on endothelilal damage in vivo.
Systemic levels of IL-6 and IL-8 play important roles in initiating ischemic vascular diseases (26). In the present study, washed DEP+LPS resulted in substantial increases in circulatory levels of IL-6 and KC compared to LPS alone (Table 3). In mice, KC is the functional homologue of IL-8 in human. These reports and the present results suggest that DEP, especially washed DEP, may be involved in the increased morbidity of ischemic cardiovascular diseases, at least partly through the elevation of circulatory IL-6 and KC.
Exaggeration of lung-to-systemic translocation of LPS correlates with high mortality in rabbit under conventional artificial ventilation (12). However, studies elucidating the direct correlation between lung-to-systemic translocation of LPS, thrombohemostatic changes, endothelial damage, and systemic inflammation have not been done. Previous work by others has shown that the alveolar space is a tight compartment with very little exchange of materials to the systemic circulation (27). In the present study, however, we found that the washed DEP + LPS group showed significantly higher plasma LPS concentrations than the LPS group alone (Table 3). The high LPS concentration was consequently paralleled by the enhanced serum concentrations of fibrinogen, vWF, IL-1ß, IL-6, KC, and E-selectin in overall trends. Our results indicate that washed DEP can enhance LPS translocation from the lung to circulation and may result in the accelerated systemic inflammatory response as well as endothelial damage and thrombohemostatic disturbance.
Pulmonary vascular permeability was also greater in the washed DEP group and the LPS group than in the vehicle group and further enhanced in the washed DEP +LPS group compared to the LPS group in the present study. Furthermore, the presence of red blood cells in the BAL fluid was occasionally observed in the LPS and the DEP-OC + LPS groups, whereas it was frequently seen in the washed DEP + LPS group (Ref. 8 and unpublished observation). Therefore, it might be hypothesized that endothelial–epithelial damage induced by washed DEP and/or LPS and additional damage by infiltrated effector leukocytes allows large molecules such as LPS to pass into the circulation, resulting in synergistic effects on systemic inflammation and coagulopathy. On the other hand, exposure to environmental particles reportedly generates local and systemic oxidative stress, which in turn induces/enhances inflammation and blood coagulation (28). Further, Nemmar and colleagues have demonstrated that nanoparticles instilled intratracheally rapidly diffuse from the lung into the systemic circulation in vivo (29). Therefore, it is also possible that intratracheally instilled particulate compounds of DEP (washed DEP in the present study) enter the circulation by themselves and contribute to high susceptibility against LPS-elicited systemic inflammation and coagulatory disturbance.
Our previous study demonstrated that washed DEP, rather than DEP-OC, predominantly contributed to the aggravation of the LPS-related lung inflammation (8). Interestingly, in that study the aggravated lung inflammation parallels the enhanced circulatory levels of fibrinogen, IL-6, and KC and decreased APC found in the present study. Therefore, we can consider washed DEP, rather than DEP-OC, as the prominent contributors to DEP toxicity also in the present study.
In conclusion, pulmonary exposure to DEP components aggravates coagulatory disturbance, including endothelial damage, systemic inflammation, and endotoxin translocation related to lung inflammation. The effects of DEP on the thrombohemostatic and inflammatory process may play a vital role in the adverse health effects of PM on the sensitive populations who have had cardiovascular and respiratory diseases, including ischemic cardiovascular diseases and respiratory infection.
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Acknowledgments |
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Footnotes |
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Received for publication January 2, 2006. Accepted for publication April 19, 2006.
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References |
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3)-beta-D-glucan in human blood. Clin Chim Acta 226:109–112, 1994.[Medline]This article has been cited by other articles:
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