点击显示 收起
Laboratory of Pneumology (Lung Toxicology) and Center for Molecular and Vascular Biology, K. U. Leuven, Leuven, Belgium
Department of Cell Biology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands
ABSTRACT
Rationale: Inflammation and thrombosis are related via interactions between leukocytes, platelets, the vasculature, and the coagulation system. However, the mechanisms behind these interactions remain poorly understood. Objectives: We have investigated the effects of the well known pulmonary inflammation induced by silica for the development of peripheral thrombogenicity in a hamster model of thrombosis. In addition, the consequences of pulmonary macrophage and circulating monocyte and neutrophil depletion on the thrombogenicity were investigated. Methods: Silica particles (2eC200 e/hamster) were intratracheally instilled, and experimental thrombosis in photochemically induced femoral vein lesions was assessed 24 hours later, in association with cellular infiltration in the lung. Measurements and main results: Intratracheally instilled silica particles (20 and 200 e/hamster) triggered pulmonary inflammation, together with stimulation of peripheral platelet-rich thrombus formation. Both the selective depletion of lung macrophages by intratracheal administration of clodronate liposomes and the combined depletion of circulating monocytes and neutrophils by intraperitoneal injection of cyclophosphamide significantly reduced silica-induced influx of macrophages and neutrophils in bronchoalveolar lavage, and reduced peripheral thrombogenicity. Silica-induced lung inflammation was accompanied by increased neutrophil elastase levels in bronchoalveolar lavage and in plasma. Specific neutrophil elastase inhibition in the lung did not affect lung inflammation but reduced peripheral thrombogenicity. Conclusion: These findings uncover pulmonary macrophageeCneutrophil cross-talk releasing neutrophil elastase into the blood circulation. Elastase, triggering activation of circulating platelets, may then predispose platelets to initiate thrombotic events on mildly damaged vasculature.
Key Words: lung macrophage neutrophil silica particles thrombosis
Both inflammation and thrombosis play a central role in the development of atherothrombosis, the underlying cause of approximately 80% of all sudden cardiac deaths (1, 2). There is growing evidence of extensive cross-talk between inflammation and thrombosis, not only for inflammation leading to activation of thrombotic events but also to show that thrombosis affects inflammatory activity. During these processes, a multitude of interactions are triggered involving different types of cells, such as platelets, leukocytes, endothelial cells, and the coagulation/anticoagulation cascades (2, 3).
On platelet activation in pathologic vascular conditions, polymorphonuclear neutrophils (PMN) may adhere to the growing thrombus, amplifying the thrombotic process by additionally activating platelets (4). Neutrophil adhesion can be accompanied by monocyte/macrophage accumulation, in turn amplifying the inflammatory process (2). PlateleteCleukocyte interactions further support vascular inflammation (5, 6). These inflammatory cellular interactions may take place not only in the systemic circulation (e.g., after contact with infectious agents, such as in sepsis [7], or with noneCself cells, such as during transplant vasculopathy [8]), but they also occur in the lung after exposure to environmental insults, such as particulate air pollution (9, 10). In this context, it has been reported that pulmonary exposure to particles triggers fibrinogen elevation (11, 12), enhances atherosclerosis (13), and increases the risk for platelet-rich thrombosis (14eC18).
We have recently shown that diesel exhaust particles (DEPs) cause lung inflammation accompanied by the development of a peripheral vascular thrombogenic tendency caused by platelet activation. We have also shown that histamine release by pulmonary mast cells plays a major role in triggering these processes (14, 18).
Experimentally, acute exposure to silica particles produces sustained pulmonary inflammation in animal models, characterized by increased macrophage and neutrophil numbers and by damage of lung tissue (19). Therefore, this well established model appeared to be appropriate for the study of the possible consequences of pulmonary macrophage and neutrophil inflammation for extrapulmonary events, such as vascular inflammation and platelet activation.
The hypotheses of this study were as follows: (1) instilled silica particles enhance peripheral vascular thrombosis in a manner similar to that by other particles studied previously (14eC18) and (2) thrombotic effects depend both on pulmonary macrophages and neutrophils. These questions were studied by depleting animals of macrophages or neutrophils by clodronate or cyclophosphamide (CP) pretreatments, respectively. Finally, the roles of neutrophil elastase, as a mediator of platelet activation by neutrophils (20, 21), and histamine were also assessed.
METHODS
This project was reviewed and approved by the Institutional Review Board of the University of Leuven, and experiments were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee.
Silica Particles
Crystalline SiO2 (Min-U-Sil), provided by Professor B. Fubini (Facolte?di Farmacia, Universite?di Torino, Italy), was suspended in sterile pyrogen-free saline (NaCl 0.9%). The median size of particles was around 2 e, as measured by means of a Coulter LS particle-size analyzer (Beckman Coulter, Fullerton, CA) at the Vlaamse Instelling voor Technologisch Onderzoek, Belgium.
To minimize their aggregation, particle suspensions were always sonicated (Branson 1200, van Waters and Rogers, Leuven, Belgium) for 15 minutes and vortexed immediately (< 1 minute) before their dilution and before intratracheal administration. Control hamsters received saline.
Intratracheal Instillation of Particles
Male or female hamsters (Syrian Gold; Iffacredo, Brussels, Belgium) weighing 100 to 110 g were anesthetized with sodium pentobarbital (60 mg/kg intraperitoneally). The tracheal zone was shaved and disinfected with ethanol (70%), and the trachea was exposed for the intratracheal administration of 120 e of saline or silica particles (2, 20, or 200 e/hamster), as well as for the intraperitoneal or intratracheal pretreatment of hamsters with cell-depleting or elastase inhibitor agents.
Experimental Thrombosis Model
Twenty-four hours after intratracheal instillation of particles or saline, in vivo thrombogenesis was assessed, as recently described (17, 22). After induction of anesthesia, hamsters were placed in a supine position on a heating pad at 37°C. A 2-F venous catheter (Portex, Hythe, UK) was inserted in the right jugular vein for the administration of Rose Bengal dye. Thereafter, the right femoral vein was exposed from the surrounding tissue and mounted on a transilluminator. Mild endothelial injury was produced in the femoral vein (17, 22), and thrombus formation and/or disappearance were monitored for 40 minutes under a microscope at 40x magnification (17, 22). The size of the thrombus was expressed in arbitrary units as the total area under the curve, when plotting light intensity against time (23). The hamsters were killed at the end of the recording.
Bronchoalveolar Lavage Fluid Analysis
Twenty-four hours after the intratracheal instillation of particles or vehicle, hamsters were killed with an overdose of sodium pentobarbital. The trachea was cannulated and lungs were lavaged three times with 1.5 ml of sterile NaCl 0.9%. The recovered fluid aliquots were pooled. No difference in the volume of collected fluid was observed between the different groups. Bronchoalveaolar lavage (BAL) fluid was centrifuged (1,000 x g x 10 minutes, 4°C). Cells were counted in a Thoma hemocytometer (van Waters and Rogers) after resuspension of the pellets and staining with 1% gentian violet. The cell differentials were microscopically performed on cytocentrifuge preparations fixed in methanol and stained with Diff Quick (Dade, Brussels, Belgium). The supernatant was stored at eC80°C until further analysis.
Preparation of Liposome-encapsulated Clodronate and Depletion of Alveolar Macrophages
Liposomes composed of phosphatidylcholine and cholesterol (molar ratio 6/1), with or without added dichloromethylene diphosphonate (clodronate; courtesy of Roche Diagnostics GmbH, Mannheim, Germany), were produced as previously described (24). Briefly, 86 mg of phosphatidylcholine and 8 mg of cholesterol were dissolved in 10 ml of chloroform and dried to a film by low-vacuum rotary evaporation. The lipids were rehydrated in 10 ml of saline or in a solution of 2.5 g of clodronate in 10 ml of saline and incubated at room temperature. The liposome suspension was then diluted in 100 ml of saline and centrifuged at 100,000 x g for 30 minutes to remove free clodronate, after which liposomes were resuspended in 4 ml of saline.
Alveolar macrophage depletion was achieved by the intratracheal instillation of 150 e of a liposome-encapsulated clodronate suspension (CL), as described by Koay and coworkers (25). Control hamsters received empty (saline-containing) liposomes (SL). Then, 24 hours later, hamsters were intracheally instilled with silica particles (20 e/ hamster) or saline. After another 24 hours, BAL was performed, and thrombosis experiments were performed as described previously (i.e., 48 hours after SL/CL administration). The extent of lung macrophage and circulating monocyte depletion was assessed by differential cell counting in BAL and blood, respectively.
Depletion of Neutrophils and Circulating Monocytes
In vivo depletion of circulating monocytes and neutrophils was achieved, as described by Lardot and colleagues (26), by a single intraperitoneal injection of CP (20 mg/animal, suspended in 100 e of sterile saline) 3 days before the administration of silica particles or saline. Twenty-four hours after the intratracheal administration of silica particles or saline (i.e., 96 hours after CP administration), the extent of cell depletion was assessed in the BAL and blood by differential cell counting. Platelets were counted on a Cell-Dyn 1800 (Abbott Laboratories, Abbott Park, IL), and thrombosis experiments were performed as described previously.
Histamine Determination in BAL and in Plasma
Histamine concentrations in BAL and in plasma were determined by means of a commercially available radioimmunoassay kit (Immunotech, Marseille, France). The lower limit of detection of this assay was 0.2 nM.
Venous blood samples collected from the abdominal vena cava on ethylenediaminetetraacetic acid (5 mM) were centrifuged (1,000 x g x 10 minutes, 4°C), and plasma samples were stored at eC80°C.
Elastase Determination in BAL and in Plasma
Neutrophil elastase activity in BAL and in plasma was determined using the highly neutrophil elastaseeCspecific chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma, St. Louis, MO) (27). Briefly, samples were incubated in 0.1 M Tris-HCl buffer (pH 8.0), containing 0.5 M NaCl and 1 mM substrate, for 24 hours at 37°C. After incubation, p-nitroaniline was measured spectrophotometrically at 405 nm, and absorbance, corrected for baseline activity, was taken as an index of neutrophil elastase activity.
Elastase Inhibition during Lung Inflammation and Thrombosis
To assess the role of neutrophil elastase on lung inflammation and peripheral thrombosis, hamsters were intratracheally instilled with methoxysuccinyl-alanyl alanyl-prolyl-valine-chloromethylketone (MeOSuc-AAPV-CMK; Calbiochem, Darmstadt, Germany) at a dose of 250 e/animal 10 minutes before silica particle or saline administration. Lung inflammation and thrombosis were assessed as outlined previously.
Statistics
Data are expressed as means ± SEM. Comparisons between groups were performed by the following methods: one-way analysis of variance (ANOVA), followed by Newman-Keuls multiple-range tests; two-way ANOVA, followed by Bonferroni multiple-range tests; or unpaired Student's t tests, as indicated. p Values less than 0.05 were considered significant.
The experiments were performed over a number of weeks. The total numbers of hamster for the control groups and for each treatment group represent pools of hamsters over the entire experimental interval.
RESULTS
Silica Particles Induce Lung Inflammation and Enhance Peripheral Thrombosis
After intratracheal instillation of saline or silica particles, cells in BAL consisted mainly of macrophages and PMN, the remainder of the cells (< 1%) being lymphocytes. The intratracheal instillation of silica particles resulted in a marked cellular influx at doses of 20 and 200 e/hamster but not at 2 e/hamster (Figure 1). Macrophages increased to a comparable degree at 20 e/hamster (fourfold, p < 0.05) and 200 e/hamster (fivefold, p < 0.01; Figure 1a). However, PMN numbers increased 30-fold at 20 e/hamster (p < 0.05) and 230-fold at 200 e/hamster (p < 0.01; Figure 1b).
The intratracheal instillation of silica particles enhanced the thrombus mass formed in a mildly photochemically injured hamster femoral vein 2.7-fold at 20 e/hamster (p < 0.05) and 3.7-fold at 200 e/hamster (p < 0.001; Figure 2); i.e., at those doses that also triggered measurable lung inflammation.
Alveolar Macrophage Depletion Reduces the Silica-induced Peripheral Thrombogenicity
The intratracheal pretreatment of control hamsters with empty liposomes (SL) did not significantly affect the baseline amount of lung macrophages, as measured in BAL (Figures 1a and 3a). Similarly, intratracheal pretreatment of hamsters with SL did not affect the macrophage infiltration induced by silica particles (20 e/hamster; Figures 1a and 3a). Correspondingly, pretreatment with SL had no effect on baseline or silica-induced PMN numbers in BAL (Figures 1b and 3b).
In contrast, the intratracheal administration of CL resulted in a reduction by 70% in the baseline macrophage numbers (SL, n = 4) in BAL fluid compared with SL (n = 4, p < 0.001; Figure 3a). Pretreatment with CL did not block the silica-triggered macrophage lung infiltration entirely, but after silica exposure, macrophage numbers in CL-pretreated hamsters only reached values comparable to baseline values in control animals (Figure 3a). CL pretreatment did not deplete monocytes from the circulation: monocyte numbers in circulating blood were similar after SL pretreatment (2.6 ± 0. 9 x 105/ml blood, n = 4) and CL pretreatment (2.8 ± 0. 6 x 105/ml blood, n = 4) at the time of thrombosis induction. However, pretreatment with CL reduced significantly the influx of PMN in BAL fluid after intratracheal silica (Figure 3b). Because circulating numbers of PMN were not affected by the intratracheal pretreatment with CL (PMN numbers after SL pretreatment: 1.6 ± 0. 4 x 105/ml blood, n = 4; after CL pretreatment: 1.5 ± 0. 3 x 105/ml blood, n = 4, p = not significant), these findings implicate that PMN influx in the lung is secondary to the activation of pulmonary macrophages (28, 29), which attract fewer PMN, when reduced in number.
Although no effect of pretreatment with SL or CL was observed on peripheral thrombus formation in saline-treated hamsters, the pretreatment of hamsters with CL strongly reduced the prothrombotic effects induced 24 hours after the intratracheal silica particle administration (Figure 4).
Depletion of Systemic PMN and Monocytes Inhibits Peripheral Thrombogenicity
Figure 5a shows that, in the saline-treated group, the number of macrophages in BAL was not affected by pretreatment with CP, whereas the expected increase in the numbers of macrophages after intratracheal silica instillation was completely inhibited. After intraperitoneal CP injection, the low PMN numbers in saline-treated hamster lungs were unaffected. However, the PMN influx caused by silica particle administration was strongly reduced after CP pretreatment (Figure 5b).
Without CP pretreatment, the mean numbers of total blood leukocytes in the saline- and silica-treated groups were 20 ± 2 x 105/ml blood (80% lymphocytes, 12% monocytes, and 8% neutrophils) and 22 ± 3 x 105/ml blood (75% lymphocytes, 13% monocytes, and 12% neutrophils), respectively (i.e., silica administration by itself had no impact on circulating monocyte and PMN numbers 24 hours later). CP pretreatment led to an 80% reduction in circulating leukocytes in both the CP plus saline group (3.7 ± 0.5 x 105/ml blood) and the CP plus silica group (4.5 ± 0.6 x 105/ml blood). There were no quantifiable neutrophils among the circulating leukocytes remaining after CP pretreatment. Circulating monocyte numbers in the saline-treated group (0.25 ± 0.03 x 1 05/ml blood, n = 4) were not different from those in the silica-treated group (0.24 ± 0.03 x 105/ml blood, n = 4), but they were reduced 10-fold as a consequence of the CP pretreatment.
Platelet numbers did not change significantly after pretreatment with CP (216 ± 15 x 103/e blood in the saline group; 220 ± 5 x 103/e in the CP + salineeCtreated group; 195 ± 19 x 103/e blood in the silica group; 178 ± 30 x 103/e in the CP + silica group).
CP pretreatment did not affect the extent of thrombosis in saline-treated hamsters, despite the strong reduction of circulating leukocyte numbers. However, pretreatment with CP significantly reduced the silica-induced stimulation of thrombosis (Figure 6).
Histamine and Elastase Determination in BAL and in Plasma
Silica particle administration had no effect on the concentrations of histamine (mean ± SEM, n = 4eC5) in BAL (2.5 ± 0.9 vs. 2.2 ± 0.7 nM, in control animals), and these levels were not affected by pretreatment with CL (2.4 ± 0.8 nM) or with CP (2.5 ± 0.7 nM). Similarly, in plasma, no effect of silica on histamine levels was observed (26.6 ± 6.7 vs. 25.5 ± 8.0 nM, in control animals), neither after pretreatment with CL (29.5 ± 3.6 nM) nor after pretreatment with CP (24.3 ± 2.5 nM).
Silica particles induced a significant increase in neutrophil elastase activity in BAL and in plasma, compared with control hamsters. Pretreatment of hamsters with CL or with CP significantly reduced this increase in BAL and in plasma (Figure 7).
Neutrophil Elastase Inhibition in Lung Inflammation and Thrombosis
The intratracheal pretreatment of control hamsters with MeOSuc-AAPV-CMK did not significantly affect total cell numbers in BAL (Figures 8a and 8b) nor the thrombosis in vivo. No effect of this pretreatment was observed on the silica-induced increase of macrophage or PMN numbers in BAL (Figures 8a and 8b). However, the intratracheal administration of MeOSuc-AAPV-CMK partially but significantly mitigated the silica-induced elevation of the thrombotic response (Figure 8c).
DISCUSSION
We have demonstrated that the intratracheal instillation of silica particles in hamsters leads to significant dose-dependent increases of macrophage and neutrophil numbers in BAL and the development of a prothrombotic tendency in circulating blood. By specifically depleting lung macrophages with CL, we found that both the influx of PMN in BAL and the peripheral thrombotic tendency were abrogated. The depletion of circulating PMN and monocytes by CP also abolished both the cellular influx in BAL and the peripheral thrombotic tendency, despite normal numbers of lung macrophages. Although silica particles did not affect histamine concentrations in BAL or plasma, they caused an increase in neutrophil elastase activity in plasma.
PMN, through the oxidant species and mediators they release, contribute to vessel injury not only by their adherence to endothelium and by diapedesis but also through interactions with platelet receptors such as P-selectin (3, 5, 30). The majority of studies have investigated the impact of inflammation on tissue injury using isolated cells (31, 32), whole blood (33), or at sites of vascular damage linked to the presence of thrombi (34). However, previously reported population-based studies have established that reduced lung function is associated with cardiovascular morbidity and mortality (35, 36). Also, it has been recently shown that particulate air pollution can cause lung inflammation and promotes systemic inflammation, atherosclerosis, and thrombosis (11, 13eC17, 37). The present study investigated the relationship between lung inflammation and thrombosis via the study of interactions between lung macrophages, monocytes, PMN, and platelets, operating in two different compartments (i.e., the respiratory and cardiovascular system). As a cardiovascular endpoint, we used a recently established and validated model of acute thrombosis in the hamster (38). In this photochemical injury model of platelet-rich thrombosis, prothrombotic tendencies can be approached experimentally (17, 23, 39).
To investigate the role of macrophages and PMN in priming platelet activation and thrombus formation, we selected silica particles as a tool to produce lung inflammation within 24 hours (19). In contrast to ultrafine particles (diameter < 0.1 e), which may translocate from the lung into the blood (40eC42), the silica particles used (2 e) were too large to translocate. It is known that extrathoracic structures, such as the liver and spleen, may be affected by exposure to silica; however, these features have been described in the clinical and pathologic literature after long-term exposure to silica particles. Such extrathoracic silicosis is always associated with pulmonary silicosis, and it is generally believed to be "metastatic" through a possible lymphatic spread (43, 44). However, such extrathoracic spread is unlikely to have occurred in the present study because of the time window (24 hours) investigated. Therefore, any systemic effect produced by this type of particle in our model must have resulted predominantly, if not exclusively, from lung inflammation and the passage of mediators released from the lung into the systemic circulation.
Silica particles caused a dose-dependent increase in the number of macrophages and neutrophils in BAL, together with enhanced thrombus formation, most likely caused by peripheral platelet activation as indicated previously.
An effect of silica particles on peripheral thrombosis has not been reported previously, but using other types of particles, we have previously reported that polystyrene ultrafine particles and DEPs cause lung inflammation and the development of peripheral thrombogenicity resulting from circulating platelet activation (14eC17). Therefore, within the time window investigated (24 hours), lung inflammation, after exposure to silica particles, appears to be a common initiating step with other particles such as DEPs.
Alveolar macrophages are the principal phagocytes mediating uptake and degradation of organisms in the lung. In addition to locomotion, phagocytosis, and microbiocidal activities, resident and infiltrating macrophages secrete a variety of chemokines and cytokines responsible for PMN recruitment (45). To assess the role of macrophages in the influx of PMN in the lung and in the development of peripheral thrombosis in response to silica particles, hamsters were depleted by intratracheal pretreatment with CL. Lung macrophage depletion in the present study was comparable to that described by Koay and coworkers (25) after intratracheal administration of CL in mice. Our results demonstrate that the selective depletion of pulmonary macrophages leads to significant inhibition of monocyte and PMN influx on administration of silica. This result confirms a primary role for macrophages in the PMN recruitment.
To further assess the role of lung macrophages, circulating PMN and monocytes were depleted using CP. CP did not affect the number and composition of cells (including macrophages) in BAL of control hamsters, nor did it affect the number of circulating platelets. The degree of thrombosis in saline-treated hamsters was not affected by the CP treatment, demonstrating that the acute thrombotic response to the photochemical injury is independent of leukocyte activation and plateleteCleukocyte interactions. These results are in agreement with studies reported in mice (26), hamsters (46), and pigs (47). However, depletion of monocytes and neutrophils with CP caused a strong inhibition of the silica particleeCdependent peripheral thrombosis. These findings indicate that lung macrophage activation by silica (28, 29) is required to trigger peripheral thrombogenicity, but that the simple activation of macrophages is insufficient to do so in the absence of a further macrophage-mediated influx of monocytes and PMN in the lung. Taken together, these results uncover the primary role of lung macrophages, which are responsible for PMN influx in the lung, but also the essential role of PMN, which further contribute to additional monocyte infiltration. Our depletion approach thus revealed that macrophageeCPMN cross-talk is an essential element in explaining the development of peripheral thrombotic events after instillation of silica particles.
Recently, we found that 24 hours after intratracheal administration of DEPs, histamine concentrations increased in BAL and in plasma, and that the pretreatment of hamsters with diphenhydramine (14), a histamine H1-receptor antagonist, or with sodium cromoglycate (18), a mast cell and basophil stabilizer, abrogated the inflammatory and thrombotic effects by antagonizing histamine H1 receptor or by blocking its release. In the present study, no histamine release was observed in BAL or plasma, thus excluding mast cell and basophil participation in the development of peripheral thrombogenicity. This striking difference between DEPs and silica may be related to the specific surface chemistry of DEPs, which carry organic compounds that may trigger histamine release in mast cells and initiate lung inflammation (48, 49). Therefore, to study the relation between pulmonary infiltrating cells and peripheral platelet activation, the silica model seems simpler, because it does not involve plasma histamineeCdependent leukocyte activation, complicating the study of the relation between lung cells and peripheral platelets. Moreover, circulating leukocyte numbers were not affected after silica administration, excluding possible systemic leukocyte activation.
Neutrophil elastase activity was elevated in BAL and in plasma in response to silica particle administration. Neutrophil elastase activity has been shown to augment on lung injury associated with neutrophil infiltration in alveolar spaces (50). In addition, neutrophil elastase and cathepsin G released from activated neutrophils were reported to contribute to platelet activation in vitro via activation of the platelet receptor PAR-4 (21, 51). To assess the role of neutrophil elastase released in the lung on the observed peripheral thrombotic events, hamsters were intratracheally instilled with MeOSuc-AAPV-CMK, a specific neutrophil elastase inhibitor (52), which has been shown to inhibit elastase-induced acute lung injury in hamsters (52). Our results confirm a potential role for pulmonary elastase in peripheral platelet activation. Furthermore, the intratracheal pretreatment of hamsters with MeOSuc-AAPV-CMK, although not affecting the silica particleeCinduced lung inflammation per se, partially but significantly inhibited the peripheral thrombotic tendency. Both neutrophil elastase and cathepsin G have been proposed as mediators of platelet activation by neutrophils (20, 21); the importance of each enzyme separately will have to be evaluated for the priming of platelets.
In conclusion, our findings provide novel evidence for a critical role of macrophageeCneutrophil cross-talk during lung inflammation, leading to the release of neutrophil elastase into the systemic circulation. Neutrophil enzymes may be responsible for the priming of platelet activation and contribute to the development of a thrombotic tendency, when such primed platelets encounter a (mildly) injured vessel wall.
Acknowledgments
The authors thank Professor Jos Vermylen for critically reading the manuscript.
REFERENCES
Albert CM, Ma J, Rifai N, Stampfer MJ, Ridker PM. Prospective study of C-reactive protein, homocysteine, and plasma lipid levels as predictors of sudden cardiac death. Circulation 2002;105:2595eC2599.
Libby P. Inflammation in atherosclerosis. Nature 2002;420:868eC874.
Levi M, van der Poll P, Buller HR. Bidirectional relation between inflammation and coagulation. Circulation 2004;119:2698eC2704.
Hagberg IA, Roald HE, Lyberg T. Adhesion of leukocytes to growing arterial thrombi. Thromb Haemost 1998;80:852eC858.
Theilmeier G, Lenaerts T, Remacle C, Collen D, Vermylen J, Hoylaerts MF. Circulating activated platelets assist THP-1 monocytoid/endothelial cell interaction under shear stress. Blood 1999;94:2725eC2734.
Kawasaki T, Dewerchin M, Lijnen HR, Vreys I, Vermylen J, Hoylaerts MF. Mouse carotid artery ligation induces platelet-leukocyte-dependent luminal fibrin, required for neointima development. Circ Res 2001;88:159eC166.
Levi M, Keller TT, van Gorp E, ten Cate H. Infection and inflammation and the coagulation system. Cardiovasc Res 2003;60:26eC39.
Pethig K, Heublein B, Kutschka I, Haverich A. Systemic inflammatory response in cardiac allograft vasculopathy—high-sensitive C-reactive protein is associated with progressive luminal obstruction. Circulation 2000;102:233eC236.
Peters A, Dockery DW, Muller JE, Mittleman MA. Increased particulate air pollution and the triggering of myocardial infarction. Circulation 2001;103:2810eC2815.
Johnson RL. Relative effects of air pollution on lungs and heart. Circulation 2004;109:5eC7.
Salvi S, Blomberg A, Rudell B, Kelly F, Sandstrom T, Holgate ST, Frew A. Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers. Am J Respir Crit Care Med 1999;159:702eC709.
Ghio AJ, Kim C, Devlin RB. Concentrated ambient air particles induce mild pulmonary inflammation in healthy human volunteers. Am J Respir Crit Care Med 2000;162:981eC988.
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;39:935eC942.
Nemmar A, Nemery B, Hoet PHM, Vermylen J, Hoylaerts MF. Pulmonary inflammation and thrombogenicity caused by diesel particles in hamsters: role of histamine. Am J Respir Crit Care Med 2003;168:1366eC1372.
Nemmar A, Hoet PH, Dinsdale D, Vermylen J, Hoylaerts MF, Nemery B. Diesel exhaust particles in lung acutely enhance experimental peripheral thrombosis. Circulation 2003;107:1202eC1208.
Nemmar A, Hoylaerts M, Hoet PH, Vermylen J, Nemery B. Size effect of intratracheally instilled ultrafine particles on pulmonary inflammation and vascular thrombosis. Toxicol Appl Pharmacol 2003;186:38eC45.
Nemmar A, Hoylaerts MF, Hoet PHM, Dinsdale D, Smith T, Xu H, Vermylen J, Nemery B. Ultrafine particles affect experimental thrombosis in an in vivo hamster model. Am J Respir Crit Care Med 2002;166:998eC1004.
Nemmar A, Hoet PHM, Vermylen J, Nemery B, Hoylaerts MF. Pharmacological stabilization of mast cells abrogates late thrombotic events induced by diesel exhaust particles in hamsters. Circulation 2004;110:1670eC1677.
Fubini B, Hubbard A. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radic Biol Med 2003;34:1507eC1516.
Trumel C, Si-Tahar M, Balloy V, Chignard M, Chap H, Payrastre B, Plantavid M, Pidard D. Phosphoinositide 3-kinase inhibition reverses platelet aggregation triggered by the combination of the neutrophil proteinases elastase and cathepsin G without impairing alpha(IIb)beta(3) integrin activation. FEBS Lett 2000;484:184eC188.
SiTahar M, Pidard D, Balloy V, Moniatte M, Kieffer N, VanDorsselaer A, Chignard M. Human neutrophil elastase proteolytically activates the platelet integrin alpha(IIb)beta(3) through cleavage of the carboxyl terminus of the alpha(IIB) subunit heavy chain—involvement in the potentiation of platelet aggregation. J Bioll Chem 1997;272:11636eC11647.
Kawasaki T, Kaida T, Arnout J, Vermylen J, Hoylaerts MF. A new animal model of thrombophilia confirms that high plasma factor VIII levels are thrombogenic. Thromb Haemost 1999;81:306eC311.
Stockmans F, Stassen JM, Vermylen J, Hoylaerts MF, Nystrom A. A technique to investigate mural thrombus formation in small arteries and veins: I. Comparative morphometric and histological analysis. Ann Plast Surg 1997;38:56eC62.
Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 1994;174:83eC93.
Koay MA, Gao X, Washington MK, Parman KS, Sadikot RT, Blackwell TS, Christman JW. Macrophages are necessary for maximal nuclear factor-kappa B activation in response to endotoxin. Am J Respir Cell Mol Biol 2002;26:572eC578.
Lardot C, Delos M, Lison D. Upregulation of urokinase in alveolar macrophages and lung tissue in response to silica particles. Am J Physiol Lung Cell Mol Physiol 1998;18:L1040eCL1048.
Yoshimura K, Nakagawa S, Koyama S, Kobayashi T, Homma T. Roles of neutrophil elastase and superoxide anion in leukotriene B-4-induced lung injury in rabbit. J Appl Physiol 1994;76:91eC96.
Driscoll KE, Lindenschmidt RC, Maurer JK, Higgins JM, Ridder G. Pulmonary response to silica or titanium-dioxide: inflammatory cells, alveolar macrophage-derived cytokines, and histopathology. Am J Respir Cell Mol Biol 1990;2:381eC390.
Tao F, Kobzik L. Lung macrophage-epithelial cell interactions amplify particle-mediated cytokine release. Am J Respir Cell Mol Biol 2002;26:499eC505.
Willerson JT, Ridker PM. 2004. Inflammation as a cardiovascular risk factor. Circulation 109:II-2eCII-10.
Evangelista V, Manarini S, Rotondo S, Martelli N, Polischuk R, McGregor JL, deGaetano G, Cerletti C. Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18. Blood 1996;88:4183eC4194.
Evangelista V, Manarini S, Sideri R, Rotondo S, Martelli N, Piccoli A, Totani L, Piccardoni P, Vestweber D, de Gaetano G, et al. Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule. Blood 1999;93:876eC885.
Li NL, Hu H, Lindqvist M, Wikstrom-Jonsson E, Goodall AH, Hjemdahl P. Platelet-leukocyte cross talk in whole blood. Arterioscler Thromb Vasc Biol 2000;20:2702eC2708.
Coller BS. Binding of abciximab to alpha V beta 3 and activated alpha M beta 2 receptors: with a review of platelet-leukocyte interactions. Thromb Haemost 1999;82:326eC336.
Coultas DB, Mapel D, Gagnon R, Lydick E. The health impact of undiagnosed airflow obstruction in a national sample of United States adults. Am J Respir Crit Care Med 2001;164:372eC377.
Engstrom G, Wollmer P, Hedblad B, Juul-Moller S, Valind S, Janzon L. Occurrence and prognostic significance of ventricular arrhythmia is related to pulmonary function—a study from "men born in 1914," Malmo, Sweden. Circulation 2001;103:3086eC3091.
Nemmar A, Hoet PH, Vermylen J, Nemery B, Hoylaerts M. Pharmacological stabilization of mast cells abrogates late thrombotic events induced by diesel exhaust particles in hamsters. Circulation 2004;110:1670eC1677.
Kawasaki T, Kaida T, Arnout J, Vermylen J, Hoylaerts MF. A new animal model of thrombophilia confirms that high plasma factor VIII levels are thrombogenic. Thromb Haemost 1999;81:306eC311.
Matsuno H, Uematsu T, Nagashima S, Nakashima M. Photochemically induced thrombosis model in rat femoral artery and evaluation of effects of heparin and tissue-type plasminogen activator with use of this model. J Pharmacol Methods 1991;25:303eC317.
Nemmar A, Hoet PH, Vanquickenborne B, Dinsdale D, Thomeer M, Hoylaerts MF, Vanbilloen H, Mortelmans L, Nemery B. Passage of inhaled particles into the blood circulation in humans. Circulation 2002;105:411eC414.
Nemmar A, Vanbilloen H, Hoylaerts MF, Hoet PH, Verbruggen A, Nemery B. Passage of intratracheally instilled ultrafine particles from the lung into the systemic circulation in hamster. Am J Respir Crit Care Med 2001;164:1665eC1668.
Oberdorster G, Sharp Z, Atudorei V, Elder A, Gelein R, Lunts A, Kreyling W, Cox C. Extrapulmonary translocation of ultrafine carbon particle following whole-body inhalation exposure of rats. J Toxicol Environ Health A 2002;65:1531eC1543.
Craighead JE, Abraham JL, Churg A, Green FHY, Kleinerman J, Pratt PC, Seemayer TA, Vallyathan V, Weill H. The pathology of asbestos-associated diseases of the lungs and pleural cavities—diagnostic-criteria and proposed grading schema—report of the Pneumoconiosis Committee of the College-of-American-Pathologists and the National-Institute-for-Occupational-Safety-and-Health. Arch Pathol Lab Med 1982;106:544eC597.
Calvert GM, Rice FL, Boiano JM, Sheehy JW, Sanderson WT. Occupational silica exposure and risk of various diseases: an analysis using death certificates from 27 states of the United States. Occup Environ Med 2003;60:122eC129.
Thomas CF, Limper AH. Pneumocystis pneumonia. N Engl J Med 2004;350:2487eC2498.
Bertuglia S, Colantuoni A. Protective effects of leukopenia and tissue plasminogen activator in microvascular ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 2000;278:H755eCH761.
Merhi Y, Llacoste L, Lam JYT. Neutrophil implications in platelet deposition and vasoconstriction after deep arterial injury by angioplasty in pigs. Circulation 1994;90:997eC1002.
Devouassoux G, Saxon A, Metcalfe DD, Prussin C, Colomb MG, Brambilla C, Diaz-Sanchez D. Chemical constituents of diesel exhaust particles induce IL-4 production and histamine release by human basophils. J Allergy Clin Immunol 2002;109:847eC853.
Kepley CL, Lauer FT, Oliver JM, Burchiel SW. Environmental polycyclic aromatic hydrocarbons, benzo(a) pyrene (BaP) and BaP-quinones, enhance IgE-mediated histamine release and IL-4 production in human basophils. Clin Immunol 2003;107:10eC19.
Kawabata K, Hagio T, Matsuoka S. The role of neutrophil elastase in acute lung injury. Eur J Pharmacol 2002;451:1eC10.
Goel MS, Diamond SL. Neutrophil cathepsin G promotes prothrombinase and fibrin formation under flow conditions by activating fibrinogen-adherent platelets. J Biol Chem 2003;278:9458eC9463.
Fletcher DS, Osinga DG, Hand KM, Dellea PS, Ashe BM, Mumford RA, Davies P, Hagmann W, Finke PE, Doherty JB, et al. A comparison of alpha-1-proteinase inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone and specific beta-lactam inhibitors in an acute model of human polymorphonuclear leukocyte elastase-induced lung hemorrhage in the hamster. Am Rev Respir Dis 1990;141:672eC677.