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American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1329-1335, (2002)
© 2002 American Thoracic Society

Original Article

Diesel Exhaust Particles Enhance Lung Injury Related to Bacterial Endotoxin through Expression of Proinflammatory Cytokines, Chemokines, and Intercellular Adhesion Molecule-1

Hirohisa Takano, Rie Yanagisawa, Takamichi Ichinose, Kaori Sadakane, Shin Yoshino, Toshikazu Yoshikawa and Masatoshi Morita

National Institute for Environmental Studies, Tsukuba; First Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto; Department of Health Science, Oita University of Nursing and Health Sciences, Oita; and Department of Pharmacology, Kobe Pharmaceutical University, Kobe, Japan

Correspondence and requests for reprints should be addressed to Hirohisa Takano, Pathophysiology Research Team, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-0053, Japan. E-mail: htakano{at}nies.go.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidemiologic studies demonstrate acute and serious adverse effects of particulate air pollution on respiratory health, especially in people who are susceptible to bacterial infection. However, the underlying mechanism remains to be elucidated. To provide experimental evidence for the epidemiologic data, we determined the effects of diesel exhaust particles (DEP), major participants in particulate pollutants, on lung injury related to bacterial endotoxin in mice. Intratracheal instillation of DEPs synergistically enhanced lung injury related to endotoxin from gram-negative bacteria, which was characterized by neutrophil sequestration, interstitial edema, and alveolar hemorrhage. In the presence of endotoxin, DEPs further activated the nuclear translocation of p65 subunit of nuclear factor-{kappa}B (NF-{kappa}B) in the lung and increased the lung expression of intercellular adhesion molecule-1, interleukin-1ß, macrophage chemoattractant protein-1, keratinocyte chemoattractant (KC), macrophage inflammatory protein-1{alpha}, and Toll-like receptors. DEPs given alone increased the lung expression of Toll-like receptor 4 and the nuclear localization of p50 subunit of NF-{kappa}B. The combined exposure to DEPs and endotoxin decreased nuclear localization of CCAAT/enhancer binding protein ß. These results provide the first experimental evidence that DEPs enhance neutrophilic lung inflammation related to bacterial endotoxin. The enhancement is mediated by the induction of proinflammatory molecules, likely through the expression of Toll-like receptors and the activation of p65-containing dimer(s) of NF-{kappa}B, such as p65/p50.

Key Words: acute lung injury • chemokines • transcription factors • lipopolysaccharide • diesel exhaust particles


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Short-term exposure to increased ambient amounts of particulate matter (PM) is associated with an increase in morbidity and daily mortality for cardiopulmonary conditions, especially in industrialized cities (1, 2). A plausible biologic mechanism linking short-term particulate exposure and pathophysiologic effects has not been established. One intriguing aspect of the epidemiologic data is that health effects of PM are primarily seen in people with predisposing factors, including bronchitis, asthma, chronic obstructive pulmonary diseases, pneumonia, compromised immune systems, and age over 65 years old (1). The coarse fractions of PM (PM10: 10 µm > particles > 2.5 µm) are dominated by natural sources, whereas the fine fractions (PM2.5: particles < 2.5 µm) are dominated by emissions that are chiefly from the combustion of fossil fuels (3). Epidemiologically, PM2.5 is more closely associated with mortality and adverse respiratory health effects than PM10 (4, 5). Diesel exhaust particles (DEPs) with a diameter of less than 1 µm, derived from diesel engine-powered automobiles, are major constituents of the atmospheric PM2.5 in metropolitan areas (6). DEPs or diesel exhaust has been linked to lung cancer, pulmonary fibrosis, chronic alveolitis, bronchitis (7), edematous changes (8), and airway inflammation with hyperresponsiveness (9). In addition, we have recently reported that DEPs or diesel exhaust enhances the manifestation of allergic asthma in a variety of murine models (10–13). These experimental data, however, cannot sufficiently explain the epidemiologic results that short-term exposure within a few days to increased ambient amounts of PM causes serious health effects, including death in the people with the predisposing factors (14, 15).

A glycolipid of gram-negative bacteria, known as endotoxin or lipopolysaccharide (LPS), stimulates host cells through activation of transcription factors (16). In animal models, intratracheal administration of LPS causes lung cytokine production, neutrophil influx, and lung injury (17). Concentrations of LPS in bronchoalveolar lavage fluid (BALF) are elevated in microbiologically confirmed gram-negative pneumonia (18). LPS is present in BALF of patients with acute respiratory distress syndrome (19), which is often associated with circulatory failure and fatal outcome. We also studied whether DEP instilled intratracheally enhances the lung inflammation related to the intratracheal inoculation of LPS in mice. In particular, we studied the role of proinflammatory cytokines, chemokines, nuclear transcription factors, and Toll-like receptors in the lung.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
We used ICR male mice that have been reported to be highly responsive to LPS rather than Balb/c, C3H/He, and A/J mice (20). They were fed and housed as previously described (10).

Study Protocol
The animals were randomized into four groups. The vehicle group received phosphate-buffered saline at pH 7.4 (Nissui Pharmaceutical Co., Tokyo, Japan) containing 0.05% Tween 80 (Nacalai Tesque, Kyoto, Japan). The LPS group received 100 µg of LPS (Escherichia coli B55: 05; Difco Lab, Detroit, MI) dissolved in the identical vehicle. The DEP group received 250 µg of suspended DEPs in the same vehicle. The suspension was sonicated for 3 minutes with an ultrasonic disrupter (UD-201; Tomy Seiko, Tokyo, Japan). The LPS + DEP group received the combined treatment of LPS and DEPs. In each group, vehicle, LPS, DEPs, or LPS + DEPs was 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). The endotoxin activity, which was determined by Limulus Amebocyte Lysate assay (Seikagaku-kogyo, Tokyo, Japan), was lower than the detection limit of 0.001 EU/ml in the vehicle solutions and DEP solutions (n = 8 in each group). The activity in LPS solutions and LPS + DEP solutions was 959 x 104 ± 41 x 104 and 949 x 104 ± 17 x 104 EU/ml, respectively, which was not significantly different between the two groups (n = 8 in each group).

Collection of DEPs
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 engine was operated by using standard diesel fuel at a speed of 1,500 rpm under a load of 10 torque (kg/m). DEPs were collected as previously described (9). The mass median aerodynamic diameter of the DEPs, as determined by Anderson Air Sampler (Shibata Science Technology, Tokyo, Japan), was 0.4 µm. Most of the particles were globular in shape.

Histologic Evaluation, Lung Water Content, Bronchoalveolar Lavage, and Measurement of Soluble Intercellular Adhesion Molecule-1, Cytokines, and Chemokines
The lungs were fixed and stained with hematoxylin and eosin as previously described (10). The lungs were weighed and dried as previously reported (8). The wet lung weight–the dry lung weight/body weight was calculated. BAL and cell counts were conducted as previously described (10). The lungs were homogenized and centrifuged as previously described (10). Enzyme-linked immunosorbent assay for tumor necrosis factor-{alpha} (TNF-{alpha}; Endogen, Cambridge, MA), interleukin-1ß (IL-1ß; Endogen), macrophage inflammatory protein-1{alpha} (MIP-1{alpha}; R&D Systems, Minneapolis, MN), macrophage chemoattractant protein-1{alpha} (MCP-1{alpha}; R&D Systems), and keratinocyte chemoattractant (KC) (R&D Systems) in the lung tissue supernatants and soluble intercellular adhesion molecule-1 (s-ICAM-1; Endogen) in the serum were conducted according to the manufacturer's instruction.

RNA Isolation and Polymerase Chain Reaction
Lung tissue was homogenized in ISOGEN (Nippon Gene, Tokyo, Japan), and total RNA was extracted according to the manufacturer's instructions. cDNA synthesis and polymerase chain reactions (PCRs) were conducted according to the manufacturer's protocol (Perkin-Elmer, Foster City, CA). The conditions for PCRs are shown in Table E1 (see online data supplement). PCR products were separated in agarose gels containing ethidium bromide and were located by fluorescence under ultraviolet light. For quantification, PCR bands in photographs of the gel were scanned by a densitometer linked to a computer analysis system (Densitograph, Atto, Japan). We used a published technique to measure relative differences in transcript amounts after normalization against levels of the reference gene ß-actin (21).

Preparation of Nuclear and Cytoplasmic Protein and Western Blot Analysis
We prepared nuclear and cytoplasmic protein extracts using methods described previously (22). Nuclear and cytoplasmic proteins were electrophoresed and blotted onto polyvinylidene difluoride membrane. The membrane was incubated with a rabbit anti-p65 subunit of nuclear factor-{kappa}B (NF-{kappa}B) (Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit anti-p50 subunit of NF-{kappa}B (Upstate Biotechnology, Lake Placid, NY), or a rabbit anti-CCAAT/enhancer binding protein ß (C/EBPß) (Santa Cruz Biotechnology). After washes, the membrane was incubated with horseradish peroxidase-conjugated donkey anti-rabbit. After washes, the membrane was developed using the enhanced chemiluminescence light detecting kit (ECL-plus; Amersham Pharmacia, Buckinghamshire, UK) according to the manufacturer's instructions. For quantification, bands in photographs were scanned by a densitometer linked to a computer analysis system (Densitograph, Atto, Japan).

Statistics
Data were reported as mean ± SEM. Differences among groups were determined using analysis of variance (Stat View, version 4.0; Abacus Concepts, Berkeley, CA) as previously described (10).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DEPs Enhance Lung Injury Related to Bacterial Endotoxin
We used ICR male mice randomized into the four experimental groups that received intratracheal inoculation of vehicle, LPS alone (100 µg), DEPs alone (250 µg), or LPS and DEPs in combination. To determine the effects of DEPs on the lung injury related to bacterial endotoxin, we evaluated the lung specimens stained with hematoxylin and eosin 24 hours after the intratracheal instillation. The infiltration of neutrophils was slight in the DEP group (Figure 1A) , but the LPS group showed moderate infiltration of neutrophils (Figure 1B). The combined treatment of LPS and DEP led to a marked recruitment of neutrophils, interstitial edema, and alveolar hemorrhage (Figure 1C). Vehicle administration alone caused no histologic changes (Figure 1D).






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  		Figure 1. DEPs enhance histologic changes in lung related to bacterial endotoxin (LPS). We used ICR male mice randomized into the four experimental groups that received intratracheal inoculation of vehicle, LPS, DEPs, or LPS and DEPs in combination. We evaluated the lung specimens stained with hematoxylin and eosin 24 hours after the intratracheal instillation. Although the infiltration of neutrophils was slight in DEP-treated mice (A), LPS treatment caused the moderate infiltration of neutrophils (B). Combined treatment of LPS and DEPs resulted in a marked recruitment of neutrophils, interstitial edema, and alveolar hemorrhage (C), whereas vehicle administration caused no histologic change (D). Original magnification, x 25.

 
To quantitate the pulmonary edema, we evaluated the lung water content in the four experimental groups 24 hours after the intratracheal treatments (Figure 2A) . The lung water content was significantly greater in the LPS group than in the vehicle group (p < 0.01) or in the DEP group (p < 0.05). The combined administration of LPS and DEPs caused a significant increase in the lung water content as compared with LPS or DEPs given alone, even at a dose of 200 or 500 µg, respectively (p < 0.01).



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  		Figure 2. DEPs enhance pulmonary edema and neutrophilic inflammation related to LPS. To quantitate pulmonary edema, we evaluated the lung water content in the four experimental groups 24 hours after the intratracheal treatments (A). *p < 0.01 versus other groups; **p < 0.01 versus vehicle. Values are the mean ± SEM of six animals in each group. To quantitate the magnitude of neutrophilic lung inflammation, we investigated the cellular profile of BALF 24 hours after the intratracheal instillation. We evaluated the number of macrophages (B) and neutrophils (C) in BALF. The total cell count was determined on a fresh fluid specimen using a hemocytometer. Differential cell counts were assessed on cytologic preparations stained with Diff-Quik. #p < 0.01 versus vehicle and p < 0.05 versus LPS; *p < 0.01 versus other groups. Values are the mean ± SEM of seven animals in each group.

 
To quantitate the magnitude of neutrophilic lung inflammation, we investigated the cellular profile of BALF 24 hours after the intratracheal instillation. The number of macrophages in BALF (Figure 2B) was significantly greater in the LPS + DEP group than in the vehicle group (p < 0.01) or in the LPS group (p < 0.05). A marked increase in the number of neutrophils was observed in the LPS group as compared with the vehicle or DEP group (p < 0.01). A combined instillation of LPS and DEP caused a further two-fold increase in BAL neutrophils as compared with LPS instillation (p < 0.01). Although the DEP administration caused an increase in neutrophils as compared with vehicle administration alone, the effects did not achieve statistical significance (Figure 2C).

DEPs Enhance Expression of Proinflammatory Molecules Related to Bacterial Endotoxin
To elucidate the role of proinflammatory molecules in enhancing effects of DEPs on the lung injury related to LPS, we measured protein amounts of TNF-{alpha}, IL-1ß, MIP-1{alpha}, MCP-1, and KC in the lung tissue supernatants, and s-ICAM-1 in the serum, 24 hours after the intratracheal treatments. The protein concentrations of IL-1ß (Figure 3A) , MIP-1{alpha} (Figure 3B), and s-ICAM-1 (Figure 3C) were significantly elevated in the LPS group as compared with the vehicle group (p < 0.01), and further increases were observed in the LPS + DEP group as compared with the LPS group (p < 0.01 for IL-1ß and MIP-1{alpha}, p < 0.05 for s-ICAM-1). The results with MCP-1 (see online data supplement, Figure E1) and KC (see online data supplement, Figure E2) were similar to those with MIP-1{alpha}. Although DEPs alone increased the amounts of IL-1ß and chemokines, the results did not achieve statistical significance. Among these proinflammatory proteins, the changes in MIP-1{alpha} most closely paralleled the increase in pulmonary neutrophilic inflammation and edema. Furthermore, the enhanced protein expression of IL-1ß, ICAM-1, and MIP-1{alpha} was associated with the increased expression of the mRNA 4 hours after the intratracheal treatments (Figure 4) . The combined treatment with LPS and DEP significantly increased the protein concentrations of TNF-{alpha} as compared with the vehicle treatment (p < 0.05; Figure 3D).




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  		Figure 3. DEPs enhance protein expression of proinflammatory molecules related to LPS. In the four experimental groups, we measured protein concentrations of IL-1ß (A), MIP-1{alpha} (B), and TNF-{alpha} (D) in the lung tissue supernatants and s-ICAM-1 (C) in the serum 24 hours after the intratracheal treatments using ELISA. #p < 0.01 versus other groups; ##p < 0.05 versus LPS; ###p < 0.01 versus vehicle and DEPs; ***p < 0.05 versus vehicle. Values are the mean ± SEM of nine animals in each group.

 


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  		Figure 4. DEPs enhance mRNA expression of proinflammatory molecules in lung related to LPS. We examined the lung expression of mRNA for IL-1ß (A), ICAM-1 (B), and MIP-1{alpha} (C) 4 hours after the intratracheal treatments in the four experimental groups using semiquantitative reverse transcription-polymerase chain reaction. The amplification cycles were 32 and 35 for IL-1ß; 20, 24, and 27 for ICAM-1; and 27, 30, and 35 for MIP-1{alpha}. In each case, ß-actin was amplified by the same protocol. For quantification, PCR bands in photographs of the gel were scanned by a densitometer linked to a computer analysis system. We used a published reverse transcription-polymerase chain reaction technique to measure relative differences in transcript amounts after normalization against amounts of the reference gene ß-actin. The top panel shows actual reverse transcription-polymerase chain reaction gel pictures of IL-1ß (A), ICAM-1 (B), and MIP-1{alpha} (C) with ß-actin. The bottom panel shows band density ratios of arbitrary density units for IL-1ß (A), ICAM-1 (B), and MIP-1{alpha} (C) to ß-actin. Each ratio represents the mean ± SEM of at least three animals per group.

 
DEPs Modulate Activation of Nuclear Transcription Factors Related to Bacterial Endotoxin
To examine the role of nuclear transcription factors in the increased expression of the chemokines, IL-1ß, and ICAM-1, we investigated the nuclear localization of NF-{kappa}B and C/EBPß in the lung 2 hours after the intratracheal administration. As compared with vehicle treatment, LPS treatment caused intense nuclear localization of the p65 subunit of NF-{kappa}B, and the combined treatment with LPS and DEPs resulted in more intense nuclear localization of p65 (Figure 5A) . In contrast, the cytoplasmic localization of p65 was decreased in the LPS + DEP group as compared with the other groups (Figure 5B). On the other hand, the nuclear localization of the p50 subunit of NF-{kappa}B was increased in the DEP group and the LPS + DEP group as compared with the vehicle group (Figure 5C). The cytoplasmic localization of p50 was not significantly different among the four experimental groups (data not shown). The nuclear expression of C/EBPß was decreased by the combined treatment with LPS and DEP as compared with the other treatments (Figure 5D). c-Jun was phosphorylated in the DEP, LPS, and LPS + DEP groups but not in the vehicle group, whereas the nuclear localization of phosphorylated protein was not different among the three experimental groups (data not shown).



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  		Figure 5. DEPs modulate activation of nuclear transcription factors related to LPS. In the four experimental groups, we investigated nuclear translocation of NF-{kappa}B (A–C) and nuclear localization of C/EBPß (D) 2 hours after intratracheal administration using Western blot analysis. LPS treatment caused intense nuclear localization of p65 subunit of NF-{kappa}B and the combined treatment of LPS and DEPs resulted in more intense nuclear localization of p65 (A). In contrast, the cytoplasmic localization of p65 was decreased in the LPS + DEP group as compared with the other groups (B). The nuclear localization of p50 subunit of NF-{kappa}B was increased in the DEP group and the LPS + DEP group as compared with the vehicle group (C). The nuclear expression of C/EBPß was decreased by the combined treatment of LPS and DEP as compared with the other treatments (D). The top panel shows actual membrane pictures of p65 (A and B), p50 (C), and C/EBPß (D). The bottom panel shows band density for p65 (A, B), p50 (C), and C/EBPß (D). Each density represents the mean ± SEM of at least three animals per group.

 
DEPs Modulate Expression of Toll-like Receptors Related to Bacterial Endotoxin
We also evaluated the lung expression of Toll-like receptors, important receptors for bacterial endotoxin, 4 hours after the intratracheal treatments. The expression of Toll-like receptor 2 was increased by LPS treatment in the presence or absence of DEP, but not by vehicle or DEP treatment (Figure 6A) . The expression of Toll-like receptor 4, however, was elevated in the DEP, LPS, and LPS + DEP groups as compared with the vehicle group (Figure 6B). The expression of Toll-like receptor 4 was more intense in the DEP group and the LPS + DEP group than in the LPS group.



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  		Figure 6. DEPs modulate expression of Toll-like receptors in the presence or absence of LPS. We evaluated the lung expression of Toll-like receptor 2 (A) and 4 (B) in the four experimental groups 4 hours after the intratracheal treatments using semiquantitative reverse transcription-polymerase chain reaction. The amplification cycles were 20, 25, and 30 for Toll-like receptor 2 and 20, 25, and 30 for Toll-like receptor 4. In each case, ß-actin was amplified by the same protocol. For quantification, PCR bands in photographs of the gel were scanned by a densitometer linked to a computer analysis system. We used a published reverse transcription-polymerase chain reaction technique to measure relative differences in transcript amounts after normalization against amounts of the reference gene ß-actin. The top panel shows actual reverse transcription-polymerase chain reaction gel pictures of Toll-like receptor 2 (A) and 4 (B) with ß-actin. The bottom panel shows band density ratios of arbitrary density units for Toll-like receptor 2 (A) and 4 (B) to ß-actin. Each ratio represents the mean ± SEM of at least three animals per group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study has demonstrated that DEP instilled intratracheally enhances lung inflammation related to bacterial endotoxin. The lung inflammatory response comprises neutrophilic inflammation, interstitial edema, and alveolar hemorrhage. This enhancement is paralleled by the increased local expression of IL-1ß, ICAM-1, and chemokines, such as MIP-1{alpha}, MCP-1, and KC. Nuclear translocation of the p65 subunit of NF-{kappa}B in vivo is concomitant with the enhancing effects of DEPs on LPS-related injury and on the expression of these proinflammatory molecules with respect to overall trends. Furthermore, DEPs modulate nuclear localization of C/EBPß and p50 subunit of NF-{kappa}B and expression of Toll-like receptors in the presence or absence of LPS.

To our knowledge, this is the first demonstration that DEPs dramatically enhance the lung inflammation related to endotoxin from gram-negative bacteria. The combined administration of LPS at a dose of 100 µg and DEPs at a dose of 250 µg caused a significant increase in the lung water content as compared with LPS or DEPs administered alone at a dose of 200 or 500 µg, respectively (Figure 2A). In addition, the number of neutrophils in BALF in the LPS + DEP group was much greater than the summed number of neutrophils in the LPS group and the DEP groups (Figure 2C). These results suggest that the effects of DEPs on LPS-related neutrophilic lung injury are synergistic rather than additive.

In our previous studies with ICR mice, intratracheal administration of DEPs at a dose of 100 or 200 µg did not induce substantial acute lung injury (9). Additionally, our repetitive intratracheal administration of 100 µg of DEPs once a week for 6 weeks ( a total of 700 µg of DEPs) resulted in the enhancement of local cytokine expression and eosinophilic airway inflammation in the presence of allergen (10). On the basis of these previous data and our preliminary experiments that single intratracheal instillation of DEPs at a dose of 250 µg did not significantly increase lung water content, we chose this dose for these experiments with acute lung injury related to LPS. Diaz-Sanchez and colleagues found that instillation of DEPs at a dose of 300 µg into each nostril enhanced local cytokine expression and Ig production in the presence or absence of allergen (23–25) and that exposure to this dose could occur from standing at a bus stop when a bus starts or being in a freeway tunnel for 10 minutes or more (26). Walley and colleagues (27) administered LPS intratracheally at a dose of 40 µg to CD-1 mice that are sensitive to LPS (20). Because the lung sequestration of neutrophils was not significant in their histologic specimens and because we used larger mice, we chose 100 µg as the intratracheal dose of LPS. Strikingly, the synergistic effects on the lung water content also were observed at combined doses of 10 µg of DEPs and 4 µg of LPS given intratracheally, as compared with DEPs or LPS given alone (data not shown).

The accumulation of leukocytes within the lung in LPS-related inflammation is probably dependent on alterations in leukocyte rigidity, the coordinated expression of proinflammatory cytokines, adhesion molecules, and the establishment of chemotactic gradients via the local generation of chemotactic factors (28). Among the proinflammatory cytokines, IL-1ß is believed to play an important role in LPS-induced acute inflammation because LPS and IL-1ß both cause acute neutrophilic inflammation in the lung after intratracheal injection and IL-1 receptor antagonist inhibits LPS-induced acute inflammation (29). In our experiments, the local expression of IL-1ß was paralleled by the magnitude of neutrophilic inflammation. IL-1ß should be an important proinflammatory cytokine in enhancing the effects of DEPs on the inflammation related to LPS. Although the concentration of TNF-{alpha} protein in the lung was significantly elevated in the LPS + DEP group as compared with the vehicle group, it did not parallel the intensity of the neutrophilic lung injury among the four experimental groups 24 hours after the treatments. Because a previous study has shown that soluble TNF-{alpha} receptor partially reduced the lung injury caused by LPS and because TNF-{alpha} is known to increase after LPS challenge (30), further studies are needed of the role of TNF-{alpha} in DEP-mediated lung inflammation. Pretreatment with DEPs has been reported to decrease the secretion of IL-1ß and TNF-{alpha} from alveolar macrophages challenged with LPS ex vivo 7 days after the pretreatment (31). However, this experiment in vivo clearly shows that simultaneous treatment with DEPs and LPS increases the expression of these cytokines in the whole lung. On the other hand, anti–ICAM-1 antibodies reportedly inhibits both neutrophil sequestration and injury in the lung (32). In this study, the serum concentration of s-ICAM-1 and the mRNA expression of ICAM-1 in the lung were concomitant with the magnitude of inflammation and neutrophil sequestration.

IL-1ß is a potent proinflammatory cytokine that triggers recruitment of adhesion molecules and chemokines, which play a role in the initiation and amplification of inflammatory responses. These are subdivided into C-X-C type, such as IL-8, and C-C type, such as MCP-1 and MIP-1{alpha}. IL-8, MCP-1, and MIP-1{alpha} significantly increase in patients with acute respiratory distress syndrome, regardless of its duration (33). Because an anti–IL-8 treatment has prevented the pulmonary edema with neutrophil infiltration induced by LPS and heat-killed Streptococcus pyogenes, IL-8 is thought to have a significant role in the induction of lung injury associated with LPS (34). Also, anti–MCP-1 antibodies have reduced acute lung injury, suggesting that MCP-1 is a potential mediator (35). A time-dependent increase in MIP-1{alpha} mRNA and protein has been detected in the lung after LPS treatment (36), and pretreatment with anti–MIP-1{alpha} antibody decreased neutrophil accumulation, lung permeability, and ICAM-1 mRNA amounts within the lung after LPS challenge (36). In our experimental groups, the expression of KC, MCP-1, and MIP-1{alpha} in the lung paralleled the lung injury and neutrophilic inflammation. The data suggest that these chemokines are likely to be involved in the enhancing effects of DEPs on LPS-related insults. The concentrations in the lung tissue supernatants of these chemokines, in particular MIP-1{alpha}, in the LPS + DEP group were greater than their summed concentrations in the LPS group and in the DEP group. The synergistic relationship between DEPs and LPS on lung inflammation might be explained, at least partly, by synergistic effects on chemokine expression.

LPS stimulates host cells through activation of transcription factors such as NF-{kappa}B. DNA-binding studies with MIP-1{alpha} gene reveal major nuclear protein binding sites, including NF-{kappa}B and C/EBP (37). NF-{kappa}B activation in the lung after intratracheal instillation of LPS has correlated with cytokine mRNA expression and neutrophilic alveolitis, supporting the idea that NF-{kappa}B activation is a pivotal event in the generation of neutrophilic lung inflammation (22). In this study, nuclear localization of the p65 subunit of NF-{kappa}B in vivo was coincident with the neutrophil influx and the expression of IL-1ß, ICAM-1, and chemokines, which were clearly induced by LPS and further enhanced by the combination with DEPs. In the LPS + DEP group, the decreased cytoplasmic localization of p65 also suggests accelerated nuclear translocation and turnover. DEPs given alone did not increase the nuclear translocation of p65, but enhanced nuclear localization of p50 subunit. It has been reported that transient overexpression of p65 can transactivate the promoter activity of ICAM-1 reporter constructs, whereas p50 overexpression has no effect on the basal amounts of ICAM-1 transcription (38). The combined treatment with LPS and DEPs resulted in the intense nuclear localization of both p65 and p50. The synergistic enhancement in the expression of the proinflammatory molecules, including chemokines in the LPS + DEP group, might be explained by the increased nuclear translocation of p65-containing dimers of NF-{kappa}B, such as p65/p50, which bind to the {kappa}B DNA target site of the enhancer region with a higher affinity than the other dimers (39). On the other hand, it has been reported that overexpression of C/EBPß inhibits basal amounts of ICAM-1 promoter activity and completely abolishes the transactivation effects of p65 when C/EBPß and p65 are cotransfected (38). In this study, the decreased localization of C/EBPß in the LPS + DEP group could augment the transactivation effects of p65, which were significantly increased in this group.

Toll-like receptor mediates signaling by similar Rel-type transcription factors, and a Toll homology domain presumably plays an important role in the activation of the NF-{kappa}B pathway (40). Toll-like receptor 2 has been shown to be involved in LPS signaling (41). Other studies have found that Toll-like receptor 4 is essential for LPS signaling in mice (42, 43). Another group has suggested that mouse Toll-like receptor 2 and mouse Toll-like receptor 4 are both involved in LPS signaling and are differently regulated (44). In this study, the expression of Toll-like receptor 2 was increased in the LPS and the LPS + DEP groups, whereas that of Toll-like receptor 4 was elevated in the DEP and the LPS + DEP groups. The intense activation of NF-{kappa}B by the combined administration of LPS and DEPs might be mediated through increased expression of Toll-like receptors 4 and 2. In addition, DEPs alone increased the expression of Toll-like receptor 4 in the lung.

In conclusion, this study has shown that DEPs enhance lung inflammation related to endotoxin. The enhancement is mediated through the increased local expression of IL-1ß, ICAM-1, and chemokines, in particular MIP-1{alpha}. Expression of Toll-like receptors, activation of NF-{kappa}B, and modification of C/EBPß are cellular mechanisms that may be important in the synergistic effects of DEPs and LPS on lung inflammation.


   FOOTNOTES
 
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form August 28, 2001; accepted in final form January 14, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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