Inhalation Toxicology, 2009; 21(11): 943–951

R e s e a R c h a R t i c l e

Particles from wood smoke and road traffic differently affect the innate immune system of the lung

Mari Samuelsen1, Unni Cecilie Nygaard1, and Martinus Løvik1,2

1Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway, 2Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway

Address for Correspondence: Mari Samuelsen, P.O. Box 4404 Nydalen, NO-0403 Oslo, Norway. e-mail: mari.samuelsen@fhi.no

(Received 10 April 2008; revised 04 September 2008; accepted 30 October 2008)

ISSN 0895-8378 print/ISSN 1091-7691 online © 2009 Informa UK LtdDOI: 10.1080/08958370802590499

abstractThe effect of particles from road traffic and wood smoke on the innate immune response in the lung was studied in a lung challenge model with the intracellular bacterium Listeria monocytogenes. Female Balb/cA mice were instilled intratracheally with wood smoke particles, particles from road traffic collected during winter (studded tires used; St+), and during autumn (no studded tires; St−), or diesel exhaust particles (DEP). Simultaneously with, and 1 or 7 days after particle instillation, 105 bacteria were inoculated intratracheally. Bacterial numbers in the lungs and spleen 1 day after Listeria challenge were determined, as an indicator of cellular activation. In separate experiments, bronchoalveolar lavage (BAL) fluid was collected 4 h and 24 h after particle instillation. All particles tested reduced the numbers of bacteria in the lung 24 h after bacterial inoculation. When particles were given simultaneously with Listeria, the reduction was greatest for DEP, followed by St+ and St−, and least for wood smoke particles. Particle effects were no longer apparent after 7 days. Neutrophil numbers in BAL fluid were increased for all particle exposed groups. St+ and St− induced the highest levels of IL-1β, MIP-2, MCP-1, and TNF-α, followed by DEP, which induced no TNF-α. In contrast, wood smoke particles only increased lactate dehydrogenase (LDH) activity, indicating a cytotoxic effect of these particles. In conclusion, all particles tested activated the innate immune system as determined with Listeria. However, differences in kinetics of anti-Listeria activity and levels of proinflammatory mediators point to cellular activation by different mechanisms.

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Introduction

Particulate matter, when inhaled into the lungs, encoun-ters the innate immune system of the mucosa. The adverse pulmonary effects of air pollution have to a large extent been linked to the capacity of particulate matter to trig-ger inflammation (Frampton, 2006). However, the com-position of ambient air particles is complex. Particle size, surface area, and associated compounds like polycyclic aromatic hydrocarbons (PAH), transition metals, minerals, and organic material such as endotoxin contribute to the inflammatory capacity and other biological effects of acti-vation of the innate immune system (Ball et al., 2000; Ma & Ma, 2002; Schwarze et al., 2007; Soukup & Becker, 2001; Stoeger et al., 2006).

People living in densely populated areas or near roads with high traffic loads often experience exposure to high levels of particulate matter (PM), and especially ultrafine combustion

particles have been linked to cardiopulmonary health prob-lems (Donaldson et al., 2005). In areas where wood is used for heating, wood smoke particles make a considerable contri-bution to particulate air pollution during the winter months. Wood smoke particles may account for as much as 15–40% of fine-sized particulate matter (PM2.5) in some urban areas, comparable to the contribution from vehicle exhaust (Saarikoski et al., 2008; Song et al., 2007; Wu et al., 2007).

Diesel exhaust particles (DEP) are a major constituent of PM2.5 in urban areas (Ho et al., 2006; Zheng et al., 2002), and the respiratory effects of these particles have been extensively studied (Ma & Ma, 2002; Nightingale et al., 2000; Salvi et al., 1999). Observed effects like mucosal inflam-mation and induction of proinflammatory cytokines and reactive oxygen species (ROS) have been attributed to the ultrafine size and metals and PAH content of DEP (Ma & Ma, 2002), characteristics which also apply to wood smoke

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particles (Kocbach et al., 2006). Although there are relatively few publications on pulmonary effects of wood smoke par-ticles, it has been reported that exposure to such particles may cause lung inflammation (Barregard et al., 2007) and lead to adverse health effects similar to traffic-generated PM (Boman et al., 2003; Orozco-Levi et al., 2006). However, some studies exposing rodents subchronically to wood smoke by inhalation have reported only mild effects with regard to pulmonary inflammation (Reed et al., 2006; Tesfaigzi et al., 2005). Interestingly, it was also reported that some of these mild effects were comparable to those observed for diesel exhaust (Barrett et al., 2006; Seagrave et al., 2005).

Previously, we have found that particle size is a major determinant of the allergy adjuvant effect (Nygaard et al., 2004), as well as activation of the innate immune system in the lung (Samuelsen et al., unpublished results), when mice are exposed to chemical-free polystyrene particles of different sizes. In the present study, the same lung exposure model was used to study how different types of combus-tion and traffic-related particles, with different chemistries, activate the innate immune system of the lung. Wood smoke particles were collected directly from a wood stove, while road traffic particles were collected in a tunnel with high traffic load (Kocbach et al., 2006). The road traffic particles were collected during two different seasons, in the winter when studded tires were used (St+), and during autumn with no use of studded tires (St−), giving different amounts of mineral particles in the two samples. DEP were used as an example of vehicle exhaust particles with no mineral parti-cle content. Wood smoke particles, St+, St−, and DEP were instilled intratracheally in Balb/cA mice simultaneously with, or prior to, intratracheal inoculation with the intracel-lular bacterium Listeria monocytogenes. Others have used Listeria to study the influence of particles on the course of infection during the first three or more days after bacterial inoculation (Steerenberg et al., 2004; Yin et al., 2002). While such studies reflect components of both the innate and the adaptive immunity, we wanted solely to study the effect of particles on innate immunity. Therefore, the number of bacteria in the lungs and spleen 1 day after Listeria chal-lenge, before the onset of acquired immunity (North, 1973), was used as an indicator of cell activation. Listeria is very sensitive to cellular activation (Campbell, 1994; Lohmann-Matthes et al., 1994), and this microbe is therefore a suitable tool to investigate aspects of particle effects on the innate immune system. To further investigate how the different par-ticles activated the innate immune system, bronchoalveolar lavage (BAL) fluid was collected 4 h and 24 h after particle exposure, and inflammatory cell influx, proinflammatory mediators, and indicators of cellular and tissue toxicity were measured.

Materials and methods

AnimalsFive to six week-old female Balb/cA mice were obtained from Taconic M&B A/S, Ry, Denmark, and were rested for 1 week

before entering the experiments. The animals were kept, 5–8 animals per cage, on BeeKay bedding (B&K Universal AS, Nittedal, Norway) in type III macrolon cages in filter cabinets. They were given sterilized tap water and pelleted food (RM1; SDS, Essex, UK) ad libitum and were exposed to a 12-h/12-h light/dark cycle.

The experiments were performed in conformity with the laws and regulations controlling experiments with live ani-mals in Norway, and were approved by the Experimental Animal Board under the Ministry of Agriculture in Norway.

ParticlesWood smoke particles and road traffic particles (St+ and St−) were collected and characterized by Kocbach et al. (2006), while DEP (Standard Reference Material® 2975; Industrial Forklift) were obtained from the National Institute of Standards and Technology (Gaithersburg, MD). As previ-ously described (Kocbach et al., 2006), wood smoke par-ticles from the burning of birch wood at high temperature (high burn rate) were collected in 2004 from a commonly used Norwegian type of wood stove with single stage com-bustion (Jøtul 3; Jøtul, Norway), placed in a laboratory at the Norwegian University of Science and Technology (NTNU, Trondheim, Norway). Particles from road traffic were col-lected in a motorway tunnel (Oslo, Norway) in 2004 during autumn before the studded tires season (St−), and dur-ing the winter season when studded tires (St+) were used. Endotoxin levels in the aqueous extract of suspended parti-cles were determined by the Limulus amebocyte lysate QCL-1000 assay (Bio Whittaker, Walkersville, MD) according to the manufacturer’s protocol (Table 1).

The physicochemical characteristics of the particles are given in Table 1. Data were provided by Kocbach et al. (2006), except total PAH for DEP which were based on data from the Certificate of analysis (National Institute of Standards and Technology, 2000) and endotoxin content for all par-ticles. The particle preparations from road traffic consisted of both combustion particles and mineral particles, and St+ contained more and larger sized mineral particles (60% of mineral particles > 2 µm) as compared to St− (20% > 2 µm) (Kocbach et al., 2006). DEP were used as well studied refer-ence particles.

Listeria monocytogenesThe Listeria strain L 242/73, type 4b, isolated from the spinal fluid from a meningitis case (Ruitenberg et al., 1976), was kindly provided by Arja de Klerk (National Institute of Public Health and the Environment, Department of Toxicology, Pathology and Genetics, Bilthoven, The Netherlands). For experimental use, two colonies of L. monocytogenes were grown overnight in brain heart infusion (BHI) broth (Becton, Dickinson and Co., Sparks, MD) at 37°C in a shak-ing incubator. The bacterial concentration was determined spectrophotometrically as optical density at 600 nm. The bacterial suspension was diluted with sterile saline to the appropriate concentration for inoculation (106 bacteria/ml). The number of bacteria in the suspension was confirmed by

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plating 100 µl from serial 10-fold dilutions onto blood agar plates, both before and after bacterial inoculation.

Particle and bacterial exposureMice were exposed to 100 µg (1 mg/ml) of DEP, St+, St−, or wood smoke particles by intratracheal instillation. All sus-pensions were made in Hank’s balanced salt solution (HBSS; PAA Laboratories GmbH, Linz, Austria), and particle suspen-sions were stirred at 18°C overnight using a magnetic stirrer and subsequently sonicated for 5 min using the ultrasonic processor Vibra-cellTM (Sonics & Materials, Inc., Newtown, CT). The animals were lightly anesthetized by inhalation of isofluorane (Baxter Healthcare Corporation of Puerto Rico, Guayama), and held in an upright position while 100 µl of HBSS or particle solution was instilled into the trachea through a curved metal cannula. Simultaneously with, 1 day after, or 7 days after particle instillation, all mice were inocu-lated intratracheally with 100,000 bacteria in 100 µl of ster-ile saline, as described above. Mice were killed by cervical dislocation 24 h after bacterial inoculation. Due to the work-load, a randomized block design (Festing et al., 2002; Ruxton & Colegrave, 2006) was used, splitting all experiments into three equal subexperiments separated in time, each with four mice per group, giving a total of 12 mice per group.

To study the capacity of the particles to create an acute inflammatory response in the lung, bronchoalveolar lavages (BALs) were performed as separate experiments 4 h or 24 h after a similar particle instillation as in the Listeria experi-ments. Again, a randomized block design was used, and the experiments were split into two equal subexperiments sepa-rated in time, each with four mice per group, giving a total of eight mice per group.

Homogenization of lungs and spleensLungs and spleens were excised and kept in sterile saline on ice, before homogenization in 2 ml of sterile saline using a tissue homogenizer (Bellco Biotechnology, Vineland, NJ). Series of four 10-fold dilutions of the tissue homogenate were plated on blood agar and incubated at 37°C overnight. Colony-forming units (cfu), an index of viable bacteria, were counted on each plate, and total cfu per lung or spleen were calculated.

Bronchoalveolar lavageMice were deeply anesthetized with thiopentone (165 mg/kg; Hospira Enterprises BV, Hoofddorp, The Netherlands) injected intraperitoneally, before lungs and trachea were

exposed by dissection. The trachea was cannulated and BAL was performed by flushing the lungs with 1 ml of ice-cold sterile Ca2+/Mg2+-free Dulbecco’s phosphate-buffered saline (PBS). The instillation and aspiration of PBS was performed three times, and the BAL fluid from the first lavage was kept separate from the later ones. All samples were kept on ice. The BAL fluid was centrifuged at 500 × g for 10 min at 4°C, and the supernatant from the first lavage was stored at −80°C for later analyses.

Differential cell countsThe cell pellets from all three lavages for each mouse were combined and resuspended in 300 µl of ice-cold PBS. The number of cells was determined on a Coulter Counter Z1 (Beckman Coulter Incorporated, FL), and the samples were diluted with PBS to yield 125,000 cells/ml. Smears were prepared by spinning 200 µl (25,000 cells) of each sample on to poly-l-lysin coated Shandon single cytoslides using a Shandon Cytospin® 4 cytocentrifuge (Thermo Fisher Scientific Inc., Waltham, MA) at 72 × g (800 rpm) for 6 min. Smears were immediately fixed in methanol for subsequent staining with a Hemacolor rapid staining of blood smears kit (Merck KGaA, Darmstadt, Germany) according to the manufacturer’s protocol. Differential cell counts were per-formed in a blinded fashion using a Zeiss Axioplan 2 light microscope (Carl Zeiss MicroImaging GmbH, Gottingen, Germany) at 400 × magnification. Two hundred cells per slide were counted.

Total protein, LDH, TNF-, IL-1, MCP-1, and MIP-2 in BAL fluidTotal protein concentration, lactate dehydrogenase (LDH) activity, and the levels of tumor necrosis factor (TNF-), interleukin 1 (IL-1), monocyte chemoattractant pro-tein 1 (MCP-1), and macrophage inflammatory protein 2 (MIP-2) were measured in the first BAL fraction using bicinchoninic acid assay (BCATM Protein Assay Kit; Pierce, Rockford, IL), TOX-7 kit (Sigma-Aldrich, St. Louis, MO), TNF- enzyme-linked immunosorbent assay (ELISA) kit (Biosource Europe S.A., Nivelles, Belgium), and IL-1, MCP-1, and MIP-2 ELISA kits (R&D Duo Sets; R&D Systems, Minneapolis, MN) respectively, all according to the manu-facturer’s protocols.

Statistical analysisStatistical analyses were performed with the SigmaStat® Statistical Analysis System for Windows Version 2.03

Table 1. Characteristics of diesel exhaust particles (DEP), road traffic particles collected during winter (use of studded tires, St+) and autumn (no use of studded tires, St−), and wood smoke particles (wood).

Sample Mean diameter (nm)a Mineral particles Carbon aggregates Total carbon (%) PAH (ng/mg) Endotoxin (ng/mg)

DEP 24 ± 7 — +++ 80.0 ± 5.1 67b <0.0006

St+ 25 ± 7 ++ + 14.3 ± 0.1 73 1.6

St– 24 ± 6 + ++ 51.0 ± 3.8 381 0.45

Wood 31 ± 7 — +++ 82.6 ± 5.8 9745 <0.0006

Note: Data, except endotoxin and DEP polycyclic aromatic hydrocarbons (PAH) values, are from Kocbach et al. (2006).aGeometric diameters of primary carbon particles.bBased on data from the Certificate of analysis (National Institute of Standards and Technology, 2000).

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(Jandel Scientific, Erkrath, Germany). For all experiments a randomized block design was used, with blocking in time (Festing et al., 2002; Ruxton & Colegrave, 2006). This means that the experiments were split into smaller, equal subex-periments (blocks) carried out at different times. The lung and spleen cfu data were log

10-transformed to obtain normal

distribution and equal variance. Total cell numbers, differ-ential cell counts, and the parameters analyzed in BAL fluid were judged to be normally distributed. All experiments were analyzed by two-way analysis of variance (ANOVA) with “day” and “treatment” as the two factors. When the ANOVA demonstrated statistically significant differences between treatments (p ≤ 0.001), Tukey’s post hoc tests were run (averaging over the two or three subexperiments), in order to determine which treatment differed from the oth-ers. Experimental groups were considered statistically dif-ferent if p ≤ 0.05.

Results

Bacterial counts in lungs and spleenThe effect of intratracheal exposure of mice to DEP, St+, St−, and wood smoke particles on the number of Listeria in the lungs and the spleen 24 h after bacterial inoculation is shown in Figure 1. When mice were simultaneously exposed to particles and Listeria, a significant reduction in bacterial numbers was seen in all particle exposed groups (p ≤ 0.001, except wood smoke particles with p = 0.044; Figure 1A). Mice exposed to DEP had significantly lower bacterial numbers in the lungs than mice exposed to the other particles (p ≤ 0.002). Further, exposure to St+ led to significantly lower bacterial numbers than exposure to wood smoke particles (p = 0.001). When particles were instilled 1 day prior to Listeria, bacte-rial numbers were reduced to similar levels in all particle exposed groups, the levels being significantly lower than in the HBSS group (p ≤ 0.001; Figure 1B). Seven days after particle exposure, particles had no significant effect on the numbers of Listeria (Figure 1C) in the lung.

Bacterial counts in the spleen showed a similar pattern to those in the lung, both when particles were given simul-taneously with and 1 day prior to Listeria inoculation, but no statistically significant differences between particle exposed groups were observed (data not shown). No effects of particles were observed in the spleen when particles were instilled 7 days prior to Listeria (data not shown).

Total cell numbers and differential cell counts in BAL fluidTo explore the lung environment created by the intratrache-ally instilled particles, about the time of inoculation with Listeria, bronchoalveolar lavage was performed 4 h and 24 h after particle exposure. Four hours after particle instillation, no differences in total cell number were seen between the different groups (Figure 2A, left panel). The number of neu-trophils, however, increased slightly in the groups exposed to St+ and St− compared to HBSS at this time point, although the increase was statistically significant only for St− (p = 0.018;

Figure 2B, left panel). Twenty-four hours after particle expo-sure, the increase in total cell numbers was significant for all particle exposed groups compared to HBSS (p < 0.001, Figure 2A, right panel). In addition, a marked increase of neutrophils was observed in all particle exposed groups com-pared to HBSS (p ≤ 0.004, Figure 2B, right panel), although the increase was not statistically significant for DEP.

TNF-, IL-1, MCP-1, MIP-2, LDH, and total protein in BAL fluidTo further explore the inflammatory state of the lungs, the levels of TNF-, IL-1, MCP-1, and MIP-2, as well as LDH activity and total protein content, were measured in the BAL fluid collected 4 h and 24 h after instillation of HBSS or the various particles (Figure 3). TNF-, MIP-2, and MCP-1 levels

Figure 1. Numbers of colony forming units (cfu) in the lungs of mice inoculated intratracheally with 105 Listeria simultaneously with (A), 1 day after (B), or 7 days after (C) intratracheal instillation with 100 µl Hank’s balanced salt solution (HBSS) or 100 µg particles in HBSS (1 mg/ml). Particles used were diesel exhaust particles (DEP), road traffic particles collected during winter (use of studded tires, St+) and during autumn (no use of studded tires, St−), and wood smoke particles (wood). One day after bacterial inoculation the numbers of cfu in the lungs were deter-mined. Values (cfu) for individual mice (circles) and median values (col-umns) for 12 mice from three individual experiments analyzed according to a block design are shown. Asterisk denotes statistically significant dif-ferences versus HBSS, and brackets denote statistically significant differ-ences between groups given the different particle preparations (p < 0.05).

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Combustion particles and lung innate immunity 947

were highest 4 h after particle exposure. At this time point, TNF- values were higher in the BAL fluid from mice exposed to St+ and St− than from mice exposed to DEP, wood smoke particles, and HBSS (p < 0.001; Figure 3A). Twenty four hours after exposure, TNF- values were low, and there were no significant differences between groups (data not shown).

Four hours after particle exposure, also the levels of the neutrophil chemoattractant MIP-2 in the BAL fluid were significantly higher after exposure to St+ and St− than after exposure to DEP, wood smoke particles, and HBSS (p < 0.001; Figure 3B). Further, the level of MIP-2 was significantly higher after exposure to DEP compared to HBSS (p = 0.022). MIP-2 levels were relatively low 24 h after particle exposure, although St+ and St− groups still had levels significantly higher than the control group (data not shown).

At the 4-h time point, MCP-1 levels were significantly higher in the BAL fluid from mice exposed to DEP, St+, and St− than from mice exposed to HBSS or wood smoke parti-cles (p < 0.001; Figure 3C). There were no differences between DEP, St+, and St−. MCP-1 levels were significantly higher for all particle exposed groups than for the control group 24 h after exposure (p ≤ 0.02; data not shown), although the overall levels were reduced compared to the 4-h time point. No significant differences were observed between particle exposed groups.

In the case of IL-1 levels, no significant increases were detected in the particle exposed groups compared to the control group 4 h after exposure (data not shown). However, 24 h after exposure, DEP, St+, and St− significantly increased the IL-1 levels compared to HBSS (p ≤ 0.022; Figure 3D),

but no differences were observed between particle exposed groups.

LDH activity measured in BAL fluid did not differ between any of the experimental groups 4 h after particle exposure. Twenty four hours after exposure, however, the level of LDH activity was significantly higher in mice exposed to wood smoke particles than in mice exposed to any of the other particles (p < 0.001; Figure 3E). No differences in total pro-tein content were observed either 4 h or 24 h after particle exposure (data not shown).

Noteworthy, wood smoke particles did not at any time point increase the levels of TNF-, IL-1, MCP-1, or MIP-2.

Discussion and conclusion

In the present work, a lung bacterial challenge model using the intracellular bacterium L. monocytogenes (van Loveren et al., 1988) was established. The number of bacteria in the lung after simultaneous or pre-exposure to wood smoke par-ticles, mixed road traffic particles (St+ and St−), and DEP was used as an endpoint to reflect particle activation of innate immunity. By terminating the experiments 24 h after bac-terial inoculation, we were able to study the particle effect before the development of an acquired immune response to the bacteria (North, 1973).

In the lung challenge model, traffic related particles and wood smoke particles appeared to have similar effects, since particle exposure 1 day prior to Listeria inoculation reduced lung bacterial numbers equally in all particle

Figure 2. Total cell numbers (A) and number of neutrophils (shaded bars) and macrophages (hatched bars) (B) in bronchoalveolar lavage (BAL) fluid. Mice were instilled intratracheally with 100 µl HBSS or 100 µg particles in HBSS (1 mg/ml) 4 h (left panel) or 24 h (right panel) prior to lung lavage. Particles used were diesel exhaust particles (DEP), road traffic particles collected during winter (use of studded tires, St+) and during autumn (no use of studded tires, St−), and wood smoke particles (wood). Values (cell numbers) for individual mice (circles) and median values (columns) for eight mice from two individual experiments analyzed according to a block design are shown. Asterisk denotes statistically significant differences versus HBSS, and brackets denote statistically significant differences between groups given the different particle preparations (p < 0.05).

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exposed groups. However, DEP had a more rapid effect, because when particles and Listeria were instilled simulta-neously, the reduction in bacterial numbers was greater in the group exposed to DEP than in the groups exposed to the other particles. The observed particle effect was transient, as no reduction in bacterial numbers was apparent when particles were instilled 7 days prior to Listeria inocula-tion. Importantly, although in our study the acute effect of particle instillation was a reduction in bacterial numbers, the long-term effect may be reduced resistance to Listeria. Specific immunity is necessary to clear Listeria infection (Nomoto et al., 1983), and reduced bacterial numbers and antigen stimulation early after bacterial inoculation leads to lower antigen load and a weaker specific immune response (Lovik & North, 1985). Resistance might be further weak-ened by other effects of particle exposure (Hiramatsu et al., 2005).

While exposure to all tested particles significantly reduced bacterial numbers and increased neutrophil numbers, the production of the proinflammatory mediators TNF-, IL-1, MIP-2, and MCP-1 varied between particle types. The two samples of mixed road traffic particles, St+ and St−, were generally the strongest inducers of the proinflammatory mediators, followed by DEP, while wood smoke particles did not increase the levels of any of the cytokines/chemokines measured. Thus, the different levels of proinflammatory mediators in BAL fluid could not explain the functional effects of the various particles seen as a reduction in bacterial numbers. However, the pattern of the mediators measured indicated that the innate immune system was activated via different mechanisms when exposed to the various particle types.

Only the mixed road traffic particle preparations St+ and St−, which both contained coarse mineral particles,

Figure 3. Concentrations of tumor necrosis factor (TNF-) (A), macrophage inflammatory protein 2 (MIP-2) (B), monocyte chemoattractant protein 1 (MCP-1) (C), interleukin 1 (IL-1) (D), and lactate dehydrogenase (LDH) (E) in bronchoalveolar lavage (BAL) fluid. Mice were instilled intratracheally with 100 µl HBSS or 100 µg particles in HBSS (1 mg/ml) 4 h or 24 h prior to lung lavage. Particles used were diesel exhaust particles (DEP), road traffic particles collected during winter (use of studded tires, St+) and during autumn (no use of studded tires, St−), and wood smoke particles (wood). Levels of TNF-, MIP-2, and MCP-1 4 h after particle exposure and levels of IL-1 and lactate dehydrogenase (LDH) 24 h after particle exposure are shown. Dotted lines indicate the upper and lower quantitative detection limits for the ELISAs. Values (pg/ml) for individual mice (circles) and median values (columns) for 6–8 mice from two individual experiments analyzed according to a block are shown. Asterisk denotes statistically significant differences versus HBSS, and brackets denote statistically significant differences between groups given the different particle preparations (p < 0.05).

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although in different amounts, induced increased levels of TNF-, known to be a key initiator of pulmonary inflam-mation (Driscoll et al., 1997). This is in accordance with our previous findings showing that coarse, but not ultrafine, polystyrene particles induced increased TNF- production after intratracheal exposure (Samuelsen et al., unpublished results). We therefore suggest that coarse particles may acti-vate the innate immune system through the conventional TNF- pathway, thereby influencing the production of other proinflammatory cytokines like MIP-2 and MCP-1 (Driscoll et al., 1997), which were also significantly increased after exposure to St+ and St−.

The composition of road traffic particles is complex. Ultrafine combustion particles, mineral particles of different sizes, and associated metals, chemicals, and organic sub-stances may all contribute to the inflammatory effect (Ball et al., 2000; Ma & Ma, 2002; Schwarze et al., 2007; Soukup & Becker, 2001; Stoeger et al., 2006). Importantly, endotoxin was detected in the St+ and St− samples. Endotoxin has pre-viously been associated with the inflammatory response to ambient air particles both in vivo and in vitro, and is likely to have contributed to the observed effects (Becker et al., 2005; Schins et al., 2004). However, polymyxin B only partially inhibited the release of inflammatory mediators in a mono-cytic cell-line exposed to St+ and St− particles, suggesting that also other particle properties are important (Kocbach et al., 2008).

Both wood smoke particles and DEP reduced bacterial numbers and induced increased neutrophil influx, even though no increase in TNF- levels was observed for these particles. Similar findings have been reported by others after DEP exposure (Rao et al., 2005; Saber et al., 2006), as well as after exposure to ultrafine polystyrene particles as observed by our group (Samuelsen et al., unpublished results). It has indeed been suggested that an increase in neutrophils and an inflammatory response in general is not dependent on TNF- production (Saber et al., 2005). Moreover, DEP have been reported to be a potent inducer of oxidative stress, and properties like the ultrafine size, metals, and the content of PAH have been proposed as explanations (Ball et al., 2000; Ma & Ma, 2002). Production of reactive oxygen species (ROS) might explain the cellular activation seen as a reduction in bacterial numbers, as well as the observed increase in the production of proinflam-matory mediators (Donaldson et al., 2001; Pourazar et al., 2005). Intra- and extracellular ROS generation may also have a direct antibacterial effect (Fang, 2004; Ohya et al., 1998), explaining the early reduction of bacterial numbers after DEP exposure.

Interestingly, wood smoke particles induced no sig-nificant increases in the proinflammatory mediators meas-ured. This is in agreement with a report that a human lung epithelial cell-line produced considerably lower levels of inflammatory cytokines after exposure to wood smoke par-ticles than to traffic related particles (Karlsson et al., 2006). Further, diesel exhaust and wood smoke have been found to differentially affect lung responses (Seagrave et al., 2005),

and only weak inflammatory responses have been reported in rat lungs after subchronic inhalation of wood smoke (Reed et al., 2006; Tesfaigzi et al., 2005), which is in accordance with our findings. Moreover, wood smoke particles were the only particles to induce increased LDH activity in BAL fluid 24 h after exposure. This indicates cytotoxicity caused by components of the wood smoke particles, candidates being PAH or zinc (Adamson et al., 2000; Kubatova et al., 2004), which are found in high amounts in the wood smoke parti-cles (Table 1; 9 µg zinc/mg wood smoke particles; Kocbach et al., unpublished results). One effect of the observed toxic-ity might be reduced ability of macrophages to phagocytose and kill bacteria (Morozzi et al., 1997; Wilson et al., 2007). This is a possible explanation for the higher bacterial num-bers observed after simultaneous exposure to wood smoke particles and Listeria than after corresponding exposures to DEP and St+. However, the neutrophil influx observed 24 h after wood smoke particles may have compensated for their cytotoxic effect, leading to equally low levels of bacteria in all particle exposed groups when particles were instilled 24 h prior to bacteria. Immigrating neutrophils have been sug-gested to play an important role in the early defense against Listeria (Cousens et al., 2000), and cytotoxicity may actually increase the release of endogenous danger signals resulting in an influx of neutrophils as observed for wood smoke par-ticles (Gallucci et al., 1999).

The neutrophil influx seen in all particle exposed groups 24 h after exposure did not correspond with the MIP-2 lev-els measured at either 4 h or 24 h after particle exposure. A similar lack of correspondence was also observed after intratracheal instillation of polystyrene particles (Samuelsen et al., unpublished results). This may possibly be explained by differences in kinetics for the two parameters, as well as by MIP-2 levels possibly peaking at different time points depending on the type of particles instilled. The early neu-trophil influx observed 4 h after exposure to St+ and St−, was probably caused by the endotoxin content of these particles (Li et al., 1998).

We cannot completely exclude that the particles have a direct toxic effect on the bacteria. This possibility seems, however, unlikely. Simultaneous exposure to Listeria and wood smoke particles, presumably the most cytotoxic com-pound used in this study, induced the smallest reduction in bacterial numbers. Further, particles and bacteria were mixed immediately before exposure when instilled simulta-neously, reducing the time for possible direct toxic interac-tions. Finally, the observed reduction in bacterial numbers was even stronger when particles were instilled 24 h prior to bacterial inoculation.

In conclusion, both wood smoke and road traffic derived particles activated the innate immune system in the lung, measured as reduced bacterial numbers and increased neutrophil influx. However, the mechanisms leading to cel-lular activation seemed to differ for the various particles, as indicated by the different kinetics of bacterial reduction and the different patterns of proinflammatory mediators in BAL fluid. Differences in allergy adjuvant effect (Samuelsen

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et al., 2008) support this notion. Wood smoke particles clearly differ from DEP with regard to inflammatory activity, and further studies are warranted to reveal the mechanisms behind the different particle induced responses. The fact that different particle types appear to activate the innate immune system via different mechanisms may have conse-quences for how particles affect allergic and cardiopulmo-nary diseases.

Acknowledgements

We thank Arja de Klerk (National Institute of Public Health and the Environment (RIVM), Department of Toxicology, Pathology and Genetics, Bilthoven, The Netherlands) for providing Listeria monocytogenes and Joseph K. H. Ma and Xuejun J. Yin (Shool of Pharmacy and School of Medicine, West Virginia University, Morgantowns, West Virginia, USA) for providing valuable information on the prepara-tion of L. monocytogenes for animal infection. The authors acknowledge Anette Kocbach for planning and administer-ing the particle sample collection and for the chemical and morphological analyses. We thank Trude Karin Olsen, Åse Eikeset, Astri Grestad, Else-Carin Groeng, Bodil Hasseltvedt, and Berit A Stensby for excellent technical assistance. The endotoxin measurements provided by Randi Jacobsen are highly appreciated.

Declaration of interest: This work was funded by a grant from the Norwegian Academy of Science and Letters and Statoil (VISTA) (6141) and The Norwegian Institute of Public Health. The authors alone are responsible for the content and writing of the paper.

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