Comparative acute lung inflammation induced by atmospheric PM and size-fractionated tire particles

Page 1

Cs

PAa

b

c

a

ARRAA

KATMLI

1

s(aateeo

0d

Toxicology Letters 198 (2010) 244–254

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

omparative acute lung inflammation induced by atmospheric PM andize-fractionated tire particles

aride Manteccaa,∗,1 , Francesca Farinab,1 , Elisa Moschinia , Daniele Gallinotti a , Maurizio Gualtieri a ,nnette Rohrc, Giulio Sancinib, Paola Palestinib, Marina Camatinia

Department of Environmental Science, POLARIS Research Center, University of Milano-Bicocca, 1 piazza della Scienza, Milan 20126, ItalyDepartment of Experimental Medicine, POLARIS Research Center, University of Milano-Bicocca, 48 via Cadore, Monza 20052, ItalyAir Quality and Health, Electric Power Research Institute (EPRI), 3420 Hillview Avenue, Palo Alto, CA 94304, USA

r t i c l e i n f o

rticle history:eceived 4 May 2010eceived in revised form 29 June 2010ccepted 2 July 2010vailable online 17 July 2010

eywords:tmospheric PMire particleice

ungnflammation

a b s t r a c t

A comparison of the effects produced by size-fractionated tire particles (TP10 and TP2.5) and similar-sizedurban particulate matter (PM10 and PM2.5), collected in Milan in 2007, on the lungs of mice has beenperformed. The focus is on early acute lung responses following intratracheal instillation of aerosolizedparticles at a 3-h recovery period. Together with bronchoalveolar lavage (BAL) conventional endpointslike total and differential cell counts, total protein, alkaline phosphatase, lactate dehydrogenase and pro-inflammatory cytokines (TNF-�, MIP-2), the expression of different stress protein markers (caspase8,Hsp70, H0-1, NF-kB) was evaluated 3 h after particle instillation into Balb/c mice. The TP2.5 fractionreached the alveolar spaces and produced an acute inflammatory response as evidenced by increasedLDH and AP activities, total protein and Hsp70 content. TNF-� and MIP-2 production was significantlyincreased and polymorphonuclear neutrophils (PMN) recruitment was apparent. The TP10 fraction dis-tributed mainly in the bronchial district and the only modified BAL parameter was the expression ofMIP-2. PM2.5 induced an inflammatory response lesser in magnitude than that produced by PM10 frac-tion. The TNF-� increase was not significant, and HO-1, though significantly increased with respect tothe control, was unable to reduce NF-kB activation, suggesting a role of the endotoxin component of

PM in stimulating a pro-inflammatory limited response. This response was maximized by the PM10 thatinduced a significant increase in MIP-2, TNF-�, and HO-1. Lung immunohistochemistry showed fine par-ticles, TPs in particular, being able to deeply penetrate and rapidly induce inflammatory events in theparenchyma, even involving endothelial cells, while PM10 produced a strong pro-inflammatory responsemediated by the bronchiolar cells and residential macrophages of the proximal alveolar sacs, likely as aconsequence of its larger dimension and endotoxin content. These results provide evidence of variable

s in m

inflammatory mechanism

. Introduction

Tire particles (TP), generated from tire tread against the roadurface, are an important source of ambient particulate matterPM), contributing to urban atmospheric pollution (Camatini etl., 2001) and to potential risk for human health (Gualtieri etl., 2005c; Wik and Dave, 2009). We have recently investigated

he in vitro toxicity of TP and TP extracts on the human alveolarpithelial cell line A549 (Gualtieri et al., 2005b,c, 2008; Berettat al., 2007), provided evidence of toxicity on Xenopus devel-pment (Gualtieri et al., 2005a,b; Mantecca et al., 2007) and of

∗ Corresponding author. Tel.: +39 02 64482916; fax: +39 02 64482996.E-mail address: [email protected] (P. Mantecca).

1 These authors contributed equally to this work.

378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2010.07.002

ouse lungs in response to both urban PM and tire particles.© 2010 Elsevier Ireland Ltd. All rights reserved.

lung toxicity in mice exposed to different sized TP (Mantecca etal., 2009). Our biochemical, cytological, and histological resultsindicated differential lung toxicity mechanisms elicited by size-fractionated TP, in agreement with other studies performed inin vivo systems that have shown that lung responses to inhaledor instilled particles are affected by particle size. We demon-strated that lung toxicity induced by the coarse fraction of TP(<10 �m) was primarily due to macrophage-mediated inflamma-tory events, while toxicity induced by the fine fraction of TP(<2.5 �m) appeared to be related more closely to cytotoxicity.These results were obtained after 24 h exposure, and the low valueexpressed by the markers of inflammation (MIP-2 and TFN-�), have

suggested the need for further investigation with shorter post-exposure time and with the analysis of parameters correlated toearly intracellular oxidative stress and inflammation (Stone et al.,2007).

Page 2

ogy Le

fbtbfstahbrta2stb11psaiD

2ttmhdcs

laoghahintshs

gircePop

2

2

tiha

P. Mantecca et al. / Toxicol

Moreover, since there is a lack of data regarding how particlesrom different sources contribute to effects on human health, andecause particles are often handled as a uniform pollutant despiteheir complex composition, we compared the effects producedy coarse and fine TP fractions with those produced by similarractions of urban PM collected in Milan in 2007. Epidemiologictudies suggest that coarse particles may be more strongly relatedo respiratory health effects while the fine fraction tends to showstronger association with cardiovascular disease and mortality;owever, the picture is not clear and the literature describing mor-idity and mortality in response to specific size fractions variesegionally (Donaldson et al., 2005). PM collected in spatially andemporally variable environments shows different inflammatorynd toxic responses in the lung (Cassee et al., 2003; Peng et al.,005); concentrations and properties of PM also vary with time,eason, and climate. Current research suggests that the possibleoxic and pro-inflammatory effects of PM are strongly influencedy its constituents, which include biological agents (Becker et al.,996; Dong et al., 1996), metals (Pope, 1989; Costa and Dreher,997; Ghio et al., 1999; Donaldson et al., 2005), and organic com-ounds (Li et al., 2002). Other physico-chemical properties besidesize and composition, such as mass, number, and available surfacerea, are also important properties that may determine the healthmpact of particulate pollutants (Dong et al., 1996; Dreher, 2000;riscoll et al., 2000).

As reported in a previous paper (Mantecca et al., 2009), at4 h post-TP exposure, mice showed significant lung inflamma-ory parameters, mainly as inflammatory cells infiltration. At thatime, early pro-inflammatory signals were not significantly aug-

ented, allowing us to hypothesize that acute lung response mayappen at earlier exposure time. Moreover several clinical and epi-emiological data support the thesis that acute respiratory andardiovascular symptoms in sensitized or debilitated subjects startoon after exposure to PM peaks.

With these assumptions we did our experiments to test earlyung toxicity markers, focusing on particles from different sourcesnd in different size fractions, and to furnish relevant evidencen the mechanism of toxicity. Several cytokines have been sug-ested to be involved in particle-induced inflammation. The focusas primarily been directed to tumor necrosis factor alpha (TNF-�)nd macrophage inflammatory protein-2 (MIP-2), both of whichave been suggested to play an important role in particle-induced

nflammation in the lung (Adamson et al., 2004). TNF itself haso chemotactic effect for neutrophils, and its chemotactic effect ishought to be mediated by the induction of chemotactic cytokinesuch as MIP-2. MIP-2 is considered to be a murine homologue of theuman interleukin-8 (Haelens et al., 1996), thought to be respon-ible for the recruitment of neutrophils in mice.

We evaluated the expression of caspase8 (involved in pro-rammed cell death), Hsp70 and heme oxygenase-1 (H0-1) asndicators of oxidative stress, and NF-kB, involved in cellularesponses to stimuli such as stress and cytokines. In addition,onventional bronchoalveolar lavage fluid (BALF) endpoints werevaluated at 3 h post-instillation with fine and coarse TP and urbanM. Pulmonary histopathological analysis completed the resultsbtained, which demonstrate the different impact of the differentarticle sources.

. Materials and methods

.1. Animals

Male BALB/c mice (7–8 weeks old) were purchased from Charles River Labora-ories (Italy); food and water were administered ad libitum. The mice were housedn plastic cages under controlled environmental conditions (temperature 19–21 ◦C,umidity 40–70%, lights on 7 a.m. to 7 p.m.). The established rules of animal carepproved by Italian Ministry of health (DL 116/92) were followed.

tters 198 (2010) 244–254 245

2.2. TP and PM sources and characterization

Tire Particles (TPs) and atmospheric particulate matter (PM) samples used inthis work were previously characterized and data are available in Camatini et al.(2001), Gualtieri et al. (2005b, 2009, 2010) and Mantecca et al. (2009).

Briefly, TPs obtained from a commercial tire recycling plant were size-fractionated in the lab (Mantecca et al., 2009) to allow the recovery of TP2.5(<2.5 �m) and TP10 (<10 �m) fractions, while PM10 and PM2.5 were collected dur-ing 2007 in a Milan urban area (Gualtieri et al., 2009). Size-fractionated PM sampleswere collected onto Teflon filters using a low volume gravimetric sampler (EU sys-tem 38.33 l min−1, FAI Instruments, Rome, Italy). For in vivo experiments particleswere recovered from filters by sequential sonications (four cycles of 20 min each) insterile water. Detached particles were dried in a dessicator, weighed, resuspendedin sterile water at a final concentration of 4 �g/�l and stored at −20 ◦C until use.

As reported in the papers mentioned above, morphological aspects of the parti-cles were investigated with a TEM Jeol-JEM1220 operating at 100 kV and equippedwith a CCD camera. Elemental analysis was conducted by an energy-dispersive typeX-ray fluorescence analyzer (XRF, Spectra QuanX, Thermo Scientific), while PAHanalysis was conducted by HPLC-FD (Shimadzu, Kyoto, Japan) after particle elutionin acetonitrile for 20 min in an ultrasonic bath.

Summarizing and comparing the results of the toxicologically relevant met-als and PAHs, the sum of the eight major PAHs accounted for 38.23 ng/mg and23.15 ng/mg for TP10 and TP2.5, respectively. These values were comparable to thoseof both PM samples used, which contained 36 ng/mg PAHs. Zn, the most significantelement in TP samples with an amount of 29 �g/mg, showed a similar concentrationin the PM samples used for instillation (30 �g/mg in PM2.5).

PM samples are a complex mixture of organic and inorganic carbon, ionic speciesand other transition metals (cfr. Gualtieri et al., 2009, 2010), while the major con-stituent of TPs was found to be isoprene polymers (Camatini et al., 2001; Gualtieriet al., 2005b).

To further characterize PM samples, and to verify that no contaminationoccurred in TP manipulation, the endotoxin content of the particles was deter-mined using a quantitative chromogenic Limulus Amebocyte Lysate (LAL) test(Pyrochrome-LAL test, Associates of Cape Cod, Inc.) following the manufacturer’sinstructions. Briefly, PM and TP extracts were diluted in apirogenic LAL reagentwater to reach 10 �g/ml concentration, mixed with pyrochrome and incubated at37 ◦C for 40 min. The reaction was stopped with 10% acetic acid. The absorbanceof the samples was determined by the use of a spectrophotometer (Ascent scanmultiplate reader, Thermo Scientific Inc.) at 405 nm. The concentration of endo-toxin was calculated from a standard of E. coli 0113:H10 LPS. The endotoxinconcentrations were expressed as endotoxin units per milligram (EU/mg) of testedparticles. Endotoxin was almost absent in TPs (<0.03 EU/mg for both TP10 and TP2.5),while they were abundant in PM2.5 (17.61 ± 0.21 EU/mg) and especially in PM10(25.94 ± 0.62 EU/mg).

Particle suspensions for animal experiments were prepared as follows. Imme-diately prior to intratracheal instillation, TP fractionated particles were suspendedin sterile saline (0.9% NaCl) supplemented with 0.01% Tween 20 and sonicated for15 min to obtain a uniform dispersion and to prevent particle aggregation. Afterbeing thawed, PM aliquots were properly diluted in sterile saline, sonicated andvortexed and immediately instilled in mice, similarly to TPs.

2.3. Experimental design

Mice were instilled with 100 �l of a solution containing 100 �g of either PM10,PM2.5, TP10, or TP2.5 and euthanized 3 h following a single instillation. Controlmice, running parallel to PM- and TP-treated animals, were instilled with 100 �l ofsaline solution or saline supplemented with 0.01% Tween 20. Bronchoalveolar lavagefluid (BALF) was collected and analyzed for cellular and biochemical inflammatorymarkers. Lungs were excised and dissected. The right lobes were cryo-preservedfor further biochemical analyses on tissue lysates, and the left lobes were fixed andprocessed for histological analyses. Animal testing was carried out by instilling threemice for each experimental group and the experiment was replicated two times.

2.4. Intratracheal TP and PM instillation

Male BALB/c mice were briefly exposed to a mixture of 2.5% isoflurane(Flurane), 70% O2 and 30% NO2 anesthetic gas and then anesthetized withTiletamine/Zolazepam-Xylazine (TZX 40 + 5 mg/kg) i.m. administered. Once a deepstage of anesthesia was reached, mice were intratracheally instilled by means ofMicroSprayer® Aerosolizer system (MicroSprayer® Aerosolizer-Model IA-1C andFMJ-250 High Pressure Syringe, Penn Century, USA) with 100 �g of PM2.5 or PM10in 100 �l of isotonic saline solution, or 100 �l of isotonic saline solution (ControlPM); alternatively, mice were intratracheally instilled by means of MicroSprayer®

Aerosolizer system with 100 �g of TP2.5 or TP10 in 100 �l of isotonic saline solu-

tion with 0.01% Tween 20, or 100 �l of isotonic saline solution with 0.01% Tween 20(Control TP). Each mouse was placed in a supine position, the mouth was opened,and the tongue was gently moved aside using a pince to better incannulate the tra-chea. The particles were suspended in the appropriate solution immediately priorto instillation according to the previously described procedure. Immediately fol-lowing instillation, treated and control mice were allowed to recover under visual

Page 3

2 ogy Le

cc

2

ata2i2a

2

Fw(2wta

22naapi(Aa

2

BaacPpapta4i

Hm

2

sfi

ecawd

oWwabT1pi(I

46 P. Mantecca et al. / Toxicol

ontrol before placing them back in plastic cages under controlled environmentalonditions.

.5. BALF collection and analyses

After 3 h, mice from each experimental group were euthanized with annesthetic mixture overdose (Tiletamine/Zolazepam-Xylazine and isoflurane), therachea was exposed, cannulated and secured with suture thread, and three in-nd-out washes with 0.6 ml of isotonic saline solution were performed (Henderson,005). The efficacy of BALF collection ranged from 50% to 90% of the total solution

njected. The BALF was centrifuged at 1500 × g for 15 min at 4 ◦C (Gilmour et al.,004) and pellets collected for cell counts. Supernatants were divided into aliquotsnd appropriately stored for subsequent biochemical analyses.

.5.1. Cell countsAfter centrifugation, the BALF pellets were resuspended in 500 �l of DMEM (10%

BS, 1% penicillin–streptomycin, 1% glutamine), and total cell counts performedith a Burker chamber, using the Trypan Blue exclusion method. A cell aliquot

240,000 cells, 800 cells/�l) was smeared in duplicate onto slides using Cytofuge(StatSpin, USA) 40 × g for 7 min at room temperature. Subsequently, the smearsere stained with Diff Quik (Medion Diagnostic) for cell differential count, according

o manufacturer instructions. Macrophages, polymorphonuclear leukocytes (PMNs)nd lymphocytes were identified by their characteristic shapes.

.5.2. Biochemical analyses

.5.2.1. The following biochemical analyses were performed on cell-free BALF super-atants. Total protein content was measured spectrophotometrically at 750 nmccording to Lowry method with bovine serum as standard. The commerciallyvailable kit for alkaline phosphatase (DALP-250 QuantiChrom Alkaline Phos-hatase Assay Kit, Gentaur Molecular) was employed according to the manufacturer

nstructions. Lactate dehydrogenase (LDH) activity was assessed using TOX-7 kitSigma–Aldrich); optical density (OD) at 490 nm was measured using a Multiskanscent plate reader (Thermo). ODs were corrected for plate background absorbancet 690 nm.

.5.3. Cytokine and defence proteins analysesThe analyses of pro-inflammatory cytokines and chemokines released in the

ALF were performed by DuoSet ELISA kits for tumor necrosis factor-� (TNF-�)nd macrophage inflammation protein (MIP-2) (R&D Systems, Minneapolis, MN)ccording to the manufacturer’s protocols. Briefly, 96-well plates were coated withapture antibody and incubated overnight at room temperature before washing withBS containing 0.05% Tween 20 (wash buffer, WB). After incubation with the sam-les (2 h at room temperature), the wells were washed with WB and the detectionntibody was added for 2 h at room temperature. After repeated washings in WB,lates were incubated with horseradish conjugated streptavidin for 20 min at roomemperature and then washed again in WB. Finally, peroxidase substrate was added,nd the reaction was stopped by adding 1 N H2SO4. Optical density was measured at50 nm with Multiskan Ascent multiplate reader (Thermo). Each sample was read

n triplicate.The heat shock protein Hsp70 was detected by immunoblotting using an anti-

sp70 monoclonal antibody (Santa Cruz Biotech, Inc., CA, USA) and following theethods reported in the next paragraph.

.6. Protein analyses from tissue lysates

The lungs, quickly excised from the chest and washed in ice cold isotonic salineolution at the end of BALF procedure, were minced at 4 ◦C and briefly centrifugedor 30 s at 11,000 rpm with an Ultra-Turrax T25 basic (IKA WERKE) in 2 ml of coldsotonic saline solution.

Then samples were submitted to trichloroacetic acid (TCA) precipitation. Anqual volume of 30% TCA was added, samples were incubated on ice for 1 h and thenentrifuged at 18,400 × g for 30 min at 4 ◦C. After one wash of the pellets with coldcetone, samples were centrifuged at 18,400 × g for 20 min at 4 ◦C. This procedureas replicated twice. The pellets were suspended in water and protein quantityetermined by BCA method (Sigma–Aldrich, USA).

Thereafter, 30 �g lung homogenates of control and treated mice were loadedn SDS-PAGE (12%-polyacrylamide) and submitted to electrophoresis, followed byestern blot. The membranes were stained with Ponceau S and the protein loadingas assessed by densitometry (BIORAD Densitometry 710, program Quantity one)

s described (Moore and Viselli, 2000; Daffara et al., 2004; Botto et al., 2008). Afterlocking, blots were incubated for 2 h with the primary antibody diluted in PBS-

/milk (anti-NF-kB, 1:200; anti-caspase8-p18, 1:200 from Santa Cruz, and anti-HO1:750, from QED Bioscience). Then, blots were incubated for 1.5 h with horseradisheroxidase-conjugated anti-rabbit IgG (1:5000) or anti-goat IgG (1:2000) diluted

n PBS-T/milk. Proteins were detected by ECL using the SuperSignal detection kitPierce, Rockford, IL). Immunoblot bands were analyzed and quantified by Kodakmage Station 2000R interfaced with Kodak Molecular Imaging Software.

tters 198 (2010) 244–254

2.7. Histological analyses

After BALF recovery, the lungs of control and TP- and PM-instilled mice werequickly excised for histological analyses. Pulmonary left lobes were immediatelyformalin fixed and processed according to routine histological procedures. Briefly,after being preserved for 24 h in the fixative, the fragments were rinsed in distilledwater, dehydrated in an ethanol series from 70% to 100% and embedded in Bio-plast tissue embedding medium. For each control and exposed sample, 7 �m serialsections were cut by a rotary microtome, mounted on slides and stained with Hema-toxylin and Eosin (HE). Slides were qualitatively screened for histological lesions inthe alveolar and the airway tissues.

Immunohistochemical techniques were also performed to visualize the spatialexpression of caspase8 and HO-1 in the PM and TP exposed lungs. Two sectionsof the lungs (5 �m thickness) were placed on gelatin-coated slides. After beingdewaxed and hydrated, sections were incubated for 20 min in 3% H2O2 in PBS forthe quenching of the endogenous peroxidases and subsequently rinsed in PBS. Tis-sues were permeabilized with 0.4% Tween 20 in PBS for 20 min and non-specificbinding was blocked with 10% Normal Horse Serum (NHS) (Elite® ABC Kit, Vec-tastain Laboratories, Burlingame, CA) in a humid chamber at 37 ◦C for 40 min. Thesections on the slide were incubated overnight at 4 ◦C with rabbit polyclonal anti-bodies against caspase8-p18 or HO-1 (Santa Cruz Biotechnology, Inc.) diluted in PBS0.5% BSA. Specificity controls were performed by incubating the lung sections withPBS 0.5% BSA. Slides were abundantly rinsed in PBS and incubated with a secondaryhorse anti-rabbit antibody conjugated to biotin (ABC Kit) for 90 min at 37 ◦C andwashed again in PBS. The avidin–biotin complex conjugated with horseradish per-oxidase (ABC Kit) was added on each section for 30 min at 37 ◦C and, after repeatedwashings with PBS, slides were incubated in 0.5 mg/ml DAB and 0.3‰ H2O2 in PBSfor 5 min. Slides were abundantly rinsed in tap and distilled water and then nucleiwere counterstained with hematoxylin for 1 min. Lastly slides were dehydrated andmounted with Eukitt. Slides were viewed under the light microscope Zeiss Axioplan40 and images taken by a Zeiss MRC5 digital camera interfaced with the AxiovisionReal 4.6 software.

2.8. Statistical analyses

For each cytological and biochemical parameter measured in controls andtreated mice, the mean values ± standard error of the mean (SEM) were calculated.Statistical differences were tested by one-way ANOVA followed by Bonferroni’spost hoc comparisons or by the non-parametric Kruskal–Wallis test (KW), followedby Dunn’s method, when variances were not uniform. Statistical differences wereconsidered to be significant at the 95% level (p < 0.05).

3. Results

3.1. Bronchoalveolar lavage fluid analysis

3.1.1. Cellular inflammatory responseCellular indicators of inflammation and the percentage of differ-

ent cell types were evaluated (Fig. 1). The number of total cells inBALF (Fig. 1A) slightly increased in TP10- and TP2.5-treated mice.Both TP fractions induced a nonsignificant decrease in macrophagepercentage (Fig. 1B) and an increase in PMN percentage (Fig. 1C)and this result was most evident in TP 2.5-treated mice.

The BALF analysis from PM-treated mice showed that onlyPM2.5 induced a decrease in the total cell number (Fig. 1E) with anevident decrease in macrophages (Fig. 1F) and a parallel increase inthe PMN percentage (Fig. 1G). No notable variations in lymphocyteswere noted after both TP and PM exposure (Fig. 1D and H).

Statistically significant differences were only observed for theTP2.5- and PM2.5-induced PMN increase (Fig. 1C and G), suggestingthat the fine particle fractions are the most effective in inducinginflammatory cell recruitment.

3.1.2. CytotoxicityTotal protein, lactate dehydrogenase (LDH) and alkaline phos-

phatase (AP) activities were measured as markers of cytotoxicity(Fig. 2).

No significant differences in these parameters were observedfor TP- and PM-treated mice. Only BALF from TP2.5-treated miceshowed a tendency toward an increase in all three cytotoxic mark-ers measured (Fig. 2A–C). In the PM10 experiments only AP wasslightly augmented (Fig. 2F).

Page 4

P. Mantecca et al. / Toxicology Letters 198 (2010) 244–254 247

F from Tg The dP ignific

3

fpiicmT

wa

3

ai

ig. 1. Histograms showing differential cell counts in the BALFs collected at 3 h p.i.rey bars = fine particles (TP2.5, PM2.5); black bars = coarse particles (TP10, PM10).arameters showing a trend need too many animals in order to reach statistically s

.1.3. Cytokine and defense protein releaseA significant increase in MIP-2 and TNF-� was evident in BALF

rom both TP- and PM-instilled mice (Fig. 3). In particular, com-aring TP and PM effects, MIP-2 secretion appeared to be largely

nduced by TP2.5 (Fig. 3A and D), while TNF-� appeared to benduced by PM10 (Fig. 3B and E). In addition, MIP-2 was signifi-antly induced by PM2.5 and PM10 exposure (Fig. 3D). In the sameanner, TNF-� was significantly increased also after TP2.5 and

P10 exposure (Fig. 3B).Hsp70, a protein induced by a wide variety of stressful stimuli,

as nonsignificantly increased only after TP2.5 treatment (Fig. 3Cnd F).

.2. Lung histology and tissue-particle interactions

Hematoxylin–eosin (HE) stained lung sections presented char-cteristic histological aspects related to the different particle typesnstilled.

P (left panel, A–D) and PM (right panel, E–H) exposed mice. Empty bars = controls;ata are mean ± standard error of the mean (SEM) of n = 6 instilled mice per group.ant differences.

PM10 and PM2.5 particles and their aggregates were not eas-ily distinguishable in the lung tissues (Fig. 4B and C), suggestingeither that they were rapidly and efficiently cleared, or that theyhad a relatively low capacity to adhere and penetrate the alveo-lar epithelium after such a brief exposure time. When comparedto controls, slight tissue lesions were observed in PM-exposedlungs, with localized epithelial cell lyses. In particular, the mostevident histological effects were observed in the terminal tractof the bronchiolar epithelia, especially after PM10 treatment(Fig. 4B), while alveolar walls were mainly affected by PM2.5,with some epithelial cells showing altered nuclear morphology(Fig. 4C).

Contrary to PM, TPs were abundantly present in airway andalveolar spaces lining the epithelial tissues (Fig. 4D and E). TP10-

instilled lungs presented large particle deposits throughout theterminal bronchioles and the proximal alveolar spaces (Fig. 4D),with associated tissue lesions. TP2.5 deeply penetrated the alveolarsacs, engulfing macrophages and adhering to the alveolar epithelialcells. Cellular debris and tissue exudate were commonly seen inside

Page 5

248 P. Mantecca et al. / Toxicology Letters 198 (2010) 244–254

F m TPg The dP ignific

tc

3

mklo

ipwsmtsl–(tit

oe

ig. 2. Histograms showing cytotoxic markers in the BALFs collected at 3 h p.i. frorey bars = fine particles (TP2.5, PM2.5); black bars = coarse particles (TP10, PM10).arameters showing a trend need too many animals in order to reach statistically s

he alveolar lumen, sometimes accompanied by diffuse necrotichanges of the alveolar walls (Fig. 4E and F).

.3. Protein analyses on lung tissues

Despite the increased level of TNF-� in the BALF of all treatedice, which suggest potential activation of the nuclear factor

appa-light-chain-enhancer of activated B cells (NF-kB p50) in theung tissue, no significant increase of this transcription factor wasbserved in TP- and PM-treated mice (Fig. 5A and B).

Caspase8 (Casp8), a pro-apoptotic protein marker, wasncreased in the lung tissues of PM-exposed mice (Fig. 5B) and inarticular of TP2.5-treated mice (Fig. 5A). In exposed lungs, TP2.5as indeed able to induce a 4X higher protein expression than in

aline instilled controls (data not shown). Control saline instilledice showed weak but remarkable positive reaction for Casp8 at

he level of the bronchiolar epithelial cells (Fig. 6A), likely as a con-equence of the mechanical stress of the instilling procedure. Theiterature has already recognized the activation of apoptotic signals

Casp8 included – in lung cells subjected to mechanical stressesSeitz et al., 2008). As reported in the control for the specificity ofhe Casp8 immunochemical reaction (Fig. 6B), no cells stained pos-

tively in the lung parenchyma. Similar results were obtained forhe HO-1 antibody specificity.

In PM10 and PM2.5 instilled lungs, the Casp8 signal in bronchi-lar cells was comparable in intensity to that of controls, while,specially after PM2.5 exposure, Casp8 expression was seen in

(left panel, A–C) and PM (right panel, D–F) exposed mice. Empty bars = controls;ata are mean ± standard error of the mean (SEM) of n = 6 instilled mice per group.ant differences.

many alveolar cells marked by brown fine granular cytoplas-mic spots. Even endothelia of some capillary vessels showed apale brown reaction (Fig. 6C–E). The most diffuse and intenseCasp8 positive reaction was observed in TP2.5-treated mice.Almost all bronchiolar cells and alveolar type 2 epithelial cellsshowed intense and uniformly stained dark brown cytoplasm.Even many vessel walls were intensely stained (Fig. 6F andG).

Heme oxygenase (H0)-1, a key protein in the defense againstoxidative and inflammatory insults, was increased after TP2.5instillation (Fig. 5A), and a very strong expression of this pro-tein was evident after PM exposure (Fig. 5B). PM10 induced thestrongest production of HO-1, with OD values ten times higher thanthose of control mice (data not shown).

Lungs of saline instilled control mice did not show immunos-taining for HO-1 except from a shadowing reaction in bronchiolesand in blood cells (Fig. 7A and B). Immunohistochemical localiza-tion of HO-1 in PM10-instilled lungs showed terminal bronchioleepithelial cells positively and intensely stained, as well as the alve-olar cells, mainly in correspondence of the proximal sacs (Fig. 7C).Residual alveolar macrophages were often HO-1 positive and bloodcells much more stained than in controls. PM2.5 induced less

intense HO-1 immunoreaction in the bronchiolar cells, but a greaternumber of positive cells in the alveolar epithelium (Fig. 7D). The fineTP fraction mainly induced HO-1 expression at the basal laminaeof the epithelial cells and in the endothelium of the blood vesselsand even in the alveolar capillaries (Fig. 7F and G).

Page 6

P. Mantecca et al. / Toxicology Letters 198 (2010) 244–254 249

F Fs colE partici

4

iieptrtHmlahia

taewf

fat

ig. 3. Histograms showing cytokine and defense protein concentration in the BALmpty bars = controls; grey bars = fine particles (TP2.5, PM2.5); black bars = coarsenstilled mice per group.

. Discussion

We acutely exposed mice to TP and PM fractions by intratrachealnstillation, which is an accepted method to determine the lungnjury sustained by inhalable particulates (Gavett et al., 2003; Costat al., 2006; Wegesser and Last, 2008; Mantecca et al., 2009). Thisrocedure has been used to facilitate a comparative study betweenhe post-exposure time considered here and the one previouslyeported (Mantecca et al., 2009). Moreover, an advantage of intra-racheal instillation is the delivery of a known dose to the lung.owever, despite these advantages, there are limitations of thisethod, including potentially unrealistically high doses, deposition

ocations that may not be representative of inhalation exposures,nd stressful stimuli to the animals. Overall, the PM experimentsere performed can easily be compared with the many in vivo stud-

es reported in the literature which deal with instillation (Gavett etl., 2003; Costa et al., 2006; Wegesser and Last, 2008, 2009).

Since it has been estimated that over 60% of respirable par-iculate matter (PM10) in urban areas comes from road transportnd that tire and brake wear are responsible for about 7% of thesemissions, we sought to compare the effects produced by TP, aell-characterized particle, with those produced by similar PM size

ractions collected in an urban site in Milan in 2007.

We have previously reported the toxic effects of size-

ractionated TP in mice 24 h post-instillation (Mantecca etl., 2009). Lung toxicity induced by TP10 was primarily dueo macrophage-mediated inflammatory events, while toxicity

lected at 3 h p.i. from TP (left panel, A–C) and PM (right panel, D–F) exposed mice.les (TP10, PM10). The data are mean ± standard error of the mean (SEM) of n = 6

induced by TP2.5 appeared to be related more strongly to a directcytotoxic effect. Moreover, mild increases in the inflammationmarkers MIP-2 and TNF-� were measured. Here we present theresults obtained 3 h post-instillation of TP and PM to elucidate thetime course of the lung inflammatory process and to better disclosethe early markers of acute lung inflammation in the BALF and in thelung parenchyma.

Mice were instilled with the dose of 100 �g of PM or TP, follow-ing the calculations performed by Gavett et al. (2003), who showedthat the concentration of 425 �g/m3 PM2.5 inhaled over an 8-hperiod would produce human doses per tracheo-bronchial surfacearea equivalent to the instilled dose of 100 �g.

The chemical and physical properties of TPs were extensivelyanalyzed (Camatini et al., 2001; Gualtieri et al., 2005a; Milani et al.,2004; Mantecca et al., 2009) and their composition was shown to berich in isoprene polymers and some metals (especially zinc). Dueto their chemical composition, TPs may be considered a simplermodel particle than ambient PM, which is composed of a hetero-geneous mixture of both inorganic and carbonaceous materials,rich in PAHs, transition metals and other compounds as previouslyreported (Gualtieri et al., 2009, 2010; Perrone et al., 2010).

The total and differential cell counts in BALF are well establishedparameters to identify inflammation events in the respiratory tract.

In healthy mice BALF pulmonary macrophages (AMs) are abundantwhile polymorphonuclear leukocytes (PMNs) are rare; an increaseof PMNs in the BALF is a sensitive indicator of lung inflammation(Henderson, 2005). The total cell count failed to be predictive of

Page 7

250 P. Mantecca et al. / Toxicology Letters 198 (2010) 244–254

F bronw g shol ing als 0 �m;

Pidcatbrilndco

P

ig. 4. Histology of PM and TP exposed lungs. (A) Control lung parenchyma showingith dispersed fine particles and slight tissutal inflammation; (C) PM2.5 instilled lun

arge particle deposits in the peri-bronchiolar region; (E) TP2.5 instilled lung showhowing abundant TP masses and degeneration of the alveolar walls. (A–E) Bars = 5

M- and TP-induced lung toxicity since we found no differencen the BALF between control and treated groups. Nevertheless theifferential cell counts disclosed an acute lung inflammatory pro-ess occurring 3 h after the intratracheal instillation of both PMnd TP. The PMN percentage increased in the BALF of TP- and PM-reated mice while the AM percentage decreased, particularly whenoth PM2.5 and TP2.5 fractions were instilled (Fig. 1). Our previousesults (Mantecca et al., 2009) outlined that both TP10 and TP2.5nduced an inflammatory response still evident 24 h after the instil-ation, while the present results suggest that coarse particles doot induce early PMN recruitment. Indeed, different sized particlesistributed through the lung in a size-dependent manner, with the

oarse particles mainly confined to the upper respiratory tract andnly the fine fraction reaching the alveolar epithelium.

The percentage of lymphocytes in the BALF of TP- andM-treated mice was not modified 3 h after the instillation,

chiolar and alveolar epithelia; (B) PM10-instilled lung showing terminal bronchiolewing alveolar cells in apoptotic and necrotic changing; (D) TP10-instilled lung withveolar spaces and macrophages engulfed by particles; and (F) TP2.5 instilled lung(F) bar = 20 �m.

demonstrating that no immuno-based inflammatory response wasinduced by different sized particles from different sources and con-firming our previous results (Mantecca et al., 2009).

The total protein content in the BALF collected 3 h after TP2.5instillation increased about twofold in comparison with TP10-treated and control mice.

Similar TP2.5-induced responses were observed for LDH release,a marker of lung cytotoxicity (Henderson et al., 1995), and AP activ-ity, a specific marker of type II cell damage and/or proliferationof the lung alveolar epithelium (Fehrenbach, 2001; Mason, 2006;Bhalla et al., 1999). Considering these results with those obtained24 h after TP instillation (Mantecca et al., 2009), a time related

progression of the damage to the alveolar/capillary barrier perme-ability could be outlined (Henderson, 2005).

LDH and AP activities as well as total protein content and thepercentage of PMNs in BALF of TP2.5-treated mice increased 3 h

Page 8

P. Mantecca et al. / Toxicology Le

Fig. 5. Immunoblottings of lung homogenate proteins. (A) TP-instilled lungs and (B)PM-instilled lungs.

Fig. 6. Casp-8 immunohistochemical localization in PM and TP exposed lungs. (A) Contrinstilled lung; (C) PM10-instilled lung; (D and E) PM2.5 instilled lung; and (F and G) TP2.5

tters 198 (2010) 244–254 251

after the instillation, indicating that the damage produced by fineTP on the alveolar epithelium elicits an early lung inflammatoryprocess.

TNF-� is one of the early biochemical mediators of pulmonaryinflammation and is released by resident macrophages to promotethe adherence of circulating inflammatory cells to the endothe-lium. It stimulates the release of chemoattractant factors such asMIP-2, which is mainly responsible of the influx of neutrophilsin the rodent lung (Becker et al., 1996; Henderson, 2005). It hasbeen suggested that TNF-� is a key player in particle-induced lunginflammation (Driscoll, 2000), and that TNF-� is an inflammatorymediator upstream of MIP-2 (Tessier et al., 1997). TNF-� and MIP-2levels in both TP- and PM-instilled mice were higher in comparison

to controls. TNF-� increased significantly in TP2.5- and PM10-treated mice, while the increase of MIP-2 was significant for TP2.5-,PM2.5-, and PM10-treated mice.

These results differ from those obtained by Saber et al. (2006),who reported on exposure of mice to high doses of diesel exhaust

ol lung; (B) negative control obtained by avoiding the primary antibody in a TP2.5instilled lung. (A–D, and F) Bar = 50 �m; (E and G) bar = 20 �m.

Page 9

252 P. Mantecca et al. / Toxicology Letters 198 (2010) 244–254

F B) Cona

pstieiaTcMbTb

iiit

ig. 7. HO-1 immunohistochemical localization in PM and TP exposed lungs. (A andnd (F and G) TP2.5 instilled lung. (A–F) Bar = 50 �m; (G) bar = 20 �m.

articles (DEP). This group found that DEPs increased the expres-ion level of MIP-2, independently of TNF status. The authors statehat MIP-2 is a cytokine induced in the early phase of DEP-inducednflammation, while TNF-� is expressed later (about a day afterxposure). In fact MIP-2 was increased at 3 h when TP2.5 wasnstilled, while at 24 h the level diminished to that of control. PM10nd PM2.5 produced significant increases in MIP-2 and TNF-�.his increase may be due to the presence of metals and organicompounds and also to endotoxin components in PM10 (Alfaro-oreno et al., 2007; Stone et al., 2003; Camatini et al., 2010). Thus

ecause of its chemical composition and ability to aggregate, fineP apparently produced a toxic response higher than that producedy PM2.5.

HSPs are a family of proteins induced in all cells upon threaten-ng alterations of the cellular environment, and among them Hsp70s mainly involved in inflammatory processes. Its major functions a protective one against the deleterious effects of the media-ors of inflammation, such as TNF-� and reactive oxygen species

trol lung; (C) PM10-instilled lung; (D) PM2.5 instilled lung; (E) TP10-instilled lung;

(ROS) (Jacquier-Sarlin et al., 1994). Hsp70 could be released whencells undergo necrosis (Svensson et al., 2006). However, Hsp70 inthe extracellular milieu acts as a cytokine to human monocytes bystimulating a pro-inflammatory signal transduction cascade thatresults in a further up-regulation of cytokines such as IL-1�, IL-6and TNF-� (Asea et al., 2000). The Hsp70 content in the BALF oftreated mice increased 3 h after TP2.5 instillation. These data are inagreement with the increased LDH activity and total protein con-tent in the BALF of TP2.5-treated mice, confirming an early lungcytotoxic effect sustained by the fine TP fraction.

The nuclear factor NF-kB is involved in regulating many aspectsof cellular activity, such as stress, injury and especially in thepathways of the immune and inflammatory response following

induction of TNF-� (Sun and Zhang, 2007). Moreover, NF-kB isreported to play an important role in mediating cell survival (Liu etal., 2008) and in preventing a chronic inflammatory response (Stoneet al., 2007), thus indicating a double role for this transcription fac-tor. Defects in NF-kB result in increased susceptibility to apoptosis,

Page 10

ogy Le

lefaiemHawi

pitPce2sctialm

dBtvaape

it

lucMp

iigfe2HtWrclwtp

tusMs

P. Mantecca et al. / Toxicol

eading to an increase in cell death (Sheikh and Huang, 2003). Wevaluated p50, one of the active subunits of the NF-kB transcriptionactor, and no significant modification of its expression 3 h after PMnd TP2.5 instillation was found, while TP10 produced a significantncrease of the factor level. A possible explanation for the differ-nt response of NF-kB-p50 related to the different sized particlesay be found in the correlation of NF-kB with the activity of theO-1. This is an inducible stress protein that confers cytoprotectiongainst oxidative stress (Kim et al., 2008; Wagener et al., 2001) andhose deficiency is associated with a chronically inflamed state and

ncreased leukocyte recruitment (Poss and Tonegawa, 1997a,b).The levels of HO-1 increased in mice treated with PM (PM10 in

articular) and the TP2.5 fraction, while they remained unchangedn TP10-treated mice. The HO-1 increase with the fine fractionreatment can explain the unmodified NF-kB-p50 levels found inM2.5- and TP2.5-treated mice. Indeed the HO-1 over-expressionan confer anti-inflammatory protection by decreasing cytokinexpression, probably involving the block of NF-kB (Zampetaki et al.,003). Since a defect in NF-kB expression results in an increasedusceptibility to apoptosis (Wong et al., 1997), the observedaspase8-p18 levels in PM2.5 and TP2.5 treatments are consis-ent with the NF-kB profile. Accordingly, both immunoblotting andmmunohistochemical findings revealed caspase8-p18 abundantlynd widely expressed in the respiratory tracts of TP2.5 instilledungs (Figs. 5A and 6F and G), testifying for the strong acute inflam-

atory potential of these fine particles.The TP2.5 fraction reached the alveolar spaces and initiated early

amage to the alveolar epithelium, as evidenced by the increase ofALF LDH and AP activities, total protein and Hsp70 content. Thenhis fraction produced an acute inflammatory status, AMs were acti-ated to digest particles, and TNF-� and MIP-2 production reachedpeak, followed by PMN recruitment. This acute process was thenpparently stopped and a possible chronic inflammation event wasrevented by the enhancement of HO-1 expression (Fredenburght al., 2007) and the inhibition of NF-kB activation.

The TP10 fraction produced less notable damage, mainly local-zed at the bronchial level, as evidenced by histological analysis andhe unchanged levels of LDH, ALP and total protein.

At 3 h, TNF-� was not significantly increased, and MIP-2 wasower than that produced by TP2.5. This observation justifies thenchanged level of HO-1 and the over-expression of NF-kB, whichontinued the transcription of pro-inflammatory genes, such asIP-2. In fact, at 24 h the MIP-2 levels were still significant in com-

arison with TP2.5 (Mantecca et al., 2009).The instillation of PM size fractions showed that PM10 had more

nflammatory and cytotoxicity potential than the fine PM. In fact,n vivo and in vitro studies have shown that the coarse fraction hasreater potency at equivalent masses when compared to the fineraction (Monn and Becker, 1999; Soukup and Becker, 2001; Schinst al., 2004; Hetland et al., 2005; Lipsett et al., 2006; Yeatts et al.,007). PM10 produced a significant increase in MIP-2, TNF-�, andO-1, and these effects are probably due to the associated endo-

oxin components (Alfaro-Moreno et al., 2007; Stone et al., 2003;egesser and Last, 2008, 2009; Fredenburgh et al., 2005). PM2.5

ecruited PMNs, even if the inflammatory response was lower inomparison with that of the coarse fraction. In fact, the TNF-�evel was not significant, and HO-1, even significantly increased

ith respect to the control, was unable to reduce NF-kB activa-ion, suggesting a role of the endotoxins of PM10 in stimulating aro-inflammatory limited response.

In conclusion, the larger TP fraction distributed mainly at

he bronchial district, the levels of LDH and AP activities werenchanged, the increase in the BALF of total protein content was notignificant, the PMN infiltration occurred early, and the increase inIP-2 was lower than that produced by TP2.5 at 3 h. This value was

ignificantly higher at 24 h, as reported in Mantecca et al. (2009),

tters 198 (2010) 244–254 253

suggesting a delay in the pro-inflammatory effects sustained byTP10.

The PM2.5 resulted in recruitment of PMNs, even though theoverall elicited lung inflammatory response was lower in compari-son with that sustained by the PM10. Indeed the increase of TNF-�was not significant and 3 h after the instillation the expression ofall the analyzed markers in the BALF was generally similar in PMand control mice.

The intratracheal instillation of a single dose of TP and PM wasfound to produce effects linked both to their size and their chemi-cal and biogenic composition. There are at present numerous dataindicating that PM generated from tires/pavement might be a con-tributing factor to the toxicity of urban particles (Lindbom et al.,2007; Wik and Dave, 2009), by increasing the amount of PM gener-ated as well as by mediating inflammatory effects. Our results addto this literature and may provide valuable insight into the healthrisks posed by urban particles.

Acknowledgment

This research was performed with the financial support ofCariplo Foundation (TOSCA project).

References

Adamson, I.Y.R., Prieditis, H., Renaud, V., 2004. Soluble and insoluble air particlefractions induce differential production of tumor necrosis factor-� in rat lung.Exp. Lung Res. 30 (5), 355–368.

Alfaro-Moreno, E., López-Marure, R., Montiel-Dávalos, A., Symonds, P., Osornio-Vargas, A.R., Rosas, I., Murray, J.C., 2007. E-Selectin expression in humanendothelial cells exposed to PM10: the role of endotoxin and insoluble fraction.Environ. Res. 103, 2221–2228.

Asea, A., Kraeft, S.K., Kurt-Jones, E.A., Stevenson, M.A., Chen, L.B., Finberg, R.W., Koo,G.C., Calderwood, S.K., 2000. HSP70 stimulates cytokine production througha CD14-dependent pathway, demonstrating its dual role as a chaperone andcytokine. Nat. Med. 6, 435–442.

Bhalla, D.K., Gupta, D.K., Reinhart, P.G., 1999. Alteration of epithelial integrity, alka-line phosphatase activity, and fibronectin expression in lungs of rats exposed toozone. J. Toxicol. Environ. Health A 57, 329–343.

Becker, S., Soukup, J.M., Gilmour, I.M., Devlin, R.B., 1996. Stimulation of human andrat alveolar macrophages by urban air particulates: effects of oxidant radicalgeneration and cytokine production. Toxicol. Appl. Pharmacol. 141, 637–648.

Beretta, E., Gualtieri, M., Botto, L., Palestini, P., Miserocchi, G., Camatini, M., 2007.Organic extract of tire debris causes localized damage in the plasma membraneof human lung epithelial cells. Toxicol. Lett. 173, 191–200.

Botto, L., Beretta, E., Bulbarelli, A., Rivolta, I., Lettiero, B., Leone, B.E., Miserocchi,G., Palestini, P., 2008. Hypoxia-induced modifications in plasma membranesand lipid microdomains in A549 cells and primary human alveolar cells. J. CellBiochem. 105, 503–513.

Camatini, M., Corvaja, V., Mantecca, P., Gualtieri, M., 2010. PM10-biogenic frac-tion drives the seasonal variation of pro-inflammatory response in A549 cells.Environ. Toxicol. doi:10.1002/tox.20611.

Camatini, M., Crosta, G.F., Dolukhanyan, T., Sung, C., Giuliani, P., Corbetta, G.M.,2001. Microcharacterization and identification of tire debris in heterogeneousspecimens. Mater. Charact. 46, 271–283.

Cassee, F.R., Fokkens, P.H.B., Lesemann, D.L.A.C., Bloemen, H.J.Th., Boere, A.J.F., 2003.Respiratory allergy and inflammation due to ambient particles (RAIAP); collec-tion and characterisation of particulate matter samples from 5 European sites.Report 863001001/2003. RIVM, Bilthoven.

Costa, D.L., Dreher, K.L., 1997. Bioavailable transition metals in particulate mattermediate cardiopulmonary injury in healthy and compromised animal models.Environ. Health Perspect. 105 (Suppl. 5), 1053–1060.

Costa, D.L., Lehmann, J.R., Winsett, D., Richards, J., Ledbetter, A.D., Dreher, K.L., 2006.Comparative pulmonary toxicological assessment of oil combustion particlesfollowing inhalation or instillation exposure. Toxicol. Sci. 91 (1), 237–246.

Daffara, R., Botto, L., Beretta, E., Conforti, E., Faini, A., Palestini, P., Miserocchi, G.,2004. Endothelial cells as early sensors of pulmonary interstitial edema. J. Appl.Physiol. 97, 1575–1583.

Donaldson, K.T., Mill, S., MacNee, W., Robinson, S., Newby, D., 2005. Role of inflam-mation in cardiopulmonary health effects of PM. Toxicol. Appl. Pharmacol. 207:2(Suppl. 1), 483–488.

Dong, W., Lewtas, J., Luster, M.I., 1996. Role of endotoxin in tumor necrosis factor

� expression from alveolar macrophages treated with urban air particles. Exp.Lung Res. 22, 577–592.

Dreher, K.L., 2000. Particulate matter physiochemistry and toxicology: in search ofcausality—a critical perspective. Inhal. Toxicol. 12, 45–57.

Driscoll, K.E., Costa, D.L., Hatch, G., Henderson, R., Oberdoster, G., Salem, H.,Schlesinger, R.B., 2000. Forum: intratracheal instillation as an exposure tech-

Page 11

2 ogy Le

D

F

F

F

G

G

G

G

G

G

G

G

G

H

H

H

H

J

K

L

L

L

L

M

M

M

54 P. Mantecca et al. / Toxicol

nique for the evaluation of respiratory tract toxicity: uses and limitations.Toxicol. Sci. 55, 24–35.

riscoll, K.E., 2000. TNF alpha and MIP-2: role in particle-induced inflammation andregulation by oxidative stress. Toxicol. Lett. 112–113, 177–183.

ehrenbach, H., 2001. Alveolar epithelial type II cell: defender of the alveolus revis-ited. Respir. Res. 2, 33–46.

redenburgh, L.E., Baron, R.M., Carvajal, I.M., Mouded, M., Macias, A.A., Ith, B.,Perrella, M.A., 2005. Absence of heme oxygenase-1 expression in the lungparenchyma exacerbates endotoxin-induced acute lung injury and decreasessurfactant protein-B levels. Cell Mol. Biol. 51, 513–520.

redenburgh, L.E., Perrella, M.A., Mitsialis, S.A., 2007. The role of heme oxygenase-1in pulmonary disease. Am. J. Respir. Cell Mol. Biol. 36, 158–165.

avett, S.H., Haykal-Coates, N., Highfill, J.W., Ledbetter, A.D., Chen, L.C., Cohen, M.D.,Harkema, J.R., Wagner, J.G., Costa, D.L., 2003. World trade center fine particulatematter causes respiratory tract hyperresponsiveness in mice. Environ. HealthPerspect. 111, 980–991.

hio, A.J., Stonehuerner, J., Dailey, L.A., Carter, J.D., 1999. Metals associated with boththe water soluble and insoluble fractions of an ambient air pollution particlecatalyze an oxidative stress. Inhal. Toxicol. 11, 37–49.

ilmour, M.I., O’Connor, S., Dick, C.A., Miller, C.A., Linak, W.P., 2004. Differentialpulmonary inflammation and in vitro cytotoxicity of size-fractionated fly ashparticles from pulverized coal combustion. J. Air Waste Manage. Assoc. 54 (3),286–295.

ualtieri, M., Andrioletti, M., Vismara, C., Milani, M., Camatini, M., 2005a. Toxicity oftire debris leachates. Environ. Int. 31, 723–730.

ualtieri, M., Andrioletti, M., Mantecca, P., Vismara, C., Camatini, M., 2005b. Impactof tire debris on in vitro and in vivo systems. Part. Fibre Toxicol. 2, 1.

ualtieri, M., Rigamonti, L., Galeotti, V., Camatini, M., 2005c. Toxicity of tire debrisextracts on human lung cell line A549. Toxicol. In Vitro 19, 1001–1008.

ualtieri, M., Mantecca, P., Cetta, F., Camatini, M., 2008. Organic compounds in tireparticle induce reactive oxygen species and heat-shock proteins in the humanalveolar cell line A549. Environ. Int. 34, 437–442.

ualtieri, M., Mantecca, P., Corvaja, V., Longhin, E., Perrone, M.G., Bolzacchini, E.,Camatini, M., 2009. Winter fine particulate matter from Milan induces morpho-logical and functional alterations in human pulmonary epithelial cells (A549).Toxicol. Lett. 188, 52–62.

ualtieri, M., Øvrevik, J., Holme, J., Perrone, M.G., Bolzacchini, E., Schwarze, P., Cama-tini, M., 2010. Differences in cytotoxicity versus pro-inflammatory potency ofvarious particulate matter in human lung cells. Toxicol. In Vitro 24, 29–39.

aelens, A., Wuyts, A., Proost, P., Struyf, S., Opdenakker, G., van Damme, J., 1996.Leukocyte migration and activation by murine chemokines. Immunobiology195, 499–521.

enderson, R.F., Scott, G.G., Waide, J.J., 1995. Source of alkaline phosphatase activityin epithelial lining fluid of normal and injured F344 rat lungs. Toxicol. Appl.Pharmacol. 134, 170–174.

enderson, R.F., 2005. Use of bronchoalveolar lavage to detect respiratory tracttoxicity of inhaled material. Exp. Toxicol. Pathol. 57, 155–159.

etland, R.B., Cassee, F.R., Lag, M., Refsnes, M., Dybing, E., Schwarze, P.E., 2005.Cytokine release from alveolar macrophages exposed to ambient particulatematter: heterogeneity in relation to size, city and season. Part. Fibre Toxicol. 2,4.

acquier-Sarlin, M.R., Fuller, K., Dinh-Xuan, A.T., Richard, M.J., Polla, B.S., 1994. Pro-tective effects of hsp70 in inflammation. Experientia 50, 1031–1048.

im, H.P., Wang, X., Lee, S.-J., Huang, M.-H., Wang, Y., Ryter, S.W., Choi, A.M.K., 2008.Autophagic proteins regulate cigarette smoke induced apoptosis: protective roleof heme oxygenase-1. Autophagy 4 (7), 887–895.

i, N., Wang, M., Oberley, T.D., Sempf, J.M., Nel, A.E., 2002. Comparison of the proox-idative and proinflammatory effects of organic diesel exhaust particle chemicalsin bronchial epithelial cells and macrophages. J. Immunol. 169, 4531–4541.

indbom, J., Gustafsson, M., Blomqvist, G., Dahl, A., Gudmundsson, A., Swietlicki, E.,Ljungman, A.G., 2007. Wear particles generated from studded tires and pave-ment induces inflammatory reactions in mouse macrophage cells. Chem. Res.Toxicol. 20, 937–946.

ipsett, M.J., Tsai, F.C., Roger, L., Woo, M., Ostro, B.D., 2006. Coarse particles and heartrate variability among older adults with coronary artery disease in the CoachellaValley, California. Environ. Health Perspect. 114 (8), 1215–1220.

iu, X., Togo, S., Al-Mugotir, M., Kim, H., Fang, Q.-H., Kobayashi, T., Wang, X.-Q., Mao,L., Bitterman, P., Rennard, S., 2008. NF-kappaB mediates the survival of humanbronchial epithelial cells exposed to cigarette smoke extract. Respir. Res. 9,66.

antecca, P., Gualtieri, M., Andrioletti, M., Bacchetta, R., Vismara, C., Vailati, G.,

Camatini, M., 2007. Tire debris organic extract affects Xenopus development.Environ. Int. 33, 642–648.

antecca, P., Sancini, G., Moschini, E., Farina, F., Gualtieri, M., Rohr, A., Miseroc-chi, G., Palestini, P., Camatini, M., 2009. Lung toxicity induced by intratrachealinstillation of size-fractionated tire particles. Toxicol. Lett. 189, 206–214.

ason, R.J., 2006. Biology of alveolar type II cells. Respirology 11, S12–S15.

tters 198 (2010) 244–254

Milani, M., Pucillo, F.P., Ballerini, M., Camatini, M., Gualtieri, M., Martino, S., 2004.First evidence of type debris characterization at the nanoscale by focused ionbeam. Mater. Charact. 52, 283–288.

Monn, C., Becker, S., 1999. Cytotoxicty and induction of proinflammatorycytokines from human monocytes exposed to fine (PM2.5) and coarse parti-cles (PM10-2.5) in outdoor and indoor air. Toxicol. Appl. Pharmacol. 155, 245–252.

Moore, M.K., Viselli, S.M., 2000. Staining and quantification of proteins trans-ferred to polyvinylidine fluoride membranes. Anal. Biochem. 279, 241–242.

Peng, R.D., Dominici, F., Pastor-Barriuso, R., Zeger, S.L., Samet, J.M., 2005. Seasonalanalyses of air pollution and mortality in 100 US cities. Am. J. Epidemiol. 161 (6),585–594.

Perrone, M.G., Gualtieri, M., Ferrero, L., Lo Porto, C., Udisti, R., Bolzacchini, E., Cama-tini, M., 2010. Seasonal variations in chemical composition and in vitro biologicaleffects of fine PM from Milan. Chemosphere 78, 1368–1377.

Pope III, C.A., 1989. Respiratory disease associated with community air pollution anda steel mill, Utah Valley. Am. J. Public Health 79, 623–628.

Poss, K., Tonegawa, S., 1997a. Reduced stress defense in heme oxygenase 1-deficientcells. Proc. Natl. Acad. Sci. U.S.A. 94, 10925–10930.

Poss, K.D., Tonegawa, S., 1997b. Heme oxygenase 1 is required for mammalian ironreutilization. Proc. Natl. Acad. Sci. U.S.A. 94, 10919–10924.

Saber, A.T., Jacobsen, N.R., Bornholdt, J., Kjærl, S.J., Dybdahl, M., Risom, L., Loft,S., Vogell, U., Wallin, H., 2006. Cytokine expression in mice exposed to dieselexhaust particles by inhalation. Role of tumor necrosis factor. Part. Fibre Toxicol.3, 4.

Schins, R.P.F., Lightbody, J.H., Borm, P.J.A., Shi, T., Donaldson, K., Stone, V., 2004.Inflammatory effects of coarse and fine particulate matter in relation to chemicaland biological constituents. Toxicol. Appl. Pharmacol. 195, 1–11.

Seitz, D.H., Perl, M., Mangold, S., Neddermann, A., Braumüller, S.T., Zhou, S., Bachem,M.G., Huber-Lang, M.S., Knöferl, M.W., 2008. Pulmonary contusion inducesalveolar type 2 epithelial cell apoptosis: role of alveolar macrophages and neu-trophils. Shock 30 (5), 537–544.

Sheikh, M.S., Huang, Y., 2003. Death receptor activation complexes: it takes two toactivate TNF receptor 1. Cell Cycle 2 (6), 550–552.

Soukup, J.M., Becker, S., 2001. Human alveolar macrophage responses to air pollu-tion particulates are associated with insoluble components of coarse material,including particulate endotoxin. Toxicol. Appl. Pharmacol. 171, 20–26.

Stone, V., Wilson, M.R., Lightbody, J., Donaldson, K., 2003. Investigating the potentialfor interaction between the components of PM10. Environ. Health Prev. Med. 7,246–253.

Stone, V., Johnston, H., Clift, M.J., 2007. Air pollution, ultrafine and nanoparticletoxicology: cellular and molecular interactions. IEEE Trans. Nanobioscience 6,331–340.

Sun, X.F., Zhang, H., 2007. NFKB and NFKBI polymorphisms in relation to sus-ceptibility of tumour and other diseases. Histol. Histopathol. 22, 1387–1398.

Svensson, P.A., Asea, A., Englund, M.C.O., Bausero, M.A., Jernas, M., Wiklund, O., Ohls-son, B.G., Carlsson, L.M.S., Carlsson, B., 2006. Major role of HSP70 as a paracrineinducer of cytokine production in human oxidized LDL treated macrophages.Atherosclerosis 185 (1), 32–38.

Tessier, P.A., Naccache, P.H., Clark-Lewis, I., Gladue, R.P., Neote, K.S., McColl, S.R.,1997. Chemokine networks in vivo: involvement of C–XC and C–C chemokinesin neutrophil extravasation in vivo in response to TNF-alpha. J. Immunol. 159,3595–3602.

Wagener, F., Eggert, A., Boerman, O.C., Oyen, W.J.G., Verhofstad, A., Abraham, N.G.,Adema, G., van Kooyk, Y., de Witte, T., Figdor, C.G., 2001. Heme is a potent inducerof inflammation in mice and is counteracted by heme oxygenase. Blood 98,1802–1811.

Wegesser, T.C., Last, J.A., 2008. Lung response to coarse PM: bioassay in mice. Toxicol.Appl. Pharmacol. 230 (2), 159–166.

Wegesser, T.C., Last, J.A., 2009. Mouse lung inflammation after instillation of partic-ulate matter collected from a working dairy barn. Toxicol. Appl. Pharmacol. 236,348–357.

Wik, A., Dave, G., 2009. Occurrence and effects of tire wear particles in theenvironment—a critical review and an initial risk assessment. Environ. Pollut.157 (1), 1–11.

Wong, H.R., Ryan, M., Wispé, J.R., 1997. Stress response decreases NF-�B nucleartranslocation and increases I-�Ba expression in A549 cells. J. Clin. Invest. 99,2423–2428.

Yeatts, K., Svendsen, E., Creason, J., Alexis, N., Herbst, M., Scott, J., Kupper, L., Williams,

R., Neas, L., Cascio, W., Devlin, R.B., Peden, D.B., 2007. Coarse particulate matter(PM2.5-10) affects heart rate variability, blood lipids, and circulating eosinophilsin adults with asthma. Environ. Health Perspect. 115 (5), 709–714.

Zampetaki, A., Minamino, T., Mitsialis, S.A., Kourembanas, S., 2003. Effect of hemeoxygenase-1 overexpression in two models of lung inflammation. Exp. Biol. Med.228, 442–446.