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(The FASEB Journal. 2006;20:2393-2395.)
© 2006 FASEB

Titanium dioxide nanoparticles induce emphysema-like lung injury in mice

Huei-Wen Chen*, Sheng-Fang Su*,{dagger}, Chiang-Ting Chien{ddagger}, Wei-Hsiang Lin§, Sung-Liang Yu{dagger}, Cheng-Chung Chou{dagger}, Jeremy J. W. Chen{dagger},||,1 and Pan-Chyr Yang{dagger},{dagger}{dagger},1,2

* Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan;

{dagger} NTU Center for Genomic Medicine, National Taiwan University, Taipei, Taiwan;

{ddagger} Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan;

§ Institute of Life Sciences, College of Life Sciences, National Chung-Hsing University, Taichung, Taiwan;

|| Institutes of Biomedical Sciences and Molecular Biology, College of Life Sciences, National Chung-Hsing University, Taichung, Taiwan; and

{dagger}{dagger} Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan

2Correspondence: Department of Internal Medicine National Taiwan University Hospital and National Taiwan University College of Medicine, No. 7 Chung-Shan South Rd., Taipei 100, Taiwan. E-mail: pcyang{at}ha.mc.ntu.edu.tw

ABSTRACT

Titanium dioxide nanoparticles (nanoTiO2) have been widely used as a photocatalyst in air and water cleaning. However, these nanoparticles inhalation can induce pulmonary toxicity and its mechanism is not fully understood. In this study we investigated the pulmonary toxicity of nanoTiO2 and its molecular pathogenesis. The adult male ICR mice were exposed to intratracheal single dose of 0.1 or 0.5 mg nanoTiO2 (19–21 nm) and lung tissues were collected at 3rd day, 1st wk, and 2nd wk for morphometric, microarray gene expression, and pathway analyses. NanoTiO2 can induce pulmonary emphysema, macrophages accumulation, extensive disruption of alveolar septa, type II pneumocyte hyperplasia, and epithelial cell apoptosis. NanoTiO2 induced differential expression of hundreds of genes include activation of pathways involved in cell cycle, apoptosis, chemokines, and complement cascades. In particular, nanoTiO2 up-regulates placenta growth factor (PlGF) and other chemokines (CXCL1, CXCL5, and CCL3) expressions that may cause pulmonary emphysema and alveolar epithelial cell apoptosis. Cultured human THP-1 cell-derived macrophages treated with nanoTiO2 in vitro also resulted in up-regulations of PlGF, CXCL1, CXCL5, and CCL3. These results indicated that nanoTiO2 can induce severe pulmonary emphysema, which may be caused by activation of PlGF and related inflammatory pathways.—Chen, H-W., Su, S-F., Chien, C-T., Lin, W-H., Yu, S-L., Chou, C-C., Chen, J. J. W., Yang, P. C. Titanium dioxide nanoparticles induce emphysema-like lung injury in mice.


Key Words: nanotechnology • chemokines • placenta growth factor • microarray • pulmonary emphysema

NANOTECHNOLOGY, NOT ONLY is widely used in industry, but also has been extensively explored for possible applications in medicine. However, the potential toxicity issues regarding these powerful nanoparticles are often ignored (reviewed in ref. 1 ). Nanoparticles are defined as particles with a diameter less than 100 nm, which may cause pulmonary toxicity (2) . Chronic inhalation studies in rats have shown that nanoparticles can induce impaired lung clearance, chronic pulmonary inflammation, pulmonary fibrosis, and lung tumors (reviewed in ref. 3 ). Previous studies (reviewed in ref. 4 ) suggested that attention should be paid to nanoparticle-induced toxicity, including the possibility that some of the nanoparticles are deposited by diffusional mechanisms in all regions of the respiratory tract when inhaled, then may undergo transcytosis across epithelial and endothelial cells into the blood and lymph circulation, and could induce various biological responses such as inflammation and free radical modulation. It is therefore important to clarify the effects of various nanoparticles on pulmonary health as well as the pathogenic mechanisms and signaling pathways involved.

Titanium dioxide nanoparticles (nanoTiO2) (<100 nm) are widely used as photocatalysts in air and water cleaning (5) . The potential pulmonary toxicity is not yet clear. Earlier studies indicated that a single intratracheal exposure to nanoTiO2 nanoparticles (2 mg per rat) is cytotoxic for pulmonary alveolar macrophages (6) . The toxic effects of TiO2 particles are dose- and size-dependent. Smaller nanoTiO2 (20 nm) cause a greater pulmonary inflammatory response in rats and mice than larger TiO2 particles (250 nm). The toxicity of nanoTiO2 correlates well with their surface area per unit mass (7) . Cocultures of human A549 epithelial cells and macrophages (differentiated THP-1 cells) show increased sensitivity to nanoparticles and increased cytokine release [interleukin-6 and interleukin-8 (IL-8)], as compared with mono-cultures of each cell type (8) . However, exposure of THP-1 cell-derived macrophages or endothelial cells to chitosan-DNA nanoparticles or other nanoparticles (PVC, TiO2, SiO2, Co, Ni) does not induce the release of proinflammatory cytokines or have cytotoxic effects (9 , 10) .

A host inflammatory or immune response to inhaled toxic gases and particles might lead to pulmonary emphysema and chronic obstructive pulmonary disease (COPD) (11) , which is a widespread illness with an increasing prevalence and mortality rate (12) . Emphysema is characterized by an increased number of alveolar macrophages, neutrophils, and cytotoxic T lymphocytes, and the release of inflammatory mediators (lipids, chemokines, cytokines, and growth factors) (13) . Overexpression of placenta growth factor (PlGF) may contribute to the pathogenesis of pulmonary emphysema in transgenic mice (14) . Although it is known that nanoTiO2 or other nanoparticles can induce serious pulmonary toxicities, the mechanisms and the molecular pathogenesis are still unclear.

This study we investigate the effect of nanoTiO2 on the induction of pulmonary toxicity and emphysema, its mechanisms, and the molecular pathogenesis.

MATERIALS AND METHODS

Animals and nano materials
Adult male ICR mice (2 months old, 30 g; Harlan Sprague-Dawley, Indianapolis, IN, USA), free of known rodent pathogens, were obtained from the National Taiwan University (Taipei, Taiwan). They were cared and used humanely according to the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Health Research Institutes (NHRI, Taiwan). This study was approved by the Institutional Review Board and the Animal Care and Use Committee of the National Chung-Hsing University (Taichung, Taiwan).

NanoTiO2 (Rutile crystal phase, ultra-fine TiO2 nanoparticles), a highly dispersed and hydrophilic fumed TiO2 with a diameter of 19–21 nm (average primary particle size 21 nm), a specific surface area of 50 ± 15 m2/g, and a purity ≥ 99.5%, purchased from Degussa (Frankfurt, Germany). NanoTiO2 readily aggregate to form microparticles in normal saline or culture medium. To avoid aggregation, the nanoTiO2 suspension was ultrasonicated before it was used to treat animals or cells. Each sample was vortexed just before an aliquot was drawn for instillation. TiO2 microparticles have a diameter of 180–250 nm and a specific surface area of 6.5 m2/g (Fisher Scientific, Springfield, NJ, USA).

Cell lines
The human monocyte cell line THP-1 (American Type Culture Collection TIB 202; ATCC, Manassas, VA, USA) and the human lung carcinoma A549 cells (American Type Culture Collection CCL-185) were grown in normal culture medium [RPMI 1640 medium (GIBCO-Life Technologies, Inc., Gaithersburg, MD, USA), supplemented with 1.5 g/liter of Na2HCO3, 4.5 g/liter of glucose (Glc), and 10% FBS (FBS; GIBCO-Life Technologies)] (15) . To collect conditioned medium to stimulate other THP-1 cells, THP-1 cells were treated for 24 h with 3.2 x 10–7 M phorbol myristate acetate (PMA; Sigma Chemical Co., St. Louis, MO, USA), washed three times with PBS, and incubated for another 24 h to eliminate the effect of PMA. The conditioned medium was then used to stimulate fresh THP-1 cells to differentiate into macrophages (THP-1-derived macrophages), which were grown in normal culture medium for 24 h, then treated with or without nanoTiO2 for 24 h.

Intratracheal instillation of nanoparticles
The intratracheal instillation procedure was modified from that in a previous study (16) . In brief, after being anesthetized with 3 to 5% isoflurane in a small chamber, individual mice were secured on an inclined plastic platform and anesthesia continued via a small nose cone. The trachea was exposed by a 1 cm incision in the ventral neck skin for instillation of normal saline (NS, control) or the nanoTiO2 suspension. The instillation procedure for nanoTiO2 [0.1 mg per mouse (low dose) or 0.5 mg per mouse (high dose) in a 50 µl aliquot] was modified from previous reports to ensure that the instilled material was delivered into the lungs of mice with good distribution (16 , 17) . The NS group underwent the same surgical procedure and intratracheal instillation with normal saline.

The mice recovered and were active within 10 min after removal of the inhalation anesthetic. The incision healed within two days, and then the animals were observed daily until their scheduled termination, including 3 days for hyperacute response, 1 wk for acute-phase, and 2 wk for chronic phase. After instillation for 3 days, 1 wk, or 2 wk, the mouse was injected intraperitoneally (i.p.) with a lethal dose (0.1 ml) of pentobarbital sodium solution (Nembutal, Abbott, North Chicago, IL, USA) and the three right lobes of the lung tissues were collected and frozen in liquid nitrogen for RNA or protein extraction. For the histological study, the lung tissues were inflated with air at constant pressure (25 cmH2O) and then fixed with 10% buffered formalin by tracheal instillation.

Morphometric analysis of mice lungs after nano TiO2 exposure
Morphometric measurements mice lungs were performed by an investigator who was unaware of the identity of the samples. The mice lung sections (5 µm) were prepared and viewed with a 20x objective and the images digitized, converted to tagged image format file, and analyzed using MetaMorph® Imaging System software (Universal Imaging Corp., Downingtown, PA, USA). To evaluate the pathological changes following treatment, enlarged alveoli, disrupted septa, and thickened epithelia were measured as in previous studies (14 , 18 , 19) . Three parameters were measured in each section. The airspace area was measured and compared between the test groups and the NS group. A second parameter, which we call septal chord length, was measured as an indication of the thickness of the septa; this is identical to the parameter called airspace wall thickness in an earlier report (18) . The third parameter, the mean linear intercept (MLI), was used as a measure of the interalveolar wall distance (19) . Five areas selected randomly of each section slide were counted and six sections were examined in each animal.

Apoptosis assay by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)
Apoptotic cells in lung tissue sections were detected with the ApopTag in situ apoptosis detection kit (Roche Diagnostics, Mannheim, Germany) as in our previous study (20) according to the protocol provided by the manufacturer. The cells were counterstained with methyl green and those that were intact and exhibited dark brown-stained nuclei (TUNEL-positive cells) were considered positive for apoptosis (21) . The number of positively stained cells was measured in five randomly selected high-power fields (x400) per slide, and the significance of differences between the nanoTiO2-treated and NS groups was examined using Student’s t test.

Immunohistochemical staining
Immunohistochemistry was performed as in previous reports (20 , 22) . Lung tissue sections were incubated with a 1:100 dilution of rabbit polyclonal anti-F4/80 antibody (Ab) (marker for mouse macrophages) or anti-PCNA Ab (marker for proliferative cells) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal anti-PlGF2 Ab (R&D Systems, Minneapolis, MN, USA). The slides were then incubated with biotinylated secondary Ab and peroxidase-labeled streptavidin (avidin-biotin complex kit; Vector Laboratories, Burlingame, CA, USA). Negative control slides that were not treated with primary Ab were included for each staining procedure. Finally, 3,3'-diamino-benzidine (DAB) was used to develop the signals (brown color), while methyl green or hematoxylin was used for counterstaining. The observers randomly selected five areas and counted positive cells (brown staining) on a x200 field (i.e., x20 objective lens and x10 ocular lens, 1.227 mm2 per field). All counts were performed by two investigators blinded to the type of sample and both had to agree as to what constituted a positive cell before any cell was included in the count.

cDNA microarray analysis
The detailed protocol for the mouse cDNA microarray analysis has been reported in our previous studies (20 , 23) . Mouse expressed sequence tag (EST) clones were obtained from the IMAGE consortium libraries through its distributor (ResGen Invitrogen, Huntsville, AL, USA) (24) . The cDNA microarray carrying 6,144 polymerase chain reaction (PCR) -amplified cDNA fragments was prepared using an arraying machine. Potential interindividual variability was minimized by pooling the mRNA samples from two mice from each group to yield a representative sample for analysis. Total RNA was extracted from the pooled lung tissues for each group using RNAzolTM B solution (Life Tech, Gaithersburg, MD, USA) and mRNAs were extracted using an mRNA isolation kit (Qiagen, Hilden, Germany), in accordance with the manufacturer’s protocol. Five micrograms of mRNA from each sample was used in each array. The microarray images were scanned, digitized, and analyzed using a flatbed scanner (PowerLook 3000; UMAX, Taipei, Taiwan) and GenePix 3.0 software (Axon Instruments, Union City, CA, USA). In designing experiments involving microarrays, we adhered to the guidelines of the Microarray Gene Expression Data (MGED) Society (www.mged.org/Workgroups/MIAME/miame_checklist.html).

Identification of pathways using the KEGG and BioCata database
Gene identification was performed to determine which biochemical pathways were altered during the nanoTiO2-induced pulmonary inflammatory response. Having identified genes on the basis of the cDNA microarray data, it was of interest to determine whether any of these genes were part of the same pathway. The approach taken was to search the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.ad.jp/kegg/pathway.html) and the BIOCARTA (http://www.biocarta.com/genes/allpathways.asp) biochemical pathway database using the genes selected from cDNA microarray analysis as described previously (25) .

Real-time quantitative RT-polymerase chain reaction (RT-PCR)
To validate the microarray data, real-time quantitative RT-PCR was used in a 96-well format according to our previous described method (26) . Total RNA from lung tissues or cell cultures with or without nanoTiO2 treatment was prepared. Primers were designed using Primer Express v2.0 Software (Applied Biosystems Inc., Foster City, CA, USA) (Table 1 ). All reactions were carried in 50 µl volumes containing 25 µl of SYBR Green PCR Master Mix (Applied Biosystems Inc., Foster City, CA, USA). The amount of test gene cDNA relative to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (mouse tissues) or TATA box binding protein (TBP) cDNA (human cells) (housekeeping controls) was measured as –{Delta} computed tomography (CT) = – [computed tomographyTested gene - CTGAPDH or TBP]. The ratio of the tested gene mRNA copies relative to those for GAPDH or TBP was defined as 2 – {Delta} CT x K (K: constant).


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  		Table 1. NanoTiO2-induced genes detected using the "homemade" cDNA microarray (n=3)

Western blotting
The detailed protocol has been previously reported (23) . PlGF expression was detected using anti-mouse PlGF2 polyclonal antibody (pAb) (1:1,000 dilution; R&D Systems) or anti-human PlGF pAb (1:500 dilution; Santa Cruz Biotechnology). Western blot data was analyzed by densitometry (Imagemaster V-DS; Amersham Biosciences, Piscataway, NJ, USA).

Quantification of cytokine levels
Serum from mice with or without nanoTiO2 treatment or cell culture medium from A549 or THP-1-derived macrophages with or without nanoTiO2 treatment was collected and stored at –80°C until analysis. Levels of PlGF, MCP-1, and MIP-1 were measured using ELISA assay (R&D Systems) as described (14) .

Statistical analysis
Detailed descriptions and an excellent discussion of the issues involved in generating the microarray data, data normalization, statistical analysis, and its interpretation are given in our previous studies (20 , 27) . Genes which up-regulated or down-regulated in response to nanoTiO2 treatment were identified and were used for pathway analysis. An up-regulated gene had to show a 1.5-fold increase in the cDNA microarray. These genes were further analyzed by our in-house data mining tool based on KEGG and BIOCARTA pathway databases (http://biochip.nchu.edu.tw/SpecificDB/mouse.html).

ANOVA (Excel, Microsoft, Taipei, Taiwan) or Student’s t test was used to determine if significant differences were seen in replicate experiments between the NS and nanoTiO2-treated groups for the numbers of F4-positive, PCNA-positive, or TUNEL-positive cells in lung tissue sections and for PlGF expression on Western blots or by ELISA analysis.

RESULTS

NanoTiO2 can induce emphysema-like lung injury in mice
We found that 1 wk after single intratracheal instillation with nanoTiO2 in mice, the lungs showed significant changes in morphology and histology (Fig. 1 A), whereas no obvious morphological changes were seen in the NS-treated control group (Fig. 1A ). Disruption of the alveolar septa and alveolar enlargement (emphysematous change), type II pneumocyte proliferation, increased alveolar epithelial thickness, and accumulation of particle-laden macrophages (Fig. 1B ) were observed in the low dose (0.1 mg/mouse) nanoTiO2-treated group and were more severe in mouse lung treated with higher dose of NanoTiO2 (0.5 mg/mouse). These pathological changes diffusely involved the entire both lungs and were considerably more severe in areas in which nanoTiO2 accumulated. The NS control group showed no significant morphological or histological changes.


Figure 1
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  		Figure 1. NanoTiO2-induced pulmonary morphological and histological changes. A) Morphological (a, b) and histological (c, d) changes (H/E, hematoxylin and eosin staining) in the mouse lung at 1 wk after intratracheal instillation with NS (normal saline, a, c), or 0.1 mg/mouse nanoTiO2 (b, d). Arrowheads indicate the nodule-like lesions caused by chronic inflammation. Original magnification x100, bar = 100 µm for H/E histological image (c, d). B) Histological changes in the mouse lung after intratracheal instillation with nanoTiO2 for 1 wk. Lung tissues were collected from NS-treated control mice (a) and nanoTiO2 (0.1 mg/mouse) -treated mice (b, c). Original magnification, x400, bar = 50 µm. Similar results were obtained in six dependent experiments. C) Morphometric measurements from lungs at 3 days, 1 wk, and 2 wk after installation with NS or nanoTiO2 (0.1 or 0.5 mg/mouse). The mean linear intercept (MLI), mean airspace area, and septal chord length were measured (n=6) as described in Materials and Methods. The data are the means ±SD *P < 0.05 in Student’s t test compared with the control (NS) group.

Serial morphometric measurements of injured mice lungs were made at 3rd day, 1st wk, and 2nd wk after intratracheal installation of nanoTiO2 (Fig. 1C ). The mean linear intercept (MLI) (a measure of the interalveolar wall distance), the airspace area, and the septal chord length (a parameter that increases with septal thickness) were usually used as the pathological markers of pulmonary emphysema and granuloma. All three parameters were slightly increased at 3rd day (hyperacute phase) and significantly increased at 1st wk (acute-phase) after instillation of nanoTiO2, and the pathological changes persisted until 2nd wk (chronic phase). The nanoTiO2 therefore can induce time- and dose-dependent pulmonary emphysema-like changes in mice.

Alveolar macrophage infiltration and pulmonary cell apoptosis and proliferation
The nanoTiO2 treated mouse lungs showed significant increase in alveolar macrophage infiltration, alveolar epithelial septal thickness, and alveolar enlargement. Figure 2 A shows that the number of alveolar macrophage was significantly increased at wk 1 in the group treated with 0.1 mg of nanoTiO2 (4.16-fold increase), indicating that a severe inflammatory response occurred. These infiltrated particle-laden macrophages accumulated in the alveolus and could be identified by mouse macrophage-specific anti-F4/80 receptor antibodies. Using PCNA as the marker for cell proliferation, nanoTiO2-stimulated abnormal proliferation of type II pneumocytes could be identified in pulmonary tissues (Fig. 2B ).


Figure 2
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  		Figure 2. Effects of nanoTiO2 on macrophage accumulation, cell proliferation, and apoptosis. The left panels show typical results, the right panels the quantified results expressed as the mean±SD (n=6); *P < 0.05 vs. controls (NS). A) Immunostaining with mouse macrophage-specific anti-F4 Ab. The red arrows show the brown colored DAB F4/80-positive cells, Original magnification x400, bar = 50 µm. B) Immunostaining with Ab against PCNA (proliferative cell nuclear antigen) for proliferating cells. The red arrows indicate PCNA-positive cells. Original magnification x 400, bar = 50 µm. C) TUNEL staining for apoptotic cells. The red arrows indicate apoptotic macrophages, the blue arrows apoptotic epithelial cells. Original magnification x 400, bar = 50 µm.

TUNEL staining showed that the number of apoptotic cells was significantly increased in the nanoTiO2-treated group; these TUNEL-positive cells were both macrophages and alveolar type II pneumocytes (Fig. 2C ). The nanoparticle-induced alveolar epithelial cell apoptosis might cause abnormal airspace enlargement, which is a major pathological change in pulmonary emphysema.

Gene expression profiles of the mice lung after nanoTiO2 treatment
Messenger RNAs from mice lung tissues with or without 1 wk of nanoTiO2 treatment (0, 0.1, or 0.5 mg per mouse) were analyzed using the mouse cDNA microarray (19 , 24) . There were 506 genes out of the 6,144 putative genes showed a statistically significant difference (1.5-fold difference) in expression at wk 1 in nanoTiO2-treated mice compared with NS-treated mice, 318 genes being up-regulated and 188 genes down-regulated (Supplemental data, Fig. S1).

Some of these nanoTiO2-regulated genes are listed and categorized by their putative functions (Table 1) . Several vascular endothelial growth factor (VEGF) -related factors, G protein-coupled receptors (GPCR), cell growth regulators, and chemotaxis and immune response factors were significantly up-regulated by nanoTiO2-treatment. These gene expression changes reflect, at the molecular level, the observed nanoTiO2-induced inflammatory response.

Pathway analysis of nanoTiO2-induced transcriptomic changes
The differentially expressed profiles of nanoTiO2-induced genes were categorized and integrated to fit the transduction signaling map using the KEGG and BIOCARTA pathway database. This provided us with new information by giving a biological interpretation of the voluminous data generated by microarray experiments. According to the pathway analysis, four major pathways were up-regulated by nanoTiO2: the cell cycle regulatory pathway and apoptosis pathway (Fig. 3 A) and the chemokines pathway and complement cascade (classical pathway) (Fig. 3B ).


Figure 3
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  		Figure 3. NanoTiO2-induced genes in different pathways according to the KEGG pathway database and BIOCARTA. The pink color indicates nanoTiO2-induced genes, the red numbers close to the genes the fold increase. A) The cell cycle and apoptosis pathways. B) The PlGF/chemokines pathway and the classic complement pathway. The hypothetical nanoTiO2-regulated signaling pathways were modified from the KEGG and BIOCARTA database, while the PlGF pathway was modified from Selvaraj SK, 2003 (28) .

As shown in Fig. 3A , the cell cycle pathway analysis showed that nanoTiO2 regulated key factors for G2/M progression by increasing the expression of cdc2a, cyclin A2 (2.30-fold), cyclin B1 and B2, cyclin D1, cyclin E1 while the apoptosis pathway analysis showed they increased expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and tumor necrosis factor-receptor1 (TNF-R1) expression, respectively. As shown in Fig. 3B , nanoTiO2 stimulated the expression of several cytokines and chemokines, including PlGF (3.56-fold), a prochemokine known to regulate the expression of MCP-1, IL-1, and TNF-{alpha} (45 , 46) , as seen in our system. Other C-C and C-X-C chemokines were also up-regulated (Ccl2, Ccl3, Ccl9, Cxcl1, Cxcl5, Cxcr4, and Gp49b).

PlGF and chemokines in nanoTiO2-treated mice lung tissues
To confirm the role of the PlGF/chemokine pathway in nanoTiO2-induced pulmonary injury, real-time quantitative RT-PCR, Western blotting, ELISA, and immunostaining were used to demonstrate PlGF and related cytokines induction in nanoTiO2-treated mice.

Real-time quantitative RT-PCR analysis showed that plgf, chemokines (cxcl1, cxcl5, and ccl3), TRAIL, and prostaglandin E receptor 4 (ptger4, EP4) were significantly up-regulated in the lung tissues of mice treated with nanoTiO2 for 1 wk, while expressing of flt-1 and flt-3 (PlGF receptors), were not significantly affected (Fig. 4 A), the results are comparable to those obtained in the microarray and pathway analyses (Fig. 3) . Fine TiO2 microparticles (microTiO2) had less effect on induction of these genes (Fig. 4A ).


Figure 4
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  		Figure 4. PlGF, chemokines and related factors expression in mice after single intratracheal instillation with nanoTiO2. A) Real-time quantitative RT-PCR for flt-1 (PlGF receptor), flt-3, plgf, chemokines (cxcl1, cxcl5, ccl3), and apoptosis-related factors (trail and ptger4). The data are expressed as the fold increase compared with the NS control ±SD *P < 0.05 vs. the NS control; **P < 0.05 vs. microTiO2 (n=4). B) Western blotting for the effect of nanoTiO2 on PlGF protein expression. The right panel shows the expression of PlGF relative to that for {alpha}-tubulin expressed as a fold increase compared with the NS control ±SD *P < 0.05 vs. NS control (n=4). C) Serum PlGF protein levels measured by ELISA. The data are expressed as the mean ±SD. *P < 0.05 vs. controls (n=4). D) Immunohistochemical staining with anti-PlGF2 Ab showing overexpression of PlGF (brown color, DAB staining) in mice lung tissues after intratracheal instillation with nanoTiO2 (0.1 mg and 0.5 mg per mice). Similar results were obtained in 4 experiments.

The nanoTiO2-induced PlGF expression was also examined at the protein level after single intratracheal instillation with nanoTiO2 (0.1 mg or 0.5 mg per mice) for 1 wk. Western blotting showed that nanoTiO2 caused significant induction of PlGF expression in a dose-dependent manner (Fig. 4B ), while ELISA analysis showed that nanoTiO2-treated mice had higher serum levels of PlGF protein (Fig. 4C ), which might be produced mainly by infiltrating macrophages and some pulmonary epithelial cells, as shown by immunostaining with PlGF-specific antibodies (Fig. 4D ).

PlGF and other chemokines expression in macrophagse and lung epithelial cells exposed to nanoTiO2 in vitro
To study the responses of different human pulmonary cells to nanoTiO2, THP-1-derived macrophages and the lung epithelial cell line, A549, were used as in vitro models. Incubation of cultured cells with nanoTiO2 showed that they caused significant dose-dependent induction of PlGF expression at both the mRNA and protein levels in THP-1-derived macrophages, as shown by Western blotting (Fig. 5 A), real-time quantitative RT-PCR (Fig. 5B ), and ELISA (Fig. 5C ), but had less effect on human A549 pulmonary epithelial cells (Fig. 5B ).


Figure 5
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  		Figure 5. A) Western blotting of PlGF protein expression in human THP-1 cell-derived macrophages treated for 24 h with nanoTiO2 (0.1, 0.2, 0.5 µg/ml) or microTiO2 (0.2 µg/ml). The right panel shows the expression of PlGF relative to that for {alpha}-tubulin expressed as a fold increase compared with the controls (n=3). The data are expressed as the mean±SD *, P < 0.05 vs. controls. B) Real-time quantitative RT-PCR analysis showing effects of nanoTiO2 or microTiO2 on PlGF expression in THP-1 cell-derived macrophages and A549 cells. C) ELISA measurements of PlGF levels in THP-1 cell-derived macrophage or A549 cells culture medium following nanoTiO2 or microTiO2 treatment. (D, E) Real-time quantitative RT-PCR analysis for cxcl5 mRNA in THP-1 cell-derived macrophages (D) and A549 cells (E). F) Expression of MCP-1 (ccl2) in THP-1 cell-derived macrophages and A549 cells following nanoTiO2 or microTiO2 treatment. The data are the mean ±SD (n=3).

To examine whether these effects on PlGF/chemokines induction pathways were specific to nanoTiO2, the macrophages THP-1 and lung epithelial cells A549 were treated with nanoTiO2 or microTiO2. The results showed that nanoTiO2 caused significant induction of PlGF expression in macrophages and lung epithelial cells at the protein level (Fig. 5A ) and mRNA level (Fig. 5B ) in cells and increased levels of secreted protein (Fig. 5C ), whereas microTiO2 had no, or only a slight, effect. Figure 5D , E shows that cxcl5 mRNA levels were increased by nanoTiO2 in both types of the cells, while microTiO2 had little effect. To study the downstream effectors of PlGF, we examined protein levels of MCP-1 (CCL2) in the culture medium and found that these chemokines were increased by nanoTiO2 in THP-1 cell-derived macrophages, but not in A549 cells, and that microTiO2 had no effect (Fig. 5F ).

DISCUSSION

The results of this study indicate that single intratracheal instillation of 0.1 mg nanoTiO2 can induce severe pulmonary inflammation and emphysema in the mouse lung. The finding of pulmonary inflammation is consistent with another report showing that nanoparticle inhalation can induce pulmonary inflammation (2) , However, the observation that nanoTiO2 can induce pulmonary emphysema after single intratracheal expose of 0.1 mg nanoTiO2 is novel. Our results indicate that the pulmonary emphysema is triggered by nanoTiO2 activation of macrophages, up-regulations of PlGF and other inflammatory cytokines that resulted in disruption of alveolar septa, alveolar epithelial injury, type II cell proliferation and apoptosis. This information may have important clinical implications regarding the safety issue, as nanoTiO2 are widely used as a photocatalyst in air and water cleaning (4) and TiO2 is used as a pigment in the paint industry. Extra caution should therefore be taken in the handling of higher dose nanoTiO2.

Pulmonary toxicity caused by nanoTiO2 inhalation has been reported (5 , 6) , but its molecular pathogenesis is not known. In this study, the microarray gene expressions and pathway analysis indicated that the cell cycle, apoptosis, chemokine, and complement pathways may be involved in nanoTiO2-induced pulmonary toxicity (Fig. 3) . The activation of cell cycle pathway suggests that nanoTiO2 can regulate key factors for G2/M progression by increasing the expression of cdc2a, cyclin A2, and cyclin B1, which may explain the increase in the number of proliferating (PCNA-positive cells) type II pneumocytes and the increase in septal thickness seen in this study. The activation of apoptosis pathway indicate that nanoTiO2 can increase TRAIL expression, which may account for the increased number of TUNEL-positive cells in nanoTiO2-treated samples, explaining the alveolar type II cell apoptosis, abnormal airspace enlargement, and pulmonary emphysema.

The pathway analysis also shows that nanoTiO2 can stimulate the expression of several cytokines and chemokines, including PlGF, a prochemokine that can regulate the expression of MCP-1, IL-1, and TNF-{alpha} (13 , 45 , 46) . These chemokines may also affect the expression of other C-C and C-X-C chemokines (Ccl3, Cxcl1, and Cxcl5) that modulate chemotaxis, neutrophil infiltration, macrophage accumulation, epithelial cell proliferation, and apoptosis to generate the inflammatory cascade, which, may lead to the pathogenesis of pulmonary emphysema.

The nanoTiO2-induced expression of many cytokines and chemokines (Fig. 3) may play an important role in the macrophage accumulation, neutrophil infiltration, cell apoptosis, lung destruction, and pulmonary emphysema seen in this study. The induction of chemokines by nanoTiO2 has some similarities with the gene expression profiles seen in a lipopolysaccharide (LPS) (lipopolysaccharide) -induced acute lung injury model (28) . CCL22 (macrophage-derived chemokine, MDC), CCL3 (MIP-1 alpha), CCL2 (MCP-1 alpha), CXCL2/3 (MIP-2), and CXCL1 (keratinocyte cell-derived chemokine, KC) were induced in both lung injury models. Among these factors, expression of MIP-1 alpha and MIP-2, has been shown to be induced by TiO2 in A549 epithelial cells and macrophages in previous studies (29 , 30) . In our study, CXCL1, CXCL5, and CCL3 were significantly induced and this was confirmed in in vivo and/or in vitro studies. CXCL1 (KC) is a potent neutrophil chemoattractant involved in several lung injury processes, and its up-regulation has been correlated with neutrophil infiltration and the development of granulomas (31) . CXCL5 (epithelial cell-derived neutrophil-activating peptide-78, ENA-78) is also a neutrophil chemoattractant involved in pulmonary inflammation (32) , while CCL3 (MIP-1 alpha) is an important chemokine involved in pulmonary host defense during infections (33 , 34) . Several of these factors were also found to be increased in patients with emphysema and {alpha}1-antitrysin deficiency-related emphysema in a microarray study (35) . These key chemokines might play important roles in nanoTiO2-induced inflammatory responses and could also be involved in the pathogenesis of pulmonary granuloma and emphysema.

NanoTiO2 caused increased expression of the classical complement pathway components, C1q, C3a, and C4, which could lead to complement activation, including C5 and its receptor, C5R1, leading to more phagocyte recruitment and chemotaxis and an inflammatory response in the lungs (36 , 37) and the destruction of pulmonary tissues. A recent study demonstrated that C5 and the C5a receptor are involved in the mycobacterial glycolipid trehalose 6,6'-dimycolate-induced pulmonary granulomatous response (38) . These findings suggest that the classical complement pathway might be involved in nanoTiO2-induced lung injury.

In this study, nanoTiO2 increased mRNA and protein levels of PlGF (a chemokine inducer) both in vivo (mice) and in vitro (human THP-1 cell-derived macrophages) and up-regulated factors downstream of PlGF [MIP-1 (CCL3), MCP-1 (CCL2), and IL-1] in a concentration-dependent manner. PlGF has been highlighted to be involved in pathophysiologic monocyte recruitment previously (39 40 41 42) . It and its receptor, Flt-1, can activate the PI3 kinase/AKT and ERK-1/2 pathways through monocytes and cause increased expression of cytokines (TNF-alpha and IL-1beta) and chemokines (monocyte chemotactic protein-1 [MCP-1, CCL2], IL-8, and macrophage inflammatory protein-1 [MIP-1, CCL3]) in both normal monocytes and the THP-1 monocytic cell line (43) . Recently, PlGF has been correlated with the pathogenesis of pulmonary emphysema, as PlGF transgenic mice show lung epithelial cell apoptosis and spontaneous pulmonary emphysema (13) . We suggest that PlGF may be involved in nanoTiO2-induced pulmonary emphysema by regulating certain inflammatory responses, including chemokines induction, macrophage infiltration, cell proliferation and apoptosis. Previous PlGF knockout mice studies have shown that the absence of PlGF could reduce vascular leakage induced by skin wounding, allergens, and neurogenic inflammation in many diseases, also the inflammatory angiogenesis and edema formation were inhibited (44 , 45) . These results indicated that the important role of PlGF in immuno-regulation. More studies will be done to confirm the central role of PlGF in nanoTiO2-induced pulmonary inflammation and emphysema via using PlGF knockout mice.

In this study, we found that a single intratracheal exposure to nanoTiO2 could induce pulmonary emphysema and severe lung injury in mice. However, no significantly pathological changes were seen using the same dose of microTiO2 (180–250 nm). This finding is consistent with other reports that nanoTiO2 (20 nm) cause a significantly greater pulmonary inflammatory response than microTiO2 (250 nm) in rats and mice (6 , 46) . The greater toxicity of nanoTiO2 might correlate with their greater surface area per unit mass. A significantly greater increase in PlGF was induced by nanoTiO2 than microTiO2 in this study, suggesting that nanoTiO2-induced pulmonary toxicity may be mediated by PlGF.

The results of this study add our understanding of nanoTiO2-induced pulmonary toxicity and pulmonary emphysema. Both are complicated multifactorial disease processes. We suggest that PlGF, chemokines, and the complement cascade may cause inflammatory cell chemotaxis, cell proliferation and apoptosis, resulting in serious lung injury. Further investigations are needed to elucidate the potential pulmonary toxicity of different nanoparticles and their pathogenesis.

ACKNOWLEDGMENTS

The authors thank Drs. Gene Alzona Nisperos, W. K. Chan, and Tom Barkas for their excellent editing. This investigation was supported by grants from the National Science Council, Taiwan (NSC94–2314-B-005–004 and NSC95–2314-B-005–003).

FOOTNOTES

1 These authors contributed equally to this work.

Received for publication May 17, 2006. Accepted for publication June 19, 2006.

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