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Published ahead of print on June 16, 2005, doi:10.1165/rcmb.2005-0003OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 290-296, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0003OC

Interleukin-25 and Interleukin-13 Production by Alveolar Macrophages in Response to Particles

Chun-Mi Kang*, An-Soo Jang*, Mi-Hyun Ahn, Jeong-Ah Shin, Ji-Hye Kim, Yun-Sung Choi, Tai-Youn Rhim and Choon-Sik Park

Genome Research Center for Allergy and Respiratory Diseases, and Division of Allergy and Respiratory Diseases, Soonchunhyang University Hospital, Bucheon, Republic of Korea

Correspondence and requests for reprints should be addressed to Dr. Choon-Sik Park, Division of Allergy and Respiratory Diseases, Department of Internal Medicine, Soonchunhyang University Hospital, 1174, Jung-dong, Wonmi-gu, Bucheon-si, Gyeonggido 420-767, Republic of Korea. E-mail: mdcspark{at}unitel.co.kr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Particle inhalation–induced lung inflammation acts as an adjuvant to allergens or respiratory viral infection in a process that is mediated by macrophages and epitheliums. The production of interleukin (IL)-4 and IL-13 by activated T cells is involved in the augmentation of Th2-type immune responses to particles, and IL-25 induces the synthesis of IL-4 and IL-13. However, whether IL-13 and IL-25 are directly regulated by particle instillation in the lung has not been studied. The aim of this study was to reveal particle induction of IL-13 and IL-25 in the lung. TiO2 instillation potently induced the mRNA expression for IL-25 and IL-13 in lung tissue extracts 24 h after treatment, as compared with the sham group. Immunostaining for IL-25 and IL-13 showed strong positivity for macrophages in the inflammatory lung lesions of TiO2-treated rats. The alveolar macrophages expressed IL-25 and IL-13 24 h after in vitro stimulation with TiO2 particles in dose- and time-dependent manners, with maximal induction at 24 and 48 h after stimulation, respectively. The sequence of the rat IL-25 gene is 95% homologous with the mouse IL-25 gene. These findings indicate that alveolar macrophages play an important role in particle-induced lung inflammation via direct induction of IL-13 and IL-25 production.

Key Words: cytokines • inflammation • lung • macrophages


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Growing epidemiologic evidence indicates that the inhalation of airborne particulate matter (PM) is associated with the adverse health outcomes of increased respiratory and cardiac mortality and morbidity (1). Patients with chronic obstructive pulmonary disease who live in communities where they are exposed to high levels of air pollution show more rapid declines in lung function than those living in areas with low levels of pollution (2). The level of environmental particles is also positively correlated with exacerbation of asthma (3). During the last decade, the composition of air pollution has changed from classical type I pollution, which consists of SO2 and large dust particles, to modern type II pollution, which is characterized in developed countries by oxides of nitrogen, organic compounds, ozone, and ultrafine particles (4). Airborne PM of < 10 µm in aerodynamic diameter (PM10) is a complex mixture of materials with a carbonaceous core and associated materials, such as organic compounds, acids, and fine particles of metals (5).

PM10 or diesel extract particles (DEP) act as adjuvants to augment lung inflammation caused by inhalant allergens or respiratory viral infection. The responses may lead to the enhancement of already existing allergies or IgE responses to neoallergens, and susceptibility to respiratory infection. This adjuvant effect is exerted by the enhanced production of inflammatory Th2 and/or Th1 cytokines (6, 7). In animal experiments and human studies, the levels of several cytokines and CC chemokines, including interleukin (IL)-4, IL-5, IL-13, granulocyte macrophage–colony-stimulating factor (GM-CSF), RANTES, monocyte chemotactic protein-3, and macrophage inflammatory protein (MIP)-1, are increased when lymphocytes and macrophages/monocytes are co-stimulated with particles in the presence of specific allergens (8–10). The immune responses can be modified in different ways according to the type of PM. DEP favor Th2-type responses, whereas macrophages that are pulsed with asbestos fiber and carbon black particles upregulate both Th1 and Th2 cytokine production from autologous antigen-stimulated lymphocytes (9).

In addition to their adjuvant effects, inhaled inert particles cause a spectrum of pulmonary responses, ranging from minimal changes to marked acute and chronic inflammation (10). The inflammation-inducing effects of PM10 have been demonstrated in experimental animal studies following direct instillation into the lung, while the human studies have shown pulmonary effects after experimental exposure to PM (11, 12). Clinically, PM10 is thought to provoke airway inflammation via the release of mediators that are capable of exacerbating lung disease in susceptible individuals (13), whereby even a single exposure compromises the host's ability to handle ongoing pulmonary infections (14). The fine and ultrafine particles directly stimulate macrophages and epithelial cells to produce inflammatory cytokines, such as tumor necrosis factor (TNF)-{alpha}, transforming growth factor-ß1, GM-CSF, platelet-derived growth factor, IL-6, and IL-8 (15–18), and reactive oxygen species (19). All of these cytokines are responsible for acute and chronic inflammation in the lung. However, there is a lack of evidence on whether particles can directly induce Th2-like cytokines, such as IL-4 and IL-13.

IL-25 (IL-17E) is a member of the IL-17 family that induces Th2-type cytokines, eosinophilia, and the development of Th2-associated histologic changes in the lungs and gastrointestinal tracts of experimental animals (20). IL-25 induces the expression of mRNA for Th2-like cytokines, notably IL-4 and IL-13, and for chemokines, such as eotaxin, in addition to stimulating airway hyperreactivity (21). Therefore, IL-25 may be a candidate cytokine for the inflammation seen in particle- or allergen-induced lung diseases. We hypothesize that particles induce IL-25 overproduction by activated macrophages in the lung, and that this leads to a Th2-like environment, with the overproduction of IL-4 and IL-13. In the current study, we demonstrate at the protein and mRNA levels, both in vivo and in vitro, that alveolar macrophages produce IL-25 and IL-13 in response to fine particles of TiO2.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Preparation of TiO2 Particles, and the Animal Model of TiO2–Induced Inflammation
Fine rutile TiO2 particles (mean diameter = 0.29 µm) were sterilized by autoclaving and suspended in serum-free medium, after coating with bovine serum albumin (BSA) to minimize particles aggregation and hydrophobicity (22). After sonication, the endotoxin concentration of the TiO2 suspension was < 0.064 ng/ml (0.32 EU/ml), as judged by the Limulus Amebocyte Lysate assay (QCL-1000; BioWhittaker, Inc. Walkersville, MD) (23). Male, 7-wk-old Sprague-Dawley rats (Orient Co., Ltd., Charles River Laboratories, Seoul, Korea) that were free of rat-specific pathogens were used. The rats were housed throughout the experiments in a laminar flow cabinet, and were fed standard laboratory chow ad libitum. All of the experimental animals used in this study were treated according to guidelines approved by the Institutional Animal Care and Use Committee of the Soonchunhyang University Medical School. The rats received 4 mg TiO2 in 0.2 ml of endotoxin-free water by the intratracheal route. The rats were killed with an overdose of pentobarbital sodium (65 mg/kg body weight, administered intraperitoneally). The chest cavity was exposed, and the catheter was carefully inserted into the trachea and secured with ligatures. Bronchoalveolar lavage (BAL) was performed by three instillations of 2 ml normal saline, followed by gentle retrieval. Cell numbers were measured using a hemocytometer, and differential cell counts were performed on slides that were prepared by cytocentrifugation and Diff-Quik staining (Scientific Products, Gibbstowne, NJ). Supernatants were separated by centrifugation (500 x g for 5 min) and kept at –70°C until use. After ligation of the right main bronchus, the left lung was fixed with 4% paraformaldehyde in phosphate-buffered saline and paraffin-embedded. The right lung was excised and immersed in TRI reagent (guanidium isothiocyanate-phenol mixture; Molecular Research Center, Inc., Cincinnati, OH), and immediately frozen in liquid nitrogen.

Isolation of Macrophages and Culturing in the Presence of TiO2 Particles
Alveolar macrophages were isolated from the BAL cells of normal, 7-wk-old, male Sprague-Dawley rats by centrifugation on Ficoll solution (Histopaque-1077; Sigma Diagnostics Inc., St. Louis, MO), with purity and viability of > 98%. Cells (5 x 105) were seeded on 24-well plates and cultured in 1 ml RPMI 1640 medium (WelGENE, Daegu, Korea) that was supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml), at 37°C in 5% CO2, in the presence or absence of various concentrations of BSA-treated TiO2 particles. The culture supernatants were removed and stored at –70°C until analysis, and RNA was extracted from the cell pellets. For the immunocytochemical staining, 1 µM monensin (Sigma) was added 6 h before the end of the culturing period, to block extracellular trafficking of cytokines. The cells (2 x 105) were cytocentrifuged and air-dried for 1 min, and then fixed in 100% EtOH for immunocytochemistry.

Histology and Immunohistochemistry of Lung Tissues
Sections of the fixed embedded tissues were cut to 4-µm thickness using a Leica model 2165 rotary microtome (Leica Microsystems Nussloch GmbH, Nussloch, Germany), and placed on glass slides. For the immunohistochemistry and immunocytochemistry of IL-13 and IL-25, the deparaffinized 4-µm-thick tissue sections or the cytocentrifuged preparations of cultured macrophages were stained using the RTU Vectastain Universal Quick Kit (Vector Laboratories Inc., Burlingame, CA). Briefly, the slides were treated with 0.3% H2O2-methanol for 30 min, to block endogenous peroxidase, and incubated with the mouse anti-rat IL-13 monoclonal antibody (1:50 dilution; Biosource International Inc., Camarillo, CA) and the goat anti-mouse IL-25 polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4°C overnight. After washing with Tris-buffered saline, the slides were incubated with avidin–biotin peroxidase complex (ABC kit; Vector Laboratories). The color reaction was developed by staining with 3,3'-diaminobenzidine tetrachloride (Zymed Laboratory Inc., San Francisco, CA).

IL-13 Assays of BAL Fluids and Culture Supernatants
The levels of IL-13 were quantified in BAL fluids and in the culture supernatants of alveolar macrophages using a sandwich enzyme-linked immunosorbent assay kit, according to the manufacturer's protocol (Biosource International, Inc.). Each sample was assayed in duplicate. The lower limit of detection was 7.8 pg/ml. Values below this limit were assigned the value of zero for the purpose of statistical analysis. The inter- and intra-assay coefficients of variance were < 10%.

Western Blotting
Cultured macrophages were detached from the plates by the addition of cold phosphate-buffered saline plus EDTA and vigorous pipetting: The cells were harvested by centrifugation (400 x g for 10 min) and treated with a solution that contained 50 mM Tris-HCl (pH 7.4), 1% NP-40, 150 mM NaCl, 1 mM EDTA, and 0.25% Na-deoxycholate on ice for 20 min. Protein concentrations were determined using the BCA kit (Pierce Biotechnology Inc., Rockford, IL) and 10 µg of protein were loaded and electrophoresed on a discontinuous 15% and 4% polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane at 70 V for 2 h. The membrane was blocked with 5% nonfat milk in Tris-buffered saline for 2 h, and incubated with the goat anti-mouse IL-25 polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology) at 4°C overnight. The membrane was incubated with horseradish peroxidase–conjugated rabbit anti-goat IgG (1:1,000) at room temperature for 2 h. The target protein was detected by enhanced chemiluminescence (ECL Kit; Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) using X-ray film.

Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted using the TRI Reagent (guanidium isothiocyanate-phenol mixture; Molecular Research Center) and chloroform, according to the manufacturer's instructions. The following primer pairs were used for the PCR: rat IL-13, 5'-ATCACACAAGACCAGAAGACTTC-3' and 5'-AACTGGGCTACTTCGATTTTGG-3'; mouse IL-25, 5'-GAGGCTTCTGGACTGCGGTGGTCC-3' and 5'-TTGACAG GCGAGGGTCACAGCTCC-3'; and rat GAPDH, 5'-GGCATTGCTCTCAATGACAA-3' and 5'-AGGGCCTCTCTCTTGCTCTC-3'. The mouse IL-25 sequence (www.ncbi.nlm.nih.gov; accession no. AF458060) was used to design the rat IL-25 primers, because the rat IL-25 sequence has not been published. The RNA was reverse-transcribed by incubation with 10 mM dNTP, 0.1 M DTT, 1 µl oligo(dT) (500 µg/ml) and 1 µl SuperScript II (200 U/µl; Life Technologies, Grand Island, NY) at 42°C for 50 min, and then heat-inactivated at 70°C for 15 min. After reverse transcription, the cDNA was aliquoted into tubes that contained specific primer pairs for the rat IL-13, mouse IL-25, and rat GAPDH genes. PCR was performed in a thermocycler with an initial denaturation step of 94°C for 5 min, followed by 28 cycles of 1 min at 94°C, 1 min at 50°C, 1 min at 72°C for the IL-13 gene, or 28 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C for the IL-25 gene, with a final extension at 72°C for 10 min in both cases. The amplified PCR products were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining, together with the 100-bp DNA ladder marker (Bioneer Co., Daejon, Korea).

PCR Amplicon Sequencing
The PCR product of the IL-25 gene was mixed with PCR primer and sequenced using the ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and the ABI PRISM 3700 DNA analyzer (Applied Biosystems). The derived sequences were compared with those of other genes using the BLAST search program of the National Institutes of Health.

Statistical Analysis
Differences between independent samples were compared using the nonparametric Kruskal-Wallis H test for continuous data. When the differences were significant, the Mann-Whitney U test was applied, to compare differences between two samples. Differences were considered to be statistically significant when the P value was < 0.05. The results are expressed as means ± SEM unless stated otherwise.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Comparison of the IL-13 and IL-25 mRNA Levels in the Lungs and the IL-13 Levels in the BAL Fluids between TiO2-Treated and Sham-Treated Rats
The expression of IL-13 and IL-25 mRNA transcripts was investigated using RT-PCR of lung tissue extracts that were obtained from TiO2-treated and sham-treated rats. The levels of IL-13 and IL-25 mRNA expression were significantly increased in the lungs 24 h after treatment with TiO2 particles, compared with those of sham-treated rats (Figure 1A). The levels of IL-13, as measured by ELISA, were significantly increased in the BAL fluids of TiO2-treated rats 72 h after treatment (n = 8), as compared with those of sham-treated rats (n = 8) (P = 0.03) (Figure 1B).



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  		Figure 1. Comparison of the IL-13 and IL-25 mRNA expression levels in the lungs (A) and the IL-13 level in the BAL fluids (B) between TiO2-treated and sham-treated rats. The levels of IL-13 and IL-25 mRNA were determined by RT-PCR (A). The IL-13 and IL-25 mRNA levels are increased in the lungs 24 h after treatment with TiO2 particles (4 mg TiO2 in 0.2 ml endotoxin-free water). Shown are representative data from five independent experiments. The IL-13 levels are increased in the BAL fluids of the TiO2-treated rats 72 h after treatment (n = 8), as compared with sham-treated rats (n = 8) (P = 0.03) (B). The results are expressed as means ± SEM. *Significant difference (P < 0.05) when compared with the sham-treated group.

 
Comparison of IL-13 and IL-25 Protein Expression in the Lungs of TiO2-Treated and Sham-Treated Rats
Lung tissues from TiO2-treated and sham-treated rats were stained with monoclonal antibodies directed against IL-13 (Figures 2A and 2B) and IL-25 (Figures 2D and 2E). Serial tissue sections from TiO2-treated rats were stained with hematoxylin-eosin (Figures 2C and 2F). Inflammatory cells, which included mononuclear cells and polymorphonuclear leukocytes, infiltrated into the alveolar interstitium and the peribronchiolar area in the TiO2-treated rats (Figures 2C and 2F). In the inflammatory lesions, macrophages were seen to engulf TiO2 particles. The particle-laden macrophages were positive for IL-13 and IL-25 protein in the immunohistochemical staining (Figures 2B and 2E). In contrast, there was no inflammation (data not shown) and no immunoreactivity for either IL-13 or IL-25 in the lung tissues of the sham-treated rats (Figures 2A and 2D). These in vivo findings suggest that lung macrophages produce IL-13 and IL-25 after the engulfment of particles.



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  		Figure 2. The expression of IL-13 and IL-25 proteins in the lungs of TiO2-treated and sham-treated rats. Lung tissues from TiO2-treated and sham-treated rats were stained with monoclonal antibodies directed against IL-13 (A and B) and IL-25 (D and E). Serial tissue sections from TiO2-treated rats were stained with hematoxylin-eosin (C and F). Inflammatory cells, including mononuclear cells and polymorphonuclear leukocytes, have infiltrated into the alveolar interstitium and the peribronchiolar area in the TiO2-treated rats (C and F). In the inflammatory lesions, macrophages are seen to engulf TiO2 particles. Note that the particle-laden macrophages (arrow) are positive for IL-13 and IL-25 protein in the immunohistochemically stained sections (B and E). In contrast, there was no inflammation (data not shown) and no immunoreactivity for the IL-13 and IL-25 proteins in the lung tissues of sham-treated rats (A and D). Bars indicate 100 µm.

 
IL-13 and IL-25 mRNA and Protein Expression by Cultured Alveolar Macrophages Stimulated In Vitro with TiO2 Particles
To confirm the production of IL-13 and IL-25 by macrophages in response to particles in vitro, purified alveolar macrophages were harvested and cultured with or without TiO2 particles (40 µg/ml) for different periods of time (24, 48, and 72 h). Under the microscope, the alveolar macrophages stimulated with TiO2 for 48 h contained numerous particles and showed positive staining for IL-13 (Figure 3B) and IL-25 (Figure 3D). The unstimulated macrophages were negative for the IL-13 (Figure 3A) and IL-25 (Figure 3C) proteins. The levels of cytokine production were validated by the increased expression of mRNA for each cytokine. RT-PCR showed that the IL-13 and IL-25 mRNA expression levels were increased in alveolar macrophages 24 h after treatment with TiO2 particles (Figure 3E). To prove that the cells producing IL-25 are macrophages, we performed an in vitro experiment using macrophage-specific antibody (CD163) and anti–IL-25. TiO2 particle ingested macrophage showed positive stain to macrophage-specific antibody (CD163) and anti–IL-25 (data not shown). These in vitro results confirm that macrophages produce IL-13 and IL-25 when stimulated with particles.



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  		Figure 3. In vitro expression levels of IL-13 and IL-25 mRNA, and protein, by cultured alveolar macrophages that were stimulated with TiO2 particles. Purified alveolar macrophage were harvested and cultured with or without TiO2 particles (40 µg/ml) for 48 h. Viewed with the microscope, numerous particles are apparent inside the alveolar macrophages, which show positive staining for IL-13 (B) and IL-25 (D) when stimulated with TiO2. The unstimulated macrophages are negative for the IL-13 (A) and IL-25 (C) proteins. The observed levels of protein synthesis are validated by the increased expression of mRNA for each cytokine. RT-PCR shows that IL-13 and IL-25 mRNA transcripts are expressed by alveolar macrophages 24 h after treatment with TiO2 particles (E). Bars indicate 50 µm.

 
Time- and Dose-Responses for IL-13 and IL-25 Production by Macrophages Exposed to TiO2 Particles
To investigate the time- and dose-dependency of macrophage IL-13 and IL-25 production, purified alveolar macrophages were stimulated with 1, 10, and 40 µg/ml TiO2 for 24, 48, and 72 h (n = 6 in each experiment). The control group (n = 6) consisted of untreated alveolar macrophages. The IL-13 levels in the supernatants of the macrophage cultures were measured by ELISA. Macrophages that were cultured for 48 h with TiO2 produced IL-13 in a dose-dependent manner (Figure 4A). TiO2 concentrations > 10 µg/ml significantly enhanced IL-13 production when compared with the control group. The production of IL-13 protein was increased in a time-dependent manner and peaked 48 h after TiO2 stimulation (Figure 4B).



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  		Figure 4. Time and dose responses of IL-13 production by macrophages exposed to TiO2 particles. Purified alveolar macrophages stimulated with 1, 10, and 40 µg/ml TiO2 for 24, 48, and 72 h (n = 6 in each experiment). The control group (n = 6) consisted of unstimulated alveolar macrophages. The IL-13 in the 48-h culture supernatants is produced in a dose-dependent manner after TiO2 treatment (A). TiO2 concentrations > 10 µg/ml significantly enhance IL-13 production when compared with the control group. The production of IL-13 protein is increased in a time-dependent manner and peaks 48 h after TiO2 stimulation (B). The results are expressed as means ± SEM. *Significant difference (P < 0.05) when compared with the control group.

 
Due to the lack of a commercially available kit for IL-25 protein, the kinetics of IL-25 production was analyzed by Western blotting and RT-PCR. Alveolar macrophages were stimulated with 40 µg/ml TiO2 for 24, 48, and 72 h. The control group consisted of untreated alveolar macrophages. IL-25 mRNA expression peaked 24 h after treatment and persisted for 72 h after stimulation, whereas it was not expressed before TiO2 stimulation or at 4 h after stimulation. The IL-25 protein levels revealed by Western blotting showed similar kinetics to the RT-PCR experiment (Figure 5).



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  		Figure 5. Kinetics of IL-25 production by alveolar macrophages that were stimulated with TiO2. Alveolar macrophages were stimulated with 40 µg/ml TiO2 for 24, 48, and 72 h. The control group consisted of untreated alveolar macrophages. The IL-25 mRNA and protein expression levels were analyzed by Western blotting and RT-PCR, respectively. IL-25 mRNA expression peaks 24 h after treatment and persists for 72 h after stimulation, whereas it is not expressed before TiO2 stimulation or at 4 h after stimulation. The IL-25 protein levels revealed by Western blotting show similar kinetics to the RT-PCR experiment. The results shown are representative of three experiments.

 
Validation of the PCR Product by Direct Sequencing
To validate the RT-PCR product of the RNA samples from the rat alveolar macrophages, the DNA sequence was analyzed using the BLAST search program after direct sequencing. The amplified fragment shared 95% sequence identity with the mouse IL-25 gene and 99% identity with the rat LOC363329 gene.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Particle exposure modulates the immune responses to respiratory viral infections and allergens (24, 25). Before the discovery of the adjuvant effects of PM, which deviate the airways of experimental animals (26) and humans (6) toward Th2 environments, macrophages and epithelial cells, as the principle targets of PM in the lung, were known to modulate immune and/or inflammatory responses via the direct production of proinflammatory or fibrogenic cytokines, such as TNF-{alpha}, transforming growth factor-ß1, GM-CSF, platelet-derived growth factor, IL-6, and IL-8 (16, 17, 19), as well as reactive oxygen species (20). Because alveolar and airway macrophages are the initial target cells for particle interaction and deposition, and are therefore the cells most likely to be affected by the toxic effects of particles (27), we focused on these cell types.

To our knowledge, the current study is the first to report on the expression of IL-13 and IL-25 mRNAs and proteins by alveolar macrophages in response to PM, both in vivo and in vitro. We demonstrate in vivo expression of both cytokines in the lung using direct instillation of TiO2 particles. The upregulation of IL-13 and IL-25 mRNA production was apparent 24 h after treatment, as assessed by RT-PCR (Figure 1). IL-13 protein was also detected in the BAL fluids. Using immunohistochemical staining, IL-13–positive and IL-25–positive cells were clearly identified as macrophages that had engulfed TiO2 particles (Figure 2). These data indicate that TiO2-engulfing macrophages are the main source of IL-13 and IL-25 in TiO2 particle–induced inflammation of the lung.

Our in vivo results were confirmed by the in vitro study using isolated alveolar macrophages that were loaded with TiO2 particles. The alveolar macrophages that engulfed TiO2 particles in vitro produced IL-13 and IL-25 protein and mRNA, whereas the unstimulated macrophages did not (Figure 3). The response of macrophages to TiO2 is not specific, because carbon black also induced the IL-13 and IL-25 production by alveolar macrophages (data not shown). Endotoxin contamination in TiO2 particle of the present study has no effect on IL-13 and IL-25 production by alveolar macrophages because lipopolysaccharide up to 10 ng/ml did not change the production of IL-25 by alveolar macrophages (data not shown).

Because the sequence of the rat IL-25 gene has not been published, we sequenced the PCR product of the RNA from stimulated rat alveolar macrophages using the mouse IL-25 primer set, and revealed 95% homology with the mouse IL-25 gene. These results indicate that alveolar macrophages are important sources of IL-25 and IL-13 in lung responses to particles.

In this study, we used rutile fine TiO2 particles coated with BSA. It is noteworthy that the TiO2 particles were readily visible under the microscope, and easily localized inside alveolar macrophages in the present study (Figure 2). TiO2 particles are found in dusty workplaces, such as those in industries involved in the crushing and grinding of the mineral ore rutile (28). Fifty percent of TiO2-exposed workers have respiratory symptoms that are accompanied by reductions in pulmonary function (29). In addition, TiO2 particles are known to cause acute and chronic inflammation of the airways and alveolar spaces, and induce procollagen synthesis (30) via the production of chemokines, such as MIP-2 and CINC (31), as well as reactive oxygen species (20). Mild inflammation that involved mononuclear and polymorphonuclear leukocytes was seen in the TiO2-treated rats of the present study (Figure 2). Thus, this animal model is valuable for the study of particle-induced lung inflammation.

In animal experiments and human studies, the levels of IL-4, IL-5, GM-CSF, RANTES, monocyte chemotactic protein-3, and MIP-1 were elevated when lymphocytes and macrophages/monocytes were stimulated with particles in the presence of specific allergens (8–10). Although IL-4 is produced by DEP or carbon black particles in allergen-sensitized models (32), we did not observe any increases in the levels of IL-4, IL-5 and TNF-{alpha} in the BAL fluids of TiO2-treated rats (data not shown). Although Th2 cells, mast cells, and basophils are major sources of IL-13 (33–35), Th2 cells are probably not involved in the TiO2-induced IL-13 production seen in the present study.

IL-13 may be critical for the regulation of inflammatory and immune responses (36). Endogenous IL-13 attenuates NF-{kappa}B activation by interfering with the breakdown of I{kappa}B{alpha} (37) and related cytokine/chemokine generation, which determine the intensity of the lung inflammatory responses in animal experiments that use immune complex deposition to trigger acute lung inflammation (38), and in granulomatous inflammation in rats (39). However, a series of recent studies demonstrates that IL-13 directly induces features of the allergic response via its actions on epithelial and smooth muscle cells (40). IL-13 acts via matrix metalloproteinase-9 and -12, to induce the accumulation of eosinophils and macrophages and alveolar remodeling (41), which may underlie chronic obstructive pulmonary disease and asthma. Based on the present study and the regulatory effect on lung inflammatory response (37, 38, 41, 42), particles are one of the stimulants for IL-13 production by macrophages, and macrophage-expressed IL-13 may be involved in the prevention of particle-associated granuloma formation and in the enhancement of a Th2-like allergic environment.

IL-25 has been recently identified, and IL-25 expression has been detected by RT-PCR in several tissues, including the brain, kidney, lung, prostate, testis, spinal cord, adrenal gland, and trachea (43). Treatment of mice with IL-25 induces Th2-type cytokines (notably IL-5, IL-13, and chemokines such as eotaxin), and increases mucus secretion and airway hyperreactivity (20). Th2 cells and mast cells have been known to be potent IL-25–producing cells till now (44). In the present study, however, we have clearly demonstrated that macrophages are potent IL-25–producing cells when stimulated with particles. Although our data do not address the interrelation between IL-25 and IL-13 production by alveolar macrophages, IL-25 production precedes IL-13 production based on the finding that the production of IL-13 protein peaked 48 h after TiO2 stimulation (Figure 4B), whereas IL-25 protein peaked 24 h after TiO2 treatment (Figure 5). Taken together, our results suggest that alveolar macrophages may act as major effectors of innate immunity to modulate immune and inflammatory responses toward a Th2-like condition via the production of IL-25 and IL-13, as seen in the adaptive immune response.

In summary, we show that alveolar macrophages produce IL-25 and IL-13 at the protein and mRNA levels in response to particles. The increased expression of IL-25 and IL-13 mRNAs, and proteins, may be involved in modulating the inflammatory response within the lung of TiO2-instilled rats. It is interesting to speculate on whether the inhibition of IL-25 would have an inhibitory effect on particle-induced inflammation; these studies are currently ongoing in our laboratory.


   Footnotes
 
This work was supported by grant R01-2003-000-10041-0 from the Basic Research Program of the Korea Science and Engineering Foundation.

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

* Chun-Mi Kang and An-Soo Jang contributed equally as co–first authors. Back

Received in original form January 4, 2005

Received in final form April 12, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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