Published ahead of print on October 18, 2007, doi:10.1165/rcmb.2007-0138OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0138OC The Role of Macrophages in the Clearance of Inhaled Ultrafine Titanium Dioxide Particles1 Institute of Anatomy, University of Bern, Bern, Switzerland; and 2 GSF—National Research Center for Environment and Health, Institute for Inhalation Biology, Neuherberg/Munich, Germany Correspondence and requests for reprints should be addressed to M. Geiser, Ph.D., Institute of Anatomy, University of Bern, Baltzerstrasse 2, CH-3000 Bern 9, Switzerland. E-mail: geiser{at}ana.unibe.ch
The role of macrophages in the clearance of particles with diameters less than 100 nm (ultrafine or nanoparticles) is not well established, although these particles deposit highly efficiently in peripheral lungs, where particle phagocytosis by macrophages is the primary clearance mechanism. To investigate the uptake of nanoparticles by lung phagocytes, we analyzed the distribution of titanium dioxide particles of 20 nm count median diameter in macrophages obtained by bronchoalveolar lavage at 1 hour and 24 hours after a 1-hour aerosol inhalation. Differential cell counts revealing greater than 96% macrophages and less than 1% neutrophils and lymphocytes excluded inflammatory cell responses. Employing energy-filtering transmission electron microscopy (EFTEM) for elemental microanalysis, we examined 1,594 macrophage profiles in the 1-hour group (n = 6) and 1,609 in the 24-hour group (n = 6). We found 4 particles in 3 macrophage profiles at 1 hour and 47 particles in 27 macrophage profiles at 24 hours. Model-based data analysis revealed an uptake of 0.06 to 0.12% ultrafine titanium-dioxide particles by lung-surface macrophages within 24 hours. Mean (SD) particle diameters were 31 (8) nm at 1 hour and 34 (10) nm at 24 hours. Particles were localized adjacent (within 13–83 nm) to the membrane in vesicles with mean (SD) diameters of 592 (375) nm at 1 hour and 414 (309) nm at 24 hours, containing other material like surfactant. Additional screening of macrophage profiles by conventional TEM revealed no evidence for agglomerated nanoparticles. These results give evidence for a sporadic and rather unspecific uptake of TiO2-nanoparticles by lung-surface macrophages within 24 hours after their deposition, and hence for an insufficient role of the key clearance mechanism in peripheral lungs.
Key Words: clearance • electron filtering transmission electron microscopy • lungs • macrophages • nanoparticles
For more than 10 years epidemiology has provided consistent evidence for the link between adverse health effect and increased concentrations of ambient fine and ultrafine particles (1–4). There are indications for a specific toxicological role for ultrafine particles (UFP, particles with diameters < 100 nm) (5, 6). Although airborne particle mass is declining, the environmental burden by UFP is more likely to increase over time (7). In addition, the nanotechnology industry daily generates new UFP, which may become aerosolized at some stage of their life cycle and may pose additional health risks. The deposition of inhaled fine particles (1–2.5 µm in diameter) in the respiratory tract is mainly caused by sedimentation and occurs primarily in the peripheral lungs, while deposition of UFP is caused by diffusional displacement and occurs efficiently on the surfaces of the entire respiratory tract. Diffusional deposition relates inversely to the UFP diameter, showing a maximum for approximately 20-nm UFP in the alveolar region (8). Moreover, micrometer-sized particles usually remain on the epithelial surface upon their deposition (9) and are subjected to clearance by mucociliary transport, cough, and/or phagocytosis by macrophages. The clearance pathways of UFP are not yet clarified. They may be cleared by mucociliary transport, but they also have the capability to rapidly penetrate the boundary membranes of the lungs and thereby get access to epithelial cells and tissues beyond (10–15). The importance of airway and alveolar macrophages in the clearance of micrometer-sized particles from the lung surface has long been known (for review see Ref. 16). Data from animal experiments have demonstrated that engulfment of particles by macrophages is rapid and that the process is essentially completed within 24 hours (17–21). Very similar results have been obtained from human studies (22, 23). Inhalation studies with radio-labeled ultrafine iridium particles in rats have shown that a major fraction of deposited particles is displaced into lung tissues, but only a minor fraction is eventually translocated into the circulation (15). Little particle translocation into the circulation was also observed in humans (24–26). Furthermore, we have observed the displacement of ultrafine TiO2 particles from the lung surface into the tissue immediately after a 1-hour inhalation using morphometric techniques (11). These displaced particles are no longer available for immediate macrophage-mediated clearance. Hence, these findings raise questions about the significance of common clearance mechanisms for this particle category, especially of that by lung surface macrophages.
The aim of this study was to examine the uptake of UFP by lung surface macrophages and their time course at the individual particle level. For this purpose rats inhaled an ultrafine TiO2 aerosol of 20 nm count median diameter (CMD) for 1 hour, resulting in a deposition of 1 to 2 µg TiO2 (i.e.,
Animals The animal experiments were conducted under German federal guidelines for the use and care of laboratory animals and were approved by the District of Upper Bavaria (Approval No. 211-2531-108/99) and by the GSF Institutional Animal Care and Use Committee, as well as in accordance with the Swiss Federal Act on Animal Protection and the Swiss Animal Protection Ordinance. Twelve young, adult, male WKY/Kyo@Rj rats (body weight [BW] 246–316 g; Centre D'Elevage R. Janvier, Le Genest St. Isle, France) (Table 1) were housed under standard conditions for animal husbandry (22°C, 55% relative humidity, 12-h day/night cycle) with access to food and water ad libitum. Animals were anaesthetized by intramuscular injection of a mixture of medetomidine (Domitor, 15 µg/100 g BW; Pfizer GmbH, Karlsruhe, Germany), midazolam (Dormicum, 200 µg/100 g BW; Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany), and fentanyl (Fentanyl, 0.5 µg/100 g BW; Janssen-Cilag GmbH, Neuss, Germany) for inhalation. Immediately after inhalation anesthesia was deepened with a mixture of xylazin (Rompun, 470 µg/100 g BW; Bayer Vital GmbH, Leverkusen, Germany) and ketamine (Ketamin 10%, 9.5 mg/100 g BW; WDT eG, Garbsen, Germany) for killing by exsanguination before BAL or for 24-hour examinations antagonized by subcutaneous injection of atipamezole (Antisedan, 75 µg/100 g BW; Pfizer GmbH), flumazenil (Anexate, 20 µg/100 g BW; Hoffmann-La Roche AG), and naloxone (Narcanti, 12 µg/100 g BW; Janssen Animal Health, Neuss, Germany). Twenty-four hours after inhalation, a mixture of xylazin and ketamine was injected before killing by exsanguination and BAL.
Aerosol Generation and Inhalation The generation and inhalation of the TiO2 aerosol used in this study has been previously described (11). Briefly, ultrafine TiO2 aerosols were generated with a Palas spark generator; quasi-neutralized by a radioactive 85Kr source; diluted; and conditioned for inhalation with respect to gas composition, humidity, and temperature. Particle size distribution and number concentration were monitored continuously by a differential electrical mobility particle sizer and a condensation particle counter. The aerosol produced had a CMD of 20 nm (geometric standard deviation 1.7) and a mean number concentration of 7.2 x 106 (SD 0.5 x 106) particles cm–3, resulting in a mass concentration of approximately 0.1 mg m–3.
Each anesthetized rat was placed in an airtight plethysmograph box. Four animals inhaled the aerosol at the same time (two each for the 1-h and 24-h examinations) for 1 hour via an endotracheal tube by negative-pressure ventilation (–1.5 kPa) at a breathing frequency of 40 min–1 (tidal volume of After aerosol exposure, two rats were immediately killed and subjected to BAL. The anesthesia of the other two animals was antagonized as described above, and they were put back into their cage for 24 hours.
BAL and Preparation of Cells The cell pellets were resuspended in fresh glutaraldehyde, post-fixed with buffered 1.0% osmium tetroxide (Simec, Zofingen, Switzerland) and 0.5% uranyl acetate (Fluka Chemie GmbH, Sigma-Aldrich, Buchs, Switzerland), dehydrated in a graded series of ethanol, and embedded in Epon (Fluka) (28), as shown in Figure 1A. From the embedded cell pellets, sections of 1 µm nominal thickness were cut and stained with toluidine blue for differential cell counting of the lavaged cells.
Ultrathin sections of 60 to 70 nm and of less than or equal to 50 nm nominal thicknesses were cut and mounted onto uncoated 200- and 600-mesh copper grids, respectively, and post-stained with uranyl acetate and lead citrate (Ultrostain; Leica, Glattbrugg, Switzerland). The former sections were used to analyze macrophages for agglomerated 20-nm TiO2 particles by TEM, the latter for titanium particle analysis by EFTEM (29), as shown in Figures 1B and 1C.
Macrophage Sampling and Analysis For EFTEM analyses of TiO2 particles, a systematic random sampling of macrophage profiles was adopted, as outlined in Figures 1B and 1C. In brief, a random hexagon was chosen as starting point at a magnification of x80 (Figure 1B). From there on the automated goniometer allowed the unbiased screening of macrophages in vertical and horizontal direction (Figure 1C). In total, 80 hexagons were analyzed per animal. Macrophages located within these hexagons were investigated for the presence and localization of TiO2 particles by elemental microanalysis as described below.
Morphologic Characterization and Elemental Microanalysis of TiO2 Particles
Statistics Group data were compared using the nonparametric Mann-Whitney Rank Sum Test. The level of significance was set at P < 0.5.
Identification of TiO2 Particles in Tissue Sections To investigate ultrafine particles in biological specimens, here in ultrathin sections of BAL macrophages, it is essential to precisely know the morphology of the particles by investigating area and section profiles of aerosol particles, as well as to confirm the elemental composition of the nanoparticles by elemental microanalysis. Figures 2A and 2B show that the generated 20-nm TiO2 aerosol particles per se consist of small agglomerates of primary particles of approximately 4 nm in diameter, as estimated previously (11). Unambiguous identification of ultrafine TiO2 particles in biological specimens is not possible solely upon recognition of the particle morphology at the ultrastructural level. Hence, each particle found in the ultrathin tissue section, which morphologically resembled an inhaled aerosol particle by its ultrastructure, was confirmed to consist of titanium by EFTEM, as described in MATERIALS AND METHODS and shown in Figure 2C.
Number and Cytology of BAL Cells
Uptake and Localization of TiO2 Particles in BAL Macrophages As shown in Table 2 and Figure 3, there were no significant differences in particle size found in both animal groups. Mean (SD) particle diameters were 31 (8) nm at 1 hour and 34 (10) nm at 24 hours, which compares well with a CMD of 20 nm determined for the aerosol particles. In addition, there was no evidence for agglomerated ultrafine 20-nm TiO2 particles in either of the two animal groups from the screening of macrophages by conventional TEM (identification by morphology only).
As shown in Table 2 and Figures 3 and 4, particles were located adjacent (within 13–83 nm) to the membrane, within vesicles containing other material like surfactant. Mean (SD) diameters of vesicles that contained particles were 592 (375) nm at 1 hour and 414 (309) nm at 24 hours. Vesicle size ranged from 98 nm to 1,485 nm. There were no significant differences found between the two animal groups, in either the size of the vesicles or the localization of particles with respect to the vesicular membrane.
EFTEM analysis allows us to assess the localization of ultrafine TiO2 particles in the different lung compartments and within surface macrophages at the individual particle level (29). In our previous study (11), we found 80% of the ultrafine TiO2 particles on the luminal side of the airways and alveoli and 20% within the lung tissue. In the present study, performed under the same experimental conditions as the previous one, we addressed the potential uptake of those ultrafine TiO2 particles being on the luminal side of the airways and alveoli by lung surface macrophages. According to our differential cell counts, showing more than 96% of the BAL cells being macrophages and less than 1% neutrophils and lymphocytes in both animal groups, the experimental conditions in this study exclude a potential bias due to an inflammatory alteration of the epithelial barrier. In the present study, the number of macrophage profiles that contained particles as well as the number of particles were extremely low: 3 out of 1,594 (i.e., 0.2%; macrophage profiles contained 4 particles at 1 h), and 27 out of 1,609 (i.e., 1.7%; macrophage profiles contained 47 particles at 24 h). Quantitative data for 3- to 6-µm particles of different materials showed an average uptake of 27.7% (SD 16.0%) of all deposited particles by macrophages within less than 1 hour after the beginning of the inhalation (16), as well as that 12 to 15% of the macrophages had phagocytosed particles at 1 hour and at 24 hours after particle inhalation (30). This suggests uptake of UFP by macrophages to be less efficient than that of larger particles. Unfortunately, unbiased stereology for direct quantitative data assessment (the disector for number estimates, e.g., 17), cannot be applied to ultrafine particles yet; hence, we have to resort to a model-based approach for data evaluation: To estimate particle uptake at 1 hour, we assume minimal particle uptake by macrophages within this short period of time, namely that macrophages phagocytose only those particles that deposited on their surface. Considering an alveolar surface area, SA = 2,500 cm2 (31), a macrophage surface area, SM = 133 µm2 (spherical macrophage with a diameter, d = 13 µm) (32) and a total number of lung surface macrophages, NM = 1.25 x 107 (15), then 0.7% of the alveolar surface area is covered by macrophages and, hence, the number of particles deposited on macrophages, NP = 4–8 x 108. When 100% of these particles are taken up by the macrophages, they distribute in a total macrophage volume of 1.44 x 1010 µm3. Screening 1,594 macrophage profiles (of a surface area, SM = 133 µm2 each) in ultrathin section with a thickness, h = 0.05 µm, we analyzed a total volume of 1.06 x 104 µm3 and found four particles. This results in an uptake of 0.65–1.3% of the particles that deposited on the macrophages at 1 hour after UFP inhalation. Considering maximal particle uptake by macrophages, namely that all (= 6–12 x 1010) deposited particles were available for phagocytosis, results in an uptake of 0.005 to 0.009% of the particles at 1 hour. This is clearly evidence for ultrafine TiO2 particles to be less efficiently engulfed by macrophages than micrometer-sized particles. Furthermore, the ultrastructural analysis showed that the ultrafine TiO2 particles were not tightly enclosed by the vesicular membrane, as it is known from phagocytic uptake of micrometer-sized particles. Instead, the UFP were located in large vesicles compared with particle size and the vesicles contained other material, like surfactant. These findings also point to a rather sporadic uptake of ultrafine TiO2 particles by lung surface macrophages, maybe during the process of phagocytic uptake of other material. The ultrafine TiO2 particles may also have entered the cells and subcellular compartments by a nonendocytic process, as we have demonstrated earlier to occur for ultrafines of different materials (11). It still remains to be shown whether passive uptake mechanisms (not triggered by receptor–ligand interactions) subsumed as "adhesive interactions" (33) and put forward by Geiser and coworkers (11) were responsible for the penetration of ultrafine TiO2 particles into cells. To estimate particle uptake at 24 hours after inhalation, we assume that macrophages had access to all particles on the lung surface. We registered 47 particles in 1,609 macrophage profiles, each 50 nm thick. Assuming again a macrophage corresponding to a 13-µm sphere (32) and a total number of 1.25 x 107 lung surface macrophages (15), this results in an estimate of 6.3 x 107 phagocytosed ultrafine TiO2 particles. The number of deposited particles in our study ranged between 6 and 12 x 1010 particles. From the results of our previous study (11), we expect 20% of the deposited particles to be displaced into the lung tissue, that is, 80% or 4.8–9.6 x 1010 particles remained on the lung surface and, therefore, were accessible for macrophages. Clearance of particles from the alveoli via the airways within 24 hours is considered negligible. Hence, this results in an estimate of 0.06 to 0.12% of the ultrafine TiO2 particles on the lung surface that were phagocytosed within 24 hours. This is in clear contrast to fine particles, which are taken up by more than 10% already within the first hour of their deposition (16) and by more than 80% within 24 hours (17–21). In addition, the size of ultrafine TiO2 particles found in macrophages did not increase within 24 hours after inhalation and the localization of particles was the same as at 1 hour after inhalation in the present study. Moreover, there was no evidence for agglomerated 20-nm TiO2 particles by supplementary macrophage screening by conventional TEM. These findings are additional evidence that macrophages did not efficiently phagocytose ultrafine TiO2 particles to keep the lung surface clean and sterile and what they are well known for after exposure to micrometer-sized particles (e.g., 16, 30). Avid endocytic uptake of "ultrafine" TiO2 was shown in vitro in the epithelial cell line A549 (34), and the phagolysosomal membranes tightly enclosed the large agglomerates of ultrafine TiO2 particles. From the experimental setup in that study it is very likely that these particles were taken up by the cells as agglomerates. In a study about the clearance of sulfur particles of 100 to 200 nm in diameter by airway macrophages in humans, we have identified sulfur particles within the cytoplasm by EFTEM. However, we were not able to unambiguously disclose the particle–vesicle relationship due to insufficient membrane contrast of the specimens (23). There is evidence for macrophage particle uptake to be size dependent (35) and for UFP to be less phagocytosed than micrometer-sized particles from inhalation studies in rats using iridum particles (12, 15) and polystyrene particles (36). In summary, this is the first time that macrophage clearance of inhaled UFP has been assessed ultrastructurally at the individual particle level by elemental microanalysis. The findings and the model-based evaluation of the data from this inhalation study with 20-nm TiO2 particles in rats give evidence that lung surface macrophages do not efficiently phagocytose these ultrafines but take them up in a rather sporadic and unspecific way. The evidence that UFP bypass the most important clearance mechanisms for particles deposited in the alveoli, namely phagocytic uptake by macrophages, requires further clarification as to whether these results are specific for the material, the size or other characteristics of the particles. The rethinking of clearance pathways for inhaled UFP is considered necessary.
The authors thank Nadine Kapp for passing on her experience in EFTEM analysis to her successors.
This study was supported by the Swiss National Science Foundation. Originally Published in Press as DOI: 10.1165/rcmb.2007-0138OC on October 18, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form April 19, 2007 Accepted in final form August 9, 2007
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Abstract
CLINICAL RELEVANCE
6–12 x 1010 particles) per animal. We harvested lung surface macrophages at 1 hour and 24 hours after inhalation by bronchoalveolar lavage (BAL), and processed the cells for microscopic analysis. We employed conventional transmission electron microscopy (TEM) to screen macrophages for particle agglomerates, and energy-filtering transmission electron microscopy (EFTEM) for elemental microanalysis of individual particles to investigate the nature of particle–cell interaction. 
50 nm) sections were cut, transferred onto 600-mesh hexagonal cupper TEM grids, and post-stained with lead citrate and uranyl acetate. Systematic random sampling of macrophage profiles: a random hexagon was chosen as starting point at a magnification of x80. (C) From this point on the automated goniometer allowed the unbiased screening of macrophage profiles in vertical and horizontal directions. In total, 80 hexagons were sampled per animal. Macrophages located within these hexagons were investigated by EFTEM for the presence of TiO2 particles.
