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Fate of inhaled particles after interaction with the lung surface
PAEDIATRIC RESPIRATORY REVIEWS (2006) 7S, S73–S75
IV. ISAM JOINT MEETING Aerosols – Good and Bad
Fate of inhaled particles after interaction with the lung surface Peter Gehr*, Fabian Blank and Barbara M. Rothen-Rutishauser Institute of Anatomy, Division of Histology, University of Bern, Baltzerstr. 2, CH-3000, Bern 9, Switzerland KEYWORDS particles; airway wall; surfactant; macrophages; dendritic cells
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Summary Inhaled particles may cause increased pulmonary and cardiovascular morbidity and mortality. The wall structures of airways and alveoli act as a series of structural and functional barriers against inhaled particles. Deposited particles are displaced and come into close association with epithelial cells, macrophages and dendritic cells. The cellular interplay after particle deposition in a triple cell co-culture model of the human airway wall was investigated by laser scanning microscopy. Furthermore, the cellular response was determined by measurement of TNF-a. Dendritic cells gained access to the apical side of the epithelium where they sampled particles and interacted with macrophages. ß 2006 Elsevier Ltd. All rights reserved.
INTRODUCTION Epidemiological studies give evidence that the inhalation of fine particles (0.1–2.5 mm) and nanoparticles (0.1 mm) may cause increased pulmonary and cardiovascular morbidity and mortality.1,2 Nanoparticles are reported to be particularly toxic.3 A huge internal surface area of 140 m2,4 is available for interaction with inhaled particles. Once deposited, particles are displaced by surface forces of the surfactant film at the air-liquid interface.5,6 This gets them into close association with epithelial cells and cells of the defense system, airway or alveolar macrophages (AM) and dendritic cells (DC). Little is known about the interaction of such particles with lung cells, how they may cross the epithelial barrier and how they may penetrate the capillaries in the pulmonary tissue. The wall structures of the airways and alveoli act as a series of structural and functional barriers which pro-
* Corresponding author. Tel.: +41 31 631 8432; Fax: +41 31 631 3807. E-mail address: [email protected] (P. Gehr). 1526-0542/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.prrv.2006.04.169
tects the respiratory system against harmful and innocuous particles.7 It was the aim of this study to find out how immunocompetent DC8,9 located in or at the base of the airway epithelium take up inhaled particulate antigens and how AM on top of the epithelium may collaborate Fig. 1.
METHODS Cellular interaction of particles of different size, material and charge was studied in a triple cell co-culture model of the human airway wall to simulate the cellular part of the epithelial barrier, represented by AM, epithelial cells, and DC.10,11 The cellular interplay after particle deposition was investigated by laser scanning microscopy (LSM) and the cellular response determined by measurement of TNF-a. Since nanoparticles have the size of small cell components their identification in the cytoplasm is difficult. Hence, we combined different microscopic techniques to visualize nanoparticles in the cytoplasm of cultured cells: (1) Fluorescent particles were analyzed by LSM combined with
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P. GEHR ET AL.
Figure 1 Tissue barrier of airways (a) and alveoli (b). Abbreviations: alveolar epithelium type I (AEPT I), alveolar epithelium type II (AEPT II), aqueous lining layer (ALL), alveolar macrophage (AM), basal membrane (BM), capillary (C), connective tissue (CT), dendritic cell (DC), epithelial cell (EP), particle (P), surfactant (S), tight junction (TJ). See text for explanation.
Figure 2 Visualization of nanoparticles. (a) Laser scanning micrograph. Single particles detected after deconvolution (arrows). (b) Transmission electron micrograph of a 0.025 mm gold particle within erythrocyte. (c) Electron energy loss spectroscopy image of TiO2 (arrow). The white open circle marks the position where energy loss analysis was performed. Corresponding energy loss spectra (black line) is shown; dotted line represents background.
Figure 3 LSM of particle exposed triple cell co-culture. DC residing underneath the insert membrane pushed processes between epithelial cells upwards into ‘‘luminal space’’ to collect 1 mm particles (arrow).
FATE OF INHALED PARTICLES AFTER INTERACTION WITH THE LUNG SURFACE
digital image restoration; (2) Gold particles were analyzed by conventional transmission electron microscopy (TEM) and energy filtering TEM (EFTEM); (3) Titanium dioxide (TiO2) particles were analyzed by EFTEM.
S75
ACKNOWLEDGMENTS This work was supported by the Swiss National Science Foundation (Nr. 32-65352.01), the Swiss Federal Office for the Environment and the Silva Casa Foundation.
RESULTS We visualized particles in epithelial cells, AM and DC, as well as in erythrocytes. The surface charge and the material of the particles did not influence their interaction with the cells; however, we found an increase of TNF-a in the supernatants after applying gold particles, but not for ultrafine polystyrene and TiO2 particles Fig. 2. Combining LSM with advanced visualisation techniques we could show in the triple cell co-culture model that DC made processes between the epithelial cells through the tight junctions or transmigrated through the epithelium towards the ‘‘luminal side’’ to capture deposited 1 mm particles on the epithelial surface Fig. 3. DC were found to interact with particle loaded AM or other DC to take particles from them and AM containing particles to form inter-epithelial processes towards the base of the epithelium.
CONCLUSIONS Using the triple cell co-culture model of the epithelial airway barrier and advanced microscopic techniques we were able to visualize nanoparticles within single cells. These particles were not membrane bound. We could demonstrate how DC gain access to the ‘‘luminal side’’ of the epithelial cells where they sample particulate antigens and interact with AM. Hence, we postulate that DC and AM as sentinels against particulate antigens form a transepithelial interacting cellular network.
REFERENCES 1. Pope CA III, Dockery DW, Schwartz J. Review of epidemiological evidence of health effects of particulate air pollution. Inhal Toxicol 1995; 7: 1–18. 2. Schulz H, Harder V, Ibald-Mulli A, Khandoga A, Koenig W, Krombach F, Radykewicz R, Stampfl A, Thorand B, Peters A. Cardiovascular effects of fine and ultrafine particles. J Aerosol Med 2005; 18: 1–22. 3. Peters A, Wichmann HE, Tuch T, Heinrich J, Heyder J. Respiratory effects are associated with the number of ultrafine particles. Am J Respir Crit Care Med 1997; 155: 1376–1383. 4. Gehr P, Bachofen M, Weibel ER. The normal human lung: Ultrastructure and morphometric estimation of diffusion capacity. Respir Physiol 1978; 32: 121–140. 5. Gehr P, Schu¨rch S, Berthiaume Y, Im Hof V, Geiser M. Particle retention in airways by surfactant. J Aerosol Med 1990; 3: 27–43. 6. Schu¨rch S, Gehr P, Im Hof V, Geiser M, Green F. Surfactant displaces particles toward the epithelium in airways and alveoli. Respir Physiol 1990; 80: 17–32. 7. Nicod LP. Lung defenses: an overview. Eur Respir Rev 2005; 14: 45–50. 8. Nicod LP. Function of human lung dendritic cells. In: Lipscomb MF, Russels SW, eds: Health and disease. New York, Basel: Marcel Dekker, Inc., 1997; pp. 311–334. 9. Holt PG. Pulmonary dendritic cells in local immunity to inert and pathogenic antigens in the respiratory tract. Proc Am Thorac Soc 2005; 2: 116–120. 10. Rothen-Rutishauser BM, Kiama SG, Gehr P. A 3D cellular model of the human respiratory tract to study the interaction with particles. Am J Respir Cell Mol Biol 2005; 32: 281–289. 11. Blank F, Rothen-Rutishauser B, Schurch S, Gehr P. An Optimized in vitro Model of the Respiratory Tract Wall to Study Particle Cell Interactions. J Aerosol Med 2006; In press.
IV. ISAM JOINT MEETING Aerosols – Good and Bad
Fate of inhaled particles after interaction with the lung surface Peter Gehr*, Fabian Blank and Barbara M. Rothen-Rutishauser Institute of Anatomy, Division of Histology, University of Bern, Baltzerstr. 2, CH-3000, Bern 9, Switzerland KEYWORDS particles; airway wall; surfactant; macrophages; dendritic cells
\
Summary Inhaled particles may cause increased pulmonary and cardiovascular morbidity and mortality. The wall structures of airways and alveoli act as a series of structural and functional barriers against inhaled particles. Deposited particles are displaced and come into close association with epithelial cells, macrophages and dendritic cells. The cellular interplay after particle deposition in a triple cell co-culture model of the human airway wall was investigated by laser scanning microscopy. Furthermore, the cellular response was determined by measurement of TNF-a. Dendritic cells gained access to the apical side of the epithelium where they sampled particles and interacted with macrophages. ß 2006 Elsevier Ltd. All rights reserved.
INTRODUCTION Epidemiological studies give evidence that the inhalation of fine particles (0.1–2.5 mm) and nanoparticles (0.1 mm) may cause increased pulmonary and cardiovascular morbidity and mortality.1,2 Nanoparticles are reported to be particularly toxic.3 A huge internal surface area of 140 m2,4 is available for interaction with inhaled particles. Once deposited, particles are displaced by surface forces of the surfactant film at the air-liquid interface.5,6 This gets them into close association with epithelial cells and cells of the defense system, airway or alveolar macrophages (AM) and dendritic cells (DC). Little is known about the interaction of such particles with lung cells, how they may cross the epithelial barrier and how they may penetrate the capillaries in the pulmonary tissue. The wall structures of the airways and alveoli act as a series of structural and functional barriers which pro-
* Corresponding author. Tel.: +41 31 631 8432; Fax: +41 31 631 3807. E-mail address: [email protected] (P. Gehr). 1526-0542/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.prrv.2006.04.169
tects the respiratory system against harmful and innocuous particles.7 It was the aim of this study to find out how immunocompetent DC8,9 located in or at the base of the airway epithelium take up inhaled particulate antigens and how AM on top of the epithelium may collaborate Fig. 1.
METHODS Cellular interaction of particles of different size, material and charge was studied in a triple cell co-culture model of the human airway wall to simulate the cellular part of the epithelial barrier, represented by AM, epithelial cells, and DC.10,11 The cellular interplay after particle deposition was investigated by laser scanning microscopy (LSM) and the cellular response determined by measurement of TNF-a. Since nanoparticles have the size of small cell components their identification in the cytoplasm is difficult. Hence, we combined different microscopic techniques to visualize nanoparticles in the cytoplasm of cultured cells: (1) Fluorescent particles were analyzed by LSM combined with
S74
P. GEHR ET AL.
Figure 1 Tissue barrier of airways (a) and alveoli (b). Abbreviations: alveolar epithelium type I (AEPT I), alveolar epithelium type II (AEPT II), aqueous lining layer (ALL), alveolar macrophage (AM), basal membrane (BM), capillary (C), connective tissue (CT), dendritic cell (DC), epithelial cell (EP), particle (P), surfactant (S), tight junction (TJ). See text for explanation.
Figure 2 Visualization of nanoparticles. (a) Laser scanning micrograph. Single particles detected after deconvolution (arrows). (b) Transmission electron micrograph of a 0.025 mm gold particle within erythrocyte. (c) Electron energy loss spectroscopy image of TiO2 (arrow). The white open circle marks the position where energy loss analysis was performed. Corresponding energy loss spectra (black line) is shown; dotted line represents background.
Figure 3 LSM of particle exposed triple cell co-culture. DC residing underneath the insert membrane pushed processes between epithelial cells upwards into ‘‘luminal space’’ to collect 1 mm particles (arrow).
FATE OF INHALED PARTICLES AFTER INTERACTION WITH THE LUNG SURFACE
digital image restoration; (2) Gold particles were analyzed by conventional transmission electron microscopy (TEM) and energy filtering TEM (EFTEM); (3) Titanium dioxide (TiO2) particles were analyzed by EFTEM.
S75
ACKNOWLEDGMENTS This work was supported by the Swiss National Science Foundation (Nr. 32-65352.01), the Swiss Federal Office for the Environment and the Silva Casa Foundation.
RESULTS We visualized particles in epithelial cells, AM and DC, as well as in erythrocytes. The surface charge and the material of the particles did not influence their interaction with the cells; however, we found an increase of TNF-a in the supernatants after applying gold particles, but not for ultrafine polystyrene and TiO2 particles Fig. 2. Combining LSM with advanced visualisation techniques we could show in the triple cell co-culture model that DC made processes between the epithelial cells through the tight junctions or transmigrated through the epithelium towards the ‘‘luminal side’’ to capture deposited 1 mm particles on the epithelial surface Fig. 3. DC were found to interact with particle loaded AM or other DC to take particles from them and AM containing particles to form inter-epithelial processes towards the base of the epithelium.
CONCLUSIONS Using the triple cell co-culture model of the epithelial airway barrier and advanced microscopic techniques we were able to visualize nanoparticles within single cells. These particles were not membrane bound. We could demonstrate how DC gain access to the ‘‘luminal side’’ of the epithelial cells where they sample particulate antigens and interact with AM. Hence, we postulate that DC and AM as sentinels against particulate antigens form a transepithelial interacting cellular network.
REFERENCES 1. Pope CA III, Dockery DW, Schwartz J. Review of epidemiological evidence of health effects of particulate air pollution. Inhal Toxicol 1995; 7: 1–18. 2. Schulz H, Harder V, Ibald-Mulli A, Khandoga A, Koenig W, Krombach F, Radykewicz R, Stampfl A, Thorand B, Peters A. Cardiovascular effects of fine and ultrafine particles. J Aerosol Med 2005; 18: 1–22. 3. Peters A, Wichmann HE, Tuch T, Heinrich J, Heyder J. Respiratory effects are associated with the number of ultrafine particles. Am J Respir Crit Care Med 1997; 155: 1376–1383. 4. Gehr P, Bachofen M, Weibel ER. The normal human lung: Ultrastructure and morphometric estimation of diffusion capacity. Respir Physiol 1978; 32: 121–140. 5. Gehr P, Schu¨rch S, Berthiaume Y, Im Hof V, Geiser M. Particle retention in airways by surfactant. J Aerosol Med 1990; 3: 27–43. 6. Schu¨rch S, Gehr P, Im Hof V, Geiser M, Green F. Surfactant displaces particles toward the epithelium in airways and alveoli. Respir Physiol 1990; 80: 17–32. 7. Nicod LP. Lung defenses: an overview. Eur Respir Rev 2005; 14: 45–50. 8. Nicod LP. Function of human lung dendritic cells. In: Lipscomb MF, Russels SW, eds: Health and disease. New York, Basel: Marcel Dekker, Inc., 1997; pp. 311–334. 9. Holt PG. Pulmonary dendritic cells in local immunity to inert and pathogenic antigens in the respiratory tract. Proc Am Thorac Soc 2005; 2: 116–120. 10. Rothen-Rutishauser BM, Kiama SG, Gehr P. A 3D cellular model of the human respiratory tract to study the interaction with particles. Am J Respir Cell Mol Biol 2005; 32: 281–289. 11. Blank F, Rothen-Rutishauser B, Schurch S, Gehr P. An Optimized in vitro Model of the Respiratory Tract Wall to Study Particle Cell Interactions. J Aerosol Med 2006; In press.