Asbestos fibers are carcinogens causing oxidative stress and inflammation, but the sources and ramifications of oxidant production by asbestos are poorly understood. Here, we show that inhaled chrysotile asbestos fibers cause increased myeloperoxidase activity in bronchoalveolar lavage fluids (BALF) and myeloperoxidase immunoreactivity in epithelial cells lining distal bronchioles and alveolar ducts, sites of initial lung deposition of asbestos fibers. In comparison with sham mice, asbestos-exposed myeloperoxidase-null (MPO−/−) and normal (MPO+/+) mice exhibited comparable increases in polymorphonuclear leukocytes, predominately neutrophils, in BALF after 9 days of asbestos inhalation. Differential cell counts on BALF revealed decreased proportions of macrophages and increased lymphocytes in all mice exposed to asbestos, but numbers were decreased overall in asbestos-exposed myeloperoxidase-null versus normal mice. Asbestos-associated lung inflammation in myeloperoxidase-null mice was reduced (P ≤ 0.05) in comparison with normal asbestos-exposed mice at 9 days. Decreased lung inflammation in asbestos-exposed myeloperoxidase-null mice at 9 days was accompanied by increases (P ≤ 0.05) in Ki-67- and cyclin D1-positive immunoreactive cells, markers of cell cycle reentry, in the distal bronchiolar epithelium. Asbestos-induced epithelial cell proliferation in myeloperoxidase-null mice at 30 days was comparable to that found at 9 days. In contrast, inflammation and epithelial cell proliferation in asbestos-exposed normal mice increased over time. These results support the hypothesis that myeloperoxidase status modulates early asbestos-induced oxidative stress, epithelial cell proliferation, and inflammation.

Increases in polymorphonuclear leukocytes (PMN), predominantly neutrophils, are features of bronchoalveolar lavage fluid (BALF) samples and lung tissues in animal models of oxidant injury (13) and in patients with inflammatory lung diseases such as asbestosis (4). Exposure to asbestos is also associated with the development of lung cancers and mesotheliomas, and inflammation is a hallmark of response to acute exposure to asbestos both in animals and humans (5, 6). One possible mediator of asbestos-associated inflammation is myeloperoxidase, a major constituent of neutrophils that generates hypochlorous acid (HOCl) and reactive nitrogen species, including nitric oxide-derived inflammatory oxidants when nitrate and nitrite become available (7, 8). Nitration of tyrosine residues to 3-nitrotyrosine, a hallmark of inflammation as well as a footprint of many types of oxidative injury, may play a role in human lung disease (9) and is increased in the lungs of rats after asbestos inhalation (10). Myeloperoxidase can contribute to 3-nitrotyrosine formation in vivo and directly modulate inflammatory responses via regulation of nitric oxide bioavailability during inflammation (11, 12). Although myeloperoxidase seems to mediate host defense reactions through its microbicidal activity, its role in the development or modulation of inflammation after inhalation of exogenous oxidants is unclear. Moreover, how myeloperoxidase status affects the hyperplastic responses of lung epithelial cells that are critical to lung remodeling and/or carcinogenesis is of relevance to a broad realm of lung diseases.

After initial injury by airborne pathogenic fibers such as asbestos, epithelial cells undergo compensatory hyperplasia and metaplasia (13). Epithelial cell survival and re-epithelialization are thought to be critical to repair of the epithelium after damage, but unchecked epithelial cell proliferation may also be a risk factor in the development of lung cancers. Epithelial cells are also important contributors to chemokine and cytokine elaboration that may play a role in the inflammation of cancers, and fibroproliferative diseases of the lung (14).

Asbestos fibers induce localized oxidative stress through multiple mechanisms including impaired phagocytosis of longer (>8 μm) fibers, by epithelial cells or alveolar macrophages, iron-dependent reactions occurring on the fiber surface, and by elicitation of an inflammatory response (15). After inhalation or intratracheal administration of asbestos fibers to rodents, an initial inflammatory response, characterized by increases in PMNs in BALF and lung tissues, is observed (1, 1618). Increases in PMNs are also observed in the BALF and lungs of workers exposed to asbestos and individuals with a variety of pneumoconiosis (19, 20). The relationship between PMN influx and the development of lung inflammation or epithelial cell changes is not well understood.

Here, we used a well-characterized murine model of inflammation and bronchiolar epithelial cell proliferation (16) to show that brief inhalation of chrysotile asbestos causes increases in myeloperoxidase activity in BALF and immunoreactive protein in distal lung epithelium. We then compared the responses of myeloperoxidase-null (MPO−/−; ref. 21) and normal (MPO+/+) mice at 9 and 30 days after initiation of asbestos inhalation. At these time points, both epithelial cell proliferation and inflammation are observed in this model (16). Our results show that asbestos-associated inflammation is delayed in myeloperoxidase-null mice. Moreover, the bronchiolar epithelial cells of asbestos-exposed myeloperoxidase-null mice exhibit significant increases in cyclin D1 immunoreactivity and Ki-67-positive epithelial cells, indications of proliferation and reentry into the cell cycle at 9 days. These novel observations show that myeloperoxidase status affects patterns of acute inflammation and cell survival and/or proliferation, processes intrinsic to epithelial cell repair and hyperplasia.

Myeloperoxidase assays. To determine whether inhalation of asbestos caused release of myeloperoxidase into lavage fluids, a series of initial experiments were done on sham and asbestos-exposed C57BL/6 male mice (8-12 weeks old) obtained from Charles River (Wilmington, MA). Sham mice or those exposed to asbestos for 9 days (n = 4-5/group) were killed using an i.p. injection of sodium pentobarbital, and lungs lavaged with sterile Ca2+- and Mg2+-free PBS (CMF-PBS) as described below in a total volume of 1 mL. Fifteen micrograms of protein in ∼100 μL of fresh bronchoalveolar lavage supernatant was immediately mixed with 100 μL of fresh 3,3′-5,51-tetramethylbenzidine reagent prepared from a stock solution of 300 mmol/L sodium acetate buffer (pH 5.4), 15 mmol/L tetramethylbenzidine prepared in dimethylformamide, and 60 mmol/L H2O2. All chemicals were from Sigma (St. Louis, MO). The oxidation of tetramethylbenzidine by myeloperoxidase was measured at 630 nm over a 20-minute period, with readings every 5 minutes.

Myeloperoxidase-null (MPO−/−) mice. Gene-targeted myeloperoxidase-null (MPO−/−) mice (21) and normal (MPO+/+) littermates were bred into the C57BL6 background (>95% congenic) to allow direct comparison with our previous experiments using this strain (16). All mice were obtained from the animal facility of the University of Alabama at Birmingham and screened serologically for the absence of mouse hepatitis virus. Eight- to 12-week-old mice were housed and allowed to acclimate for 1 week in a HEPA-filtered clean air environment under controlled conditions of temperature, humidity, and light, and provided food and water ad libitum before the initiation of inhalation exposures.

Inhalation protocol. Previous time course experiments using chrysotile asbestos in inhalation experiments with C57BL/6 mice showed that neutrophilic influx in BALF samples peaked at 10 days, whereas proliferation of bronchiolar epithelium and alveolar duct cells was maximal between 14 and 30 days (16). Based on these observations, MPO−/− and normal (MPO+/+) littermates were exposed to either ambient air or National Institutes of Environmental Health Sciences reference samples of chrysotile asbestos for 6 hours per day, 5 days a week, for a total of 9 or 30 days. The chemical and physical characteristics of National Institutes of Environmental Health Sciences chrysotile asbestos have been previously described (22). Asbestos fibers were aerosolized using a modified Timbrell generator to generate a target concentration of 7 to 10 mg/m3 air, as previously described (23). Aerosol characteristics and concentrations were measured daily using a Sierra cascade impactor. All procedures were approved by the University of Vermont Institutional Animal Care and Use Committee.

Bronchoalveolar lavage procedures and assays on bronchoalveolar lavage fluids. Following asbestos exposure for 9 days, four groups of mice (MPO−/− sham, MPO+/+ sham, MPO−/− asbestos, and MPO+/+ asbestos; n = 4/group) received a lethal dose of pentobarbital, and the trachea was cannulated with polyethylene tubing. Lungs were then lavaged with sterile CMF-PBS in a total volume of 1 mL. Total cells in bronchoalveolar lavage were enumerated, and 2 × 104 cells were centrifuged onto glass slides at 800 rpm. Cytospins were stained using the Hema3 kit (Biochemical Sciences, Inc., Swedesboro, NJ), and differential cell counts were done on 500 cells/mouse. Total protein in BALF was determined on cell-free BALF supernatant stored at −80°C with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). A lactate dehydrogenase assay to indicate lytic activity was done on BALF supernatant using the Cytotox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI) according to the manufacturer's protocol. Results were quantified by spectrophotometry.

Histopathology and scoring of inflammation. The lungs from additional mice (n = 4/group/time point) at both 9 and 30 days were instilled through the trachea with CMF-PBS at a constant pressure of 14 cm H2O and placed in 4% paraformaldehyde at 4°C overnight for fixation of the tissue before embedding of tissue blocks in paraffin. Lung sections were cut at a 5-μm thickness for immunohistochemistry as described below, or stained with H&E. The 30-day lung sections were also stained using Masson's trichrome technique for the detection of collagen as an indication of fibrosis (16). Inflammation was scored by a certified pathologist (K. Butnor) using a blind code for identification of slides and an inflammation scale from 1 to 4; with 1 indicating absent inflammation, 2 showing mild predominantly lymphocytic inflammation restricted to peribronchiolar regions, 3 indicating moderate peribronchiolar mononuclear neutrophilic inflammation with minimal extension into adjacent tiers of alveoli, and 4 as severe mixed peribronchiolar and adjacent alveolar inflammation.

Ki-67 immunoperoxidase technique. The expression of Ki-67 protein is a requirement for progression through the cell division cycle and is an accurate marker of cell proliferation (24). To quantitate bronchiolar epithelial cell proliferation, lung sections from 9-day sham- and asbestos-exposed MPO−/− and MPO+/+ mice were deparaffinized in xylene, rehydrated through a series of graded ethanols, and equilibrated in water as described above. Antigen retrieval was then done using a 1:10 dilution in PBS of 10× DAKO target retrieval solution (DAKO, Carpinteria, CA) in a 95°C water bath for 40 minutes followed by 20 minutes of cooling to room temperature. Sections were then treated with DAKO peroxidase block for 30 minutes followed by a 5-minute wash in TBS before incubation in DAKO serum-free protein block for 30 minutes. Sections were then immersed in 50 μL of a 1:25 dilution of monoclonal rat anti-mouse Ki-67 primary antibody (DAKO), a 1:600 dilution of biotinylated anti-rat IgG secondary antibody (Vector Laboratories, Burlingame, CA), a 1:25 dilution of rat whole serum (Zymed, South San Francisco, CA), and 1% bovine serum albumin in PBS at room temperature for 30 minutes before blocking of excess secondary antibody with normal rat serum for 1.5 hours. Negative controls were incubated in PBS without primary antibody, and all sections incubated overnight at 4°C in a humidified chamber. The following day, sections were washed thrice in 1× TBS, treated for 30 minutes with horseradish peroxidase streptavidin (Vector) and incubated in 3,3′-diaminobenzidine (DAKO) for 3 minutes. Sections were then rinsed in double-distilled water, counterstained for 30 seconds in hematoxylin, dehydrated through increasing concentrations of ethanol, and washed in xylene twice for 15 minutes before coverslips were mounted in Histomount (Zymed). Slides were then examined by light microscopy using an Olympus BX50 upright microscope (Olympus America, Inc., Lake Success, NY) with associated MagniFier software. The number of Ki-67-positive immunoreactive epithelial cells was determined on a total of at least five distal bronchioles on duplicate lung sections per animal.

Myeloperoxidase and cyclin D1 immunofluorescence in lung sections. To confirm the negative status of MPO−/− mice (with respect to expression of myeloperoxidase) and to determine if myeloperoxidase and cyclin D1 immunoreactivity was associated with epithelial cells of MPO+/+ mice, lung sections from sham and asbestos-exposed MPO−/− and MPO+/+ mice were deparaffinized in xylene for 5 minutes (thrice), rehydrated through a graded series of ethanols, and equilibrated in water. Slides were then boiled in 1× DAKO target retrieval solution for 40 minutes, and cooled for 20 minutes. After a wash in 1× PBS for 5 minutes, sections were blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in PBS. A polyclonal rabbit anti-myeloperoxidase 562 (a kind gift from Marie Luise Brennan, Cleveland Clinic, Cleveland, OH) or a polyclonal rabbit anti-cyclin D1 (Labvision Corporation, Fremont, CA) were diluted in 1% bovine serum albumin in PBS, and slides were incubated overnight at 4°C. After washing with PBS, slides were incubated with a 1:400 dilution of AlexaFluor 647-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) in PBS for 1 hour at room temperature. SYTOX Green (1:1,000, Molecular Probes) in PBS was used as a counterstain for nuclei. Controls included slides incubated with secondary antibody alone. Sections of mouse intestinal epithelium were used as positive controls for cyclin D1 immunoreactivity. Negative controls for cyclin D1 included sections of normal lungs and intestine incubated with secondary antibody alone. Slides were examined by using a Bio-Rad MRC 1024ES confocal scanning laser microscope system (Bio-Rad Laboratories).

Statistical analysis. Data were analyzed using two-way ANOVA and Student-Newman-Keul's tests to adjust for multiple pair-wise differences. P ≤ 0.05 between groups were considered significant.

Myeloperoxidase activity is increased in bronchoalveolar lavage fluids and observed in distal bronchiolar epithelial cells in normal (MPO+/+) mice after inhalation of asbestos. To determine if myeloperoxidase activity was increased in BALF from C57/BL6 mice after inhalation of asbestos, we did a number of experiments to optimize detection of myeloperoxidase in cell-free BALF at 9 days, i.e., the peak time of asbestos-induced inflammation (16). Myeloperoxidase activity was not detected in BALF from sham mice using protein concentrations of up to 50 μg (Fig. 1, top, solid lines). Conversely, fresh lavage samples from four asbestos-exposed mice showed linear reactions over a 20-minute period (Fig. 1, top, dashed lines).

Figure 1.

Myeloperoxidase activity in BALF (top). Fifteen micrograms of protein in 100 μL of BALF supernatant from sham- and asbestos-exposed C57Bl/6 mice (n = 4/group) was mixed with 100 μL tetramethylbenzidine reagent, and the oxidation of tetramethylbenzidine was measured at 630 nm over a 20-minute period. All sham mice showed no increases in activity (solid lines), whereas the four asbestos-exposed mice showed linear release kinetics over time (dashed lines). Myeloperoxidase immunolocalization in bronchiolar epithelial cells (bottom). Anti-myeloperoxidase reactivity (red), and nuclei stained with SYTOX Green (green). Representative images of lung tissue sections stained for myeloperoxidase and examined by confocal scanning laser microscope. A, 9-day sham-exposed normal (MPO+/+); B, 9-day asbestos-exposed normal (MPO+/+); C, 9-day sham-exposed (MPO−/−); D, 9-day asbestos-exposed (MPO−/−). All original magnifications, ×400.

Figure 1.

Myeloperoxidase activity in BALF (top). Fifteen micrograms of protein in 100 μL of BALF supernatant from sham- and asbestos-exposed C57Bl/6 mice (n = 4/group) was mixed with 100 μL tetramethylbenzidine reagent, and the oxidation of tetramethylbenzidine was measured at 630 nm over a 20-minute period. All sham mice showed no increases in activity (solid lines), whereas the four asbestos-exposed mice showed linear release kinetics over time (dashed lines). Myeloperoxidase immunolocalization in bronchiolar epithelial cells (bottom). Anti-myeloperoxidase reactivity (red), and nuclei stained with SYTOX Green (green). Representative images of lung tissue sections stained for myeloperoxidase and examined by confocal scanning laser microscope. A, 9-day sham-exposed normal (MPO+/+); B, 9-day asbestos-exposed normal (MPO+/+); C, 9-day sham-exposed (MPO−/−); D, 9-day asbestos-exposed (MPO−/−). All original magnifications, ×400.

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We then focused on whether myeloperoxidase protein could be detected in bronchiolar or alveolar duct epithelial cells of normal mice, as these are the cell types encountering asbestos fibers at sites of deposition and progenitor cells of asbestos-induced lung cancer. Immunohistochemistry revealed that sham control mice showed no detectable myeloperoxidase in bronchiolar epithelium or surrounding tissue (Fig. 1A,, bottom), but after inhalation of asbestos, myeloperoxidase was observed in the cytoplasm of distal bronchiolar epithelial cells (Fig. 1B,, bottom). No myeloperoxidase was observed in either sham or asbestos-exposed null mice (Fig. 1C  and D, bottom).

Patterns of asbestos-associated inflammation are delayed in MPO(−/−) null mice. Chrysotile asbestos-induced markers of inflammation in BALF have been previously characterized in our murine model at various time periods after initial exposures to asbestos. PMN influx in BALF peaks at 9 days (16). In line with earlier data, total protein in BALF and lactate dehydrogenase levels, an indication of lung permeability reflecting dead or dying cells as well as plasma proteins, were elevated (P ≤ 0.05) in asbestos-exposed MPO+/+ and MPO−/− mice in comparison with sham mice (Fig. 2A and B). In comparison with respective sham groups, total inflammatory cell numbers in BALF also did not change significantly in asbestos-exposed normal or MPO-null mice (Fig. 2C). However, fewer cells were noted in BALF samples from myeloperoxidase-null in comparison with normal (MPO+/+) sham mice (P ≤ 0.05). This was due to a significant decrease (P ≤ 0.05) in alveolar macrophages in MPO−/− mice (Fig. 2D). Compared with sham controls, both normal and myeloperoxidase-null asbestos-exposed mice exhibited increases (P ≤ 0.05) in the number of neutrophils and lymphocytes. However, myeloperoxidase-null mice showed lower (P ≤ 0.05) numbers of macrophages and lymphocytes than normal mice in response to asbestos (Fig. 2D).

Figure 2.

Markers of cell injury and inflammation in bronchoalveolar lavage. Total protein (A) and lactate dehydrogenase (B) are significantly increased (P < 0.05) in asbestos-exposed (+Asb) animals in comparison with sham controls at 9 days. Total (C) and differential cell counts (D) on bronchoalveolar lavage samples from sham- and asbestos-exposed (+Asb) mice at 9 days. Columns, mean; bars, ± SE; *, significantly different (P < 0.05) from shams in each group; †, significantly different (P < 0.05) from respective MPO+/+ group.

Figure 2.

Markers of cell injury and inflammation in bronchoalveolar lavage. Total protein (A) and lactate dehydrogenase (B) are significantly increased (P < 0.05) in asbestos-exposed (+Asb) animals in comparison with sham controls at 9 days. Total (C) and differential cell counts (D) on bronchoalveolar lavage samples from sham- and asbestos-exposed (+Asb) mice at 9 days. Columns, mean; bars, ± SE; *, significantly different (P < 0.05) from shams in each group; †, significantly different (P < 0.05) from respective MPO+/+ group.

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Lung histopathology at 9 days supported the results of BALF studies. In normal MPO+/+ mice, inhalation of asbestos caused a characteristic influx of macrophages into alveolar tissue and peribronchiolar mononuclear and neutrophilic inflammation that was not observed in sham mice (Figs. 3B and 4A). The lungs of myeloperoxidase-null mice showed no inflammatory responses to asbestos at this time point (Figs. 3C and 4A). However, at 30 days, inflammation scores of both normal and myeloperoxidase-null asbestos-exposed mice were comparable and significantly increased (P ≤ 0.05; Figs. 3E,, F, and 4B) in comparison with respective sham controls (Figs. 3D and 4B).

Figure 3.

Histopathologic findings in lungs at 9 (A-C) and 30 (D-F) days (H&E stained sections). A, 9-day sham MPO+/+ lung shows no inflammation; B, 9-day asbestos-exposed MPO+/+ lung showing mild peribronchiolar mononuclear inflammation and scattered alveolar macrophages; C, 9-day asbestos-exposed MPO−/− lung demonstrating essentially absent inflammation; D, 30-day sham MPO−/− lung is devoid of inflammation; E, 30-day asbestos-exposed MPO+/+ lung features moderate neutrophilic and mononuclear inflammation extending from the bronchiolar epithelium into surrounding alveoli. Moderate intra-alveolar macrophage infiltration is also present; F, 30-day asbestos-exposed MPO−/− lung exhibits moderate mixed peribronchiolar inflammation. All original magnifications, ×200.

Figure 3.

Histopathologic findings in lungs at 9 (A-C) and 30 (D-F) days (H&E stained sections). A, 9-day sham MPO+/+ lung shows no inflammation; B, 9-day asbestos-exposed MPO+/+ lung showing mild peribronchiolar mononuclear inflammation and scattered alveolar macrophages; C, 9-day asbestos-exposed MPO−/− lung demonstrating essentially absent inflammation; D, 30-day sham MPO−/− lung is devoid of inflammation; E, 30-day asbestos-exposed MPO+/+ lung features moderate neutrophilic and mononuclear inflammation extending from the bronchiolar epithelium into surrounding alveoli. Moderate intra-alveolar macrophage infiltration is also present; F, 30-day asbestos-exposed MPO−/− lung exhibits moderate mixed peribronchiolar inflammation. All original magnifications, ×200.

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Figure 4.

Grading of peribronchiolar inflammation in lungs at 9 (A) and 30 (B) days. Columns, mean; bars, ± SE; *, significantly different (P < 0.05) from shams in each group; †, significantly different (P < 0.05) from respective MPO+/+ group.

Figure 4.

Grading of peribronchiolar inflammation in lungs at 9 (A) and 30 (B) days. Columns, mean; bars, ± SE; *, significantly different (P < 0.05) from shams in each group; †, significantly different (P < 0.05) from respective MPO+/+ group.

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Asbestos-associated epithelial cell proliferation is increased in the distal bronchioles of MPO(−/−) null mice in comparison with normal mice. We and others have shown that incorporation of 5′ bromodeoxyuridine is increased in the bronchiolar epithelium of rodents after inhalation of chrysotile asbestos (16, 25). This technique requires preinjection of mice or implantation of mini-pumps containing bromodeoxyuridine and does not differentiate between unscheduled, unsuccessful, DNA synthesis or cell proliferation. In contrast, incorporation of Ki-67 is a sensitive and specific immunocytochemical method not requiring injection of a precursor into animals and measuring the number of epithelial cells progressing through the cell division cycle (24). In comparison with sham control groups (Fig. 5), increases in the number of Ki-67-positive bronchiolar epithelial cells were observed in both asbestos-exposed groups at 9 days—whereas numbers of Ki-67-positive cells doubled in normal mice, myeloperoxidase-null mice showed ∼4-fold increases (P ≤ 0.05) in the number of proliferating cells. At 30 days, the number of Ki-67-positive cells were increased in asbestos-exposed MPO+/+ mice and comparable to levels in asbestos-exposed myeloperoxidase-null mice.

Figure 5.

Ki-67 immunoperoxidase staining to show replicating distal bronchiolar epithelial cells in lungs of mice at 9 days. A, sham MPO+/+; B, asbestos-exposed MPO+/+; C, sham MPO−/−; D, asbestos-exposed MPO−/−; L, bronchiolar lumen. All original magnifications, ×400. Quantitation of Ki-67-positive distal bronchiolar epithelial cells at 9 (E) and 30 (F) days. Columns, mean; bars, ± SE; *, significantly different (P < 0.05) from sham groups; †, significantly different (P < 0.05) from asbestos-exposed MPO+/+ group.

Figure 5.

Ki-67 immunoperoxidase staining to show replicating distal bronchiolar epithelial cells in lungs of mice at 9 days. A, sham MPO+/+; B, asbestos-exposed MPO+/+; C, sham MPO−/−; D, asbestos-exposed MPO−/−; L, bronchiolar lumen. All original magnifications, ×400. Quantitation of Ki-67-positive distal bronchiolar epithelial cells at 9 (E) and 30 (F) days. Columns, mean; bars, ± SE; *, significantly different (P < 0.05) from sham groups; †, significantly different (P < 0.05) from asbestos-exposed MPO+/+ group.

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Cyclin D1 is a marker of cell cycle progression that has been previously uncharacterized in models of lung injury. To confirm our quantitative results using Ki-67 to delineate cells not in G0, we also examined immunolocalization of cyclin D1 comparatively in the distal bronchiolar region and alveolar duct junctions, sites of initial fiber deposition (25), in sham and asbestos-exposed mice. As shown in Fig. 6, dual fluorescence studies using SYTOX Green to demonstrate nuclei, showed that both nuclear (yellow indicating colocalization of cyclin D1, with nucleus) and cytoplasmic fluorescence (red) occurred predominantly in distal bronchiolar epithelial cells. Cyclin D1 immunofluorescence was occasionally observed in sham normal (Fig. 6A) and myeloperoxidase-null mice (Fig. 6B). However, it was more predominant in asbestos-exposed groups (Fig. 6C and D), particularly in myeloperoxidase-null mice, where it was also observed in mesothelial cells (arrowhead) and at sites of alveolar duct junctions (arrow). Although quantitation of cyclin D1 cells in epithelium was not feasible due to its infrequent expression and colocalization in both the nucleus and cytoplasm, trends of expression in various groups of mice were similar to those observed with Ki-67 labeling.

Figure 6.

Immunofluorescent localization of cyclin D1 by confocal scanning laser microscopy in lung sections at 9 days. Representative images of lung tissue sections stained with an antibody to cyclin D1 (red) or SYTOX Green (green) to stain nuclei. Yellow, overlap of signals indicating colocalization (see arrow and arrowhead). A, sham MPO−/−; B, sham MPO+/+; C, asbestos-exposed MPO−/−; D, asbestos-exposed MPO+/+; E, mouse intestine (+, control); F, lung section stained with secondary antibody alone (−, control); L, lumen of distal bronchiole; P, pleural space. All original magnifications, ×400.

Figure 6.

Immunofluorescent localization of cyclin D1 by confocal scanning laser microscopy in lung sections at 9 days. Representative images of lung tissue sections stained with an antibody to cyclin D1 (red) or SYTOX Green (green) to stain nuclei. Yellow, overlap of signals indicating colocalization (see arrow and arrowhead). A, sham MPO−/−; B, sham MPO+/+; C, asbestos-exposed MPO−/−; D, asbestos-exposed MPO+/+; E, mouse intestine (+, control); F, lung section stained with secondary antibody alone (−, control); L, lumen of distal bronchiole; P, pleural space. All original magnifications, ×400.

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We show for the first time that myeloperoxidase is an important factor in the acute inflammatory response to inhaled asbestos. Because asbestos is an oxidative stress (15) and a potent inflammatory agent in lung and pleura (4, 26, 27), our findings may be relevant to the inflammation and proliferation of cells observed after exposure to a number of carcinogenic environmental toxicants. Our initial interest in the role of myeloperoxidase in asbestos-induced inflammation stemmed from observations that PMNs are the predominant cell type infiltrating the lung after brief inhalation or intratracheal exposures to asbestos (4). Although neutrophils produce a plethora of chemokines and cytokines after inhalation of oxidant gases and oxidant-generating particulates such as asbestos or silica, myeloperoxidase production by neutrophils has been classically linked to its microbicidal and inflammatory effects via the generation of the halogenating oxidant, HOCl. Although HOCl is the major reactive species generated by myeloperoxidase, recent work shows that myeloperoxidase also participates in nitrotyrosine formation in some, but not all, neutrophil-rich animal models of inflammation (28). Because asbestos is known to induce inducible nitric oxide synthase in alveolar macrophages (29) and nitrotyrosine formation in rat lungs which subsequently develop inflammation and fibrosis (10), the myeloperoxidase-catalyzed formation of nitric oxide–derived inflammatory oxidants might also be critical in the early pathogenesis of asbestos-associated diseases.

Here, we show that myeloperoxidase enzyme activity occurs in PMN-rich BALF after inhalation of asbestos by mice. Moreover, we show that myeloperoxidase protein is found in the distal bronchiolar epithelium at alveolar duct bifurcations, sites of asbestos fiber deposition. Increased myeloperoxidase activity in homogenized lung tissues in an amiodarone-induced rat model of pulmonary fibrosis and its decrease with indicators of fibrosis (lung hydroxyproline, transforming growth factor-β1 expression, etc.), have been documented after gastric intubation of the antioxidant and antiinflammatory agent, curcumin (30), but the cell types expressing myeloperoxidase under these circumstances were unclear. Our results support the hypothesis that myeloperoxidase is generated predominantly by PMNs, but has the capacity to cross the epithelium, presumably by pinocytosis or phagocytosis as do asbestos fibers (31). This model is supported by a study showing myeloperoxidase immunoreactivity in the alveolar epithelial compartment of lung tissues from patients with sickle cell disease or those undergoing lung transplant rejection (32).

A number of in vitro models show that high concentrations of oxidants cause cytotoxicity or cytostasis, whereas lower concentrations cause cell proliferation and transformation (33, 34). Likewise, asbestos is an agent causing cell injury at higher concentrations and proliferation at lower concentrations in airway epithelial cells in vitro (35). The fact that myeloperoxidase and asbestos might cooperatively contribute to oxidant-associated cell damage is supported by many observations showing that these agents increase oxidative stress in lung tissue (17, 34). For example, myeloperoxidase in the sputum of patients with cystic fibrosis enhances cell death after addition of generating systems of hydrogen peroxide to human tracheobronchial epithelial cells (36). In asbestos-exposed myeloperoxidase-null mice, initial oxidant profiles in lung tissue may be lower than those in asbestos-exposed MPO+/+ mice, thus favoring an environment promoting cell survival and mitogenesis. Our data thus support a model whereby acute epithelial cell proliferation by asbestos is initially curtailed under increased oxidative stress in MPO+/+ mice. However, as inhalation of asbestos ensues from 9 to 30 days, it is known that oxidative stress–inducing fibers accumulate and inflammation increases (1, 6, 9, 37). These factors may explain why the number of Ki-67 immunoreactive epithelial cells are comparable in both normal and myeloperoxidase-null mice at 30 days.

Cyclin D1 and its partner, cyclin-dependent kinase 4, promote G1 to S phase progression via phosphorylation of the retinoblastoma protein. The exciting discovery that cyclin D1 is expressed in pulmonary epithelial cells, and that expression corresponds with quantitative trends in asbestos-induced proliferation, as assayed by Ki-67 labeling, provides some insight into the molecular mechanisms of oxidant-induced proliferation. We have previously shown that initial injury and subsequent proliferative effects of asbestos in vitro and in vivo are linked to stimulation of the extracellular signal-regulated kinases, ERK1/2 and ERK5 in epithelial cells (3841). Increased ERK phosphorylation is linked causally to increased activation and expression of the activator protein-1 (AP-1) family members, c-fos, fra-1 (ERK1/2) and c-Jun (ERK5) in pulmonary cells as well as elevated AP-1 activity (4143). HOCl also activates ERK1/2, growth arrest, and apoptosis in human umbilical vein endothelial cells, and loss of viability is enhanced when the survival pathway, ERK1/2, is inhibited (44). These results also suggest the importance of AP-1-mediated gene expression in HOCl-induced cell responses. Because cyclin D1 is an AP-1-dependent gene (45), it may be a key player in the induction of asbestos-induced cell cycle reentry and progression. Our immunolocalization results in lung epithelium in vivo are consistent with recently published observations in neonatal rat cardiomyocytes in vitro where cyclin D1 localization is predominantly cytoplasmic (46). However, when cyclin D1 is ectopically expressed in the nucleus of postnatal cardiocytes in vivo, cell cycle reentry, as evaluated by the expression of Ki-67, is increased. This observation and recent work from our group (47) suggests that trafficking and accumulation of cell signaling proteins and transcription factors in the nucleus may be important in the induction of responses to oxidants.

In summary, our results show that myeloperoxidase activity is increased in lavage fluid after inhalation of asbestos fibers. Myeloperoxidase also localizes in bronchiolar epithelial cells in asbestos-exposed mice where it may act directly to cause alterations in epithelial cells. Our data reveal that myeloperoxidase status is functionally important not only in the control of epithelial cell proliferation in response to asbestos but also in the induction of asbestos-induced inflammation. The inflammatory profiles of BALF in asbestos-exposed normal and myeloperoxidase-null mice did not reflect differences in the number of PMNs, but rather decreases in alveolar macrophages and lymphocytes in myeloperoxidase-null mice. It is therefore tempting to speculate that myeloperoxidase catalyzed HOCl directly or through signaling pathways involving tyrosine nitration affects redox-sensitive transcription factors, such as AP-1 or nuclear factor κB, that are linked to chemokine elaboration and influx of macrophages and lymphocytes. The fact that myeloperoxidase plays a critical role in initial asbestos-associated epithelial cell repair responses and inflammation may be important in the prevention of asbestos- and oxidant-related lung cancers.

Grant support: PO1 grant HL67004 from the National Heart, Lung, and Blood Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Maria Stern from the Cell Imaging Core and Microscopy Imaging Center (University of Vermont) for technical assistance with immunocytochemistry. Daniel Bullard, PhD (Department of Microbiology, University of Alabama at Birmingham) kindly provided and typed the mice for studies here. Veronique Andriessen, Maximilian MacPherson, Beth Langford-Corrigan, and Laurie Sabens provided valuable technical assistance.

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