Quantitation and Localization of Pulmonary Manganese Superoxide Dismutase and Tumor Necrosis Factor
following Exposure to Ozone and Nitrogen Dioxide
Barbara L. Weller1,
Hanspeter Witschi and
Kent E. Pinkerton
Center for Comparative Respiratory Biology and Medicine, California Regional Primate Research Center, Institute of Toxicology and Environmental Health, University of California, One Shields Avenue, Davis, California 95616
Received August 5, 1999;
accepted December 14, 1999
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ABSTRACT
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Tumor necrosis factor
(TNF
) and manganese superoxide dismutase (MnSOD) are thought to play critical roles in the process of lung injury, repair, and disease. The induction of TNF
and MnSOD were examined in a model of progressive pulmonary fibrosis along the length of the alveolar duct in rats exposed for 1, 5, and 8 weeks to a combination of 0.8 ppm ozone and 14.4 ppm nitrogen dioxide. This oxidant injury model results in a triphasic response with an initial inflammatory stage during weeks 13, followed by a partial resolution at weeks 45, and a final stage of rapidly progressive fibrosis during weeks 68. Changes in TNF
and MnSOD labeling for the proximal and distal alveolar ducts of the lungs were quantified using immunohistochemistry and morphometric techniques at 1, 5, and 8 weeks of exposure. A significant elevation in MnSOD was noted in alveolar macrophages and interstitial cells of the proximal and distal portions of the alveolar duct following 8 weeks of exposure. Labeling for TNF
only in the proximal region of the alveolar duct, was significantly increased in alveolar macrophages after 1 and 8 weeks of exposure, while a significant increase in TNF
labeling of interstitial cells in proximal regions was noted at all time points. We conclude that MnSOD is elevated in areas of focal injury as well as the more distal protected areas of the lungs, while TNF
correlates strongly with both the temporal and spatial aspects of greatest cellular injury in the lungs.
Key Words: lung; fibrosis; cytokine; antioxidant; tumor necrosis factor
(TNF
); manganese superoxide dismutase (MnSOD).
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INTRODUCTION
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Pulmonary fibrosis, the sequela to a variety of toxic exposure and injury/repair processes, typically involves the cytokine tumor necrosis factor
(TNF
). Increased expression of TNF
has been established in sarcoidosis (Bost et al., 1994
; Ishioka et al., 1996
), asbestosis (Zhang et al., 1993
), beryllium disease (Bost et al., 1994
), coal workers' pneumoconiosis (Schins and Borm, 1995
; Vanhée et al., 1996
), idiopathic pulmonary fibrosis (Kapanci et al., 1995
; Zhang et al., 1993
), and adult respiratory distress syndrome (Schutte et al., 1996
). A number of animal models of fibrosis using exposure to compounds such as silica (Kang et al., 1996
), bleomycin (Phan and Kunkel, 1992
; Piguet et al., 1989
; Zhang et al., 1997
), asbestos (Driscoll et al., 1995
; Lemaire and Ouellet, 1996
), and amiodarone (Futamura, 1996
) also have increased levels of TNF
. Although pulmonary fibrosis is predominately an interstitial disease, the primary cells in the lungs which demonstrate increased levels of TNF
include epithelial type II cells (Kapanci et al., 1995
) and alveolar macrophages (Bost et al., 1994
; Ishioka et al., 1996
; Vanhée et al., 1996
; Zhang et al., 1993
). However, no studies have examined the precise anatomical distribution of TNF
within cells of the lungs and those areas undergoing fibrotic change. Transgenic mice over-expressing TNF
have been found to develop fibrotic lung lesions similar to those seen in idiopathic pulmonary fibrosis (Miyazaki et al., 1995
), suggesting an important role for overproduction of this cytokine leading to excessive deposition of collagen and subsequent remodeling of the lungs.
TNF
has also been noted to increase the expression of the antioxidant manganese superoxide dismutase (MnSOD). MnSOD has been linked to human diseases (Dobashi et al., 1993
; Lakari et al., 1998
), as well as oxidant and fibrotic injury in animals (Ho et al., 1998
; Parizada et al., 1991
; Quinlan et al., 1995
; Wispé et al., 1992
), although its role is obscure. MnSOD is known to be upregulated by TNF
(Das et al., 1995
; Visner et al., 1990
; Warner et al., 1991
; White and Ghezzi, 1989
), but a recent study suggests that MnSOD and TNF
may be controlled at least in part by differing mechanisms (Tian et al., 1998
). To our knowledge, no studies have examined in a quantitative manner the presence and distribution in the lungs of MnSOD and TNF
during the process of evolving injury and fibrosis.
Despite the established involvement of TNF
in progressive pulmonary fibrosis, the mechanisms initiated by this cytokine in promoting fibrosis and its association with MnSOD have yet to be clarified. In this study, the production of both TNF
and MnSOD was examined in a rat model of progressive pulmonary fibrosis, using simultaneous exposure to 0.8 ppm ozone and 14.4 ppm nitrogen dioxide for up to 8 weeks. Injury in this model results in a triphasic response (Farman et al., 1997
), beginning with an inflammatory phase (weeks 13), followed by partial resolution and stabilization (weeks 46), and ending with rapidly progressing fibrosis (weeks 78). The expression of TNF
and MnSOD was studied with immunohistochemical techniques, following exposure of rats to ozone and nitrogen dioxide for 1, 5, and 8 weeks. The cellular distribution of TNF
and MnSOD were quantified over each of the 3 phases of this injury and correlated with cellular proliferation to better understand the interactive roles of TNF
and MnSOD in progressive pulmonary fibrosis.
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MATERIALS AND METHODS
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Animals
Male Sprague-Dawley rats, 1012 weeks of age, were purchased from Charles River Labs (Portage, MI). Rats were housed in wire cages and received food (Purina Rat Chow, Ralston-Purina, St. Louis, MO) and water ad libitum. Rats were randomly screened and found free of respiratory pathogens. Animals were maintained on a 12-h light/dark cycle in accordance with NIH animal care guidelines.
Exposures
All exposures took place at the California Regional Primate Research Center Inhalation Facility. Rats were exposed for 6 h/night, 7 nights/wk (from 6 P.M. to 12 A.M.) for 1, 5, or 8 weeks to a combined target concentration of 0.8 ppm ozone and 14.4 ppm nitrogen dioxide. Actual exposure concentrations are shown in Table 1
. Animals were exposed in 4.2 m3 glass and stainless steel chambers using 30 changes of air per h. Nitrogen dioxide was generated by instilling nitrogen through a tank of pure liquid dimer, dinitrogen tetroxide, at 0°C (Freeman et al., 1974
) and was transported by stainless steel lines to the mixing inlet of the exposure chamber. A chemiluminescent monitor (model 2108, Dasibi Corp., Glendale, CA) was used to monitor exposure concentrations and calibrated using a Dasibi gas calculator model 1005-CE-2. Ozone was produced by infusing medical grade oxygen through a silent arc discharge ozonizer (Erwin Sander Corp., Griessen Germany). Ozone concentrations were monitored by Dasibi ultraviolet photometric monitors (model 1003- AH, Dasibi Corp.) calibrated against a standard reference photometer at the California Air Resources Board Quality Assurance Laboratory. The chambers were sampled at the cage level using probes inserted into sampling ports, and were monitored every 8 min. A total of 24 animals were studied with 4 controls and 4 treated animals examined following 1, 5, or 8 weeks of exposure.
Antibodies
MnSOD antiserum (Kinnula et al., 1994
; Weller et al., 1997
), a generous gift from Dr. James D. Crapo (National Jewish Medical and Research Center, Denver, CO), is rabbit anti-human manganese superoxide dismutase made using recombinant human MnSOD supplied by Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT). The antiserum, which cross-reacts with rat MnSOD, is monospecific for a single protein at a molecular weight corresponding to MnSOD on immunoblots and is abolished by co-incubation with the antigen. The TNF
antiserum is a polyclonal rabbit anti-mouse TNF
from Genzyme Pharmaceutical (Cambridge, MA). This antiserum is known to cross-react with rat TNF
(Gossart et al., 1996
).
Immunohistochemistry
Following cessation of exposure, animals were anesthetized with sodium pentobarbital (450 mg/kg). The lungs were inflation-fixed in situ for 1 h with 4% paraformaldehyde at 30 cm of pressure, dehydrated in ethanol, and embedded in paraffin. Sections 3 µm thick were labeled with antisera to MnSOD (1:300) or TNF
(1:500) using the avidin-biotin peroxidase method with reagents from Vector Laboratories (Burlingame, CA) visualized with 3,3' diaminobenzidine tetrahydrochloride from Sigma (St. Louis, MO) and counterstained with methyl green. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide prior to labeling. Sections in which the primary antibody was replaced with phosphate buffered saline served as a negative control.
Electron Microscopy
A subset of animals exposed for 8 weeks were anesthetized with sodium pentobarbital, and the lungs were fixed by intratracheal infusion of Karnovsky's fixative at 30 cm pressure for 30 min. Sections of the lungs were embedded in araldite, ultrathin (5060 nm thick) sections prepared, and mounted on copper grids. Grids were stained with uranyl acetate and lead, and were photographed with a Zeiss 10C electron microscope. Additional sections 1.5 µm thick were stained with toluidine blue for light microscopy.
Quantitative Light Microscopy
Sections labeled with antisera to MnSOD or TNF
were examined by light microscopy. All longitudinally oriented bronchiole-alveolar duct junctions (BADJs) on each slide were identified. Three BADJs were selected by random number generation for analysis from each animal. Low magnification images of the BADJs were captured using a Macintosh IIci computer and an Olympus BH-2 microscope with a Dage MTI video camera (Michigan City, IN). Images were overlaid with a concentric circle bullseye grid with a spacing of 100 µm using the level of the first alveolar outpocketing as the geometric reference point. Epithelial cells and alveolar macrophages labeled for MnSOD or TNF
were counted, along with total numbers of epithelial cell nuclei and total alveolar macrophage profiles. A rigorous definition of labeling was used to reduce variability from immunohistochemical staining. A cell was considered to be positively labeled for MnSOD or TNF
only if the cytoplasm of the cell was strongly labeled above the background level. All cell counts were performed with the bullseye pattern of circles to aid in defining the distance from the terminal bronchiole in the analysis of cellular changes. All cell count data from the 3 selected BADJ's were averaged for each animal. A total of 4 animals per group per time point were studied.
Statistical Analysis
All cell counts per animal for each treatment group and time point were compared by analysis of variance (ANOVA). Multiple comparisons over the time course of the study were performed using the Fisher least significant difference test. A value of p < 0.05 was considered significant.
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RESULTS
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Oxidant induced cellular changes were examined in the lungs of animals exposed to 0.8 ppm ozone (O3) and 14.4 ppm nitrogen dioxide (NO2), 6 h/night, 7 nights/week for 1, 5, and 8 weeks using morphometric techniques. The injury response to combined oxidant exposure based on location in the lung parenchyma is summarized in Table 2
. A significant increase in the number of alveolar macrophages in alveolar ducts within the first 400 µm of the terminal bronchiole was noted in exposed animals, compared to animals breathing only filtered air. Alveolar macrophage numbers were not significantly different from control values in the more distal alveolar ducts 400600 µm from the terminal bronchiole. An exception to this pattern was found in the significantly elevated numbers of alveolar macrophages in the distal alveolar ducts of animals exposed for 8 weeks. Epithelial cell numbers were unchanged during all times examined following exposure in alveolar ducts 0400 µm from the terminal bronchiole, when compared with filtered air controls. Examination of more distal alveolar ducts 400600 µm from the terminal bronchiole in animals exposed to ozone and nitrogen dioxide revealed a similar pattern.
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TABLE 2 Time Course of Total Epithelial and Alveolar Macrophage Numbers per 100 µm of Basement Membrane Length in the Proximal and Distal Alveolar Ducts following Exposure to Ozone and Nitrogen Dioxide
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Immunohistochemical techniques were used to determine the level and cellular location of both TNF
and MnSOD in the lungs of rats exposed to both oxidant gases or to filtered air. All labeling for both MnSOD and TNF
was determined to be intracellular; no extracellular labeling for either protein was seen. Only cells that were strongly labeled were used in the counting study. No nonspecific labeling was found in the negative controls. Lungs from control animals labeled with MnSOD antiserum had an occasional, rare alveolar macrophage labeled in the most proximal alveolar duct, as well as labeled Clara cells in the terminal bronchioles (Fig. 1A
). All control lungs had little labeling for TNF
in the proximal alveolar ducts, with some labeling of Clara cells in terminal bronchioles (Fig. 1B
). Following 1 week of exposure to the combined oxidants, increased labeling for both TNF
(data not shown) and MnSOD (Fig. 1C
) was seen in epithelial cells and alveolar macrophages in the alveolar ducts 0400 µm from the terminal bronchiole. After 5 weeks of exposure, labeling for TNF
(Fig. 1D
) and MnSOD (data not shown) was further increased and included labeling of interstitial cells as well as epithelial cells and alveolar macrophages. A dramatic increase in labeling for both MnSOD (Fig. 1E
) and TNF
(Fig. 1F
) was seen following 8 weeks of exposure, with increases in labeling for alveolar macrophages, epithelial cells, and interstitial cells. The increase in labeling for both MnSOD and TNF
, seen in the 8 week exposed animals, was due in large measure to an increase in the number of cells expressing these proteins. Due to the increase in cellularity of the interstitium, small cell profiles could be found lightly labeled for TNF
in the 8 week, oxidant exposed animals (Fig. 1F
). This diffuse interstitial labeling was not seen in the filtered air controls (Fig. 1B
). These small cell profiles were not counted as labeled, due to their diffuse intensity of labeling for TNF
. Labeling intensity for TNF
and MnSOD in cells identified as positive for the counting study was similar for all 3 exposure time points.

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FIG. 1. Light micrographs of rat lungs exposed to filtered air or to 0.8 ppm ozone and 14.4 ppm nitrogen dioxide for 1, 5, or 8 weeks. Sections containing BADJ's were labeled with antisera to either MnSOD (1:300) or TNF (1:500). (A) Control rat lung labeled with antisera to MnSOD. Clara cells were labeled in the terminal bronchiole (small arrow). Occasionally labeled alveolar macrophages were found in alveolar ducts (arrowhead), detailed in the higher magnification insert (Bar = 10 µm). Bar = 30 µm. (B) Control rat lung labeled with TNF antisera. Clara cells were labeled in terminal bronchioles (small arrow). Little labeling was found in alveolar ducts. An alveolar macrophage (arrowhead) is detailed in higher magnification in the insert (Bar = 10 µm). Bar = 30 µm. (C) Increased labeling for MnSOD was found in rat lungs following 1 week of exposure to ozone and nitrogen dioxide. Alveolar macrophages (arrowhead) and epithelial cells (arrow) labeled for MnSOD are detailed at higher magnification in the insert (Bar = 10 µm). Bar = 30 µm. (D) After 5 weeks of exposure to combined oxidants, increased labeling for TNF was seen in alveolar macrophages (arrowhead), epithelial cells (arrow), and interstitial cells (triangle). A labeled interstitial cell is detailed at higher magnification in the insert (Bar = 10 µm). Bar = 30 µm. (E) A dramatic increase in labeling for MnSOD was found after 8 weeks of exposure to ozone and nitrogen dioxide. Increased expression of MnSOD was found in alveolar macrophages (arrowhead), epithelial cells (arrow), and interstitial cells (triangle). Bar = 30 µm. (F) A significant increase in TNF labeling was seen in rat lungs exposed to combined oxidants for 8 weeks. Increased labeling for TNF antisera was noted in epithelial cells (arrow), alveolar macrophages (arrowhead), and interstitial cells (triangle). Bar = 30 µm.
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Mast cells were identified using sections stained with toluidine blue, which results in a metachromatic staining of the mast cell granules. Mast cells were present in interstitial lesions in the BADJ's of oxidant-exposed animals following 5 weeks of exposure, and were increased in number after 8 weeks of exposure (Fig. 2B
). Mast cells within these lesions were found to express both MnSOD (Fig. 2C
) and TNF
(Fig. 2D
). No mast cells were found in the BADJ's of filtered air controls (Fig. 2A
). In an identical study of rats exposed for 8 weeks to 0.8 ppm ozone and 14.4 ppm nitrogen dioxide, an increase in interstitial macrophages and monocytes could be seen (Fig. 2E
), which was confirmed using transmission electron microscopy (Fig. 2F
).

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FIG. 2. Light micrographs of rat lungs exposed to filtered air or 0.8 ppm ozone and 14.4 ppm nitrogen dioxide for 8 weeks. All micrographs were taken from BADJ's within the first 400 µm of the terminal bronchiole. (A) Control-rat lung stained with toluidine blue. No mast cells were seen in the interstitium. The nuclei of interstitial cells (triangle) and alveolar macrophages (arrowhead) stained blue. (B) Rat lung exposed to the combined oxidants and stained with toluidine blue reveals an increase in mast cells (arrows) and other interstitial cells (triangle). (C) The effect of exposure to combined oxidants in a rat lung labeled with antisera to MnSOD. Increased interstitial cell labeling was found (triangle), including cells identified as mast cells (arrow). Increased labeling was found in alveolar macrophages (arrowhead) with exposure. (D) Exposure to combined oxidants resulted in increased labeling for TNF antisera. Labeled interstitial cells (triangle) and cells identified as mast cells (arrows) were seen as well as labeled alveolar macrophages (arrowhead). (E) A light micrograph of a rat lung exposed to the combined oxidants for 8 weeks, embedded in glycol methacrylate, and stained with toluidine blue. Increased numbers of inflammatory cells can be seen in the interstitium (triangles) as well as mast cells (arrows). Bar =10 µm. (F) A transmission electron micrograph of a rat lung exposed to 0.8 ppm ozone and 14.4 ppm nitrogen dioxide for 8 weeks. Macrophages (arrowheads) and monocytes (triangle) can be seen in the interstitium. Bar = 3 µm.
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To quantify the response seen for both TNF
and MnSOD with oxidant injury, morphometric techniques were used to determine changes in the total number of labeled cells and changes in cell populations labeled for these proteins. Changes in the total number of cells expressing TNF
and MnSOD were normalized to the alveolar basement membrane length to eliminate variations in sample size. Substantial differences in the pattern of labeling for the two proteins were seen following oxidant exposure in epithelial cells, alveolar macrophages, and interstitial cells. An increase in epithelial cells labeled with antiserum to MnSOD was noted in exposed animals, which reached significance following 8 weeks of exposure (Fig. 3A
). No significant change in labeling for TNF
was found in epithelial cells following exposure (Fig. 3B
). Oxidant exposure caused an increase in both the absolute number and the percent of alveolar macrophages labeled for MnSOD and TNF
, although the temporal pattern of labeling was different for both proteins. A significant increase in alveolar macrophage labeling for MnSOD was found after 5 weeks of oxidant exposure, which was maintained following 8 weeks of exposure (Fig. 4A
). There was a significant increase in labeling for TNF
in animals exposed for 1 week, which returned to control values after 5 weeks and then increased again at 8 weeks (Fig. 4B
). A slight increase in MnSOD labeling was found in the interstitial cells of animals exposed for 5 weeks and this increase became dramatic following 8 weeks (Fig. 5A
). Interstitial cells labeled for TNF
increased after 1 week and remained elevated for the duration of the exposure (Fig. 5B
). In the distal alveolar ducts 400600 µm from the terminal bronchiole, an increase in alveolar macrophage and interstitial cell labeling for MnSOD was seen in the 8 week exposed animals (Table 3
). Animals exposed for 5 weeks also had an increase in MnSOD labeling in interstitial cells (Table 3
). There were no significant changes in TNF
, compared with filtered air controls, in alveolar macrophages or in interstitial cells in the distal alveolar ducts at any time during exposure (data not shown).

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FIG. 3. (A) Changes in the number of MnSOD-labeled epithelial cells within the first 400 µm of the terminal bronchiole. All data are mean ± SEM. Cell counts from animals exposed to filtered air (solid bars) or oxidants (hatched bars) are normalized to the basement membrane length. A significant increase in labeling was seen in animals exposed for 8 weeks compared to controls. The asterisk denotes p < 0.05. (B) The number of epithelial cells labeled for TNF in the proximal alveolar ducts in animals exposed to filtered air (solid bars) or the combined oxidants (hatched bars). All cell counts are normalized to the basement membrane length. Although there is some variability in the count data, there is no significant difference between animals exposed to the combined oxidants and their age matched controls.
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FIG. 4. (A) Alveolar macrophages labeled for MnSOD in proximal alveolar ducts. Cell counts from animals exposed to filtered air (solid bars) or the combined oxidants (hatched bars) are normalized to the basement membrane length. All data are mean ± SEM. A significant increase in labeled cells was seen in animals exposed for 5 and 8 weeks compared to controls. The asterisk denotes p < 0.05. (B) Alveolar macrophages labeled for TNF in proximal alveolar ducts from animals exposed to filtered air (solid bars) or to ozone and nitrogen dioxide (hatched bars) normalized to the epithelial basement membrane length. A significant increase in labeled alveolar macrophages was seen in animals exposed for 1 week. This increase was reduced to levels equal to control values in animals exposed for 5 weeks and then significantly increased following 8 weeks of exposure, compared to controls. All data are mean ± SEM. An asterisk denotes p < 0.05.
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FIG. 5. (A) Changes in interstitial cell labeling for MnSOD in proximal alveolar ducts from animals exposed to filtered air (solid bars) or the combined oxidants (hatched bars) normalized to the epithelial basement membrane length. A significant increase in the number of labeled interstitial cells was noted in animals exposed for 5 and 8 weeks compared to controls. All data are means ± SEM. An asterisk denotes p < 0.05. (B) Alterations in the interstitial cell labeling for TNF in proximal alveolar ducts from animals exposed to filtered air (solid bars) or the oxidants (hatched bars) normalized to the epithelial basement membrane length. All exposure groups were significantly increased above control values, but were not significantly different from each other. All data are mean ± SEM. Asterisk denotes p < 0.05.
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TABLE 3 Time Course of Labeling for MnSOD in Alveolar Macrophages and Interstitial Cells in Distal Alveolar Ducts following Exposure to Ozone and Nitrogen Dioxide
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DISCUSSION
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The progressive injury in this model has been previously described in detail (Farman et al., 1997
). The tissue response consists of a triphasic pattern, with an initial inflammatory phase during weeks 13, a temporal resolution of the injury at weeks 45, then followed by rapidly progressing interstitial fibrosis at weeks 68. This study examined animals exposed to 0.8 ppm ozone and 14.4 ppm nitrogen dioxide for 1, 5, and 8 weeks to determine the changes in TNF and MnSOD at all stages of the model. Exposure to these combined oxidants caused a focal response in the lung that targeted the bronchiole-alveolar duct junction (BADJ) and proximal alveolar ducts 0400 µm from the terminal bronchiole (Table 2
), in agreement with previous studies (Farman et al., 1999
). Due to the focal nature of the lung response in this oxidant injury model, we concentrated our study of TNF
and MnSOD distribution within this same region. More distal areas of the alveolar ducts, 400600 µm from the terminal bronchiole, were also examined to confirm the focal nature of the response (Table 2
). The total number of alveolar macrophages was significantly increased compared with controls in the proximal alveolar region at all exposure times, but did not change with progressive exposure to these oxidants (Table 2
). In the more distal alveolar ducts, there was a significant increase, in alveolar macrophages only, following 8 weeks of exposure, which may be reflective of the increased damage found in the 8 week exposed animals (Table 2
). No significant change in total epithelial cell number per length was found at any time following exposure (Table 2
). The lack of a change in epithelial cell number is not an indication of an absence of response in this tissue compartment since metaplasia was found in animals exposed to O3 and NO2 beginning at 5 weeks, and progressed with continued exposure out to 8 weeks. These changes consisted of a transition in cell type from squamous alveolar epithelial type I cells to an undifferentiated cuboidal epithelium, most notably observed in the proximal alveolar ducts 0200 µm from the terminal bronchiole.
The progressive injury in our exposure model was most prominent in the proximal alveolar ducts 0400 µm from the terminal bronchiole and was accompanied by an increase in staining for MnSOD in alveolar macrophages, epithelial cells, and interstitial cells (Figs. 1C and 1E
). In addition to the expression of MnSOD in mast cells (Fig. 2C
), interstitial macrophages, and monocytes may also be significant sources of MnSOD during periods of oxidant stress to the lungs (Figs. 2E and 2F
). Epithelial cells increased in MnSOD labeling, which attained significance in animals exposed for 8 weeks (Fig. 3A
) and alveolar macrophages had a significant increase in labeling in the proximal alveolar ducts following 5 and 8 weeks of exposure (Fig. 4A
). In addition, the increase in alveolar macrophages following 8 weeks of exposure was also noted in more distal alveolar ducts (Table 3
). An increase was found in interstitial-cell labeling for MnSOD, in the proximal alveolar ducts following both 5 and 8 weeks of exposure (Fig. 5A
), and this increase was also noted in the more distal alveolar ducts 400600 µm from the terminal bronchiole (Table 3
), where less injury was seen. A previous study of this oxidant injury model has shown that the area of damage in alveolar ducts is similar after 7 or 90 days of exposure to both ozone and nitrogen dioxide (Farman et al., 1999
). This absence of further extension of injury down alveolar ducts may be associated with the protection offered by MnSOD. However, since MnSOD was also found to be elevated in the areas of greatest injury, the increase in MnSOD found in the distal alveolar ducts could also represent a marker of cellular response to oxidant gas exposure.
Labeling for TNF
was predominantly found in alveolar macrophages and interstitial cells (Figs. 1D and 1F
). Mast cells (Fig. 2D
) and possibly macrophages and monocytes (Figs. 2E and 2F
) are the most likely cells contributing to the increase in interstitial labeling. While labeling for MnSOD mainly increased with duration of oxidant exposure, the pattern of labeling for TNF
, in general, followed the triphasic cellular response to oxidant injury. This response was characterized by a significant increase in TNF
labeling in the proximal alveolar ducts in alveolar macrophages of animals exposed for 1 week (Fig. 4B
) during the inflammatory phase of injury. At 5 weeks of exposure, during the transitional phase characterized by decreased inflammation and partial resolution of cellular injury, TNF
labeling declined to control levels (Fig. 4B
). During rapid fibrotic proliferation after 8 weeks of exposure, TNF
labeling was significantly increased clearly above levels noted after 1 week of exposure (Fig. 4B
). However, interstitial cell labeling in proximal alveolar ducts, was significantly increased for TNF
at all times during exposure, and these increases were not significantly different from each other over the 8-week period (Fig. 5B
). The presence of increased levels of TNF
within the interstitium during all phases of the model is of interest and highlights the complexity of the role of TNF
in the development of fibrosis, with increased levels of TNF
preceding the development of interstitial fibrosis. Increased levels of TNF
associated with oxidant injury were found only at sites of greatest injury, readily apparent by histologic examination of the centriacinar regions of the lungs (Figs. 1D and 1F
).
TNF
is well documented as a critical cytokine in the development of fibrosis, although the role of TNF
in interstitial disease is still unclear. Increases in TNF
with interstitial lung disease were found to be dependent on the stage of the disease and were increased in patients with progressive disease (Ziegenhagen et al., 1998
). We found that increases in TNF
in our disease model were site-specific within focal fibrotic lesions, and closely followed the overall temporal pattern of cellular injury. MnSOD elevation was found in conjunction with reduced cellular damage only in the absence of increased levels of TNF
. Further study using this oxidant injury model needs to investigate the reduction in TNF
and augmentation of MnSOD as potential approaches that may lead to new therapeutics in the treatment of interstitial lung diseases.
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ACKNOWLEDGMENTS
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This investigation was supported by NIEHS ES00628, NIEHS ES05707, and RR00169. B.L.W. is the recipient of an NRSA award. The authors are grateful for the technical assistance of Brian Tarkington and Tim Duvall in the operation and characterization of exposure conditions. The authors also appreciate the dedicated assistance of Dr. Ya Mei Zhou, Diana Perez, and Daniel Chamberlain.
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NOTES
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1 To whom correspondence should be addressed. Fax: (530) 752-5300. E-mail: blweller{at}ucdavis.edu. 
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