* Center for Comparative Respiratory Biology and Medicine
Center for Health and the Environment, and
California Regional Primate Research Center, University of California, Davis, California 95616
Received October 11, 2001; accepted April 16, 2002
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ABSTRACT |
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Key Words: IL-6; ADSS; ozone; lung inflammation and injury; CCSP.
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INTRODUCTION |
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The inflammatory process in the lungs is characterized by the production of leukotrienes, histamine, bradykinin, and a variety of cytokines and chemokines by tissues and migrating cells. Among the proinflammatory cytokines, interleukin-6 (IL-6) is considered to contribute to the initiation and extension of the inflammatory process. IL-6 is a multifunctional cytokine that is produced by a wide range of cells (Baumann et al., 1984; Jirik et al., 1989
; Nijsten et al., 1987
), usually at sites of tissue inflammation. IL-6 acts on a wide range of cells in different systems, but the role of IL-6 in the pathophysiology of inflammation is still controversial. It induces fever, activates B and T lymphocytes, and stimulates hepatocytes to produce acute phase proteins. Recent studies have shown that IL-6 also has potent antiinflammatory and protective properties. These include the ability to inhibit the production of tumor necrosis factor (TNF), IL-1, and macrophage inflammatory protein-2; to decrease neutrophil sequestration; to increase levels of IL-1 receptor antagonist and TNF-soluble receptor; to stimulate the production of metalloproteinase inhibitors; to reduce intracellular superoxide production; to reduce tissue matrix degradation; and to inhibit cellular apoptosis (Jordan et al., 1995
; Mizuhara et al., 1994
; Rollwagen et al., 1998
; Shingu et al., 1994
, 1995
; Tilg et al., 1994
; Ulich et al., 1991
; Xing et al., 1998
). In a previous study, we demonstrated that the ability of alveolar macrophages to produce IL-6 in vitro was altered following exposure to ozone or to ADSS followed by ozone (ADSS/ozone; Yu et al., 2002
). This study implicated that a strong correlation may exist between IL-6 expression and pulmonary inflammation/injury.
An extensive review of the literature suggests that the role of IL-6 in lung inflammation and injury induced by exposure to air pollutants has not been extensively investigated. In the lung, a variety of cells such as alveolar macrophages, lymphocytes, alveolar epithelial cells, and endothelial cells are capable of producing IL-6 (Arcangeli et al., 2001; Bankey et al., 1995
; Frampton et al., 1999
; Koyama et al., 1998
; Mosmann and Sad, 1996
; Yu et al., 2002
). Therefore, to examine only alveolar macrophage-derived IL-6 is not sufficient to elucidate the role of IL-6 in the process of inflammation/injury in the lung following exposure to air pollutants. However, the use of IL-6 knockout (KO) mice makes it possible to investigate the role of this cytokine in various models of inflammation.
The purpose of this study was to characterize the role of IL-6 in pulmonary inflammation and injury induced by exposure to air pollutants. IL-6 KO mice and wild-type (WT) mice were exposed to filtered air, ADSS, ozone, or ADSS/ozone following identical protocols. Bronchoalveolar lavage (BAL) was performed to evaluate the inflammatory response in the lung. The magnitude of lung injury within the terminal bronchiolar epithelium and proximal alveolar regions of the lungs was examined using cell proliferation as identified by bromodeoxyuridine (BrdU) cell-labeling techniques. WT mice treated with recombinant IL-6 antibodies to block the activities of endogenous IL-6 were also used to confirm the role of endogenous IL-6 in epithelial damage and repair. Clara cell secretory protein (CCSP), an important secretory protein produced by Clara cells present in the distal conducting airways of the lungs, is considered to possess antiinflammatory properties (Camussi et al., 1990; Miele et al., 1988
). Therefore, immunohistochemical detection of CCSP was also performed to monitor the function of airway epithelial cells following pollutant exposure.
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MATERIALS AND METHODS |
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Exposure system.
The exposure system used to generate ADSS was derived from the system originally described by Teague et al(1994). Briefly, ADSS was formed by burning Kentucky 1R4F reference cigarettes in a smoking machine with standardized 35-ml puffs of 2-s duration, once every minute, for a total of eight puffs per cigarette. The mainstream cigarette smoke, which was determined to contribute 11% of total suspended particulate matter (TSP), was directed into the sidestream cigarette smoke, which contributed 89% of TSP (Witschi et al., 1998). The mixture of sidestream and mainstream cigarette smoke was introduced into a conditioning chamber to dilute and age the cigarette smoke. To reach the desired target concentration, the cigarette smoke was further diluted with filtered air before it was introduced into the exposure chamber. Exposure conditions were monitored for carbon monoxide (CO), nicotine, and TSP. TSP was determined by gravimetric measurement of samples collected from the chamber onto preweighed filters. CO was measured using a Model 880 nondispersive-infrared analyzer (Beckmann Industries, La Habra, CA). Nicotine concentrations were measured by drawing air samples through sorbent tubes, extracting the nicotine, and performing analysis by gas chromatography.
Cigarettes.
Reference cigarettes (Kentucky 1R4F) were purchased from the Tobacco Research Institute, University of Kentucky, Lexington, KY. The 1R4F is a filtered cigarette designed to deliver about 11 mg tar and 0.8 mg nicotine per cigarette (Davis et al., 1984). The cigarettes were stored at 4°C until needed. At least 48 h prior to use, cigarettes were placed in a closed chamber at 23°C along with a mixture of glycerin/water (mixed in a ratio of 0.76/0.24) to establish a relative humidity of 60%.
Ozone exposures.
Ozone exposure was performed at the exposure facility of the California Regional Primate Research Center. Mice were placed in sets of four in stainless steel wire cages with free access to food and water during the exposures. Cages were placed inside a 4.2 m3 glass and stainless steel chamber with an airflow of 30 changes/h. Ozone was produced from vaporized liquid medical-grade oxygen with a silent arc discharge ozonizer (Erwin Sander Corp., Giessen, Germany) located upstream of the exposure chamber. During the period of exposure, the chamber concentration was set at 0.5 ppm (ozone) or 0.0 ppm (filtered air) for 24 h. Ozone concentration was measured using a Dasibi 1003AH ozone analyzer (Dasibi Environmental Corp., Glendale, CA) that was calibrated against a Dasibi UV Photometer Model 1008 PC, which was calibrated to a National Bureau of Standards reference photometer located at the California Air Resources Board Quality Assurance Standards Laboratory, Sacramento, CA. Each chamber was monitored for ozone concentration for a minimum of 15 min/h. Nominal exposure concentrations were within 5% of target values.
Experimental protocol.
WT and IL-6 KO mice were randomly divided into four groups each for exposure to filtered air, ADSS, ozone, or ADSS/ozone. In each treatment group of WT mice, five mice were used for bronchoalveolar lavage (BAL) and five mice were used for immunohistochemical examination of the pulmonary proliferative response using BrdU labeling. For IL-6 KO mice, four mice were used in each treatment group for pulmonary lavage and five mice were used to examine the pulmonary proliferative reaction following each exposure regimen. Exposure to ADSS was 6 h/day (9:00 A.M. to 3:00 P.M.) for three consecutive days, at 10 mg/m3 of TSP. Exposure to ozone was for 24 h at 0.5 ppm starting at 9 oclock the morning following ADSS exposure. Pulmonary lavage was performed immediately after ozone exposure, followed by examination of BAL cellular changes and total protein levels in response to each exposure. To examine proliferative response in the lungs, BrdU pulse labeling was performed by an intraperitoneal injection of BrdU solution 2 h prior to the sacrifice of animals. The mice were euthanized 24 h after ozone exposure, followed by intratracheal instillation of 4% paraformaldehyde to fix the lungs.
IL-6 antibody treatment.
WT mice were randomly divided into four groups that were exposed to filtered air, ADSS, ozone, and ADSS/ozone, respectively. Five mice from each group received IL-6 antibody treatment, while the remaining mice were sham treated with sterile saline as controls. Intraperitoneal injection of rat antimouse IL-6 antibody (PharMingen, San Diego, CA) was done daily immediately following ADSS exposure for a total of three times, at a dose of 2.5 µg IL-6 antibody/0.2 ml normal saline/mouse/day.
BAL, total protein, and cell preparation.
Within 2 h after exposure to ozone, mice were deeply anesthetized with an overdose of sodium pentobarbital. The left lung was ligated and removed, while the right lung lobes were lavaged two times sequentially with identical aliquot volumes (35 ml/kg body weight) of Hanks balanced salt solution (pH 7.27.4). Recovered bronchoalveolar lavage fluid (BALF) was immediately cooled at 4°C. Recovered aliquots of BALF were pooled together. BAL cells were pelleted by centrifugation (500 x g at 4°C) for 10 min. The supernatant was used to measure total protein concentration as an indicator of lung permeability. A bovine serum albumin protein assay kit (Pierce, Rockford, IL) was used to measure BAL protein. The cell pellet was resuspended in 1 ml phosphate-buffered saline (PBS). A volume of 100 µl of the cell suspension was mixed with 100 µl 4% trypan blue (Gibco, Grand Island, NY) to determine total cell number and viability. To determine the cell differential in BALF, 5 x 104 cells (in 100 µl PBS) were mounted on a slide by cytospin centrifugation (Shandon Southern Instruments, Pittsburgh, PA) and stained with Diff-Quik (Baxter Healthcare, Miami, FL). Cell differential was determined by counting at least 400 BAL cells per animal, according to standard cytological techniques.
Tissue preparation.
One day following exposure to ozone and 2 h before the sacrifice of animals, BrdU pulse labeling was done by a single intraperitoneal injection of BrdU solution (in phosphate balanced solution, 50 mg/kg body weight). Mice were sacrificed by intraperitoneal injection of an overdose of sodium pentobarbital. The lungs (five mice per group) were inflated through cannulating the trachea and instilling 4% paraformaldehyde solution in PBS (pH 7.4) under a constant pressure of 30 cm water. After 1 h, the lungs were removed from the thoracic cavity and immersed in 4% paraformaldehyde for 1 h. A piece of gut was removed, perfused with 4% paraformaldehyde, and immersed in fixative for 2 h. Tissues were subsequently embedded in paraffin, and 5-µm sections were cut and mounted on superfrost plus glass slides (Fisher Scientific, Pittsburgh, PA).
BrdU immunohistochemical staining of lung sections.
Sections were deparaffinized for 15 min in xylene, dehydrated in 100, 95, and 70% ethanol for 5 min, respectively, and washed with PBS for 5 min. Endogenous peroxidase activity was blocked with 0.5% H2O2 in PBS for 30 min. For BrdU staining, slides were treated for 20 min with 0.1% trypsin (Zymed, San Francisco, CA) at 37°C and washed under running tap water. After treatment in 4 M HCl for 20 min, sections were neutralized for 5 min in sodium borate (0.1 M, pH 8.5) and washed in PBS for 10 min. After applying the blocking solution (5% horse serum in PBS) for 15 min, sections were incubated with a mouse anti-BrdU antibody (1/100, Roche, Minneapolis, MN) for 90 min at 37°C. After rinsing in PBS for 10 min, the sections were incubated with biotinylated horse antimouse antibody (1/200, Vector Inc., Burlingame, CA) for 60 min, avidin-biotinylated horseradish peroxidase complex (1/200, Vector Inc., Burlingame, CA) for 30 min, and diaminobenzidine (DAB) substrate for 3 min, then counterstained with nuclear fast red.
Evaluation of BrdU cell labeling.
All slides were coded and counted without knowledge of the exposure regimen for each group. Two anatomical sites, terminal bronchiolar epithelium and the adjoining proximal alveolar region, were chosen to examine the nuclear incorporation of BrdU. Terminal bronchioles were identified as airways opening directly into alveolar regions. The proximal alveolar region was defined as that area immediately beyond the terminal bronchiole consisting of all alveoli within an average of 8 alveolar diameters from the terminal bronchiole. All sites were by random selection to determine the labeling index in each anatomical site. The epithelial nuclear labeling index was expressed as a percentage of BrdU-positive nuclei versus total nuclei of the epithelial cells in terminal bronchioles. In contrast, BrdU labeling in proximal alveolar regions included many cell types (i.e., epithelial, interstitial, and endothelial cells). Appropriate systemic distribution of the BrdU labeling was confirmed by intense BrdU staining in the gut of all animals, which also served as the positive control of BrdU labeling. No staining was observed when the primary antibody was omitted. Counting of cells was done at magnification x400 for a minimum of 600 cells/anatomical category/animal.
Immunohistochemistry for CCSP.
The presence of CCSP protein was detected using rabbit antiserum specific for rat CCSP, which was a generous gift from Dr. Gurmukh Singh (Veterans Affairs Medical Center, Pittsburgh, PA). Sections were deparaffinized for 15 min in xylene, dehydrated in 100, 95, and 70% ethanol for 5 min, respectively, then washed with PBS for 5 min. Endogenous peroxidase activity was blocked with 0.5% H2O2 in PBS for 30 min. To eliminate nonspecific binding of the primary antibody, sections were blocked with 1% normal goat serum for 30 min. All sections were subsequently incubated with rabbit polyclonal antirat CCSP antiserum (1:1000) overnight at 4°C. Slides were then rinsed and incubated sequentially at room temperature in biotin-conjugated goat antirabbit IgG (1:50, Vector Labs, Burlingame, CA) for 30 min and ABC complex (Vector Labs) for 30 min. Bound peroxidase activity was visualized by incubation with 0.02% DAB and 0.01% H2O2 in PBS. Control of the specificity of immunolabeling included the omission of the primary antibody or the substitution of the primary antibody with nonimmune serum. In each case, these assays confirmed the specificity of CCSP immunolabeling in tissue sections.
Statistical analysis.
Data were expressed as mean ± SD. The effects of exposure (ADSS, ozone, and ADSS/ozone) on inflammatory (recovered cells and total protein in BALF) and epithelial injury (epithelial proliferation in terminal bronchioles and the proximal alveolar regions) were assessed using analysis of variance (ANOVA; StatView 4.5, Abacus Concepts, Inc., Berkeley, CA). Differences among experimental groups were further examined using Tukey-Kramer multiple comparison tests. Comparison of data between WT and KO littermates following the identical exposure protocol was done using unpaired Student t-test. A p value of less than 0.05 was considered significant.
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RESULTS |
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BAL-Recovered Leukocytes and Cell Viability
The right lung lobes were used for BAL. In IL-6 KO mice exposed to filtered air, the number of total cells recovered by BAL was significantly higher than that of their WT littermates exposed to filtered air. A significant decrease in cell viability was also observed in IL-6 null mice compared with their WT littermates (Table 1). Only a significant increase in total cell number in BALF was observed in WT mice exposed to ADSS. A significant decrease in viability of BALF cells was found in WT mice exposed to ADSS, ozone, and ADSS/ozone, compared with control mice exposed to filtered air. In IL-6 KO mice, there was no significant difference in the viability of BALF cells observed between control mice and mice exposed to ADSS, ozone, or ADSS/ozone (Table 1
).
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CCSP Protein Expression
In the lungs of WT mice exposed to filtered air, CCSP-immunoreactive epithelial cells intensely stained, represented the predominant epithelial cell type, and were evenly distributed throughout the bronchiolar epithelium. Following exposure to ADSS, ozone, or ADSS/ozone, the bronchiolar epithelium was notably lacking the normal CCSP-immunoreactive distribution of cells in WT mice. In contrast, in the lungs of IL-6 KO mice following exposure to ADSS, ozone, or ADSS/ozone, no changes were noted in the pattern or distribution of CCSP-immunoreactive cells throughout the bronchiolar epithelium (Figs. 5 and 6).
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DISCUSSION |
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Examination of BALF is an excellent method to evaluate the inflammatory response in the lung. An increase in the proportion of monocytes in BALF cells actively contributes to the process of inflammation and lung injury by producing proinflammatory cytokines, reactive oxygen species (ROS), and proteolytic enzymes (Jones et al., 1992; Rosseau et al., 2000
), as well as expanding the alveolar macrophage pool (Hance et al., 1985
; Hoogsteden et al., 1989
; Kiemle-Kallee et al., 1991
; Wassermann et al., 1994
). Besides promoting the differentiation of monocytes toward macrophages (Mitani et al., 2000
), IL-6 can activate peripheral blood mononuclear cells to secrete monocyte chemoattractant protein (MCP)-1, a C-C chemokine involved in mediating lung injury by activating monocytes, increasing production of ROS, and regulating cell adhesion properties (Sozzani et al., 1993
; Vaddi and Newton, 1994
; Warren et al., 1993
). Increased IL-6 levels have also been found to coincide with enhanced production of MCP-1 (Bachert et al., 2001
; Kinter et al., 2001
; Leonard and Yoshimura, 1990
; Yaszay et al., 2001
). Therefore, a slight but significant increase in the proportion of monocytes recovered in BALF in WT mice exposed to ozone alone, with an even higher monocyte percentage following sequential exposure to ADSS and ozone in WT mice, correlates with an enhanced degree of lung inflammation and injury. This pattern of injury was confirmed by a significant elevation in BrdU cell labeling in WT mice. The decrease in the proportion of monocytes in BALF observed in IL-6 KO mice exposed to ozone or to ADSS/ozone was associated with a slight but significant attenuation of the inflammatory response and less subsequent injury, as observed by a reduced BrdU labeling index in the lungs of IL-6 KO mice.
Increased levels of total protein, as well as greater numbers of neutrophils in BALF, are classical features noted in the lungs of mice immediately following exposure to ozone (Bhalla, 1999). In the present study, these same findings were observed in both strains of mice following exposure to ozone alone or ADSS/ozone. The effect of prior exposure to ADSS on the proportion of neutrophils in BALF leukocytes observed in B6C3F1 mice in our previous study (Yu et al., 2002
) was not found in WT C57BL/6J mice in the present study, following a similar timing protocol for pulmonary lavage. We speculate this may be due to strain differences and/or a decrease in the concentration of ADSS (10 mg/m3 vs. 30mg/m3). Bhalla (1999) observed the influx of neutrophils into the lung continues to increase over a period of time following ozone exposure. It is possible that the timing used for BAL analysis in the present study may have been prior to the peak of neutrophil influx into the lungs, which may have obscured the effect of IL-6 deficiency for neutrophil recruitment into the lung air spaces in this study.
A significant increase in protein levels found in BALF reflects epithelial barrier disruption and tissue injury, resulting in increased transmucosal permeability (Bhalla, 1999; Kleeberger et al., 2001
). Tissue barrier disruption after an acute injury is transient in nature, with the barrier function of tissues returning to normal over time (Bhalla, 1999
). BAL was performed within 2 h after the ozone exposure in this study. Therefore, the protein level measured in BALF at this time may reflect the disruption of the epithelial barrier and direct damage of airway and alveolar epithelial cells caused by ozone or ADSS/ozone. IL-6 deficiency would not block this type of direct oxidative damage to cells or to the epithelial barrier. Prior exposure to ADSS may have also contributed to greater epithelial and tissue barrier damage in the lungs of these mice.
Epithelial injury may also be a consequence of damage by reactive oxygen metabolites and proteolytic enzymes released from the influx of inflammatory cells. IL-6 deficiency in mice could alter production of proinflammatory cytokines, chemokines, and other mediators, thus contributing to the attenuation of lung inflammation and cell injury. Tyburski and coworkers (2001) found a net gradient increase in IL-6 levels in blood drawn from the lung (samples taken from arterial and venous vessels, respectively) in patients with lung disorders. This finding implies that the lung is a major producer of IL-6 in patients with active lung inflammation. There is evidence that TNF- and IL-1 also help to propagate the extension of a local or systemic inflammatory process. In a murine model of pulmonary inflammation, a number of investigators (Alonzi et al., 1998
; Cuzzocrea et al., 1999
; Utsunomiya et al., 1991
) reported the production of proinflammatory cytokines TNF-
and IL-1 to be significantly decreased in IL-6 null mice.
Exposure to oxidant gases could increase the level of ROS in the lung, contributing to tissue damage (Cuzzocrea et al., 1999; Oh-Ishi et al., 1989
). ROS could further trigger mechanisms of DNA repair and activate the nuclear enzyme poly (adenosine diphosphate-ribose) synthetase (PARS), resulting in activation of the PARS suicide pathway, which plays a critical role in inflammation (Szabo et al., 1997
, 1998
; Cuzzocrea et al., 1998a
, 1998b
).
Recently, significantly attenuated activation of PARS during the process of lung inflammation was found in IL-6 null mice as well as WT mice pretreated with antibodies against IL-6 (Cuzzocrea et al., 1999). These results provide a mechanistic basis for the findings of our study to confirm that ozone-induced lung injury would be attenuated in IL-6 KO mice as well as in WT mice treated with IL-6 antibodies.
Incorporation of BrdU into DNA during cell replication remains a sensitive and simple technique to identify proliferating cells (Doolittle et al., 1992). Cell proliferation within the centriacinar regions of the lung is a typical pattern found in ozone-injured lungs (Pinkerton et al., 1992
; Rajini et al., 1993
). In a recent study, we demonstrated that prior exposure to ADSS significantly increased ozone-induced lung injury, identified by a significant increase in BrdU labeling of cells within the pulmonary centriacinar region in B6C3F1 mice (Yu et al., 2002
). In the present study, the proliferative response was measured separately in terminal bronchiolar epithelium and the proximal alveolar regions of the lungs. Together, these regions comprise the centriacinar region of the lung. These anatomical regions represent two of the most critical sites in the lungs affected by exposure to ozone. Therefore, examination of both anatomical regions could better identify the relative sensitivity of cells to injury following exposure to ADSS and/or ozone. We found cells in the proximal alveolar region were more sensitive than cells in the terminal bronchioles of WT C57BL/6J mice following exposure to ADSS/ozone, based on the BrdU labeling index for each region. Therefore, cell proliferation in the proximal alveolar region is the primary anatomical site that is most sensitive to ADSS-potentiated responses in ozone-injured lungs.
In the present study, the magnitude of increase in BrdU labeling in KO mice following exposure to ozone or to ADSS/ozone was significantly lower than that observed in their respective WT littermates. In addition, we found ADSS preexposure enhanced cell proliferation in the proximal alveolar region following subsequent exposure to ozone in WT mice, but not in IL-6 KO mice. This observation provides strong support that IL-6 could be involved in the potentiation of ADSS to further augment ozone injury in the lungs. Therefore, we propose IL-6 deficiency is associated with attenuated lung injury. Further evidence in support of this hypothesis was found by treating WT mice with an antibody to block the activities of endogenous IL-6, resulting in a similar effect observed in IL-6 KO mice for both the terminal bronchiole and proximal alveolar regions of the lungs.
The speculation may be raised that reduced cell proliferation could reflect an impairment of epithelial repair following injury. IL-6 could be postulated to be involved in regulating cell proliferation and differentiation in the process of tissue repair. A number of recent studies suggest that IL-6 deficiency may cause delayed wound healing (Gallucci et al., 2000, 2001
; Sugawara et al., 2001
; Swartz et al., 2001
), or be responsible for the lack of inflammation seen during fetal wound healing (Liechty et al., 2000
). In our study, if reduced cell proliferation in the terminal bronchiolar epithelium or proximal alveolar regions was due to an impaired tissue repair process, we would speculate that a damaged epithelial barrier would have been associated with an even higher total protein level in the BALF of IL-6 KO mice exposed to ozone or ADSS/ozone compared with their WT littermates. Necrotic epithelial cells within lavage fluid or the presence of denuded basal lamina would also serve as strong indicators of the absence of epithelial repair (King et al., 1999
). However, none of these features were observed to support a lack of epithelial repair in IL-6 KO mice following exposure to ozone or to ADSS/ozone.
The detection of CCSP expression in bronchiolar epithelium following exposure to environmental air pollutants provides additional insights for the potential role of IL-6 in lung inflammation and injury/repair. CCSP can inhibit the activity of phospholipase A2, an enzyme involved in the production of arachidonic acid and its metabolites linked to the inflammatory process (Lesur et al., 1995; Levin et al., 1986
). CCSP can also inhibit the chemotaxis and phagocytosis of neutrophils and monocytes, respectively (Mukherjee et al., 1988
, 1999
). Therefore, loss of CCSP-immunoreactive cells in proximal and distal airways may enhance the inflammatory response and epithelial damage. Infiltration of monocytes and neutrophils in the lung parenchyma was more extensive in CCSP-deficient mice following adenoviral infection and was accompanied by upregulation of IL-6 and TNF-
(Harrod et al., 1998
). In a hyperoxic lung injury model, survival of CCSP-deficient mice was reduced compared with control mice. Furthermore, expression of proinflammatory cytokines IL-6 and IL-1 was increased in the lungs of CCSP-deficient mice (Johnston et al., 1997
). In our study, we found exposure of WT mice to ozone or ADSS/ozone decreased the expression of CCSP in bronchiolar epithelial cells. In contrast, sustained CCSP expression was found in IL-6 KO mice following identical exposures. These observations suggest that reduction in CCSP protein expression is associated with epithelial cell damage, whereas IL-6 deficiency is associated with an increased resistance to lung injury following exposure to ozone or ADSS/ozone. Exposure of WT mice to ADSS reduced CCSP protein expression, which may be associated with a loss in cellular protection to subsequent environmental insults. However, a decrease in CCSP expression following exposure to ADSS alone was not associated with a significant increase in epithelial cell damage, as indicated by BrdU labeling. In contrast, exposure to ADSS/ozone in the presence of impaired CCSP expression may be key to augmenting ozone-induced epithelial damage. Therefore, we propose that the sustained CCSP expression in IL-6 KO mice to maintain the protective function of this secretory protein may aid in attenuating epithelial damage observed in IL-6 null mice exposed to ozone or to ADSS/ozone.
In summary, this study has demonstrated that endogenous IL-6 plays a critical role in the inflammatory response and injury in the lungs of mice following exposure to environmental air pollutants. IL-6 also appears to facilitate ADSS-enhanced lung injury to ozone. IL-6 is further involved in the mediation of CCSP expression in airway epithelial cells. Using antibodies to block endogenous IL-6 activity attenuates lung injury, suggesting that IL-6 antibody treatment may be an effective means to reduce pulmonary inflammation and lung injury.
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ACKNOWLEDGMENTS |
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NOTES |
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