Ozone Exposure Enhances Expression of Cell-Surface Molecules Associated with Antigen-Presenting Activity on Bronchoalveolar Lavage Cells in Rats

Eiko Koike*, Takahiro Kobayashi{dagger},1 and Nobuhiro Shimojo{ddagger}

* Department of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan; {dagger} Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba 305-8506, Japan; and {ddagger} Institute of Community Medicine, University of Tsukuba, Tsukuba 305-8575, Japan

Received January 26, 2001; accepted June 11, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we investigated the effects of ozone (O3) exposure on expression of cell-surface molecules associated with antigen presentation and on accessory activity of bronchoalveolar lavage cells (BAL cells). Rats were exposed to 1 ppm O3 for 3 days. Expression of cell-surface molecules was measured by flow cytometry. Accessory activity of BAL cells was assessed by the allogeneic mixed lymphocyte reaction (MLR) and specific antigen-presenting activity. O3 exposure increased the expression of Ia, B7.1, B7.2, and CD11b/c on BAL cells. Morphological and immunological studies showed the Ia-positive cells to have monocyte-like features. Peripheral blood monocytes expressed Ia, B7.1, B7.2, and CD11b/c. The Ia expression on the monocytes was further increased by treatment of them with BAL fluid from O3-exposed rats (O3-BALF). Resident alveolar macrophages, however, did not express Ia antigen, and the Ia expression was not increased by O3-BALF. Neutrophils, which also infiltrated in response to O3 exposure, did not express Ia, B7.1, and B7.2. Therefore, infiltrating monocytes may have caused the increased expression of Ia and B7 molecules on BAL cells exposed to O3. The accessory activity of BAL cells in terms of MLR and specific antigen-presenting activity was also enhanced by O3 exposure. The present study suggests that monocytes infiltrating in response to O3 exposure caused enhancements of the expression of Ia and costimulatory molecules on and the accessory activity of BAL cells.

Key Words: ozone; bronchoalveolar lavage cells; accessory activity; Ia antigen; B7 molecules; CD11b/c; monocytes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, allergic asthma and rhinitis appear to be increasing in many countries, and previous reports have shown that these allergic disorders are associated with air pollutants (Koren, 1995Go; Leikauf et al., 1995Go). Epidemiological studies have also shown a relationship between O3 and the prevalence of asthma (Balmes, 1993Go). Accordingly, O3 may induce an attack of airway allergic disorders or aggravation of symptoms. Airway hyperresponsiveness and enhancement of IgE production are features of airway allergic disorders. The mechanism of airway hyperresponsiveness induced by O3 exposure has been analyzed from various points of view (Kobayashi, 1996Go). Exposure to O3 enhances the activity of IgE-containing cells in the lungs (Gershwin et al., 1981Go). There are many factors that contribute to IgE production, such as antigen-presenting activity, differentiation and proliferation of type II helper T cells and B cells, cytokines, and chemokines. It is thus necessary to examine the effect of O3 exposure on these factors in order to clarify the mechanisms of enhancement of IgE production.

Antigen-laden bronchoalveolar lavage (BAL) cells may present the antigen to helper T cells in the lungs and lymph nodes after migration. Therefore, an increase in the antigen-presenting activity of BAL cells would play an important role in the recruitment of eosinophils and/or enhancement of IgE production. To our knowledge, however, there are no reports that show the effect of O3 on the antigen-presenting activity of BAL cells. It has been reported that monoclonal antibodies against MHC class II (Ia) antigen can suppress the T-cell proliferation elicited by mixed lymphocyte reaction (MLR; Gotze et al., 1975) and specific antigen presentation (Niederhuber and Shreffler, 1977Go). Therefore, the expression of Ia is essential to antigen presentation. Resident alveolar macrophages (AM) function poorly as antigen-presenting cells. It was reported, however, that Ia expression in BAL cells was increased by exposure to silica, asbestos, and tobacco smoke (Hartmann et al., 1984Go; Higashimoto et al., 1994Go; Struhar et al., 1989Go). Monoclonal antibodies against costimulatory molecules such as B7.1, B7.2, CD11b, or CD11c could also suppress the T-cell proliferation caused by MLR (Landry et al., 1990Go; Lanier et al., 1995Go; Van Gool et al., 1994Go; Xu et al., 1992Go) or by specific antigen presentation (Freeman et al., 1993Go; Lenschow et al., 1993Go; Meunier et al., 1994Go). These data suggest that Ia antigen and costimulatory molecules are necessary for MLR as they are for antigen presentation. Therefore, the accessory function in the MLR can equally estimate that in antigen presentation. Exposure to O3 increases the markers of allergic lung disease such as IgE-containing cells (Gershwin et al., 1981Go) or eosinophils (Bassett et al., 2000Go) in the lungs. O3-induced stimuli in the lungs may also increase the expression of cell-surface molecules associated with antigen-presenting activity in BAL cells. Then the BAL cells expressing these cell-surface molecules may transit to the lymph nodes and present the antigen to T cells. As there are no reports that show the effect of O3 on the expression of Ia and costimulatory molecules on BAL cells, it is thus important to elucidate whether O3 affects the expression of these cell-surface molecules on the BAL cells.

Alveolar macrophages are accounted for primarily by the recruitment of circulating blood monocytes (Struhar et al., 1990Go; Tryka et al., 1984Go). The number of BAL cells increases in response to a variety of inflammatory stimuli, and these infiltrating cells express Ia antigen to a greater degree than resident AM (Blusse et al., 1983Go; Bowden and Adamson, 1980Go). Therefore, O3 exposure may increase the expression of cell-surface molecules associated with antigen-presenting activity on BAL cells by affecting the infiltrating monocytes.

In this study, we investigated (1) the effect of O3 exposure on the expression on BAL cells of cell-surface molecules associated with antigen presentation (Ia antigen, B7.1, B7.2, and CD11b/c), (2) whether infiltration of monocytes may reflect the O3-induced changes in the expression of BAL cell-surface molecules, and (3) the effect of O3 exposure on the accessory activity of BAL cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Specific pathogen-free, male Wistar rats (4-weeks old) and Fisher rats (4-weeks old) were obtained from Japan Charles River Inc. (Yokohama, Japan) and were used at 8–10 weeks of age (weighing 310–380 g and 200–250 g, respectively). The animals were given sterile distilled water and CE-2 food (CLEA JAPAN Inc., Tokyo, Japan). Wistar rats were used in all experiments except for the preparation of allogeneic lymphocytes in the MLR.

Exposure to O3.
Wistar rats were placed in stainless-steel cages and exposed to 1 ± 0.1 ppm O3 or filtered air for 3 days (24 h/day) in 2 identical chambers (volume 1.16 m3) made of stainless-steel and glass. Food and water was provided ad libitum to the animals during the exposure period. The conditions in the chambers were as follows: temperature, 25 ± 1°C; humidity, 55 ± 10%; pressure, –5 mm H2O relative to atmospheric pressure; total air flow rate, 90–100 m3/h. O3 was generated by a silent discharge apparatus (ML9811, 22245 Network Place, Chicago, IL). The concentration of O3 was continuously monitored with a chemiluminescence-based O3 analyzer (Model 8410, Monitor Lab., Inc.). Rats were exposed to O3 or filtered air for 3 days in all experiments except the time-course study.

Preparation of BALF and BAL cells.
Air- or O3-exposed Wistar rats were anesthetized with sodium pentobarbital (Dainippon Pharmaceutical Co., Osaka, Japan) given intraperitoneally (50 mg/kg), and exsanguinated from the abdominal aorta. An incision was made between the cartilaginous rings of the trachea, and a cannula was inserted into the trachea and secured with a suture. Lungs and trachea were lavaged with 5 ml/300 g body wt of 37°C RPMI 1640 medium (Dainippon Pharmaceutical Co., Osaka, Japan). The lavage was done with the same RPMI 1640 medium repeatedly by slowly instilling and withdrawing the instillation 10 times. The lavage fluids were recovered about 80 and 60% from air- and O3-exposed rats, respectively. The recovered BALF was centrifuged at 400 x g for 10 min at 4°C, and the cells were removed. The acellular lavage fluid was then sterilized by filtration through a MILLEX-HA (0.45 µm: Millipore, Bedford, MA). Ten percent heat-inactivated fetal bovine serum (FBS; Dainippon Pharmaceutical Co., Osaka, Japan), 100 µg/ml penicillin, and 100 U/ml streptomycin (Sigma, St. Louis, MO) were added to each filtrate of the BALF from Wistar rats exposed to either air or O3 for 3 days, and these final solutions were designated as Air- or O3-BALF. There was no difference in the pH of the BALF between the O3- and air-exposed rats. For preparation of BAL cells, lungs and trachea were lavaged twice with 10 ml of 37°C Dulbecco's calcium and magnesium-free, phosphate-buffered saline (PBS [-]; Dainippon Pharmaceutical Co., Osaka, Japan). BAL cells were collected by centrifugation at 400 x g for 10 min at 4°C and resuspended in R10, which is RPMI 1640 medium containing 10% heat-inactivated FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin. The numbers of viable cells were determined by the trypan blue (Gibco Laboratories, Grand Island, NY) exclusion method.

Preparation of lymph node cells (LNC).
LNC for use in the allogenic MLR experiments were derived from a pool of superficial, facial, and posterior mediastinal lymph nodes from normal Fisher rats (2 rats). The lymph nodes were pushed through a sterile stainless wire mesh (200 mesh), and the resulting cells were suspended in 37°C PBS [-]. Cells were collected by centrifugation at 400 x g for 10 min at 20°C and resuspended in R10. The numbers of viable cells were determined by the trypan blue exclusion method.

Preparation of ovalbumin (OVA)-sensitized T cells.
OVA-sensitized T cells for use in the experiments on the antigen-presenting activity of BAL cells were derived from a pool of superficial, facial, and posterior mediastinal lymph nodes from OVA-sensitized Wistar rats (2 rats). Rats were immunized with 100 µg OVA (Seikagaku, Co., Tokyo, Japan) and 3 mg Al(OH)3 in 500 µl of saline. Lymph nodes were harvested on day 14, and LNC were prepared as described above. LNC (1 x 108) in 1 ml of R10 were loaded onto a nylon fiber column (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and the column was incubated for 45 min at 37°C in a 5% CO2/95% air atmosphere. After incubation, the column was washed with R10, and the fraction of nonadherent cells (T cells) was collected. These T cells were collected by centrifugation at 400 x g for 10 min at 20°C and resuspended in R10. The numbers of viable cells were determined by the trypan blue exclusion method.

Preparation of monocytes.
Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood from normal Wistar rats by Ficoll Paque (Amersham Pharmacia Biotech, Sweden) gradient centrifugation, and monocytes were purified from PBMC by adherence to plastic dishes. Monocytes were derived from a pool of 3 animals. Heparinized blood was diluted 1:2 in PBS [-], layered over Ficoll-Paque, and centrifuged at 400 x g for 20 min at 20°C. The PBMC at the interface were recovered and washed 3 times with PBS [-]. Then, the PBMC were resuspended in R10 and incubated in plastic culture dishes for 1 h at 37°C in a 5% CO2/95% air atmosphere. Nonadherent cells were removed by gently flushing the dishes 3 times with R10. Adherent cells were incubated with 0.5% EDTA in PBS [-] containing 5% FBS for 30 min at 4°C. After incubation, the liberated monocytes were collected and resuspended in R10. The numbers of viable cells were determined by the trypan blue exclusion method.

Preparation of neutrophils.
Neutrophils that had infiltrated the BAL cell population were isolated from O3-exposed rats by Percoll (Sigma, St. Louis, MO) discontinuous gradient centrifugation. Neutrophils were derived from a pool of 3 animals. Percoll was diluted from 100% stock with PBS [-] and 10% FBS to strengths of 30, 40, 50, 60, 70, and 80%. Five-step discontinuous gradients (40–80%) were prepared. BAL cells were suspended in 30% Percoll, layered onto 40% Percoll layer, and then centrifuged at 400 x g for 20 min at 20°C. Neutrophils (> 80% pure) were recovered from the 70–80% Percoll fraction, washed 3 times with PBS [-], and resuspended in R10.

Immunohistochemical staining of BAL cells.
BAL cells were centrifuged onto glass slides for 5 min at 500 rpm. The slides were allowed to air dry. Endogenous peroxidase activity was blocked by incubation for 30 min with methanol containing 0.3% H2O2. The cells were then treated with monoclonal mouse anti-rat Ia IgG1 (Harlan Sera-Lab, Loughborough, UK), biotinylated anti-mouse IgG, and Streptavidin-horseradish peroxidase conjugate (Amersham, UK) for 30 min for each reagent. Thereafter, 3,3'-diaminobenzidine tetrahydrochloride (2.8 mM) and 0.05% H2O2 in PBS [-] were added as a substrate of the peroxidase for staining. Differential cell counts were determined by use of Diff-Quik (International Reagents, Kobe, Japan) stain.

FACS analysis.
For FACS analysis, the following monoclonal antibodies were used: FITC-labeled Ox6 (anti-rat Ia), PE-labeled 3H5 (anti-rat B7.1), PE-labeled 24F (anti-rat B7.2) and PE-labeled Ox42 (anti-rat CD11b/c; Pharmingen, San Diego, CA). Cells (1 x 106) were resuspended in 100 µl PBS [-] with 0.3% bovine serum albumin (BSA; Sigma, St. Louis, MO) and 0.05% sodium azide (Wako Pure Chemical Industries, Osaka, Japan) and incubated with 1 µg FITC-Ox6/PE-3H5, FITC-Ox6/PE-24F or FITC-Ox6/PE-Ox42 for 30 min on ice. After incubation, the cells were washed, and the fluorescence was measured by a FACScan flow cytometer (Becton Dickinson, Tokyo, Japan). Fluorescence data were expressed as the percentage of positive cells.

Culture of AM and monocytes with BALF.
AM and peripheral blood monocytes were prepared from normal Wistar rats. Cells of each type (5 x 105) were cultured with R10 or BALF in 96-well flat-bottom plates (Nunc, Denmark). Treatment with BALF was performed in 200 µl of R10, Air-BALF, or O3-BALF in triplicate for 2 days at 37°C in a 5% CO2/95% air atmosphere. After incubation, Ia expression on AM or monocytes was measured by FACScan flow cytometry. Fluorescence data were expressed as the percentage of Ia positive cells.

MLR.
LNC as responder cells were prepared from Fisher rats and BAL cells as stimulator cells were prepared from air- or O3-exposed Wistar rats. BAL cells were treated with 50 µg/ml mitomycin C (Kyowa, Tokyo, Japan) for 30 min in water bath at 37°C. Then the cells were washed 3 times with R10 and resuspended in R10. LNC (4 x 105) were cocultured or not with BAL cells (3 x 103 – 2 x 105) in 96-well flat-bottom plates. The MLR was performed in 200 µl of R10 in triplicate for 4 days at 37°C in a 5% CO2/95% air atmosphere. LNC proliferation was measured after a 4-day culture period.

OVA-specific AP activity of BAL cells.
BAL cells were treated with mitomycin C as described above. OVA-sensitized T cells (4 x 105) were cocultured or not with BAL cells (1.2 x 104 – 4.8 x 104) in the presence of OVA (40 µg) in 96-well flat-bottom plates. These cell cultures were performed in 200 µl of R10 in triplicate for 4 days at 37°C in a 5% CO2/95% air atmosphere. T-cell proliferation was measured after a 4-day culture period.

Measurement of cell proliferation.
Cell proliferation was measured with a Cell-Proliferation-ELISA Kit (Boehringer Mannheim, Mannheim, Germany), as described previously (Koike et al., 1998Go, 1999Go). This technique is based on the incorporation of the pyrimidine analogue 5-bromo-2'-deoxyuridine (BrdU) instead of thymidine into the DNA of proliferating cells. BrdU incorporated into DNA is measured by a sandwich-type enzyme immunoassay using monoclonal anti-BrdU antibody. This technique is at least as sensitive as the traditional counting of [3H]-thymidine (Porstmann et al., 1985Go). Cell proliferation was measured by adding BrdU to each well 18 h before the measurement. Absorbance of the samples was measured in an ELISA reader (ImmunoReader NJ-2000, Inter Med., Tokyo, Japan) at the wavelength of 450 nm (reference wavelength: 620 nm).

Statistical analysis.
Data were represented as the mean ± SEM of 3 animals from 1 experiment representative of 3 experiments. For statistical analysis, the unpaired Student's two-tailed t-test was used. A p value of < 0.05 was considered to indicate a significant difference between the 2 groups.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Changes in the Differential BAL Cell Counts and the Percentage of Ia-Positive Cells by O3 Exposure
Figure 1aGo shows the changes in the numbers of the different types of BAL cells during a 3-day exposure to 1 ppm O3. The total number of cells decreased until 12 h, then recovered and significantly increased on day 3. The number of AM and monocytes decreased until 12 h and then tended to recover. The number of neutrophils increased time dependently through day 2 of exposure. The numbers of eosinophils and lymphocytes only slightly increased. Figure 1bGo shows the changes in the percentage of Ia-positive cells during the 3-day exposure to 1 ppm O3. BAL cells from control rats did not express Ia antigen. The percentage of Ia-positive BAL cells increased in a time-dependent manner during O3 exposure.



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FIG. 1. (a) Differential BAL cell counts. The changes in the number of AM and monocytes, neutrophils, eosinophils, and lymphocytes during 0 to 3-day exposure to 1 ppm O3 were examined. BAL cells were isolated from filtered air- or 1 ppm O3-exposed rats. Differential cell counts was determined by Diff-Quik staining after cytospin preparation. Solid, shaded, hatched, dotted, and open bars represent total BAL cells, AM and monocytes, neutrophils, eosinophils, and lymphocytes, respectively. (b) Changes in the percentage of Ia-positive BAL cells during a 3-day exposure to 1 ppm O3 BAL cells were isolated from filtered air- or 1 ppm O3-exposed rats. The cells were treated with mouse anti-rat Ia monoclonal antibody and stained by the indirect avidin-biotin immunoperoxidase method. Values are means ± SEM of 3 animals. (*p < 0.05, **p < 0.01 compared with BAL cells from air-exposed rats.)

 
Effect of O3 on the Expression of Ia Antigen and B7 Costimulatory Molecules on BAL Cells
Figures 2a and 3aGoGo show typical examples of the effects of O3 on the expression of Ia, B7.1, and B7.2 on BAL cells in dot plots. Figures 2b and 3bGoGo show the percentage of expression of Ia, B7.1, and B7.2 on BAL cells. Most of the BAL cells from air-exposed rats did not express these cell-surface molecules. The expression of Ia, B7.1, and B7.2 molecules on BAL cells was increased by O3 exposure. The number of double-positive cells also increased in the BAL cells from O3-exposed rats.



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FIG. 2. Effect of O3 exposure on the expression of Ia and B7.1 on BAL cells. BAL cells (106 cells) were incubated with FITC-Ox6 and PE-3H5 antibodies and analyzed by FACS. (a) Examples of typical Ia and B7.1 expression on BAL cells in dot plots. (b) Percentages of each cell type in the BAL cells. Open and solid bars represent BAL cells from rats exposed to air and 1 ppm O3, respectively, for 3 days. Data represent the mean ± SEM of 3 animals from 1 experiment representative of 3 experiments. (*p < 0.05, ** p < 0.01 compared with BAL cells from air-exposed rats.)

 


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FIG. 3. Effect of O3 exposure on the expression of Ia and B7.2 on BAL cells. BAL cells (106 cells) were incubated with FITC-Ox6 and PE-24F antibodies and analyzed by FACS. (a) Examples of typical Ia and B7.2 expression of BAL cells in dot plots. (b) Percentages of each cell population in BAL cells. Open and solid bars represent BAL cells from rats exposed to air and 1 ppm O3, respectively, for 3 days. Data represent the mean ± SEM of 3 animals from 1 experiment representative of 3 experiments. (*p < 0.05, **p < 0.01 compared with BAL cells from air-exposed rats.)

 
Effect of O3 on the Expression of Ia Antigen and CD11b/c on BAL Cells
Figures 4a and 4bGo show that the expression of Ia antigen and CD11b/c on BAL cells was increased by O3 exposure. These figures show that double-positive cells also increased in number in the BAL cell population from O3-exposed rats. Infiltration of cells expressing Ia and costimulatory molecules and increases in the expression induced by the changes in the microenvironment of the lungs may have caused the increases in the expression of these molecules on BAL cells. Thus, these possibilities were examined next.



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FIG. 4. Effect of O3 exposure on the expression of Ia and CD11b/c on BAL cells. The cells (106 cells) were incubated with FITC-Ox6 and PE-Ox42 antibodies and analyzed by FACS. (a) Examples of typical Ia and CD11b/c expression of BAL cells in dot plots. (b) Percentages of each cell type in the BAL cells. Open and solid bars represent BAL cells from rats exposed to air and 1 ppm O3, respectively, for 3 days. Data represent the mean ± SEM of 3 animals from 1 experiment representative of 3 experiments. (*p < 0.05, **p < 0.01 compared with BAL cells from air-exposed rats.)

 
Morphology of Ia-Positive BAL Cells Increased by O3 Exposure
Figure 5Go shows photomicrographs of BAL cell population. Figures 5a and 5bGo are only Diff-Quik-stained BAL cells. O3 exposure increased the number of neutrophils or monocyte-like cells. Figures 5c and 5dGo are BAL cells immunostained with Ox6 antibody. Normal BAL cells were Ia negative, but in the BAL cells from O3-exposed rats, Ia-positive cells had increased in number. The Ia-positive cells were small-sized and looked like monocytes morphologically.



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FIG. 5. Morphological study of Ia-positive BAL cells. Light micrographs of BAL cells isolated from rats exposed to filtered air (a, c) or 1 ppm O3 for 3 days (b, d). The cells were stained with Diff-Quik stain with (c, d) or without (a, b) immunostaining for Ia antigen. Arrows in b show monocyte-like cells. Arrows in d show an Ia-positive cell (brown). Original magnification x300.

 
Expression of Cell-Surface Molecules of Infiltrating Neutrophils in the BAL Cell Population
Figure 6Go shows that neutrophils, that had infiltrated the lungs during O3 exposure did not express Ia, B7.1, or B7.2 molecules. However, approximately 90% of the neutrophils expressed CD11b/c.



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FIG. 6. Expression of cell-surface molecules on neutrophils that infiltrated in the lungs of O3-exposed rats. Neutrophils that had infiltrated the lungs were isolated by Percoll discontinuous gradient centrifugation. The neutrophils (106 cells) were incubated with FITC-Ox6 and PE-3H5, PE-24F, or PE-Ox42 antibodies and analyzed by FACS. Data represent those of 3 animals in 1 of 4 similar experiments.

 
Expression of Ia Antigen and Costimulatory Molecules on Peripheral Blood Monocytes
The expression of these molecules on peripheral blood monocytes before the cells had infiltrated the alveoli was examined. Both monocytes and AM were obtained from normal rats, and the expression on the monocytes was compared with that on the AM. Figures 7a and 7bGo show that monocytes highly expressed Ia, B7.1, B7.2, and CD11b/c molecules in comparison with resident AM. These figures also show that double-positive monocytes were observed.



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FIG. 7. Expression of Ia and B7.1, B7.2, or CD11b/c on monocytes. Peripheral blood monocytes were prepared from normal rats, and the cells (106 cells) were incubated with FITC-Ox6 and PE-3H5, PE-24F, or PE-Ox42 antibodies and analyzed by FACS. (a) Examples of typical Ia and B7.1, Ia and B7.2, and Ia and CD11b/c expression on monocytes in dot plots. (b) Percentages of each cell population. Open and solid bars represent resident AM and monocytes, respectively. Data represent the mean ± SEM of 3 animals from 1 experiment representative of 3 experiments. (*p < 0.05, **p < 0.01 compared with resident AM.)

 
Effect of O3-BALF on the Expression of Ia Antigen on AM and Monocytes
Figure 8aGo shows that the Ia expression on the resident AM was very minimal and was not affected by culture with either Air-BALF or O3-BALF for 2 days. However, the Ia expression on the peripheral blood monocytes, which was found on approximately 20% of the cells, was increased by culture with O3-BALF but not Air-BALF (Fig. 8bGo).



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FIG. 8. Effect of O3-BALF on the expression of Ia antigen on AM and monocytes. Resident AM and peripheral blood monocytes were prepared from normal rats. Cells of each type (5 x 105) were cultured with R10, Air-BALF, or O3-BALF for 2 days. Then these cultured cells were incubated with FITC-Ox6 antibody and analyzed by FACS. Fluorescence data are expressed as the percentage of Ia-positive cells in AM (a) and monocytes (b). Data represent those of 1 of 3 similar experiments.

 
Accessory Activity of BAL Cells
Figure 9aGo shows that proliferation of LNC was increased by the addition of BAL cells from O3-exposed rats. Figure 9aGo also shows that BAL cells from air-exposed rats (> 99% AM) tended to suppress the proliferation of LNC in a BAL cell number-dependent manner. The accessory activity of BAL cells in the MLR was thus enhanced by O3 exposure. The capacity of BAL cells to serve as antigen-presenting cells for OVA-sensitized T cells was also examined. Figure 9bGo shows that OVA-specific proliferation of T cells was stimulated by BAL cells. BAL cells from O3-exposed rats, compared with those from air-exposed rats, caused increased proliferation of T cells. The OVA-specific antigen-presenting activity of BAL cells was thus also enhanced by O3 exposure.



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FIG. 9. Accessory activity of BAL cells. BAL cells were prepared from rats exposed to 1 ppm O3 or filtered air for 3 days. (a) MLR. LNC (2 x 106/ml) were cultured in R10 (200 µl/well) in the presence of graded numbers (0.75 to 50% of the LNC number) of BAL cells from rats exposed to air (open bar) or O3 (solid bar). Cells were cultured for 4 days, and BrdU was added 18 h before measuring cell proliferation. Data represent the mean ± SEM of 3 animals from 1 experiment representative of 3 experiments (*p < 0.05, **p < 0.01 compared with BAL cells from air-exposed rats, {dagger}p < 0.05, {dagger}{dagger}p < 0.01 compared with LNC alone). (b) OVA-specific antigen presenting activity. OVA-sensitized T cells (4 x 105) were cultured in R10 (200 µl/well), in the presence of OVA (200 µg/ml) and graded numbers (3.125 to 12.5% of the T-cell number) of BAL cells from rats exposed to air (open bar) or O3 (solid bar). Cells were cultured for 4 days, and BrdU was added 18 h before measuring cell proliferation. Data represent the mean ± SEM of 3 animals from 1 experiment representative of 3 experiments. (*p < 0.05 compared with BAL cells from air-exposed rats, {dagger}p < 0.05, {dagger}{dagger}p < 0.01 compared with T cells with OVA.)

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Figure 1aGo showed that the total number of BAL cells decreased until 12 h. This decrease in the recovery of BAL cells may have been caused by adherence of cells to the alveolar epithelium. It is well known that recovery of BAL cells is decreased by short-term exposure to O3 (Dowell et al., 1970Go). Figure 1bGo showed that most resident AM (BAL cells from air-exposed rats) did not express Ia antigen or costimulatory molecules, B7.1, B7.2, and CD11b/c. It has been reported that resident AM function poorly as antigen-presenting cells and rather suppress T-cell activation (Koike et al., 1998Go, 1999Go; Strickland et al., 1993Go). In contrast, Figure 1bGo showed that expression of Ia antigen on BAL cells was increased by exposure to O3 for 3 days, time dependently. Previous reports have shown that the expression of Ia antigen on BAL cells is increased by exposure to silica, asbestos, and tobacco smoke (Hartmann et al., 1984Go; Higashimoto et al., 1994Go; Struhar et al., 1989Go). Figures 2–4GoGoGo showed that the expression of B7.1, B7.2, and CD11b/c was also increased by O3 exposure. It has been reported that BAL cells in lung inflammation caused by asthma showed increased expression of B7.1 and B7.2 (Agea et al., 1998Go; Burastero et al., 1999Go). Therefore, O3-induced changes in the microenvironment of the lungs caused the increase in expression of Ia antigen and costimulatory molecules on BAL cells. Furthermore, double-positive cells, which expressed Ia/B7.1, Ia/B7.2, and Ia/CD11b/c, were increased in number by the O3 exposure (Figs. 2–4GoGoGo). This article is the first report that O3 exposure enhanced the number of BAL cell double positives for Ia antigen and costimulatory molecules associated with antigen presentation. This accumulation of the cells could contribute to antigen presentation and subsequent antibody production.

There are many factors that induce expression of Ia antigen and costimulatory molecules on BAL cells during O3 exposure. Firstly, O3-induced changes in the microenvironment of the lungs may affect the expression of these cell-surface molecules on resident AM. It has been reported that IFN-{gamma} induced Ia expression on AM (Struhar et al., 1989Go). TH1 cytokines, such as IFN-{gamma} and IL-15, induced expression of both B7.1 and B7.2 (Agostini et al., 1999Go), and IL-16 induced only B7.1 expression on AM (Hermann et al., 1999Go). TH2 cytokine such as IL-4 and GM-CSF induced only B7.2 expression on AM (Agea et al., 1998Go). GM-CSF also induced expression of CD11b and CD11c on AM (Eischen et al., 1992Go; Rivier et al., 1994Go). Figure 8aGo showed that expression of Ia antigen on the very few positive resident AM was not increased by treatment with either Air-BALF or O3-BALF. These results suggest that O3-induced changes in the microenvironment of the lungs may not affect the expression on resident AM of cell-surface molecules associated with antigen-presentation. Alveolar epithelial cells (AEC) desquamated by O3 exposure might affect the enhancement of Ia, B7.1, and B7.2 expression on BAL cells. It has been reported that Ia antigen, B7.1, and B7.2 are aberrantly expressed on AEC in idiopathic pulmonary fibrosis (Kaneko et al., 2000Go), and Ia antigen on AEC is also increased by exposure to silica (Struhar et al., 1989Go). Thus, O3 might enhance the expression of these cell-surface molecules on AEC. There are no reports, however, that show O3 enhancement of the expression of these cell-surface molecules on AEC as far as we know. After 3 days of O3 exposure, the percentage of AEC in BAL cells as measured by keratin staining was about 5% (data not shown). In contrast, the percentage of Ia-positive cells was about 35%. These data suggest that AEC desquamated by O3 exposure can hardly account for the increased number of Ia-positive BAL cells.

Secondly, infiltration of cells expressing Ia antigen and costimulatory molecules may cause the increase in the number of BAL cells expressing these cell-surface molecules. It has been reported that small-sized cells (Mochitate and Miura, 1989Go) and CD11b-positive cells (Bhalla, 1996Go) in the BAL cell population are increased by O3 exposure. Actually, Fig. 5bGo showed that small-sized cells, which had monocyte-like feature and neutrophils, were increased by O3 exposure. Most of the infiltrating neutrophils expressed CD11b/c, but did not express Ia antigen, B7.1, or B7.2 (Fig. 6Go). Accordingly, infiltrating neutrophils did not contribute to the increase in the number of BAL cells expressing Ia antigen, B7.1, and B7.2 by O3 exposure. In contrast, peripheral blood monocytes generally expressed Ia antigen, B7.1, B7.2, and CD11b/c (Fig. 7Go). Figure 5dGo also demonstrated that the Ia-positive cells were monocyte-like cells. These results suggest that monocytes infiltrating into the alveoli would cause the O3-induced enhancement of the number of BAL cells expressing cell-surface molecules associated with antigen presentation.

There are many cytokines that induce the expression of these cell-surface molecules on monocytes. IFN-{gamma}, GM-CSF, and tumor necrosis factor-{alpha} are known to enhance the expression of Ia antigen on monocytes (Alvaro-Gracia et al., 1989Go; Chang and Lee, 1986Go). A TH1 cytokine, IFN-{gamma}, enhanced the expression of both B7.1 and B7.2; and 2 TH2 cytokines, IL-4 and IL-10, up-regulated B7.1 and down-regulated B7.2 expression (Creery et al., 1996Go; Debs et al., 1988Go). Figure 8bGo showed that expression of Ia antigen on peripheral blood monocytes was increased by treatment with O3-BALF but not Air-BALF. Accordingly, O3-induced changes in the lung microenvironment may have enhanced the expression of cell-surface molecules associated with antigen-presentation on monocytes that had infiltrated the lungs.

As for the effect of O3 exposure on the functions of BAL cells, Figure 9aGo showed that the accessory activity of BAL cells in the MLR was enhanced by O3 exposure. Furthermore, O3 exposure also enhanced OVA-specific antigen-presenting activity of BAL cells for sensitized T cells (Fig. 9bGo). These enhancements of the accessory activity of BAL cells in terms of MLR and OVA-specific antigen-presentation were associated with the O3-induced increase in the number of Ia antigen and costimulatory molecules on the BAL cells. Such enhancements could also be associated with O3-induced inhibition of the immunosuppressive activity of BAL cells. Our previous reports showed, however, that O3 inhibited the suppressive activity of BAL cells toward T-cell function through changing the microenvironment of the lungs. These studies also showed that O3 did not change the immunosuppressive activity of the BAL cells themselves (Koike et al., 1998Go, 1999Go). Accordingly, the enhanced accessory activity of the BAL cells may not be attributed to the immunosuppressive activity of the BAL cells.

In summary, the number of BAL cells expressing cell-surface molecules associated with antigen presentation was increased by O3 exposure. Morphological and immunological studies suggested that the Ia-positive BAL cells were infiltrating monocytes. The accessory activity of BAL cells in MLR and OVA-specific antigen-presentation was also enhanced by O3 exposure. These enhancements of accessory activity may be caused by the increase in the number of BAL cells expressing Ia antigen, B7.1, B7.2, and CD11b/c. Furthermore, the enhancement of these cell-surface molecules on BAL cells may be associated with the infiltration of monocytes expressing these molecules and/or the increase in their expression on infiltrating monocytes caused by O3-induced changes in the microenvironment of the lungs. On the basis of the present results, we need to conduct further research on the effect of exposure to the ambient level of O3 on the expression of cell-surface molecules associated with antigen-presenting activity of BAL cells.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +81-298-50-2439. E-mail: takakoba{at}nies.go.jp. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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