Abstract
Exposure to diesel exhaust (DE) increased airway inflammatory responses and airway responsiveness to allergen challenge. To clarify the roles of T cells in DE exposure–induced early inflammation, we studied the effect of CD4 and CD8 cells on the effect DE might have on allergic inflammation by using monoclonal antibody-mediated cellular depletion assays. In the bronchoalveolar lavage (BAL) fluid, the numbers of inflammatory cells from 3 mg/m3 DE-exposed and ovalbumin (OVA)–immunized mice markedly increased. Depletion of CD4+ cells resulted in reduced accumulation of inflammatory cells. DE exposure to OVA-immunized mice significantly increased interleukin (IL)-1 β production but decreased IL-12 production. DE exposure significantly enhanced production of the macrophage inflammatory proteins (MIP)-1 α and MIP-2, but not monocyte chemoattractant protein (MCP)-1 and regulated upon activation normal T cells expressed and secreted (RANTES). Treatment with anti-CD4 and anti-CD8 mAbs abrogated the adverse effect of DE exposure. In CLN cells from OVA + DE-exposed mice, CD45R/B220-, CD3-, CD4-, and CD8-positive cells were significantly increased, but the OVA-stimulated cytokine production remained at the same levels with OVA-immunized mice. These findings suggest that the induction of early inflammatory responses by DE exposure may initially be related to the modulated function of lymphocyte subpopulations.
Keywords: diesel; inflammation; lymphocyte
It is well established that exposure to particulate air pollutants affects the incidence of allergic diseases such as allergic rhinitis and asthma by modulating respiratory defense mechanisms (1-3). In Japan, for example, there is a positive correlation between the increase in the number of diesel exhaust vehicles and the increased incidence of Japanese cedar pollinosis along the roadside (4), and the prevalence of asthmatic symptoms and chest congestion in school children has been correlated significantly with the levels of suspended particulate matter (5, 6), suggesting that diesel exhaust particles (DEPs) may aggravate respiratory inflammation.
Animal experiments have revealed many effects on defense mechanisms following exposure to DEPs or diesel exhaust (DE): exposure to DE enhanced susceptibility to infection and decreased interferon (IFN) production in mice (7, 8). The number of antibody-forming cells and lymphocytes in lung-associated lymph nodes increased in rats exposed to 3.5 and 7.0 mg/m3 DE (9). The inhalation of 6.0 mg/m3 DE by mice was reported to significantly increase production of antiovalbumin (OVA) immunoglobulin E (IgE) in plasma and production of interleukin (IL)-4 and IL-10 in supernatants from cultured spleen cells (10). Recently, exposure to 1.0 mg/m3 DE for 12 wk combined with OVA sensitization was reported to enhance anti-OVA IgE and IgG1 production in the serum and increase the number of eosinophils and mast cells in the lungs of mice (11). From physiologic examinations of the upper respiratory organs, it has been reported that in guinea pigs, short-term exposure to 3.2 mg/m3 DE increased sneezing and histamine-induced nasal secretion (12), and 4-wk exposure to 3.2 mg/m3 DE induced nasal mucosal hyperresponsiveness (13). These findings indicate that DE exposure affects both local and systemic immune responsiveness, resulting in induction of allergic inflammation of the airway. In contrast, no significant changes were noted in the mitogenic response of splenic T cells or plaque-forming cells in the spleens of rats exposed to 2.0 mg/m3 DE (14).
In human volunteers, short-term exposure to 0.3 mg/m3 DE significantly increased neutrophils and B lymphocytes in material recovered after airway lavage, and bronchial biopsies showed an increased number of mast cells and CD4+ and CD8+ T lymphocytes (15). After in vivo challenge of human subjects with DEP alone, the levels of nasal macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant protein (MCP)-3, and regulated upon activation normal T cells expressed and secreted (RANTES) were significantly elevated (16). In human bronchial epithelial cells, DEP induced the production of IL-8, granulocyte–macrophage colony-stimulating factor (GM-CSF), RANTES, and soluble intercellular adhesion molecules-1 (sICAM-1) (17-19). It is therefore important to evaluate the effects of DE exposure on the roles of lymphocyte subsets and cytokine/chemokine production involved in induction of allergic inflammation.
Since Mosmann and coworkers (20) reported the existence of CD4+ T cell subsets and their respective functions, not only CD4+ T cell subsets but also CD8+ T cell subsets have been associated with cell-mediated and humoral immunity by cross-regulation. Humoral antibody production, particularly IgE production, is regulated by the balance of the Th1/Th2 cytokine profile. Although DEPs seem to induce pathways from a Th0- to a Th2-like response, the mechanism by which DEPs facilitate the development of the Th2-like response and induce eosinophilic inflammation is unclear.
To determine the early effects of DE exposure on induction of allergic inflammation, mice were treated with anti-CD4 or anti-CD8 monoclonal antibodies (mAbs) to clarify the contribution of CD4+ and CD8+ T cells in modulating the early effects of DE inhalation. After DE inhalation for 4 wk with a challenge of OVA at 3 wk, we studied the changes in cytokine and chemokine production in bronchoalveolar lavage (BAL) fluid, changes in cellularity, and in vitro cytokine production in cervical lymph node (CLN) and spleen cells, and changes in antibody production in plasma.
Male BALB/c mice (5 wk old) were purchased from Japan SLC Inc. (Shizuoka, Japan). Six-week-old mice were used in all experiments. Food and water were given ad libitum. This study was performed with the approval of the National Institute for Environmental Studies Ethics Committee for Experimental Animals.
DE was generated by a four-cylinder, 2.74-L, Isuzu diesel engine under computer control. The details on DE inhalation were previously described (10, 21).
Anti-CD4 (Clone GK1.5) and anti-CD8 (Clone 3.155) mAbs were purchased from Leinco Technologies, Inc. (St. Louis, MO). Isotype-matched negative antibody control (rat IgG) was obtained from Chemicon International, Inc. (Temecula, CA).
As shown in Figure 1, the AIR group of mice was exposed to fresh filtered air without immunization. The DE group of mice was exposed to DE without immunization. The OVA group of mice was exposed to fresh filtered air with OVA immunization. The OVA+DE group of mice was exposed to DE with OVA immunization. Each group of five mice was injected intraperitoneally with 100 μg anti-CD4, 100 μg anti-CD8, or 100 μg rat IgG (controls) on Days –1 and 1. On Day 0, mice were immunized intraperitoneally with 10 μg OVA immediately before DE inhalation. On Day 21, mice were boosted by exposure to 1% OVA in sterile saline for 6 min (22).
Fig. 1. Experimental design for treatment with anti-CD4 or anti-CD8 mAb, immunization with OVA, and exposure to DE. Four groups of mice (AIR, exposed with fresh filtered air without immunization; DE, exposed to DE without immunization; OVA, exposed to fresh filtered air with OVA immunization; OVA+DE, exposed to DE with OVA immunization) received anti-CD4 mAb, anti-CD8 mAb, or vehicle control (rat IgG). Both OVA- and OVA+DE-exposed mice were injected intraperitoneally with OVA on Day 0 and were exposed to aerosolized OVA on Day 21.
[More] [Minimize]On Day 28, mice were killed under ether anesthesia, and BAL fluid, CLNs, spleens, and blood samples were collected as described previously (10, 23). For cytokine production, CLN cells were cultured with OVA in the presence of APCs. Spleen cells were cultured with or without OVA. Culture supernatants were collected 48 h after OVA stimulation, centrifuged, and frozen at −70° C until assayed for cytokines.
Using commercially available mouse ELISA kits, we quantitated cytokine and chemokine production of tumor necrosis factor (TNF)-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12p70 (Endogen, Inc., Woburn, MA), interferon (IFN)-γ (Amersham International Plc, England), MIP-1α, MIP-2, MCP-1, and RANTES (R&D Systems, Minneapolis, MN). Eotaxin was quantified by ELISA using anti-mouse eotaxin Ab. We performed ELISA to quantify anti-OVA IgE and total IgE antibodies in plasma (10). Anti-OVA IgG1 and IgG2a antibodies in plasma were measured by ELISA using horseradish peroxidase-labeled goat anti-mouse IgG1 and IgG2a (Southern Biotechnology Associates, Inc., Birmingham, AL).
We analyzed the immunophenotypes of CLN and spleen cells from each group of mice using flow cytometry (24). We purchased the following mAbs from PharMingen (San Diego, CA): phycoerythrin (PE)– labeled rat IgG anti-CD4 (clone GK1.5), fluoroscein isothiocyanate (FITC)–labeled rat IgG anti-CD8a (clone 53-6.7), PE-labeled hamster IgG anti-CD3 (clone 145-2C11), FITC-labeled rat IgG anti-CD45R/ B220 (clone RA3-6B2), and corresponding isotype-matched controls. Cell samples were analyzed on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, Mansfield, MA).
All data are presented as the mean ± standard error (SE), which are indicated by bars in the figures. Statistical analysis was performed with the Statcel statistical analysis system for Microsoft Excel version 5.0 (Seiunsha Inc., Tokyo, Japan). Differences between group means were analyzed using ANOVA with post hoc test (Bonferroni/Dunn) (p < 0.05 or p < 0.01).
To examine the effect of DE exposure on allergic inflammation, we determined the total number of BAL cells immediately after exposure to DE for 4 wk (Figure 2). DE exposure significantly increased the accumulation of inflammatory cells. The numbers of macrophages, neutrophils, and eosinophils were all elevated in the OVA+DE-exposed group compared with other groups. Although the total number of BAL cells, macrophages, and neutrophils from vehicle-treated DE-exposed mice (Group D, E, F) significantly increased compared with that in the AIR group (Group A, B, C), treatment with anti-CD4 and anti-CD8 mAbs did not result in any change in inflammatory cell numbers (Figure 3a). Treatment with anti-CD4 and anti-CD8 mAbs in OVA-immunized mice did not affect the induction of inflammatory cells in BAL fluid (Figure 3b). However, DE exposure to immunized mice markedly increased the number of macrophages, neutrophils, and eosinophils in BAL fluid (Figure 3b). Depletion of CD4-positive cells significantly reduced the induction of inflammatory cells in BAL fluid of control mice (Group D versus Group E). Treatment with anti-CD8 mAb in OVA+DE-exposed mice decreased the number of macrophages and eosinophils (Group D versus Group F).
Fig. 2. Changes in the number of inflammatory cells in BAL fluids. On Day 28, BAL fluids were collected from the mice. The numbers of macrophages, neutrophils, and eosinophils were calculated by total cell count using a hemocytometer and smears of lavaged cells stained with Diff-Quik. Each value is mean ± SE of five mice. AIR, air-exposed mice without immunization; DE, DE-exposed mice without immunization; OVA, air-exposed mice with OVA immunization; OVA+DE, DE-exposed mice with OVA immunization. *p < 0.05 versus AIR group; ** p < 0.01 versus AIR group; §§ p < 0.01 versus DE group; ##p < 0.01 versus OVA group.
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Fig. 3. Changes in the number of inflammatory cells in BAL fluids from anti-CD4 or anti-CD8 mAb-treated mice. (a) BAL fluids were collected from DE-exposed and AIR-exposed mice treated with anti-CD4 mAb, anti-CD8 mAb, or rat IgG (control). The numbers of macrophages and neutrophils were calculated by total cell count using a hemocytometer and smears of lavaged cells stained with Diff-Quik. Each value is mean ± SE of five mice. Group A, vehicle-treated air-exposed mice; Group B, anti-CD4 mAb-treated air-exposed mice; Group C, anti-CD8 mAb-treated air-exposed mice; Group D, vehicle-treated DE-exposed mice; Group E, anti-CD4 mAb-treated DE-exposed mice; Group F, anti-CD8 mAb-treated DE-exposed mice. **p < 0.01 versus AIR group. (b) BAL fluids were collected from OVA-immunized and OVA+DE-exposed mice treated with anti-CD4 mAb, anti-CD8 mAb, or rat IgG (control). The numbers of macrophages, neutrophils, and eosinophils were calculated by total cell count using a hemocytometer and smears of lavaged cells stained with Diff-Quik. Each value is mean ± SE of five mice. Group A, vehicle-treated OVA-immunized mice; Group B, anti-CD4 mAb-treated OVA-immunized mice; Group C, anti-CD8 mAb-treated OVA-immunized mice; Group D, vehicle-treated OVA+DE-exposed mice; Group E, anti-CD4 mAb-treated OVA+DE-exposed mice; Group F, anti-CD8 mAb-treated OVA+DE-exposed mice. **p < 0.01 versus AIR group (a); ††p < 0.01 versus Group D.
[More] [Minimize]To clarify the interaction of the increased numbers of inflammatory cells with cytokine production, we measured proinflammatory cytokines in BAL fluid by ELISA. Although IL-2, IL-6, and TNF-α production in OVA+DE-exposed mice remained at the same levels with OVA-immunized mice, the production of IL-1β was significantly increased in OVA+DE-exposed mice (Figure 4a, Group D). Treatment with anti-CD4 or anti-CD8 mAbs abrogated the increased IL-1β production. In contrast, IL-12p70 production in BAL fluid from OVA+ DE-exposed mice significantly decreased, but treatment with anti-CD4 or anti-CD8 mAb inhibited the decrease (Figure 4b). There was no increase in IL-1β and IL-12 in BAL fluid from DE and AIR groups of mice. Chemokine production, which induces the migration and attraction of inflammatory cells, also may play a role in induction of inflammation in mice exposed to DE. DE inhalation markedly increased MIP-1α and MIP-2 production in BAL fluid from OVA+DE-exposed mice (Figure 5a and 5b). Treatment with anti-CD4 and anti-CD8 mAbs abrogated the increased MIP-1α production in BAL fluid. Treatment with anti-CD8 mAb abrogated the increased MIP-2 production in BAL fluid from OVA+DE-exposed mice compared with anti-CD4 mAb. We detected no increase in RANTES, eotaxin, or MCP-1 production in DE-exposed mice (data not shown).
Fig. 4. Cytokine production in BAL fluid in OVA-immunized and OVA+DE-exposed mice. On Day 28, BAL fluids were collected from OVA-immunized and OVA+DE-exposed mice treated with anti-CD4 mAb, anti-CD8 mAb, or rat IgG (control). IL-1β (a) and IL-12 (b) production was determined by ELISA. Each value is mean ± SE of five mice. Group A, vehicle-treated OVA-immunized mice; Group B, anti-CD4 mAb-treated OVA-immunized mice; Group C, anti-CD8 mAb-treated OVA-immunized mice; Group D, vehicle-treated OVA+DE-exposed mice; Group E, anti-CD4 mAb-treated OVA+DE-exposed mice; Group F, anti-CD8 mAb-treated OVA+DE-exposed mice. *p < 0.05 versus Group A.
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Fig. 5. Chemokine production in BAL fluid from DE-exposed mice. On Day 28, BAL fluids were collected from AIR, DE, OVA, and OVA+DE-exposed groups of mice treated with anti-CD4 mAb (B), anti-CD8 mAb (C), or rat IgG (control) (A). MIP-1α (a) and MIP-2 (b) production was determined by ELISA. Each value is mean ± SE of five mice. *p < 0.05 versus AIR group; **p < 0.01 versus AIR group; †p < 0.05 versus OVA+DE group (A); † †p < 0.01 versus OVA+DE group (A).
[More] [Minimize]To investigate the effect of DE exposure on local immunity, we examined changes in the cellularity and function of CLN cells. The total number of CLN cells from OVA+DE-exposed mice was significantly increased compared with other groups of mice (Figure 6). The numbers of CD45R/B220-positive cells significantly increased in OVA and OVA+DE-exposed groups. The numbers of CD3, CD4, and CD8-positive cells in the CLN cells also significantly increased in DE, OVA, and OVA+DE-exposed groups of mice. Changes in the percentage of CD45R/B220- and CD3-positive cells and the ratio of CD4/CD8 are shown in Table 1. Treatment with anti-CD4 mAb clearly reduced the ratio of CD4/CD8, but the comparison of values between DE- and AIR-exposed mice showed no significant difference. Similarly, treatment with anti-CD8 mAb markedly increased the ratio of CD4/CD8, but the values between DE- and AIR-exposed mice showed no difference, indicating that in CLN cells, DE exposure increased the lymphocyte subpopulation, but did not change the CD4/CD8 ratio. We next investigated the cell proliferation response to OVA in CLN cells from DE-exposed and control mice; however, we observed no significant difference between OVA and OVA+DE-exposed or between vehicle-treated and anti-CD4 or anti-CD8 mAb-treated mice (data not shown).
Fig. 6. Changes in the total number of CLN cells and the lymphocyte subpopulations from DE-exposed mice. On Day 28, CLN cells were collected from AIR, DE, OVA, and OVA+DE groups of mice. The number of CLN cells in each lymphocyte subpopulation was determined by cell count and flow cytometric analysis. Values are mean ± SE of five mice. *p < 0.05 versus AIR group; **p < 0.01 versus AIR group.
[More] [Minimize]| DE (mg/m 3) | OVA | Treatment | CD45R/B220 (%) | CD3 (%) | CD4/CD8 | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| 0 | − | Rat IgG | 12.0 ± 0.8* | 76.8 ± 2.7 | 3.25 ± 0.09 | |||||
| 0 | − | Anti-CD4 | 18.4 ± 1.0 | 69.7 ± 1.5 | 0.76 ± 0.07 | |||||
| 0 | − | Anti-CD8 | 12.5 ± 0.5 | 76.4 ± 1.8 | 8.49 ± 0.39 | |||||
| 3.0 | − | Rat IgG | 13.9 ± 1.2 | 83.1 ± 1.3 | 2.59 ± 0.03 | |||||
| 3.0 | − | Anti-CD4 | 19.0 ± 1.6 | 76.4 ± 1.9 | 0.57 ± 0.12 | |||||
| 3.0 | − | Anti-CD8 | 13.8 ± 1.1 | 82.6 ± 0.9 | 7.17 ± 0.41 | |||||
| 0 | + | Rat IgG | 19.2 ± 2.4 | 79.0 ± 2.6 | 3.24 ± 0.15 | |||||
| 0 | + | Anti-CD4 | 17.8 ± 1.6 | 80.4 ± 1.6 | 1.20 ± 0.04 | |||||
| 0 | + | Anti-CD8 | 18.9 ± 1.0 | 79.1 ± 1.1 | 10.57 ± 0.51 | |||||
| 3.0 | + | Rat IgG | 20.9 ± 1.1 | 77.4 ± 1.2 | 2.75 ± 0.22 | |||||
| 3.0 | + | Anti-CD4 | 30.9 ± 3.8 | 67.1 ± 3.9 | 0.97 ± 0.06 | |||||
| 3.0 | + | Anti-CD8 | 22.4 ± 2.1 | 76.0 ± 2.2 | 9.90 ± 0.72 |
The effect of DE exposure on the spleen as a secondary lymphoid organ was investigated. We observed no significant difference between total number of spleen cells in DE-exposed mice and control mice (Figure 7). The numbers of CD45R/ B220-positive cells in spleen cells from OVA+DE-exposed mice and of CD8-positive cells in spleen cells from DE-exposed mice were significantly greater than that of the AIR control; however, the numbers of other lymphocyte subpopulations in the DE-exposed mice were similar to those of the AIR-exposed mice. We observed no significant difference in the ratio of CD4/CD8 between DE- and AIR-exposed or between vehicle-treated and anti-CD4 or anti-CD8 mAb-treated mice (data not shown). Therefore, the effect of DE exposure may be less severe on the cellularity of spleen cells than on CLN cells.
Fig. 7. Changes in the total number of spleen cells and lymphocyte subpopulations from DE-exposed mice. On Day 28, spleen cells were collected from AIR, DE, OVA, and OVA+DE groups of mice. The number of spleen cells in each lymphocyte subpopulation was determined by cell count and flow cytometric analysis. Values are mean ± SE of five mice. **p < 0.01 versus AIR group.
[More] [Minimize]To investigate the effect of DE exposure on functional properties of lymphocytes in the CLNs and spleen, we examined antigen-induced cytokine production in CLN and spleen cells from in vitro stimulation with OVA. Forty-eight hours after in vitro OVA stimulation, IL-4 and IL-5 production in supernatants of cultured CLN cells from DE-exposed mice remained at the same levels as those from control mice (Figure 8a and 8b). Because IFN-γ production in culture supernatants of CLN cells showed no significant difference between exposed and control mice (Figure 8c), cytokine-producing ability in CLN cells was not severely affected by DE exposure.
Fig. 8. Cytokine production in CLN and spleen cells by in vitro antigenic stimulation. CLN cells from OVA-immunized and OVA+DE-exposed mice were stimulated with 0 and 100 μg/ml OVA in the presence of APC. Spleen cells from mice exposed to DE and controls were stimulated with 0 and 100 μg/ml OVA. Culture supernatants were harvested 48 h after OVA stimulation. (a) IL-4, (b) IL-5, and (c) IFN-γ production in CLN cells measured by ELISA. (d ) IL-4, (e) IL-5, and (f ) IFN-γ production in spleen cells measured by ELISA. Values are mean ± SE of five mice. **p < 0.01 versus OVA group.
[More] [Minimize]Although spleen cells from DE-exposed and control mice showed no significant difference in the production of IL-4 and IL-5 (Figure 8d and 8e), IFN-γ production was significantly greater in spleen cells from DE-exposed mice than it was in the control (Figure 8f). Treatment with anti-CD4 mAb abrogated the increased IFN-γ production by DE exposure (data not shown).
To determine the systemic effect of DE exposure, antibody production in plasma was determined by ELISA. DE exposure to OVA-immunized mice induced no significant difference in total or OVA-specific IgE production compared with levels in AIR-exposed, OVA-immunized mice (Figure 9a and 9b; Group A versus Group D). Anti-OVA IgE production in plasma of anti-CD4 mAb-treated DE-exposed and control mice decreased significantly, but those of anti-CD8 mAb-treated mice did not show a decrease (Figure 9b). Because DE exposure failed to induce a significant difference in anti-OVA IgG2a production (Figure 9c), as well as anti-OVA IgG1 production (data not shown), DE exposure showed no significant effects on systemic immunity.
Fig. 9. Effects of DE inhalation on IgE and IgG antibody production in DE-exposed mice. On Days −1 and 1, each group of mice was injected intraperitoneally with 100 μg of anti-CD4, 100 μg of anti-CD8, or 100 μg of rat IgG (control). On Day 0, mice were immunized with 10 μg of OVA. On Day 21, each mouse was exposed to aerosolized 1% OVA. On Day 28, plasma was collected from each treatment group and levels of (a) total IgE, (b) anti-OVA IgE, and (c) anti-OVA IgG2a were measured using ELISA. Group A, vehicle-treated OVA-immunized mice; Group B, anti-CD4 mAb-treated OVA-immunized mice; Group C, anti-CD8 mAb-treated OVA-immunized mice; Group D, vehicle-treated OVA+ DE-exposed mice; Group E, anti-CD4 mAb-treated OVA+ DE-exposed mice; Group F, anti-CD8 mAb-treated OVA+ DE-exposed mice. Values are mean ± SE of five mice. *p < 0.05 versus Group A; **p < 0.01 versus Group A.
[More] [Minimize]We studied the early effects of DE inhalation on local and systemic immune response. In particular, we focused on the contribution of lymphocyte subpopulations to the induction of migration and recruitment of inflammatory cells following DE exposure. Mindful of potential effects on local immunity, we investigated not only accumulation of inflammatory cells and cytokine and chemokine production in BAL fluid but also the cellularity and cytokine production in CLN cells in both DE-exposed and control mice. Our present study showed that DE exposure to OVA-immunized mice markedly increased MIP-1α and MIP-2 production in BAL fluid accompanied by an increase of IL-1β production. This is the first report to indicate that treatment with anti-CD4 or anti-CD8 mAb abrogated the increased MIP-1α and MIP-2 production in BAL fluid from OVA+DE-exposed mice. It is not known why treatment with anti-CD4 or anti-CD8 mAb reduced the increased production of MIP-1α and MIP-2 induced by DE exposure. In vitro studies by Krakauer (25) showed that human peripheral blood mononuclear cells (PBMC) stimulated with enterotoxin B (EB) induced high levels of MIP-1α, MIP-1β, and MCP-1. Krakauer also observed that monocytes separated from PBMC are a source of these chemokines and that addition of purified T cells to the monocytes amplifies the levels of chemokines produced, suggesting that cognate interaction of EB bound to antigen-presenting cells (APCs) with T cells contributes to chemokine production. Therefore, one possibility is that DE exposure may act on alveolar macrophages or other APCs in concert with lymphocytes to enhance the production of chemokines. Another possibility is that CD4+ and CD8+ T cells activated by DE exposure directly released the chemokines; however, we observed no marked increase in the number of lymphocytes in BAL fluid.
Regarding the balance of Th1-/Th2-type immune responses, previous human and animal data (10, 26-29) suggest that DE inhalation and DEP instillation enhance the cytokine production of Th2 type helper T cells, resulting in increased IgE antibody production. The mechanism by which DE inhalation activates Th2-type helper T cells has yet to be clarified. However, the recruitment of eosinophils to the lung may not necessarily be correlated with the increase of IgE antibody production in DE-exposed mice (30). Treatment with anti-CD4 and anti-CD8 mAbs significantly reduced the increased number of inflammatory cells in BAL fluid from OVA+DE-exposed mice. The balance of Th-/Th2-type cytokine and chemokine profile in CD4+ T cells may be related to the balance of Tc1-/ Tc2-type cytokine and chemokine profile in CD8+ T cells. Our recent studies showed that DEP injection in the presence of OVA affected the function of both CD4- and CD8-positive T cell subtypes (31). Our present results showing that the number of CD4- and CD8-positive cells in CLNs of DE-exposed mice was markedly increased are consistent with the study of Salvi and coworkers (15), indicating that short-term exposure to 0.3 mg/m3 DE significantly increased B lymphocytes in airway lavage fluid and caused an increase in CD4+ and CD8+ T lymphocytes in bronchial biopsies. These increases in T cell subsets by DE exposure may contribute to induction and aggravation of allergic airway inflammation via the different T cell–mediated mechanisms. T cell–mediated dissociation of airway eosinophilia from the development of airway hyperreactivity was recently indicated by Hogan and coworkers (32). Chemokine production regulated by CD4+ and CD8+ T cells may be a sensitive marker for further investigation into the effects of DE exposure on the initiation of local immune responses to inhaled allergens.
DE exposure to OVA-immunized mice significantly increased the number of inflammatory cells such as macrophages, neutrophils, and eosinophils in BAL fluid. A significant increase in proinflammatory cytokines in BAL fluid from DE-exposed mice was also seen in IL-1β production but not in TNF-α or IL-6 production. When rat alveolar macrophages were cultured with DEP, or when alveolar macrophages isolated from DE-exposed rats were cultured, IL-1 production in the culture supernatants was increased in vitro (33, 34). Moreover, it has been suggested that IL-1 increases the production of chemokines such as IL-8 and MCP-1 by pulmonary epithelial cell lines (35) and induces the release of MIP-2 and MCP-1 by a variety of cell types such as macrophages, epithelial cells, and fibroblasts (36). In humans, an intranasal challenge of DEP increased mRNA and protein expression for RANTES, MIP-1α, and MIP-3 in nasal lavage fluid (37). In vitro studies showed that polyaromatic hydrocarbons associated with DEP increased the production of mRNA transcripts for IL-8, MCP-1, and RANTES in the PBMC of healthy subjects (38). Our present study showing that DE exposure increased the release of chemokines such as MIP-1α and MIP-2 in BAL fluid is in agreement with the recent reports described above (37, 38). DEP can induce the production of GM-CSF, IL-8, and RANTES by human airway epithelial cells (17, 18). Mouse MIP-2, which is classified as a member of the CXC chemokine family, is a functional homologue of human IL-8 and may be a key mediator of neutrophil recruitment (39). Although we observed no increase in chemoattractants for eosinophils such as RANTES or eotaxin in BAL fluid in DE-exposed mice, the number of eosinophils was increased slightly. MIP-1α as well as RANTES are major eosinophil chemotactic factors produced during allergic responses (40, 41). Increased MIP-1α production by DE exposure may be related to the slight eosinophil increment. A marked increase in the number of eosinophils in lung tissue and BAL fluids by DE exposure was observed by Miyabara and coworkers (42, 43). Since the mice were immunized with OVA in the presence of alum in their experiments, the use of alum may enhance the severeness in induction of eosinophils. Expression of adherent molecules, which induces the recruitment of inflammatory cells to the sites of inflammation, may be relevant to DEP-induced inflammation: DEP-enhanced expression of ICAM-1 in human bronchial epithelial cells (19), and Krakauer (35) has reported that IL-1β induced the expression of ICAM-1 by pulmonary epithelial cell lines. Comparison of cytokine production by human bronchial epithelial cells from subjects with and without asthma showed that DEPs induced greater amounts of sICAM-1 in subjects with asthma than in subjects without asthma (44). Therefore, exposure to DEPs and DE may induce proinflammatory features in the airway and lung via the increased cytokine and chemokine production or the expression of ICAM-1 by alveolar macrophages and other resident cells, such as bronchial epithelial cells and fibroblasts.
In spleen and CLN cells, B cell populations were significantly increased by DE exposure. However, we did not observe polyclonal activation in total IgE antibody production. No significant differences in antigen-specific IgE, IgG1, or IgG2a antibody production in plasma were seen between DE-exposed and control mice. The contribution of increased numbers of B cells in CLNs and spleens of DE-exposed mice in inducing allergic inflammation is unknown. Exposure to 3.0 mg/m3 DE for 4 wk might be insufficient to enhance systemic immune responses. Similar results were observed by Miyabara and coworkers (42), showing that exposure to DE combined with an aerosolized OVA challenge for 5 wk enhanced infiltration of eosinophils and neutrophils to airways and airway resistance, but did not significantly increase antigen-specific IgE and IgG1 antibody production in plasma compared with levels in OVA-challenged mice exposed to air.
In summary, the cellular mechanisms by which DE might increase allergic inflammation were investigated by using in vivo antibody-mediated cell depletion. DE exposure leads to increased release of IL-1β and to increased production of MIP-1α and MIP-2. The effects were abrogated by treatment with anti-CD4 or anti-CD8 mAbs. The induction of early inflammatory responses by DE exposure may initially be related to the modulated function of lymphocyte subpopulations.
The authors thank Dr. T. Kobayashi for his encouragement and Mmes. Miyoko Wada, Sachiyo Shimizu, and Keiko Mishima for their excellent technical assistance. The authors thank Dr. H. Nagai (Gifu Pharmaceutical University) for providing standard anti-OVA IgE antibodies.
This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan, and in part from special coordination funds from the Science and Technology Agency of the Japanese Government for promoting science and technology.
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