help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by FUJIMAKI, H.
Right arrow Articles by ENDO, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by FUJIMAKI, H.
Right arrow Articles by ENDO, T.
Am. J. Respir. Crit. Care Med., Volume 164, Number 10, November 2001, 1867-1873

Induction of Inflammatory Response of Mice Exposed to Diesel Exhaust Is Modulated by CD4+ and CD8+ T Cells

HIDEKAZU FUJIMAKI, NAOYA UI, and TOMOHIKO ENDO

National Institute for Environmental Studies, Onogawa, Tsukuba, Ibaraki, Japan; and the Jikei University School of Medicine, Minato-ku, Tokyo, Japan




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-1beta production but decreased IL-12 production. DE exposure significantly enhanced production of the macrophage inflammatory proteins (MIP)-1alpha 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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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). 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)-1alpha , 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). 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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

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 Inhalation

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).

Study Protocol

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).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 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.

Collection of BAL, CLN, and Spleen Cells and Cell Culture

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.

Assay for Cytokine and Chemokine Production, and for OVA-specific IgE, IgG1, and IgG2a in Plasma

Using commercially available mouse ELISA kits, we quantitated cytokine and chemokine production of tumor necrosis factor (TNF)-alpha , IL-1beta , IL-2, IL-4, IL-5, IL-6, IL-12p70 (Endogen, Inc., Woburn, MA), interferon (IFN)-gamma (Amersham International Plc, England), MIP-1alpha , 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).

Flow Cytometric Analysis

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).

Statistical Analysis

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in the Number of BAL Cells in DE-exposed Mice Treated with Anti-CD4 and Anti-CD8 mAbs

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).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 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.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 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); dagger dagger p < 0.01 versus Group D.

Cytokine and Chemokine Production in BAL Fluid from DE-exposed Mice

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-alpha production in OVA+DE-exposed mice remained at the same levels with OVA-immunized mice, the production of IL-1beta was significantly increased in OVA+DE-exposed mice (Figure 4a, Group D). Treatment with anti-CD4 or anti-CD8 mAbs abrogated the increased IL-1beta 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-1beta 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-1alpha 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-1alpha 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).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 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-1beta (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.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 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-1alpha (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; dagger p < 0.05 versus OVA+DE group (A); dagger  dagger p < 0.01 versus OVA+DE group (A).

Changes in the Number of B and T Cell Subpopulations in CLNs and Spleens from DE-exposed Mice

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).



View larger version (41K):
[in this window]
[in a new window]
  		Figure 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.

                              
View this table:
[in this window]
[in a new window]
 

TABLE 1

 EFFECT OF DE INHALATION ON B AND T CELL SUBPOPULATIONS IN CLNS

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.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 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.

Cytokine Production in CLN and Spleen Cells

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-gamma 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.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 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-gamma production in CLN cells measured by ELISA. (d ) IL-4, (e) IL-5, and (f  ) IFN-gamma production in spleen cells measured by ELISA. Values are mean ± SE of five mice. **p < 0.01 versus OVA group.

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-gamma 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-gamma production by DE exposure (data not shown).

Antigen-specific Antibody Titers in Plasma from DE-exposed and Control Mice

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.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1alpha and MIP-2 production in BAL fluid accompanied by an increase of IL-1beta production. This is the first report to indicate that treatment with anti-CD4 or anti-CD8 mAb abrogated the increased MIP-1alpha 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-1alpha 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-1alpha , MIP-1beta , 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) 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-1beta production but not in TNF-alpha 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-1alpha , 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-1alpha 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-1alpha as well as RANTES are major eosinophil chemotactic factors produced during allergic responses (40, 41). Increased MIP-1alpha 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-1beta 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-1beta and to increased production of MIP-1alpha 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.


    Footnotes

Correspondence and requests for reprints should be addressed to Dr. Hidekazu Fujimaki, Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2, Onogawa, Tsukuba, Ibaraki 305-0053, Japan. E-mail: fujimaki{at}nies.go.jp

(Received in original form September 27, 2000 and accepted in revised form August 20, 2001).

Acknowledgments: 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.


    References
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1. Pope CA, Dockery WD, Spengler JD, Raizenne ME. Respiratory health and PM10 pollution: a daily time series analysis. Am Rev Respir Dis 1991; 144: 668-674 [Medline].

2. Schwartz J, Slater T, Larson T, Pierson WE, Koenig JQ. Particulate air pollution and hospital emergency room visits for asthma in Seattle. Am Rev Respir Dis 1993; 147: 826-831 [Medline].

3. Romieu I, Meneses F, Ruiz S, Sienra JJ, Huerta J, White MC, Etzel RA. Effects of air pollution on the respiratory health of asthmatic children living in Mexico City. Am J Respir Crit Care Med 1996; 154: 300-307 [Abstract].

4. Ishizaki T, Koisumi K, Ikemori R, Ishiyama Y, Kushibiki E. Studies of prevalence of Japanese cedar pollinosis among the residents in a densely cultivated area. Ann Allergy 1987; 58: 265-270 [Medline].

5. Nitta H, Sato T, Nakai S, Maeda K, Aoki S, Ono M. Respiratory health associated with exposure to automobile exhaust: 1. Results of cross-sectional studies in 1979, 1982, and 1983.  Arch Environ Health 1993; 48: 53-58 [Medline].

6. Kagawa J. Atmospheric pollution due to mobile sources and effects on human health in Japan. Environ Health Perspect 1994;102(Suppl. 4):93-99.

7. Campbell KI, George EL, Washington IS Jr.. Enhanced susceptibility to infection in mice after exposure to dilute exhaust from light duty diesel engines. Environ Int 1981; 5: 377-382 .

8. Hahon N, Booth JA, Green F, Lewis TR. Influenza virus infection in mice after exposure to coal dust and diesel engine emissions. Environ Res 1985; 37: 44-60 [Medline].

9. Bice DE, Mauderly JL, Jones RK, McClellan RO. Effects of inhaled diesel exhaust on immune responses after lung immunization. Fundam Appl Toxicol 1985; 5: 1075-1086 [Medline].

10. Fujimaki H, Saneyoshi K, Shiraishi F, Imai T, Endo T. Inhalation of diesel exhaust enhances antigen-specific IgE antibody production in mice. Toxicology 1997; 116: 227-233 [Medline].

11. Miyabara Y, Ichinose T, Takano H, Sagai M. Diesel exhaust inhalation enhances airway hyperresponsiveness in mice. Int Arch Allergy Immunol 1998; 116: 124-131 [Medline].

12. Kobayashi T, Ikeue T, Ito T, Ikeda A, Murakami M, Kato A, Maejima K, Nakajima T, Suzuki T. Short-term exposure to diesel exhaust induces nasal mucosal hyperresponsiveness to histamine in guinea pigs. Fundam Appl Toxicol 1997; 38: 166-172 [Medline].

13. Kobayashi T, Ikeue T, Ikeda A. Four-week exposure to diesel exhaust induces nasal mucosal hyperresponsiveness to histamine in guinea pigs. Toxicol Sci 1998; 45: 106-112 [Abstract/Free Full Text].

14. Mentnech MS, Lweis DM, Olenchock SA, Mull JC, Koller WA, Lewis TR. Effects of coal dust and diesel exhaust on immune competence in rats. J Toxicol Environ Health 1984; 13: 31-41 [Medline].

15. Salvi S, Blomberg A, Rudell B, Kelly F, Sandstrom T, Holgate ST, Frew A. Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers. Am J Respir Crit Care Med 1999; 159: 702-709 [Abstract/Free Full Text].

16. Diaz-Sanchez D, Jyrala M, Ng D, Nel A, Saxon A. In vivo nasal challenge with diesel exhaust particles enhances expression of the CC chemokines RANTES, MIP-1alpha , and MCP-3 in humans. Clin Immunol 2000; 97: 140-145 [Medline].

17. Ohtoshi T, Takizawa H, Ozaki H, Kawasaki S, Takeuchi N, Ohta K, Ito K. Diesel exhaust particles stimulate human airway epithelial cells to produce cytokines relevant to airway inflammation in vitro. J Allergy Clin Immunol 1998; 101: 778-785 [Medline].

18. Hashimoto S, Gon Y, Takeshita I, Matsumoto K, Jibiki I, Takizawa H, Kudoh S, Horie T. Diesel exhaust particles activate p38 MAP kinase to produce interleukin 8 and RANTES by human bronchial epithelial cells and N-acetylcysteine attenuates p38 MAP kinase activation. Am J Respir Crit Care Med 2000; 161: 280-285 [Abstract/Free Full Text].

19. Takizawa H, Abe S, Ohtoshi T, Kawasaki S, Takami K, Desaki M, Sugawara I, Hashimoto S, Azuma A, Nakahara K, Kudoh S. Diesel exhaust particles up-regulated expression of intercellular adhesion molecule-1 (ICAM-1) in human bronchial epithelial cells. Clin Exp Immunol 2000; 120: 356-362 [Medline].

20. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone: I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986; 136: 2348-2357 [Abstract].

21. Sagai M, Saito H, Ichinose T, Kodama M, Mori Y. Biological effects of diesel exhaust particles. 1. In vitro production of superoxide and in vivo toxicity in mouse. Free Radic Biol Med 1993; 14: 37-47 [Medline].

22. Imai T, Shimizu F, Fujimaki H, Watanabe N. Enhancement of IgE antibody production by ovalbumin aerosol in mice. Int Arch Allergy Immunol 1983; 70: 368-372 .

23. Fujimaki H, Shiraishi F, Aoki Y, Saneyoshi K. Modulated cytokine production from cervical lymph node cells treated with B(a)P and PCB. Chemospere 1997; 34: 1487-1493 .

24. Nohara K, Ushio H, Tsukumo S, Kobayashi T, Kijima M, Tohyama C, Fujimaki H. Alterations of thymocyte development, thymic emigrants and peripheral T cell population in rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicology 2000; 145: 227-235 [Medline].

25. Krakauer T. Induction of CC chemokines in human peripheral blood mononuclear cells by staphylococcal exotoxins and its prevention by pentoxifylline. J Leukocyte Biol 1999; 66: 158-164 [Abstract].

26. Takafuji S, Suzuki S, Koizumi K, Tadokoro K, Miyamoto T, Ikemori R, Muranaka M. Diesel-exhaust particulates inoculated by the intranasal route have an adjuvant activity for IgE production in mice. J Allergy Clin Immunol 1987; 79: 639-645 [Medline].

27. Fujimaki H, Nohara O, Ichinose T, Watanabe N, Saito S. IL-4 production in mediastinal lymph node cells in mice intratracheally instilled with diesel exhaust particulates and antigen. Toxicology 1994; 92: 261-268 [Medline].

28. Fujimaki H, Saneyoshi K, Nohara O, Shiraishi F, Imai T. Intranasal instillation of diesel exhaust particulates and antigen in mice modulated cytokine productions in cervical lymph node cells. Int Arch Allergy Immunol 1995; 108: 268-273 [Medline].

29. Diaz-Sanchez D, Tsien A, Fleming J, Saxon A. Combined diesel exhaust particulate and ragweed allergen challenge markedly enhances human in vivo nasal ragweed-specific IgE and skews cytokine production to a T helper cell 2-type pattern. J Immunol 1997; 158: 2406-2413 [Abstract].

30. Ichinose T, Takano H, Miyabara Y, Sagai M. Long-term exposure to diesel exhaust enhances antigen-induced eosinophilic inflammation and epithelial damage in the murine airway. Toxicol Sci 1998; 44: 70-79 [Abstract/Free Full Text].

31. Fujimaki H, Ui N, Ushio H, Nohara K, Endo T. Roles of CD4+ and CD8+ T cells in adjuvant activity of diesel exhaust particles in mice. Int Arch Allergy Immunol 2001; 124: 485-496 [Medline].

32. Hogan SP, Matthaei KI, Young JM, Koskinen A, Young IG, Foster PS. A novel T cell-regulated mechanism modulating allergen-induced airways hyperreactivity in BALB/c mice independently of IL-4 and IL-5. J Immunol 1998; 161: 1501-1509 [Abstract/Free Full Text].

33. Yang HM, Ma JY, Castranova V, Ma JK. Effects of diesel exhaust particles on the release of interleukin-1 and tumor necrosis factor-alpha from rat alveolar macrophages. Exp Lung Res 1997; 23: 269-284 [Medline].

34. Yang HM, Barger MW, Castranova V, Ma JK, Yang JJ, Ma JY. Effects of diesel exhaust particles (DEP), carbon black, and silica on macrophage responses to lipopolysaccharide: evidence of DEP suppression of macrophage activity. J Toxicol Environ Health 1999; 58: 261-278 .

35. Krakauer T. Pentoxifylline inhibits ICAM-1 expression and chemokine production induced by proinflammatory cytokines in human pulmonary epithelial cells. Immunopharmacology 2000; 46: 253-261 [Medline].

36. Driscoll KE, Hassenbein DG, Carter J, Poynter J, Asquith TN, Grant RA, Whitten J, Purdon MP, Takigiku R. Macrophage inflammatory proteins 1 and 2: expression by rat alveolar macrophages, fibroblast, and epithelial cells and in rat lung after mineral dust exposure. Am Rev Respir Cell Mol Biol 1993; 8: 311-318 .

37. Nel AE, Diaz-Sanchez D, Ng D, Hiura T, Saxon A. Enhancement of allergic inflammation by the interaction between diesel exhaust particles and the immune system. J Allergy Clin Immunol 1998; 102: 539-554 [Medline].

38. Fahy O, Tsicopoulos A, Hammad H, Pestel J, Tonnel AB, Wallaert B. Effects of diesel organic extracts on chemokines production by peripheral blood mononuclear cells. J Allergy Clin Immunol 1999; 103: 1115-1124 [Medline].

39. Schall TJ. The chemokines. In: A Thomson, editor. The cytokine handbook, 2nd ed. New York: Academic Press; 1994. p. 419-460.

40. Lukacs NW, Strieter RM, Shaklee CL, Chensue SW, Kunkel SL. Macrophage inflammatory protein-1 alpha influences eosinophil recruitment in antigen-specific airway inflammation. Eur J Immunol 1995; 25: 245-251 [Medline].

41. Lukacs NW, Standiford TJ, Chensue SW, Kunkel RG, Strieter RM, Kunkel SL. C-C chemokine-induced eosinophil chemotaxis during allergic inflammation. J Leukocyte Biol 1996; 60: 573-578 [Abstract].

42. Miyabara Y, Ichinose T, Takano H, Lim H-B, Sagai M. Effects of diesel exhaust on allergic airway inflammation in mice. J Allergy Clin Immunol 1998; 102: 805-812 [Medline].

43. Miyabara Y, Takano H, Ichinose T, Lim H-B, Sagai M. Diesel exhaust enhances allergic airway inflammation and hyperresponsiveness in mice. Am J Respir Crit Care Med 1998; 157: 1138-1144 [Abstract/Free Full Text].

44. Devalia JL, Bayrum H, Abdelaziz MM, Sapsford RJ, Davies RJ. Differences between cytokine release from bronchial epitherial cells of asthmatic patients and non-asthmatic subjects: effect of exposure to diesel exhaust particles. Int Arch Allergy Immunol 1999; 118: 437-439 [Medline].




This article has been cited by other articles:


Home page
 Eur Respir JHome page
B-M. Larsson, M. Sehlstedt, J. Grunewald, C. M. Skold, A. Lundin, A. Blomberg, T. Sandstrom, A. Eklund, and M. Svartengren
Road tunnel air pollution induces bronchoalveolar inflammation in healthy subjects
Eur. Respir. J., April 1, 2007; 29(4): 699 - 705.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. Senechal, P. de Nadai, N. Ralainirina, A. Scherpereel, H. Vorng, P. Lassalle, A.-B. Tonnel, A. Tsicopoulos, and B. Wallaert
Effect of Diesel on Chemokines and Chemokine Receptors Involved in Helper T Cell Type 1/Type 2 Recruitment in Patients with Asthma
Am. J. Respir. Crit. Care Med., July 15, 2003; 168(2): 215 - 221.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. J. TOBIN
Chronic Obstructive Pulmonary Disease, Pollution, Pulmonary Vascular Disease, Transplantation, Pleural Disease, and Lung Cancer in AJRCCM 2001
Am. J. Respir. Crit. Care Med., March 1, 2002; 165(5): 642 - 662.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by FUJIMAKI, H.
Right arrow Articles by ENDO, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by FUJIMAKI, H.
Right arrow Articles by ENDO, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2001 American Thoracic Society