Mechanism of TCDD-Induced Suppression of Antibody Production: Effect on T Cell-Derived Cytokine Production in the Primary Immune Reaction of Mice

Tomohiro Ito*,{dagger}, Kaoru Inouye*,{ddagger}, Hidekazu Fujimaki*,{dagger}, Chiharu Tohyama*,{dagger} and Keiko Nohara*,{dagger},1

* Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba 305-8506, Japan; {dagger} CREST, JST (Japan Science and Technology), Kawaguchi 332-0012, Japan; and {ddagger} Japan Society for the Promotion of Science (JSPS), Tokyo 102-8471, Japan

Received June 18, 2002; accepted August 5, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress antigen-specific antibody production in humoral immune reactions, but the precise mechanism remains unclear. Since T cell activation and subsequent production of type 2 helper T (Th2) cell-derived cytokines are required for antigen-specific antibody production in humoral immunity, we examined the effects of TCDD on splenic T cell numbers, T cell growth factor IL-2 production, and Th2 cell-derived cytokine production. C57BL/6N mice were orally given TCDD (20 µg/kg) or vehicle, and immediately intraperitoneally immunized with ovalbumin (OVA) adsorbed to alum, and cellular changes in the spleen and cytokine production by spleen cells were investigated from Day 1 to Day 14. In vehicle-control mice the numbers of splenic CD3+ T cells increased from Day 7 onward, but no increase was observed in the TCDD-exposed mice. When spleen cells from control mice were cultured and restimulated with OVA ex vivo, a significant amount of IL-2 was found from Day 1, but it decreased on Day 7, whereas TCDD exposure promptly suppressed the increase on Day 4. TCDD exposure significantly suppressed the production of Th2 cell-derived cytokines IL-4, IL-5, and IL-6, which were prominently increased from Day 4 onward in control mice. The dose-dependent study showed that IL-5 production was significantly suppressed in a dose-dependent manner starting at 1 µg/kg TCDD. Moreover, separation and reconstitution studies showed that the TCDD-induced suppression of IL-5 production was due to the impaired function of T cells rather than that of antigen-presenting cells. The results of this study suggest that TCDD-induced suppression of T cell activation and Th2-type cytokine production is involved in the impairment of antigen-specific antibody production.

Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; Th2 cell-derived cytokine; T cell growth factor; antibody production; primary immune reaction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been found to be an extremely toxic congener among the halogenated aromatic hydrocarbons that are widespread and persistent in the environment (Tohyama, 2002Go; Van den Berg et al., 1998). It is well known that TCDD suppresses both humoral and cellular immunity in mice (Holsapple et al., 1991Go; Kerkvliet, 1995Go, 2002Go; Vos et al., 1997–1998Go) in an aryl hydrocarbon receptor (AhR)-dependent manner (Vorderstrasse et al., 2001Go).

With regard to the suppressive effect of TCDD on antibody production, primary effect on B cells or on T cells has been independently reported by previous studies using different experimental systems (reviewed by Holsapple et al., 1991Go; Kerkvliet, 2002Go). Holsapple et al. used in vitro approach, where unprimed splenocytes obtained from vehicle control or TCDD-administered mice were primed in vitro by culture with antigen in the presence or absence of TCDD, and the antibody production was examined by plaque-forming cell assay (reviewed in Holsapple et al., 1991Go). Based on the results obtained in the studies using T-dependent and T-independent antigens, and cell separation/reconstitution assays, they showed that TCDD directly affects B cells, rather than T cells or macrophages, and suppresses antibody response (Dooley and Holsapple, 1988Go; Dooley et al., 1990Go). A recent study by Sulentic et al. (2000)Go reported the possibility that TCDD directly suppresses µ gene expression by inducing the binding of AhR to the xenobiotic responsive element (XRE) within the Ig heavy chain 3‘{alpha}-enhancer in B cells. While these studies have shown the primary effect of TCDD on B cells, another target cell type has been demonstrated to function in TCDD-induced suppression of immune reaction in vivo. Kerkvliet and Brauner (1987)Go reported using T-dependent and T-independent antigens in vivo or in vivo immunization approaches with T cell-deficient nude mice, that administration of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin primarily affects regulatory T cells in the antibody suppression, although the chemical also suppresses B cell function with higher doses. Tomar and Kerkvliet (1991)Go further indicated that immunization to TCDD-treated mice resulted in reduced helper T cell activity using cell separation/reconstitution system. From these results, T cell function seems to be a susceptible target of TCDD toxicity in antigen response in vivo, although the precise linkage between the deteriorated T cell function and suppression of antibody production has not been clarified.

Helper T cells play a pivotal role in antibody production against T-dependent antigens. Upon encountering an antigen presented by antigen-presenting cells (APCs), naive helper T cells with antigen-specific receptors (TCRs) are induced to undergo activation, clonal expansion, and differentiation. T cell growth factor IL-2 efficiently induces clonal expansion of activated helper T cells in an autocrine fashion. The activated helper T cells directly interact with antigen-specific B cells via CD40-CD154 interaction, leading to induction of several B cell functions (Grewal and Flavell, 1998Go). Subsequently, the activated helper T cells differentiate into type 1 helper T (Th1) cells or Th2 cells (Mosmann and Sad, 1996Go). IL-4 induces a commitment of T cells to develop into Th2 cells, which can secrete cytokines, including IL-4, IL-5, and IL-6, and these Th2-type cytokines induce the proliferation of antigen-specific B cells, isotype switching, and their differentiation into plasma cells (Finkelman et al., 1990Go; Mosmann and Sad, 1996Go; Paul and Seder, 1994Go).

We have previously reported that TCDD suppresses the production of T cell-derived cytokines, IL-2, IL-4, and IL-5 by spleen cells in the secondary immune reaction following ovalbumin (OVA) immunization (Fujimaki et al., 2002Go; Nohara et al., 2002Go), suggesting that TCDD-induced suppression of T cell-derived cytokine production is involved in the reduction of antigen-specific antibody production. While T cell-derived cytokines are indispensable for naive T cells and B cells to acquire effector functions in the primary immune reaction, how TCDD affects T cell-derived cytokine production in the primary immune reaction is largely unknown. In the present study, we investigated the time course and dose-dependent effects of TCDD on T cells in the primary immune reaction, focusing on T cell-derived cytokine production by spleen cells. Furthermore, we investigated the target cells responsible for the TCDD immunotoxicity by separation and reconstitution assay of spleen cells using the Th2-derived cytokine production as the index.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Female C57BL/6N mice (5 weeks old) were purchased from Clea Japan (Tokyo) and acclimatized to the environment for 1 week prior to use. They were given food and distilled water ad libitum and maintained under controlled conditions with a temperature of 24 ± 1°C, humidity of 50 ± 10%, and a 12/12 h light/dark cycle. The mice were handled in a humane manner according to NIES guidelines for animal experiments.

Reagents.
2,3,7,8-TCDD was purchased from Cambridge Isotope Laboratories (Andover, MA), and various concentrations of TCDD were prepared with corn oil containing 4% nonane. OVA (Albumin, Chicken egg, Grade VII) was purchased from Sigma (St. Louis, MO). For immunization, 1 mg/ml OVA in phosphate buffered saline (PBS) was mixed with an equal volume of 9% (w/v) AlK(SO4)2, and the pH of the mixture was adjusted to 6.5 with KOH. OVA adsorbed to AlK(SO4)2 (OVA/alum) was washed with PBS three times and then resuspended with PBS at 0.5 mg/ml.

Experimental design.
In order to evaluate the effect of TCDD on T cells, we performed three types of studies: time course study, dose dependency study, and separation and reconstitution study. In the time course study and dose dependency study, the results obtained in each study were confirmed in another independent experiment and a set of representative results was shown in the Results section.

In the time course study, TCDD in a single dose of 20 µg/10 ml/kg body weight was administered to the mice by gavage, and they were immediately intraperitoneally immunized with OVA/alum (100 µg OVA/mouse). Control mice were given vehicle (corn oil containing 4% nonane) and immunized with OVA/alum. Spleens were excised on different days after administration, and single cell suspensions were prepared from the spleens of the TCDD-exposed and control mice as described previously (Nohara et al., 2002Go). Spleen cell population and cytokine production were determined as described below.

In the dose dependency study, mice were given TCDD in a single dose of 0, 0.2, 1, 5, or 20 µg/kg body weight and intraperitoneally immunized with OVA/alum. Ten days after administration, blood was collected by heart puncture with a heparinized syringe, and the spleen was excised. Plasma was collected by centrifuging the blood and stored at –80°C until measurement of OVA-specific IgG1 antibody by ELISA. Spleen cell population and cytokine production were determined as described below.

In the separation and reconstitution study, mice were given TCDD in a single dose of 0 or 20 µg/kg body weight and intraperitoneally immunized with OVA/alum. Seven and 10 days after the treatment, separation and reconstitution study was performed as described below.

Flow cytometric analysis.
Percentages of T cells and B cells in the spleen were measured by flow cytometry with FACSCalibur (Becton-Dickinson, Mountain View, CA) as described previously (Nohara et al., 2002Go). T cells and B cells were stained with phycoerythrin (PE)-labeled antimouse CD3 (17A2) and fluorescein isothiocyanate (FITC)-labeled antimouse CD45R/B220 (RA3-6B2), respectively. Percentages of CD4+ and CD8+ T cells were determined by staining with PE-labeled antimouse CD4 (GK1.5) and FITC-labeled antimouse CD8 (53–6.7). All antibodies were purchased from PharMingen (San Diego, CA).

Cell culture.
Spleen cells were cultured at 2 x 106 cells/200 µl in complete medium with or without OVA (100 µg/ml) for 2 days under 5% CO2 (Nohara et al., 2002Go). The culture medium was centrifuged, and the supernatant was stored at –80°C until measurement of the cytokine concentration by ELISA.

Cell separation and reconstitution.
T cells and non-T cells were separated with a MACS separation system (Miltenyi Biotec, Berrgisch Gladbach) according to the manufacturer’s instructions. Briefly, spleen cells (5 x 107 cells) pooled from 5 mice were stained with FITC-labeled antimouse CD45R/B220 and PE-labeled antimouse CD11b (M1/70, Southern Biotechnology Associates Inc., Birmingham, AL). The cells were washed and then labeled with antirat IgG conjugated microbeads (Miltenyi Biotec). After washing, labeled (non-T cell) and unlabeled cells (T cell) were separated with a LS+ separation column (Miltenyi Biotec). Flow cytometric analysis showed that the T cell fraction contained 88–95% T cells and < 0.2% B cells. The non-T cell fraction contained < 5% T cells, 72–76% B cells, and 15–20% CD11b+ cells.

T cells and non-T cells separated from the spleen cells of TCDD-exposed and control mice were reconstituted in various combinations. Because flow cytometric analysis showed that the spleen cells from each group of mice contained approximately 30% T cells and 50% B cells, the separated cells were reconstituted at a ratio of 30% T cells and 50% B cells and then cultured to measure IL-5 as described above.

ELISA.
ENDOGEN Matched Antibody Pair (ENDOGEN, Woburn, MA) was used according to the manufacturer’s instructions to determine IL-2, IL-4, IL-5, IL-6, and IFN-{gamma} in culture supernatant. OVA-specific IgG1 in plasma was determined as previously described (Nohara et al., 2002Go).

Statistical analysis.
Differences in means between control and TCDD-exposed groups in the time course experiment were assessed by Student’s t-test. The dose-dependent effects of TCDD were analyzed by one-way ANOVA followed by Fisher’s PLSD using StatView (ver. 4.57, SAS Institute, Cary, NC).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of TCDD on Body Weight and Spleen Weight
Table 1Go summarizes the data for the time course of the body weight and spleen/body weight ratio data of the control and TCDD-exposed mice after administration and immunization. TCDD exposure had no effect on body weight from Days 1 to 14 when compared with vehicle treatment. The spleen/body weight ratio of the control mice gradually increased after immunization and peaked on Day 10, whereas TCDD exposure significantly reduced the increase on Day 14.


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TABLE 1 Effect of TCDD on Body Weight and Spleen Weight
 
Time Course of the Numbers of Total Cells and T Cells in Spleen after TCDD Administration
In the control mice, the numbers of total cells (Fig. 1aGo) and CD3+ T cells (Fig. 1bGo) in the spleen greatly increased on Day 7 after immunization and then gradually decreased, whereas in the TCDD-exposed mice the increases were completely suppressed, and the suppression was statistically significant on Days 7 and 14. Similarly, the numbers of splenic CD4+ T cells, CD8+ T cells, and CD45R/B220+ B cells in the control mice were also increased on Day 7, and the increases were completely suppressed by TCDD exposure (data not shown). The percentage of T cells in the spleen significantly increased in the TCDD-exposed mice, compared with control mice, on Days 2, 7, and 10 (Fig. 1cGo), and the increases were due to the decrease in CD45R/B220-CD3- cells as a result of TCDD exposure (data not shown).



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FIG. 1. Effect of TCDD on the numbers of total cells and CD3+ T cells in spleen. Mice were given TCDD (20 µg/kg body weight) or vehicle alone and immediately intraperitoneally immunized with OVA/alum. On various days thereafter (a) total number of spleen cells, (b) number of splenic CD3+ T cells, and (c) percentage of CD3+ T cells in spleen cells were measured. Each point and vertical bar indicate mean ± SE (n = 4 or 5). Asterisks indicate statistically significant difference (*p < 0.05, **p < 0.01) from the corresponding control by Student’s t-test.

 
Effect of TCDD on T Cell-Derived Cytokine Production by Spleen Cells following Restimulation Ex Vivo
Cytokine production by spleen cells responding to the antigen was evaluated by culturing the same number of the cells from each mouse in the presence of OVA. As shown in Fig. 2aGo, IL-2 production in the control mice showed biphasic changes after immunization with OVA/alum: after producing a significant amount of IL-2 on Days 1 to 4, the production decreased on Day 7, and then increased again. In the TCDD-exposed mice, IL-2 production rapidly decreased and was significantly lower on Day 4, compared to control mice. IL-4, IL-5, and IL-6 production in the control mice markedly increased on Day 4, indicating that a Th2 reaction had occurred after immunization with OVA/alum (Figs. 2b, 2c, and 2dGoGoGo). In the TCDD-exposed mice, production of all these cytokines, IL-4, IL-5, and IL-6, was significantly suppressed on Day 4, but the degree of suppression of these Th2-type cytokines by TCDD differed. IL-5 production was most sensitive to TCDD exposure, and decreased to the basal level through Days 4 to 14 in the TCDD-exposed mice (Fig. 2cGo). IL-6 was also sensitive to TCDD exposure, and production was greatly inhibited on Days 4 and 7, with recovery up to control levels by Day 10 (Fig. 2dGo). IL-4 production was less sensitive to TCDD exposure than IL-5 and IL-6 production (Fig. 2bGo), and it was only partially suppressed by TCDD on Day 4. We also assessed the production of the Th1-type cytokine IFN-{gamma} by spleen cells, but no differences between the TCDD-exposed and control mice were found (data not shown).



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FIG. 2. Effect of TCDD on T cell-derived cytokine production by spleen cells restimulated with OVA ex vivo. Mice were given TCDD (20 µg/kg body weight) or vehicle alone and immediately intraperitoneally immunized with OVA/alum. On various days thereafter, equal numbers of spleen cells from TCDD-exposed and control mice were cultured for two days with OVA. The concentrations of (a) IL-2, (b) IL-4, (c) IL-5, and (d) IL-6 in the supernatant were measured by ELISA. Each point and vertical bar indicate mean ± SE (n = 4 or 5). Asterisks indicate statistically significant difference from the corresponding control (*p < 0.05, **p < 0.01, ***p < 0.001) by Student’s t-test.

 
Among the Th2 cell-derived cytokines, the suppression of IL-4 and IL-6 production by TCDD was apparently reversed by Day 10, in contrast to the complete suppression of IL-5 through the experiment, as described above. These findings suggest that TCDD affects these cytokine productions by Th2 cells in different ways. However, there was another possibility that the apparent recovery in production of IL-4 and IL-6 was caused by non-OVA-responding cell types other than OVA-responding Th2 cells, including dendritic cells, macrophages, and NKT cells (Hope et al., 1995Go; Shirai et al., 1993Go; Yoshimoto and Paul, 1994Go). To assess this point, we examined the cytokine productions obtained in the presence and absence of OVA in the culture about the typical two types of cytokines, IL-5 and IL-6. In order to calculate net production by OVA-responding Th2 cells, IL-5 and IL-6 production obtained in the presence of OVA in the culture was subtracted by cytokine production obtained in the absence of OVA. As shown in Figs. 3a and 3bGoGo, the correction showed that both IL-5 and IL-6 production were similarly inhibited by TCDD throughout the experiment. These findings suggest that TCDD suppresses each Th2-type cytokine production by a common mechanism.



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FIG. 3. Effect of TCDD on net production of IL-5 and IL-6 by Th2 cells. Spleen cells from TCDD-exposed and control mice on various days were cultured for two days in the presence and absence of OVA. (a) IL-5 and (b) IL-6 production was corrected by subtracting cytokine concentration in the absence of OVA from that in the presence of OVA. Each point and vertical bar indicate mean ± SE (n = 4 or 5). Asterisks indicate statistically significant difference from the corresponding control (*p < 0.05, **p < 0.01, ***p < 0.001) by Student’s t-test.

 
Dose-Dependent Effect of TCDD on the Primary Immune Reaction of OVA/Alum-Immunized Mice
Next, we evaluated the dose-dependent effect of TCDD on the primary immune reaction of mice immunized with OVA/alum. Because OVA-specific IgG1 production was induced beginning on Day 10 after immunization with OVA/alum (Inouye et al., manuscript in preparation), mice were given TCDD in a single dose of 0.2, 1, 5, or 20 µg/kg and then immunized, and the changes in T cell numbers and cytokine production were examined on Day 10, at the same time as OVA-specific IgG1 production. Table 2Go summarizes the dose-dependent effects of TCDD on body weight and the spleen/body weight ratio. Body weight was unaffected by any dose of TCDD as compared with the control. The spleen/body weight ratio decreased in a dose-dependent manner, with statistical significance at 20 µg/kg TCDD. OVA-specific IgG1 production in plasma dose-dependently decreased starting at 1 µg/kg TCDD, and the decreases were statistically significant at 5 and 20 µg/kg TCDD (Fig. 4Go). In nonimmunized mice, OVA-specific IgG1 production was below the detection limit (data not shown).


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TABLE 2 Dose-Dependent Effect of TCDD on Body Weight and Spleen Weight
 


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FIG. 4. Dose-dependent effect of TCDD on OVA-specific IgG1 production. Mice were given TCDD in a single dose of 0.2, 1, 5, or 20 µg/kg body weight and immediately intraperitoneally immunized with OVA/alum. Ten days later, OVA-specific IgG1 level in plasma was determined by ELISA. Each column and vertical bar indicate mean ± SE (n = 3 or 5). Asterisks indicate statistically significant difference from the corresponding control (*p < 0.05, **p < 0.01) by ANOVA followed by Fisher’s PLSD.

 
On the other hand, numbers of total and CD3+ T cells in the spleen significantly decreased in a dose-dependent manner starting at 1 µg/kg TCDD (Figs. 5a and 5bGoGo). The percentage of T cells was unaffected by any dose of TCDD (data not shown). Because IL-5 production was most sensitive among Th2 cell-derived cytokines measured in the present study and was adequately suppressed on Day 10 (Fig. 2Go), IL-5 production was measured as an index of a dose-dependent effect of TCDD on Th2-type cytokine production. The same number of spleen cells from each mouse exposed to various doses of TCDD were cultured with OVA and IL-5 production was determined. IL-5 production was suppressed in a dose-dependent manner starting at the dose of 0.2 µg/kg TCDD, and the suppression was statistically significant at the 1 µg/kg TCDD and higher doses (Fig. 6Go). These results demonstrate that T cell functions, especially IL-5 production, are highly sensitive to TCDD in the primary immune reaction.



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FIG. 5. Dose-dependent effect of TCDD on total and T cell numbers in spleen. Mice were given TCDD in a single dose of 0.2, 1, 5, or 20 µg/kg body weight and immediately intraperitoneally immunized with OVA/alum. Ten days later, (a) total number of spleen cells and (b) number of splenic CD3+ T cells were measured. Each column and vertical bar indicate mean ± SE (n = 4 or 5). Asterisks indicate statistically significant difference from the corresponding control (*p < 0.05, **p < 0.01) by ANOVA followed by Fisher’s PLSD.

 


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FIG. 6. Dose-dependent effect of IL-5 production by spleen cells restimulated with OVA ex vivo. Mice were given TCDD in a single dose of 0.2, 1, 5, or 20 µg/kg body weight and immediately intraperitoneally immunized with OVA/alum. Ten days later, equal numbers of spleen cells from each mouse were cultured for 2 days with OVA, and IL-5 concentration in the supernatant was measured by ELISA. Each column and vertical bar indicate mean ± SE (n = 4 or 5). Asterisks indicate statistically significant difference from the corresponding control (*p < 0.05, **p < 0.01) by ANOVA followed by Fisher’s PLSD.

 
The Suppression of Cytokine Production by TCDD Is Mainly Due to a Defect in T Cells, Not in APCs
To produce cytokines upon restimulation with OVA ex vivo, effector T cells with antigen-specific TCR need to recognize the antigen presented by APCs, such as B cells, macrophages, and dendritic cells. We therefore addressed the question of whether the suppression of cytokine production observed in the present study is due to suppressive effects of TCDD on the ability of T cells to produce cytokines or of APCs to present antigens. On Day 7, when IL-5 production was most suppressed by TCDD exposure, T cells and non-T cells (APCs) were separated from the spleen cells of TCDD-exposed and control mice and then reconstituted at various combinations. In the nonseparated spleen cells, TCDD exposure markedly suppressed IL-5 production (Fig. 7Go, left panel), a finding that was consistent with the data shown in Fig. 2cGo. The response after reconstitution with T cells and non-T cells both from control mice was almost the same as that of the nonseparated spleen cells from control mice. Likewise, the TCDD-induced suppression of IL-5 production was reproduced after separation and reconstitution of T cells and non-T cells, both of which were from TCDD exposed mice. Thus, these results confirmed that the procedures for separation and reconstitution did not alter the ability of T cells and non-T cells to produce cytokines. When TCDD-exposed T cells and control non-T cells were used for reconstitution, IL-5 production was clearly suppressed to the level of the nonseparated spleen cells from TCDD-exposed mice (Fig.7, right panel). By contrast, when a combination of control T cells and TCDD-exposed non-T cells was examined, only slight suppression of IL-5 production was found. These results demonstrate that the suppression of Th2 cell-derived cytokine production by TCDD is due to a defect in T cells, not in APCs.



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FIG. 7. Cell separation and reconstitution of T cells and non-T cells. Mice were given TCDD (20 µg/kg body weight) or vehicle alone and immediately intraperitoneally immunized with OVA/alum. Seven days later, T cells and non-T cells were separated from spleen cells as described in Materials and Methods, and T cells and non-T cells from TCDD-exposed and control mice were reconstituted at the indicated combinations. Reconstituted spleen cells were cultured for two days with OVA, and IL-5 concentration in the supernatant was measured by ELISA.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we evaluated the effects of TCDD on T cell functions in the primary immune reaction, focusing on T cell-derived cytokine production. First, we investigated the time course of the effects of TCDD from Days 1 to 14 following immunization. We demonstrated here that TCDD exposure significantly suppressed the production of Th2 cell-derived cytokines, IL-4, IL-5, and IL-6 starting on Day 4 (Fig. 2Go). The TCDD-induced suppression of these cytokine productions is not due to a decrease in the percentage of splenic T cells, because the percentage of T cells in the spleen was increased by TCDD exposure (Fig. 1cGo). In the same system, we found that TCDD suppressed OVA-specific IgG1 production starting on Day 10 and germinal center formation starting on Day 7 (Inouye et al., manuscript in preparation), indicating that TCDD-induced suppression of T cell-derived cytokine production precedes that of antigen-specific IgG1 production. These observations strongly suggest that TCDD-induced suppression of Th2 cell-derived cytokine production is involved in the impaired antibody production.

With regard to the cause of the suppression of Th2 cell-derived cytokine production by TCDD exposure, the present study showed that TCDD suppresses the production of T cell growth factor IL-2 by spleen cells and the increases in T cell numbers in the spleen following immunization. These results suggest that TCDD suppresses activation of antigen-specific T cells and their subsequent expansion, which leads to the suppression of Th2 cell development and cytokine production by them. These effects of TCDD on T cells are consistent with a previous study in which prior TCDD exposure was shown to suppress lymph node cell proliferation following immunization and IL-2 production by spleen cells restimulated ex vivo (Lundberg et al., 1992Go). The present study also demonstrated by a separation and reconstitution assay that TCDD impairs the function of helper T cells more than that of APCs in cytokine production (Fig. 7Go). All these findings suggest that TCDD exposure suppresses T cell proliferation following immunization, leading to decreases in antigen-specific T cell numbers in the spleen and suppression of Th2 cell-derived cytokine production.

In contrast to the suppression of T cell expansion observed in our study, Shepherd et al. (2000)Go reported that TCDD exposure did not affect clonal expansion of CD4+ T cells in the spleen, but enhanced deletion of CD4+ T cells after the expansion following immunization in OVA-specific TCR transgenic DO11.10 CD4+ T cell adoptive transfer system. Although the reason for the discrepancy between Shepherd et al. and our results for the effect of TCDD on the splenic T cell response following immunization is unclear, it may be due to the difference in experimental conditions. Shepherd et al. monitored the expansion of antigen-specific CD4+ T cells, while we measured total CD3+ or CD4+ T cells, and there may be a difference between the effect of TCDD on antigen-specific and total CD4+ T cells. Alternatively, the difference in the effect of TCDD on clonal expansion may be attributable to the distinct function of the adjuvant. We used alum as an adjuvant, because it effectively induces Th2-type immune reaction (Brewer et al., 1999Go), whereas Shepherd et al. used complete Freund’s adjuvant, which has been found to be very effective in inducing a potent immune reaction (Billiau and Matthys, 2001Go).

In order to evaluate the effects of TCDD at lower doses on cytokine and antigen-specific antibody, we measured IL-5 production by spleen cells and OVA-specific IgG1 in plasma after TCDD exposure at several doses. OVA-specific IgG1 production was dose-dependently suppressed starting at 5 µg/kg TCDD and by 40% at 20 µg/kg TCDD exposure (Fig. 4Go). The antigen-specific IgG1 in plasma we measured was considered to be produced by long-lived antibody forming cells (AFCs), since it was detected on Day 10 and increased thereafter (Inouye et al., manuscript in preparation). The long-lived AFCs play an important role in long-term protective immunity. Although the suppression of IgM response to sheep red blood cells (SRBC), which is produced by short lived AFCs, is known to be a very sensitive endpoint of TCDD toxicity with an ED50 of around 0.7 µg/kg in C57BL/6 mice (Davis and Safe, 1988Go; Kerkvliet et al., 1990Go; Smialowicz et al., 1994Go), the antigen-specific IgG1 in plasma was not so much susceptible to TCDD. Similar results on the suppressive effect of TCDD on OVA-specific IgG1 in plasma were reported by Shepherd et al. (2000)Go. On the other hand, IL-5 production was more sensitive to TCDD exposure than IgG1 in plasma, and it was significantly inhibited in a dose-dependent manner starting at only 1 µg/kg TCDD (Fig. 6Go). Moreover, the degree of TCDD-induced suppression of IL-5 production was greater than that of OVA-specific IgG1 production. These results showed that IL-5 production is a good index for predicting the TCDD toxicity on antibody production by long-lived AFCs.

TCDD is known to be contained in exhaust particles from gasoline and diesel engine motor vehicles (Miyabara et al., 1999Go). These particles are main air pollutants in urban areas and have been suggested to be blamed for increases in allergic diseases such as asthma (Norris et al., 1999Go; Pandya et al., 2002Go). Diesel exhaust particles (DEP) have been reported to preferentially enhance antigen-induced Th2 responses, thereby exacerbating antigen-induced allergic asthma (Fujimaki et al., 1994Go; Takano et al., 1997Go). Among the chemicals contained in the vehicle exhaust particles, benzo[a]pyrene and lead have been reported to enhance Th2 cell-derived cytokine production (Fujimaki et al., 1997Go; Heo et al., 1996Go). By contrast, we have demonstrated that TCDD suppresses Th2 responses, suggesting that TCDD is not involved in the exacerbation of allergic immune responses. Consistent with this, previous studies have shown that TCDD suppresses allergic immune reactions in response to house dust mites (Luebke et al., 2001Go) or OVA (Fujimaki et al., 2002Go) and following atopic dermatitis.

In conclusion, we have demonstrated that TCDD exposure suppresses Th2 cell-derived cytokine production by spleen cells prior to the suppression of antigen-specific IgG1 production, which seems to lead to suppression of antibody production. Our results also suggest that the TCDD-induced suppression of T cell activation and expansion followed by lowering of the numbers of antigen-specific T cells are involved in the causation of reduced Th2 cell-derived cytokine production. The involvement of AhR in TCDD-induced immunotoxicity has been demonstrated by previous studies (Staples et al., 1998Go; Vorderstrasse et al., 2001Go). To fully understand the mechanism of its toxicity, further study is necessary to identify the AhR-dependent gene expressions that are responsible for the suppression of T cell activation and expansion. In addition, the direct action of TCDD on cytokine gene expressions also remains to be clarified, since cytokine genes like mouse IL-5 and IL-6 (GenBank accession number, D14461 and M20572) include XRE in their promoter regions and are possibly modulated by TCDD-activated AhR.


    ACKNOWLEDGMENTS
 
We thank M. Matsumoto and K. Nakazawa for their excellent technical and secretarial assistance, respectively.


    NOTES
 
1 To whom correspondence should be addressed at National Institute for Environmental Studies, Environmental Health Sciences Division, 16-2 Onogawa, Tsukuba 305-8506, Japan. Fax: +81-298-50-2574. E-mail: keikon{at}nies.go.jp. Back


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