* Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba 305-8506, Japan;
CREST, JST (Japan Science and Technology), Kawaguchi 332-0012, Japan; and
Japan Society for the Promotion of Science (JSPS), Tokyo 102-8471, Japan
Received June 18, 2002; accepted August 5, 2002
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ABSTRACT |
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Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; Th2 cell-derived cytokine; T cell growth factor; antibody production; primary immune reaction.
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INTRODUCTION |
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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., 1991; Kerkvliet, 2002
). 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., 1991
). 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, 1988
; Dooley et al., 1990
). A recent study by Sulentic et al. (2000)
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
-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)
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)
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, 1998). Subsequently, the activated helper T cells differentiate into type 1 helper T (Th1) cells or Th2 cells (Mosmann and Sad, 1996
). 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., 1990
; Mosmann and Sad, 1996
; Paul and Seder, 1994
).
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., 2002; Nohara et al., 2002
), 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.
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MATERIALS AND METHODS |
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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., 2002). 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., 2002). 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 (536.7). 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., 2002). 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 manufacturers 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 8895% T cells and < 0.2% B cells. The non-T cell fraction contained < 5% T cells, 7276% B cells, and 1520% 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 manufacturers instructions to determine IL-2, IL-4, IL-5, IL-6, and IFN- in culture supernatant. OVA-specific IgG1 in plasma was determined as previously described (Nohara et al., 2002
).
Statistical analysis.
Differences in means between control and TCDD-exposed groups in the time course experiment were assessed by Students t-test. The dose-dependent effects of TCDD were analyzed by one-way ANOVA followed by Fishers PLSD using StatView (ver. 4.57, SAS Institute, Cary, NC).
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RESULTS |
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DISCUSSION |
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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., 1992). 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. 7
). 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) 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., 1999
), whereas Shepherd et al. used complete Freunds adjuvant, which has been found to be very effective in inducing a potent immune reaction (Billiau and Matthys, 2001
).
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. 4). 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, 1988
; Kerkvliet et al., 1990
; Smialowicz et al., 1994
), 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)
. 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. 6
). 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., 1999). 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., 1999
; Pandya et al., 2002
). Diesel exhaust particles (DEP) have been reported to preferentially enhance antigen-induced Th2 responses, thereby exacerbating antigen-induced allergic asthma (Fujimaki et al., 1994
; Takano et al., 1997
). 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., 1997
; Heo et al., 1996
). 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., 2001
) or OVA (Fujimaki et al., 2002
) 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., 1998; Vorderstrasse et al., 2001
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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