* Department of Environmental and Molecular Toxicology and Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331
Received September 18, 2002; accepted December 5, 2002
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ABSTRACT |
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Key Words: dendritic cells; TCDD; immunotoxicity; antigen presentation; T cell activation.
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INTRODUCTION |
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Suppression of T cell-dependent immune responses is one of the primary toxicities associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reviewed by Kerkvliet, 1998). However, despite intensive study, the immunological mechanisms of suppression remain poorly understood. Until recently, DC have been overlooked as a potential target for TCDD. Yet, it has been known for some time that TCDD and related congeners must be given during the early stages of the immune response in order for suppression to result (Kerkvliet and Brauner, 1987
; Kerkvliet et al., 1996
; Luster et al., 1988
). This suggests that events occurring early in the generation of the response, such as activation of T cells by DC, are altered by exposure to TCDD. Additionally, immune responses that require antigen processing appear to be more sensitive to suppression than are mitogen- or superantigen-induced responses, which partially or completely bypass the need for normal APC function (Kerkvliet and Brauner, 1987
; Neumann et al., 1993
; Prell and Kerkvliet, 1997
; Vecchi et al., 1980
). Taken together, these data support the hypothesis that DC function is altered by TCDD.
Previously, we reported that, in the absence of antigen, TCDD exposure caused an apparent activation of DC (Vorderstrasse and Kerkvliet, 2001). Specifically, splenic DC from TCDD-treated mice produced more IL-12 and showed enhanced expression of ICAM-1, B7-2, CD24, and CD40, cell surface molecules which are characteristically upregulated following activation of the cells. In addition, DC from TCDD-treated mice enhanced T-cell proliferation and cytokine production when cultured with allogeneic T cells. Due to the immunosuppressive properties of TCDD, and the highly sensitive nature of T cell-dependent immune responses in particular, these results were unexpected and difficult to explain.
One possible explanation is that inappropriate preactivation of DC by TCDD negatively affects the function of the cells when processing and presentation of antigen is required. For example, activation of DC with stimuli such as TNF-, CD40L, IL-1ß, and LPS diminishes their ability to internalize antigen (Henderson et al., 1997
;Reis e Sousa et al., 1993
; Sallusto et al., 1995
). Furthermore, DC that have been activated with TNF-
in vitro or LPS in vivo prior to antigen exposure are significantly suppressed in their ability to subsequently activate antigen-specific T cells (De Smedt et al., 1996
; Sallusto and Lanzavecchia, 1994
).
In the studies presented here we have examined the effects of TCDD on DC function, specifically those involving antigen processing and presentation. We hypothesized that TCDD would suppress the ability of DC to activate T cells when processing and presentation of antigen was required. To test this hypothesis, we isolated DC from TCDD-treated mice and examined their ability to stimulate antigen-specific T cells ex vivo. In addition, the phagocytic activity of DC was evaluated using fluorescent beads. Finally, DC from mice injected with allogeneic tumor cells were examined to determine if TCDD exposure affects the expression of costimulatory molecules on DC from mice responding to antigen differently than DC from naïve mice.
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MATERIALS AND METHODS |
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TCDD exposure.
TCDD (Cambridge Isotope Laboratories, Inc., Woburn, MA) was dissolved in anisole and diluted in peanut oil. The vehicle control consisted of an equivalent amount of anisole in peanut oil. Mice were treated with vehicle or 15 µg/kg TCDD by gavage. This dose of TCDD is known to suppress immune responses in mice (Kerkvliet et al., 1996), and was chosen based on a previous dose-response study examining the effects of TCDD on DC phenotype (Vorderstrasse and Kerkvliet, 2001
).
Preparation of DC
Depending on the experimental design, DC were isolated from the spleens of vehicle- and TCDD-treated mice by various procedures described below. No consistent effect of TCDD on DC recovery was observed with any of the methods. Since the cells were obtained from TCDD-treated mice within 5 days of exposure, the loss of DC that occurs later (Vorderstrasse and Kerkvliet, 2001) was not a factor in these studies. Likewise, the median channel fluorescence of CD11c expression on the positive cells was not affected by treatment with TCDD.
For flow cytometric analysis.
DC were enriched from spleens using the method of Swiggard et al. (1992) with modifications as described in Inaba et al. (1997)
. Briefly, splenic tissue was digested with collagenase D (Boehringer Mannheim, Indianapolis, IN) at 37°C for 4560 min to release DC from the capsule and increase recovery. Recovered cells were spun over a BSA gradient (1.080 g/ml) and cells in the low-density fraction were collected. These freshly isolated DC-enriched preparations were then stained for flow cytometric analysis. Typically, 1525% of the low-density cells expressed high levels of CD11c (CD11cHI), a marker for splenic DC (Crowley et al., 1990b
)
For use as APC for 10.5.17 cells.
Low density spleen cells were obtained from C57Bl/6 mice as indicated above, and were further enriched for DC by overnight culturing based on their property of transient adherence to plastic (Swiggard et al., 1992). In this procedure, the low density cells were cultured in plastic dishes for 90 min to allow for the DC to adhere. The nonadherent contaminating cells were then washed away, and the remaining adherent cells left in culture overnight. DC become nonadherent during this culture period and were collected from media the following day. Purity after this final enrichment was greater than 80% CD11cHI cells.
For use as APC for DO11.10 T cells.
Spleens from Balb/c mice were digested with collagenase as indicated above. DC were enriched by incubating cell preparations with CD11c MicroBeads (Miltenyi Biotec, Auburn, CA) and subjecting the cells to magnetic separation following the manufacturers standard protocol. The concentration of DC was determined by flow cytometry. CD11cHI cells comprised 1347% of the recovered cells. Cell suspensions were irradiated (1500 rads) and cell concentrations were adjusted such that a constant number of DC were added to each well.
10.5.17 T Cells
A TH2 cloned T-cell line (10.5.17), derived from a C57Bl/6 mouse and specific for keyhole limpet hemocyanin (KLH), was obtained from Barbara Fox (Immunologic Pharmaceutical Corp., Waltham, MA). For normal maintenance, 10.5.17 cells were restimulated every 1014 days with irradiated C57Bl/6 spleen cells and KLH as previously described (Li and Fox, 1993). For proliferation and cytokine analysis, 10.5.17 cells were used 11 days after the last activation.
DC-Mediated Activation of 10.5.17 Cells
DC (1 x 104 cells per well) were cultured with 10.5.17 cells (1 x 104 per well) in standard 96-well tissue culture plates. KLH (Calbiochem, San Diego, CA) was either provided in vitro at 20 µg/ml or in vivo. For in vivo studies, DC were isolated from mice which had been injected ip with 4 mg KLH (in PBS) 3 h prior to sacrifice, as previously described (Crowley et al., 1990a). T cell proliferation was measured by incorporation of 3H-TdR that was added (1 µCi/well) during the last 20 h of culture. For IL-4 analysis, 60 µl supernatant was removed from each well just prior to addition of 3H-TdR (in 60 µl).
DC-Mediated Activation of DO11.10 T Cells
T cells were purified from spleens of DO11.10 T cell receptor transgenic mice using Cellect T-cell enrichment columns (Cytovax Biotechnologies Inc., Edmonton, Canada). Final T-cell purity was 80% CD4+ and 11% CD8+, and no B cells or MHC class II+ cells were detected. Various numbers of DC-enriched cells from Balb/c mice were cultured with T cells (3 x 105) and OVA protein (100 µg/ml, Sigma, St. Louis, MO) in 96-well Costar Ultra Low Cluster plates (Corning, Inc., Corning, NY). T-cell proliferation was measured by incorporation of 3H-TdR (1 µCi/well) that was added during the last 20 h of culture.
In Vivo Proliferation of DO11.10 T Cells
CFSE-labeling of DO11.10 splenocytes.
The proliferation of OVAspecific DO11.10 cells in vivo was monitored by labeling cells with 5- and 6-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Lyons and Parish, 1994). This dye freely diffuses into cells and becomes fluorescent and nonpermeable upon cleavage of the carboxyl groups by cellular esterases. CFSE is equally divided among daughter cells during cell division, thereby decreasing the fluorescence by half with each division. Single-cell suspensions of spleen cells were prepared from DO11.10 mice and were labeled by incubating the cells in 10 µM CFSE (Molecular Probes, Eugene, OR) for 8 min at room temperature in the dark. After labeling, the cells were adoptively transferred into syngeneic Balb/c hosts as described below.
Adoptive transfer.
Balb/c mice were treated with vehicle or TCDD 1 day prior to injection of DO11.10 cells as described previously (Kearney et al., 1994; Shepherd et al., 2000
). Briefly, spleen cells from untreated DO11.10 mice were collected, and the percentage of CD4+/KJ1-26+ cells determined by flow cytometry. Balb/c recipient mice (sex and age matched) were injected iv with 5 x 106 CD4+/KJ1-26+ T cells (total spleen cells injected
2 x 107) in a volume of 0.5 ml. The adoptively transferred mice were rested for 2 days and then immunized ip with 2 mg OVA emulsified in complete Freunds adjuvant (OVA-CFA).
Flow cytometric analysis of cell division.
Spleen cells from adoptively transferred mice were collected 1 or 3 days after injection of OVA-CFA. Cells were stained with anti-CD4 and KJ-126 antibodies to identify the DO11.10 cells, and analyzed by flow cytometry as described below. The CFSE content of the adoptively transferred cells was examined by establishing a gate on the CD4+/KJ1-26+ cells.
Flow Cytometry
Cells were incubated in 96-well plates with optimized concentrations of antibodies. Biotinylated-CD11c, as well as fluorochrome-conjugated antibodies to CD40, ICAM-1 (CD54), LFA-1 (CD11a), CD24, CD4, CD8, CD19, and I-Ab (MHCII) were obtained from BD PharMingen (San Diego, CA). Red670- and Red613-streptavidin were obtained from Gibco (Gaithersburg, MD). The KJ1-26 hybridoma (used to identify DO11.10 cells) was provided by Dr. M. Jenkins. KJ1-26 antibody was purified from culture supernatant using Protein A sepharose Fast Flow columns (Amersham Pharmacia) and biotinylated using N-hydroxysuccinimido-biotin (Sigma, St. Louis, MO). Pre-incubating the cells with rat, hamster, and/or mouse IgG blocked nonspecific antibody binding. Appropriately labeled isotype controls were used to determine nonspecific fluorescence. A viable cell gate was established based on propidium iodide exclusion. For DC cell surface molecule evaluation, 10,000 viable CD11cHI cells were analyzed. Listmode data were collected on a Coulter Epics XL flow cytometer and analyzed using WinList software (Verity Software House, Inc., Topsham, ME). WinList was also used occasionally for compensation during data analysis.
P815 Tumor Response
C57Bl/6 mice (H-2b) were injected ip with 1 x 107 allogeneic P815 mastocytoma cells (H-2d). Mice were sacrificed on days 14 postinjection for analysis of splenic DC. P815 cells were maintained by propagation in syngeneic DBA hosts.
Cytokine Analysis
Culture supernatants were analyzed using antibody sandwich ELISA techniques. IL-2, IFN-, and IL-4 antibody pairs were purchased from BD PharMingen, and antibodies to the p40 subunit of mouse IL-12 were purchased from Genzyme (Cambridge, MA). The secondary biotinylated antibodies were complexed with avidin-peroxidase and visualized with 2,2'-azinobis[3-ethylbenzthiazoline-6-sulfonic acid] as substrate. Absorbance was read at 405 nm using a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT).
In Vivo Uptake of Fluorescent Beads
FITC-fluorescent 2 µm latex beads (1 x 109; Polysciences, Warrington, PA) were injected via the tail vein. Twenty-four h later, spleens were digested with collagenase as described above, and uptake of beads by DC was analyzed by flow cytometry. Preliminary experiments were conducted to optimize the route of injection, dose, and optimum time for detection of the beads after injection.
Statistical Analysis
For most experiments, a Students t-test was used to compare means of vehicle-treated groups to TCDD-treated groups. For comparison of cell surface marker expression across multiple groups, analysis of variance modeling (ANOVA) was performed using SAS statistical software (SAS Institute, Inc., Cary, NC), and comparisons between means were made using the least significant difference (LSD) multiple comparison t-test. For all analyses, values of p 0.05 were considered significant.
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RESULTS |
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Accessory molecule expression was evaluated on days 1 through 4 following tumor injection, as the activation of CD4+ T cells has been shown to occur during this time (Kerkvliet et al., 1996). Vehicle- and TCDD-treated nonimmune animals (not injected with P815 cells) were also included as controls.
Figure 1 shows representative histograms of accessory molecule expression on DC evaluated 3 days after tumor injection. Similar results were observed on the other days examined. Injection of P815 cells alone did not significantly alter DC phenotype. Except for a small and transient increase in ICAM-1 expression on day 3, DC profiles from nonimmune mice treated with vehicle (Fig. 1A
, dotted line) and P815-injected mice treated with vehicle (Fig. 2B
, dotted line) were similar.
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Effect of TCDD on ability of DC to stimulate KLH-specific 10.5.17 cells.
In previous studies we found that TCDD enhanced the ability of DC to stimulate T-cell proliferation and cytokine production in an allogeneic mixed lymphocyte response (MLR), a response which does not require antigen processing. To evaluate DC function in a response that requires processing and presentation of antigen, we examined the ability of DC to activate KLH-specific cloned T cells (10.5.17). DC from vehicle- or TCDD-treated mice were cultured with 10.5.17 cells and KLH, and proliferation and IL-4 production were measured. As shown in Figure 2, TCDD exposure did not influence the ability of DC to process and present KLH to 10.5.17 T cells. Neither the proliferation of antigen-specific T cells nor the production of IL-4 was significantly different in cultures containing DC from vehicle- or TCDD-treated mice.
The method used to isolate DC in these experiments included an overnight culturing step, a procedure that has been shown to activate DC and reduce their ability to internalize antigen (Inaba et al., 1994;Reis e Sousa et al., 1993
). Therefore it was possible that T-cell activation resulted from presentation of peptide fragments on surface MHC, and did not reflect the ability of DC to internalize antigen. To better assess the potential for TCDD to alter the uptake of antigen, we also tested the APC function of DC which had been exposed to KLH antigen in vivo. In these experiments, vehicle- and TCDD-treated mice were injected with PBS or KLH and sacrificed 3 h later. Splenic DC were isolated and cultured with 10.5.17 cells without additional antigen. As shown in Figure 3
, DC from KLH-, but not PBS-injected mice stimulated proliferation of 10.5.17 cells, indicating that the DC had effectively phagocytized and processed KLH antigens in vivo. As in the experiments where KLH was provided in vitro, TCDD did not suppress the ability of DC from KLH-injected mice to stimulate proliferation of 10.5.17 cells. These results confirm that TCDD does not suppress the uptake and processing of antigen by DC.
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As shown in Figure 4, the proliferation of OVA-specific T cells was not suppressed in the presence of DC from TCDD-treated mice, but was instead significantly enhanced compared to the response with DC from vehicle-treated mice. This enhancement of proliferation occurred independent of the time of assessment or number of DC added to the cultures. On the other hand, as shown in Figure 5
, the production of T cell-derived cytokines, IL-2 and IFN-
, and the production of DC-derived IL-12 were not significantly different in cultures containing DC from vehicle- or TCDD-treated mice
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DISCUSSION |
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In the current studies we examined the effects of TCDD on DC surface markers in mice challenged with antigen, and tested the ability of DC from TCDD-treated mice to present antigen to antigen-specific T cells. Interestingly, the results of the current studies do not support the hypothesis that exposure to TCDD suppresses the ability of DC to process and present antigen to T cells. Antigen-dependent T-cell proliferation and cytokine production was either unaffected or enhanced in the presence of DC from TCDD-treated mice. Likewise, the ability of DC to internalize latex beads was not affected by TCDD. These observations are consistent with results from other recent studies where TCDD did not suppress the early proliferation or phenotypic activation of OVA-specific CD4+ T cells in vivo in adoptively transferred mice (Shepherd et al., 2000).
Contrary to suppression of T-cell activation, exposure to TCDD enhanced the ability of DC to stimulate proliferation of antigen-specific T cells in vitro when naïve DO11.10 T cells were used as responders. Similarly, enhanced OVA-specific T-cell proliferation was observed in vivo when CFSE-labeled DO11.10 T cells were adoptively transferred into TCDD-treated Balb/c mice. Interestingly, recent studies indicate that following the initial T-cell expansion, an increased percentage of the DO11.10 T cells from TCDD-treated mice undergo apoptosis (Funatake et al., manuscript in preparation), suggesting that TCDD may induce a type of activation-induced cell death (AICD). If true, this conclusion would be consistent with the significantly reduced numbers of D011.10 T cells observed in TCDD-treated mice at later time points during the anti-OVA response (Shepherd et al., 2000). Similar results have been shown in mice treated in vivo with polyclonal T-cell activators (anti-CD3 or Staphyloccus enterotoxin A), where TCDD exposure resulted in enhanced T-cell proliferation followed by an apparent increase in death of CD4+ T cells (Camacho et al., 2001
, 2002
; Dearstyne and Kerkvliet, 2002
; Prell et al., 1995
). It is also possible that TCDD enhances the deletion of DC, since, once activated, DC are committed to undergo apoptosis (De Smedt et al., 1996
, 1998
; Winzler et al., 2000). Since DC and T cells must remain in close association for T cells to become fully activated, premature death of DC may contribute to premature death or incomplete activation of T cells (Josien et al., 2000
; Miga et al., 2001
). This possibility is supported by previous studies showing that exposure to TCDD significantly reduces the number of DC in the spleen (Vorderstrasse and Kerkvliet, 2001
).
Although DC from TCDD-treated Balb/c mice enhanced the proliferation of the DO11.10 T cells relative to DC from vehicle-treated animals, the production of cytokines was not altered. This finding is in agreement with in vivo studies, where TCDD had little effect on the production of various T cell cytokines (Shepherd et al., 2000). However, these results contrast with our previous observations where DC from TCDD-treated C57Bl/6 mice produced more IL-12 and stimulated enhanced production of IFN-
and IL-2 when cultured with allogeneic T cells (Vorderstrasse and Kerkvliet, 2001
). The reasons for these conflicting results are not clear. However, C57Bl/6 and Balb/c mice may respond to TCDD differently in terms of cytokine production since these strains are considered to be predisposed toward Th1 (C57Bl/6) and Th2 (Balb/c) responses (Brenner et al., 1994
). Further studies will be needed to clarify such differential effects. Alternatively, different methods were used to enrich DC in the current study (magnetic beads) than in the previous studies using cells from C57Bl/6 mice (adherence to plastic). Thus it is possible that the method by which the DC were isolated in the different experiments contributed to the difference in cytokine production. For example, Vremec et al. (2000)
recently reported that particular subsets of DC were selectively depleted when splenic DC were isolated by adherence to plastic. It is therefore possible that TCDD enhances IL-12 production in a subpopulation of DC that was selectively enriched in the experiments with C57Bl/6 mice. Alternatively, cytokine production may have been influenced by some other cell population, which contaminated the DC preparations used to activate the DO11.10 T cells. However, this is unlikely as we failed to find a relationship between T cell activation and the other potential antigen-presenting cells present in the DC preparations.
TCDD exposure did not suppress the expression of costimulatory molecules on splenic DC isolated from mice responding to challenge with allogeneic P815 tumor cells. This result was somewhat unexpected since previous studies have shown that TCDD suppresses the expression of CD86 on B220+ and Mac-1+ cells in the same model (Prell and Kerkvliet, 1997; Shepherd et al., 2001
). However, the effect of TCDD on CD86 expression was not seen until later in the response, suggesting that the changes were not associated with the disruption of early T-cell activation events. In contrast to suppression, in the current studies, TCDD significantly increased the expression of CD40, CD24, ICAM-1, and MHC class II on DC on days 14 after P815 tumor challenge. This increased expression was identical to the increased expression of accessory molecules on DC from naïve mice treated with TCDD, suggesting that the changes noted were not antigen-induced. Since injection of P815 cells alone did not alter accessory molecule expression, it also suggests that the subpopulation of DC that are directly involved in processing and presenting P815 alloantigens to CD4+ T cells was below detection limits. Therefore it remains possible that TCDD influences accessory molecule expression on the actual antigen-bearing DC, which might influence T-cell activation in this model.
In summary, although we hypothesized that the inappropriate activation of DC by TCDD would suppress their ability to internalize antigen and activate T cells, results from the current studies do not support this conclusion. Instead we have found that TCDD-induced activation of DC occurs both with and without antigen and does not appear to inhibit the ability of the DC to activate T cells. Because treatment with TCDD caused a phenotypic activation of DC and enhanced their ability to stimulate the proliferation of naïve DO11.10 T cells, further studies are warranted to determine whether TCDD-induced activation of DC plays a role in suppression of T cell-dependent immune responses.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: NeoRx Corporation, 300 Elliott Avenue West, Suite 500, Seattle, WA 98119.
3 To whom correspondence should be sent at 1007 Agricultural and Life Sciences Building, Oregon State University, Corvallis, OR 97331. Fax: (541) 737-0497. E-mail: nancy.kerkvliet{at}orst.edu.
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