2,3,7,8-Tetrachlorodibenzo-p-dioxin affects the number and function of murine splenic dendritic cells and their expression of accessory molecules

Primary T cell-mediated immune responses are highly susceptible to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, yet direct effects of TCDD on T cells have been difficult to demonstrate. Since the activation of naive T cells has been shown to be initiated primarily by dendritic cells (DC), these cells represent a potential target for TCDD immunotoxicity. In this report, we have examined the influence of TCDD exposure on splenic DC phenotype and function in the absence of antigenic stimulation. Results showed that DC from TCDD-treated mice expressed higher levels of several accessory molecules including ICAM-1, CD24, B7-2, and CD40, whereas the expression of LFA-1 was significantly reduced. These effects were dose-dependent and persisted for at least 14 days after exposure. The effects were also dependent upon the aryl hydrocarbon receptor (AhR), as similar effects were observed in AhR+/+ C57Bl/6 and Balb/c mice but not in AhR-/- mice. When DC from TCDD-treated mice were cultured with allogeneic T cells, the proliferative response and production of IL-2 and IFN-gamma by the T cells were increased. Production of IL-12 by the DC was likewise enhanced in comparison to cells from vehicle-treated mice. Interestingly, however, the number of DC recovered from TCDD-treated mice was significantly decreased. Taken together, these results suggest that, in the absence of antigen, TCDD provides an activation stimulus to DC that may lead to their premature deletion. Since the survival of DC has been shown to influence the strength and duration of the immune response, these results suggest a possible novel mechanism for TCDD-induced immune suppression.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cell-derived cytokine production in ovalbumin (OVA)-immunized C57Bl/6 mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress both cellular and humoral immunity. Effector T cell-derived type-2 cytokines, including IL-4 and IL-5, play pivotal roles in humoral immunity. Herein, we studied whether TCDD affects type-2 cytokine productions during the immune response. C57Bl/6 mice were intraperitoneally immunized with ovalbumin (OVA) and orally administered 5 or 20 microg TCDD/kg on Day 0, and then challenged with OVA on Day 21. Seven days later (Day 28), antigen-specific antibodies in plasma, and T cell-derived cytokines produced by splenocytes and proliferation of splenocytes upon ex vivo re-stimulation with OVA were investigated. The quantities of IgM class and IgG1 class OVA-specific antibodies in plasma were reduced by 5 or 20 microg TCDD/kg and by 20 microg TCDD/kg, respectively. While thymus weight and cellularity were reduced by 20 microg TCDD/kg, spleen weight and cellularity were not changed by either 5 or 20 microg TCDD/kg. The proportions of B and T cells in the spleen were not affected by TCDD exposure. On the other hand, splenocytes from mice treated with 5 or 20 microg TCDD/kg were shown to produce less IL-4 or IL-5 upon ex vivo re-stimulation with OVA. Production of the T cell growth factor IL-2 was also decreased in splenocytes from TCDD-treated mice. In contrast, the type-1 cytokine IFN-gamma was increased by TCDD. Twenty micrograms of TCDD/kg suppressed OVA- or T cell mitogen (Con A)-stimulated proliferation of splenocytes, but did not affect B cell mitogen (LPS)-stimulated proliferation. These results suggested compromised T cell activation and suppressed type-2 cytokine production by T cells to be involved in the impaired humoral immunity associated with TCDD exposure.     

Early consequences of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on the activation and survival of antigen-specific T cells.

TCDD is a potent immunotoxicant that suppresses adaptive immunity by mechanisms that are not well defined. To gain insight at the level of the T cell, we used the DO11.10 transgenic T-cell receptor (TCR) mouse model in an adoptive transfer approach to characterize the influence of TCDD on the responsiveness of antigen-specific CD4+ T cells in vivo. Flow cytometry was used to track the response of the OVA-specific transgenic CD4+ T cells in syngeneic recipients using an antibody specific for the transgenic TCR (KJ1-26 [KJ]). Consistent with a previous report, exposure of the recipient mice to TCDD (15 microg/kg po) did not alter the initial expansion of the CD4+KJ+ T cells in the spleen following immunization with OVA but resulted in a significant decline in the number of cells present on and after day 4. The degree of decline was dependent on the dose of TCDD. On day 3 after OVA injection, a higher percentage of the CD4+KJ+ T cells in the spleens of TCDD-treated mice had down-regulated the expression of CD62L, a phenotype associated with T-cell activation. Also on day 3, an increased number of CD4+KJ+ T cells were found in the blood of TCDD-treated mice. However, as in the spleen, the number of CD4+KJ+ T cells in the blood rapidly declined on day 4. CD4+KJ+ T cells in both the spleen and blood of TCDD-treated mice failed to up-regulate CD11a, an adhesion molecule important for sustained interaction between T cells and DC whereas the up-regulation of the adhesion molecule CD49d was not altered. Based on analysis of cell division history, CD4+KJ+ T cells in vehicle-treated mice continued to divide through day 4 whereas CD4+KJ+ T cells in TCDD-treated mice showed no further division after day 3. Increased annexin V staining on CD4+KJ+ T cells in TCDD-treated mice was also observed but not until days 5 and 6. Fas-deficient CD4+KJ+ T cells were depleted from the spleen of TCDD-treated mice in a manner similar to wild-type CD4+KJ+ T cells, suggesting that Fas signaling does not play a critical role in this model. On the other hand, gene array analysis of purified CD4+KJ+ T cells on day 3 showed that the expression of several genes associated with cell survival/death were altered by TCDD. Taken together, the results are consistent with our hypothesis that TCDD provides an early but inappropriate activation signal to the antigen-specific T cells that allows, and possibly enhances, the initial activation and proliferation of the T cells, yet at the same time, interferes with the vital expression of certain adhesion/costimulatory molecules that serve to enhance the survival of the T cells. These changes result in truncated proliferation, increased T-cell death, and suppression of the adaptive immune response.     

Anti-CD40 Treatment of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed C57Bl/6 mice induces activation of antigen presenting cells yet fails to overcome TCDD-induced suppression of allograft immunity

We have previously demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the induction of the costimulatory molecule CD86 (B7-2) on B220+ and Mac-1+ spleen cells following the injection of allogeneic P815 tumor cells. In this study, TCDD exposure was shown to suppress CD54 and major histocompatibility complex (MHC) class II expression on B220+, Mac-1+, and CD11c+ splenic antigen presenting cells (APC). Furthermore, interleukin-12 (IL-12) production by spleen cells from P815-immunized mice was significantly decreased following exposure to TCDD. To determine if exogenous costimulation could enhance the activation of APC, vehicle- and TCDD-treated mice were injected with an agonistic antibody to murine CD40. Stimulation with anti-CD40 increased the expression of CD86, CD54, and MHC class II on splenic APC and greatly enhanced the production of interleukin-12. TCDD treatment had minimal effects on the anti-CD40-induced expression of accessory molecules on splenic APC. TCDD exposure had no effect on anti-CD40-induced IL-12 in the plasma but suppressed its production from cultured spleen cells. Surprisingly, although stimulation via CD40 increased the activation of APC, allograft effector functions were not restored in TCDD-treated mice, perhaps due to persistent defects in antigen processing and presentation, cytokine production, T cell function, or CD40-independent pathways of APC activation.     

Enhanced activation-induced cell death as a mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity in peripheral T cells

T cells upon activation undergo apoptosis, a process termed activation-induced cell death (AICD). In the current study, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases AICD and whether this constitutes one of the mechanisms by which TCDD induces immunotoxicity. To this end, C57BL/6+/+, C57BL/6 gld/gld (Fas ligand-defective) and C57BL/6 lpr/lpr (Fas-deficient) mice were injected with TCDD (50 microg/kg body weight, ip) or the vehicle (corn oil) and with anti-CD3 mAbs into the footpads. 3 days later, inguinal and popliteal lymph node cells were harvested, pooled and enumerated. Cells were cultured in vitro with anti-CD3 mAbs and cell proliferation was measured. Also, such cells were studied for their ability to undergo apoptosis upon in vitro culture with either tissue culture medium alone or with anti-CD3 mAbs. The data demonstrated that lymph nodes from TCDD-treated wild-type (+/+) mice showed a decrease in cellularity and the T cells exhibited decreased responsiveness to anti-CD3 mAbs when compared to the vehicle-treated control group. Furthermore, such cells from TCDD-treated mice exhibited increased levels of apoptosis upon in vitro culture when compared to the cells from vehicle-treated mice. In contrast, activated lymph nodes from TCDD-treated C57BL/6 gld/gld and C57BL/6 lpr/lpr mice showed normal cellularity and T cell responsiveness to anti-CD3 stimulation when compared to the vehicle controls. In addition, the activated lymph node T cells from the TCDD-treated C57BL/6 gld/gld and C57BL/6 lpr/lpr mice failed to exhibit increased apoptosis when compared to the controls. The current study demonstrates that the immunotoxic effects of TCDD in activated peripheral T cells may result from increased AICD mediated through Fas-Fas ligand interactions.     

Elucidation of cellular targets responsible for tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of antibody responses: II. The role of the T-lymphocyte

Various immunological assays have been applied by our laboratory in an attempt to assess the role of the T-cell in the TCDD-induced suppression of the antibody response by murine B6C3F1 splenic lymphocytes. Animals were treated in vivo (via gavage) with 1.0 microgram/kg TCDD in corn oil for 5 days before in vitro analysis of splenocyte immunocompetence and T-cell function. To study the effects on T-helper cell function, alterations in the proliferative responses of T-cells following TCDD exposure were investigated. Results show no significant difference in [3H]thymidine uptake between vehicle- and TCDD-treated whole splenocytes 24 h after in vitro stimulation with the T-cell mitogen Con A. This is consistent with the finding that IL-2 production at either 24 or 48 h after Con A stimulation of TCDD-treated lymphocytes was not significantly different from that of vehicle-treated controls. The possibility of the induction of a suppressor T-cell by TCDD was also investigated. Titration of T-cells from TCDD-treated mice into naive splenocyte cultures did not suppress the humoral response to either a T-dependent (SRBC) or a T-independent (DNP-Ficoll) antigen. In contrast, titration of cells stimulated in vitro with Con A for 48 h (a positive control for the induction of a suppressor T-cell) inhibited humoral responses of naive cells to both types of antigen. Likewise, T-cells plus macrophages from TCDD-treated mice did not suppress the in vitro humoral responsiveness of naive B-cells plus macrophages to a T-independent antigen (DNP-Ficoll). These results would indicate that an alteration in T-cell function following TCDD exposure does not play a role in the suppression of the antibody response elicited by antigen stimulation of murine B6C3F1 splenocytes.     

FEWER T LYMPHOCYTES AND DECREASED PULMONARY INFLUENZA VIRUS BURDEN IN MICE EXPOSED TO 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD)

The immune system is a sensitive target for the toxicity of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In mice, immunotoxicity associated with exposure to TCDD includes suppression of humoral and cell-mediated immunity and impaired host resistance to infectious agents. However, the underlying mechanisms for these effects of TCDD are unknown. We previously reported that treatment of C57Bl/6 mice with TCDD and influenza A virus increases mortality, impairs cytokine production, and suppresses T cell expansion and the generation of cytotoxic T lymphocytes (CTL) in the draining lymph node. However, the overall virus-specific cytolytic activity in the lung is unaffected. This enigmatic finding left several questions unanswered, including whether decreased CD8+ lymphocytes in the lung are the consequence of either delayed or suppressed recruitment of cells to the lung; whether exposure to TCDD affects the recruitment of CD4+ cells to the lung; and what effect TCDD treatment has on pulmonary virus burden. To compare the kinetics of the response in vehicle- and TCDD-treated mice, we examined the number of bronchoalveolar lavage (BAL) cells and CTL activity in the lung through d 10 postinfection. We found that the peak day for cellular influx and cytolytic activity in the lung is 9 d after infection. Immunophenotypic analysis of BAL cells shows that, when compared with BAL cells from infected controls, exposure to TCDD caused a 50% decrease in the percentage and number of both CD4+ and CD8+ cells. When the pulmonary virus burden was examined over time, we found that on d 1-5 postinfection, lungs from mice exposed to TCDD generally had lower virus titers than lung homogenates from vehicle-treated controls. By d 9 postinfection, no influenza virus was detected in lung homogenates from either vehicle- or TCDD-treated mice. These findings are likely not related to the observed decrease in CD4+ and CD8+ BAL cells; moreover, the diminished CD4+ population in the lung indicates that CD4+ cells are probably not compensating for the decreased CTL generation in TCDD-treated mice. Our observation that mice exposed to TCDD and infected with influenza virus do not have an increased pulmonary virus burden suggests either that TCDD treatment alters the host response to infection, creating a cellular environment that is less supportive for viral growth, or that exposure to TCDD directly affects influenza virus, leading to impaired virus replication within lung epithelial cells.     

Functional alterations in CD11b(+)Gr-1(+) cells in mice injected with allogeneic tumor cells and treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure results in an increased percentage of CD11b(+) (Mac-1(+)) cells in the spleens of mice challenged with P815 tumor cells, coincident with a failure of the mice to generate allospecific CD8(+) CTL activity. Since CD11b(+)Gr-1(+) myeloid suppressor cells (MSC) have been described as that which prevent cytotoxic T lymphocyte (CTL) development in a variety of disease states, we hypothesized that TCDD promoted MSC development, leading to suppression of CTL activity. The purpose of the present studies was to compare the phenotypic and functional characteristics of CD11b(+) cells in vehicle- and TCDD-treated mice during the P815 tumor allograft response to determine their potential to function as MSC. Initial studies showed that virtually all splenic CD11b(+) cells in both vehicle- and TCDD-treated mice co-expressed Gr-1. Consistent with MSC activity, CD11b(+)Gr-1(+) cells isolated from TCDD- but not vehicle-treated mice suppressed the development of CTL activity when added in vitro to mixed lymphocyte-P815 tumor cell cultures. Also consistent with MSC activity, this suppressive effect in vitro required cell-to-cell contact. Surprisingly, however, in vivo depletion of CD11b(+)Gr-1(+) cells failed to affect TCDD-induced suppression of the CTL response, arguing against an immunoregulatory role for the cells in vivo. Immunohistochemical analysis of the spleen showed that CD11b(+)Gr-1(+) cells were localized in the red pulp, and physically separated from the T cells in the white pulp. The localization of CD11b(+)Gr-1(+) cells in the red pulp was indicative of extramedullary myelopoiesis and suggested that TCDD enhanced myelopoiesis. A significantly enhanced neutrophilia in the blood of TCDD-treated mice supported this conclusion. CD11b(+)Gr-1(+) cells isolated from the blood or spleen of TCDD-treated mice produced up to fivefold higher levels of superoxide following PMA stimulation when compared with cells from vehicle-treated mice. However, unlike vehicle-treated mice, CD11b(+)Gr-1(+) cells from TCDD-treated mice were unable to kill YAC-1 target cells. These results indicate that TCDD exposure alters the host response to allogeneic tumor growth, resulting in enhanced myelopoiesis perhaps as a compensatory response to the suppressed T cell-mediated immunity in the face of an increasing P815 tumor burden. Furthermore, within the context of the P815 response, TCDD appears to alter the functional capabilities of mature neutrophils, by enhancing their oxidative burst capacity but reducing their tumoricidal response.     

Mechanism of TCDD-induced suppression of antibody production: effect on T cell-derived cytokine production in the primary immune reaction of mice.

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 microg/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 microg/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.     

Effect of 2,3,7,8-tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin (TCDD) on maternal immune response during pregnancy

Whether pregnancy-induced immunosuppression when combined with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could exacerbate immunotoxicity has not been previously investigated. The current study evaluated the immune status of C57BL/6 pregnant and virgin mice following exposure to TCDD. To this end, syngeneically pregnant or virgin female mice were injected intraperitoneally with 10 µg/kg TCDD. Pregnancy alone significantly decreased thymic cellularity and J11d expression as well as induced changes in T-cell subsets. TCDD treatment caused significant thymic atrophy in pregnant mice as early as 48 h after exposure, but this effect was apparent in virgin mice only after 72 h. TCDD treatment also caused more marked alterations in thymic T-cell subpopulations of pregnant mice when compared to the virgin mice, with a decrease in the percentage of double-positive T cells and an increase in the percentage of single-positive (sP CD4⁺ or sP CD8⁺) and double-negative T cells. Moreover, the proliferative responses of thymocytes, but not splenocytes, to mitogens were significantly altered in TCDD-treated pregnant mice when compared to the TCDD-treated virgin mice. Furthermore, no significant changes in the expression of CD4, CD8, B220 and NK1.1 markers were found in splenocytes from TCDD-treated virgin and pregnant mice. Immunization of mice with a superantigen caused a similar immunotoxic response in TCDD-treated pregnant and virgin mice with a decreased lymph node cellularity and lower percentages and cell numbers of V3⁺ and V11⁺ T cells. Together, the results of the current study demonstrate for the first time that pregnancy augments the sensitivity to TCDD-induced immunotoxicity in the thymus, but not in secondary lymphoid organs.     

Suppression of splenic accessory cell function in mice exposed to gallium arsenide.

Acute exposure of mice to a single intratracheal dose of gallium arsenide (50, 100, and 200 mg/kg) depresses the primary IgM antibody response to the T-dependent antigen sheep red blood cells (SRBC) through alterations in the function of splenic accessory cells. To determine the mechanism by which GaAs exposure influences splenic accessory cells, the cells were isolated by adherence and their functional capability investigated 24 hr following GaAs exposure in the animal. Splenic adherent cells from GaAs-exposed mice were greatly impaired in their ability to process and present the particulate antigen SRBC to a SRBC-primed T-cell population. However, GaAs exposure did not inhibit phagocytosis of fluorescent covaspheres by these cells, nor did it inhibit in vivo phagocytosis of 51Cr-labeled SRBC, indicating that the findings reported here were not due to decreased uptake of antigen by the accessory cells. Furthermore, production of IL-1 by these cells from exposed mice was not different from control and addition of exogenous IL-1 to cultures did not reverse GaAs-induced inhibition of the primary antibody response. GaAs exposure did not affect the percentage of Ia positive macrophages (F4/80 positive cells), but the amount of cell surface IAk molecules expressed was significantly decreased as measured by flow cytometry. In contrast to the SRBC response, GaAs did not suppress the ability of adherent splenocytes to process and present the antigen pigeon cytochrome c to the helper/inducer T cell clone F1.A.2 or the antigen KLH (keyhole limpet hemocyanin) to KLH-primed T cells. Therefore, GaAs exposure interferes with the capacity of splenic macrophages to process and/or present the particulate antigen SRBC, but not the soluble protein antigens pigeon cytochrome c or KLH.     

The effects of TCDD on the activation of ovalbumin (OVA)-specific DO11.10 transgenic CD4(+) T cells in adoptively transferred mice.

Exposure to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses the generation of T cell-dependent immunity, both humoral and cell-mediated. However, the mechanism of TCDD-induced immune suppression remains to be defined. We hypothesized that exposure to TCDD suppresses the activation of naive CD4(+) T cells and prevents their expansion and differentiation into effector T-helper cells capable of driving T cell-dependent immune responses. To test this hypothesis, we adoptively-transferred DO11.10 OVA-specific T-cell receptor (TCR) transgenic T cells into syngeneic recipients and used a TCR-specific monoclonal antibody to track the in vivo activation of naive CD4(+) T lymphocytes following exposure to OVA. The production of OVA-specific antibodies was suppressed in a dose-dependent manner in adoptively transferred mice that had been exposed to TCDD. Although TCDD exposure had little effect on the expansion or activation of the adoptively transferred, OVA-specific CD4(+) T cells, these cells disappeared from the spleen more rapidly in TCDD-treated mice and produced significantly decreased levels of the T cell-derived cytokines IL-2 and IL-10. There was also a trend towards reduced IFN-gamma and IL-4 production following in vitro re-stimulation. These data suggest that TCDD may interfere with the survival and/or differentiation of OVA-specific T-helper cells. These results demonstrate for the first time the potential of the DO11.10 adoptive transfer system to directly assess immunotoxic effects of xenobiotics on antigen-specific CD4(+) T cells in vivo.     

Examining the relationship between impaired host resistance and altered immune function in mice treated with TCDD

Exposure to TCDD suppresses the immune response to numerous antigens, including bacterial and viral pathogens. Although we administer a non-lethal infection with influenza A virus, we often observe significant mortality in TCDD-treated animals. With the goal of identifying which TCDD-induced defects impair host resistance, we conducted a dose response study to examine whether alteration of particular immunological endpoints could be correlated with mortality. C57Bl/6 mice were treated with vehicle control, or 1, 2.5, 5, 7.5 or 10 microg/kg TCDD 1 day prior to intranasal (i.n.) infection with influenza virus. Survival was monitored for 9 days, when remaining mice were sacrificed and multiple endpoints evaluated. Lymphocyte migration to the lung and the production of virus-specific IgG2a, IgG1, and IgG2b antibodies were significantly diminished, even at the lower doses. IgA was enhanced in all groups treated with TCDD. In contrast, T cell expansion in the lymph node, and the production of IFNgamma and IL-12 were relatively resistant to suppression. Treatment with TCDD also enhanced pulmonary neutrophilia in infected mice. These results suggest that decreased antibody production and hyperinflammation may contribute to the death of TCDD-treated mice, and underscore the importance of evaluating numerous endpoints before concluding that a chemical is or is not immunotoxic.     

Thymosins alpha 1 and beta 4 potentiate the antigen-presenting capacity of macrophages

The immunomodulatory effects of thymosins on the Ag-presenting capacity of macrophages were investigated. Using an in vitro antigen (Ag)-specific macrophage-dependent T-cell proliferation system, we found that both thymosin alpha 1 (T alpha 1) and thymosin beta 4 (T beta 4) augment the Ag-presenting capacity of macrophages. Macrophage monolayers were pulsed with keyhole limpet hemocyanin (KLH) in the absence or presence of thymosins, washed and overlaid with spleen cells. Splenocytes were collected, mitomycin C-treated and injected into syngeneic mice. Draining lymph node cells were tested for Ag-specific response by measuring proliferation, interleukin 2 (IL-2) secretion and expression of IL-2 receptors (IL-2R) on their cell surface. We found that the presence of thymosins during the pulsing of macrophages with KLH led to significantly enhanced lymph node cell proliferation responses to KLH was correlated to increased IL-2 production and IL-2R expression. The concentrations of T alpha 1 and T beta 4 required for amplification were 10(-8) to 10(-10) M, well within the physiological range of activity of most peptide hormones. The observed enhancement of IL-2 secretion was not accompanied by interleukin 4 (IL-4) production. This study is the first to demonstrate that thymic hormones have the ability to increase the efficiency of antigen presentation by macrophages. The results suggest that an initial step in the regulation of the immune function by T alpha 1 and T beta 4 may involve activation of the macrophages at the time of antigen presentation.     

Enhancement of human T lymphocyte functions by prothymosin α. I. Augmentation of mixed lymphocyte culture reactions and soluble protein-induced proliferative responses

Prothymosin α(ProTα), a 115-amino-acid thymic polypeptide, was tested for its effect on soluble antigen, allo- and auto-antigen-induced human T-cell proliferation. ProTα enhanced the secondary T-cell proliferative response to ovalbumin (OVA)- and keyhole limpet haemocyanin (KLH)-pulsed antigen-presenting cells (peripheral blood monocytes). Maximum enhancement (20-fold for OVA and 23-fold for KLH) occurred when suboptimal concentrations of either OVA or KLH were employed. Subset depletion experiments showed that the helper/inducer T-cell subpopulation was responsible for the observed enhancement. In the mixed lymphocyte reaction (MLR), ProTα enhanced autoantigen- (autoMLR; 9- to 14-fold) as well as the alloantigen- (alloMLR; 8- to 10-fold) induced T-cell proliferation when suboptimal ratios of the participating cells were used. Preincubation of the stimulating (autologous or allogeneic monocytes) with ProTα induced significantly higher T-cell proliferation in both primary and secondary MLR responses as compared to that induced by non-treated monocytes. In contrast, T lymphocytes pre-incubated with ProTα did not show enhanced proliferative activity when tested subsequently in the MLR. Suboptimal numbers of T cells exhibited high proliferative activity when pre-incubated with ProTα in the presence of autologous monocytes. These studies suggest that ProTα potentiates T-cell proliferative responses not directly, but via monocytes which are included in the cultures either as antigen-presenting cells or accessory and/or stimulator cells. The importance of ProTα in pathologically occuring defective cellular immune responce systems is discussed.     

Fewer T lymphocytes and decreased pulmonary influenza virus burden in mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The immune system is a sensitive target for the toxicity of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In mice, immunotoxicity associated with exposure to TCDD includes suppression of humoral and cell-mediated immunity and impaired host resistance to infectious agents. However, the underlying mechanisms for these effects of TCDD are unknown. We previously reported that treatment of C57BI/6 mice with TCDD and influenza A virus increases mortality, impairs cytokine production, and suppresses T cell expansion and the generation of cytotoxic T lymphocytes (CTL) in the draining lymph node. However, the overall virus-specific cytolytic activity in the lung is unaffected. This enigmatic finding left several questions unanswered, including whether decreased CD8+ lymphocytes in the lung are the consequence of either delayed or suppressed recruitment of cells to the lung; whether exposure to TCDD affects the recruitment of CD4+ cells to the lung; and what effect TCDD treatment has on pulmonary virus burden. To compare the kinetics of the response in vehicle- and TCDD-treated mice, we examined the number of bronchoalveolar lavage (BAL) cells and CTL activity in the lung through d 10 postinfection. We found that the peak day for cellular influx and cytolytic activity in the lung is 9 d after infection. Immunophenotypic analysis of BAL cells shows that, when compared with BAL cells from infected controls, exposure to TCDD caused a 50% decrease in the percentage and number of both CD4+ and CD8+ cells. When the pulmonary virus burden was examined over time, we found that on d 1-5 postinfection, lungs from mice exposed to TCDD generally had lower virus titers than lung homogenates from vehicle-treated controls. By d 9 postinfection, no influenza virus was detected in lung homogenates from either vehicle- or TCDD-treated mice. These findings are likely not related to the observed decrease in CD4+ and CD8+ BAL cells; moreover, the diminished CD4+ population in the lung indicates that CD4+ cells are probably not compensating for the decreased CTL generation in TCDD-treated mice. Our observation that mice exposed to TCDD and infected with influenza virus do not have an increased pulmonary virus burden suggests either that TCDD treatment alters the host response to infection, creating a cellular environment that is less supportive for viral growth, or that exposure to TCDD directly affects influenza virus, leading to impaired virus replication within lung epithelial cells.     

CTL hyporesponsiveness induced by 2,3,7, 8-tetrachlorodibenzo-p-dioxin: role of cytokines and apoptosis

Studies have shown that blocking B7-mediated costimulation induces T cell tolerance via anergy or apoptosis. Provision of exogenous IL-2 can reverse or prevent the induction of tolerance. We have previously shown that TCDD-induced suppression of the CTL response to allogeneic P815 tumor cells is accompanied by decreased expression of CD86 (B7-2) as well as suppressed IL-2 and IFNgamma production. In the present studies, the role of IL-2 and IFNgamma and the analysis of inappropriate deletion of CD8(+) cells was examined. Administration of IL-2 on days 7-9 relative to the injection of P815 tumor cells dose-dependently increased the CTL activity and the generation of CD8(+) CTL effector cells in TCDD-treated mice. This increased CTL response was not due to recruitment of naive CTL precursors (CTLp), suggesting that a small pool of activated CTLp in TCDD-treated mice could respond to the IL-2. A much larger pool of activated CTLp in control mice was also expanded by IL-2 treatment. In contrast, treatment with IFNgamma during the same time period did not alter CTL activity in control or TCDD-treated mice. To address the possibility that insufficient IL-2 early in the response was responsible for the reduced pool of activated CTLp in TCDD-treated mice, IL-2 was administered on days 1-3 after P815 injection. However, not only did early treatment with IL-2 fail to restore the response in TCDD-treated mice, it suppressed the CTL response of non-TCDD-treated mice. To test whether exposure to TCDD induced apoptosis of activated CD8(+) T cells, phosphatidylserine (PS) expression was measured on various days after P815 tumor challenge. Surprisingly, the percentage of apoptotic CD8(+) T cells was significantly lower in TCDD-treated mice compared to controls throughout the allograft response. Similarly, exposure to TCDD failed to enhance peripheral deletion of Vbeta3(+)CD8(+) T cells after injection of the superantigen Staphylococcal enterotoxin A (SEA). Taken together, the data indicate that TCDD induces an early defect in CTLp activation that is not due to insufficient IL-2 or deletion of CD8(+) cells and may implicate a novel mechanism by which ligands of the Ah receptor disrupt CTL precursor activation.     

Archaeosomes as self-adjuvanting delivery systems for cancer vaccines

Archaeal ether glycerolipid vesicles (archaeosomes) efficiently deliver exogenous antigen for induction of humoral and cell-mediated immunity. Because induction of CD8 cytotoxic T cells is critical for protective vaccination against tumors, we compared the ability of various archaeosome lipid compositions to evoke a strong CD8 CTL response to entrapped antigen. Subcutaneous immunization of mice with ovalbumin (OVA) entrapped in all archaeosome lipid compositions evoked a primary (day 10) splenic CTL response indicating processing for MHC class I presentation. Interestingly, several polar lipid compositions from halophilic archaea were very potent to adjuvant this early CTL response. Despite this, the lytic units reduced substantially by weeks 6-7. More importantly, at >50 weeks, only Methanobrevibacter smithii and Thermoplasma acidophilum both rich in bipolar membrane-spanning caldarchaeols, demonstrated recall memory CTLs. Immunization of mice with OVA entrapped in M. smithii, Halobacterium salinarum or T. acidophilum vesicles provided prophylactic protection against challenge with OVA-expressing solid tumors at 6 weeks. Even a dose of 3 microg OVA in archaeosomes significantly delayed tumor growth. Tumor protection was also noted in a therapeutic design wherein OVA-archaeosomes were injected concurrent with the tumor challenge. Interestingly, antigen-free T. acidophilum but not antigen-free H. salinarum archaeosomes provided innate therapeutic protection. Vaccination with a CTL peptide epitope from the melanoma differentiation antigen, tyrosinase-related protein 2, in archaeosomes induced a protective CD8 response against B16OVA metastasis, indicating potential for targeting self, tumor antigens. Thus, lipid structural properties of archaea may differentially modulate primary, long-term and/or innate immunity, impacting adjuvant choice for vaccine design.     

Activation of the aryl hydrocarbon receptor by TCDD inhibits mammary tumor metastasis in a syngeneic mouse model of breast cancer

Treatment with aryl hydrocarbon receptor (AhR) agonists can slow or reverse the growth of primary mammary tumors in rodents, which has fostered interest in developing selective AhR modulators for treatment of breast cancer. However, the major goal of breast cancer therapy is to inhibit metastasis, the primary cause of mortality in women with this disease. Studies conducted using breast cancer cell lines have demonstrated that AhR agonists suppress proliferation, invasiveness, and colony formation in vitro; however, further exploration using in vivo models of metastasis is warranted. To test the effect of AhR activation on metastasis, 4T1.2 mammary tumor cells were injected into the mammary gland fat pad of syngeneic Balb/c mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Primary tumor growth was monitored for 4 weeks, at which time metastasis was determined. TCDD treatment suppressed metastasis by approximately 50%, as measured both in the lung and in mammary glands at sites distant from the primary tumor. Primary tumor growth was not suppressed by TCDD exposure nor was proliferation of 4T1.2 cells affected by TCDD treatment in vitro. Taken together, these results suggest that the protective effect of AhR activation was selective for the metastatic process and not simply the result of a direct decrease in tumor cell proliferation or survival at the primary site. These observations in immunologically intact animals warrant further investigation into the mechanism of the protective effects of AhR activation and support the promise for use of AhR modulators to treat breast cancer.     

Mode of metabolism is altered in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats

Male Sprague-Dawley rats were fed either a high-fat (HF) or a high-carbohydrate (HC) diet and subsequently injected with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (125 micrograms/kg) or vehicle (pair-fed controls). In all TCDD-treated animals, a reduction in caloric intake was evident as early as 1 day after dosage. Respiratory quotients (RQ) were determined at 5-day intervals. Their pattern for the HC-fed but not for the HF-fed TCDD-treated rats was different from that of the corresponding pair-fed controls. After an initial parallel decrease the RQ values remained low for TCDD-treated rats whereas they increased again for pair-fed controls. Serum total thyroxine (T4) was significantly lower in TCDD-treated animals and this reduction was not influenced by the composition of the diet. Serum triiodothyronine (T3) was neither altered by diet nor by TCDD. Thymic atrophy was as severe in pair-fed as in TCDD-treated rats fed the HC diet but not in rats fed the HF diet. Our results suggest that TCDD-treated rats are in a different mode of metabolism from pair-fed rats and that this difference is related to gluconeogenesis.