Inhibition of TC-1 cytokine production, effector cytotoxic T lymphocyte development and alloantibody production by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant and prototypic ligand for the aryl hydrocarbon receptor, is a potent immunotoxicant. To understand the underlying mechanisms of TCDD immunotoxicity, we have characterized the time course of changes in CTL, alloantibody, and cytokine responses to the P815 tumor allograft in C57B1/6 mice treated with 0 or 15 microg TCDD/kg. Suppression of CTL activity by TCDD directly correlated with reduced numbers of splenic CTL effector cells identified by their CD8+CD44 high CD45RB low phenotype, while suppression of the alloantibody response correlated with a lack of expansion of the B220+ splenocyte population. Cytokine production was differentially modulated following TCDD treatment. Although type 1 cytokine production (IFN-gamma, IL-2, and TNF) was initially induced in TCDD-treated mice, production failed to increase normally after day 5. In contrast, the production of IL-1 beta, IL-4, and IL-6 was mostly unaffected by TCDD exposure. This differential effect of TCDD on cytokine production was reflected in the degree of suppression of specific alloantibody isotypes. TCDD abrogated the production of IgG2a (promoted by IFN-gamma), but had much less effect on the level of IgG1 (promoted by IL-4). IgM Ab titers were also highly suppressed. CD8+ cells were the exclusive producers of IFN-gamma and IL-2 when spleen cells from P815-injected mice were cultured in vitro on days 4 to 7 after P815 injection. However, CD4+ cells were shown to play a crucial role in the generation of both CTL and alloantibody responses, since their depletion in vivo abolished both responses. Based on similar temporal effects produced by TCDD and anti-CD4 Ab on alloimmune responses, we postulate that TCDD interferes with the initial activation of CD4+ T cells, which leads to downstream inhibition of the activation and/or differentiation of CD8+ T cells and B cells. In addition, since delayed treatment with either anti-CD4 Ab or TCDD suppressed the alloantibody but not the CTL response, TCDD may also affect later CD4+ T helper-B cell interactions.

Polychlorinated biphenyl-induced suppression of cytotoxic T lymphocyte activity: role of prostaglandin-E2.

Prostaglandin-E2 (PGE2) was investigated for its role in suppression of splenic cytotoxic T lymphocyte (CTL) activity following exposure to 3,3',4,4',5,5'-hexachlorobiphenyl (HxCB) in mice. Following i.p. alloantigen injection, PGE2 levels significantly increased in peritoneal fluid and in spleen cell culture supernatants. HxCB exposure (1) significantly elevated PGE2 levels above control in peritoneal fluid, (2) significantly reduced production of PGE2 by spleen cells, and (3) did not alter PGE2 production by peritoneal cells. The levels of PGE2 observed were below (> 100-fold) those shown by others to cause immune suppression, and splenic CTL activity was unaltered by indomethacine treatment sufficient to reduce peritoneal PGE2 to undetectable levels. We conclude that altered PGE2 production is not involved in suppression of CTL activity by HxCB.

Acute inflammatory response to sheep red blood cells in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: the role of proinflammatory cytokines, IL-1 and TNF.

Recent studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure of C57Bl/6 mice results in an enhanced inflammatory response to intraperitoneal injection of sheep red blood cells (SRBC). This response is characterized by an increase in total peritoneal cells (PEC) as well as an increase in relative and absolute numbers of neutrophils (PMN) harvested 16 to 40 hr following injection of SRBC. The mechanisms whereby TCDD increases cellular influx are unknown. In the present studies, the role of the proinflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) in TCDD-induced hyperinflammation was examined. Intraperitoneal administration of recombinant IL-1 beta (0.4 U) or TNF alpha (10 ng) resulted in an enhanced peritoneal inflammatory response compared to phosphate-buffered saline-injected control animals measured 20 hr following injection of SRBC. The effect of exogenous cytokines mimicked the effects of exposure to 5 micrograms/kg TCDD. When endogenous IL-1 activity was blocked using an IL-1 receptor antagonist (IL-1ra, 1 mg every 3 hr), the PMN influx was significantly decreased in control animals but not in animals exposed to 20 micrograms/kg TCDD. When endogenous TNF activity was blocked using a TNF-soluble receptor (rhuTNFR:Fc, 100 micrograms), the numbers of total PEC and macrophages (MAC) harvested from control mice were reduced, while in mice exposed to 20 micrograms/kg TCDD, inhibition of TNF activity dramatically reduced the numbers of PEC, MAC, and PMN. Following rhTNFR:Fc treatment, there was no difference between TCDD-treated and control mice in inflammatory cell influx. These results demonstrate that TNF plays a major role in mediating TCDD-induced hyperinflammation. In support of this conclusion, a dose-dependent increase in plasma TNF alpha was measured by ELISA in TCDD-treated mice following SRBC injection.

Immunologic and endocrine effects of the flame-retardant pentabromodiphenyl ether (DE-71) in C57BL/6J mice.

Polybrominated diphenyl ethers are manufactured for use as flame retardants in commercial plastics and textiles in Europe and North America. These studies investigated the acute and subchronic immunotoxicity and endocrine effects of a commercial pentabromodiphenyl either mixture, DE-71, in female C57BL/6 mice. Mice were orally exposed to acute single doses of DE-71 of 0, 0.8, 4.0, 20, 100, or 500 mg/kg, or to subchronic daily doses totaling 0, 250, 500, or 1000 mg/kg over a 14 day period. Immunotoxicity was assessed by measuring the plaque-forming cell response to sheep erythrocytes (SRBC) and natural killer cell (NKC) activity (basal and poly I:C stimulated) to YAC-1 target cells. Liver cytochrome P450 content and activities (ethoxyresorufin-o-deethylase (EROD) and pentoxyresorufin-o-deethylase (PROD)) as well as corticosterone (CS) and thyroxine (T4) concentrations were also measured. PROD activity was induced 3-5-fold in mice exposed acutely or subchronically to DE-71 at doses > 250 mg/kg. EROD activity and total microsomal cytochrome P450 content were significantly induced only in mice treated subchronically with DE-71; maximum induction of EROD was 3.3-fold. Total serum T4 concentrations were significantly lower in mice treated acutely with DE-71 at all doses except the 100 mg/kg dose. Total and free T4 concentrations were dose-dependently decreased in DE-71-treated mice following subchronic exposure. Plasma CS levels were elevated following subchronic exposure to DE-71. The elevation of CS was correlated with order of capture at necropsy, suggesting an interactive effect of DE-71 and stress. In regard to immunotoxicity, significant suppression of the anti-SRBC response was seen only in mice exposed subchronically to 1000 mg DE-71/kg, an exposure that also resulted in decreased thymus weight. NKC activity was not altered by exposure to DE-71.

Polychlorinated biphenyl-induced immune suppression: castration, but not adrenalectomy or RU 38486 treatment, partially restores the suppressed cytotoxic T lymphocyte response to alloantigen.

The cytotoxic T lymphocyte (CTL) response to allogeneic P815 tumor in C57bl/6 mice is dose-dependently suppressed after treatment with 3,3',4,4',5,5'-hexachlorobiphenyl (HxCB). Elevation of plasma corticosterone (CS) is also observed coincident with CTL suppression. Because immune suppression is inducible by glucocorticoid administration, the role of elevated CS was investigated as an indirect mechanism of HxCB-induced immunotoxicity. In multiple experiments, HxCB treatment (10 mg/kg b.w.) consistently reduced CTL activity by 70 to 85% in male mice. Adrenalectomy failed to alter the suppression of CTL activity by HxCB. However, the mortality rate was high (> or = 70%) in these experiments and plasma CS elevation persisted in HxCB-treated adrenalectomy survivors. Therefore, the use of adrenalectomized mice was inadequate to determine whether CS elevation leads to CTL suppression after HxCB treatment. Daily administration of the glucocorticoid receptor antagonist 17-beta-hydroxy-11-beta-(4-dimethylaminophenyl)-17-alpha-(propanyl )-estra- 4,9-dien-3-one (RU 38486) (150 mg/kg b.w., p.o.) also failed to alter the suppression of CTL activity in HxCB-treated mice; however, spleen cellularity was significantly increased, suggesting functional GCR antagonism. Male mice were more sensitive to HxCB-induced CTL suppression than female mice, and HxCB-induced plasma CS elevation was greater in male mice. Castration failed to reduce the elevation of plasma CS in HxCB-treated male mice. However, castration partially alleviated CTL suppression in HxCB-treated male mice.(ABSTRACT TRUNCATED AT 250 WORDS)

2,3,7,8-Tetrachlorodibenzo-p-dioxin pretreatment of female mice altered tissue distribution but not hepatic metabolism of a subsequent dose.

Lipid partitioning of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inadequately explains its tissue distribution since higher concentrations occur in liver than fat except at high doses. This study provides in vivo evidence that an inducible, saturable system plays a predominant role in disposition of [14C]TCDD in female mice at doses between 5 and 20 micrograms/kg. Female C57BL/6J mice were gavaged with 0, 5, or 15 micrograms TCDD/kg, received a subsequent gavage of 5 or 20 micrograms [14C]TCDD after 6 days, and were killed 1 day later. In mice pretreated with 5 and 15 micrograms TCDD/kg and subsequently dosed with 20 micrograms [14C]TCDD/kg, liver weight and [14C]TCDD concentration increased. Total liver [14C]TCDD burden increased about 50% in both pretreatment groups. Concentrations of [14C]TCDD in kidney, fat, heart, lung, gastrointestinal tract, but not plasma or splenic lymphocytes, decreased in a reciprocal manner. Alterations in absorption, concentrations of polar metabolites of [14C]TCDD in liver, and hepatic lipid content failed to explain these results. About 97% of hepatic 14C was hexane extractable. HPLC of this extract indicated [14C]TCDD was the only significant nonpolar form of radiolabel in liver. In mice pretreated with 5 micrograms TCDD/kg and subsequently dosed with 5 micrograms [14C]TCDD/kg, a more marked pretreatment disposition response was observed. These results are consistent with a predominant role for an inducible, high affinity, low capacity system in whole animal pharmacokinetics of TCDD.

Role of the Ah locus in suppression of cytotoxic T lymphocyte activity by halogenated aromatic hydrocarbons (PCBs and TCDD): structure-activity relationships and effects in C57Bl/6 mice congenic at the Ah locus.

Previous studies have shown that the generation of cytotoxic T lymphocytes (CTL) following allogeneic tumor challenge is suppressed in Ah-responsive C57Bl/6 mice treated with a single oral dose of the toxic, Ah receptor-binding 3,4,5,3',4',5'-hexachlorobiphenyl (HxCB). The present studies have examined the specific role of the Ah receptor in this immunotoxic response by utilizing HxCB isomers of known, varied affinity for the Ah receptor as well as by comparing effects of high-affinity Ah receptor ligands (3,4,5,3',4',5'-HxCB and 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) on the CTL response of mice that differ only at the Ah locus, that is, Ah-responsive (Ahbb) and Ah-nonresponsive (Ahdd) congenic C57Bl/6 mice. Correlative changes in thymic weight, serum corticosterone (CS) levels, and spleen cellularity were also measured. The potency of HxCB congeners (3,4,5,3',4',5'-; 2,3,4,5,3',4'-; 2,4,5,2',4',5'-) and 2,3,7,8-TCDD to suppress the CTL response, to reduce spleen cellularity, to cause thymic atrophy, and to elevate serum CS levels was directly correlated with the binding affinity of the congener for the Ah receptor. Furthermore, these parameters of immunotoxicity in Ahdd C57Bl/6 mice were significantly more resistant to alterations induced by either 3,4,5,3',4',5'-HxCB or 2,3,7,8-TCDD as compared to Ahbb C57Bl/6 mice. These results strongly support an Ah receptor-dependent immunotoxic mechanism in suppression of the CTL response following acute exposure to halogenated aromatic hydrocarbons.

The broiler chicken as a model for immunotoxicity assessment. 1. Standardization of in vitro immunological assays.

The broiler chicken was developed as an alternative animal model to laboratory rodents for immunotoxicologic assessment. In vivo treatment with 100-200 mg/kg cyclophosphamide (CY) was used as a known immunosuppressive treatment to standardize the assay systems. Protocols for assessing specific immunological functions were developed in specific pathogen-free (SPF) broilers to measure lymphocyte blastogenesis to T-cell (concanavalin A and phytohemagglutinin) and B-cell (Staphylococcus aureus cells) mitogens, delayed-type hypersensitivity (DTH) to tuberculin, natural killer (NK) cell cytotoxicity, plaque-forming cell (PFC) response to sheep red blood cells (SRBC), and serum antibody titers to SRBC. CY was an effective immunosuppressant in the broiler system for assessment of lymphocyte responsiveness to mitogenic stimulation, DTH reactivity, and the antibody response to SRBC as assessed by PFC and serum antibody titers. NK cytotoxicity was not altered on a cellular level following treatment with CY at a dose that produced greater than 75% depletion of spleen cellularity. However, under these conditions, it must be assumed that the capacity of CY-treated birds to mediate NK effector functions would be reduced. These results demonstrate the applicability of the broiler chicken as an animal model for immunotoxicity testing.

Suppression of allograft immunity by 3,4,5,3',4',5'-hexachlorobiphenyl. I. Effects of exposure on tumor rejection and cytotoxic T cell activity in vivo.

There are conflicting reports in the literature concerning the immunotoxic effects of polychlorinated biphenyls (PCB) on cell-mediated immune responses. In the studies reported here, we show that the in vivo generation of cytotoxic T lymphocytes (CTL) in response to allogeneic tumor challenge is sensitive to suppression by 3,4,5,3',4',5'-hexachlorobiphenyl[(345)2-HxCB], a poorly metabolized, toxic, Ah receptor-binding PCB isomer. C57B1/6 mice treated with a single oral dose of (345)2-HxCB two days prior to the i.p. injection of allogeneic P815 tumor cells exhibited a dose-dependent reduction in peak CTL activity in the spleen. When examined on a kinetic basis, the CTL response was reduced in magnitude on all days examined with no evidence for a shift in the kinetics of the response induced by (345)2-HxCB exposure. (345)2-HxCB exposure prior to antigen challenge (day -14, -7, or -1 relative to P815 injection on day 0) produced significant suppression of the CTL response. (345)2-HxCB exposure 6 weeks prior to antigen challenge was still significantly suppressive, although the reduced degree of suppression suggested that recovery was in progress. When (345)2-HxCB exposure occurred after antigen challenge, significant suppression was produced only when exposure occurred within the first three days of the response, suggesting that, as the CTL matured, their sensitivity to (345)2-HxCB diminished. Clearance of the allogeneic tumor cells from the peritoneal cavity was delayed in (345)2-HxCB-treated mice and was associated with an altered composition of the white blood cell infiltrate in the peritoneal cavity. Symptoms of overt toxicity as well as immunotoxicity were apparent at lower doses of (345)2-HxCB in male as compared to female mice. In addition, interactive effects of (345)2-HxCB exposure and P815 antigen challenge on body weight and thymic involution were observed in both male and female mice. Possible mechanisms for the altered CTL response in (345)2-HxCB-exposed mice are discussed.

Suppression of allograft immunity by 3,4,5,3',4',5'-hexachlorobiphenyl. II. Effects of exposure on mixed lymphocyte reactivity and induction of suppressor cell activity in vitro.

Previous studies in our laboratory have established the sensitivity of the in vivo allogeneic cytotoxic T lymphocyte (CTL) response to suppression by 3,4,5,3',4',5'-hexachlorobiphenyl[(345)2-HxCB], a toxic, Ah receptor-binding polychlorinated biphenyl isomer. The present studies have examined possible cellular mechanisms for this suppression. A modest dose-dependent suppression of the proliferative response to alloantigen in mixed lymphocyte culture (MLC) was observed with lymphocytes from B6 mice exposed to 10 or 100 mg/kg (345)2-HxCB while the CTL response generated in MLC was significantly suppressed only following exposure to 100 mg/kg (345)2-HxCB. The amount of time between treatment with (345)2-HxCB and sacrifice, which ranged from 2 to 23 days, did not appear to influence the degree of immunosuppression produced by (345)2-HxCB exposure. Mitomycin C-treated lymphocytes from B6 mice treated with (345)2-HxCB were not suppressive when added as third party cells to an independent MLC. However, if the mice were alloimmune, lymphocyte-mediated suppression of the MLC response was observed and directly correlated with the magnitude of the CTL response present in the same population. Thus, (345)2-HxCB-treated mice which had less CTL activity as compared to vehicle-treated mice also had less suppressor activity. Further analysis indicated that stimulator cell lysis by the CTL was likely to be responsible for the inhibitory activity of the alloimmune lymphocytes rather than suppressor cells per se. Avoidance of stimulator cell lysis by using H-2-incompatible MLC stimulator cells revealed the existence of antigen-nonspecific suppressor activity that was greater with lymphocytes from vehicle-treated than from (345)2-HxCB-treated mice, suggesting that both CTL and suppressor cell activities were suppressed by (345)2-HxCB exposure. Direct addition of (345)2-HxCB to lymphocyte cultures in vitro indicated a lack of direct toxicity of (345)2-HxCB on lymphoproliferative responses to mitogen or alloantigen at concentrations equal to or less than 1 x 10(-6) M. Thus, the in-vitro functional integrity of lymphocytes obtained from (345)2-HxCB-treated mice coupled with the lack of a direct lymphotoxic effect of (345)2-HxCB in vitro suggest an indirect mechanism of action for (345)2-HxCB-mediated suppression of CTL activity in vivo. Previous reports implicating suppressor cell induction and/or activation by Ah-receptor-binding halogenated aromatic hydrocarbons that mediate the inhibition of CTL generation were not confirmed in these studies.

Effects of dietary technical pentachlorophenol exposure on T cell, macrophage and natural killer cell activity in C57Bl/6 mice.

The effects of technical grade pentachlorophenol (T-PCP) exposure on several immunological parameters were examined in adult C57Bl/6 mice following eight weeks of dietary exposure. Immune function tests included mitogen-induced lymphocyte blastogenesis, mixed lymphocyte reactivity (proliferation and cytotoxicity), spontaneous and boosted levels of natural killer (NK) cytotoxicity, and phagocytic activity of resident, thioglycollate-induced, and P815-tumor activated peritoneal macrophages. Thymic and splenic weights, spleen cellularity, percentages of splenic T and B cells, and bone marrow cellularity were also determined. The only statistically significant functional alteration observed in T-PCP exposed mice in these studies was a reduction in the lymphoproliferative response in mixed lymphocyte culture which occurred in the absence of any apparent effect on the generation of cytotoxic cells. Mitogen responses, NK cytotoxicity and macrophage phagocytosis were unaltered by exposure to T-PCP. No changes were observed in spleen or thymus weights or in spleen or bone marrow cellularity. A dose-responsive trend toward reduced T cell and increased B cell percentages in the spleen of T-PCP exposed mice was noted. The apparent functional resistance of T cells, macrophages, and NK cells to T-PCP is in contrast to the marked sensitivity of the humoral immune response to T-PCP induced suppression. The results are discussed in relation to the dioxin contaminants present in T-PCP.

Immunotoxicology studies on lead: effects of exposure on tumor growth and cell-mediated tumor immunity after syngeneic or allogeneic stimulation.

Chronic exposure of C57BL/6 mice to lead acetate in the drinking water enhanced the growth of primary Moloney sarcoma virus (MSV)-induced tumors. Regression of MSV-induced tumors was not prevented by lead exposure and lead-treated animals were more resistant to late sarcoma development following primary tumor regression. The primary cell-mediated cytotoxic response in the spleen or lymph nodes of MSV-tumor bearing mice was significantly augmented by lead exposure. This augmentation appeared to reflect the increased antigenic stimulation resulting from the enhanced primary tumor growth in lead-exposed animals. Using an allogeneic tumor model, under conditions of similar antigenic stimulation, little effect of lead on T cell-mediated cytotoxicity was observed. On the other hand, macrophage phagocytic activity was significantly depressed in lead-exposed mice. Coupled with a decrease in the total number of macrophages recovered from lead-exposed mice, the results suggested significant impairment of macrophages function by lead. The influence of lead-induced macrophage dysfunction on tumor growth is discussed.

Immunotoxicity of technical pentachlorophenol (PCP-T): depressed humoral immune responses to T-dependent and T-independent antigen stimulation in PCP-T exposed mice.

The effects of chronic dietary exposure to technical pentachlorophenol (PCP-T) on humoral immune responses in mice were examined. Primary and secondary splenic antibody responses to the T-dependent antigen, sheep red blood cells (SRBC), were examined in Swiss-Webster mice using our recently developed screening technique, the Hemolytic Antibody Isotope Release (HAIR) assay. To assess direct effects of PCP-T on B cells, the splenic plaque-forming cell response and serum antibody titers to the T-independent antigen, dinitrophenyl (DNP)-Ficoll, were examined. PCP-T exposure altered both the kinetics and the magnitude of the humoral antibody responses to SRBC and DNP-Ficoll. Peak splenic antibody production and serum antibody titers were delayed and the magnitude of the antibody responses were dose-dependently suppressed by PCP-T exposure. IgM responses appeared to be more sensitive to PCP-T-induced suppression than the IgG response. Significant depression of the IgM anti-SRBC splenic HAIR response was apparent as early as 2 weeks after initiation of PCP-T exposure and persisted for at least 8 weeks after termination of PCP-T feeding. Liver weight and serum lactate dehydrogenase (LD-L) and alanine aminotransferase (ALT) levels were significantly elevated during PCP-T exposure and returned to control levels after a 4-6 week recovery period. The immunotoxic effect of PCP on humoral immunity was observed only in animals exposed to technical grade PCP known to be contaminated with significant levels of other chlorinated phenols as well as non-phenolic impurities including chlorinated dioxins, furans, and diphenyl ethers. Animals exposed to analytical grade PCP did not exhibit depressed humoral immunity.

The haemolytic antibody isotope release (HAIR) assay: an efficient alternative technique to conventional plaque assays.

The haemolytic antibody isotope release (HAIR) assay quantitates antibody production by splenic antibody-producing cells by lysis of chromium-51-labelled sheep red blood cells. The amount of antibody quantitated by the HAIR assay directly correlates with the number of antibody-producing cells measured by a conventional plaque assay. The HAIR assay is an easy, sensitive, and reproducible technique that is especially useful when large numbers of animals are required for testing.