Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing.

Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing and RNA synthesis. Like other gene-silencing technologies, PMO are poorly taken up by primary leukocytes without the use of physical or chemical delivery techniques. We sought an alternative delivery mechanism of PMO into immune cells that eliminates the need for such manipulations. Here we demonstrate the first use of arginine-rich cell-penetrating peptides (CPPs) to deliver PMO (P-PMO) directly into primary murine leukocytes for inhibition of gene expression and promotion of altered pre-mRNA splicing. We compared the P-PMO delivery efficacy of four arginine-rich CPPs including HIV Tat and penetratin, and one histidine rich CPP, and found that the (RXR)(4) peptide was the most efficacious for PMO delivery and targeted antisense effect. The delivery and antisense effects of P-PMO are time- and dose-dependent and influenced by the activation and maturation states of T cells and dendritic cells, respectively. Targeted expression of several genes using P-PMO is shown including surface signaling proteins (CD45 and OX-40), a cytokine (interleukin-2), and a nuclear transcription factor (Foxp3). Considering the abundance of naturally occurring alternatively spliced gene products involved in immune regulation, P-PMO offer an effective method for modulating gene activity for immunological research and applications beyond traditional antisense approaches.

Effect of cadmium exposure on primary tumor growth and cell-mediated cytotoxicity in mice bearing MSB sarcomas.

In vivo MSB tumor growth and cell-mediated cytotoxicity (CMC) to MSB tumor cells in vitro were studied in male C57BL/6 mice exposed to 0, 3, 30, or 300 ppm Cd as CdCl2 in their drinking water for 21 weeks prior to and during tumor growth. CMC was assessed on days 5, 12, and 19 post injection with the use of both a 51Cr release assay and a 51Cr post-label assay. Cd exposure significantly inhibited the growth of MSB tumors in vivo and enhanced the levels of CMC in the tumor-bearing hosts. Peak levels of CMC on day 12 post tumor injection were significantly increased in Cd-exposed animals. However, whereas the inhibition of tumor growth was directly dependent on the dose of Cd, the enhancement of CMC was inversely related to dosage. These data suggested that other mechanisms in addition to increased CMC were involved in tumor growth inhibition. Possible factors such as direct inhibition of tumor growth by Cd and decreased serum blocking levels in Cd-exposed animals are discussed.

Anti-androgen flutamide suppresses hepatocellular carcinoma cell proliferation via the aryl hydrocarbon receptor mediated induction of transforming growth factor-ß1.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and a member of the basic helix-loop-helix PER/ARNT/SIM family of chemosensors and developmental regulators. The AhR is widely known as a mediator of dioxin toxicity; however, it also suppresses cancer cell proliferation and recent findings have implicated its role as a tumor suppressor. We conducted a chemical library screen to identify nontoxic AhR ligands with anti-cancer effects and discovered flutamide (Eulexin) as a putative AhR ligand. Flutamide is an androgen receptor (AR) antagonist approved by the United States Food and Drug Administration for the treatment of prostate cancer. We found that flutamide inhibited the growth of several cancer cell lines independent of AR status, and that suppression of AhR expression reversed the anti-proliferative effects of flutamide. We investigated the AhR-dependent mechanism of action of flutamide in human hepatocellular carcinoma cells and identified that transforming growth factor-ß1 (TGF-ß1) is induced by flutamide in an AhR-dependent manner. In contrast, the potent AhR agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin had no effect on TGF-ß1 expression, indicating the ligand specificity of AhR activation. We also determined that TGF-ß1 induction is required for the AhR-dependent growth inhibitory effects of flutamide. Therefore, flutamide may be effective in AhR-positive cancers that are sensitive to TGF-ß1 signaling, such as hepatocellular carcinoma.

Pyridoxine supplementation: effect on lymphocyte responses in elderly persons.

The effect of pyridoxine supplementation on lymphocyte responsiveness was investigated in 15 persons aged 65-81 y. Eleven subjects received 50 mg/d pyridoxine HCl (PN). Four subjects received a placebo. Lymphocyte proliferation to T and B cell mitogens, lymphocyte subpopulations with monoclonal antibodies, and plasma pyridoxal 5'-phosphate (PLP) were measured before and after 1 and 2 mo of supplementation. After 1 and 2 mo plasma PLP levels increased by 195 +/- 88 nM and 201 +/- 84 nM, respectively, in subjects receiving PN. With PN supplementation, lymphocyte proliferation increased significantly in response to phytohemagglutinin (p less than 0.01), pokeweed mitogen (p less than 0.01), and Staphylococcus aureus (Cowain I) (p less than 0.05). For PN-treated subjects with low presupplement plasma PLP levels, lymphocyte blastogenesis also increased significantly (p less than 0.01) in response to concanavalin A. Percentages of T3+ and T4+ but not T8+ cells increased significantly (p less than 0.05) in PN-treated subjects. These results suggest that improving vitamin B-6 status is important in stimulating immunocompetence in the elderly.

Glucocorticoid effects on natural and humoral immunity in mallards.

Two studies were conducted to determine the effects of dexamethasone (DEX) on immune function in mallard ducks. Each day ducks were injected intramuscularly with DEX at doses ranging from 0.2-4.0 mg/kg for 28-30 days. Physiologic effects consistent with high dose glucocorticoid (GC) treatment were observed at the 4 mg/kg dose, and included significant body weight loss, lowered hematocrit, and elevated alanine aminotransferase (ALT) activity. At all doses, effects of DEX on the immune system were observed. When DEX was given at 0.2 mg/kg/day, significant suppression of primary IgG antibody titers to sheep erythrocytes (SRBC) was observed. At 1 mg/kg/day, primary IgM and secondary IgM and IgG titers were suppressed as well. These doses of DEX also produced significant elevation in natural killer cell (NKC) activity of peripheral blood mononuclear cells (PBMNC). Removal of adherent cells from the PBMNC prior to NKC assay eliminated the enhancement in NKC activity. Based on these results, it was postulated that the elevation in NKC activity may be due to suppression by DEX of monocyte production of prostaglandin-E2 (PGE-2) resulting in the release of NKC activity from the inhibitory effects of PGE-2. This hypothesis was supported by a measured decrease in PGE-2 production during the NKC assay by cells from DEX-treated birds. Furthermore, an enhanced NKC activity could be reproduced in vitro with the addition of indomethacin or DEX to NKC cultures containing adherent cells from PBMNC. Direct effects of DEX on nonadherent cell NKC activity and lymphocyte viability were only observed at high concentrations (10(-4) M) of DEX, while the phagocytic activity of adhered blood monocytes was inhibited at 10(-6) M DEX. The suppressed phagocytic activity may contribute to the suppressed antibody responses observed in DEX-treated birds. Together, these results support an indirect immunomodulatory effect of DEX on NKC activity and perhaps antibody responses in vivo via altered monocyte function in mallard ducks.

Immunological effects of chlorinated dibenzo-p-dioxins.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally similar halogenated aromatic hydrocarbons cause a broad range of immunologic effects in experimental animals including decreased host resistance to infectious disease and suppressed humoral and cell-mediated immune responses. In the mouse, TCDD immunotoxicity has been shown to be an aryl hydrocarbon (Ah) receptor-dependent process. However, despite considerable research, the biochemical and molecular alterations that occur subsequent to Ah receptor activation that lead to altered immune reactivity remain to be elucidated. In addition to immune suppression, TCDD promotes inflammatory responses. This effect may result from an upregulation of the production of inflammatory cytokines such as interleukin-1 and tumor necrosis factor. Nonhuman primates exposed to TCDD show suppressed antibody responses and changes in lymphocyte subsets in the peripheral blood. The immunotoxic effects of TCDD in humans are poorly characterized, and few studies have examined the immune status of individuals with known, documented exposure to TCDD. It is important for laboratory research to focus on defining TCDD-sensitive immunologic biomarkers in animal models that can also be used in human subjects. Understanding the mechanisms that underlie species differences in TCDD immunotoxicity is also of critical importance for extrapolation of effects seen in laboratory animals to man.

Role of altered arachidonic acid metabolism in 2,3,7, 8-tetrachlorodibenzo-p-dioxin-induced immune suppression in C57Bl/6 mice.

One of the most sensitive targets of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is the immune system. Many arachidonic acid (AA) metabolites are potent immunoregulatory molecules, and in other systems, TCDD has been shown to alter AA metabolism. Furthermore, the genes for cyclooxygenase (cox) contain a dioxin response element, suggesting that exposure to TCDD may directly alter cox levels and prostaglandin (PG)E2 production. To test the hypothesis that TCDD induces immune suppression by altering the production of immunomodulatory AA metabolites, we examined the effects of TCDD on splenic AA release, LTB4 and PGE2 production, and cox-1 and cox-2 expression. Exposure of C57Bl/6 mice to TCDD (15 microg/kg) resulted in a 2-fold increase in the release of AA from spleen cell membranes, a 1.4-fold enhancement of LTB4 and PGE2 production in the spleen, and 3-fold higher PGE2 levels in the peritoneal cavity during the immune response to allogeneic P815 tumor cells. We examined the direct induction of cox-1 and cox-2 by TCDD and the indirect induction of cox-2 via TCDD-induced IL-1. Interestingly, exposure to TCDD did not alter message or protein levels of cox-1, cox-2, or IL-1 over the course of the response to P815. Various metabolic inhibitors were then used to address the in vivo role of TCDD-induced changes in AA metabolism. While these inhibitors blocked AA metabolism, they failed to affect the TCDD-induced suppression of either the cytotoxic T lymphocyte response to P815 tumor cells or antibody formation in response to sheep red blood cells. The lack of effect of TCDD on cox expression, combined with the failure of metabolic inhibitors to reverse the suppression caused by TCDD, supports the conclusion that TCDD immunotoxicity is likely not mediated by a direct effect on the production of immunomodulatory AA metabolites.

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.

Role of glutathione and reactive oxygen intermediates in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced immune suppression in C57Bl/6 mice.

Recent developments in basic immunology have revealed the importance of glutathione (GSH) and cellular redox balance in the generation of an immune response. In the liver, it has been shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters cellular GSH and reactive oxygen intermediate (ROI) production. We have tested the hypothesis that TCDD mediates the suppression of the cytotoxic T lymphocyte (CTL) response to alloantigen by increasing oxidative stress. Total cellular GSH, GSSG, and GSH-protein adducts were analyzed by HPLC. Changes in intracellular GSH and ROI were simultaneously measured in isolated hepatocytes and individual subpopulations of spleen cells (CD4+, CD8+, B220+, and Mac-1+) following in vivo exposure to TCDD and antigenic challenge with P815 mastocytoma cells. Monochlorobimane was utilized to measure GSH levels, and two fluorescent probes were used to evaluate ROI levels: dichlorofluoroscein diacetate to monitor peroxides and dihydroethidine to assess superoxide anion. In hepatocytes, in vivo treatment with TCDD resulted in a transient, 2-fold increase in GSH, a 50% decrease in peroxide levels and a small (20-40%) decrease in superoxide anion levels. Although alloantigen challenge resulted in increased GSH and peroxide in spleen cells, in vivo exposure to TCDD had no effect on splenic ROI levels, nor did it consistently alter GSH levels in any subpopulation of spleen cells examined. Moreover, in vivo treatment with the antioxidant N-acetyl cysteine failed to affect the immune suppression caused by TCDD. These results suggest to us that although TCDD perturbs cellular redox balance in the liver, it does not exacerbate or diminish the normal increased GSH and ROI which occur in the spleen in response to antigenic challenge.

Toxicology of protein allergenicity: prediction and characterization.

The ability of exogenous proteins to cause respiratory and gastrointestinal allergy, and sometimes systemic anaphylactic reactions, is well known. What is not clear however, are the properties that confer on proteins the ability to induce allergic sensitization. With an expansion in the use of enzymes for industrial applications and consumer products, and a substantial and growing investment in the development of transgenic crop plants that express novel proteins introduced from other sources, the issue of protein allergenicity has assumed considerable toxicological significance. There is a need now for methods that will allow the accurate identification and characterization of potential protein allergens and for estimation of relative potency as a first step towards risk assessment. To address some of these issues, and to review progress that has been made in the toxicological investigation of respiratory and gastrointestinal allergy induced by proteins, a workshop, entitled the Toxicology of Protein Allergenicity: Prediction and Characterization, was convened at the 37th Annual Conference of the Society of Toxicology in Seattle, Washington (1998). The subject of protein allergenicity is considered here in the context of presentations made at that workshop.

Tier-2 studies on monocrotaline immunotoxicity in C57BL/6 mice.

Monocrotaline (MCT) is a member of a class of naturally occurring phytotoxins known as pyrrolizidine alkaloids, and is a toxicological concern to both man and his livestock. The purpose of these studies was to evaluate the effect of a 14-day oral MCT (0-100 mg/kg per day) exposure on the functional integrity of various immunocyte effector systems in C57BL/6 mice, as well as to investigate potential mechanisms for its immunotoxicity. Decreases in lymphoid organ weights and cellularity, and resident peritoneal exudate cell (PEC) number were only observed after exposure to the highest dose of 100 mg/kg MCT. This dose also inhibited NK cell cytotoxicity, while the total number of NK lytic units per spleen was decreased (-53%) after exposure to 50 mg/kg MCT. Following i.p. injection of SRBC, the percentage of PEC macrophages containing engulfed SRBC was significantly increased in MCT-exposed mice, while the percentage of large vacuolated (activated) macrophages was decreased in a dose-dependent manner. Exposure to MCT significantly decreased the total number of Ig+ cells without altering the number of CD4+ and CD8+ cells. The antibody responses (PFC/10(6) spleen cells) to two T cell-independent antigens, TNP-LPS and DNP-Ficoll, were significantly decreased at all MCT doses, and the degree of suppression of both responses was identical at coincident doses. MCT exposure (25 mg/kg) significantly suppressed the blastogenic response to the T cell mitogen concanavalin A (-38%), and to the B cell mitogen lipopolysaccharide (-58%). These results indicate that exposure to MCT can alter the functional integrity of various immune effector responses in a dose-dependent manner, and suggest that the B cell may be a relatively more sensitive target of MCT immunotoxicity compared to T cells, macrophages and NK cells.

Mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced decrease in anti-CD3-activated CD4(+) T cells: the roles of apoptosis, Fas, and TNF.

The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suppresses T cell functions and reduces T cell numbers in multiple models of immune stimulation. However, the underlying mechanism(s) by which TCDD induces these changes has yet to be elucidated. We hypothesized that TCDD affects T cells through the induction or augmentation of apoptosis. In these studies, we used antibody to CD4, annexin V, and 7-AAD in three-color flow cytometric analyses to examine the relationship between the decrease in CD4(+) T cells and cell death in mice treated with anti-CD3 and TCDD. In addition, we examined two signaling pathways, Fas and TNF, in order to elucidate a potential mechanism by which TCDD increases cell death. Our results show that the TCDD-induced decrease in CD4(+) T cell number correlated with an increase in the percentage of dead cells, but not with cells expressing an early apoptotic phenotype. The TCDD-induced decrease in CD4(+) T cells was attenuated in Fas- and FasL-deficient mice (lpr and gld, respectively), but not by treatment with a neutralizing antibody to TNF. While these results suggest that the Fas pathway may be important in TCDD-induced T cell death, however, the effect of TCDD on the Fas pathway remains unclear. Taken together, our data suggest that TCDD-induced suppression of CD4(+) T cells involves, in part, increased cell death that may be mediated by Fas/FasL interaction.

Suppression of cytotoxic T lymphocyte activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin occurs in vivo, but not in vitro, and is independent of corticosterone elevation.

Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent immunosuppressive compound. In our laboratory, TCDD and structurally related polychlorinated biphenyls (PCBs) have been shown to suppress alloantigen-specific cytotoxic T lymphocyte (CTL) activity in C57B1/6 mice. PCB-induced CTL suppression occurs coincident with significant elevation of plasma glucocorticoid (GC) levels (> 500 ng/ml). Since GC elevation can cause immune suppression, this study was conducted to determine if TCDD-induced CTL suppression is correlated with elevation of plasma corticosterone (CS), the major GC in mice. Single oral doses of TCDD (2.5-40 micrograms/kg) induced a dose-dependent suppression of CTL activity with a calculated 50% immunosuppressive dose (ID50) occurring at 7.2 micrograms/kg. When total lytic units (LU)/spleen were calculated, the ID50 was 2.8 micrograms/kg. In contrast, plasma CS levels were not significantly altered at doses below 40 micrograms/kg. These data suggest that TCDD-induced CTL suppression is not dependent on CS elevation. The direct effect of TCDD on CTL generation was tested by adding TCDD at 10(-13)-10(-9) M to in vitro mixed lymphocyte-tumor cell (MLTC) cultures. No alteration of CTL activity was observed after 5 days of culture at any TCDD concentration. In contrast, CS alone significantly suppressed CTL activity in vitro. CS-induced CTL suppression in vitro was neither enhanced nor inhibited by the presence of TCDD. These results suggest that TCDD causes CTL suppression in vivo by a mechanism that does not involve CS.

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.

Humoral immunotoxicity of polychlorinated diphenyl ethers, phenoxyphenols, dioxins and furans present as contaminants of technical grade pentachlorophenol.

Previous studies have demonstrated that the humoral immune response in mice as measured by the splenic IgM response to sheep erythrocytes (SRBC) is highly sensitive to suppression by technical grade (86%) pentachlorophenol (T-PCP) whereas analytical grade (greater than 99%) PCP is not immunosuppressive. In the present studies, we have examined several contaminant fractions and purified isomers from T-PCP for their humoral immunosuppressive effect. C57BL/6 mice were treated with a single oral dose of the various contaminants 2 days prior to SRBC challenge and the peak splenic IgM antibody response was measured 5 days later. Under these exposure conditions, T-PCP produced a dose-related suppression of the antibody response whereas analytical grade PCP was without effect. The dose of T-PCP producing 50% immunosuppression relative to the vehicle-treated control (ID50) was 83 mg/kg. Results from studies using contaminant fractions extracted from T-PCP indicated that a chlorinated dioxin/furan fraction was significantly immunosuppressive, whereas a chlorinated phenoxyphenol fraction and a chlorinated diphenyl ether fraction were without effect when administered at dose levels expected to occur in the ID50 dose of T-PCP. Several purified phenoxyphenol isomers representing the major pre- and isopredioxins in T-PCP were also not immunosuppressive, nor was octachlorodibenzo-p-dioxin. The 1,2,3,4,6,7,8-hexachlorodioxin (HxCDD), 1,2,3,4,6,7,8-heptachlorodioxin (HpCDD), and 1,2,3,4,6,7,8-heptachlorofuran (HpCDF) isomers were all significantly immunosuppressive. The single, oral ID50s were 7.1, 85 and 208 micrograms/kg for HxCDD, HpCDD and HpCDF, respectively. Coadministration of HxCDD and HpCDD produced an additive immunosuppressive effect suggesting that the toxic dioxin and furan isomers present in T-PCP function in concert to produce the degree of immune suppression observed following T-PCP exposure. When analytical grade PCP was coadministered with HpCDD, the degree of immune suppression was equivalent to that produced by HpCDD alone, indicating no significant influence of PCP on dioxin-induced immunosuppression. The enhanced susceptibility of Ah-responsive C57BL/6 mice to T-PCP induced immune suppression as compared to Ah-nonresponsive DBA/2 mice and the correlation of immune suppression with P1-450 associated monoxygenase induction provided further evidence for the role of the toxic Ah-interactive dioxin and furan contaminants in T-PCP as the mediators of T-PCP immunotoxicity.

Role of metabolism in monocrotaline-induced immunotoxicity in C57BL/6 mice.

Monocrotaline (MCT) is a pyrrolizidine alkaloid which has been shown to induce immunotoxicity in mice. We hypothesized that metabolic activation of MCT by mixed-function oxygenases (MFO) to dehydromonocrotaline (MCTP) is a prerequisite for its immunotoxicity, as has been shown for other toxic effects of MCT. To test this hypothesis, we compared the in vitro immunotoxic potency of MCT and MCTP to suppress the in vitro antibody response to SRBC and the blastogenic response to B and T cell mitogens. In addition, the effects of in vivo modulation of MFO activities on the immunotoxicity of MCT was examined using phenobarbital (PB) to increase and chloramphenicol (CP) to decrease MCTP production. Results showed that in vitro exposure of splenic lymphocytes to MCT or MCTP produced significant suppression of the antibody and blastogenic responses. MCTP was 200-400-fold more potent than MCT. No metabolism of MCT by splenic cells was detectable, suggesting that unmetabolized MCT is capable of inducing immunotoxicity. In vivo studies showed that, while treatment of mice with PB or CP produced significantly increased and decreased MCTP production by liver microsomes, neither PB or CP treatment significantly altered the immunotoxic potency of MCT. Thus, while the MCTP metabolite is directly immunotoxic in vitro and much more potent than MCT, a role for the MCTP metabolite in MCT immunotoxicity in vivo could not be demonstrated.

Reduced T-helper cell function in mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Antibody production to the T-dependent antigen SRBC is highly sensitive to suppression by polychlorinated dibenzo-p-dioxins. The present study provides evidence for a defect in T-helper (TH) cells in TCDD-exposed mice. Because spleen cells from nonimmune TCDD-exposed mice did not show suppressed antibody responses when adoptively transferred to irradiated hosts, we used a hapten-carrier (TNP-SRBC) system with cell separation/reconstitution techniques to determine the effects of TCDD on carrier-specific TH cells. In vitro cultures of spleen cells from SRBC-primed TCDD-treated (5 micrograms/kg) mice produced fewer anti-TNP plaque-forming cells (PFC) when immunized with TNP-SRBC, as compared to cells from primed vehicle-treated controls. A reduced anti-TNP PFC response was also observed in experiments where non-immune B-cells were induced to produce anti-TNP PFC by TH-cells obtained from carrier-primed TCDD-exposed mice, as compared to carrier-primed vehicle-exposed mice. Removal of Lyt-2+ (suppressor) T-cells in these experiments did not alter the anti-TNP PFC response. These results provide direct evidence for reduced activity of TH-cells after exposure to TCDD.

Inhibition of tumor necrosis factor activity fails to restore 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of the antibody response to sheep red blood cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) immunotoxicity is characterized in part by a profound suppression in T cell-dependent antibody production to sheep red blood cells (SRBC); however the mechanisms involved in antibody suppression are not fully understood. Recent studies from several different laboratories have suggested that increased tumor necrosis factor (TNF) activity may mediate some of the toxicity associated with TCDD exposure. The current studies were designed to evaluate the role TNF plays in TCDD-induced suppression of the antibody response. We examined the effects of exogenous TNF alpha and the effects of blocking TNF activity with a soluble TNF receptor (rhuTNFR:Fc) on antibody production in control and TCDD exposed C57B1/6 mice. Results indicate that under certain conditions, increased TNF can suppress antibody production to SRBC, but TNF does not appear to mediate TCDD-induced antibody suppression.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on tumor necrosis factor (TNF) production by peritoneal cells.

Recent studies in mice have demonstrated that TNF plays a critical role in mediating the TCDD-induced enhanced inflammatory response to intraperitoneal (i.p.) sheep red blood cells. The current studies were designed to evaluate the effects of TCDD on TNF production by ex-vivo peritoneal cells and a peritoneal macrophage cell line (IC-21) stimulated with LPS. In support of the hypothesis that TCDD can act directly on the peritoneal macrophage to increase TNF production, following pretreatment with TCDD, both ex-vivo peritoneal cells and IC-21 cells produced increased levels of bioactive TNF when stimulated with LPS. Flow cytometric analyses of IC-21 cells indicate that TCDD exposure increases intracellular production and secretion of TNF but does not alter levels of membrane associated TNF.

Immunologic effects of cocaine in prenatally exposed rats and mice.

The immunotoxicity of prenatal cocaine exposure was investigated using Sprague-Dawley rats and C57B1/6 mice. Pregnant animals were injected twice a day with cocaine or saline from gestation day 5 until the day before parturition. The immune system of the rat offspring was evaluated at 8 weeks of age by measuring the antibody response to SRBC (plaque assay and serum IgM), delayed-type hypersensitivity response to KLH, and lymphocyte subpopulations in the spleen and thymus using flow cytometry. The immune system of the mice offspring was evaluated at 4 weeks of age by measuring spleen cell proliferation in response to KLH, LPS, and alphaCD3 and IgG production to KLH. From the differences observed between cocaine exposed animals and controls, we conclude that prenatal cocaine exposure does not cause lasting detrimental effects on the immune system, but instead, may enhance B-cell responsiveness.