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There are conflicting reports in the literature regarding the role of the Ah locus in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) immunotoxicity. The present studies have utilized two congenic strains of C57Bl/6 mice that differ only at this locus to assess its influence on TCDD-induced suppression of antibody responses. Mice were given a single oral dose of TCDD 2 days prior to challenge with sheep red blood cells (SRBC) or trinitrophenyl-lipopolysaccharide (TNP-LPS). The subsequent dose-dependent effects of TCDD on the amount of antibody produced by splenic plasma cells were measured using the hemolytic antibody isotope release assay. In addition, the relative importance of the Ah genotype of lymphoid versus nonlymphoid tissue was examined in adoptive transfer experiments. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced in Ahbb mice by a dose of 0.5 micrograms/kg TCDD and maximally induced by a dose of 2 micrograms/kg. Ahdd mice required 10-fold higher doses of TCDD to induce comparable levels of AHH. The degree of thymic involution and liver hypertrophy induced by TCDD was also influenced by the Ah genotype of the animals. Both Ahbb and Ahdd mice exhibited dose-dependent suppression of the anti-TNP response following TCDD exposure. The ID50 was 7.0 micrograms/kg in Ahbb mice and 30.8 micrograms/kg in Ahdd mice. Suppression of the antibody response to SRBC was also dependent on the Ah locus. The ID50 in Ahbb mice was 0.6 micrograms/kg TCDD. However, an apparent biphasic dose response for suppression of the anti-SRBC response in Ahdd mice suggested the involvement of an Ah-independent component of suppression as well. In adoptive transfer studies, lymphocytes were identified as an Ah-dependent component of the response. The Ah-independent component of the response was not identified, and could be either lymphoid or nonlymphoid in nature. The possibility that T helper cells represent the Ah-independent component is discussed.

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The objective of the present studies was to determine if acute exposure to an immunotoxic dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces alterations in the expression of lymphocyte surface markers as measured by multiparameter flow cytometry. The immunotoxicity of a single oral dose of TCDD was assessed by the anti-SRBC PFC response; an ED50 of 0.74 micrograms/kg was determined. Subpopulations in the spleen and thymus of C57B1/6 mice were analyzed 2 days following exposure to 2 micrograms/kg TCDD. In addition, splenic lymphocyte subsets were examined on Days 1-4 following SRBC challenge of mice treated with 0, 2, or 5 micrograms/kg TCDD. T and B cells were identified by single parameter analysis of Thy 1.2 and Ig expression. T cell subsets were defined by dual parameter analysis of CD4 and CD8 expression. In TCDD-treated mice, the percentage and the total number of double-positive CD4+ CD8+ thymocytes were significantly decreased while the percentage but not the total number of double-negative CD4- CD8- thymocytes was significantly increased. No changes in the percentage or total number of single positive (CD4+ CD8- or CD4- CD8+) thymocyte subsets were observed. In contrast to the thymus, lymphocyte subsets in the spleen were not significantly altered in percentage or total number 2 days following acute TCDD exposure. When splenic lymphocytes were analyzed daily following SRBC challenge, Ig+, Thy 1.2+, and CD4+ CD8- subpopulations remained relatively unchanged in both control and TCDD-treated animals. A small but significant decrease in the percentage of CD4- CD8+ T cells was observed on Day 3 in mice treated with 2 or 5 micrograms/kg TCDD when compared to that of vehicle-treated mice. The total number of CD4- CD8+ splenocytes was also significantly lower in the 5-micrograms/kg group on Day 3. However, this effect appeared to result from an elevation of the CD4- CD8+ subset in the controls rather than from a reduction in the TCDD-treated groups. Double-positive (CD4+ CD8+) lymphocytes were not detected in either control or TCDD-treated spleens. These results indicate that an acute dose of TCDD which reduced the splenic anti-SRBC response by 65-80% did not cause detectable changes in major splenic lymphocyte subpopulations. This is an important finding from the standpoint of utilizing lymphocyte subset analysis to screen for potential immunotoxic effects of TCDD. Specifically, the absence of subset changes does not preclude the presence of functional immunosuppression.

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The antibody response of C57B1/6 mice to sheep erythrocytes (SRBCs), a macrophage and T-cell-dependent antigen, is highly sensitive to suppression by 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD), a major immunotoxic contaminant of pentachlorophenol. The present studies have used several in vivo approaches to characterize the humoral immune suppression produced by an acute oral exposure to HpCDD and to assess the potential cellular defects responsible for the suppression. Administration of HpCDD at various times prior to or following antigen challenge indicated that HpCDD induced rapid yet prolonged suppression of the antibody response. Timing studies also suggested that initial processing of antigen by the macrophage and antibody production by the mature plasma cell were resistant to HpCDD. The demonstration that antibody responses could not be improved by either increasing or decreasing the antigen dose indicated that the effects of HpCDD were not related to the level of antigenic stimulation per se. In addition, there was no evidence for a shift in the kinetics of the antibody response of HpCDD-treated mice at any antigen dose that might have reflected a delay in the development of the response. The dose-response effects of HpCDD on antibody responses to T-helper-cell-dependent (SRBC) and T-helper-cell-independent type 1 (TNP-LPS) and type 2 (DNP-Ficoll) antigens indicated that sensitivity to HpCDD-induced suppression directly correlated with the sensitivity of the response to T-cell regulation. Nonspecific activation of T-amplifier cells with concanavalin A was capable of reconstituting the depressed antibody response of mice treated with 100 micrograms/kg HpCDD but not of mice treated with 500 micrograms/kg HpCDD, further suggesting that regulatory T cells are most sensitive to HpCDD. Definitive evidence for the role of regulatory T cells in mediating HpCDD-induced suppression of the antibody response was obtained in studies using congenitally T-cell-deficient nude (nu/nu) mice. The antibody response to DNP-Ficoll was was assayed since the anti-DNP response proceeds in the absence of T-helper cells in nu/nu mice yet the response can be modulated by changes in the activity of T-amplifier and T-suppressor cells present in nu/+ mice. Results of these studies showed that nu/nu mice were significantly more resistant to the immunosuppressive effects of HpCDD as compared with their nu/+ littermates.(ABSTRACT TRUNCATED AT 400 WORDS)

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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.

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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.

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Fasciola hepatica infections of lambs (250 or 500 metacercariae) were shown to alter the proliferative responses of peripheral blood lymphocytes (whole blood culture) to mitogens at specific times postinfection (PI). Responses to concanavalin A (Con A) were significantly suppressed at weeks 4, 8, 10, and 11 PI whereas suppressed responses to phytohemagglutinin (PHA) occurred at weeks 4, 10, 11, and 16 PI. Only on weeks 4 and 6 PI were responses to pokeweed mitogen (PWM) suppressed. The fluke-induced modulation of responses appeared to be related more to specific phases of infection rather than to worm burdens.

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Mice were exposed to 0, 1,5 and 10 ppm methylmercury for 10 weeks. After exposure, splenic lymphocytes were collected and examined for the ability of their antibody (Fc) and complement receptors to rosette sheep red blood cells (SRBC) in the presence of anti-SRBC antibody. Peritoneal exudate cells were also collected and examined for Fc rosetting and phagocytic properties. The largest dosage of methylmercury activated the complement receptor of B lymphocytes to rosette SRBC. The lower dosages did not affect this property of B cells nor did methylmercury alter the Fc receptors on B lymphocytes and macrophages or phagocytosis of peritoneal macrophages.

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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.

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Mice fed 1, 5, and 10 ppm methylmercury plus 6 ppm selenium for 10 wk had a significant increase in antibody synthesis. Since methylmercury singly depresses antibody synthesis and the response was greater than that produced by selenium alone, synergism between methylmercury and selenium occurred. In this case, the synergism is considered to be advantageous to a host, while exposure by other combinations of environmental contaminants may be detrimental. Mercury concentrations in the kidney were markedly elevated when methylmercury and selenium were administered simultaneously compared to when methylmercury was given without selenium supplement. These results indicate that data collected from individual pollutants may not be of value in predicting responses to multiple exposure.

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Mice were exposed to 0, 13, or 1,300 ppm lead in drinking water for 18 months. The immunological assays examined were mitogen (lipopolysaccharide E. coli, concanavalin A, and phytohemagglutinin-P) stimulation of lymphocytes; erythrocyte-antibody (EA), erythrocyte-antibody-complement (EAC), and phagocytosis of macrophages; and EAC of splenic lymphocytes. As measured by the majority of these assays, the low dosage (13 ppm) of lead tended to stimulate certain immune responses (lymphocyte mitosis, EA, and EAC) while the high dosage (1,300 pm) did not provoke an appreciable alteration. The results were interpreted by comparing data on aged mice with data on young adult mice. It was apparent from this comparison that the aged mice were naturally immunosuppressed. Therefore, the results obtained from lead-exposed mice were unpredictable.

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