* Unité de recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL-CHUQ, Québec, QC, Canada G1V 4G2;
Division dHématologie-Oncologie, Hôpital Sainte-Justine, Montréal, QC, Canada H3T 1C5;
Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, QC, Canada G1K 7P4; and
Unité de Recherche en Santé Publique, Centre de Recherche du CHUL-CHUQ, Université Laval, Québec, QC, Canada G1V 5B3
Received June 20, 2003; accepted September 5, 2003
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ABSTRACT |
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Key Words: organochlorine insecticides; polychlorinated biphenyls; immune system; swine; prenatal exposure.
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INTRODUCTION |
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Populations that rely on species from the aquatic food chain for subsistence display high body burdens of several OCs and may be particularly at risk of immunotoxic effects. The Inuit population of Nunavik (Northern Quebec, Canada) displays a relatively high body burden of several OCs due to their high consumption of sea mammal fat as part of their traditional diet (Dewailly et al., 1993). We previously reported an association between prenatal exposure to several OCs (p,p'-DDE, hexachlorobenzene and dieldrin) and the risk of otitis media during the first year of life in Inuit infants from Nunavik (Dewailly et al., 2000
). However, the possible confounding effects of smoking and omega-3 fatty acids may have influenced our results. In the Inuit population, women who smoke during pregnancy also tend to consume more traditional foods and are therefore more exposed to OCs than nonsmokers (Muckle et al., 2001a
,b
). Hence, adequate control of these confounders may be difficult to achieve in an epidemiologic design. Experimental studies in laboratory animals are needed to avoid these potential methodological problems and identify mechanisms of action that may be involved in mediating OC-induced immunotoxicity.
The objective of the present study was to evaluate the immunotoxicity of in utero and lactational exposure to a reconstituted mixture of organochlorine compounds, designed to resemble that found in sea mammal fat consumed by the Inuit, using the pig as an animal model. Sows were exposed for several months before insemination and throughout gestation and lactation to either corn oil or a mixture of OCs (low, medium, or high doses). The following parameters were determined in the piglets: (1) innate immunity by evaluating phagocytosis by polymorphonuclear cells (PMNs) and complement component C2 function; (2) cell-mediated immunity by evaluating the lymphoproliferative response to mitogenic stimulation and the expression of T-cell markers; and (3) humoral immunity by evaluating the specific antibody response to Mycoplasma hyopneumoniae vaccination.
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MATERIALS AND METHODS |
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Immune System Parameters
Phagocytosis.
PMNs were recovered from the red cell pellet after Ficoll-Hypaque density gradient centrifugation of peripheral blood samples collected from piglets at 1, 5, and 8 months of age. The red cells were lysed with Immunolyse (Coulter, Hialeah, FL), and the PMNs were washed and resuspended in culture medium (RPMI-1640 medium). PMN concentration was adjusted to 1 x 106 cells/ml. Phagocytosis was assayed using the Vybrant Phagocytosis Assay Kit (Molecular Probes, Eugene, OR) according to the manufacturers instructions and the technique of Wan et al. (1993), with some modifications. A 100-µl volume of the E.coli (FITC-labeled) suspension was prewarmed at room temperature, briefly sonicated to disperse any aggregates, and added to 100 µl of cells. Following a 1-h incubation at 37°C, 5% CO2, the cells were washed, incubated for 1 min with 100 µl of Trypan Blue (250 µg/ml, pH 4.4), and placed on ice to stop the reaction. Tubes containing fluorescent particles with or without Trypan Blue were used as controls to indicate complete quenching or to eliminate background, respectively. The fluorescence intensity was measured by flow cytometry, and the results were expressed in percentage (%) of PMNs showing phagocytic activity.
Complement activity.
The functional activity of the second component (C2) of the classical pathway of complement activation was assessed in pigs at 1, 5, and 7 months after birth. C2 titration was performed as described by Ngan et al. (1977), with minor modifications. Serial dilutions of pig plasma were added to optimally sensitized sheep erythrocytes, to which an optimal dilution of C2-deficient guinea pig serum was added. The dilution of pig plasma that induces one lytic event per cell (Mayer et al., 1961
) represents the C2 functional titer. All plasma samples from a given pig were tested simultaneously using the same reagents. The results were expressed as the percentage of lytic activity displayed by an internal control (normal pig plasma) to compensate for minor day-to-day variations in the haemolytic assay.
Lymphoproliferative response.
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation, washed, and resuspended in culture medium (RPMI-1640 medium with 7.5% FCS, penicillin, streptomycin, and glutamine). The cells were distributed in triplicate (2 x 105 cells/well) in a 200-µl volume and incubated for 3 days at 37°C, 5% CO2, in the presence of phytohemagglutinin (PHA) (Sigma, Oakville, Ontario, Canada). A PHA concentration of 60 µg/ml was selected, which proved to yield optimal proliferation in preliminary studies. Twenty hours before the end of the incubation period, 1 µCi 3H-thymidine was added to each well. The cells were harvested on a filter (Tomtec apparatus, Wallac, Turku, Finland), and a melt-on scintillator sheet was applied (Meltilex A; Wallac, Turku, Finland). After drying, the radioactivity transferred on to the gel was measured in a micro-ß isotope counter (LKB Pharmacia, Piscataway, NJ). The lymphoproliferative response was evaluated at 1, 2, 4, and 6 months of age and expressed as total stimulation (in c.p.m.): 3H-thymidine uptake by the mitogen-stimulated lymphocytes minus the corresponding uptake by unstimulated lymphocytes.
Evaluation of T Lymphocyte Subsets
Monoclonal antibodies.
Lymphocyte subsets (CD4+, CD8+, CD4+CD8+) were evaluated at 1, 2, 4, 6, and 8 months of age. We also measured the swine leukocyte antigen SLA-DR on cytotoxic T cells since most of CD8+ cells bear this antigen in this species (Lunney et al., 1987; Thisthethwaite et al., 1983). Clones that produce monoclonal antibodies (mAb) specific for porcine CD4 (74-12-4, mouse IgG2b
) and CD8 (76-12-11, mouse IgG2a
) were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Anti-porcine CD4 and CD8 mAbs were purified by passage through a recombinant protein G-Agarose column (Gibco, Burlington, Ontario, Canada). Anti-CD4 and -CD8 mAbs were conjugated with fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively, using the FluoReporter Fluorescein-Ex protein labeling kit and the Protein-Protein cross-linking kit (Molecular Probes, Eugene, OR). Monoclonal antibodies specific for the SLA-DR (1053h2-18-1, mouse IgG2a
) were purchased from Research Diagnostics Inc. (Flanders, NJ). Anti-SLA-DR mAbs were used in indirect immunofluorescence with anti-mouse IgG (H+L)-FITC (Immunotech, Coulter, Hialeah, FL).
Flow cytometric analysis.
Flow cytometric analysis was performed by single and dual color fluorescence. Briefly, 1 x 106 PBMCs in 200 µl of phosphate buffered saline were incubated with an optimal concentration of fluorochrome-labeled monoclonal antibodies to identify the following lymphocyte subsets: (1) helper T cells: CD4-FITC; (2) cytotoxic T cells: CD8-PE; (3) double positive T lymphocytes: CD4-FITC and CD8-PE; and (4) cytotoxic T cells with SLA-DR expression: CD8-PE and SLA-DR-anti mouse IgG-FITC. The stained cells were washed, the red cells were lysed with a commercial lysing solution (Immunolyse, Beckman Coulter, Miami, FL), and the cells were then fixed with paraformaldehyde. After two washes, the cell pellets were suspended in 200 µl of Isoton II and analyzed on a Profile II flow cytometer (Coulter, Hialeah, FL). Cell labeling was measured on 5000 lymphocytes on a log fluorescence scale. The instrument performance was standardized daily with Immunocheck and Flow-set calibration beads (Beckman Coulter, Miami, FL).
Antibody Response to Mycoplasma hyopneumoniae
The piglets were vaccinated against Mycoplasma hyopneumoniae by 1-ml intramuscular injections of Mycoplasma hyopneumoniae bacterin at 5 and 7 weeks of age. An ELISA assay was performed by Biovet laboratory (St-Hyacinthe, Quebec, Canada) to determine anti-Mycoplasma hyopneumoniae IgG titers in plasma samples collected monthly from the piglets. The results were expressed as the percentage of animals showing a positive antigen-specific titer (i.e., >6 mg/l).
Statistical Analysis
All statistical analyses were performed using the SAS software (v 8.0, SAS Institute, Cary, NC). For continuous variables, the differences between mean values observed at several time points between experimental groups were tested using an analysis of variance (ANOVA) for repeated measurements (MIXED procedure). For the vaccine response (dichotomic variable), a logistic regression analysis for repeated measurements was used (GENMOD procedure) to test the differences between groups in the percentage of animals showing antibody titers above 6 mg/l at different time points. If the time x dose interaction term was statistically significant, multiple comparisons were made between the dose groups within each time stratum. In the absence of an interaction between time and dose, the interaction term was removed from the model and the differences between dose groups were tested globally for all time points. The level of statistical significance was set at 0.05.
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RESULTS |
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Lymphoproliferative Response.
The capacity of T lymphocytes, isolated from peripheral blood at 1, 2, 4, and 6 months, to proliferate upon mitogenic stimulation (PHA, 60 µg/ml) is presented in Figure 2. The ANOVA for repeated measurements revealed a statistically significant interaction between time and dose. Additional ANOVAs for each time point separately indicated a lack of treatment-related effect at 1 and 2 months of age. However, at 4 and 6 months of age, a statistically significant, dose-dependent increase in PHA-induced lymphocyte proliferation was observed. The medium dose was the minimal effective dose in the proliferation assay.
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DISCUSSION |
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This developmental study is unique because of the animal model used and the composition of the OC mixture fed to the animals. We could not find any study in the literature pertaining to immune system effects induced by a developmental exposure to a reconstituted complex OC mixture of relevance to the Arctic. Ross et al. (1997) administered orally to female rats during pregnancy and lactation one of the following treatments: (1) oil extracted from herring caught in the relatively uncontaminated Atlantic Ocean (control group); (2) oil extracted from herring caught in the contaminated Baltic Sea; or (3) oil extracted from Atlantic herring spiked with 2,3,7,8-TCDD (positive control group). In the positive control group, the authors reported immunosuppression characterized by decreased thymocyte and splenocyte proliferative responses to mitogens as well as decreased virus-associated natural killer (NK) cell activity and specific antibody responses. A similar pattern of effects was observed in rat pups exposed only to the Baltic Sea herring oil, but the effects were generally less pronounced (Ross et al., 1997
). Interestingly, F1 rats from the control group treated with the Atlantic herring oil, the Baltic Sea herring oil group, or the positive control TCDD-exposed group that were infected with rat cytomegalovirus (RCMV) showed similar RCMV-specific IgG titers 12 days after infection. However, at 59 days postinfection, the rats in the Baltic sea herring oil group and the TCDD group had significantly lower titers than the animals in the control group. Hence, the long-term antibody response to virus infection, but not the primary response, was affected by treatment with the OC mixture, similar to the results obtained in our study following Mycoplasma hyopneumoniae vaccination.
Two studies with rodents investigated the effects on the immune system of a postnatal exposure to a mixture of contaminants present in the blubber (adipose tissue) of a heavily contaminated beluga whale from the St. Lawrence River (Fournier et al., 2000; Lapierre et al., 1999
). In the first study (Lapierre et al., 1999
), Fisher 344 rats were fed for 2 months a diet in which lipids were replaced by either blubber from the St. Lawrence beluga whale (high dose), blubber from a relatively clean Arctic beluga whale (control group), or a 50:50 mixture of blubbers from the St. Lawrence beluga whale and the Arctic beluga whale (medium dose). The authors did not report any difference between treatments for the various immune parameters evaluated: lymphoblastic transformation, NK cell activity, plaque forming cells, phagocytosis, oxidative burst, and immunophenotyping (Lapierre et al., 1999
). In the second study (Fournier et al., 2000
), groups of C57B1/6 mice were fed for a period of 90 days diets in which the fat consisted of either corn oil, beef oil, or blubber from a St. Lawrence beluga whale and an Arctic beluga whale in the following proportions (five groups): 100:0, 75:25, 50:50, 25:75, and 0:100. Mice in groups fed diets containing blubber from beluga whales were not different than the control mice for blastic transformation of splenocytes, NK cell activity, and weight of the spleen and the thymus. However, groups receiving diets with blubber from beluga whales showed decreased CD8+ T cells in the spleen, reduced phagocytosis by peritoneal macrophages, and reduced humoral response of splenic cells against sheep red blood cells (Fournier et al., 2000
). In the latter study, the fact that similar immunosuppressive effects were observed in all groups of mice fed diets containing blubber from beluga whales, irrespective of their level of contamination, suggests a possible role of the lipid moiety, more specifically omega-3 fatty acids (n-3 PUFAs) that are present in high concentration in beluga whale fat and are well-known immunomodulators (Blok et al., 1991; Endres, 1996
; Harbidge, 1998
; Hardardottir and Kinsella, 1992
). We avoided this problem by using a reconstituted OC mixture rather than lipids extracted from marine mammal blubber.
Increases in the proportion of peripheral blood CD4+CD8+ T cells, phagocytosis, and lymphoproliferative response were observed in our study, in contrast to the results described above. In addition to differences in mixture composition, the animals in our study were exposed during the period of immune system development, which can produce qualitative differences in immune responses compared to postnatal exposure (Holladay and Smialowicz, 2000). Elevation in some immune parameters can be linked to targeted immunosuppression in other parameters, as observed for heavy metals that can alter the balance between Th1- and Th2-lymphocytes (Fournié et al., 2002
; Heo et al., 1996
). Finally, the unique character of the immune system in swine may also explain differences in immune responses between our study and those performed with rodents, especially with regard to the lymphoproliferative response. Indeed, the swine immune system is not fully developed at birth (Becker et al., 1993
), and spontaneous lymphocyte proliferation increases in neonatal pigs during the first weeks of life (Hoskinson et al., 1990
).
The decrease in the secondary vaccinal response noted in the high-dose group in our study indicates that the OC mixture affected the production of antibodies by B cells and the memory response. This result increases the biological plausibility of associations noted previously in epidemiological studies between developmental OC exposure and decreased humoral immunity. Weisglas-Kuperus et al. (2000) reported that prenatal PCB exposure was associated with lower antibody levels to mumps and measles after primary vaccination in Dutch pre-school children. We previously reported an association between prenatal exposure to OCs and the risk of otitis media during the first year of life in Inuit infants (Dewailly et al., 2000
). We also observed a lower concentration of IgM in newborns from a fish-eating population, living in the remote Lower North Shore region of the Gulf of St. Lawrence, compared to a reference population with lower prenatal OC exposure (Belles-Isles et al., 2002
).
A trend toward delayed effects was noted for most of the changes in immune parameters induced by treatment to the OC mixture in the present study. Indeed, treatment-induced changes were only seen in animals 4 months of age and older. This suggests that developmental exposure to the OC mixture did not affect innate immunity but rather acquired immunity, perhaps through interference with the antigen presentation process and mechanisms involved in memory response. One might speculate that the decrease in antibody response to the vaccine could be related to the concomitant decrease in the expression of SLA-DR on CD8+ T cells, which was noted in piglets at 6 months of age. In the swine, a MHC class II molecule is expressed constitutively on all T cells (Lunney et al., 1987; Thisthethwaite et al., 1983). Furthermore, 90% of CD4+/CD8+ and CD4-/CD8+ T cells express the SLA-DR. In this species, CD4+/CD8+ T cells can potentially present antigens to other cells (Zuckermann et al., 1996
). A decrease in expression of SLA-DR might affect antigen presentation and, in turn, stimulation of B cells to produce antibodies. A proper humoral immune response requires the interaction of three major immune cell types: antigen-presenting cells, helper T cells, and B cells. Alteration or dysfunction in any of these cells or cell interactions may result in aberrant antibody production (Luster et al., 1988
). However, decreases in SLA-DR expression occurred to the same extent in all OC-treated groups, while the antibody response to the vaccine was decreased only in the high-dose group. This suggests that the expression of SLA-DR is not the only factor involved in the suppression of the antibody response to the vaccine. Additional studies are required to further elucidate the mechanisms involved in OC-induced suppression of humoral immunity.
Plasma concentrations of OCs in piglets were comparable with those observed in human populations exposed to the same compounds in Quebec (Canada). Indeed, Muckle et al., (2001b) reported in Inuit women giving birth in Nunavik a mean concentration of PCBs (sum of 14 congeners) in plasma lipids of 397 µg/kg, with values ranging from 72 to 1951 µg/kg. In our study, mean plasma lipid PCB concentrations in sows at delivery were 153, 1425, and 11,485 µg/kg for the low-, medium-, and high-dose groups, respectively. The mean plasma lipid concentration of p,p'-DDE in Inuit women was 386 µg/kg (range = 60 to 2260 µg/kg). The mean p,p'-DDE concentrations in plasma lipids from sows at delivery were 152, 1538, and 13,756 µg/kg. The mean concentrations of these major OCs in sows in the high-dose group are only five to six times higher than the maximal concentrations recently reported in Inuit women giving birth in Nunavik. Therefore, the body burdens of OCs achieved in the present study are relevant to the Inuit population of Nunavik. The concentrations of PCBs achieved in our study are also relevant to maternal PCB exposure in various environmentally exposed populations (Longnecker et al., 2003
).
In summary, we observed in the pig model that gestational and lactational exposure to a mixture of OCs relevant to the Arctic alters the activity of several aspects of the immune system. Developmental exposure to OCs enhanced the phagocytic activity of PMNs and the proliferative response of T cells to mitogenic challenges. We also noted a suppression of SLA-DR expression by CD8+ T cells that may be related to the decreased humoral response observed in piglets from the high-dose group. Further studies are underway to investigate the mechanism by which developmental exposure to the OC mixture can interfere with normal humoral response, with a special focus on antigen-presenting cells.
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ACKNOWLEDGMENTS |
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NOTES |
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