Increased Tumor Necrosis Factor- Production by Peripheral Blood ...

60, 327–337 (2001) Copyright © 2001 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Increased Tumor Necrosis Factor-␣ Production by Peripheral Blood Leukocytes from TCDD-Exposed Rhesus Monkeys Sherry E. Rier,* ,1 Christopher L. Coe,† Andrine M. Lemieux,† Dan C. Martin,‡ Richard Morris,§ George W. Lucier,§ and George C. Clark§ ,2 *Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756; †Harlow Center for Biological Psychology; University of Wisconsin, Madison, Wisconsin 53715; ‡Department of Obstetrics and Gynecology, University of Tennessee, Memphis, Tennessee 38103; and §National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received September 19, 2000; accepted November 20, 2000

Previous work has shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with a dose-dependent increase in the incidence and severity of endometriosis in the rhesus monkey. Studies also suggest that immune mechanisms participate in TCDD-mediated toxicity and the pathogenesis of endometriosis. Thirteen years after TCDD treatment was terminated, we characterized the phenotypic distribution of peripheral blood mononuclear cells (PBMC) from TCDD-exposed and -unexposed rhesus monkeys and determined the ability of these cells to produce cytokines and exert cytolytic activity against NK and T-cell-sensitive cell lines. We also determined whether elevated serum levels of TCDD, dioxin-like PHAH congeners, and triglycerides correlated with changes in PBMC phenotype or function. For all animals, TCDD exposure correlated with increased PBMC tumor necrosis factor-alpha (TNF-␣) secretion in response to stimulation by T-cell mitogen and decreased cytolytic activity against NK-sensitive target cells. Furthermore, increased production of this cytokine by PHA-stimulated leukocytes was associated with elevated serum triglyceride levels. Leukocyte TNF-␣ secretion in response to viral antigen and PBMC production of interferon gamma (IFN␥), IL-6, and IL-10 following exposure to mitogen or antigen were unaffected by previous TCDD treatment. Although TCDD exposure was not associated with changes in PBMC surface antigen expression, elevated serum concentrations of TCDD, 1,2,3,6,7,8-hexachlorodibenzofuran and 3,3ⴕ,4,4ⴕ,5-pentachlorobiphenyl correlated with increased numbers of CD3ⴙ/ CD25- and CD3-/CD25ⴙ leukocytes and enhanced secretion of TNF-␣ by mitogen-stimulated PBMC. These findings indicate that TCDD-exposed rhesus monkeys with endometriosis exhibit long-term alterations in systemic immunity associated with elevated serum levels of specific PHAH congeners. Key Words: endometriosis; rhesus; environmental toxicants; dioxin; TCDD; PCBs (polychlorinated biphenyls); dioxin-like chemicals.

1

To whom correspondence should be addressed at Vanderbilt University Medical Center, Department of Obstetrics and Gynecology, B-1100 Medical Center North, Nashville, TN 37232. Fax: (615) 343-7913. E-mail: [email protected]. 2 Present address: Xenobiotic Detection Systems, 1610 E. Greer St., Suite S, Durham, NC 27704.

Endometriosis is classically defined as the growth of endometrial glands and stroma at sites outside the uterus. Characterized by infertility, chronic pain, and adhesion formation, it is a common disease which may affect 10% of reproductive-age women (Wheeler, 1992). Endometriosis occurs exclusively in menstruating species, including humans and nonhuman primates, with spontaneous development in rhesus monkeys closely resembling human disease (Fanton and Golden, 1991). Although the etiology is unknown, it is postulated that endometrial cells implant and proliferate at ectopic sites following retrograde menstruation or arise from primitive progenitor cells of the coelomic epithelium (Witz and Schenken, 1997). Disease appears to be estrogen-dependent and medical therapies consist of hormonal regimens that limit the action of endogenous estrogen. Immune and endocrine dysregulation has been proposed as a mechanism that results in ectopic endometrium since a unique, hormonally-responsive immune system within the uterus plays a key role in the cyclic growth and break-down of normal uterine endometrium (Rier and Yeaman, 1997). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and dioxinlike polyhalogenated aromatic hydrocarbon (PHAH) chemicals are present in the environment and accumulate in serum and tissues of exposed individuals and the general population (DeVito et al., 1995). These chemicals are associated with a spectrum of toxic effects on the reproductive, immune, and endocrine systems (Kerkvliet, 1995; Peterson et al., 1993; Whitlock, 1990, 1994). Evidence indicates that TCDD and dioxin-like PHAHs can act additively via specific binding to the aryl hydrocarbon receptor (AhR) and the potency of various congeners relates to AhR affinity (Safe, 1990). Recent reports suggest that acute TCDD exposure in rodents increases peritoneal and peripheral blood (PB) leukocyte production of inflammatory cytokines, tumor necrosis factor-alpha (TNF-␣), and interleukin (IL)-6 (Clark et al., 1991; Moos et al., 1994, 1997) and decreases lymphocyte anti-tumor cytolytic activity (Kerkvliet et al., 1990, 1996). Previous work has shown that chronic TCDD exposure in the rhesus is associated with a dose-dependent increase in the incidence and severity of en-

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dometriosis 10 years after termination of TCDD exposure (Rier et al., 1993). Recent studies have demonstrated that peritoneal and PB leukocytes from women with endometriosis exhibit increased TNF-␣, IL-6 (Braun et al., 1996; Halme, 1989; Rana et al., 1996; Rier et al., 1994) and IL-10 production (Ho et al., 1996). Moreover, leukocytes and endometrial cells within ectopic lesions exhibit increased expression of IFN␥ mRNA (Klein et al., 1993) and IL-6 protein relative to uterine cells from women without disease (Rier et al., 1995, Tseng et al., 1996; Zarmakoupis et al., 1995). Decreased anti-endometrial cytolytic activity by peritoneal and PB leukocytes has also been observed in women with endometriosis (Oosterlynck et al., 1991) and has been attributed to decreased NK cell activity (Oosterlynck et al., 1991, 1992). In this study, the immune status of a group of rhesus monkeys was investigated 13 years after termination of TCDD exposure. The phenotypic distribution and function of PB leukocytes were examined. The data were analyzed for an association with endometriosis and elevated serum concentrations of TCDD and dioxin-like PHAHs. Since chronic exposure to high levels of TNF-␣ and IL-6 in vivo is associated with elevated serum triglycerides (Feingold and Grunfeld, 1992), we determined whether leukocyte secretion of these cytokines in vitro correlated with serum triglyceride levels. Changes in specific immune parameters strongly correlated with TCDD exposure, elevated serum PHAH levels, or endometriosis. MATERIALS AND METHODS All experimental protocols using rhesus monkeys were performed in accordance with the regulations in the “Guide for Care and Use of Laboratory Animals” and the amended Animal Welfare Act (7USC 2131 et Sec.), and were approved by the Animal Care and Use Committee of the University of Wisconsin. Peripheral blood was obtained by femoral venipuncture after animals were lightly anesthetized with ketamine HCl (10 mg/kg). Rhesus study population. Detailed methods for TCDD exposure of these animals have been reported previously (Bowman et al., 1989; Rier et al., 1993; 2001; Schantz and Bowman, 1989; Schantz et al., 1986). In brief, 24 feral female rhesus monkeys (Macaca mulatta) approximately 6 to 10 years of age were obtained in 1977 (Hazelton Research Animals, Reston, VA). The animals were maintained at the University of Wisconsin Biotron under climate conditions that simulated their natural breeding season. Monkeys were randomly assigned to 3 groups of 8 animals each. Control animals were not exposed to TCDD, animals in the low-dose group were exposed to 5 parts per trillion (PPT) TCDD, and monkeys in the high-dose group were exposed to 25 PPT TCDD for approximately 4 years. Dosing was by ingestion in the animal feed. Following termination of TCDD exposure, animals were transferred to Harlow Primate Laboratory in early 1983. Fat samples were obtained from all animals by biopsy at several time points during and after exposure; bioaccumulation of TCDD was quantified in selected animals by analyzing TCDD content in fat (Bowman et al., 1989). Twenty animals (0 PPT TCDD [n ⫽ 6], 5 PPT TCDD [n ⫽ 7], and 25 PPT TCDD [n ⫽ 7]) comprised the study group in the first report of endometriosis (Rier et al., 1993). Thirteen years post-TCDD treatment, serum concentrations of TCDD and 19 dioxin-like PHAH congeners were determined in related studies (Rier et al., 2001). Animals evaluated in the present study consisted of 15 surviving monkeys from this colony (24 to 28 years of age) and 12 additional animals of similar age (23 mean years of age) with no previous TCDD or PHAH exposure. All animals were generally healthy with the exception of endometriosis, and none exhibited symptoms of

acute or chronic infection or cachexia; none of the animals experienced significant weight loss in the 6 months preceding the initiation of these studies. Serum levels of estradiol and follicle-stimulating hormone were similar among animal groups (data not shown). These 27 animals were grouped prospectively according to TCDD exposure: 0 PPT TCDD control group (n ⫽ 18), 5 PPT TCDD (n ⫽ 6), and 25 PPT TCDD (n ⫽ 3), and ALL TCDD (n ⫽ 9). Animals were grouped retrospectively as follows: No TCDD and No endometriosis (-TCDD/-EM; n ⫽ 14), No TCDD with endometriosis (-TCDD/⫹EM; n ⫽ 4), TCDD exposure and No endometriosis (⫹TCDD/-EM; n ⫽ 2) and TCDD exposure with endometriosis (⫹TCDD/⫹EM; n ⫽ 7). Mixing and quantitation of TCDD diets. For the surviving animals from the original TCDD-treated colony (0 PPT TCDD [n ⫽ 6], 5 PPT TCDD [n ⫽ 6], and 25 PPT TCDD [n ⫽ 3]), diets were prepared as follows: 2,3,7,8tetrachlorodibenzo-p-dioxin (Dow Chemical, Midland, MI) was prepared as a stock solution by diluting 19.8 ␮g dioxin in 1.0 ml benzene. One part stock solution was then diluted with 3550 parts acetone and 200 ml of the resulting solution was mixed with 8 kg of monkey chow (Ralston-Purina Co., Inc., St. Louis, MO). Additional normal meal was then added to yield 22.7 kg (50 lb) of chow with a final concentration of 50 PPT dioxin. This pre-mix (5- and 25-lb portions) was added to normal meal (final weight 50 lb) to make the 5-PPT and 25 PPT-diets, respectively. Diets were pelleted by the addition of 2 liters of water and 1 liter of glycerine per 50-lb bag, which served as binders. Control (0 PPT TCDD) chow was prepared without addition of TCDD, using benzene, acetone and glycerine, as described above. Dioxin was administered by addition to the daily allotment of 200 g of monkey chow. Food records documented that the animals consumed an average of 95% of their daily diet. Dioxin content in the feed was verified by gas chromatograph/mass spectrophotometer (GC/MS) analysis of selected samples over the 4-year treatment period. Diets for the additional control animals with no previous PHAH exposure (n ⫽ 12) consisted of commercial monkey chow. Diagnostic laparoscopies. The presence and severity of endometriosis in these animals was determined by diagnostic laparoscopy as described (Rier et al., 1993). The presence of endometrial glands and stroma in ectopic endometrial tissue from all animals was confirmed by histological studies performed the following year at animal sacrifice. No postoperative complications occurred in any of the animals; the absence of infection was confirmed by normal complete blood cell counts (CBC). The severity of endometriosis was determined according to human criteria, using the revised American Fertility Society (rAFS) classification system (American Fertility Society, 1985). Although the presence of endometriosis was determined previously in 15 monkeys (Rier et al., 1993), disease status was re-evaluated in these animals at the time of immune studies. Additional control animals with no previous PHAH exposure were also evaluated for endometriosis (n ⫽ 12). Staining and flow cytometric analysis of rhesus peripheral blood leukocytes. The phenotypic distribution and activational status of PB leukocytes were determined by multiparameter, flow-cytometric analysis. Leukocytes from PB were stained with monoclonal antibodies (mAbs) specific for human leukocyte surface antigens expressed at different stages of cellular activation using a whole-blood lysis method. The majority of primary mAbs were labeled either with fluorescein isothiocyanate (FITC) or phycoerythrin (PE); primary unlabeled mAbs to cluster designation (CD) 28 and CD3 (FN 18) were also used. Briefly, mAbs were added to 75 ␮l of heparinized blood at the recommended concentrations and incubated for 20 min at room temperature. Cells were washed once with 2 ml of wash buffer consisting of 0.5-g sodium azide and 0.2-g bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, MO) in 500 ml of calcium- and magnesium-free Dulbecco’s phosphate-buffered saline (DPBS, Gibco BRL, Grand Island, NY). When indirect staining was performed, cells were then incubated for 20 min at room temperature with 20 ␮l of goat anti-mouse-IgG conjugated to FITC or to PE diluted 1:20 in buffer solution (0.5 g sodium azide, 0.2 g BSA, and 18.5 ml EDTA in 81.5 ml DPBS; Sigma Chemical Co.) and then washed. Red blood cells were lysed using a commercially available lysing reagent (Whole Blood Lysing Kit, Coulter Corp.) according to the manufacturer⬘s recommendations. After 2 additional

INCREASED LEUKOCYTE TNF-␣ PRODUCTION IN TCDD-EXPOSED MONKEYS washes in 2 ml DPBS, cells were resuspended in 0.5 ml DPBS/1% formalin and refrigerated until flow-cytometric analysis. Samples were analyzed on an EPICS-C flow cytometer (Coulter Corp.) using the 488-nm laser line from an argon laser to excite FITC or PE. The leukocyte acquisition gate was set on side and log of forward-angle, lightscatter parameters and verified to include predominantly mononuclear cells, using leukocyte-specific mAbs. For each sample, a minimum of 5000 cells within the leukocyte acquisition gate was analyzed using the EPICS Elite software program (Coulter Corp.). The following mAbs specific for human leukocyte antigens were used in immunophenotyping studies: CDs 8, 14, 16, 19, 28, and 45 RO (Immunotech, Westbrook, ME), and CDs 4, 25 (Exalpha, Boston, MA), 56 (Becton Dickinson, Mountain View, CA) and 3 (FN 18 obtained from Dr. F. J. Nooij). Isotype-specific control mAbs were obtained from Immunotech, Exalpha, and Becton Dickinson. Previous primate studies have demonstrated that these mAbs cross-react with rhesus leukocyte antigens (Nooij et al., 1986; Reimann et al., 1994). Because commercially available human CD45-FITC does not cross-react with rhesus antigens, the total mononuclear cell count was obtained from the complete blood cell counts (CBC). Cell counts on ethylenediaminetetraacetic (EDTA)-anticoagulated rhesus blood samples were performed using a semi-automated Coulter Z f Hematology Cell Counter (Coulter Corp., Hialeah, FL) in which cell size discriminators were optimized for this species. Leukocyte differential counts were performed manually by counting 100 white blood cells (WBCs) on Wright’s stained blood smear. Flow cytometric analyses are expressed as mean (SE) median number of cells positive for each antigen for each group (percent positive cells ⫻ the total mononuclear cell count). Cytokine production by rhesus peripheral blood leukocytes. Cytokine production by rhesus PB leukocytes in response to stimulation by phytohemagglutinin (PHA, T-cell mitogen; Sigma) or polyinosinic acid-polycytidylic acid (poly I:C or PIC, viral antigen/double-stranded RNA; Pharmingen, San Diego, CA) was determined. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density centrifugation, washed twice, and resuspended at a concentration of 1 ⫻ 10 6 cells per ml in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 50 ␮g/ml gentamycin sulfate, and 2 mM L-glutamine (complete medium; Gibco). Leukocytes (2 ⫻ 10 6 cells per well) were incubated overnight with complete medium in 24-well microtiter plates (Costar, Cambridge, MA). The following day, cells received either media alone (unstimulated), 5 ␮g/ml PHA (Sigma) or 10 ␮g/ml Poly I:C (Pharmingen). Cell culture supernatants were harvested at 48 h and were stored at –70°C until assayed for cytokines. The levels of TNF-␣, IL-6, IFN␥ and IL-10 present in the PBMC supernatants were determined using commercially available enzyme-linked immunosorbent assays (ELISA) kits (TNF-␣, Genzyme Corporation, Cambridge MA; IL-6, and IL-10, R&D Systems, Minneapolis, MN and rhesus IFN␥, Biosource International, Camarillo, CA). These ELISAs contain mAbs which are specific for rhesus cytokine (IFN␥) or demonstrate known cross-reactivity (TNF-␣, IL-6 and IL-10) (Villinger et al., 1993). The detection limits for these assays are 10 pg/ml TNF-␣, 4 pg/ml IFN␥, 3 pg/ml IL-6, and 8 pg/ml IL-10. The intra- and interassay coefficients of variation for all ELISAs were less than 8%. ELISA calculations are expressed as mean (SE) median cytokine (pg/ml) for each group. Cytolytic activity of rhesus peripheral blood leukocytes. A standard chromium-release assay was conducted simultaneously against 2 target cell lines: K562 (erythroleukemic cell) and RAJI (Epstein-Barr virus-transformed B cell). Target cell cultures were maintained and passaged twice weekly in RPMI 1640 supplemented with 10% FBS (Gibco) and periodically tested for mycoplasma contamination. The target cells were incubated for 1–2 h in 125 ␮Ci of sodium chromate (Cr-51) and subsequently washed twice in supplemented RPMI. The concentration of target cells was held constant at 4 ⫻ 10 4 cells/ml, while the concentration of effector cells was varied to produce triplicate wells of 3 effector-to-target (E:T) ratios (100:1, 50:1, and 25:1) from each subject’s sample. After a 5-h incubation, 200 ␮l of supernatant was harvested using a Skatron harvesting system, and the amount of chromium released by the lysed cells quantified with a Beckman gamma counter, expressed as counts per minute (cpm). Results of cytolytic assays were calculated as % specific lysis

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and lytic units. Percent specific lysis of target cells at each of the 3 E:T ratios was calculated by the formula: % lysis ⫽

test cpm ⫺ spontaneous release cpm ⫻ 100 maxium release cpm ⫺ spontaneous cpm

Test cpm refers to each sample’s count; spontaneous and maximum release counts refer to the control wells. Maximum control wells consisted of target cells and a 2% solution of centrimide detergent; spontaneous wells contained target cells suspended in supplemented RPMI. Spontaneous release of the chromium was consistently less than 8% in all cytolytic assays. The methods used to calculate the lytic units in these studies were based on the formula by Whiteside, Bryant, Day, and Herberman (Whiteside et al., 1990). Briefly, lytic units were used to represent lytic activity that increases with potency per lytic batch, where a lytic batch equals 10 7 effector cells. An estimate of lysis at 20% was calculated from a regression line plotting % lysis by effector:target ratios (E:T20) by using the following formula: 10 7/(E:T20)(5 ⫻ 10 3) where 10 7 was the standard number of effector cells and 5 ⫻ 10 3 was the standard number of targets. Cytolytic data calculations are expressed as mean (SE) median lytic units. Statistical analysis. The relationship between immune variables (PBMC phenotypic distribution, cytokine production, cytotoxic activity) and exposure/ serum variables (TCDD exposure, serum PHAH, or triglyceride concentrations) was determined using Spearman’s correlation coefficient. The relationship between TNF-␣ vs. IFN␥ or IL-6 production also was evaluated using Spearman’s correlation coefficient. Significant differences between animal groups were determined by Wilcoxon’s rank-sum test. In the following text, all values are medians. For calculations, ␪ was used to denote not detected (ND) values.

RESULTS

The goal of this study was to determine the long-term effects of TCDD exposure on systemic immunity in the rhesus monkey with regard to 4 end points: (1) the immune status of these animals as assessed by PBMC phenotype (flow cytometric data) and function (cytokine and cytolytic data); (2) the presence and severity of endometriosis; (3) the serum concentrations of TCDD and related PHAH chemicals; and (4) the serum lipid content. The data were collected from TCDD-treated and untreated monkeys 13 years following the termination of treatment as previously described (Rier et al. 1993). The TCDDtreated group includes animals dosed at either 5- or 25-PPT TCDD and is referred to as “ALL TCDD” or “exposed”. Control 0 PPT TCDD animals are referred to as “unexposed”. Figure 1 and Table 1 present PBMC cytokine production; Table 2 presents PBMC phenotypic distribution; and Figure 4 shows PBMC cytolytic activity. When appropriate, immune variables were correlated with cumulative exposure and the serum concentrations of PHAHs or lipids as described in the text and Figures 2– 4. Cytokine production and cytolytic activity by peripheral blood leukocytes from TCDD-treated rhesus monkeys. PBMC from unexposed animals secreted low or undetectable levels of TNF-␣ in vitro following mitogen exposure (Table 1). In contrast, PBMC production of TNF-␣ in response to T-cell mitogen (TNF-␣-PHA) was significantly increased in a dosedependent manner in TCDD-treated animals (Fig. 1). For these

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FIG. 1. Increased production of TNF-␣ in response to stimulation by T-cell mitogen by peripheral blood mononuclear cells (PBMC) from TCDDexposed animals. PBMC were incubated for 48 h in the absence or presence of PHA (TNF␣-PHA). The concentration of immunoreactive cytokine in cell culture supernatants was determined by ELISA. Unstimulated PBMC did not produce detectable levels of TNF␣. Results are expressed as mean ⫾ SE. TNF␣ pg/ml, with median value given above bar representing each animal group. PBMC production of TNF␣ was examined in TCDD-exposed and unexposed monkeys: 0 PPT TCDD (n ⫽ 10), 5 PPT TCDD (n ⫽ 6), 25 PPT TCDD (n ⫽ 3), ALL TCDD (n ⫽ 9), -TCDD/-EM (n ⫽ 7), -TCDD/⫹EM (n ⫽ 3), ⫹TCDD/-EM (n ⫽ 2), ⫹TCDD/⫹EM (n ⫽ 7). PBMC production of TNF␣-PHA was increased with TCDD dose (p ⬍ 0.01; Jonckheere’s Test). Significant differences between groups were evaluated using the Wilcoxon’s rank-sum test. ap ⬍ 0.04 5-PPT or 25-PPT group compared with corresponding value for 0-PPT group; bp ⬍ 0.01 ALL-TCDD group compared with corresponding value for 0-PPT group; cp ⫽ 0.067 non-significant trend for ⫹TCDD/-EM group versus -TCDD/-EM group; and dp ⬍ 0.01 ⫹TCDD/ ⫹EM group compared with corresponding value for -TCDD/-EM group. ND, not detected.

animals, cumulative TCDD exposure correlated with enhanced PBMC TNF-␣-PHA production (p ⬍ 0.04; data not shown). Among unexposed animals with and without endometriosis, PBMC TNF-␣-PHA production was similar (Fig. 1). TNF-␣PHA secretion by PBMC was significantly increased in TCDD-exposed animals with disease (128 pg/ml) relative to unexposed controls without disease (not detected). PBMC from TCDD-exposed animals that did not have endometriosis also tended to produce elevated levels of TNF-␣-PHA relative to unexposed controls without disease (trend, p ⫽ 0.067). Thus, the observed increase in leukocyte TNF-␣-PHA production was associated with TCDD exposure rather than endometriosis. In the presence of viral antigen, similar amounts of TNF-␣ were released by PBMC from all animal groups (Table 1). PBMC secretion of IFN␥, IL-6 and IL-10 in response to mitogen or antigen stimulation was similar among exposed and unexposed animals (Table 1), although a strong trend in increased PHA-stimulated IFN␥ production by leukocytes from TCDD-exposed animals, as compared to controls, was noted (trend p ⫽ 0.056). Overall, leukocytes that released increased levels of TNF-␣ produced higher levels of other inflammatory cytokines following mitogen exposure. Enhanced PBMC TNF␣-PHA secretion correlated with increased PHA-stimulated production of IFN␥ and IL-6 (p ⬍ 0.05; data not shown).

As shown in Figure 2, serum levels of TCDD, 1,2,3,6,7,8hexachlorodibenzofuran (HxCDF) and 3,3⬘,4,4⬘,5-pentachlorobiphenyl (PnCB) correlated directly with PBMC secretion of TNF␣-PHA (p ⬍ 0.03; p ⬍ 0.04; p ⬍ 0.03, respectively). Moreover, increased leukocyte TNF-␣-PHA production was associated with elevated serum triglycerides (Fig. 3; p ⬍ 0.05). Secretion of IL-6 by antigen or mitogen-stimulated PBMC was unrelated to serum triglyceride levels (data not shown). As shown in Figure 4A, PBMC cytoxicity against NK-sensitive RAJI cells was decreased in TCDD-treated animals as compared to unexposed animals; however, this trend did not reach statistical significance. NK cytolytic activity was similar among unexposed animals with and without disease. Cumulative TCDD exposure correlated with decreased leukocyte cytolytic activity against RAJI target cells (Fig. 4B; p ⬍ 0.03). Thus, decreased NK cytolytic activity by PBMC was associated with prior TCDD treatment and not the presence of endometriosis. Leukocyte cytolytic activity against K562 cells was not different between animal groups (data not shown). Furthermore, serum levels of TCDD or dioxin-like PHAHs were not associated with changes in PBMC cytolytic activity directed against RAJI or K562 cells (data not shown). Phenotypic distribution of peripheral blood leukocytes from TCDD-treated rhesus monkeys. The phenotypic distribution and the activational status of PBMC from TCDD-exposed and unexposed controls were examined utilizing multiparameter flow cytometry. Neither the concentration of WBCs nor the TABLE 1 Cytokine Production by Leukocytes from TCDD-Exposed Monkeys Cytokine-stimulus

0 PPT

ALL TCDD

TNF-␣-PHA TNF-␣-PIC IFN␥-PHA IFN␥-PIC IL-6-PHA IL-6-PIC IL-10-PHA IL-10-PIC

35 (21) 0 (n ⫽ 10) 285 (47) 335 (n ⫽ 9) 670 (111) 699 (n ⫽ 9) 534 (122) 323 (n ⫽ 9) 2867 (451) 2813* (n ⫽ 11) 4539 (469) 4930 (n ⫽ 10) 227 (62) 147 (n ⫽ 18) 265 (51) 183 (n ⫽ 15)

209 (77) 128** (n ⫽ 9) 376 (102) 329 (n ⫽ 9) 1641 (422) 1000 a,* (n ⫽ 9) 477 (105) 346 (n ⫽ 9) 3378 (752) 3303 (n ⫽ 9) 6286 (1181) 5548 (n ⫽ 9) 388 (111) 308 (n ⫽ 9) 207 (42) 153 (n ⫽ 9)

Note. Peripheral blood leukocytes were incubated for 48 h in the absence or presence of PHA or PIC. Levels of cytokines spontaneously released by cells were subtracted from total cytokine produced in the presence of mitogen or antigen. Unstimulated PBMC did not secrete TNF-␣ or IFN␥ while low levels of spontaneous PBMC IL-10 secretion were detected in two animals. PBMC from all animals spontaneously secreted similar, but variable, median levels of IL-6 (unexposed, 24.2 pg/ml; TCDD-exposed, 6.1 pg/ml). The concentration of immunoreactive cytokines in cell culture supernatants was determined by ELISA. Results are expressed as mean cytokine levels pg/ml (SE) median. a Non-significant trend for PHA-stimulated IFN␥ value compared with corresponding value for 0 PPT-TCDD group; p ⫽ 0.056. *Within subgroup PHA-stimulated cytokine was determined to be significantly different when compared with the corresponding value for PIC-stimulated cytokine; p ⬍ 0.05. **Determined to be significantly different when compared with value for 0 PPT-TCDD group; p ⬍ 0.01.

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TABLE 2 Peripheral Blood Leukocyte Distribution in TCDD-Exposed Monkeys Cell type/receptor T cell IL-2 receptor

Th Naı¨ve Th

Tc,s Co-stimulatory receptor

CD4/CD8 ratio NK cell FcR␥ receptor

Monocyte B cell

Marker

0 PPT

ALL TCDD

CD3⫹ CD3⫹/CD25⫹ CD3⫹/CD25CD3-/CD25⫹ CD4⫹ CD4⫹/CD45RA⫹ CD4⫹/CD45RACD4-/CD45RA⫹ CD8⫹ CD8⫹/CD28⫹ CD8⫹/CD28CD8-/CD28⫹ CD4⫹/CD8⫹ CD56⫹ CD16⫹ CD16⫹/CD56⫹ CD16⫹/CD56CD16-/CD56⫹ CD14⫹ CD19⫹

2329 (452) 1981 2062 (289) 2005 290 (184) 14 1462 (192) 1243 1194 (137) 1167 480 (69) 405 714 (87) 684 1527 (386) 1109 1875 (442) 1400 241 (31) 221 1629 (446) 1157 344 (59) 308 0.8 (0.07) 0.8 1014 (396) 309 245 (49) 181 91 (26) 37 157 (31) 116 923 (387) 289 93 (50) 39 109 (25) 84

2019 (329) 1835 1899 (307) 1742 121 (46) 91 2041 (325) 2187 1387 (207) 1269 571 (146) 519 816 (149) 816 1536 (222) 1649 1647 (217) 1802 246 (109) 220 1401 (224) 1493 333 (81) 341 0.9 (0.06) 0.8 457 (54) 431 374 (111) 243 120 (31) 99 254 (97) 145 336 (45) 361 127 (59) 61 101 (46) 20

Note. The distribution of peripheral blood leukocytes was determined by flow cytometric analysis in TCDD-exposed (ALL TCDD; n ⫽ 9) and unexposed animals (n ⫽ 18). Results are expressed as mean (SE) median number of positive cells per ml blood. Among animals from the original colony of TCDD-exposed (n ⫽ 9) and 0 PPT TCDD animals (n ⫽ 6), a significant increase was observed in the number of CD16⫹/CD56⫹ NK cells in animals treated with 5 PPT or 25 PPT TCDD (controls, 19; 5 PPT TCDD, 141.5; 25 PPT TCDD, 61 ⫽ median values). In addition, the number of CD3-/CD25⫹ cells was increased in TCDD-treated animals (controls, 884; exposed, 2187 ⫽ median values). Th, T helper cell; Tc,s, T cytotoxic, suppressor cell.

percentages of leukocyte subsets differed between animal groups (data not shown). CD3⫹ T cells were the predominant PBMC population in all animals (Table 2). Significant numbers of CD56⫹ NK cells were present with smaller populations of CD19⫹ B-cells and CD14⫹ monocyte/macrophages. Unlike humans, where the ratio of CD4/CD8 cells is generally greater than 1, the CD4/CD8 ratio in all animals was 0.8 – 0.9, similar to that reported previously for the rhesus (Rappocciolo et al., 1992). The phenotypic distribution of PB leukocytes and surface antigen expression consistent with activation (CD16/ Fc␥III receptor, CD25/IL-2 receptor, CD28/B7 co-stimulatory receptor) or naive cells (CD45RA) did not differ between animals treated with 5 PPT or 25 PPT TCDD relative to 0 PPT controls. PBMC surface antigen expression was also similar in animals with and without endometriosis (exposed and unexposed groups, data not shown). In all animal groups, the IL-2 receptor was highly expressed on CD3⫹ T cells and CD3- cells while the naive antigen CD45RA was absent on the majority of CD4⫹ cells, suggesting activation of CD3⫹, CD3- and CD4⫹ cell populations. Although TCDD exposure was not associated with changes in leukocyte surface antigen expression, elevated serum concentrations of TCDD, 1,2,3,6,7,8-HxCDF and 3,3⬘,4,4⬘,5-PnCB correlated with increased numbers of CD3⫹/ CD25- and CD3-/CD25⫹ leukocytes in PB (TCDD, 1,2,3,6,7,8-HxCDF or 3,3⬘,4,4⬘,5-PnCB vs. CD3⫹/CD25- p ⬍

0.02; TCDD, 1,2,3,6,7,8-HxCDF, or 3,3⬘,4,4⬘,5-PnCB vs. CD3-/CD25⫹ p ⬍ 0.05; data not shown). DISCUSSION

The work described herein demonstrates that TCDD exposure in the rhesus monkey was associated with long-term changes in PBMC TNF-␣ production in vitro. Furthermore, elevated serum levels of TCDD, 1,2,3,6,7,8-HxCDF and 3,3⬘,4,4⬘,5-PnCB, resulting from TCDD exposure, correlated with aberrant PBMC TNF-␣ responses and an altered activational status of PB leukocyte subpopulations. The data suggest that endometriosis and specific changes in immune status, accompanied by increased serum PHAH levels, occur following TCDD exposure. Our finding of enhanced mitogen-stimulated TNF-␣ production by leukocytes from TCDD-treated animals with endometriosis confirms and extends previous studies suggesting that TNF-␣ may play a role in TCDD toxicity (Clark et al., 1991; Moos et al., 1994, 1997; Taylor et al., 1990, 1992 ) and the pathogenesis of endometriosis (Braun et al., 1996; Halme, 1989; Rana et al., 1996). Moreover, the present studies indicate that alterations in leukocyte TNF-␣ production following exposure to T-cell mitogen are cytokine- and stimuli-specific. In the presence of viral antigen, leukocyte secretion of TNF-␣ was similar among animal groups. These findings suggest a

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cytokines. Following leukocyte PHA stimulation, increased TNF-␣ secretion correlated with enhanced IFN␥ and IL-6 production and PBMC from TCDD-treated animals tended to produce increased levels of IFN␥ (trend, p ⫽ 0.056). Since TNF-␣ is a potent inducer of IL-6 and IFN␥ expression (Vassalli, 1992), PB leukocytes from TCDD-exposed animals may produce increased levels of IFN␥ and IL-6 as a result of changes in leukocyte activation accompanying chronic TNF-␣ secretion. A number of recent studies suggest that the long-recognized hypersensitivity of TCDD- and PCB-treated animals to endotoxin may be related to increased TNF-␣ production in TCDDexposed animals. TCDD treatment of AhR-responsive mice results in a dose-dependent increase in serum TNF␣ levels following endotoxin exposure, when compared to AhR-deficient mice (Clark et al., 1991). Moreover, production of TNF-␣ and IL-6 by leukocytes and liver cells may be increased in rodents exposed to PHAH chemicals, followed by endotoxin challenge (Hoglen et al., 1992; Taylor et al., 1990). These reports and others have noted that many of the pathophysiologic features of TCDD- or TNF-␣-mediated toxicity are similar. Animal studies have shown that acute TCDD treatment (Taylor et al., 1992) or chronic exposure to TNF-␣-secreting tumors (Spiegelman and Hotamisligil, 1993) are associated with cachexia or wasting syndrome, characterized by severe weight loss. Alterations in lipid and glucose metabolism are also found in animals exposed to TCDD (Zinkl et al., 1973) or high TNF␣ levels (Spiegelman and Hotamisligil, 1993). The ability of dexamethasone or TNF antibody to reverse the mortality and weight loss associated with treatment with TCDD and endotoxin is consistent with a role for inflammatory reFIG. 2. Increased serum levels of TCDD and dioxin-like PHAH congeners correlated with PHA-stimulated tumor necrosis factor-alpha (TNF-␣PHA) production by peripheral blood mononuclear cells (PBMC) from TCDDexposed animals. (A) Serum TCDD levels in parts per quadrillion (PPQ) vs. PBMC production of TNF-␣-PHA; p ⬍ 0.04. (B) Serum 1,2,3,6,7,8-HxCDF levels PPQ vs. PBMC production of TNF-␣-PHA; p ⬍ 0.03. (C) Serum 3,3⬘,4,4⬘,5-PnCB levels PPQ vs. PBMC production of TNF-␣-PHA; p ⬍ 0.04. TCDD was administered to animals in their feed for approximately 4 years. Thirteen years after termination of TCDD exposure, TNF-␣ production by PBMC and serum PHAH levels were determined in TCDD-treated (n ⫽ 9) and unexposed (n ⫽ 6) animals. The relationship between serum PHAH levels and leukocyte TNF-␣-PHA production was analyzed using Spearman’s rank correlation coefficient.

T-cell role in aberrant TNF-␣ secretion by leukocytes from TCDD-treated animals and are consistent with work demonstrating that T cells play an integral role in TCDD-mediated effects (Kerkvliet et al., 1996). These data are also in agreement with reports indicating that the nature of the antigenic stimulus may be an important factor in the sensitivity of immune responses to TCDD toxicity (Kerkvliet et al., 1990). Overall, leukocytes which released higher levels of TNF-␣ in vitro also produced elevated amounts of other inflammatory

FIG. 3. Increased production of PHA-stimulated tumor necrosis factoralpha (TNF-␣-PHA) by peripheral blood mononuclear cells (PBMC) from TCDD-exposed rhesus monkeys correlated with elevated levels of serum triglycerides. Serum triglyceride levels versus PBMC production of TNF-␣PHA; p ⬍ 0.05. TCDD was administered to animals in their feed for approximately 4 years. Thirteen years after termination of TCDD exposure, TNF-␣ production by PBMC and serum triglyceride levels were determined in TCDDtreated (n ⫽ 9) and unexposed animals (n ⫽ 6). The relationship between leukocyte TNF␣ production and serum triglyceride levels was analyzed using Spearman’s rank correlation coefficient.

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FIG. 4. Peripheral blood mononuclear cell (PBMC) cytolytic activity against NK-sensitive target cells negatively correlated with TCDD exposure. (A) PBMC cytolytic activity was generally decreased in TCDDexposed animals. (B) Cumulative TCDD dose correlated with decreased PBMC cytotoxicity; cumulative TCDD dose vs. RAJI lytic units p ⬍ 0.03; cumulative TCDD dose vs. % specific lysis at 100:1, 50:1, and 25:1 E:T ratios: p ⫽ 0.027, p ⫽ 0.012, and p ⫽ 0.013, respectively. PBMC cytolytic activity was determined using the RAJI cell line and a standard 51Cr assay in TCDD-exposed and unexposed animals: 0 PPT TCDD (n ⫽ 6), 5 PPT TCDD (n ⫽ 6), 25 PPT TCDD (n ⫽ 3), ALL TCDD (n ⫽ 9), -TCDD/-EM (n ⫽ 4), -TCDD/⫹EM (n ⫽ 2), ⫹TCDD/-EM (n ⫽ 3), ⫹TCDD/⫹EM (n ⫽ 6). Results (A) are expressed as mean ⫾ SE lytic units, with median value given above bar representing each animal group. Significant differences between groups were evaluated using the Wilcoxon’s rank-sum test. The relationship between cumulative TCDD exposure and PBMC cytotoxicity was analyzed using Spearman’s rank correlation coefficient.

sponses and enhanced expression of TNF-␣ in TCDD toxicity (Taylor et al., 1992). More recently, a series of studies have investigated the effect of TCDD and TNF-␣ on peritoneal leukocyte responses. TCDD or TNF-␣ administration in rodents enhances leukocyte inflammatory responses and cellular infiltration of macrophages and neutrophils into the peritoneal cavity following antigen challenge (Kerkvliet and Oughton, 1993). TCDD-induced peritoneal hyperinflammation can be blocked by neutralization of endogenous TNF activity (Alsharif et al., 1994; Kerkvliet and Oughton, 1993). Following pretreatment of mice with TCDD, TNF-␣ secretion by endotoxin-stimulated peritoneal leukocytes ex vivo is increased (Moos et al., 1997). Moreover, a peritoneal macrophage cell line (IC-21) produces increased levels of TNF-␣ and exhibits enhanced intracellular expression of this cytokine in vitro following exposure to TCDD and endotoxin (Moos et al., 1997). These findings have led Kerkvliet and colleagues to postulate that TNF-␣ is a key mediator of TCDD-enhanced

peritoneal leukocyte responses to antigenic challenge and that TCDD can act directly on peritoneal leukocyte subpopulations to increase TNF-␣ expression in response to stimulation (Kerkvliet and Oughton, 1993; Moos et al., 1997). Because TCDD exposure was associated with increased leukocyte TNF-␣ secretion in vitro, we questioned whether elevated serum concentrations of TCDD and dioxin-like PHAHs in vivo correlated with this response. Interestingly, increased TNF-␣ secretion by mitogen-stimulated PBMC was dependent upon the original TCDD exposure dose and levels of TCDD, 1,2,3,6,7,8-HxCDF and 3,3⬘,4,4⬘,5-PnCB present in serum when cytokine assays were performed. In related studies with this colony of monkeys 13 years post TCDD treatment (Rier et al., 2001), TCDD exposure correlated with increased serum levels of TCDD, 1,2,3,6,7,8-HxCDF, 3,3⬘,4,4⬘-tetrachlorobiphenyl (TCB) and 3,3⬘,4,4⬘,5-PnCB. Moreover, animals with elevated serum levels of the dioxin-like PCB congeners 3,3⬘,4,4⬘-TCB and 3,3⬘,4,4⬘,5-PnCB had a high

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prevalence of endometriosis. These findings suggest that serum levels of TCDD and specific PHAH chemicals depend upon previous TCDD exposure dose and that elevated serum levels of dioxin-like PCBs may be associated with endometriosis and alterations in immune function many years after termination of exposure. Serum levels of TCDD and PCBs in TCDD-treated monkeys and unexposed animals are similar to, or lower than, human background levels of these toxicants (Rier et al., 2001; DeVito et al., 1995). Thus, aberrant leukocyte TNF-␣ production may result from a limited time period of TCDD exposure and persists after serum dioxin levels return to background. Importantly, the observation that increased leukocyte TNF-␣ secretion in vitro correlated with elevated serum triglyceride levels provides evidence of overexpression of this cytokine in vivo by leukocytes from TCDD-treated animals. Hypertriglyceridemia reported in TCDD-exposed animals (Zinkl et al., 1973) and humans (Calvert et al., 1996) may be mediated by increased TCDD-induced TNF-␣ expression in vivo since TNF-␣ increases serum levels of lipids by stimulating hepatic lipogenesis and increased lipolysis (Spiegelman and Hotamisligil, 1993). In the current study, enhanced leukocyte TNF-␣ production and elevated serum triglycerides in TCDD-treated animals were not associated with cachexia. These findings are consistent with studies demonstrating that chronic administration of TNF-␣ often results in persistent hypertriglyceridemia without inducing weight loss (Spiegelman and Hotamisligil, 1993). Because the majority of TCDD-treated animals had endometriosis, and studies have shown that PB leukocytes from women with this disease secrete increased levels of TNF-␣ (Braun et al., 1996), aberrant production of this cytokine may be a consequence of endometriosis, rather than TCDD-treatment. However, PHA-stimulated leukocytes from TCDD-exposed animals without endometriosis tended to produce increased levels of this cytokine relative to cells from unexposed females without disease. In addition, PBMC secretion of TNF-␣ following mitogen exposure was similar in unexposed animals with and without endometriosis. These findings suggest that TCDD exposure, and not endometriosis, was associated with increased PBMC production of TNF-␣ in response to T-cell stimuli. It is possible that TCDD-treated animals without endometriosis may not have been susceptible to disease, exhibiting many of the biological effects of TCDD treatment while genetically protected from the effects of overexpression of TNF-␣, perhaps by increased serum glucocorticoids. Recent studies indicate that TCDD exposure (Kerkvliet et al., 1990, 1996) and endometriosis (Oosterlynck et al., 1991, 1992) are associated with decreased leukocyte cytotoxicity. Because cytolytic activity by leukocyte subpopulations may be important in limiting ectopic endometrial growth, we investigated the ability of PB leukocytes from TCDD-treated animals to exert cytolytic activity against NK-and T cell-sensitive target cells. Previous work has shown that K562 and RAJI target cell lines are differentially susceptible to NK- and T-cell cy-

tolytic activity in the rhesus monkey (Lemieux et al., 1996). In contrast to human cells, RAJI targets are lysed primarily by the CD3- cell subset while CD3⫹, CD8⫹, and CD3- cells are responsible for lysis of K562 targets. In the current study, increased cumulative TCDD exposure correlated with decreased PB leukocyte cytolytic activity directed at the NKsensitive RAJI cells. Lymphocytoxicity against RAJI cells was generally decreased in TCDD-treated animals with and without endometriosis as compared to unexposed animals with and without disease. Thus, the observed trend in decreased PB lymphocytoxicity was again associated with TCDD treatment, rather than endometriosis. PBMC cytolytic activity against K562 cells was unaltered by TCDD treatment or the presence of endometriosis. These findings are consistent with previous studies with this animal colony demonstrating that lymphocytoxicity against K562 cells is not affected by TCDD exposure (Hong et al., 1989). The negative correlation between cumulative TCDD exposure and NK cytolytic activity observed in these studies may result from chronic leukocyte expression of TNF-␣, since reports indicate that increased leukocyte expression of this cytokine accompanying aging is associated with low cytolytic activity (Mysliwska et al., 1997). Alterations in PBMC secretion of TNF-␣ and cytolytic activity by these cells in TCDD-treated animals occurred in the absence of quantitative changes in lymphocyte subsets or the expression of activation antigens on these cells. These findings are in agreement with human (Anonymous, 1988) and rodent studies (Kerkvliet and Brauner, 1990; Moos et al., 1994) indicating that TCDD exposure may alter leukocyte function without modulating the distribution or surface antigen expression of leukocyte subpopulations. However, increased serum levels of TCDD, 1,2,3,6,7,8-HxCDF and 3,3⬘,4,4⬘,5-PnCB, resulting from TCDD exposure, correlated with decreased IL-2 receptor expression on T cells and increased IL-2 receptor expression on CD3- mononuclear cells (NK cells, macrophages, or B cells). These findings suggest that increased serum levels of TCDD and dioxin-like PCBs may be associated with decreased activation of T-cells accompanied by enhanced activation of CD3- cells. This evidence is in agreement with studies demonstrating suppression of T-cell function (Kerkvliet et al., 1990, 1996) coupled with increased peritoneal inflammatory responses by myeloid cells in TCDD-treated mice (Alsharif et al., 1994; Kerkvliet and Oughton, 1993). In agreement with limited human studies finding no differences in the phenotypic distribution of PB leukocytes in women with and without endometriosis (Hill et al., 1988; Oosterlynck et al., 1994), no alterations in PBMC phenotype were found in animals with endometriosis relative to animals without disease. The potential role of TCDD and TNF-␣ in the pathogenesis of endometriosis remains to be elucidated. Evidence indicates that TNF-␣, IL-6 and IFN␥ play a critical role in normal endometrial growth activity and the expression of these cytokines appears to be regulated by sex hormones (Rier and Yeaman, 1997; Tabibzadeh, 1994). TNF-␣ has been recently

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implicated as delivering the key signal required for apoptosis of endometrial cells (Tabibzadeh et al., 1995). Studies demonstrating that aberrant expression of TNF is associated with endocrine-responsive tumor growth (Wu et al., 1993) highlight the importance of the stringent control of the expression of this cytokine in growth regulation. Recent reports suggest that increased expression of TNF-␣, IL-6, and IFN␥ by peritoneal leukocytes and endometrial cells may result in unregulated ectopic endometrial growth. Peritoneal leukocytes from women with endometriosis secrete increased levels of TNF-␣ and IL-6 (Halme, 1989; Rana et al., 1996; Rier et al., 1994) and ectopic endometrial cells exhibit aberrant expression of IL-6 and IFN␥ as compared to uterine cells from women with disease (Klein et al., 1993; Rier et al., 1995; Tseng et al., 1996; Zarmakoupis et al., 1995). Chronic TNF-␣ secretion by peritoneal and uterine leukocytes, therefore, may induce IL-6 and IFN␥ secretion by retrograde immune and endocrine endometrial cell populations within the peritoneal fluid resulting in aberrant adhesion, vascularization, and proliferation of these cells at extrauterine sites. Studies have shown that TNF-␣ increases the adhesion of endometrial stromal cells to peritoneal mesothelium (Zhang et al., 1993) and promotes angiogenesis (Leibovich et al., 1987). TCDD may target PB, peritoneal, and endometrial leukocyte populations, inducing chronic expression of TNF-␣ in response to stimuli resulting in disruption of growth-regulatory processes. TCDD and other dioxin-like PHAH chemicals may directly affect expression of this cytokine via a DRE in the TNF-␣ gene as found in TCDD induction of IL-1␤ (Sutter et al., 1991). Alternatively, TCDD may influence the secretion of this cytokine indirectly by disrupting the hormonal regulation of TNF-␣ expression, since this toxicant modulates endocrine homeostasis (Whitlock, 1994). Receptors for sex steroids have been demonstrated in PB leukocytes, and peritoneal and uterine cells (Rier and Yeaman, 1997; Tabibzadeh, 1994). Thus, immune and endocrine cell populations at these sites may represent particularly hormonally sensitive targets for TCDD. We postulate that the combined exposure to TCDD and dioxin-like PCBs alters leukocyte activation and TNF-␣ secretion by subpopulations of PB and uterine cells, resulting in the aberrant growth of ectopic endometrium in susceptible rhesus monkeys. In summary, these studies demonstrate that TCDD exposure and serum levels of TCDD and dioxin-like PHAHs in the rhesus are associated with increased leukocyte TNF-␣ production in response to T-cell mitogen in vitro, a cytokine important in both immune responsiveness and endometrial growth regulation (Tabibzadeh, 1994, 1995). Consistent with overexpression of leukocyte TNF-␣ in vivo, increased PHA-stimulated PBMC production of this cytokine correlated with elevated serum triglycerides. This work provides evidence suggestive of dysregulated TNF-␣ production and decreased cytolytic activity by leukocytes in peripheral blood of TCDD-exposed animals more than 13 years after termination of TCDD treatment, lending further support to the hypothesis that long-term sys-

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temic immune alterations are associated with exposure to this toxicant. ACKNOWLEDGMENTS We are grateful to Mary Lou Ballweg of the Endometriosis Association for her leadership in the protection of this colony of animals; and Joseph Karaszewski, Fang Li, Sharon Swink, and Eric Snowdeal III for their excellent technical expertise. This work was supported by The Endometriosis Association, Tracy H. Dickinson, The Fairleigh S. Dickinson, Jr. Foundation, Inc., and NIH grant ES08545– 01 from the National Institute of Environmental Health Sciences. S.E.R. holds the Tracy H. Dickinson Research Chair of the Endometriosis Association.

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