Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferator, perfluorooctanoic acid

International Immunopharmacology 2 (2002) 389 – 397 www.elsevier.com/locate/intimp

Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferator, perf luorooctanoic acid Qian Yang a,*, Manuchehr Abedi-Valugerdi b, Yi Xie a, Xiao-Yan Zhao b, Go¨ran Mo¨ller b, B. Dean Nelson a, Joseph W. DePierre a a

Unit for Biochemical Toxicology, Department of Biochemistry and Biophysics, Wallenberg Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden b Department of Immunology, Stockholm University, SE-106 91 Stockholm, Sweden Received 14 April 2001; received in revised form 2 October 2001; accepted 12 October 2001

Abstract In a previous investigation, we demonstrated that severe thymus and spleen atrophy occurs in mice upon dietary exposure to several potent peroxisome proliferators (PPs). In the present investigation, the effects of the potent PP perfluorooctanoic acid (PFOA) on the adaptive immunity of mice was evaluated both in vivo and ex vivo. The in vivo immune response examined involved immunization of mice with horse red blood cells (HRBCs), displaying T-cell-dependent antigens after pre-treatment with a PFOA-containing diet for 10 days. Subsequent quantitation of the primary humoral response was performed employing both the plaque-forming cell (PFC) assay and determination of the antibody titer by ELISA. The results clearly demonstrate that oral administration of PFOA prevents both the increases in plaque formations by anti-IgM and -IgG and in serum levels of IgM and IgG normally evoked by such immunization. Ex vivo spleen cells proliferation (assayed as incorporation of 3H-thymidine) in response to both T- and B-cell activators was attenuated by dietary treatment with PFOA, although the analogous in vitro treatment of mouse spleen cells with this same compound had no such effects. Thus, the relatively metabolically inert PP PFOA may exert adaptive immunosuppression in mice by an indirect mechanism. The possible relevance of this immunosuppression to the alterations in plasma lipids caused by PPs is discussed. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Peroxisome proliferation; Peroxisome proliferators; Immunotoxicology; Immunosuppression; Mice

1. Introduction

Abbreviations: PFOA, perfluorooctanoic acid; HRBC, horse red blood cells; PFC, plaque forming cell; ConA, concanavalin A; LPS, lipopolysaccharide; PPs, peroxisome proliferators. * Corresponding author. Tel.: +46-8-164239; fax: +46-8153024. E-mail address: [email protected] (Q. Yang).

There is increasing awareness that xenobiotics (i.e., drugs and other foreign chemicals) have profound deleterious effects on the immunological defences of exposed organisms, via either direct or indirect mechanisms (for a review, see Ref. [1]). Dysfunction of the immune system, e.g., caused by xenobiotics, may

1567-5769/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 ( 0 1 ) 0 0 1 6 4 - 3

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result in immunosuppression and an increased incidence and severity of infection and tumor frequency, or in immunoenhancement and an increased reactivity toward self-competent and development of autoimmunity disorders [2]. Peroxisome proliferators (PPs) constitute a large and growing family of widespread foreign compounds (1000 at present). These include many industrial chemicals (e.g., plasticizers such as phthalates and surfactants such as perfluoro fatty acids), agrochemicals (e.g., pesticides such as phenoxyacetic acids) and important clinical drugs (e.g., non-steroidal antiinflammatory drugs and hypolipidemic agents such as fibrate derivatives) (for a review, see Ref. [3]). The most extensively characterized effects of PPs on rodents are increases in the number and size of peroxisomes, hypertrophy of the liver and a potent up-regulation of hepatic peroxisomal fatty acid boxidation. Prolonged treatment of rodents with PPs results in the formation of liver tumors (for reviews, see Ref. [4,5]). Recently, it has been demonstrated in our laboratory that severe thymus and spleen atrophy in mice results from dietary exposure to several potent peroxisome proliferators [6]. This finding suggests that peroxisome proliferators might disturb the functions of the immune system. The major objective of the present study was to test this suggestion. Specifically, we examined the effects of the potent and relatively inert PP, perfluorooctanoic acid (PFOA), on specific humoral immunological responses in mice. Furthermore, ex vivo splenocyte proliferation in response to the T- and B-cell activators concanavalin A (ConA) and lipopolysaccharide (LPS), respectively, was also evaluated. Finally, in attempt to elucidate whether the effects of PFOA on lymphocytes are direct, lymphocyte proliferation in the presence of this compound in vitro was determined. The findings reveal that PFOA is potently immunosuppressive, possibly via an indirect pathway.

(B&K Universal, Sweden). The animals were randomly divided into groups of four to six, which were then housed individually in steel cages. They were maintained with a 12-h light/dark cycle at 25 C with free access to water and laboratory chow R3 (5% fat, 24% protein, and 49% carbohydrate (Astra Ewos, So¨derta¨lje, Sweden)). All animals were acclimated to this condition for at least 1 week before commencement of the experiment. The diet containing 0.02% (w/w) PFOA (Aldrich Chemical, Steinheim, Germany) was prepared as described previously [7]. Briefly, PFOA was dissolved in 20 ml acetone and mixed with 500 g powdered R3 laboratory chow, after which the chow was dried in a ventilated hood until no smell of acetone was detectable (>24 h). Control chow was mixed with acetone alone.

2. Materials and methods

2.3. The protein-A plaque assay

2.1. Animals and treatment

The numbers of spleen cells secreting antibodies of different Ig classes and subclasses were determined by employing a protein-A plaque assay (plaque-forming cells, PFC) as described previously [8]. Rabbit anti-

All experiments were performed on male C57BL/ 6 mice weighing 22– 28 g (about 8 –10 weeks old)

2.2. PFOA treatment and immunization with HRBCs In this experiment, animals were randomly divided into five groups (four to six mice/group). Groups 2, 4 and 5 were fed diets containing PFOA for 10 days whereas groups 1 and 3 received normal food. Subsequently, mice in groups 3, 4 and 5 were injected intravenously (i.v.) with 200 ml Earle’s balanced solution (EBSS) containing 5 – 10  107 horse red blood cells (HRBCs). Thereafter, group 4 continued to receive the PFOA-containing diet, while groups 3 and 5 received normal chow. These treatments are documented in Table 1. Six days after HRBC immunization, the animals in all groups were bled by retro-orbital puncture under light diethyl ether anesthesia and subsequently sacrificed by cervical dislocation. The spleens were removed and single splenocyte suspensions prepared by teasing the organ apart gently with forceps in EBSS. Blood samples were allowed to clot at room temperature, and the serum was separated by centrifugation and stored at 20 C prior to antibody analysis.

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Table 1 Suppression of the PFC response of mice to HRBC by PFOA Diet during the initial 10-day period Control PFOA Control PFOA PFOA

Immunize with HRBC

+ + +

Diet during the subsequent 6-day period

IgM (%)

IgG1 (%)

IgG2b (%)

IgG3 (%)

Control PFOA Control PFOA Control

10000 ± 2000 (100) 2667 ± 1155 (27) 27500 ± 8699 (275) 7600 ± 3286** (76) 26000 ± 2828 (260)

280 ± 110 (100) 200 ± 0 (71) 60000 ± 22684 (21400) 320 ± 268*** (114) 2320 ± 522*** (829)

533 ± 416 (100) 267 ± 115 (50) 6150 ± 2306 (1150) 320 ± 268*** (60) 2320 ± 610*** (435)

300 ± 200 (100) 200 ± 0 (67) 4450 ± 681 (1480) 840 ± 518*** (280) 3040 ± 1228 (1010)

C57BL/6 mice were treated with PFOA and immunized with HRBC. Splenic antibody-producing cells were quantitated employing a plaqueforming assay as described in detail in Materials and Methods. Data are the mean numbers of PFC/spleen ± SD for four or five animals. ** p < 0.01. *** p < 0.001 compared with the corresponding immunized control group.

mouse IgM, IgG1, IgG3 (Organon Teknika, Durham, NC, USA) and IgG2b (Nordic Immunological Laboratories, Tilburg, Netherlands) were used as the developing reagents. 2.4. ELISA assay for anti-HRBC IgM and IgG1 antibodies Specific IgM and IgG1 antibodies against HRBC were measured utilizing an ELISA procedure as described [9,10]. Briefly, flexible U-bottomed 96-well polystyrene plates (Corning Costar, Cambridge, MA, USA) were coated with HRBC and the sera containing anti-HRBC IgM and IgG1 antibodies were incubated with these HRBC antigens. Alkaline phosphatase-conjugated goat antimouse IgM or IgG1 (Southern Biotechnology, Birmingham, AL, USA; all conjugated antibodies were diluted 1:2000 in PBS – Tween) were then bound to the primary antibodies and the development of color was measured on a microplate reader (SPECTRAmax 250, Molecular Devices) at 405 nm utilizing p-nitrophenyl phosphate as a substrate. 2.5. Lymphoproliferative assay In this experiment, the mice were randomly divided into two groups, one of which received dietary 0.02% (w/w) PFOA for 7 days and the other normal chow. At the end of treatment, the animals were killed by cervical dislocation, their spleens removed and single splenocyte suspensions prepared (as above). Lymphoprolif-

eration in response to the T- and B-cell activators ConA and LPS, respectively, was measured as described earlier [11]. For the ex vivo test, the same numbers of splenocytes from either PFOA-treated or control mice were used. For the in vitro test, PFOA (dissolved in dimethylsulfoxide (DMSO)) was added to the medium at the indicated concentrations at the beginning of the experiment. In this case, the same amount of DMSO alone (0.05% final concentration) was added to the control medium. 2.6. 3H-thymidine incorporation as a measure of DNA synthesis 3

H-thymidine (Amersham International, UK) was added to cell cultures at different time points (2  10 6 Ci/ml). Cells were harvested 12 h later using a Skatron cell harvester (Lier, Norway). Incorporated radioactivity was counted in a Rack-Beta Scintillation Counter (LKB Pharmacia, Uppsala, Sweden). 2.7. Cellularity and cell viability Cells were counted employing a haemocytometer. Cell viability, as determined on the basis of trypan blue exclusion, was always >90%. 2.8. Statistical analysis Each experimental group contained four to six animals. Data are expressed as means ± SD. The

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results of statistical analysis using the Student’s t-test are presented where appropriate.

3. Results 3.1. Immune response to HRBC immunization To elucidate whether PFOA treatment causes immunosuppression in mice, the specific humoral immunological responses of PFOA-treated mice to HRBC were first evaluated. The protein-A plaque assay (PFC) (which quantitates the number of B-cells producing HRBC-specific antibodies e.g., IgM and IgG) represents a relative holistic approach to assessment of the humoral immune response. An enzyme-linked immunosorbent assay (ELISA), designed to quantitate the HRBC-specific antibodies produced in response to immunization is another end-point for this same response. In this study, both the PFC assay and ELISA procedures were employed. In the case of the PFC assay, male C57BL/6 mice were administered diets containing no or 0.02% (w/w) PFOA for 10 days prior to immunization with HRBC for 6 days. Such immunization of normal mice induced a strong humoral immune response, with the numbers of IgM, IgG1, IgG2b and IgG3 antibodyproducing cells in the spleen all being increased (Table 1). The most pronounced increase occurred in IgG1-producing cells and the smallest increase in IgM-producing cells. It can also be noted that the IgM anti-HRBC titer is quite high in non-immunized animals, which may reflect the presence of crossreacting ‘natural’ antibodies. In contrast, upon immunization of PFOA-treated mice with HRBC, the numbers of splenic cells producing antibodies of all isotypes (IgM and IgG) were almost the same as in non-immunized animals (Table 1). When PFOA-treated mice received normal chow following immunization with HRBC, there was significant recovery of the numbers of these specific antibody-producing cells (Table 1). Indeed, under these circumstances, the numbers of IgM- and IgG3-producing cells recovered almost totally, whereas the numbers of IgG1- and IgG2-producing cells were still strongly depressed. Clearly, PFOA treatment decreases the humoral immune response of mice to HRBC and this decrease requires the con-

tinued presence of PFOA, at least for some cell types. The ELISA assay revealed that following immunization of normal mice with HRBC for 6 days, the serum levels of anti-HRBC IgM and IgG1 antibodies were dramatically increased, as expected (Fig. 1a,b). Within a wide range (20 – 640 fold) of dilution, significant increases in the serum levels of these IgG1 and IgM were observed, in agreement with the increased numbers of IgM- and IgG-producing cells (Table 1). After immunization of PFOA-treated mice with HRBC, the serum levels of anti-HRBC IgM and IgG1 antibodies were significantly decreased in comparison to immunized control animals (Fig. 1a,b), the extent of decrease being considerably greater for IgG1 than IgM. Again, in PFOA-treated mice administered normal chow after immunization with HRBC, significant recovery was observed. Thus, findings with the ELISA assay further confirmed that the humoral immune response was suppressed in mice exposed to PFOA. The decreases in the serum levels of HRBC-specific antibodies and in the numbers of cells producing HRBC-specific antibodies were similar. 3.2. Lymphocyte proliferation upon exposure to PFOA both ex vivo and in vitro Next, it was of interest to determine whether PFOA treatment inhibits the unspecific proliferative response of lymphocytes to stimulation by the polyclonal T- and B-cell specific activators ConA and LPS, respectively. The incorporation of radiolabelled thymidine into splenocyte DNA was monitored in ex vivo and in vitro experiments. In the ex vivo experiments, splenocytes isolated from both control and PFOA-treated (0.02%, 7 days) mice were exposed to ConA or LPS. As expected, both ConA and LPS caused a dramatic increase in thymidine incorporation into the DNA of control splenocytes, whereas prior PFOA treatment attenuated these responses (Fig. 2a,b). The effects of PFOA treatment on the response to LPS appeared to be more pronounced than in the case of ConA. In order to determine if PFOA causes direct inhibition of lymphocyte proliferation, its effects were studied in vitro on splenocytes from untreated mice stimulated with ConA or LPS. The concentrations of

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Fig. 1. Effects of PFOA treatment on specific IgM (a) and IgG1 (b) anti-horse red blood cells (anti-HRBCs) titres in C57BL/6 mice. PFOA treatment and immunization is same as in Table 1. (-^-): None; (-&-): PFOA; (-~-): None + HRBC; (-.-): PFOA + HRBC + PFOA; (--): PFOA + HRBC + Normal. Six days later after immunization, sera collected from the blood of four to five mice were measured for serum levels of specific IgM and IgG1 anti-HRBC antibodies by using a specific ELISA method. The results shown are means ± SD for four animals. * p < 0.05, * * p < 0.01, * * * p < 0.001 compared to the control group.

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PFOA (1– 200 mM) added to the culture medium are similar to the plasma levels reached in rodent receiving 0.02% PFOA (corresponding to 20 mg/kg/day) [12]. Lymphocyte proliferation was determined after 48 h of exposure to ConA or LPS, when the peak

Fig. 3. The effect of PFOA in vitro treatment on lymphocyte proliferation. The spleen cell from normal mice was cultured in medium alone and in ConA (2.5 mg/ml) (a) or LPS (50 mg/ml) (b) as negative and positive controls, respectively. PFOA in indicated concentration were added to the medium. Thymidine incorporation at peak time (day 2) is presented as mean CPM from triplicated cultures. The results shown are means ± SD for four animals. * p < 0.05, * * p < 0.01, * * * p < 0.001 compared to the control group.

Fig. 2. Kinetics of lymphocyte proliferation upon PFOA in vivo treatment. The PFOA treatment was described as in Materials and Methods. Thereafter, spleen cells were cultured in medium alone and in ConA (2.5 mg/ml) (a) or LPS (50 mg/ml) (b) as negative and positive controls, respectively. (-.-): Control; (-&-): PFOA treated. On the indicated days, [3H] thymidine was pulsed and incorporated radioactivity was counted. Mean CPM from triplicated cultures of control and PFOA treated are shown. The results shown are means ± SD for four animals. * p < 0.05, * * p < 0.01, * * * p < 0.001 compared to the control group.

response is obtained (see Fig. 2). PFOA did not alter lymphocyte proliferation under these conditions (Fig. 3a,b).

4. Discussion A number of observations indicate a potential role for PPs in regulating innate immune responses, e.g., in anti-inflammatory response via w- and b-oxidation of fatty acid-derived mediators of inflammation, meta-

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bolic processes regulated by PPARa (for reviews, see Refs. [13 – 15]). At the same time, the inflammatory response is also controlled by adaptive immunity (e.g., cytokines produced by subsets of T-cells control macrophage activation, smooth muscle proliferation and other aspects of inflammation) [16,17]. The effects of PPs on such adaptive immune responses have not been examined. In our previous studies, the potent PPs PFOA, WY14,643, nafenopin and di(2-ethylhexyl)phthalate were all found to cause severe atrophy of the thymus and spleen in mice, reflecting decreased numbers of both thymocytes and splenocytes in mice [6]. In order to eliminate the possible influence of metabolites, the metabolically inert PP PFOA was chosen for more detailed investigation. This study reveal that the thymus and spleen atrophy caused by PFOA is doseand time-dependent, and that hepatic peroxisome proliferation occurred prior to this immunotoxic effect [18]. Exposure to various xenobiotics, both in animal experiments and in clinical trials, has been found to decrease the weights of lymphoid organs (primarily the thymus, spleen and lymph nodes). Such atrophy is sometimes associated with disturbances in the normal histological architecture of the organ, as well as various functional alterations in the immune response, leading to impaired resistance to experimental infections and/tumor growth [19,20]. In the present investigation, the dose of PFOA and the period of treatment determined previously to produce significant thymus and spleen atrophy but few other effects was employed [18]. Treatment of mice with PFOA under these conditions dramatically decreased both the numbers of splenocytes producing antibodies directed towards HRBC and the titres of specific anti-HRBC antibodies in the sera (as determined by the PFC and ELISA assays, respectively). Thus, it is evident that PFOA causes a pronounced suppression of adaptive immunity in mice. Furthermore, the extent of decrease in the numbers of splenic cells producing antibodies of different isotypes is much more pronounced than the decreases in the total numbers of T- and B-cells, as described previously [6]. This observation indicates that the immunosuppression by PPs reflects not only a reduction in splenocyte number but also impairment of splenocyte function.

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Usually, the IgG or IgM antibody response to HRBC can be divided into three principle stages. Stage I involves antigen recognition, uptake and processing by the macrophage. In stages II and III (the effector phase), antigen-specific T- and B-cell activation, proliferation and differentiation lead typically to antigenspecific antibody synthesis and secretion by B-cells [21]. The sensitivity of the PFC and ELISA assays for the detection of immunotoxicity reflects the fact that three separate cell types — the macrophage, T-lymphocyte and B lymphocyte— as well as several physiological processes, are required for generation of the antibody response to a T-dependent antigen, e.g., HRBC [22]. Thus, the pronounced changes obtained with both the PFC and ELISA assays indicate that PFOA may impair several steps in the immune response. Under unstimulated conditions, most lymphocytes remain in the resting phase prior to encountering the appropriate antigen(s). In order to participate in an adaptive immune response, these naive lymphocytes must be induced to proliferate and differentiate. In present investigation, we found that the ability of mouse lymphocytes to proliferate in response to Tand B-cell antigens in vitro is reduced after in vivo treatment with PFOA. This indicates that inhibition of lymphocyte proliferation may contribute, at least in part, to the immunosuppression cause by PFOA treatment. On the other hand, cytokines secreted by cells of the immune system constitute another form of communication between these cells. The biological activities of cytokines are pleiotropic and play important roles in connection with cellular and humoral immune responses (for a review, see Ref. [23]). The possible effects of PPs on cytokine production, both in vivo and in vitro, remain to be examined. The relative metabolic inertness of PFOA and the fact that in vitro treatment of mouse lymphocytes with this compound did not alter their proliferative response to stimulation indicate an indirect effect. This is in good agreement with our previous observation that PFOA does not exert a direct effect on thymocyte proliferation [6]. Furthermore, the significant recovery of the immune response in PFOA-treated mice administered normal chow during HRBC immunization is also in agreement with our previous findings that the atrophy of the thymus and spleen are rapidly and

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completely reversed after withdrawal of PFOA from the diet [18]. It is now well established that PPs regulate lipid and lipoprotein metabolism by activating PPARa. It is likely that the hypolipidemic actions of PPs (e.g., decreases in serum levels of triglyceride and cholesterol) are, at least in part, due to the increase in hepatic fatty acid b-oxidation, resulting in enhanced fatty acid mobilization and utilization by the liver, where PPARa is primarily expressed [24 – 28]. At the same time, external lipid is required for the development and physiological responses of the immune system [29 – 33] and perturbation of both external lipids and the composition and structure of the lymphocyte plasma membrane will attenuate TCR signal transduction and interface with this cell’s functions [34 –36]. Indeed, our previous studies have demonstrated that the induction of hepatic peroxisome proliferation occurs prior to the thymus and spleen atrophy caused by PPs [18], suggesting that immunosuppression by PPs may involve activation of PPARa. This hypothesis is also supported by our recent findings that the severe thymus and spleen atrophy observed in wildtype C57BL/6 mice is not seen in PPARa-knockout mice (unpublished data). Thus, immunosuppression may be due to competition between the liver and peripheral tissues for serum lipids, resulting in a disturbance of the membrane lipid composition and structure (reflected in the ex vivo experiment) and a reduction in the supply of external lipids (most severe effect reflected in the in vivo experiment) to lymphocytes. PFOA is used in increasing amounts as a corrosion inhibitor, anti-wetting agent, surfactant and in fire extinguishers. To date, only a few epidemiological studies concerning the possible effects of PFOA on occupationally exposed workers have been conducted and these have failed to detect any significant toxicity [37 – 39]. In comparison with the dose employed here, the levels of human exposure are considerably lower. The molecular mechanism(s) underlying the ability of PPs to cause immunosuppression in rodents is now under further investigation in our laboratory. Further studies on the effects of PPs on rodents may improve our understanding of the relationship between lipid metabolism and the immune system. Such studies may also help elucidate a new mechanism by which non-genotoxic compounds enhance tumor develop-

ment, as well as improve our assessment of the possible risks posed by PPs to human health.

Acknowledgements The Knut and Alice Wallenberg Foundation (Stockholm) and the Environmental Fund of the Swedish Association of Graduate Engineers (Stockholm) have supported this study. The technical assistance of Lena Israelsson in connection with some of this work is gratefully appreciated.

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