Nutritional Immunology Antigen-Driven Murine CD4؉ T Lymphocyte Proliferation and Interleukin-2 Production Are Diminished by Dietary (n-3) Polyunsaturated Fatty Acids1,2 Lisa J. Pompos and Kevin L. Fritsche3 Department of Animal Sciences, Nutritional Sciences and the Comparative Medicine Program, University of Missouri, Columbia, MO 65211 ABSTRACT This study is the first to describe the impact of consuming a diet rich in (n-3) polyunsaturated fatty acids (PUFA) from fish oil on antigen-driven activation of naive CD4ϩ T lymphocytes. To accomplish this, we used lymphocytes isolated from T cell receptor (TCR) transgenic mice (i.e., DO11.10). A large portion of the T lymphocytes from these mice expresses a TCR specific for a peptide within the ovalbumin (OVA) molecule d (OVA323–339). When this antigen is presented in the context of major histocompatibility complex I-A with costimu- lation, these naive CD4ϩ T cells become activated, produce interleukin (IL)-2 and clonally expand. (n-3) PUFA enrichment was accomplished by feeding DO11.10 mice one of two nutritionally complete experimental diets that differed only in the source of fat: lard or menhaden fish oil [high in (n-3) PUFA]. After 2 wk of consuming the experimental diets, lymphocytes were isolated from the spleen of each mouse, then cultured in the presence of antigen (i.e., OVA323–339) or concanavalin A (Con A), a nonspecific, polyclonal T cell stimulus. IL-2 production and lymphocyte proliferation were determined after 48 and 72 h, respectively. Naive CD4ϩ T lymphocytes from fish oil-fed mice stimulated with antigen produced less IL-2 (ϳ33%; P Ͻ 0.001) and proliferated to a lesser extent (ϳ50%; P Ͻ 0.0001) than the same cells from lard-fed DO11.10 mice. When stimulated with Con A, (n-3) PUFA did not affect either proliferation or IL-2 production. In summary, we report for the first time that feeding mice a diet enriched with (n-3) PUFA reduces in vitro antigen-stimulated production of IL-2 and subsequent proliferation of naive CD4ϩ T lymphocytes. J. Nutr. 132: 3293–3300, 2002. KEY WORDS: ● (n-3) fatty acids ● mice ● T lymphocyte ● interleukin-2 ● proliferation Dietary fish and supplemental fish oil have been shown to improve the clinical course of ongoing autoimmune disease as have a beneficial effect on the clinical course of a number of well as reduce the risk of developing autoimmunity in the first autoimmune diseases, such as systemic lupus erythematosus place. and rheumatoid arthritis (1,2). These benefits are associated Consumption of fish or fish oil supplements rich in (n-3) with an increased intake of the (n-3) polyunsaturated fatty PUFA has been shown to reduce lymphocyte proliferation in acids (PUFA)4, eicosapentaenoic acid (EPA) and docosa- numerous species, including mice, rats, chickens and humans hexaenoic acid (DHA). The most frequently cited mecha- (8–13). The evidence to date suggests that (n-3) PUFA re- nisms for the immunomodulation by (n-3) PUFA are reduced duce lymphocyte proliferation by reducing the biosynthesis of proinflammatory eicosanoid and cytokine production and di- interleukin (IL)-2, an essential T cell growth factor. These minished lymphocyte proliferation (3–7). Reduced prolifera- studies involve the isolation of immune cells from either the tion of autoreactive T lymphocytes would be expected to peripheral blood or secondary lymphoid tissues such as the spleen and the subsequent culturing of these cells in vitro. Lymphocyte proliferation and cytokine production were in- 1 Presented in part at the 2002 Experimental Biology Meeting [Pompos, L. J., duced with a variety of nonantigenic stimuli (e.g., plant lec- O’Brien, A. & Fritsche, K. L. (2002) Antigen-stimulated lymphocyte proliferation tins, chemicals or cross-linking antibodies). Existing data and IL-2 production are diminished by N-3 fatty acids. FASEB J. 16: A621]. clearly demonstrate that the impact of (n-3) PUFA observed 2 Supported in part by U.S. Department of Agriculture Grant 00-35200-9115 and National Institutes of Health Grant T32 RR07004. may vary depending on how cells are stimulated and their 3 To whom correspondence should be addressed. culture conditions (reviewed in Ref. 14). E-mail: [email protected]. Despite the widespread acceptance that (n-3) PUFA reduce 4 Abbreviations used: 2-ME, 2-mercaptoethanol; APC, antigen-presenting cell; CFSE, carboxyfluorescein diacetate succinimidyl ester; Con A, concanavalin lymphocyte proliferation, our knowledge of how (n-3) PUFA A; DHA, docosahexaenoic acid; DTH, delayed-type hypersensitivity; ELISA, en- affect T cell responses to antigen stimulation is quite limited. zyme-linked immunosorbent assay; EPA, eicosapentaenoic acid; FAME, fatty Most of the evidence for (n-3) PUFA reducing antigen-spe- acid methyl ester; IL, interleukin; MHC, major histocompatibility complex; OVA, ovalbumin; PBS, phosphate-buffered saline; PUFA, polyunsaturated fatty acid; cific lymphocyte responses is based on a few reports in which TBHQ, tertiary butyl-hydroquinone; TCR, T cell receptor. researchers measured delayed-type hypersensitivity (DTH) re- 0022-3166/02 $3.00 © 2002 American Society for Nutritional Sciences. Manuscript received 21 June 2002. Initial review completed 8 July 2002. Revision accepted 9 August 2002. 3293 3294 POMPOS AND FRITSCHE sponses to recall antigens. Researchers have reported that TABLE 1 (n-3) PUFA from fish oil can either reduce DTH in mice (15,16) and humans (17) or have no effect (18,19). DTH Fatty acid composition of experimental diets1 reactions require the coordinated response of both antigen- specific T cells and monocytes/macrophages. This in vivo Dietary treatment groups response involves a complex array of inflammatory mediators, Fatty acids2 Low PUFA (lard) (n-3) PUFA (fish oil) chemokines and cells. Thus, the ability of (n-3) PUFA to reduce DTH responses provides only indirect evidence that g/100 g total fatty acids antigen-specific T lymphocyte function is altered. Studies of antigen-specific primary lymphocyte responses have been 14:0 — 4.6 technically difficult due to the relative low frequency of naive 16:0 15.9 17.7 ϳ 5 6 16:1(n-7) 1.1 9.9 lymphocytes to any given antigen ( 1in10–10 lympho- 18:0 19.0 3.6 cytes) (20). To overcome this experimental limitation we used 18:1(n-7) & (n-9) 51.9 16.8 DO11.10 mice that express a transgenic T cell receptor 18:2(n-6) 10.8 12.7 (TCR). This transgenic TCR is specific for a known peptide 18:3(n-3) 0.4 1.6 d 20:5(n-3) (EPA) — 15.9 within ovalbumin (i.e., OVA323–339) bound to I-A class II major histocompatibility complex (MHC) molecules (21). 22:5(n-3) — 2.9 22:6(n-3) (DHA) — 12.0 The use of these mice over the past decade has greatly ex- panded our understanding of antigen-specific cell signaling 1 ϩ The two semipurified diets used in this study contained 18 g/100 and responses of naive CD4 T lymphocytes. The purpose of g lard or 16 g/100 g menhaden fish oil and 2 g/100 g corn oil (i.e., fish this study was to use these transgenic mice to determine the oil). impact of dietary (n-3) PUFA on antigen-specific in vitro 2 Fatty acids are denoted by the number of carbons: the number of responses of naive CD4ϩ T lymphocytes. double bonds, followed by the position of the first double bond relative to the terminal methyl group (n-). MATERIALS AND METHODS Immune cell isolation. After being anesthetized with an intra- muscular injection of ketamine (200 mg/kg) and xylazine (16 mg/kg), Mice. DO11.10 mice (a generous gift from Dr. Marc Jenkins, mice were humanely killed by exsanguination. After they were bled, University of Minnesota, Minneapolis, MN) were bred and main- spleens were aseptically removed and placed in 5 mL of sterile tained at the Office of Animal Resources, University of Missouri phosphate-buffered saline (PBS) at room temperature. Spleens were (Columbia, MO). While in this barrier facility, DO11.10 mice were forced through a sterile tissue sieve into a single cell suspension. housed in autoclaved individually ventilated polycarbonate cages Erythrocytes in the spleen-derived lymphocyte preparation (hereafter containing autoclaved recycled paper bedding (Paperchip; Canbrands referred to as splenocytes) were removed by gradient centrifugation International, Ontario, Canada). The room was maintained on a using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) as described 12:12 h light:dark cycle at 23°C and 40–50% relative humidity. Mice previously (23). Immune cells were enumerated electronically with a had free access to commercial irradiated rodent diet (PicoLab Rodent Coulter Counter (model ZM; Beckman Coulter, Fullerton, CA), then Diet 20; Purina Mills, Richmond, IN) and acidified water in drilled resuspended in HEPES-buffered RPMI medium (GIBCO-BRL, water bottles. The mice were serologically negative for the following Grand Island, NY) containing 50,000 U/L penicillin, 50 mg/L strep- pathogens: mouse hepatitis virus, minute mouse virus, mouse parvo- tomycin, 2 mmol/L L-glutamine and 50 mL/L fetal bovine serum virus, Sendai virus, Mycoplasma pulmonis, Theiler’s murine encepha- (herein referred to as complete medium) at 1 ϫ 109 cells/L. lomyelitis virus, mouse rotavirus, pneumonia virus of mice, reovirus 3, Homologous serums collection. Blood that had been collected by lymphocytic choriomeningitis virus, ectromelia virus, mouse adeno- cardiac puncture using a 1-ml insulin syringe (BD Biosciences, Frank- virus 1 and 2 and polyomavirus. Mice were negative by culture for the lin Lakes, NJ) was immediately placed in 4 mL clot-activator serum- following bacteria: Pasteurella pneumotropica, M. pulmonis, Salmonella separator tubes (BD Biosciences). Blood was allowed to clot in the spp. and Pseudomonas aeruginosa. Mice were free of external and tube at room temperature for ϳ1 h, then was centrifuged (Ther- internal parasites.