The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 498-504

Dietary Long-Chain (n-3) Fatty Acids Facilitate Immune Cell Activation in Sedentary, but not Exercise-Trained Rats^1,2,3

Lindsay E. Robinson and Catherine J. Field⁴

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Dietary long-chain (n-3) fatty acids from fish oil and low intensity exercise have been reported, independently, to inhibit tumor growth in rats. The mechanism for these effects is not known but may be related to diet and exercise-induced alterations in immune function. To study the individual and combined effects of these interventions on anticancer immune responses, healthy Fischer 344 rats were fed, for 4 wk, one of two semi-purified diets (polyunsaturated to saturated fatty acid ratio = 0.9), which differed only in the composition of fat (200 g/kg) and provided long-chain (n-3) fatty acids at 0 or 33 g/kg of total fat. Rats were randomly assigned to groups in a 2 × 2 experimental design to swim 3 h/d or to remain sedentary. For sedentary rats, dietary (n-3) fatty acids increased (P < 0.05) splenic natural killer (NK) cell cytotoxicity and the percentage of activated (CD71⁺) T and B cells and macrophages in spleen after concanavalin A stimulation. For exercise-trained rats, feeding the high (n-3) diet decreased (P < 0.05) the percentage of CD71⁺ T helper and B cells after stimulation. NK cell cytotoxicity, and the percentages of CD71⁺ T cells, B cells and macrophages after stimulation in the high (n-3)-fed exercise-trained group were not different than those of the low (n-3)-fed sedentary group. Thus individually, but not in combination, long-chain (n-3) fatty acids and low intensity exercise may be advantageous by augmenting cell-mediated immune function and NK cell cytotoxicity in healthy rats.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Components of both the innate (nonspecific) and acquired (specific) immune systems are important contributors to host defense against invading pathogens and spontaneously arising tumor cells (Robins 1986, Whiteside and Herberman 1995). T lymphocytes, part of the specific cell-mediated immune system, can be activated by interaction with different stimuli such as antigens and mitogenic lectins. Activated T helper (CD4⁺) cells can mature into a subset of CD4⁺ cells (Th1) that release interleukin-2 (IL-2),⁵ resulting in further activation and proliferation of T cells and expression of the IL-2 receptor (Cantrell and Smith 1984) and transferrin receptor (Neckers and Cossman 1983) on the cell surface. The appearance of both types of receptor is critical for the subsequent proliferation of activated T cells (Neckers and Cossman 1983). In addition, IL-2 upregulates activation of cytotoxic natural killer (NK) cells (Britten et al. 1984) and macrophages (Taub and Cox 1995). Immune activation of CD8⁺ T cells can generate a population of effector cells with lytic capability, cytotoxic T lymphocytes, which have important roles in the recognition and destruction of altered self-cells (Robins 1986).

In experimental cancer models, feeding high levels of fish oil-derived long-chain (n-3) fatty acids, eicosapentaenoic acid [20:5(n-3), EPA] and docosahexaenoic acid [22:6(n-3), DHA], inhibits tumor growth (Karmali et al. 1984) and low to moderate intensity exercise increases host resistance to tumorigenesis (Baracos 1989). Although it has been suggested that these effects occur via alterations in the host immune system, their precise cellular and molecular mechanisms are not known. Furthermore, the combined effect of dietary (n-3) fatty acids and exercise on immunocompetence has not been assessed.

There is evidence from both human and animal studies that diets rich in EPA and DHA can affect immune cell activation in both the innate and specific immune systems (Jolly et al. 1997, Meydani et al. 1991, Yaqoob and Calder 1993). For example, animals fed high levels of fish oil have suppressed NK cell cytotoxic activity (Sanderson et al. 1995, Yaqoob et al. 1994a) and T lymphocyte proliferation in response to mitogen (Sanderson et al. 1995). In mice, the feeding of highly purified EPA or DHA ethyl esters has also been shown to affect the process of immune cell activation by suppressing T cell IL-2 secretion and subsequent proliferation in vitro (Jolly et al. 1997). However, the physiologic relevance of these effects is not clear, because many of these studies involved diets that were deficient in the essential (n-6) fatty acid linoleic acid [18:2(n-6)] and contained levels of (n-3) fatty acids far above those consumed by most humans. The specific dietary balance of long-chain (n-3) fatty acids (EPA and DHA) and (n-6) fatty acids necessary to promote immunocompetence has not been established.

Both human and animal studies have shown that exercise-induced immunomodulation may occur through an alteration in the type of immune cells present in different lymphoid organs and functional changes at the cellular level (Hoffman-Goetz and Pedersen 1994, Nieman and Nehlsen-Cannarella 1994). For example, exercise changes the proportion of different immune cells within blood (Field et al. 1991), decreases lymphocyte proliferation (Tvede et al. 1989) and increases NK cell cytotoxicity (Nieman et al. 1993). Compared with the number of studies investigating chronic exercise training (involving bouts of high intensity exercise) that have been associated with both deleterious and beneficial effects on immunocompetence (Hoffman-Goetz and Pedersen 1994, Lin et al. 1993), fewer studies have reported on the immunomodulatory effects of low to moderate intensity exercise training, such as that experienced during regular nonexhaustive exercise.

Using a low intensity exercise training protocol in rats, Shewchuk et al. (1997a) demonstrated that nonexhaustive swim training increased T cell mitogenic response, suggesting enhanced cell-mediated immune function. Although lymphocyte proliferation studies provide indirect evidence of immune cell activation, there is little direct evidence concerning the effect of exercise on the expression of cell surface markers of immune activation, such as the transferrin receptor (CD71). In addition, because the majority of exercise immunology studies have been conducted using immune cells from blood, little is known about the effect of exercise training on other lymphoid organs. Furthermore, many exercise studies have not controlled for dietary fat, a factor that modulates immune function.

In animal studies, exercise and diet can be precisely controlled so as to define relationships among diet, exercise and components of the immune system that are inaccessible in humans, such as spleen. The objectives of this study were to determine the individual and combined effects of dietary long-chain (n-3) fatty acids and low intensity exercise (swim training) on immunocompetence in healthy rats.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals, diets, and exercise.  Experiments were reviewed and approved by the Faculty of Agriculture and Forestry Animal Policy and Welfare Committee and were conducted in accordance with the Canadian Council on Animal Care guidelines. Twenty-eight male (152 ± 7 g) and twenty-eight female (108 ± 3 g) adult Fischer 344 rats (Charles River Laboratories, St. Constant, Canada) were housed individually in plastic shoebox cages in a temperature-controlled room (23°C) maintained on a 12-h light:dark cycle. Body weight and food intake were recorded every third day throughout the study.

Rats were randomly assigned to be fed one of two nutritionally complete, semipurified diets (Teklad Test Diets, Madison, WI) containing (per kg) 270 g high protein casein, 408 g carbohydrate (200 g starch, 208 g dextrose) and 200 g fat. The diets were supplemented with the following (per kg): 1.4 g choline, 6.2 g inositol, 2.5 g L-methionine, 10 g AOAC vitamin mix, 51 g Bernhart-Tomerelli mineral mix and 50 g nonnutritive cellulose. The compositions of the mineral and vitamin mixes have been previously reported (Clandinin and Yamashiro 1980). Both experimental diets provided a polyunsaturated to saturated fatty acid ratio (P/S) of ~0.9, as determined by gas-liquid chromatography (Field et al. 1988). The fatty acid composition of the diets is presented in Table 1. The two diets differed only in the composition of polyunsaturated fat, providing two different levels of long-chain (n-3) fatty acids from a mixed fish oil source (P-28 Nisshin lot #28020, Nisshin Flour Milling, Tokyo, Japan) i.e., low (0 g/kg) or high (33 g/kg of total fat). The only source of (n-3) fatty acids in the low (n-3) diet was -linolenic acid [18:3(n-3), 12 g/kg of total fat], provided by linseed oil (Galaxy Enterprises, Edmonton, Canada). The high (n-3) diet contained both 18:3(n-3) (2 g/kg of total fat) and long-chain (n-3) fatty acids provided by fish oil (per kg of total fat) as follows: 23 g 20:5(n-3), 2 g 22:5(n-3) and 8 g 22:6(n-3). All animals were given free access to food and water for 4 wk.

At the start of the study, rats in each diet group were randomly assigned to one of two activity groups. Rats either remained sedentary or followed a low intensity swimming program for 4 wk, training progressively to swim a maximum of 3 h/d (6 d/wk) as previously described (Baracos 1989). To control for the rats' response to water, those in the sedentary group were subjected to an identical period of water exposure by standing daily in 10 cm of water (33-35°C). Exercise was performed at the end of the dark cycle.

Rats were food deprived overnight before being killed. On experiment days, rats swam for 2 h (or remained sedentary) and were subsequently killed by CO₂ asphyxiation and cervical dislocation. All assays were performed on individual rats (n = 14 per treatment group unless otherwise indicated) with the exception of the NK cell cytotoxicity assay (sedentary, n = 12 per diet; exercise-trained, n = 5 per diet).

Splenocyte isolation and activation.  After CO₂ asphyxiation and cervical dislocation, the spleen from each animal was removed and weighed. Splenocytes were isolated aseptically as previously described (Field et al. 1990) in Krebs-Ringer HEPES buffer (pH 7.4) supplemented with bovine serum albumin (5 g/L). Isolated splenocytes (2.5 × 10⁹ cells/L) in complete culture media [RPMI 1640 (Fisher Scientific, Edmonton, Canada) supplemented with 4% (v/v) heat-inactivated fetal calf serum (ICN, Montreal, Canada), penicillin (1 × 10⁵ U/L), streptomycin (100 mg/L), amphotericin B (25 mg/L), glutamine (4 mmol/L), HEPES (25 mmol/L) and 2-mercaptoethanol (2.5 µmol/L)] were incubated in 25 cm² sterile culture flasks for 48 h in a humidified atmosphere at 37°C in the presence of 5% CO₂. The cell culture medium either contained no mitogen (unstimulated cells) or was supplemented with concanavalin A (Con A), a polyclonal T lymphocyte mitogen (5 mg/L; ICN). After 48 h, cells were centrifuged at 228 × g (Beckman J2-HC Centrifuge, Beckman Instruments, Palo Alto, CA) for 10 min (4°C). The pelleted cells were then washed twice in PBS supplemented with bovine serum albumin (20 g/L). Indirect single- and double-label immunofluorescence analyses (described below) were then performed on both unstimulated and stimulated splenocytes.

Indirect immunofluorescence (phenotype) assay.  Lymphocyte subsets from freshly isolated splenocytes and cultured splenocytes (unstimulated and stimulated) were identified by indirect immunofluorescence assay (Field 1995) by using supernatants from hybridomas secreting mouse monoclonal antibodies specific for the different rat mononuclear cell subsets (Barclay et al. 1993). The following monoclonal antibodies were used: OX19 (CD5), which recognizes a glycoprotein on the surface of thymocytes, T lymphocytes and macrophages; w3/25 (CD4), which recognizes rat T helper lymphocytes and macrophages; OX8 (CD8), which recognizes T cytotoxic/suppressor lymphocytes and NK cells; OX12, which recognizes a determinant on the rat  chain of immunoglobulin (Ig) on B lymphocytes; OX42, which reacts with a receptor found on most monocytes and macrophages; 3.2.3, which reacts with rodent NK cells; w3/13, which recognizes pan T cell, polymorphs, plasma cells and NK cells; and OX26 (CD71), which recognizes the transferrin receptor on activated T and B lymphocytes, and macrophages. All antibodies were kindly provided by A. Rabinovitch, University of Alberta, Edmonton, Canada, with the exceptions of 3.2.3, w3/13, and OX26 (Cedarlane Laboratories, Hornby, Canada). All monoclonal antibodies were mouse anti-rat IgG. For indirect single-label (one color) phenotype analysis, cells (5 × 10⁵) were incubated for 30 min at 4°C with an antibody, washed three times in 200 µL of PBS containing fetal calf serum (40 g/L), and incubated for another 30 min at 4°C with 50 µL of a 1:300 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (FIT-C, Organon Teknika, Scarborough, Canada) that has no cross reaction to rat IgG. To determine background fluorescence due to nonspecific binding of FIT-C, samples were incubated for 30 min at 4°C with FIT-C alone. The functional state of immune cells can be measured by performing an indirect double-label (two color) immunofluorescence assay using a monoclonal antibody against the transferrin receptor (CD71), a marker of cellular activation (Neckers and Cossman 1983), plus various monoclonal antibodies identifying lymphocyte subpopulations. For this assay, the phenotypic antibody (OX19, w3/25, OX8, OX12 or OX42) was incubated with FIT-C. After FIT-C incubation and washing, an aliquot of the activation marker antibody (OX26) was added to each well and incubated for 30 min at 4°C. Cells were then washed three times before incubation for 30 min at 4°C with 10 µL of a 1:25 dilution of phycoerythrin-conjugated goat anti-mouse IgG (Cedarlane Laboratories). As with FIT-C, samples were incubated for 30 min at 4°C with phycoerythrin alone to determine background fluorescence due to nonspecific binding. Cells were fixed in paraformaldehyde (10 g/L in PBS), and relative fluorescence intensities for each antibody were determined by flow cytometry (FACScan, Becton Dickinson, Sunnyvale, CA). The resulting percentages were corrected for background fluorescence by using the analysis of cells incubated with FIT-C or phycoerythrin alone. Unwanted events (dead cells and debris) were detected on the basis of forward scatter and side scatter and were excluded from subsequent phenotype analyses by electronic gating of the viable splenocyte population. Viable events (1 × 10⁴) were collected in list mode and all subsequent immunofluorescence analyses were performed on only these cells.

NK cell cytotoxicity assay.  A sodium chromate (⁵¹Cr, 5.55 MBq, Amersham Canada, Oakville, Canada) release assay was performed using NK cell-sensitive YAC-1 cells (American Type Culture Collection, Rockville, MD) as targets and freshly isolated splenocytes as effector cells, as previously described (Field 1995). Briefly, splenocytes were added in triplicate to the wells of 96-well V-bottom microtiter plates containing YAC-1 cells to achieve effector:target ratios between 5:1 and 100:1. After 4 h at 37°C, an aliquot of the supernatant was counted in a gamma counter (Beckman gamma 8000, Beckman Instruments, Mississauga, Canada) to determine the extent of target cell lysis. Spontaneous ⁵¹Cr release was determined by incubation of labeled target cells alone. Maximum release was determined from detergent lysis of labeled target cells. The percentage lysis of the target cells was calculated using the following formula: specific lysis (%) = 100 × [mean experimental ⁵¹Cr release (cpm)  mean spontaneous ⁵¹Cr release (cpm)]/[mean maximum ⁵¹Cr release (cpm)  mean spontaneous ⁵¹Cr release (cpm)]. Results were also calculated on a per cell basis using the number of NK cells present in the effector cell samples, as determined by indirect immunofluorescence assay (described above) with the use of the 3.2.3 monoclonal antibody. This was expressed as lytic units with one lytic unit being equal to the number of effector cells (×10³) required to cause 20% lysis of target cells.

Statistical analysis.  Results are presented as means ± SEM. All statistical analyses were conducted using the SAS statistical package (Version 6.11, SAS Institute, Cary, NC). The effect of sex was determined by a three-way ANOVA. If no effect of sex was found, the main effects (diet and exercise) were analyzed by a two-way ANOVA. The method of least-squares means was used to identify significant differences (P < 0.05) among treatment groups. NK cell cytotoxicity, food intake and body weight were compared among groups by a two-way split-plot (repeated measures) ANOVA (Steel and Torrie 1980). For phenotype data, a paired t test was used to compare freshly isolated splenocytes with those cultured without Con A for 48 h (significant effect of culture, P < 0.05). Similarly, a paired t test was used to compare splenocytes cultured without Con A for 48 h with those cultured with Con A for 48 h (significant effect of stimulation, P < 0.05).

	RESULTS

Abstract Introduction Methods Results Discussion References

Animal characteristics.  Diet did not significantly affect food intake, final body weight, weight change, spleen weight or the number of spleen cells per gram of spleen; therefore, rats in the low and high (n-3) diet groups within each activity group were combined to examine the effect of exercise (Table 2). Exercise training did not significantly affect average daily food intake relative to body weight or final body weight in male or female rats (Table 2). Sedentary males gained significantly (P < 0.01) more weight than exercise-trained males, whereas weight gain did not differ between sedentary and exercise-trained females (Table 2). For both male and female rats, absolute and relative spleen weights were significantly lower (P < 0.0001) in the exercise-trained group (Table 2). However, the number of immune cells isolated per gram of spleen (377 ± 12 × 10⁶, n = 56) did not differ by gender or activity (Table 2).

Single-label phenotypes in fresh and cultured (with and without mitogen) splenocytes.  Because there was no significant effect of gender on immune variables in this study, male and female rats were combined within each treatment group. Neither diet nor exercise training significantly affected the proportion (% of total) of lymphocyte subsets in spleen; therefore, treatment groups were combined within each cell culture condition for this variable (Table 3). The relative percentages of T helper cells (CD4⁺), B cells and macrophages were significantly higher in splenocytes cultured without Con A for 48 h than in freshly isolated splenocytes (Table 3). The relative percentages of total T cells (CD5⁺ and w3/13⁺) were significantly lower in splenocytes cultured with Con A for 48 h than in those cultured without mitogen, whereas the relative percentages of T suppressor/cytotoxic cells (CD8⁺) and macrophages were significantly higher after culture with Con A (Table 3).

Double-label phenotypes in fresh and cultured (without mitogen) splenocytes.  For freshly isolated splenocytes and those cultured for 48 h without mitogen, neither diet nor exercise training significantly affected the proportion (% of total) of CD71⁺ (transferrin receptor positive) splenocytes identified as CD5⁺, CD4⁺, CD8⁺ or macrophages. Therefore, treatment groups were combined within each cell culture condition for double-label phenotypes (Table 4). The relative percentage of CD71⁺ macrophages was significantly lower in splenocytes cultured without Con A for 48 h than in freshly isolated splenocytes (Table 4), whereas the relative percentages of CD71⁺ T cells (CD5⁺), CD71⁺ T helper cells (CD4⁺) and CD71⁺ T suppressor/cytotoxic cells (CD8⁺) were significantly increased following culture without Con A for 48 h (Table 4).

Double-label phenotypes in cultured (with mitogen) splenocytes.  Double-label phenotyping analysis of splenocytes after 48 h stimulation with Con A showed that, in the sedentary group, rats fed the high (n-3) diet had a significantly higher proportion (% of total cells) of CD5⁺, CD4⁺, CD8⁺, B cells and macrophages that were CD71⁺ compared with low (n-3)-fed rats (Fig. 1). However, exercise-trained rats fed the high (n-3) diet had a significantly lower proportion of CD4⁺ and B cells that were CD71⁺ after Con A stimulation compared with low (n-3)-fed rats (Fig. 1). In the low (n-3) diet group, rats that were exercise trained had a significantly higher proportion of CD5⁺, CD4⁺ and B cells that were CD71⁺ after stimulation with Con A than did sedentary rats. However, in the high (n-3) diet group, rats that were exercise trained had a significantly lower proportion of CD4⁺, CD8⁺ and B cells that were CD71⁺ after Con A stimulation compared with sedentary rats (Fig. 1).

View larger version (30K): [in this window] [in a new window]   Fig 1. The effect of dietary long-chain (n-3) fatty acids and exercise training in rats (SED, sedentary, n = 5 per diet; EX, exercise-trained, n = 5 per diet) on the proportion (% of total cells) of immune phenotypes (CD5+, CD4+, CD8+, B cells and macrophages) expressing the transferrin receptor (CD71) after splenocytes were stimulated with concanavalin A for 48 h. Values are means ± SEM. For each phenotype, the main effects (diet and exercise) were analyzed by a two-way ANOVA and the method of least-squares means was used to determine significant (P < 0.05) differences among groups. For each phenotype, values that do not have a common superscript are significantly different (P < 0.05).

Natural killer (NK) cell cytotoxic activity.  There was a significant effect of diet, activity and diet by activity interaction on NK cell cytotoxic activity as determined by a two-way split-plot (repeated measures) ANOVA. At the 5:1 and 25:1 effector:target ratios, sedentary rats fed the high (n-3) diet had a higher (P < 0.02) percentage of specific lysis of target YAC-1 cells (5.1 ± 0.6%; 13 ± 1%, respectively, n = 12) relative to sedentary rats fed the low (n-3) diet (3.2 ± 0.3%; 10 ± 1%, respectively, n = 11). At the 5:1 and 25:1 ratios, there was no effect of diet on NK cell cytotoxicity when rats were exercise trained (data not shown). For cells from sedentary rats, at the 50:1 and 100:1 ratios, those fed the high (n-3) diet had higher (P < 0.001) NK cell cytotoxic activity against the target cells compared with those fed the low (n-3) diet (Fig. 2). For exercise-trained rats, the dietary (n-3) level did not significantly affect NK cell activity (Fig. 2). When rats were fed the low (n-3) diet there was no significant effect of exercise training on NK cell cytotoxicity (Fig. 2). When rats were fed the high (n-3) diet, exercise-trained rats had lower (P < 0.001) NK cell cytotoxic activity compared with sedentary rats (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig 2. The effect of dietary long-chain (n-3) fatty acids on splenocyte natural killer cell cytotoxic activity (against YAC-1 cells) of sedentary (n = 12 per diet) and exercise-trained (n = 5 per diet) rats at the 50:1 and 100:1 effector:target ratios. Natural killer cell cytotoxic activity is expressed as the percentage of specific lysis, which is equal to 100 × (mean experimental 51Cr release from labeled YAC-1 cells  mean spontaneous 51Cr release)/(mean maximum 51Cr release  mean spontaneous 51Cr release). Values are means ± SEM. For each effector:target cell ratio, the main effects (diet and exercise) were analyzed by a two-way ANOVA and the method of least-squares means was used to determine significant (P < 0.05) differences among groups. For each effector:target cell ratio, values that do not have a common superscript are significantly different (P < 0.001).

The relative percentage of NK cells (3.2.3⁺) in fresh spleen was not affected by diet or exercise training (5.1 ± 0.3%, n = 29). Sedentary rats fed the high (n-3) diet had lower (P < 0.05) lytic units (the number of effector cells × 10³) required to cause 20% lysis of YAC-1 cells) than sedentary rats fed the low (n-3) diet (Fig. 3). Diet did not significantly affect lytic units for cells from exercise-trained rats (Fig. 3). When rats were fed the low (n-3) diet, exercise training did not significantly affect lytic units (Fig. 3). However, in the high (n-3) diet group, exercise-trained rats had higher (P < 0.05) lytic units than sedentary rats.

View larger version (11K): [in this window] [in a new window]   Fig 3. The effect of dietary long-chain (n-3) fatty acids on splenocyte natural killer cell cytotoxic activity (against YAC-1 cells) expressed as lytic units [the number (×103) of 3.2.3+ cells required to cause 20% lysis of target cells] of sedentary (n = 12 per diet) and exercise-trained (n = 5 per diet) rats. Values are means ± SEM. The main effects (diet and exercise) were analyzed by a two-way ANOVA and the method of least-squares means was used to determine significant (P < 0.05) differences among groups. Values that do not have a common superscript are significantly different (P < 0.05).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

In experimental cancer, feeding high levels of fish oil-derived long-chain (n-3) fatty acids, EPA and DHA, inhibits tumor growth (Karmali et al. 1984), as does regular exercise of low to moderate intensity (Baracos 1989). The mechanism for these inhibitory effects is not known, but may involve alterations in the host immune system. This is the first study to assess the combined effect of dietary long-chain (n-3) fatty acids and low intensity swim training on immunocompetence in healthy rats. Our major finding is that feeding a semipurified diet (P/S = 0.9) containing 33 g/kg of the total fat as long-chain (n-3) fatty acids facilitates immune cell activation in sedentary, but not exercise-trained rats.

It has been hypothesized that diet and exercise-induced alterations in immunity might be determined by assessing changes in the proportion of cells within lymphocyte subsets (T and B cells, NK cells and macrophages) (Hoffman-Goetz and Pedersen 1994). In agreement with previous studies (Jeffery et al. 1996, Payan et al. 1986, Yaqoob et al. 1994b), long-chain (n-3) fatty acids did not significantly alter the distribution of immune cell types present in spleen (Table 3). Despite no effect of diet and exercise, culturing splenocytes altered the proportion of immune cell types (Table 3). As expected, the proportion of macrophages was significantly increased in both the unstimulated and stimulated condition (Table 3). Surprisingly, the proportion of macrophages that were activated (transferrin receptor positive, CD71⁺) after culture without mitogen was decreased (Table 4). This may have been due to the adherence of activated macrophages to the plates during culture because it is difficult to recover activated cells that represent <10% of the total cell population.

Immune status can be monitored by measuring the activation state of immune cells. Cell activation is a complex process, involving a number of plasma membrane-associated events, including activation of phospholipase C (with generation of the second messengers 1,4,5-inositol triphosphate and diacylglycerol), Ca²⁺ mobilization and activation of protein kinase C (Hadden 1988). These processes ultimately result in proliferation, target cell lysis, production of cytokines and the expression of immune cell activation markers such as the transferrin receptor (CD71) (Neckers and Cossman 1983). In vitro, the functional state of immune cells can be assessed by measuring the expression of CD71, after lymphocytes have been stimulated with a mitogen such as Con A (Neckers and Cossman 1983). Compared with the number of studies investigating the effect of (n-3) fatty acids on lymphocyte proliferation (Yaqoob and Calder 1993), fewer studies have provided evidence concerning the effect of EPA and DHA on the expression of CD71. In an in vitro study, Calder and Newsholme (1992) showed that CD71 expression on the surface of Con A-stimulated rat lymphocytes was inhibited by culture with polyunsaturated fatty acids. In another study, Yaqoob et al. (1994b) demonstrated that feeding rats a diet containing very high levels of fish oil had no effect on the proportion of CD71⁺ cells after Con A stimulation of rat splenocytes. In contrast, feeding dietary long-chain (n-3) fatty acids to sedentary rats resulted in a higher proportion of T and B cells and macrophages that were activated (CD71⁺) after Con A stimulation (Fig. 1), suggesting an upregulation of the cell-mediated immune response. These results may be of importance because activated T helper (CD4⁺) cells produce IL-2, which induces activation of NK cells, an important anticancer defense mechanism (Britten et al. 1984, Whiteside and Herberman 1995). In this study, the cytotoxic activity of splenocytes was significantly higher for sedentary rats fed the high (n-3) diet compared with the other three groups (Fig. 2). Because the percentage of NK (3.2.3⁺) cells present in the splenocyte population was not affected by diet, the increased cytotoxicity in high (n-3)-fed sedentary rats may be due to a functional enhancement at the cellular level (lower lytic units, Fig. 3). These findings contradict many previous reports in the literature (Fritsche and Johnstone 1990, Meydani et al. 1988, Sanderson et al. 1995, Yaqoob et al. 1994a). For example, Yaqoob et al. (1994a) reported that rats fed a diet containing 100% fat from fish oil had significantly decreased NK cell activity. However, other studies indicate that this result may be a product of feeding extremely high (n-3) levels, because feeding 10-12 g/100 g of total fat in the diet as long-chain (n-3) fatty acids significantly increased NK cell cytotoxicity (Brouard and Pascaud 1993). The level of dietary long-chain (n-3) fatty acids used by Brouard and Pascaud (1993) was approximately three times the level in our experimental diet, but was substantially lower than that in earlier studies, which fed diets containing fish oil as the only fat source. Thus, it appears that very high levels of fish oil-derived eicosapentaenoic acid and docosahexaenoic acid, which would be difficult to achieve in the human diet, may be inhibitory (Fritsche and Johnstone 1990, Meydani et al. 1988, Sanderson et al. 1995, Yaqoob et al. 1994a), whereas lower, more physiologic, levels of long-chain (n-3) fatty acids may be stimulatory in terms of NK cell cytotoxicity.

Exercise has also been shown to modulate immune function (Hoffman-Goetz and Pedersen 1994). For example, changes in the proportion of cells within immune cell subsets in peripheral circulation (Hoffman-Goetz and Pedersen 1994), but not spleen (Lin et al. 1993), have been related to exercise intensity and duration. High intensity exercise is associated with transient increases in the absolute number and relative proportion of NK, B, and T suppressor/cytotoxic (CD8⁺) cells and decreases in T helper (CD4⁺) cells in human blood (Field et al. 1991). Less is known about the effect of low intensity exercise training on immune phenotypes. Consistent with previous work using an identical swimming protocol (Shewchuk et al. 1997b), the proportion of splenocytes within lymphocyte subsets was not affected when exercise-trained rats were killed immediately postexercise (Table 3).

In rats fed the low (n-3) diet, exercise resulted in an increased proportion of T and B cells that were activated (CD71⁺) after Con A stimulation (Fig. 1), an observation indicative of immune enhancement. The expression of the transferrin receptor on the cell surface is crucial for the subsequent proliferation of activated T cells (Neckers and Cossman, 1983). We have previously shown that swim training increases the proliferative response of T cells to Con A stimulation (Shewchuk et al. 1997a), providing further support for our finding that cell-mediated immune function is enhanced immediately postexercise in swim-trained rats. Interestingly, immune activation after exercise may depend, in part, on the type of fat in the diet because the proportion of immune cells that were activated (CD71⁺) after Con A stimulation was suppressed when exercise-trained rats were fed long-chain (n-3) fatty acids (Fig. 1). These findings suggest the importance of controlling dietary fat in studies investigating the role of exercise in modulating immune cell activation in healthy rats.

This study confirms the previous finding that low intensity exercise training does not significantly affect NK cell cytotoxic activity when rats are fed diets without long-chain (n-3) fatty acids (Fig. 2) (Shewchuk et al. 1997b). However, surprisingly, exercise resulted in suppressed NK cell activity when rats were fed EPA and DHA (Fig. 2). Again, our results suggest that the ability of exercise to alter immune function in rats may be modified by the type of fat in the diet.

Although we found that dietary long-chain (n-3) fatty acids and exercise training were immunostimulatory on an individual basis, the combination of dietary (n-3) fatty acids and low to moderate intensity exercise training did not enhance the immune parameters assayed. Thus, our results suggest that the immune responses produced by the high (n-3) diet and exercise training are not synergistic. To our knowledge, this is the first report of the interaction between dietary (n-3) fatty acids and exercise training with respect to immunocompetence. The mechanism underlying the interaction between dietary long-chain (n-3) fatty acids and exercise training requires further investigation.

This study has shown that providing long-chain (n-3) fatty acids in the diet, at a level achievable by segments of the human population, modulates immune function. Critically, however, the immune response to (n-3) polyunsaturated fatty acids depends on the level of physical activity: immunostimulatory effects of long-chain (n-3) fatty acids are observed in sedentary, but not exercise-trained rats. These findings may help to explain current controversies in exercise immunology literature, because diet is infrequently controlled in exercise studies. Furthermore, because there is an increasing amount of evidence implicating both dietary long-chain (n-3) fatty acids and regular, low intensity exercise as having beneficial roles in cancer prevention and treatment, our findings may be important when developing clinical strategies for cancer treatment.

	ACKNOWLEDGMENTS

The authors gratefully acknowledge J. Aldrich for animal care as well as S. Goruk and G. Parsons for excellent technical and editorial assistance, respectively.

	FOOTNOTES

Manuscript received 14 July 1997. Initial reviews completed 19 August 1997. Revision accepted 11 November 1997.

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