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Articles

R3230AC Rat Mammary Tumor and Dietary Long-Chain (n-3) Fatty Acids Change Immune Cell Composition and Function during Mitogen Activation¹

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

²To whom correspondence should be addressed. E-mail: Catherine.field{at}ualberta.ca

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Cytokine production DISCUSSION REFERENCES

 
Because anticancer immunity declines progressively with tumor growth, a major focus of current research in tumor immunology is the development of means to stimulate the host immune system. This study determined the effects of dietary long-chain (n-3) fatty acids and tumor burden on immune cell phospholipid composition and membrane-mediated immune defense in rats implanted with the R3230AC mammary adenocarcinoma. Fischer 344 rats (145 ± 2 g) were fed one of two semipurified diets (20 g/100 g fat) for 21 d before and 17 d after tumor implantation. Diets provided long-chain (n-3) fatty acids at 0 or 50 g/kg of total fat. Mammary tumor growth was 31% lower (P = 0.1) in rats fed long-chain (n-3) fatty acids. Dietary long-chain (n-3) fatty acids had beneficial effects on several host immune defenses, including activation of CD8⁺ T cells and type-1 cytokine (interferon- and tumor necrosis factor-) production (P < 0.05). Upregulated immune function in tumor-bearing rats fed the high (n-3) diet occurred concurrently with specific changes in the major membrane phospholipids phosphatidylcholine and phosphatidylethanolamine in high (n-3)-fed rats. Because membrane composition plays a critical role in immune function, additional work is needed to determine the relationship between alterations in the phospholipid composition of immune cells during cancer and subsequent upregulation of host defense in the tumor-bearing state.

KEY WORDS: • dietary (n-3) fatty acids • R3230AC mammary tumor • immune • mitogen • phospholipids • rats

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Cytokine production DISCUSSION REFERENCES

 
Anticancer immune defense involves components of both the innate (natural) and acquired (specific) cell-mediated immune systems, including natural killer (NK)³ cells, macrophages, CD4⁺ T helper (Th) and CD8⁺ T suppressor/cytotoxic cells (Adams et al. 1982, Robins 1986 , Whiteside and Herberman 1995 ). Because anticancer immunity declines progressively with tumor growth (Kiessling et al. 1999 , Shewchuk et al. 1996 ), a major focus of current research in tumor immunology is the development of means to stimulate the host immune system. For example, specific dietary nutrients have received considerable attention for their potential immunoenhancing properties (Atkinson et al. 1998 , Weimann et al. 1998 ).

Diets rich in fish oil-derived long-chain (n-3) fatty acids, eicosapentaenoic [C20:5(n-3)] and docosahexaenoic [C22:6(n-3)] acids, can affect components of both the innate and acquired cell-mediated immune systems (Robinson and Field 1998 ). The mechanism for the immunomodulatory effect of (n-3) fatty acids may involve changes in membrane-mediated functions through alterations in membrane lipid composition (Calder 1998 ). Changes in dietary fat composition can induce significant alterations in the composition and function of immune cell membranes (Field et al. 2000 , Hosack-Fowler et al. 1993, Peterson et al. 1998 ). In lymphocytes, membrane-associated events play a pivotal role in signal transduction (Hosack-Fowler et al. 1993 ), the expression of surface markers (Jenski et al. 1995 ) and cellular activation (Calder et al. 1994 ), all of which are important in immune cell function. Thus, changing the membrane composition of such cells, through modulating dietary lipids, may influence immune responses important in anticancer defense. Less is known about how the lipid composition of immune cell membranes is altered during tumor growth, when immune cells are activated or suppressed by the presence of the tumor. Furthermore, it is not currently known how dietary fat and tumor growth interact to affect immune cell membrane composition and function. The objectives of this study were to determine the effects of dietary long-chain (n-3) fatty acids and tumor burden on immune cell membrane phospholipid composition and membrane-mediated immune defense in rats implanted with the R3230AC mammary adenocarcinoma.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Cytokine production DISCUSSION REFERENCES

 
Materials.

RPMI 1640 culture media, fetal calf serum, glutamine, antimycotic-antibiotic solution (1 x 10⁵ U/L penicillin, 100 mg/L streptomycin, 25 mg/L amphotericin B) and HEPES were purchased from Gibco BRL (Burlington, ON, Canada). Bovine serum albumin, 2-mercaptoethanol, ionomycin, hyaluronidase, 8-anilino-1-naphthalene-sulfonic acid and phospholipid standards were obtained from Sigma (St. Louis, MO). Phorbol myristate acetate (PMA) was purchased from ICN (Montréal, QB, Canada). The OX19, w3/25, OX8, OX12 and OX42 monoclonal antibodies were kindly provided by A. Rabinovitch (University of Alberta, Edmonton, Canada). All monoclonal antibodies were mouse anti-rat immunoglobulin G (IgG). Phycoerythrin-conjugated goat anti-mouse IgG and all other monoclonal antibodies (except JJ319) were purchased from Cedarlane Laboratories (Hornby, ON, Canada). Fluorescein isothiocyanate-conjugated goat anti-mouse IgG was obtained from Organon Teknika (Scarborough, ON, Canada). Antibody JJ319 was purchased from PharMingen (Mississauga, ON, Canada). Antibodies and reagents for the interferon (IFN)- and tumor necrosis factor (TNF)- assays were obtained from Genzyme Diagnostics (Cambridge, MA) and R&D Systems (Minneapolis, MN), respectively. For cytokine assays, Immulon® high binding flat-bottom microtiter plates were obtained from Dynex Technologies (Chantilly, VA).

Animals and diets.

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. Thirty-three female Fischer 344 rats (145 ± 2 g) were obtained from a colony maintained at the University of Alberta and were housed in individual wire-mesh cages in a temperature controlled room (23°C) maintained on a 12-h light/dark cycle. Body weight and food intake were recorded every 3rd d throughout the study. Rats were randomly assigned to be fed nutritionally complete semipurified diets (Teklad Test Diets, Madison, WI) containing (per kg) 270 g high protein casein, 408 g carbohydrate and 200 g fat. Both diets met the (n-6) and (n-3) fatty acid requirements of growing rats. The complete nutrient composition of the diets has been reported (Robinson and Field 1998 ). The dietary polyunsaturated to saturated fatty acids ratio was 0.35 as determined by gas-liquid chromatography (Field et al. 1988 ). The two diets differed only in the composition of 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): low (0 g/kg) or high (50 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)], provided by linseed oil (Galaxy Enterprises, Edmonton, Canada). The fatty acid composition of the diets is presented in Table 1 . All rats were given free access to food and water. After 21 d of feeding, a freshly harvested R3230AC mammary tumor from a rat implanted 2–3 wk earlier was finely chopped under sterile conditions to prepare a tumor brei and 50 µL were injected subcutaneously in the inguinal region of experimental rats. Rats were killed by CO₂ asphyxiation and cervical dislocation 17 d after tumor implantation. At necropsy, the tumor and spleen were removed, weighed and used for the measurements described below. There were 14 tumor-bearing rats (7/diet) and 19 healthy (control) rats [9 rats were fed the low (n-3) diet and 10 rats were fed the high (n-3) diet].

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 (3.0 x 10⁹ cells/L) in complete culture media [RPMI 1640 supplemented with 4% (v/v) heat-inactivated fetal calf serum, 1% (v/v) antimycotic-antibiotic solution, glutamine (4 mmol/L), HEPES (25 mmol/L), and 2-mercaptoethanol (2.5 µmol/L)] were incubated in 24-well sterile plates 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 PMA (30 µg/L) plus ionomycin (0.75 µmol/L). After 48 h, splenocyte culture supernatants were collected and stored at -70°C for subsequent cytokine analysis. Splenocytes that had been cultured with PMA plus ionomycin were washed twice in Krebs-Ringer HEPES buffer (pH 7.4) supplemented with bovine serum albumin (5 g/L) and used for indirect immunofluorescence analyses or frozen at -70°C for subsequent lipid analysis.

Splenocyte mitogenic response (proliferation) assay.

Splenocytes (1.25 x 10⁹ cells/L) were cultured in triplicate in 96-well microtiter plates in complete culture media with or without PMA (30 µg/L) plus ionomycin (0.75 µmol/L) for 66 or 78 h as previously described (Shewchuk et al. 1996 ). Twelve hours before harvesting the cells, each well was pulsed with 18.5 kBq of [methyl-³H]-thymidine.

Indirect immunofluorescence (phenotype) assay.

Immune cell subsets in mitogen-stimulated splenocytes were identified by indirect immunofluorescence assay as previously described (Robinson and Field 1998 ). The following monoclonal antibodies were used: OX19 (CD5), w3/25 (CD4), OX8 (CD8), OX12 (B cells), OX42 (CD11b/c), 3.2.3 (CD161), OX39 (CD25) and JJ319 (CD28). Because the monoclonal antibodies were not prelabeled with a fluorescent marker, they were incubated with either fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or phycoerythrin-conjugated goat anti-mouse IgG. The percentage of cells expressing each marker was determined by flow cytometry (FACScan; Becton Dickinson, Sunnyvale, CA) and was corrected for background fluorescence using the analysis of cells incubated with FITC or phycoerythrin alone. FITC and phycoerythrin background fluorescence were 5% and 0%, respectively (data not shown). Unwanted events (dead cells and debris) were detected by forward scatter and side scatter and were excluded from subsequent phenotype analyses by electronic gating of the viable splenocyte population. We have expressed the mitogen-stimulated phenotyping data as the percentage of live cells remaining after culture.

Cytokine production.

The concentrations of IFN- and TNF- in culture supernatants collected from unstimulated and stimulated splenocytes were determined by enzyme-linked immunosorbent assay. Briefly, flat-bottom microtiter plates were coated overnight with appropriately diluted purified rabbit anti-rat IFN- or TNF-. After washing, plates were blocked with PBS plus 10% (v/v) fetal calf serum to prevent nonspecific binding. Recombinant standards and appropriately diluted splenocyte culture supernatants were then added in triplicate at 100 µL per well, incubated for 4 h at room temperature, washed and further incubated with appropriately diluted biotinylated mouse anti-rat IFN- or TNF-. After extensive washing, the plates were incubated for 30 min with horseradish peroxidase avidin D. The absorbance was measured at 450 nm (IFN-) or 405 nm (TNF-) in a plate reader (Bio-Tek Instruments, Burlington, VT). The recombinant standard concentrations used were 20–1620 ng/L for IFN- and 15–2000 ng/L for TNF-.

Splenocyte fatty acid analysis.

Lipids were extracted from splenocytes by a modified Folch procedure as previously described (Field et al. 1988 ). Individual phospholipids were separated on thin layer chromatography plates (HPK silica gel 60 nm 10 x 10 cm; Whatman, Clifton, NJ) as previously described (Touchstone et al. 1980 ). Separated phospholipids were visualized with 8-anilino-1-naphthalene-sulfonic acid and identified under ultraviolet light with appropriate standards. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fatty acid methyl esters were prepared from the scraped silica band using 140 g/L (wt/v) BF₃/methanol reagent (Morrison and Smith 1964 ) and separated by automated gas liquid chromatography (Vista 6010; Varian Instruments, Georgetown, ON) on a fused silica BP20 capillary column (25-m x 0.25-mm internal diameter; Varian Instruments) as previously described (Field et al. 1988 ).

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 effects of diet and tumor were determined by two-way ANOVA followed by a Duncan’s multiple range test to identify significant (P 0.05) differences between individual treatments (Steele and Torrie 1980 ). Body weight changes and food intake were compared among groups by repeated-measures ANOVA (Steele and Torrie 1980 ). Paired t tests were used to compare cytokine production by immune cells with or without mitogen.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Cytokine production DISCUSSION REFERENCES

 
Food intake, body weight and spleen weight.

Neither dietary long-chain (n-3) fatty acids nor the tumor significantly affected food intake (overall mean = 63 ± 1 g · kg body^-1 · d^-1; n = 29), final body weight (overall mean = 158 ± 2 g; n = 33), weight increase (overall mean = 13 ± 1 g; n = 33), relative spleen weight (overall mean = 2.8 ± 0.1 g/kg body; n = 33), or the number of spleen cells (x10⁶) isolated per gram of spleen (overall mean = 433 ± 13; n = 32).

R3230AC mammary tumor weight.

Final tumor weight tended to be lower (-31%; P = 0.1) when rats were fed long-chain (n-3) fatty acids (0.9 ± 0.1 vs. 1.3 ± 0.2 g/100 g body; n = 7/diet).

Splenocyte mitogenic response.

Neither diet nor the tumor affected [³H]thymidine incorporation by unstimulated splenocytes at 66 and 78 h (overall 66-h mean = 2422 ± 251 dpm, n = 33; overall 78-h mean = 1898 ± 244 dpm, n = 33). Diet also did not significantly affect [³H]thymidine uptake by PMA plus ionomycin-stimulated cells at 66 and 78 h; therefore, rats in the low and high (n-3) diet groups within either the healthy or tumor-bearing group were combined for statistical analysis to examine the effect of tumor burden (Fig. 1 ). Splenocytes from tumor-bearing rats had a lower response to PMA plus ionomycin at 66 and 78 h (P < 0.01) compared with cells from healthy rats (Fig. 1) .

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of R3230AC mammary tumor on splenocyte [3H]thymidine incorporation in response to 66- and 78-h PMA plus ionomycin stimulation. Splenocytes were cultured for 66 or 78 h with PMA (30 mg/L) plus ionomycin (Iono; 0.75 mmol/L) as described in Materials and Methods. Diet did not significantly affect [3H]thymidine uptake; therefore, rats in the low and high (n-3) diet groups within either the healthy or tumor-bearing group were combined. Bars represent means ± SEM (healthy rats, n = 19; tumor-bearing rats, n = 14). For each incubation time, the asterisk indicates a significant (P < 0.01) effect of the tumor as determined by Student’s t test.

There was a significant (P 0.01) diet by tumor interaction on the proportion of CD8⁺ T suppressor/cytotoxic cells and CD25⁺CD8⁺ T cells after splenocytes were stimulated with PMA plus ionomycin (Table 2 ). In rats fed the high (n-3) diet, those bearing tumors had a significantly higher proportion of CD8⁺ and CD25⁺CD8⁺ T cells and a significantly lower CD4:CD8 ratio compared with healthy rats (Table 2) . In contrast, tumor burden did not significantly affect the proportion of CD8⁺ or CD25⁺CD8⁺ T cells or the CD4:CD8 ratio when rats were fed the low (n-3) diet (Table 2) . Furthermore, tumor-bearing rats fed the high (n-3) diet had a significantly higher proportion of CD25⁺CD8⁺ T cells compared with those fed the low (n-3) diet (Table 2) . In both diet groups, splenocytes from tumor-bearing rats had a significantly lower proportion of CD4⁺ Th cells and B cells after mitogen stimulation compared with healthy rats (Table 2) . Diet did not significantly affect CD25⁺ expression on other cell types; therefore, rats in the low and high (n-3) diet groups within either the healthy or tumor-bearing group were combined for statistical analysis to examine the effect of tumor burden (Fig. 2 ). Tumor-bearing rats had a significantly lower proportion of CD25⁺CD4⁺ Th cells, CD25⁺ B cells and CD25⁺ macrophages after mitogen stimulation compared with healthy rats (Fig. 2) .

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of R3230AC mammary tumor on IL-2 receptor (CD25) expression after PMA plus ionomycin stimulation. Isolated splenocytes were stimulated for 48 h with PMA (30 mg/L) plus ionomycin (0.75 mmol/L) and immune cell phenotypes were identified by indirect immunofluorescence assay and flow cytometry as described in Materials and Methods. Diet did not significantly affect CD25 expression; therefore, rats in the low and high (n-3) diet groups within either the healthy or tumor-bearing group were combined. Bars represent means ± SEM (healthy rats, n = 19; tumor-bearing rats, n = 14). For each immune cell phenotype, the asterisk indicates a significant (P < 0.05) effect of tumor burden as determined by Student’s t test.

	Cytokine production

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Cytokine production DISCUSSION REFERENCES

IFN-.

Splenocytes from all groups cultured for 48 h without mitogen (unstimulated) produced undetectable levels of IFN- in the culture supernatants (results not shown). In tumor-bearing rats, IFN- production by splenocytes stimulated with PMA plus ionomycin was significantly higher when rats were fed the high (n-3) diet compared with the low (n-3) diet (Fig. 3 ).

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of long-chain (n-3) fatty acids and tumor burden on splenocyte IFN- and TNF- production. Splenocytes were cultured with PMA (30 mg/L) plus ionomycin (0.75 mmol/L) for 48 h. Concentrations of IFN- and TNF- in cell culture supernatants were determined by enzyme-linked immunosorbent assay as described in Materials and Methods. Bars represent means ± SEM (n 7/group). The effects of diet and tumor were analyzed by two-way ANOVA. For each cytokine, bars that do not share a letter are significantly different (P < 0.05) as identified by a Duncan’s multiple range test.

TNF-.

Neither diet nor the tumor significantly affected TNF- production by splenocytes cultured for 48 h without mitogen (overall mean = 31 ± 6 ng · L^-1 · 10⁶ cells^-1; n = 24). There was a significant diet by tumor interaction (P 0.01) on splenocyte TNF- production after PMA plus ionomycin stimulation (Fig. 3) . In both healthy and tumor-bearing rats, TNF- production was greater (P < 0.0001) in rats fed the high (n-3) diet compared with the low (n-3) diet (Fig. 3) . Furthermore, tumor-bearing rats fed the high (n-3) diet produced more (P < 0.0001) TNF- than healthy rats fed the same diet (Fig. 3) .

Fatty acid composition of immune cells after PMA plus ionomycin stimulation.

The proportions of fatty acids from 14:0 to 24:1(n-9) in PC and PE were measured, but only major fatty acids are reported (Table 3 ).

Both healthy and tumor-bearing rats fed the high (n-3) diet had a greater proportion of 22:5(n-3) and total (n-3) fatty acids in PC in splenocytes stimulated with PMA plus ionomycin compared with those fed the low (n-3) diet (Table 3) . Healthy rats fed the high (n-3) diet also had a significantly lower proportion of 20:2(n-6), 22:5(n-6) and 22:4(6) and a lower (n-6):(n-3) fatty acid ratio in PC compared with those fed the low (n-3) diet (Table 3) . However, diet did not affect the proportions of these fatty acids in tumor-bearing rats. The tumor affected only the fatty acid composition of PC when rats were fed the low (n-3) diet. For example, low (n-3)-fed tumor-bearing rats had a significantly lower percentage of 20:2(n-6), 20:3(n-6) and 22:4(n-6) and total (n-6) fatty acids and a lower (n-6):(n-3) fatty acid ratio in PC compared with healthy rats fed the same diet (Table 3) .

PE.

Both healthy and tumor-bearing rats fed the high (n-3) diet had a significantly lower (n-6):(n-3) fatty acid ratio in PE in splenocytes stimulated with PMA plus ionomycin than in rats fed the low (n-3) diet (Table 3) . Tumor-bearing rats fed the high (n-3) diet had a significantly higher proportion of 20:5(n-3), 22:5(n-3), 22:6(n-3) and total (n-3) fatty acids compared with those fed the low (n-3) diet (Table 3) . The tumor only affected the fatty acid composition of PE when rats were fed the high (n-3) diet. For example, high (n-3)-fed tumor-bearing rats had a significantly higher percentage of 22:5(n-3) and 22:6(n-3) and total (n-3) fatty acids, and a significantly lower monounsaturated fatty acid content in PE compared with healthy rats fed the same diet (Table 3) .

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Cytokine production DISCUSSION REFERENCES

 
The lipid composition of a cell membrane is a major determinant of its physical properties and also modulates important membrane-mediated cell functions, such as integral enzyme activity, membrane receptor function and eicosanoid production (Clandinin et al. 1985 ). It is well known that feeding long-chain (n-3) fatty acids significantly alters the (n-6) and (n-3) fatty acid composition of immune cell phospholipids (Field et al. 2000, Hosack-Fowler et al. 1993 , Peterson et al. 1998 ). However, less is known about membrane phospholipid composition when immune cells are in an activated state. In vitro stimulation with mitogens provides a useful tool for studying immune cells in an activated state. For example, PMA, which activates protein kinase C, and ionomycin, a Ca²⁺ ionophore that increases intracellular calcium concentration, act together to stimulate proliferation of a variety of cell types, including T and B cells (Berry et al. 1989 , Truneh et al. 1985 ). In this study, we determined the fatty acid composition of membrane phospholipids in PMA plus ionomycin-activated splenocytes from healthy and tumor-bearing rats. Although we observed some diet-induced changes in (n-6) and (n-3) fatty acids in mitogen-activated splenocytes, overall these changes were not as reflective of dietary lipid intake as those reported for nonactivated immune cells (Field et al. 2000 , Hosack-Fowler et al. 1993 , Peterson et al. 1998 ). This may have been partially due to the effect of mitogen activation itself on membrane composition (Calder et al. 1994 , Goppelt-Strübe and Resch 1987 ), which was not assessed in the present study. It is known that various human cancers result in changes in the composition of host tissues (Baro et al. 1998 , Engan et al. 1995 ). For example, colorectal cancer patients have abnormal plasma and erythrocyte fatty acid profiles characterized by decreased levels of most saturated, monounsaturated and essential fatty acids, as well as their polyunsaturated metabolites (Baro et al. 1998 ). It has been suggested that such abnormalities are likely due to metabolic changes induced by the cancer per se, as opposed to malnutrition (Baro et al. 1998 ). Less is known about tumor-induced alterations in membrane lipid composition of other host tissues, such as immune cells. Furthermore, it is not currently known whether dietary fat composition and tumor growth interact to affect immune cell membrane composition. Overall, we observed minimal effects of the R3230AC mammary tumor on the lipid composition of mitogen-activated splenocytes. Interestingly, the limited tumor-induced lipid changes that we observed differed with the fat composition of the diet. For example, rats bearing the R3230AC mammary tumor had lower (34%) levels of (n-6) fatty acids in splenocyte PC, but only in the low (n-3) diet group. In splenocyte PE, tumor-bearing rats had a 100% increase in the long-chain (n-3) fatty acids 22:5(n-3) and 22:6(n-3), but only when rats were fed the high (n-3) diet. Because many functions of the immune system depend on interactions between activated immune and target cell membranes, changing the membrane composition of such activated splenocytes may influence antitumor immune responses.

Immune surveillance against tumors involves various effector cells, such as T and B cells, NK cells and macrophages that are able to recognize tumor antigens and mediate tumor cell killing (Adams et al. 1982 , Robins 1986 , Whiteside and Herberman 1995 ). In particular, tumor immunity is mediated, to a large extent, by activated CD8⁺ cytotoxic T lymphocytes that induce apoptotic death in tumor cells (Kiessling et al. 1999 ). The activation and function of CD8⁺ cytotoxic T cells is supported by type-1 cytokines, such as IL-2 and IFN- and TNF-, which are produced by CD4⁺ Th1 cells and CD8⁺ T cells (Pardoll and Topalian 1998 , Mosmann and Sad 1996 ). T cell activation requires at least two independent signals, one via the T cell receptor and a second via CD28, which provides a signal critical to for T cell proliferation, cytokine production and expression of cytokine receptors, such as the IL-2 receptor CD25 (June et al. 1994 ). In the present study, tumor-bearing rats had a lower proportion of activated (CD25⁺) CD4⁺ Th cells, CD28⁺ cells, B cells and macrophages compared with healthy rats after splenocytes were stimulated in vitro with PMA plus ionomycin. Activated macrophages play a key role in anticancer defense through production of TNF- and IL-1. Although the precise biological importance of IL-2 receptor expression on macrophages has not been established, Pleau and Hancock (1989 ) showed that IL-2 binding to IL-2 receptor-positive macrophages increased IL-1 production. Overall, the observed decrease in activation marker expression in tumor-bearing rats was accompanied by suppressed [³H]thymidine incorporation by splenocytes. Our results suggest that tumor-bearing rats did not respond as well to mitogen stimulation compared with healthy rats, supporting that there is impaired cell-mediated immune function in these rats at 17 d postimplantation. However, despite the observed decrease in activation marker expression and splenocyte proliferation in tumor-bearing rats, production of the cytokines IFN- and TNF- was higher in tumor-bearing rats compared with healthy rats. Although the proportion of activated CD4⁺ Th cells, which produce IFN- and TNF-, was decreased with tumor growth, we cannot determine whether there was a potential shift in CD4⁺ Th1 and Th2 cell subsets during tumor growth, which may have altered cytokine production. It is possible that more CD4⁺ Th1 versus Th2 cells were present in spleen at 17 d postimplantation, resulting in increased IFN- and TNF- in tumor-bearing rats. As well, these type-1 cytokines are produced by other cell types such as CD8⁺ T cells, macrophages and NK cells (Mosmann and Sad 1996 ).

Although there are limited data on the effect of the R3230AC mammary tumor on host immune function, other work supports suppressed host immunity with tumor growth (Kiessling et al. 1999 , Shewchuk et al. 1996 ). Although energy restriction alters both tumor growth (Kritchevsky 1990 ) and immune function (Corman 1985 ), it is unlikely that the observed tumor-induced immune changes were due to energy malnutrition because tumor-bearing rats had similar weight gain and food intake compared with control (healthy) rats at 17 d postimplantation. We suggest that immune changes induced by the tumor were mediated via signals at the primary tumor site and are unlikely caused by metastasized tumor cells as the R3230AC mammary adenocarcinoma in a nonmetastasizing rodent tumor (Hilf et al. 1965 ). However, additional work is needed to determine whether our observed changes in immune measures are important in growth of the R3230AC tumor model in vitro.

A major focus of current research in immunology and oncology is the development of methods to augment host antitumor immune defense. Our goal was to determine whether dietary fish oil-derived long-chain (n-3) fatty acids could enhance immune function in rats implanted with the R3230AC mammary adenocarcinoma. Dietary long-chain (n-3) fatty acids did not affect the suppressed mitogenic response in tumor-bearing rats. Others have shown that long-chain (n-3) fatty acids inhibit mitogen-induced lymphocyte proliferation (Calder 1998 , Meydani et al. 1991 ). This seemingly contradictory observation may be due to the content of polyunsaturated fats in the diets fed in these studies, because we have found that feeding long-chain (n-3) fatty acids reduces splenocyte proliferation in healthy rats when fed in a high polyunsaturated fat diet (unpublished data). In the present study, long-chain (n-3) fatty acids were supplemented in a diet with a low polyunsaturated fat level (more similar to the dietary patterns of humans) and they did not affect proliferation of immune cells isolated from either healthy or tumor-bearing rats. This suggests that the immunomodulatory effects of (n-3) fatty acids are dependent on the content polyunsaturated fat in the diet. We think that these results are of importance because the current conception that fish oil-derived (n-3) fatty acids are generally immunosuppressive (Calder 1998 ) has been difficult to overcome. We have previously shown that feeding healthy rats long-chain n-3 fatty acids enhanced NK cell cytotoxicity and the proportion of activated (CD71⁺) immune cells (Robinson and Field 1998 ). Certain immunostimulatory effects of long-chain (n-3) fatty acids were also observed in the present study. Feeding the high (n-3) diet to tumor-bearing rats significantly increased the proportion of activated (CD25⁺) CD8⁺ T cells and the production of IFN- and TNF-. The marked increase in splenocyte TNF- production in tumor-bearing rats fed the high (n-3) is consistent with previous reports on lipopolysaccharide-stimulated macrophage production in healthy, fish-oil-fed mice (Chang et al. 1992 ). Upregulated mitogen-induced IFN- and TNF- production in tumor-bearing rats fed the high (n-3) diet occurred concurrently with specific changes in (n-6) and (n-3) fatty acid levels in immune cell phospholipids during mitogen activation in rats fed the high (n-3) diet. However, whether this contributes to the observed diet-induced changes in immune function in tumor-bearing rats needs to be studied further. It is not known whether alterations in immune cell lipid composition are directly associated with changes in eicosanoid production, receptor or enzyme function, cell permeability, or second messenger pathways involved in cytokine production in this model. However, dietary fat modulation of eicosanoids (Peterson et al. 1998 ) and intracellular signaling pathways (Hosack-Fowler et al. 1993 ) has been reported in other studies.

Although the length of time that diets were fed in the present study (17 d postimplantation) was sufficient to alter immune cell membrane composition, increase activated CD8⁺ T cells and enhance splenocyte IFN- and TNF- production, it was perhaps not long enough for these mechanisms to impact on tumor growth. R3230AC mammary tumor growth was 31% lower in rats fed long-chain (n-3) fatty acids, but this effect was not statistically significant. Because tumor growth was not significantly inhibited by long-chain (n-3) fatty acid supplementation, the potential importance of diet-associated changes in the measured immune variables is not clear. However, our results do not preclude benefits of long-chain (n-3) fatty acids and/or enhanced immunity on later cancer stages, such as improved response to chemotherapy (de Salis and Meckling-Gill 1995 ), reduction of tumor metastasis (Rose et al. 1995 ), or prevention of cancer cachexia (Barber et al. 1999 ), or on earlier stages, such as cancer prevention (Noguchi et al. 1997 ). Overall, our results suggest that feeding long-chain (n-3) fatty acids in a low polyunsaturated fat diet may have beneficial effects on several host immune defenses, including activation of CD8⁺ T cells and IFN- and TNF- production. These immune benefits occur simultaneously with specific changes in the major membrane phospholipids in activated immune cells from high (n-3)-fed rats. Because membrane composition plays a critical role in immune function, additional work is needed to determine the relationship between diet-induced alterations in the phospholipid composition of immune cells during cancer and subsequent upregulation of immune responses.

	ACKNOWLEDGMENTS

 
We thank J. Aldrich for animal care, and S. Goruk and A. Wierzbicki for excellent technical assistance.

	FOOTNOTES

 
¹ Supported by a grant from the Natural Sciences and Engineering Research Council of Canada to C.J.F. L.E.R. is a recipient of a Natural Sciences and Engineering Research Council of Canada Scholarship and an Alberta Heritage Foundation for Medical Research Studentship.

³ Abbreviations used: NK, natural killer; Th, T helper; PMA, phorbol myristate acetate; IgG, immunoglobulin G; IFN, interferon; TNF, tumor necrosis factor; FITC, fluorescein isothiocyanate; PC, phosphatidylcholine; PE, phosphatidylethanolamine; IL, interleukin.

Manuscript received December 27, 2000. Initial review completed February 5, 2001. Revision accepted March 28, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Cytokine production DISCUSSION REFERENCES

 

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