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Influences of dietary polyunsaturated or saturated fats and of concanavalin-a upon proliferation of spleen lymphoid cells from rats
NUTRITION RESEARCH, Vol. 6, pp. 1219-1227, 1986
0271-5317/86 $3.00 + .00 Printed in the USA. Pergamon Journals Ltd. All rights reserved.
INFLUENCES OF DIETARY POLYUNSATURATED OR SATURATEDFATS AND OF CONCANAVALIN-A UPON PROLIFERATION OF SPLEEN LYMPHOID CELLS FROM RATS Tim R. Kramer I , Mary Briske-Anderson1~ Susan B. Johnson 2 and Ralph T. Holman ~ Iunited States Department of Agriculture, Agricultural Research Service Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota 58202. ~The Hormel Institute, University of Minnesota, Austin, Minnesota 55912
ABSTRACT The effects of dietary polyunsaturated and saturated fats upon several metabolic phenomena were studied in rats. These included phospholipid (PL) levels in unstimulated spleen lymphoid cells (SLC) and serum; in vitro proliferation of unstimulated SLC from the two groups of rats; the influence of mltogenlc eoncanavalln-A (Con-A) on in vitro proliferation of SLC from the two groups of rats; and the influence of serum from the two groups of rats on SLC proliferation. Weanling male Long-Evans rats were fed ad libltum either a polyunsaturated fatty acid (PUFA)-adequate (safflower oil, SO) or PUFA-deflcient (coconut oil, CNO) diet for 21 days. Growth was significantly less in the CNO group than in the SO group. Food intake was not different for the dietary groups. Unstimulated SLC and serum of CNO rats showed changes in fatty acid composition of PL typical of tissue PL in essential fatty acid-deflcient (EFA-D) rats. Unstimulated SLC from the two groups cultured in medium containing serum from the same groups showed equivalent rates of in vitro proliferation. In vitro proliferations of SLC were influenced: by the dietary source of SLC but not by the dietary source of serum, used in the culture medium, when the cells were stimulated by suboptimal doses of Con-A; by the dietary source of serum but not by the dietary source of SLC when the cells were stimulated by the optimal dose of Con-A; and by an interaction between source of SLC and serum, when the cells were stimulated by a supra-optlmal dose of Con-A. The present study indicates that in vitro proliferation of SLC from PUFA-adequate and PUFA-deflclent rats is influenced by both the dietary source of serum used in the culture medium and the concentration of Con-A used for stimulation of the SLC. KEY WORDS: polyunsaturated fatty acids, acids, spleen lymphoid cells, concanavalln-A
1219
phospholipld
fatty
1220
T.R. KRAMERet al.
INTRODUCTION In vlvo and in vitro studies suggest that fatty acids modulate immune responses ~-1-4). Mice fled diets enriched with PUFA showed prolonged survival of skin allografts and allogenelc tumor grafts (5). Mertin and Hunt (6) demonstrated that increased administration of llnoleic acid (18:2~6) prolonged survival of skin allografts in mice , which was attributed to immunoinhibition by the increased PUFA. Immunosuppresslon was also used as an explanation for the increased growth of allogeneic BI 6 melanoma cells in mice fed high fat diet (7). Increased dietary fat, particularly PUFA, decreased lymphocyte-mediated cytotoxiclty to the murine melanoma (7); whereas immunoenhancement was found in mice fed a diet containing ~inimal essential fatty acids (EFA) (8). In addition to antlgen-specific induced lymphocyte activities, dietary fats influence the proliferation of mitogen-stimulated T-lymphocytes (9-I 2). Erickson et al. (9) reported an influence by both saturation and concentration of dietary fat on in vitro proliferation of SLC stimulated by an optimal dose of Con-A. SLC from 6---~-~-eek-old mice fed a diet containing 8% safflower oil showed higher levels of proliferation than SLC from mice fed diets containing 24% safflower oil or 8% coconut oil, but SLC from mice fled a diet containing 24% coconut oll showed the highest activity (9). Locnlskar et al. (10) reported that the modulating effect of fat on proliferation of Con-A stimulated SLC depended on the duration of feeding, and on the type of fat fled. As with mice (9), SLC from rats fed a diet containing 24% safflower oil showed lower proliferation, following stimulation with an optimal dose of Con-A, than did SLC from rats fed a diet containing 5% mixed fat (10). Similar to Erickson et al. (9), Marshall and Johnston (12) reported that enhanced Con-A stimulated SLC proliferation was associated with a greater degree of saturation of dietary fat. SLC from male and female weanling rats fed a diet containing 10% hydrogenated coconut oil showed higher proliferation, following stimulation with suboptimal and optimal concentrations of Con-A for peak proliferation, than did SLC from rats fed a diet containing 10% corn oil (12). In vitro proliferation of Con-A stimulated SLC is optimal when the cells are cultured in medium containing serum. King and Spector (13) reported, however, that cells cultured in serum assume a membrane lipid profile similar to that of the serum. Loomis et al. (14) and Kollmorgen et al. (11) reported an influence by the source of serum used in the culture medium on the in vitro SLC functions of rats. Using a heterologous serum (fetal calf serum) a~-10-'~'~ the culture medium, Erickson et al. (9) reported lower proliferation by Con-A stimulated SLC from mice fed a diet containing 24% safflower oil than by SLC from mice fed lower levels of safflower oil or by an equivalent level of coconut oll (24%). Locniskar et al. (10), also using a heterologous serum (fetal bovine serum) at 10% in the cell culture medium, reported a lower rate of proliferation of SLC, stimulated with Con-A, from rats fled high PUFA (24%) than by SLC from rats fed a mixed fat at 5%. The suppressed proliferation of SLC from rats fed high PUFA cultured in heterologous serum was not apparent when the SLC were cultured in medium containing 10% rat serum from different dietary fat groups (10). This apparent lack of difference reported by Locniskar et al. (10) Is in contrast to that reported by Marshall and Johnston (12). They reported higher proliferation by SLC from rats fed 10% hydrogenated coconut oil, cultured in medium containing 6.25% serum from rats fed coconut oil, than by SLC from rats fed 10% corn oil, cultured in medium containing 6.25% serum from rats fed corn oil. Reasons for these apparent differences reported by Locniskar et al. (10) and Marshall and Johnston (12) remain unclear. The present report describes the effects of dietary polyunsaturated and saturated fat diets on PL levels in unstimulated SLC and serum, on the in vitro
PUFA AND T-CELL PROLIFERATION
1221
proliferation of SLC from PUFA-adequate and PUFA-deficient rats, and on the influence of serum f r o m these groups of rats on SLC proliferation. The influence of Con-A concentration on the interactions between cells and serum from these dietary groups during in vitro proliferation of SLC is also studied. MATERIALS AND METHODS
Weanling (age, 21 days) outbred, male Long-Evans (Crl:(LE)BR) rats were purchased from Charles River Breeding Laboratories, Inc., Wilmington, MA. Upon arrival, rats were placed in stainless steel cages and fed ad libitum a non-purified diet (Purina Rodent Laboratory Chow No. 5001, St. Louis, MO) and tap water for 72-96 hours. At age 28 days, the rats were matched by weight, placed in two groups (twenty animals per group), housed individually in suspended stainless steel cages, and given delonlzed water ad libitum. The SO rats were given free access to a diet containing 6% safflower oil (Teklad, Teklad Test Diets, Madison, WI). The CNO rats were given free access to a diet containing 6% unhydrogenated coconut oil (Teklad, Teklad Test Diets, Madison, WI). Daily food intakes and weekly weight changes of the animals were recorded. On day 21 of the dietary regimen (age, 49 days), and for the next 4 days, equal numbers of rats from each dietary group were anesthetized with ether between 0800 and 0900 hours, bled by cardiac puncture, and killed. Designated tissues were removed. Diets The purified safflower oll and unhydrogenated coconut oil diets contained 20% egg white protein, and other nutrients as listed in Table I. The level of linoleic acid present in the unhydrogenated coconut oil was 6.7%, and in the safflower oil it was 78% of the total fatty acids. Thus, the CNO diet provided 0.4%, and the SO diet 4.7%, of calories as llnolelc acid. Specimens Collected and Fatty Acid Analysis Serum from each rat was stored at -20~ SLC w~re isolated (15), and pooled from 4 rats fed each diet. A pelleted pool of 10 ~ SLC was necessary for satisfactory fatty acid analysis. All specimens were stored at -20~ for I week prior to shipment on dry ice from Grand Forks, ND to the Hormel Institute, Austin, MN. Tissues were extracted and fatty acid methyl esters (FAME) of PL were analyzed as described previously (16). Spleen Lymphoid Cell Collection and Cultures SLC were isolated using minor modifications of a method previously described (15). Spleens were removed and freed of extraneous tissue and teased with sterile stainless steel needles in Hanks Balanced Salt Solution (HBSS). SLC were separated from other spleen cells by density gradient centrifugatlon according to the Fico!l sodium metriozate method of Boyum (17). Ten ml of the spleen cell suspension in HBSS were placed on 3 ml of Ficoll-Hypaque (Histopaque 1077, Sigma Chemical Co., St. Louis, MO) and centrifuged at room temperature at 400 x g for 30 minutes. The isolated SLC suspensions were washed 3 times with HBSS, and viable cells (Trypan blue exclusion) were counted. SLC were cultured in RPMI-1640 tissue culture medium containing 2% heat-inactivated, filtered pooled serum from SO or CNO rats. The culture medium also contained L-glutamine (2.0 mmoles/L) penicillin (100 unit~/ml) and streptomycin (100 ug/ml). In a total volume of 200 ~I/well, 2 x 10 J SLC were incubated in a flat bottom Falcon Micro-Test plate (Micro-Test II, Falcon Plastics, Oxnard, CA) for 72 hours at 37~ in a 5% C02, 95% humidified air incubator.
1222
T.R. KRAMERet al.
Triplicate control (without mitogen) cultures were evaluated for each SLC sample; triplicate test (with mltogen) cultures were also used for each mitogen dose tested. Con-A (Sigma Chemical Co., St. Louis, MO) was added to the test cultures at O. 1, 0.2, 0.39 and 0.78 ~g/culture, After 48 hours of incubation, 1.0 ~Ci of 3H-TdR ([methyl-3H] thymidine, 6.7 Ci/mmol, Dupont New England Nuclear Products, Boston, MA) was added to each control and test culture. Following 24 hours of isotope labeling, the cultures were removed from the incubator and harvested with a Titertek Cell Harvester (Flow laboratories, Inc., Rockville, MD). The cell culture fluid, cells, and distilled water rinses were collected o n glass-fiber harvester filters (Flow Laboratories, Inc.). Distintegrations per minute (DPM) in the harvested cells were determined by standard liquid scintillation counting procedures using a Beckman LS 6800 scintillation counter. Statistics Fatty acid data were analyzed by the Student's t-test. Data for fatty acids are presented as percentages of total acyl groups in a given lipid because the influence of PUFA upon the function of membranes is dependent upon the proportions of the acyl groups in the membrane llpids. Data for SLC proliferation were analyzed by 2x2 repeated measures ANOVA (18). TABLE I Composition of Basal Diet Dietary Oils Ingredient
Coconut
Safflower
g/kg Sucrose* Egg-whlte protein + Fibrous cellulose ~owder # Zinc-free s ~ t mix v Vitamin mix Biotin mix ++ Coconut oll (~hydrogenated) ## Safflowe ,~il ~~ Zinc mix ~
615 200 50 35 10 10 60 -20
615 200 50 35 10 10 -60 20
Crystal Sugar, American Crystal Co., Moorhead, MN. #Teklad Mills Divlsion of ARS Sprague-Dawley, Madison, WI. Whatman CF11, W. and R. Balston Ltd., London, England. 6Modlfied AIN-76 Mineral Mix (without zlnc carbonate powder) cat. no. 170915, Teklad Test Diets, Madison, WI. See reference 15 for composition of modified .~Ineral mix. Mineral mlx was designed to be used at 3.5% of diet (23). Teklad (Vitamin fortification mix) cat. no. 40060, Teklad Test Diets, Madison, WI. See reference 15 for composition of vitamin mix. ++Biotin, crystalline (200 mg biotin in 1000 g of cornstarch), Grand Island Biological Co., Grand Island, NY. Final concentration of biotin in diets was #5.44 ~g/g diet. ~Coconut oll (unhydrogenated), Teklad, cat. no. 190120, Teklad Test Diets, Madison, WI. 86Safflower oil, Teklad, cat. no. 160470, Teklad Test Diets, Madison, WI. ***Zinc acetate, 3.35 g plus 996.65 g cornstarch. +
PUFA AND T-CELL PROLIFERATION
1223
RESULTS On day 21 of the dietary regimen, mean animal weight changes (grams • SD) showed that growth was slower in CNO (142• rats than in SO (162214) rats (p
Fatty Acids
18:2~6 20:3m6 20:4~6 22:4m6 22:5m6 16:1m7 18:1~9 20:3~9
Diet Groups Tissue
SLC + Serum SLC Serum SLC Serum SLC Serum SLC Serum SLC Serum SLC Serum SLC Serum
CNO
3.38 5.76 1.40 1.21 13.32 4.72 1.19 0.11 0.90 1.41 1.45 1.47 13.18 11.72 6.98 9.24
• • • • • • • • • • • • • • • •
0.22 1.18 0.18 0.16 1.62 0.64 0.20 0.19 0.10 0.26 0.40 0.45 0.59 1.14 0.61 1.27
SO
(4) # (8) (4) (8) (4) a (8) (4) (8) (4) (7) (4) (8) (4) (8) (4) (8)
10.99 16.07 1.10 0.48 15.78 18.84 2.92 0.76 1.24 4.96 0.88 0.85 5.84 3D44
• 0.83 + 1.93 + 0.10 • 0.13 _+ 1.63 • 2.06 + 0.97 + 0.13 • 0.25 • 1.16 + 0.12 + 0.34 + 0.36 + 0.42
(5) (7) (5) (7) (5) a (7) (5) (6) (5) (7) (5) (7) (5) (7)
O.O3 • O.O4 (7)
*CNO rats received ad libltum a diet containing 6% unhydrogenated coconut oil. SO rats received ad llbltum a diet containing 6% safflower oil. +Spleen lymphoid cells. #Mean • SD of relative % fatty acid composition of phospholiplds. Value in parenthesis indicates number of samples tested. Values marked with s u p e r s c r i p t "a" indicate not significantly different at p
1224
T.R. KRAMERet al.
Fats high in polyunsaturated (safflower oil) and saturated (coconut oil) fatty acids, fed as 6% of the diet, did not cause differences in proliferation of unstlmulated SLC. Unstimulated SLC from CNO and SO rats cultured in medium containing 2% serum from CNO or SO rats showed equivalent in vitro proliferation (Table 3). Dietary source of SLC was the main influence on in vitro proliferation (log 10 dpm • standard error) of cells stimulated with suboptimal concentrations of Con-A for peak SLC proliferation (0.1 and 0.2 ~g, Table 3). SLC from CNO rats stimulated with 0.1 pg (5.22 • 0.02 vs. 4.82 • 0.02; p = 0.0001) and 0.2 ~g (5.61 • O.01 vs. 5.37 • 0.01; p = 0.002) of Con-A showed higher proliferations than SLC from SO rats. Effects of serum source on levels of in vitro proliferation were observed in SLC from CNO and SO rats stimulated with optimal (0.39 ~g) and supra-optlmal (0.78 ~g) concentrations of Con-A. SLC from CNO and SO rats stimulated with 0.39 pg Con-A and cultured in medium containing serum from SO rats (5.62 • 0.006) showed higher (p = 0.0004) proliferation than SLC from CNO and SO rats cultured in medium containing serum from CNO (5.59 • 0.006) rats. An in vitro interaction between dietary source of SLC and serum was observed in cells stimulated with the supra-optimal (0.78 ~g) concentration of Con-A. SLC from SO rats stimulated with 0.78 ~g of Con-A and cultured in medium containing serum from CNO rats showed lower proliferation than SLC from SO rats cultured in medium containing serum from SO rats, and lower than SLC from CNO rats cultured in medium containing serum from either CNO or SO rats (Table 3).
DISCUSSION Changes in fatty acid composition of nonlymphoid and lymphoid tissues of CNO rats were in agreement with those reported for liver of EFA-D rats: increased 18:1m9, and decreased 18:2~6 and 20:4~6 (19,20). Tsang, et al. (21) reported similar changes in fatty acid composition in plasma and SLC of EFA-D rats. In agreement with Marshall and Johnston (12), SLC from EFA-D rats (CNO) stimulated with suboptimal concentrations of Con-A showed higher proliferation than SLC from EFA-A rats (SO). Marshall and Johnston (12) demonstrated this activity in SLC cultured in medium containing serum from rats fed the same diet as the source of SLC undergoing proliferation. In the present study SLC from CNO rats stimulated with 0.1 and 0.2 ~g Con-A showed enhanced proliferation when cultured in medium containing serum from either CNO or SO rats (see text of Results for values). Thus, according to the ANOVA, the main influence on enhanced in vitro proliferation by suboptlmally Con-A stimulated SLC from CNO rats was caused by the dietary source of SLC and not by the dietary source of serum used in the culture medium. In contrast to suboptimal dose Con-A stimulated SLC, the main influence on in vitro proliferation of SLC stimulated with the optimal (0.39 ~g) and supra-optimal (0.78 ~g) doses of Con-A was the dietary source of serum used in the culture medium and not the dietary source of SLC (see text of Results for values). SLC from CNO or SO rats, stimulated with the optimal and supra-optimal doses of Con-A, showed higher proliferation when cultured in medium containing serum from SO rats than from CNO rats. The mechanism of in vitro interaction between sources of SLC and serum on proliferation of supra-optimal dose Con-A stimulated SLC remains to be elucidated. Lymphoid cells stimulated in vitro by Con-A show increased polyunsaturated fatty acid incorporation (22). Altered PUFA patterns in serum from CNO rats may be the reason for reduced proliferation by supra-optimally Con-A stimulated SLC from SO rats cultured in serum from CNO rats. It is of
PUFA AND T-CELL PROLIFERATION
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1226
T.R. KRAMERet al.
interest that reduced proliferation occurred by SLC from EFA-A (SO) rats cultured in serum from EFA-D (CNO) serum, but not by SLC from EFA-D rats. cultured in serum from either EFA-A or EFA-D rats. The present study suggests that in vitro proliferation of SLC from EFA-D and EFA-A rats is influenced by the dietary source of serum used in the culture medium and the concentration of mitogenlc Con-A used for stimulation of the SLC. These in vitro observations, also, appear to agree with earlier in vivo studies (5-7)'~-wh--i-ch suggest enhanced cell-mediated (T-lymphocyte) functions in low PUFA fed animals. ACKNOWLEDGEMENTS The authors thank LuAnn Johnson for statistical evaluation of the data, Jean Klava for preparation of the diets, Nancy Drlscoll and staff for daily care of animals, and Trell Kulju and Elaine Westerlund for technical assistance.
This study was supported in part by grant HL 08214 from Projects Branch, Extramural Programs, National Heart Institute Hormel Foundation.
the and
Program by the
PROPRIETARY STATEMENT Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. REFERENCES I.
Meade CJ, Mertln 16:127-165.
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Vitale JJ, Broitmann 41:3706-10.
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Mertin J. Essential Res. 1981; 20:851-6.
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Gurr MI. The role of lipids in the regulation of the immune system. Prog. Lipid Res. 1983; 22:257-87.
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Santiago-Delphln EA, Szepsenwol J. Prolonged survival of skin and tumor allografts in mice on high-fat diets: Brief Communication. J. Natl. Cancer Inst. 1977; 59:459-61.
6.
Mertin J, Hunt R. Influence of polyunsaturated fatty acids on survival of skin allografts and tumor incidence in mice. Proc. Natl. Acad. Sci. USA. 1976; 73:928-31.
7.
Erickson KL. Dietary fat influences on murine melanoma growth and lymphocyte-mediated cytotoxlcity. J. Natl. Cancer Inst. 1984; 72:115-20.
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and
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Liplds and immune function.
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PUFA AND T-CELL PROLIFERATION
1227
8.
Erickson KL, Adams DA, McNeill CJ. Dietary lipid modulation of immune responsiveness. Liplds 1983; 18:468-74.
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Erickson KL, McNeill CJ, Gershwln ME, Ossmann JB. Influence of dietary fat concentration and saturation of immune ontogeny in mice. J. Nutr. 1980; 110:1555-72.
10. Locnlskar M, Nauss KM, Newberne PM. The effect of quality and quantity of dietary fat on the immune system. J. Nutr. 1983; 113:951-61. 11. Kollmorgen GM, Sansing WA, Lehman AA, Fischer G, Longley RE, Alexander SS Jr., King MM, McCay PB. Inhibition of lymphocyte funtlon in rats fed hlgh-fat diets. Cancer Res. 1979; 39:3458-62. 12. Marshall LA, Johnston PV. The influence of dietary essential fatty acids on rat immunocompetent cell prostaglandin synthesis and mltogen-induced blastogenesis. J. Nutr. 1985; 115:1572, 1580. 13. King MA, Spector AA. Lipid metabolism in cultured cells. In: Waymouth, C., Ham, R. and Chapple, P.J. eds. The Growth Requirements of Vertebrate Cells In Vitro. New York: Cambridge University Press, 1981: 293-312. 14. Loomis RJ, Marshall LA, Johnston PV. Sera fatty acid effects on cultured rat splenocytes. J. Nutr. 1983; 113:1292-98. 15. Kramer TR. Reevaluation of zinc deficiency on concanavalln-A induced rat spleen lymphocyte proliferation. J. Nutr. 1984; 114:953-63. 16. Kramer TR, Briske-Anderson M, Johnson SB, Holman RT. Influence of reduced food intake on polyunsaturated fatty acid metabolism in zlnc-deficlent rats. J. Nutr. 1984; 114:1224-30. !
17. Boyum A. Separation of leucocytes from blood and bone marrow. J. Clin. Lab. Invest. 1968; 21: (Suppl. 97) 7. 18. Winer BJ. Statistical Principles in Experimental Design, 2nd ed. New York: McGraw-Hill Inc., 1971: 518-39. 19. Mohrhauer H, Holman RT. The effect of low level of essential fatty acids upon fatty acid composition of rat liver. J. Lipid Res. 1963; 4:151-9. 20. Boissonneault GA, Johnston PV. Essential fatty acid deficiency, prostaglandln synthesis and humoral immunity in Lewis rats. J. Nutr. 1983; 113:1187-94. 21. Tsang WM, Belin J, Smith AD. Levels of polyunsaturated fatty acids in lymphocytes of rats fed on diets varying in polyunsaturated fatty acid content. Br. J. Nutr. 1980; 43:367-373. !
22. Goppelt M. Kohler L, Resch K. Functional role of lipid metabolism activated T-lymphocytes. Biochim. Biophys. Acta. 1985; 833:463-72.
in
23. American Institute of Nutrition. Report of the American Institute of Nutrition Committee on Standards for Nutritional Studies. J. Nutr. 1977; 107:1340-8. Accepted for publication August 20, 1986.
0271-5317/86 $3.00 + .00 Printed in the USA. Pergamon Journals Ltd. All rights reserved.
INFLUENCES OF DIETARY POLYUNSATURATED OR SATURATEDFATS AND OF CONCANAVALIN-A UPON PROLIFERATION OF SPLEEN LYMPHOID CELLS FROM RATS Tim R. Kramer I , Mary Briske-Anderson1~ Susan B. Johnson 2 and Ralph T. Holman ~ Iunited States Department of Agriculture, Agricultural Research Service Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota 58202. ~The Hormel Institute, University of Minnesota, Austin, Minnesota 55912
ABSTRACT The effects of dietary polyunsaturated and saturated fats upon several metabolic phenomena were studied in rats. These included phospholipid (PL) levels in unstimulated spleen lymphoid cells (SLC) and serum; in vitro proliferation of unstimulated SLC from the two groups of rats; the influence of mltogenlc eoncanavalln-A (Con-A) on in vitro proliferation of SLC from the two groups of rats; and the influence of serum from the two groups of rats on SLC proliferation. Weanling male Long-Evans rats were fed ad libltum either a polyunsaturated fatty acid (PUFA)-adequate (safflower oil, SO) or PUFA-deflcient (coconut oil, CNO) diet for 21 days. Growth was significantly less in the CNO group than in the SO group. Food intake was not different for the dietary groups. Unstimulated SLC and serum of CNO rats showed changes in fatty acid composition of PL typical of tissue PL in essential fatty acid-deflcient (EFA-D) rats. Unstimulated SLC from the two groups cultured in medium containing serum from the same groups showed equivalent rates of in vitro proliferation. In vitro proliferations of SLC were influenced: by the dietary source of SLC but not by the dietary source of serum, used in the culture medium, when the cells were stimulated by suboptimal doses of Con-A; by the dietary source of serum but not by the dietary source of SLC when the cells were stimulated by the optimal dose of Con-A; and by an interaction between source of SLC and serum, when the cells were stimulated by a supra-optlmal dose of Con-A. The present study indicates that in vitro proliferation of SLC from PUFA-adequate and PUFA-deflclent rats is influenced by both the dietary source of serum used in the culture medium and the concentration of Con-A used for stimulation of the SLC. KEY WORDS: polyunsaturated fatty acids, acids, spleen lymphoid cells, concanavalln-A
1219
phospholipld
fatty
1220
T.R. KRAMERet al.
INTRODUCTION In vlvo and in vitro studies suggest that fatty acids modulate immune responses ~-1-4). Mice fled diets enriched with PUFA showed prolonged survival of skin allografts and allogenelc tumor grafts (5). Mertin and Hunt (6) demonstrated that increased administration of llnoleic acid (18:2~6) prolonged survival of skin allografts in mice , which was attributed to immunoinhibition by the increased PUFA. Immunosuppresslon was also used as an explanation for the increased growth of allogeneic BI 6 melanoma cells in mice fed high fat diet (7). Increased dietary fat, particularly PUFA, decreased lymphocyte-mediated cytotoxiclty to the murine melanoma (7); whereas immunoenhancement was found in mice fed a diet containing ~inimal essential fatty acids (EFA) (8). In addition to antlgen-specific induced lymphocyte activities, dietary fats influence the proliferation of mitogen-stimulated T-lymphocytes (9-I 2). Erickson et al. (9) reported an influence by both saturation and concentration of dietary fat on in vitro proliferation of SLC stimulated by an optimal dose of Con-A. SLC from 6---~-~-eek-old mice fed a diet containing 8% safflower oil showed higher levels of proliferation than SLC from mice fed diets containing 24% safflower oil or 8% coconut oil, but SLC from mice fled a diet containing 24% coconut oll showed the highest activity (9). Locnlskar et al. (10) reported that the modulating effect of fat on proliferation of Con-A stimulated SLC depended on the duration of feeding, and on the type of fat fled. As with mice (9), SLC from rats fed a diet containing 24% safflower oil showed lower proliferation, following stimulation with an optimal dose of Con-A, than did SLC from rats fed a diet containing 5% mixed fat (10). Similar to Erickson et al. (9), Marshall and Johnston (12) reported that enhanced Con-A stimulated SLC proliferation was associated with a greater degree of saturation of dietary fat. SLC from male and female weanling rats fed a diet containing 10% hydrogenated coconut oil showed higher proliferation, following stimulation with suboptimal and optimal concentrations of Con-A for peak proliferation, than did SLC from rats fed a diet containing 10% corn oil (12). In vitro proliferation of Con-A stimulated SLC is optimal when the cells are cultured in medium containing serum. King and Spector (13) reported, however, that cells cultured in serum assume a membrane lipid profile similar to that of the serum. Loomis et al. (14) and Kollmorgen et al. (11) reported an influence by the source of serum used in the culture medium on the in vitro SLC functions of rats. Using a heterologous serum (fetal calf serum) a~-10-'~'~ the culture medium, Erickson et al. (9) reported lower proliferation by Con-A stimulated SLC from mice fed a diet containing 24% safflower oil than by SLC from mice fed lower levels of safflower oil or by an equivalent level of coconut oll (24%). Locniskar et al. (10), also using a heterologous serum (fetal bovine serum) at 10% in the cell culture medium, reported a lower rate of proliferation of SLC, stimulated with Con-A, from rats fled high PUFA (24%) than by SLC from rats fed a mixed fat at 5%. The suppressed proliferation of SLC from rats fed high PUFA cultured in heterologous serum was not apparent when the SLC were cultured in medium containing 10% rat serum from different dietary fat groups (10). This apparent lack of difference reported by Locniskar et al. (10) Is in contrast to that reported by Marshall and Johnston (12). They reported higher proliferation by SLC from rats fed 10% hydrogenated coconut oil, cultured in medium containing 6.25% serum from rats fed coconut oil, than by SLC from rats fed 10% corn oil, cultured in medium containing 6.25% serum from rats fed corn oil. Reasons for these apparent differences reported by Locniskar et al. (10) and Marshall and Johnston (12) remain unclear. The present report describes the effects of dietary polyunsaturated and saturated fat diets on PL levels in unstimulated SLC and serum, on the in vitro
PUFA AND T-CELL PROLIFERATION
1221
proliferation of SLC from PUFA-adequate and PUFA-deficient rats, and on the influence of serum f r o m these groups of rats on SLC proliferation. The influence of Con-A concentration on the interactions between cells and serum from these dietary groups during in vitro proliferation of SLC is also studied. MATERIALS AND METHODS
Weanling (age, 21 days) outbred, male Long-Evans (Crl:(LE)BR) rats were purchased from Charles River Breeding Laboratories, Inc., Wilmington, MA. Upon arrival, rats were placed in stainless steel cages and fed ad libitum a non-purified diet (Purina Rodent Laboratory Chow No. 5001, St. Louis, MO) and tap water for 72-96 hours. At age 28 days, the rats were matched by weight, placed in two groups (twenty animals per group), housed individually in suspended stainless steel cages, and given delonlzed water ad libitum. The SO rats were given free access to a diet containing 6% safflower oil (Teklad, Teklad Test Diets, Madison, WI). The CNO rats were given free access to a diet containing 6% unhydrogenated coconut oil (Teklad, Teklad Test Diets, Madison, WI). Daily food intakes and weekly weight changes of the animals were recorded. On day 21 of the dietary regimen (age, 49 days), and for the next 4 days, equal numbers of rats from each dietary group were anesthetized with ether between 0800 and 0900 hours, bled by cardiac puncture, and killed. Designated tissues were removed. Diets The purified safflower oll and unhydrogenated coconut oil diets contained 20% egg white protein, and other nutrients as listed in Table I. The level of linoleic acid present in the unhydrogenated coconut oil was 6.7%, and in the safflower oil it was 78% of the total fatty acids. Thus, the CNO diet provided 0.4%, and the SO diet 4.7%, of calories as llnolelc acid. Specimens Collected and Fatty Acid Analysis Serum from each rat was stored at -20~ SLC w~re isolated (15), and pooled from 4 rats fed each diet. A pelleted pool of 10 ~ SLC was necessary for satisfactory fatty acid analysis. All specimens were stored at -20~ for I week prior to shipment on dry ice from Grand Forks, ND to the Hormel Institute, Austin, MN. Tissues were extracted and fatty acid methyl esters (FAME) of PL were analyzed as described previously (16). Spleen Lymphoid Cell Collection and Cultures SLC were isolated using minor modifications of a method previously described (15). Spleens were removed and freed of extraneous tissue and teased with sterile stainless steel needles in Hanks Balanced Salt Solution (HBSS). SLC were separated from other spleen cells by density gradient centrifugatlon according to the Fico!l sodium metriozate method of Boyum (17). Ten ml of the spleen cell suspension in HBSS were placed on 3 ml of Ficoll-Hypaque (Histopaque 1077, Sigma Chemical Co., St. Louis, MO) and centrifuged at room temperature at 400 x g for 30 minutes. The isolated SLC suspensions were washed 3 times with HBSS, and viable cells (Trypan blue exclusion) were counted. SLC were cultured in RPMI-1640 tissue culture medium containing 2% heat-inactivated, filtered pooled serum from SO or CNO rats. The culture medium also contained L-glutamine (2.0 mmoles/L) penicillin (100 unit~/ml) and streptomycin (100 ug/ml). In a total volume of 200 ~I/well, 2 x 10 J SLC were incubated in a flat bottom Falcon Micro-Test plate (Micro-Test II, Falcon Plastics, Oxnard, CA) for 72 hours at 37~ in a 5% C02, 95% humidified air incubator.
1222
T.R. KRAMERet al.
Triplicate control (without mitogen) cultures were evaluated for each SLC sample; triplicate test (with mltogen) cultures were also used for each mitogen dose tested. Con-A (Sigma Chemical Co., St. Louis, MO) was added to the test cultures at O. 1, 0.2, 0.39 and 0.78 ~g/culture, After 48 hours of incubation, 1.0 ~Ci of 3H-TdR ([methyl-3H] thymidine, 6.7 Ci/mmol, Dupont New England Nuclear Products, Boston, MA) was added to each control and test culture. Following 24 hours of isotope labeling, the cultures were removed from the incubator and harvested with a Titertek Cell Harvester (Flow laboratories, Inc., Rockville, MD). The cell culture fluid, cells, and distilled water rinses were collected o n glass-fiber harvester filters (Flow Laboratories, Inc.). Distintegrations per minute (DPM) in the harvested cells were determined by standard liquid scintillation counting procedures using a Beckman LS 6800 scintillation counter. Statistics Fatty acid data were analyzed by the Student's t-test. Data for fatty acids are presented as percentages of total acyl groups in a given lipid because the influence of PUFA upon the function of membranes is dependent upon the proportions of the acyl groups in the membrane llpids. Data for SLC proliferation were analyzed by 2x2 repeated measures ANOVA (18). TABLE I Composition of Basal Diet Dietary Oils Ingredient
Coconut
Safflower
g/kg Sucrose* Egg-whlte protein + Fibrous cellulose ~owder # Zinc-free s ~ t mix v Vitamin mix Biotin mix ++ Coconut oll (~hydrogenated) ## Safflowe ,~il ~~ Zinc mix ~
615 200 50 35 10 10 60 -20
615 200 50 35 10 10 -60 20
Crystal Sugar, American Crystal Co., Moorhead, MN. #Teklad Mills Divlsion of ARS Sprague-Dawley, Madison, WI. Whatman CF11, W. and R. Balston Ltd., London, England. 6Modlfied AIN-76 Mineral Mix (without zlnc carbonate powder) cat. no. 170915, Teklad Test Diets, Madison, WI. See reference 15 for composition of modified .~Ineral mix. Mineral mlx was designed to be used at 3.5% of diet (23). Teklad (Vitamin fortification mix) cat. no. 40060, Teklad Test Diets, Madison, WI. See reference 15 for composition of vitamin mix. ++Biotin, crystalline (200 mg biotin in 1000 g of cornstarch), Grand Island Biological Co., Grand Island, NY. Final concentration of biotin in diets was #5.44 ~g/g diet. ~Coconut oll (unhydrogenated), Teklad, cat. no. 190120, Teklad Test Diets, Madison, WI. 86Safflower oil, Teklad, cat. no. 160470, Teklad Test Diets, Madison, WI. ***Zinc acetate, 3.35 g plus 996.65 g cornstarch. +
PUFA AND T-CELL PROLIFERATION
1223
RESULTS On day 21 of the dietary regimen, mean animal weight changes (grams • SD) showed that growth was slower in CNO (142• rats than in SO (162214) rats (p
Fatty Acids
18:2~6 20:3m6 20:4~6 22:4m6 22:5m6 16:1m7 18:1~9 20:3~9
Diet Groups Tissue
SLC + Serum SLC Serum SLC Serum SLC Serum SLC Serum SLC Serum SLC Serum SLC Serum
CNO
3.38 5.76 1.40 1.21 13.32 4.72 1.19 0.11 0.90 1.41 1.45 1.47 13.18 11.72 6.98 9.24
• • • • • • • • • • • • • • • •
0.22 1.18 0.18 0.16 1.62 0.64 0.20 0.19 0.10 0.26 0.40 0.45 0.59 1.14 0.61 1.27
SO
(4) # (8) (4) (8) (4) a (8) (4) (8) (4) (7) (4) (8) (4) (8) (4) (8)
10.99 16.07 1.10 0.48 15.78 18.84 2.92 0.76 1.24 4.96 0.88 0.85 5.84 3D44
• 0.83 + 1.93 + 0.10 • 0.13 _+ 1.63 • 2.06 + 0.97 + 0.13 • 0.25 • 1.16 + 0.12 + 0.34 + 0.36 + 0.42
(5) (7) (5) (7) (5) a (7) (5) (6) (5) (7) (5) (7) (5) (7)
O.O3 • O.O4 (7)
*CNO rats received ad libltum a diet containing 6% unhydrogenated coconut oil. SO rats received ad llbltum a diet containing 6% safflower oil. +Spleen lymphoid cells. #Mean • SD of relative % fatty acid composition of phospholiplds. Value in parenthesis indicates number of samples tested. Values marked with s u p e r s c r i p t "a" indicate not significantly different at p
1224
T.R. KRAMERet al.
Fats high in polyunsaturated (safflower oil) and saturated (coconut oil) fatty acids, fed as 6% of the diet, did not cause differences in proliferation of unstlmulated SLC. Unstimulated SLC from CNO and SO rats cultured in medium containing 2% serum from CNO or SO rats showed equivalent in vitro proliferation (Table 3). Dietary source of SLC was the main influence on in vitro proliferation (log 10 dpm • standard error) of cells stimulated with suboptimal concentrations of Con-A for peak SLC proliferation (0.1 and 0.2 ~g, Table 3). SLC from CNO rats stimulated with 0.1 pg (5.22 • 0.02 vs. 4.82 • 0.02; p = 0.0001) and 0.2 ~g (5.61 • O.01 vs. 5.37 • 0.01; p = 0.002) of Con-A showed higher proliferations than SLC from SO rats. Effects of serum source on levels of in vitro proliferation were observed in SLC from CNO and SO rats stimulated with optimal (0.39 ~g) and supra-optlmal (0.78 ~g) concentrations of Con-A. SLC from CNO and SO rats stimulated with 0.39 pg Con-A and cultured in medium containing serum from SO rats (5.62 • 0.006) showed higher (p = 0.0004) proliferation than SLC from CNO and SO rats cultured in medium containing serum from CNO (5.59 • 0.006) rats. An in vitro interaction between dietary source of SLC and serum was observed in cells stimulated with the supra-optimal (0.78 ~g) concentration of Con-A. SLC from SO rats stimulated with 0.78 ~g of Con-A and cultured in medium containing serum from CNO rats showed lower proliferation than SLC from SO rats cultured in medium containing serum from SO rats, and lower than SLC from CNO rats cultured in medium containing serum from either CNO or SO rats (Table 3).
DISCUSSION Changes in fatty acid composition of nonlymphoid and lymphoid tissues of CNO rats were in agreement with those reported for liver of EFA-D rats: increased 18:1m9, and decreased 18:2~6 and 20:4~6 (19,20). Tsang, et al. (21) reported similar changes in fatty acid composition in plasma and SLC of EFA-D rats. In agreement with Marshall and Johnston (12), SLC from EFA-D rats (CNO) stimulated with suboptimal concentrations of Con-A showed higher proliferation than SLC from EFA-A rats (SO). Marshall and Johnston (12) demonstrated this activity in SLC cultured in medium containing serum from rats fed the same diet as the source of SLC undergoing proliferation. In the present study SLC from CNO rats stimulated with 0.1 and 0.2 ~g Con-A showed enhanced proliferation when cultured in medium containing serum from either CNO or SO rats (see text of Results for values). Thus, according to the ANOVA, the main influence on enhanced in vitro proliferation by suboptlmally Con-A stimulated SLC from CNO rats was caused by the dietary source of SLC and not by the dietary source of serum used in the culture medium. In contrast to suboptimal dose Con-A stimulated SLC, the main influence on in vitro proliferation of SLC stimulated with the optimal (0.39 ~g) and supra-optimal (0.78 ~g) doses of Con-A was the dietary source of serum used in the culture medium and not the dietary source of SLC (see text of Results for values). SLC from CNO or SO rats, stimulated with the optimal and supra-optimal doses of Con-A, showed higher proliferation when cultured in medium containing serum from SO rats than from CNO rats. The mechanism of in vitro interaction between sources of SLC and serum on proliferation of supra-optimal dose Con-A stimulated SLC remains to be elucidated. Lymphoid cells stimulated in vitro by Con-A show increased polyunsaturated fatty acid incorporation (22). Altered PUFA patterns in serum from CNO rats may be the reason for reduced proliferation by supra-optimally Con-A stimulated SLC from SO rats cultured in serum from CNO rats. It is of
PUFA AND T-CELL PROLIFERATION
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1226
T.R. KRAMERet al.
interest that reduced proliferation occurred by SLC from EFA-A (SO) rats cultured in serum from EFA-D (CNO) serum, but not by SLC from EFA-D rats. cultured in serum from either EFA-A or EFA-D rats. The present study suggests that in vitro proliferation of SLC from EFA-D and EFA-A rats is influenced by the dietary source of serum used in the culture medium and the concentration of mitogenlc Con-A used for stimulation of the SLC. These in vitro observations, also, appear to agree with earlier in vivo studies (5-7)'~-wh--i-ch suggest enhanced cell-mediated (T-lymphocyte) functions in low PUFA fed animals. ACKNOWLEDGEMENTS The authors thank LuAnn Johnson for statistical evaluation of the data, Jean Klava for preparation of the diets, Nancy Drlscoll and staff for daily care of animals, and Trell Kulju and Elaine Westerlund for technical assistance.
This study was supported in part by grant HL 08214 from Projects Branch, Extramural Programs, National Heart Institute Hormel Foundation.
the and
Program by the
PROPRIETARY STATEMENT Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. REFERENCES I.
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Santiago-Delphln EA, Szepsenwol J. Prolonged survival of skin and tumor allografts in mice on high-fat diets: Brief Communication. J. Natl. Cancer Inst. 1977; 59:459-61.
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Mertin J, Hunt R. Influence of polyunsaturated fatty acids on survival of skin allografts and tumor incidence in mice. Proc. Natl. Acad. Sci. USA. 1976; 73:928-31.
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Erickson KL. Dietary fat influences on murine melanoma growth and lymphocyte-mediated cytotoxlcity. J. Natl. Cancer Inst. 1984; 72:115-20.
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PUFA AND T-CELL PROLIFERATION
1227
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Erickson KL, Adams DA, McNeill CJ. Dietary lipid modulation of immune responsiveness. Liplds 1983; 18:468-74.
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Erickson KL, McNeill CJ, Gershwln ME, Ossmann JB. Influence of dietary fat concentration and saturation of immune ontogeny in mice. J. Nutr. 1980; 110:1555-72.
10. Locnlskar M, Nauss KM, Newberne PM. The effect of quality and quantity of dietary fat on the immune system. J. Nutr. 1983; 113:951-61. 11. Kollmorgen GM, Sansing WA, Lehman AA, Fischer G, Longley RE, Alexander SS Jr., King MM, McCay PB. Inhibition of lymphocyte funtlon in rats fed hlgh-fat diets. Cancer Res. 1979; 39:3458-62. 12. Marshall LA, Johnston PV. The influence of dietary essential fatty acids on rat immunocompetent cell prostaglandin synthesis and mltogen-induced blastogenesis. J. Nutr. 1985; 115:1572, 1580. 13. King MA, Spector AA. Lipid metabolism in cultured cells. In: Waymouth, C., Ham, R. and Chapple, P.J. eds. The Growth Requirements of Vertebrate Cells In Vitro. New York: Cambridge University Press, 1981: 293-312. 14. Loomis RJ, Marshall LA, Johnston PV. Sera fatty acid effects on cultured rat splenocytes. J. Nutr. 1983; 113:1292-98. 15. Kramer TR. Reevaluation of zinc deficiency on concanavalln-A induced rat spleen lymphocyte proliferation. J. Nutr. 1984; 114:953-63. 16. Kramer TR, Briske-Anderson M, Johnson SB, Holman RT. Influence of reduced food intake on polyunsaturated fatty acid metabolism in zlnc-deficlent rats. J. Nutr. 1984; 114:1224-30. !
17. Boyum A. Separation of leucocytes from blood and bone marrow. J. Clin. Lab. Invest. 1968; 21: (Suppl. 97) 7. 18. Winer BJ. Statistical Principles in Experimental Design, 2nd ed. New York: McGraw-Hill Inc., 1971: 518-39. 19. Mohrhauer H, Holman RT. The effect of low level of essential fatty acids upon fatty acid composition of rat liver. J. Lipid Res. 1963; 4:151-9. 20. Boissonneault GA, Johnston PV. Essential fatty acid deficiency, prostaglandln synthesis and humoral immunity in Lewis rats. J. Nutr. 1983; 113:1187-94. 21. Tsang WM, Belin J, Smith AD. Levels of polyunsaturated fatty acids in lymphocytes of rats fed on diets varying in polyunsaturated fatty acid content. Br. J. Nutr. 1980; 43:367-373. !
22. Goppelt M. Kohler L, Resch K. Functional role of lipid metabolism activated T-lymphocytes. Biochim. Biophys. Acta. 1985; 833:463-72.
in
23. American Institute of Nutrition. Report of the American Institute of Nutrition Committee on Standards for Nutritional Studies. J. Nutr. 1977; 107:1340-8. Accepted for publication August 20, 1986.