KENT L. ERICKSON, M. ERIC GERSHWIN,CARLA J. McNEILL, JOHN R. OSSMANN ANDNANCY L. CANOLTY Department of Human Anatomy and Section of Rheumatology-Clinical Immunology, School of Medicine, and Department of Nutrition, University of California, Davis, California 95616 ARSTRACT Temporal changes in lymphocyte blastogenesis were studied using spleen cells from syngeneic melanoma-bearing and control mice fed various levels of purified diets containing 6, 10 or 30% casein. T-cell blastogenesis was stimulated by the presence of the tumor and these responses changed with the duration of feeding. In addition, protein con centration did not affect T-cell transformation but the level of energy intake influenced concanavalin A induced DNA synthesis. In contrast, the grow ing melanoma did not influence R-cell transformation whereas a very low level of dietary protein, a low level of energy intake and duration of the dietary manipulation influenced these cells. Tumor weights were generally not affected by the diet except in mice receiving a very low level of energy intake. Thus, we have found that R-celi responses were affected more than those of T-cells and that moderate protein deficiency did not enhance cellular immune responses in syngeneic tumor-bearing and control mice. J. Nutr. 109: 1893-1900, 1979. INDEXING KEY WORDS melanoma •protein energy malnutrition •lymphocyte transformation Epidemiologie surveys indicate that diet may be related to 50% of all cancers in humans (1). Indeed, dietary factors intrinsic to an individual and the environment can interact both positively and negatively to modulate and modify cellular transformation. Thus, diet may alter individual organ susceptibility and responsiveness to neoplastic processes. For example, energy restriction has been shown to inhibit tumorigenesis (2), and deficiency of some essential amino acids can produce decrements in the incidence, growth and metastatic Spread , c . , Other ammo acids ranrtrronoci'e I Ã-\ CinogeneslS \t).

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Protein energy malnutrition is believed .« .1 . n • ••TT to significantly influence immunity. How-

ever, the degree of malnutrition has been reported to cause either an increase (5) or decrease (6) in immune responsiveness, We (7) have previously found that mitogen induced T-cell blastogenesis in mice was dependent upon dietary protein concentration, whereas R-cell blastogenesis was dependent upon the level of energy intake. Moreover, T- and R-lymphocyte transformation was stimulated by the presenee of a growing melanoma. Other inKeceived for publication February 20 1970. Supported in part bv funds from the Cancer Research Coordinating Committee and Biomédical Research Support, University of California, Kroc Foundation and National Institute of Health Grant

KO,2¥£)8Vi; Recipient of Research AIOOIUS. 1893

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Effects of Low Dietary Protein Concentration and Energy Deprivation on Lymphocyte Transformation in Melanoma-Bearing Mice1

1894

ERICKSON

ET AL.

TABLE 1 Composition of experimental diets

Tumor cells. The cell line P51 was estab lished from a transplantable B16 murine malignant melanoma and propagated in culture (9, 10). The growth and mainte

3 Grand Island Biological Company. Grand Island, New York. ' Calbiochem, La Jolla, California. 5 Difco Laboratories. Detroit. Michigan. 6 New England Nuclear, Boston, Massachusetts.

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nance has been previously described (7). Animals, tumor production and diets. Six-week old female C57BL/6J mice were Diet (No.) fed a purified diet containing 30% casein ( table 1 ) with water ad libitum for 1 week before the start of the experiments. On day 0, tumors were transplanted into 135 mice by subcutaneous injection of 2.5 X IO6 Casein1Salt mix2Vitamin viable P51 cells suspended in sterile phos mix3Dextrose4Corn phate buffered saline (PBS) into the an terior thigh; 140 mice were injected with oil56.06.01.578.58.010.06.01.574.58.030.06.01.554.58.0 only PBS. Immediately after injection all 1 Nutritional Biochemical Co., Cleveland, Ohio. mice were individually housed and both 2The salt mix provides the following amounts per tumor-bearing and control mice were di kg of diet: CaCO3, 1.80 g; K2 HPO,, 1.93 g; NaCl, vided into three diet groups of 45 each. 1.01 g ; MgSO4-7H2O, 0.75 g; KI, 4.8 mg; ZnCO3, The diets contained 6, 10 or 30% casein 1.5 mg; CuSO4-5H2O, 1.8 mg; MnSO4-H2O, 13.8 mg; FeSO4-7H2O, 15 mg. 3The vitamin mix pro and in each diet group 15 mice were pro vides the following amounts per kg of diet : retinyl vided daily with either 2.5, 3.5 or 4.5 g of palmi tate, 2,000 lU; cholecalciferol, 900 IU; dl-afeed plus water ad libitum. Thus, absolute tocopheryl acetate, 60 IU; menodione 1.0 mg; protein feedings each day included groups thiamin, 2.5 mg; riboflavin, 4.0 mg; panthothenic acid, 12.0 mg; niacin, 70.0 mg; pyridoxine, 4.0 mg; receiving 1.35, 1.05, 0.75, 0.45, 0.35, 0.27, folacin, 2.0 mg; biotin, 0.1 mg; cyanocobalamin, 0.25, 0.21 and 0.15 g. For each feeding 0.01 mg; choline, 1.9 g. 'Corn Products Co., level of each diet, five tumor-bearing and Englewood Cliffs, New Jersey. * "Mazóla," Best five control mice were used for the mitogen Foods, Englewood Cliffs, New Jersey. assay after being fed the experimental diets for 1, 2 or 3 weeks. Tumors were vestigators (8) have shown that chronic protein insufficiency enhanced cell-medi removed in 3 weeks after transplantation ated immunity. For example, phytohaeand weighed after removal of the sur magglutin responses were higher for mice rounding connective tissue. receiving an 8r/c protein diet than those Mitogen assay. On the same day as receiving 27%. In contrast, we (7) have tumor cells were injected (day 0) five observed that PHA responses were not PBS-injected animals were killed for the significantly different for mice fed 15 or mitogen assay. Additionally, 90 mice were 45% protein. Likewise, a decrease in the killed on days 7, 14 and 21. At the time of killing, spleens were removed and single kinetics of the primary cellular response was reported for mice fed less than 10% cell suspensions prepared in RPMI-1640 protein and this diminution progressed to with 25 mm HEPES buffer,3 10% fetal calf serum,3 penicillin 3 and streptomycin.3 a profound depression of cellular responses at a 37c protein dietary level (5). Because Quadruplicate cultures from each mouse were set up using either mitogen or media. of these issues, experiments were designed Mitogens studied include concanavalin A * to determine the influence of low dietary (Con A), 2.5 fig/ml, phytohaemagglutin P protein concentration and energy intake (PHA),5 0.1% v/v, pokeweed mitogen upon mitogen response of spleen cells from (PW),3 1.0% v/v, and Salmonella typhosa tumor-bearing and nontumor-bearing con (LPS),5 500 /¿g/ml. trol mice. This paper reports the effects of lipopolysaccharide protein-energy malnutrition upon host im Samples were incubated for 72 hours at 37°in 5% CO2; 4 hours before harvest mune responses to a syngenic melanoma in each culture received 1 /¿Ci of [methyl-3H]mice. thymidine (specific activity 6.7 Ci/mmol).6 Samples were harvested onto glass fiber MATERIALS AND METHODS

DIETARY INFLUENCE ON LYMPHOCYTE TRANSFORMATION

week (table 3) then declined after the third week (table 4) to 9,550 cpm, a level no different than that of week 1 ( table 6 ). With PHA induced lymphocyte transfor mation neither protein concentration nor level of energy intake modified the response. However, the duration of feeding, tumor growth or both together had a significant influence on blastogenesis (table 5). PHA induced T-cell responses of 6,490 and 15,900 cpm after the first and second week, respectively, are significantly increased, then decreased to 3,950 cpm, a level no greater than control animals who had received no experimental diets (table 6). Moreover, when significant interaction occurred be tween multiple variables, the presence or absence of the tumor was always a neces sary factor. Examples of multiple variables interacting to produce significant lympho cyte transformation include tumor X time and tumor X protein X level for Con A and tumor X level for PHA (table 5). For the mixed but predominantly B-cell RESULTS mitogen, pokeweed, neither presence nor For these experiments mice were divided absence of tumor, protein concentration, into groups according to presence or ab nor level of energy intake influenced lym sence of tumor, percentage of protein in phocyte transformation (table 5). As with their diet, level of energy intake or length all mitogens tested, however, the duration of time on the diet. For the tumor-bearing the mice received the experimental diets animals the latter parameter was also a greatly influenced the response to this measure of the duration of tumor growth. mitogen. The highest level of 30,300 cpm The responses of these groups to the mito- was measured after 2 weeks of the ex gens tested are indicated in tables 2 perimental dietary regime, whereas no dif through 4. Lymphocyte transformation in ference was observed between levels mea duced by either of the T-cell mitogens, Con sured after 1 and 3 weeks (table 6). A or PHA, was significantly influenced by Lymphocyte transformation induced by the presence of a growing tumor. Thus, the B-cell mitogen, LPS, was influenced mitogen responses were significantly by all variables except presence or absence greater for tumor-bearing mice than con of a tumor ( table 5 ). Mice fed a diet with trols (table 5). Neither Con A nor PHA 6r/f protein concentration had a lower level induced transformation, however, was in of splenic lymphocyte transformation in fluenced by dietary protein concentration response to LPS than animals fed either whereas the level of energy intake influ 10 or 30% ; no difference was observed be enced Con A but not PHA responses (table tween the later two protein levels. LPS5). Mice receiving 3.5 g/day of diet had induced blastogenesis was also influenced the highest level of Con A induced lympho by the level of energy intake of the murine cyte transformation (29,300 cpm) which host. Splenic lymphocytes of mice fed 2.5 was significantly greater than the 15,300 g/day had 11,600 cpm, the lowest level, cpm for animals fed 2.5 g/day (table 6) those fed 3.5 g/day had 12,800 cpm, a but all T-cell responses changed signifi higher level, and those fed 4.5 g/day had cantly with time. The responses induced by Con A did not change after the first week 7 Otto Hlller Co.. Madison, Wisconsin. (table 2) but increased to 46,800 cpm, a * "Oiimlfluor." New England Nuclear. Boston. significantly higher level, after the second Massachusetts.

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filters,7 placed into vials with toluene and scintillato! s and counted in a liquid scin tillation spectrometer. Statistical methods. Data are reported as a mean total of counts per minute (cpm) plus the standard error for each group. Examination of the data indicated that the distribution was highly skewed relative to the mean. To stabilize the variance, analy ses were conducted using logarithmic trans formation. The data were then subjected to a four-way analysis of variance with dis proportionate sub-classes and multiple re gression analysis (11). Analysis of either raw counts or stimulation indices produced similar results. The latter were calculated: stimulation index = cpm,,, —cpmn,t./cpmmc where cpmn, is counts of a culture after mitogen stimulation and cpm,,,c is counts of a culture with no mitogen. Multiple pairwise comparisons were made using the Bonferroni inequality ( 12).

1895

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