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[CANCER RESEARCH 49, 1931-1936, April 15, 1989] Effects of Fish Oil and Corn Oil Diets on Prostaglandin-dependent and Myelopoiesis-associated Immune Suppressor Mechanisms of Mice Bearing Metastatic Lewis Lung Carcinoma Tumors1 M. Rita I. Young2 and Melvin E. Young Department if Research Services, Edward J. Hiñes,Jr., Veterans Administration Hospital, Hiñes,Illinois 60141 [M. K. 1. Â¥.,M. E. Y.J, and Department of Pathology, Loyola Univi rsity Stritch School of Medicine, Maywood, Illinois 601 S3 [M. R. I. Y.J ABSTRACT The effe -Is of a fish oil diet on the myelopoietic and immunological parameters of normal mice and of mice bearing metastatic Lewis lung carcinoma i LLC-C3) tumors were compared to the effects of a corn oil or a mixec-fat rodent chow diet. This was studied soon after tumor appearance, on Day 17, when immune suppression was mediated by prostaglant in E2 (PGE2)-producing suppressor cells, and late in tumor development, on Day 28 when immune suppression was associated with myelopoiesis and the appearance of bone marrow-derived suppressor cells whose activity was not dependent on PGE2. Feeding a fish oil diet from Days 10 to 17 of tumor growth partially restored splenic T-cell blastogene? is, reduced spleen cell secretion of PGE2, and alleviated splenic suppressor activity. When fed from Days 21 to 28 of tumor growth, a f sh oil diet neither restored T-cell blastogenesis nor alleviated suppressor cell activity. The fish oil diet increased the frequency of myeloid progenitor cells in normal mice and in mice bearing small or large tumors. Concurrently, the fish oil diet stimulated the appearance of bone marr jw-derived suppressor cells. When administered after the establishm -nl of palpable primary tumors, a fish oil diet also increased the formation of pulmonary lung nodules. In contrast to the fish oil stimulatior of myelopoiesis and the associated suppressor cells, feeding a corn oil diet to tumor-bearing mice during Days 21 to 28 after tumor implantation reduced myelopoiesis and the presence of the associated bone marrow suppressor cells. These data show that a fish oil diet can minimize t le immune suppression in tumor bearers when suppression is mediated by PGE2-producing suppressor cells, but can also induce mye lopoietic siimulation leading to the appearance of bone marrow-derived suppressor cells and increased tumor metastasis. INTRODUCTION Modul itors which regulate immune responses, in many in stances, also influence myelopoiesis (1-9). Attention has been focused on arachidonic acid metabolites, predominantly PGE2,3 as mediators of both immune suppression and as negative regulators of myelopoiesis. PGE2 has been shown to be inhibi tory to the activities of T-cells, B-cells, and macrophages (1014). In addition, PGE2 is myelosuppressive as it is inhibitory to the g-owth of progenitor cells including monocytic and granulocytic-monocytic CFC (1,2, 15). Dietary fats may be capable of modifying both immunological and mye opoietic parameters. Prostaglandin precursors, when administered in the diet, P.O., or by s.c. injection, have resulted in increased PGE2 production (16, 17). Dietary administration of corn oil, which is rich in n-6 prostaglandin precursors, to rats resulted in a reduced blastogenic response to PHA as Receivec 9/28/88; revised 1/6/89; accepted 1/16/89. The costs of publication of this article were defrayed in part by the payment of page chi rges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported in part by the Medical Research Services of the Veterans Administration and by Grant CA-45080 from the NIH. 2 To whom requests for reprints should be addressed, at Pathology Research (151Z2), HiñesV.A. Hospital, Hiñes,IL 60141. 3The abbreviations used are: PGE2, prostaglandin E2; CFC, myeloid progeni tor cells; PHA, phytohemagglutinin; EPA, eicosapentaenoic acid; CSF, colonystimulating factor; FBS, fetal bovine serum; Con A, concanavalin A; LLC-C3, Lewis lung carcinoma, C-3 variant. compared to that of rats fed a rodent chow diet (18). The substitution of the dietary corn oil with increasing amounts of fish oil, which is rich in n-3 polyunsaturated fats, increased PHA responsiveness. Humans treated with EPA, a major n-3 polyunsaturated fat of fish oil, had increased T-cell responses to PHA (19). EPA is a poor substrate for cyclooxygenase and competitively inhibits the formation of dienoic prostaglandins (20, 21). The administration of fish oil to mice reduced the amount of PGE2 which their macrophages secreted (22). The modulation of immune competence by corn oil or fish oil diets has also been expressed in terms of their effects on growth of transplanted tumors. Growth of mammary adenocarcinomas was hastened in rodents which had been maintained on corn oil diets and was reduced in animals fed fish oil diets (23, 24). Studies of the effects of dietary polyunsaturated fats on tumor growth have centered on the ability of the fats to modulate PGE2 levels and, in turn, PGE2-dependent suppressor mecha nisms. However, the suppressed immune competence in tumor bearers has been associated with several different types of immune suppressor mechanisms such as PGE2-secreting mac rophages (25-27) and suppressor T-lymphocytes (28, 29). Re cently, the suppressed immune competence of tumor bearers has been associated with tumor stimulation of myelopoiesis and the appearance of bone marrow-derived immune suppressor cells (30-32). Tumors have been shown to stimulate myelo poiesis by their secretion of CSFs (30, 32-36). Tumor bearers have splenomegaly (30, 31, 37, 38), increased numbers of myeloid CFC in the bone marrow and spleen (30, 31, 34, 39), granulocytosis (34, 37), and reduced proportions of lympho cytes (36, 40). The effects of dietary fats on myelopoiesis or on the appearance of the associated bone marrow-derived suppres sor cells have not been studied in either normal or tumorbearing animals. Our previous characterizations of the immune suppressor mechanisms in mice bearing metastatic LLC-C3 tumors have shown that the tumor cells do not secrete immune suppressive factors, such as PGE2 (41). Instead, the LLC-C3 tumors stim ulate the sequential appearance of several immune suppressor cell populations during the progressive growth of tumor. In mice bearing small and medium sized LLC-C3 tumors, immune suppression was associated with increased production of PGE2 by splenic adherent macrophages (27,42). In mice bearing large LLC-C3 (>3 g), myelopoiesis was stimulated and, concurrently, there was the presence of bone marrow-derived immune sup pressor cells (30, 31). In the current study, the effects of diets containing either corn oil, fish oil, or mixed fats on myelopoiesis and on the two immune suppressor mechanisms of mice bearing metastatic LLC-C3 tumors were investigated. MATERIALS AND METHODS Mice. Six- to 8-wk-old male C57BL/6 mice were used for all studies. The mice were obtained from Cumberland View Farms (Clinton, TN) 1931 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research. FATS, MYELOPOIESIS, and then housed, in groups of 5 mice per cage, at the HiñesV. A. animal research facility. Medium. The medium used to culture tumor cells and for blastogenesis and suppressor cell assays was RPMI-1640 containing 100 units/ ml of penicillin, 100 ng/ml of streptomycin, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid buffer solution, 5 x IO"5 M 2-mercaptoethanol. 2 HIM L-glutamine, and 10% endotoxin-free FBS (Hyclone Laboratories, Logan, UT). Diets. Diets containing 5% fat were administered to tumor-bearing or control mice between Days 10 and 17 or Days 21 and 28 of tumor growth. The diets differed only in fat composition and not in fat quantity. The following diets were used: fat-free basal diet mix contain ing either 5% corn oil or 4% fish oil plus 1% corn oil; or rodent chow containing 5% mixed fats. The basal diet was a vitamin- and mineralsupplemented fat-free mix (TD 84010, Teklad Test Diets, Madison, WI). The fish oil (Zapata Haynie Corp., Reedville, VA), derived from menhaden fish, was rich in eicosapentaenoic acid as well as in other n 3 polyunsaturated fatty acids. Corn oil, 1%, was added to this fish oil diet as a source of essential fatty acids. Fresh food was given daily. Characteristics of Tumor Cells (41). The cloned metastatic LLC-C3 cells were used in all studies. The LLC-C3 variant was chosen for this study of host immune suppressor mechanisms of tumor bearers because the tumor cells do not themselves secrete immune suppressive factors, such as PGE2. Briefly, the LLC-C3 variant was isolated from a lung nodule of a mouse bearing a s.c. implanted parental LLC tumor. Injection of LLC-C3 cells s.c. typically results in formation of approx imately 10 to IS metastatic lung nodules. Metastatic dissemination to other sites has never been detected. In the currently described studies, the LLC-C3 tumor cells were implanted into mice by dorsal s.c. injection of 5 x 10' cells. Experimental diets were administered between Days 10 and 17 after tumor injection, the first week after appearance of a palpable tumor, or between Days 21 and 28. when tumors were large. Assays were conducted on the last day of administration of experimental diets. Femoral Bone Marrow and Spleen Cellularity. Single cell suspensions were prepared from the spleen and femoral bone marrow of each mouse from at least 5 mice per group. The nucleated cells were counted on a hemacytometer. Soft Agar CFC Assay (9). Into each 35- x 10-mm tissue culture dish were placed 7.5 x IO4bone marrow cells or 7.5 x IO5spleen cells in I ml of semisolid supplemented RPMI-1640 medium containing 20% FBS and 0.3% agar (Bacto Agar; Difco Laboratories, Detroit, MI). Supernatant of pokeweed mitogen-stimulated spleen cells was used as a CSF source at a concentration of 5% (43). This preparation of murine CSF, which was free of known inhibitory molecules, stimulated colonies containing mainly granulocytes and granulocytes plus macrophages. The colonies (>50 cells) were counted after 7 days of culture. T-I.ymphocyte Blastogenesis to Con A. Spleen cells were cultured for 3 days in microtiter plates at a density of 2 x IO5 cells in 0.2 ml of medium containing 3 ^g/ml of Con A. For the last 18 h, l /iCi of [3H]thymidine was added. The amount of [3H]thymidine incorporated by the spleen cells was measured in a liquid scintillation counter. All assays were conducted in triplicate on individual mice from groups of at least 5 mice. Data were expressed as mean ±SEM (cpm) of 5 mice per group assayed individually. Suppressor Cell Assay. Suppressor cell activity was measured by the capacity to suppress normal spleen cell blastogenesis to Con A. Sup pressor cells were first irradiated with 2500 R. They were then added to 2 x IO5normal responder spleen cells in a T-cell blastogenesis assay AND SUPPRESSOR CELLS removed and frozen. A radioimmunoassay (New England Nuclear, Boston, MA) was used to quantitate the PGE2 levels. Plasma was diluted 1:20, and the supernatants were diluted 1:2 with assay buffer prior to being added to the radioimmunoassay. Briefly, into each polypropylene tube were mixed 0.1 ml of anti-PGE2, 0.1 ml of 12!IPGE2, and 0.1 ml of PGE2 standard or PGE2-containing sample. Tubes were refrigerated overnight. A polyethylene glycol solution (16%, M, 6000) was added to precipitate immune complexes, and the radioactiv ity in the precipitate was counted in a gamma counter. We have previously shown that there is no nonspecific interference in this assay since similar amounts of PGE2 were quantitated by this method in diluted plasma and by radioimmunoassay of PGE2 which was purified from plasma by high-performance liquid chromatography (44). Tumor Growth and Metastasis. Mice which were implanted with LLC-C3 tumor cells were maintained on a mixed-fat rodent chow diet until the primary tumors became palpable at approximately 10 days. These mice were then randomly divided into two groups of 15 mice each. One group remained on a mixed-fat rodent chow diet, while the other group was switched to a fish oil diet for the remaining 2.5 wk of tumor growth. Diameters of primary tumors were measured during tumor development. After 4 wk of tumor growth, all mice were sacri ficed. The lungs were filled with a nigrosin solution, removed, and washed in a mixture of 70% ethanol, 3.7% formaldehyde, and 5% acetic acid. White metastatic nodules on the black lung background were counted. Data were expressed as both the range and the median number of lung nodules. Analysis of Data. The significance of the differences between values was determined by the Student / test. All myelopoietic and immunological data were expressed as the mean ±SD of triplicates or the mean ±SEM from at least 5 mice with triplicate determinations for each mouse. RESULTS Effects of Fish Oil Diet, Corn Oil Diet, or Mixed-Fat Rodent Chow on T-Lymphocyte Blastogenesis of Tumor-bearing Mice. The effects of administering diets containing various fats on Tcell competence of normal mice or of mice bearing small or large tumors were assessed (Fig. 1). The Con A blastogenic response of normal mice was reduced after feeding a corn oil diet (P < 0.001) and stimulated after feeding a fish oil diet (P < 0.01) as compared to the response of mice fed a mixed-fat rodent chow diet. In comparison to the response of control mice, the Con A blastogenic response by spleen cells of mice bearing LLC-C3 tumors and fed rodent chow was suppressed at both 17 and 28 days after tumor implantation (P < 0.01). On Day 17 after tumor implantation, the blastogenic response 200 -, , ^ _ Chow D Corn Fish with 3 Mg/m' of Con A at a responder spleen cell:suppressor cell 1:1 ratio. In some experiments, indomethacin was also added to yield 5 x 10~6M(<0. l % ethanol). In all of the described experiments, the amount of ['Hjthymidine which was incorporated in control cultures containing only irradiated cells and Con A was less than 5000 cpm. Data were expressed as mean ±SD (cpm) of triplicates. Normals LLC—C3 Normals LLC—C3 Quantitation of PGE2 in Plasma or Secreted by Spleen Cells. Mice Days 10-17 Days 21-28 were exsanguinated by retroorbital puncture, and the blood was placed Fig. Con A blastogenic response of spleen cells from normal mice and of into IKp.u in coaled tubes. Plasma was collected and frozen until as LLC-C3 tumor-bearing mice fed rodent chow, corn oil, or fish oil diets during sayed for PGE2. Spleen cells were seeded into microtiter wells at a Days 10 to 17 or 21 to 28 of tumor growth. Spleen cell blastogenesis was assessed density of 5 x 10V0.2 ml. After 4 h of incubation, supernatants were on the last day of diet administration. 1'ilK.thymidine. 1932 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research. FATS, MYELOPOIESIS, AND SUPPRESSOR of tumor-tearing mice which had been fed a corn oil diet during the precec ing 7 days was equally suppressed as that of mice which had been fed mixed-fat chow. When, instead, a fish oil diet was administered to the mice, the immune suppression was abolished. The effect of administering the fish oil diet to mice during I); ys 21 to 28 of tumor growth differed as it had no immune-r>:storative effects on the T-lymphocyte responsiveness to Con A. In fact, the blastogenic responses of tumor bearers which were fed either rodent chow, corn oil diet, or fish oil diet during Days 21 to 28 were equally suppressed. Immune Suppressor Cell Activities of Normal or LLC-C3bearing M ice Fed Fish Oil or Rodent Chow Diets during Days 10 to 17 ifter Tumor Implantation. Studies were conducted to support our previous investigations (27, 30, 42) of whether spleen ani bone marrow cells of mice bearing small tumors mediate their suppressive activities by a prostaglandin-dependent mechanism. At 17 days after tumor implantation, the spleen cells of tu Tior-bearing mice fed rodent chow had more suppres sor cell activity than did spleen cells of control normal mice (/' < 0.001) (Table 1; Fig. 2). As shown in Table 1, this splenic suppressor activity was prostaglandin dependent as it was ablated by the addition of indomethacin. Tumor-induced bone marrow sjppressor activity was not yet apparent on Day 17. The effect of feeding a fish oil or rodent chow diet on the suppressiwe activities of spleen and bone marrow cells was assessed ¡ Fig. 2). The splenic suppressor activity of normal mice fed a fish oil diet was slightly stimulated (P < 0.05) in comparison to when a mixed-fat rodent chow diet was fed. The administration of a fish oil diet to tumor bearers during Days 10 to 17 alleviated the splenic suppressor cell activity. In fact, there was no difference in the suppressive activities of spleen cells of n .trmal mice irr.vw.vtumor-bearing mice fed a fish oil diet. Since the immune suppressive activity of spleen cells obtained from mie i at Day 17 after tumor implantation was indometh acin sensitive, the amounts of PGE2 which were secreted by spleen ce Is and which were in plasma of tumor-bearing mice fed either rodent chow or fish oil diet were quantitated. Spleen cells of tumor bearers fed a rodent chow diet secreted 53% more (P < 0.01) PGE2 (490 ±23 pg of PGE2/106 cells/4 h) than did spleen cells of control normal mice (323 ±17 pg of PGE2/10i cells/4 h). The administration of a fish oil diet reduced t tie amount of PGE2 which was secreted by spleen cells of tumor bearers by 31% (P< 0.05) to 347 ±16 pg/106 cells/ 4 h. Likewise, plasma PGE2 levels of tumor bearers were 2-fold CELLS higher (P < 0.001) than were plasma PGE2 levels of control normal mice (Table 2). The administration of a fish oil diet reduced the amounts of PGE2 in plasma of tumor bearers by 57% (P < 0.001) and of normal mice by 52% (P < 0.001). During this same time period of Days 10 to 17 after tumor implantation, the effects of a fish oil diet and a mixed-fat chow diet on the immune regulatory activities of bone marrow cells were compared. At Day 17, the tumor-induced bone marrow suppressor cells had not yet become prominent in mice fed a rodent chow diet (Table 1; Fig. 2). However, administration of a fish oil diet to tumor bearers stimulated the appearance of bone marrow suppressor cells. Immune Suppressor Cell Activities of Normal or LLC-C3bearing Mice Fed Fish Oil or Rodent Chow Diets during Days 21 to 28 after Tumor Implantation. We previously showed that the immune suppression in mice bearing large tumors was associated with myelopoiesis and the consequential appearance of bone marrow-derived suppressor cells first in the bone mar row and then in the spleen (30, 31). At Day 28 after tumor implantation, the bone marrow cells of tumor bearers exhibited prominent suppressor activity (Table 1; Figs. 2 and 3). The administration of a fish oil diet further exaggerated the degree of bone marrow suppressor activity (P < 0.02) (Figs. 2 and 3). In contrast, diet containing corn oil significantly reduced the level of bone marrow suppressor activity (P < 0.05) (Fig. 3). The suppressor activity of spleen cells from rodent chow-fed mice bearing large tumors was also prominent (Table 1; Figs. 2 and 3). This immune suppressive activity was prostaglandin independent as it was unaffected by the presence of indometh acin (Table 1). Administration of either a fish oil or corn oil diet between Days 21 and 28 of tumor growth did not alleviate the tumor-induced splenic suppressor activity (Figs. 2 and 3). In fact, the splenic suppressor activity of mice fed the mixedfat rodent chow diet or the fish or corn oil diets remained comparable. Modulation of Myeloid CFC Frequency and the Number of Nucleated Cells in the Spleen and Femoral Bone Marrow of Tumor Bearers Fed Diets Containing Various Fats. In mice bearing large tumors, the appearance of bone marrow-derived suppressor cells was associated with tumor stimulation of mye lopoiesis; myelopoietic stimulation was not readily apparent in mice bearing small tumors (30, 31). When a fish oil diet was fed instead of rodent chow to mice during Days 10 to 17 after tumor implantation, there resulted an increase in the frequency of CFC per 7.5 x 10" bone marrow cells from 106 ±7 to 196 Table 1 Indomethacin sensitivity of tumor-induced suppressor cells Normal spleen cells were cultured alone or with irradiated spleen or bone marrow cells obtained from normal mice or from mice bearing LLC-C3 tumors at Day 17 or 28 after tumor implantation. To the cultures were also added 3 ¿ig/mlof Con A and either diluent or 5 x 10"' M indomethacin. Assay day cells17 marrow28 by normal spleen andDiluent123,775cells with added cells value"NSC Added None Normal spleen LLC-C3 spleen Normal bonw marrow LLC-C3 bone ±12,701* 120,778 ±1,209 64,645 ±8,461 (48)'' 95,41 7 ±7,769 (23) (30)136,793 86,976 ±8,535 23,835 ±10,406 127,785 ±17,499 118,400 ±12,586 122,316 ±2,732 ±9,601140,1 116,286 NS <0.01 <0.01 <0.02NSNS 18 ±4,274 None ±7,414 Normal spleen 138,362 ±7,056 141,623 ±1,722 LLC-C3 spleen 66,400 ±18,470(51) 69,321 ±19,817(49) NS <0.01NS Normal bone marrow 101,665 ±5,565 (26) 141,855 ±11,188 LLC-C3 bone marrowBlastogenesis 49,850 ±9,270 (64)Indomethacin1 63,812 ±3,349 (53)P " Signifirance of the difference between the blastogenic response of spleen cells in the presence versus absence of indomethacin. * Mean :: SD of cpm of triplicates. ' NS, no i significant. * Numlx rs in parentheses, significant (P < 0.05) suppressor cell activity shown as the percentage of suppression of normal spleen cell blastogenesis. 1933 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research. FATS, MYELOPOIESIS, AND SUPPRESSOR 150 •¿ Chow E3 Fish Kl °100 1 50 I I Kl Norm LLC Spleen None Days Norm LLC Bone morrow Norm LLC Spleen 1O-17 Days Added Norm LLC Bone morrow 21-28 cells Fig. 2. Suppressor cell activity of spleen cells or bone marrow cells of normal or LLC-C3 tumor-bearing mice which were fed rodent chow or fish oil diet during Days 10 to 17 or 21 to 28 of tumor growth. Suppressor cell activity was measured by the capacity of cells to inhibit the Con A blastogenic response of normal spleen cells. TdR, thymidine. Table 2 Plasma PGE¡levels of tumor-bearing mice fed varying fat diets Plasma l'( ,1 levels of normal or LLC-C3 tumor-bearing mice which were fed rodent chow, corn oil, or fish oil diets during Days 10 to 17 or 21 to 28 of tumor growth. Plasma PGEj levels of mice respectively, as compared to that for control normal mice (Fig. 4). The administration of a fish oil diet during Days 21 to 28 to normal and to tumor-bearing mice caused a respective in crease in the frequency of splenic CFC by 63% (P < 0.01) and 36% (P < 0.02), and of bone marrow CFC by 34% (P < 0.02) and 18% (P < 0.01). In contrast, administration of a corn oil diet to normal and to tumor-bearing mice caused a respective reduction in the frequency of splenic CFC by 40% (P < 0.02) and 16% (not statistically significant), and of bone marrow CFC by 15% (P < 0.02) and 18% (P < 0.02). The effects of diets on the cellularity of the femoral bone marrow and spleens of both normal mice and of tumor-bearing mice were also measured (Fig. 5). Neither the tumor presence nor the various diets caused significant alteration in the number of nucleated cells in the femoral bone marrow. Hence, the fish oil-induced increases or corn oil-induced decreases in femoral CFC frequencies described above were similar in magnitude to the changes in the absolute numbers of CFC per femur. In contrast, the development of a large tumor in mice fed rodent chow was associated with an increase in spleen cellularity by 2.1-fold (P< 0.01). The number of nucleated cells in the spleen of tumor bearers was slightly reduced (not statistically signifi cant) by the administration of a corn oil diet and was increased by 77% (P < 0.05) following the administration of a fish oil diet. Therefore, the splenic CFC-stimulatory effects of fish oil bearers1099 day1728DietsRodent chow Fish oilRodent ±67° 276 (52)*586 ±62 CELLS 250 -, 200 - ±161 (57)535 626 ±74 chow ±58 ±63 Corn oil 911 ±44(155) 1269± 118(237) Fish oilNormals531347 ±79 (59)LLC-C3 367 ±96 (69) " Mean ±SEM determined in triplicate for each mouse and shown as pg of PGE2 per ml of plasma of 5 mice per group. * Numbers in parentheses, significant (/' < 0.05) effects of the com oil or fish oil diets on plasma 1'( 11 levels shown as the percentage of the value for mice fed the mixed-fat rodent chow diet. 150 •¿ Chow D Corn E Fish ^H 100 o: o H I "i ^H Y///ÃŒ Normals LLC-C3 Normals LLC-C3 ^H H Spleen cells Bone marrow cells Fig. 4. Frequency of myeloid progenitor cells (CFC) per 7.5 X IO4 bone marrow cells or 7.5 x 1(1'spleen cells of normal or LLC-C3 tumor-bearing mice which were fed rodent chow, corn oil, or fish oil diets during Days 21 to 28 of tumor growth. 50 - None Normals Spleen LLC-C3 cells Added Normals LLC-C3 Bone morrow cells cells Fig. 3. Suppressor cell activity of spleen cells or bone marrow cells of normal or LLC-C3 tumor-bearing mice which were fed rodent chow, corn oil, or fish oil diets during Days 21 to 28 of tumor growth. Suppressor cell activity was measured by the capacity of cells to inhibit the Con A blastogenic response of normal spleen cells. TdR, thymidine. ±19 (P < 0.02). The effects of feeding rodent chow, fish oil, or corn oil diets on the myelopoietic parameters of tumor bearers were more extensively evaluated in mice bearing large tumors. The frequency of CFC in the spleen and in the bone marrow of tumor-bearing mice fed a rodent chow diet during Days 21 to 28 was increased 2.3-fold (P < 0.01) and 1.5-fold (P < 0.001), «-» Normals Spleen LLC-C3 cells Normals Bone LLC-C3 marrow cells Fig. 5. Number of nucleated cells in the spleen (:<!()') or in the femoral bone marrow (x IO") of normal or LLC-C3 tumor-bearing mice which were fed rodent chow, com oil, or fish oil diets during Days 21 to 28 of tumor growth. 1934 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research. FATS, MYELOPOIESIS. AND SUPPRESSOR diets were magnified by the stimulatory effects on both the splenic CFC frequency and the spleen cellularity. Plasma 1 GK> Levels of Mice Bearing Large Tumors and Fed Various Fai Diets. Plasma PGE2 levels of mice fed either corn oil or fish cil diets during Days 21 to 28 of tumor growth were measured and compared to the plasma PGE2 levels of mice fed a mixed-fat rodent chow diet (Table 2). The concentrations of PGE2 in the plasma of rodent chow-fed normal mice and of mice bearir g large tumors were similar. The administration of a corn oil diet from Days 21 to 28 elevated the plasma PGE2 levels of normal mice by 55% (P< 0.001) and of tumor-bearing mice by 137% (P < 0.001). In contrast, the administration of fish oil diet to either normal or tumor-bearing mice resulted in a reductior in plasma PGE2 concentrations. The fish oil diet reduced th>; plasma PGE2 level of normal mice by 41% (P < 0.01) and c f tumor-bearing mice by 31% (P< 0.02). Effect of Fish Oil Diet on Growth of Subcutaneous and Melastatic Lung Tumors. The sizes of the primary s.c. tumors and the number of lung nodules of mice which were placed on either a fish oil d et or retained on a mixed-fat rodent chow diet were measured. There were no differences in the sizes of the primary tumors of mice which were fed the mixed-fat rodent chow versus the fish oil diet from the time of tumor appearance. In contrast, mice fed t le fish oil diet had approximately a 4-fold greater number of lung nodules than did mice fed the rodent chow (P < 0.001). The median number of lung nodules of mice fed the rodent chow diet was 10 with a range of 6 to 20 nodules/mouse. The median number of lung nodules of mice fed the fish oil diet was 3Õ1 with a range of 34 to 58 nodules/mouse. CELLS probably due to a decrease in the amount of PGE2 produced by splenic macrophages. In fact, at Day 17 after tumor implanta tion, not only was the splenic suppressor activity abolished, but the amount of PGE2 secreted by spleen cells of tumor bearers fed a fish oil diet was also reduced. In contrast to fish oil's abolishment of splenic suppressor DISCUSSION activity of mice bearing small tumors, the fish oil diet stimulated the suppressive activity of bone marrow cells. The fish oil diet also caused a stimulation of myelopoiesis in normal and tumorbearing mice. Since PGE2 is known to be a negative regulator of myelopoiesis (1, 2, 15), this stimulation of bone marrow suppressive activity in LLC-C3 tumor bearers was attributed to a PGE2-reducing effect of fish oil in mice bearing this CSFsecreting tumor (30) allowing for additional stimulation of myelopoiesis and the appearance of the associated bone mar row-derived suppressor cells. This was indirectly supported by previous studies showing that the reduction in PGE2 levels in normal mice by indomethacin administration resulted in mye lopoietic stimulation (47-49). The effects of the corn oil diet in mice bearing large tumors, when immune suppression was associated with myelopoiesis and the appearance of bone marrow-derived suppressor cells, were opposite of the effects of fish oil. Corn oil, being rich in the «-6polyunsaturated fat precursors for prostaglandins, in creased the levels of PGE2 and subsequently moderated mye lopoiesis. The depression of myelopoiesis in mice bearing large tumors was the probable explanation for the parallel diminish ment in the presence of bone marrow-derived suppressor cells. This is in agreement with our recent studies showing that injection of a stable analogue of PGE2 into mice bearing large LLC-C3 tumors reduced myelopoiesis and the associated sup pressor cell activity (50). In contrast to corn oil's diminishment Immune suppression in mice with progressively growing LLC-C3 tumors is mediated by two sequentially appearing immune sjppressor mechanisms. In mice bearing small or medium sized tumors, immune suppression is mediated by PGE2-secrîtingadherent macrophages (27, 42). When tumors become la'ge, the macrophage production of PGE2 declines, and the adherent macrophages are no longer immune suppressive. Instead, the development of large metastatic LLC-C3 tumors is accompanied by myelopoietic stimulation and the appearanc; of bone marrow-derived suppressor cells, first in the bone narrow and then in the spleen, resembling immature cells of the monocyte lineage (30). In the present study, we determine i how myelopoiesis, immune competence, and these immune suppressor mechanisms were modulated in normal and tumor-bearing mice by a corn oil diet, which is rich in n-6 polyunsattirated fat precursors, or a fish oil diet, which is rich in the n-3 polyunsaturated EPA. The diets were all composed of 5% fat )ut differed in the source of fat which was used. The adriinistration of a corn oil diet to normal mice reduced Con A blnstogenesis and stimulated the immune suppressive activity of their spleen cells. This is in agreement with studies by others showing that increased levels of dietary n-6 polyun saturated ats resulted in increased splenic PGE2 production (6, 17). The immune suppressive activities of PGE2 are well known (10-14, 45, 46). The corn oil diet had no effect on the T-cell blastogenic response of mice bearing small tumors, when PGE2 secretion n spleen cells was already elevated and suppression was medi ited by PGE2-producing macrophages. Substitution of fish oil for the fat in the diet increased Con A blastogenesis by spleen cells of normal mice and restored the blastogenic response ay spleen cells of mice bearing small tumors. The restoration in immune responsiveness by a fish oil diet was of bone marrow suppressor activity of mice bearing large tu mors, the corn oil diet had no effect on either splenic T-cell blastogenesis or on splenic suppressor activity. This could have been due to a shift in suppressor mechanisms from myelopoiesis-associated suppressor cells to PGE2-secreting suppres sor cells. However, studies to evaluate this possibility have not yet been conducted. A variety of studies have been conducted to assess the effects of corn oil or fish oil diets on the establishment and subsequent growth of transplanted tumors in mice (17, 23, 24). Such studies have generally shown that a corn oil diet increases the capacity of a tumor to become established and then to grow, while a fish oil diet decreases the capacity of a tumor to become established and to grow. In contrast to these studies, we assessed the effects of a fish oil versus a mixed-fat rodent chow diet on the growth and metastasis of tumors which had already become established. Switching mice to a fish oil diet from the time of appearance of a palpable s.c. tumor had no effect on the size of the primary tumor. However, the formation of lung nodules was increased in mice which had been switched to a fish oil diet. The fish oil stimulation of tumor metastasis is consistent with the fish oil stimulation of myelopoiesis-associated immune suppressor cells in tumor bearers. In conclusion, the results of this study showed that, when fed to normal mice or to tumor-bearing mice, fish oil diets were myelopoiesis stimulatory, while corn oil diets were myelopoiesis suppressive. In addition, this study showed the capacities of these diets to modulate the two immune suppressor mecha nisms which develop during progressive growth of LLC-C3 tumors in mice. The fish oil diet alleviated the splenic suppres sor activity when it was mediated by the production of PGE2. However, when immune suppression was associated with mye- 1935 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research. FATS, MYELOPOIESIS, AND SUPPRESSOR CELLS lopoiesis-associated suppressor cells, a fish oil diet exacerbated the extent of bone marrow suppressor activity, while a corn oil diet alleviated the bone marrow suppressor cell activity. REFERENCES 1. Pelus, L. M., (Miniami. O. G., and Nocka, K. H. Synergistic inhibition of human marrow granulocyte-macrophage progenitor cells by prostaglandin E and recombinant ¡nterferon-alpha,-beta, and -gamma and an effect mediated by tumor necrosis factor. J. Immunol., 140:479-484, 1988. 2. Gentile, P. S., and Pelus, L. M. In vivo modulation of myelopoiesis by prostaglandin E¡.II. Inhibition of granulocyte-monocyte progenitor cell (CFU-GM) cell-cycle rate. Exp. Hematol., IS: 119-126, 1987. 3. Dinarello, C. A. Biology of interleukin 1. FASEB J., 2: 108-115, 1988. 4. Vogel, S. N., Douches, S. D., Kaufman, E. N., and Neta, R. Induction of colony stimulating factor in vivo by recombinant interleukin 1-alpha and recombinant tumor necrosis factor-alpha. J. Immunol., 138: 2143-2148, 1987. 5. Santoli. D., Yang, Y.-C., Clark, S. C., Kreider, B. L., Caracciolo, D., and Rovera, G. Synergistic and antagonistic effects of recombinant human inter leukin (IL) 3, IL-alpha. granulocyte and macrophage colony-stimulating factors (G-CSF and M-CSF) on the growth of GM-CSF-dependent leukemic cell lines. J. Immunol., 139: 3348-3354, 1987. 6. Chen, L., Suzuki, Y., and Wheelock, E. F. Interferon-gamma synergizes with tumor necrosis factor and with interleukin 1 and requires the presence of both monokines to induce antitumor cytotoxic activity in macrophages. J. Immunol., 139:4096-4101, 1987. 7. Morrissey, P. J., Bressler, L., Charrier, K., and Alpert, A. Response of resident murine peritoneal macrophages to in vivo administration of granu locyte-macrophage colony-stimulating factor. J. Immunol., 140:1910-1915, 1988. 8. Papiernik. M., Lepault, F., and Pontoux, C. Synergistic effect of colonystimulating factors and IL-2 on prothymocyte proliferation linked to the maturation of macrophage/dendritic cells within L3T4" Lyt-2" la" Mac" cells. J. Immunol., 140: 1431-1434, 1988. 9. Broxmeyer, H. E., Cooper, S., Rubin, B. Y., and Taylor, M. W. The Synergistic influence of human interferon-gamma and interferon-alpha on suppression of hematopoietic progenitor cells is additive with the enhanced sensitivity of these cells to inhibition by interferons at low oxygen tension in vitro. J. Immunol.. 135: 2502-2506, 1985. 10. Goodwin, J. S., Bankhurst, A. I ).. and Messner, R. P. Suppression of human I ii-ll mitogenesis by prostaglandin. Existence of a prostaglandin-producing suppressor cell. J. Exp. Med., 146: 1719-1734, 1977. 11. Jordan, M. L., Hoffman. R. A., and Simmons, R. L. Further characterization of the subset-specific effects of prostaglandin E2 on T-lymphocyte clone function. Transplant. Proc., 19: 307-309, 1987. 12. Simkin, N. J., Jelinek, D. F., and Lipsky, P. E. Inhibition of human B-cell responsiveness by prostaglandin E¡.J. Immunol.. 138: 1074-1081, 1987. 13. Kunkel, S. L., Chensue, S. W., and Phan, S. H. Prostaglandins as endogenous mediators of interleukin 1 production. J. Immunol., 136: 186-192, 1986. 14. Tripp, C. S., Wyche, A., Unanue, E. R., and Needleman, P. The functional significance of the regulation of macrophage la expression by endogenous arachidonate metabolites in vitro. J. Immunol., 137: 3915-3920, 1986. 15. Lu, L., Pelus, L. M., and Broxmeyer, H. E. Modulation of the expression of HLA-DR (la) antigens and the proliferation of human erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells by prostaglandin E. Exp. Hematol., 12: 741-748, 1984. 16. Marshall. L. A., and Johnston, P. V. The influence of dietary essential fatty acids on rat immunocompetent cell prostaglandin synthesis and mitogeninduced blastogenesis. J. Nutr., 115: 1572-1580. 1985. 17. Kollmorgen. G. M., King, M. M., Kosanke, S. D., and Do, C. Influence of dietary fat and indomethacin on the growth of transplantable mammary tumors in rats. Cancer Res., 43: 4714-4719, 1983. 18. Karmali, R. A., Doshi, R. U., Adams. L., and Choi, K. Effect of n-3 fatty acids on mammary tumorigenesis. Adv. Prostaglandin Thromboxane Leukotriene Res., 17: 886-889, 1987. 19. Payan, D. G., Wong, M. Y. S., Chernov-Rogan, T., Valone, F. H.. Picke«, F. H., Blake, F. H., Gold, W. M., and Goetzl, E. J. Alterations in human leukocyte function induced by ingestion of eicosapentaenoic acid. J. Clin. Immunol., 6: 402-410, 1986. 20. Kelley, V. E., Ferretti, A., Izui, S., and Strom, T. B. A fish oil diet rich in eicosapentaenoic acid reduces cyclooxygenase metabolites, and suppresses lupus in MRL-lpr mice. J. Immunol., 134: 1914-1919, 1985. 21. Yoshino, S., and Ellis, E. F. Effect of a fish-oil-supplemented diet on inflammation and immunological processes in rats. Int. Arch. Allergy Appi. Immunol., 84: 233-240, 1987. 22. Leslie, C. A., Gonnerman, W. A., and Cathcart, E. S. Gender differences in eicosanoid production from macrophages of arthritis-susceptible mice. J. Immunol.. 138: 413-416, 1987. 23. Gabor, H., and Abraham, S. Effect of dietary menhaden oil on tumor cell loss and the accumulation of mass of a transplantable mammary adenocar- cinoma in BALB/c mice. J. Nail. Cancer Inst., 76: 1223-1229, 1986. 24. Kort, W. J., Weijma, 1. M., Bijman, A. M., van Schalkwijk, W. P., Vergroesen. A. J., and Westbroek, D. L. Omega-3 fatty acids inhibiting the growth of a transplantable rat mammary adenocarcinoma. J. Nati. Cancer Inst., 79: 593-599, 1987. 25. Wanebo, H. J., Riley, T., Katz, D., Pace, R. C., Johns, M. E., and Cantrell, R. W. Indomethacin sensitive suppressor-cell activity in head and neck cancer patients. Cancer (Phila.), 61:462-474, 1988. 26. Fujii, T., Igarashi, T., and Kishimoto, S. Significance of suppressor macro phages for immunosurveillance of tumor-bearing mice. J. Nati. Cancer Inst., 78: 509-517, 1987. 27. Young, M. R., Wheeler, E., and Newby, M. Macrophage-mediated suppres sion of natural killer cell activity in mice bearing Lewis lung carcinoma. J. Nati. Cancer Inst., 76: 745-750, 1986. 28. Hoon, D. S. B., Bowker, R. J., and Cochran, A. J. Suppressor cell activity in melanoma-draining lymph nodes. Cancer Res., 47: 1529-1533, 1987. 29. Bear, H. D. Tumor-specific suppressor T-cells which inhibit the in vitro generation of cytolytic T-cells from immune and early tumor-bearing host spleens. Cancer Res.. 46: 1805-1812, 1986. 30. Young, M. R., Newby, M., and Wepsic, H. T. Hematopoiesis and suppressor bone marrow cells in mice bearing large metastatic Lewis lung carcinoma tumors. Cancer Res., 47: 100-105, 1987. 31. Young, M. R., Young, M. E., and Wepsic, H. T. The effect of recombinant murine ¡nlerferon-gamma on the hematopoietic and immunological param eters of mice bearing metastatic Lewis lung carcinoma tumors. Exp. Hema tol., 16: 295-301, 1988. 32. Tsuchiya, Y., Igarashi, M., Suzuki, R., and Kumagai, K. Production of colony-stimulating factor by tumor cells and the factor-mediated induction of suppressor cells. J. Immunol., 141: 699-708, 1988. 33. Devlin, J. J., Devlin, P. E., Myambo, K., Lilly, M. B., Rado, T. A., and Warren, M. K. Expression of granulocyte colony-stimulating factor by human cell lines. J. Leuk. Biol., 41: 302-306, 1987. 34. Hardy, C. L., and Balducci, L. Early hematopoietic events during tumor growth in mice. J. Nail. Cancer Inst., 76: 535-540, 1986. 35. Pessina, A., Neri, M. G., Muschiato, A., Brambilla, P., Marocchi, A., and Mocarelli, P. Colony-stimulating factor produced by murine adrenocortical tumor cells. J. Nati. Cancer Inst., 76: 1096-1099, 1986. 36. Schwartz, R., and Monner, D. A. Constitutive production of colony-stimu lating factor by mouse lymphoma cell lines is correlated with granulocytosis in vivo. Exp. Hematol., 14: 615-620, 1986. 37. Ishikawa, M., Hosokawa, M., Oh-hara, N., Niho, Y., and Kobayashi, H. Marked granulocytosis in C57BL/6 mice bearing transplanted BMT-11 fibrosarcoma. J. Nati. Cancer Inst., 78: 567-571, 1987. 38. Nicoletti, G., Brambilla, P., De Giovanni, C., Lollini, P.-L., Del Re, B., Marocchi, A., Mocarelli, P., Prodi, G., and Nanni, P. Colony-stimulating activity from the new metastatic TS/A cell line and its high- and lowmetastatic clonal derivatives. Br. J. Cancer, 52: 215-222, 1985. 39. Kovacs, C. J., Emma, D. A., Evans, M. J., Johnke, R. M., and Scarantino, C. W. Haemopoietic modulation in tumor-bearing animals: enhanced pro genitor-cell production in femoral marrow. Cell. Tissue Kind.. 18: 238-246, 1985. 40. Lee, M. Y., and Rosse, C. Depletion of lymphocyte subpopulations in primary and secondary lymphoid organs of mice by a transplanted granylocytosisinducing mammary carcinoma. Cancer Res., 42:1255-1260, 1982. 41. Young, M. R., Meunier, J., and Newby, M. Relationships between morphol ogy, dissemination, migration, and prostaglandin E2 secretion by cloned variants of Lewis lung carcinoma. Cancer Res., 45: 3918-3923, 1985. 42. Young, M. R., and Newby, M. Differential induction of suppressor macro phages by cloned Lewis lung carcinoma variants in mice. J. Nati. Cancer Inst., 77: 1255-1260, 1986. 43. Metcalf, D., Johnson, G. R., and Mandel, F. E. Colony formation in agar by multipotential hemopoietic cells. J. Cell. Physiol., 98:401-410, 1979. 44. Young, M. R., and Hoover, C. S. Inhibition of spleen cell cytotoxic capacity toward tumor by elevated prostaglandin E2 levels in mice bearing Lewis lung carcinoma. J. Nati. Cancer Inst., 77:425-429, 1986. 45. Goodwin, J. S. Immunological effects of nonsteroidal anti-inflammatory agents. Med. Clin. N. Am., 69: 793-804, 1985. 46. Janniger, C. K., and Racis, S. P. The arachidonic acid cascade: an inumino logically based review. J. Med., 18:69-80, 1987. 47. Nikcevich, D. A., Young, M. R., Ellis, N. K., Newby, M., and Wepsic, H. T. Stimulation of hematopoiesis in untreated and cyclophosphamide treated mice by the inhibition of prostaglandin synthesis. J. Immunopharmacol., A1.299-313, 1986. 48. Boorman, G. A., Luster, M. I., Dean, J. H., and Luebke, R. W. Effect of indomethacin on the bone marrow and immune system of the mouse. J. Clin. Lab. Immunol., 7: 119-126, 1982. 49. Fontagne, J., Adolphe, M., Semichon, M., Zizine, L., and Lechal, P. Effect of in vivo treatment with indomethacin on mouse granulocyte-macrophage colony-forming cells in culture (<T'l ', ). Possible role of prostaglandins. Exp. Hematol., 8:1157-1164, 1980. 50. Young, M. R., Young, M. E., and Kim, K. Regulation of tumor-induced myelopoiesis and lung the associated suppressor cells in miceRes., bearing metastatic Lewis carcinomasimmune by prostaglandin !'.> Cancer 48: 6826-6831,1988. 1936 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research. Effects of Fish Oil and Corn Oil Diets on Prostaglandin-dependent and Myelopoiesis-associated Immune Suppressor Mechanisms of Mice Bearing Metastatic Lewis Lung Carcinoma Tumors M. Rita I. Young and Melvin E. Young Cancer Res 1989;49:1931-1936. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/49/8/1931 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research.