Bioclmni,'aetB,,~phr.*icaActu. I(FIS(19rH)I87 1'~5 ,() ]ggl ElsevierNciencei'nlnlishe~ n.v All ri!,hts rese~ed m67 48N'~./()1/$0350
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Tumor necrosis factor production by murinc resident peritoneal macrophages is enhanced by dietary n-3 polyunsaturated fatty acids Ingibj6rg H a r d a r ~ o t t i r i a n d J o h n E. Kinse!la ~'-' i Lipid~ Research Laborawry. Suwking Hall ('orm'/I U,Uter, it~. Ithaca. ,'~Y ( U .;'A ~ and ( ?,llek~"o f Agrlculture" and Em'ir~ment,d S¢'ie,we~. Mr,d. Hull. U,,i, ,'r,#v ' 4 c'ahfi,r,t,,. D,,, t,. (~.t rU.S A )
IRcceivcd 28 March it)t)ll
Keywords: Tumor necrosisfactor: a-3
Fatty
acid: Pt,tVunsaturatedfatty:mid;Prostaglandin:Dietaryfat
Tumor necrosis factor (TNF) is a macrophage deriveO peptide that has an antitumor attion and modulates immune and inflammatory reactions. Dietary fatty acids may modulate TNF production as dietary n-3 polyunsaturated fatty acids suppress human monocyte TNF production, but enhantx its secretion by routine peritoneal macrughages. Mice were maintained for 5 weeks on diets containing different amourl~s of n-3 and n-6 fatty acids. TNF, PGE z and 6-hero PGF|a production was monitored following in vitro stimulation of resident i~ritoneal macrophages with lipopolysaceharide. Maerophagcs from mice fed the high n - 3 diet produced g-fold more TNF and half the PGE 2 produced hy maeraphages from mice on the other diets. Indomethacin caused an increase in the TNF production by macrophages from mice on all diets but maeraphages from mice on the high n - 3 di¢4 produced mare TNF than maerophages from mice on the other diets. Exogenous PGE z II00 nM) greatly decreased TNF production by macraphages from mice on all diets, hut maerophages from mice on the high n-3 diet secreted 70% more TNF than maerophages ilrom mice fed the other diets, Mdicatlng that PGE z is only partly responsible for the effects observed. The resulis show that feeding n-3 polyunsaturated fatty acids may cause enhanced TNF production by resident peritoneal maerophages and that PGE 2 is partly responsible for the effect.
Tumor necrosis factor (TNF)/cachectin is a macrophage derived cytokine with diverse biological activities [1,2]. It induces hemorrhagic necrosis of certain tumors [3,4] and exerts antiproliferative effects on several tumor cell lines [5]. TNF may be responsible for the blockade of the lipoprotein lipase in adipocytes [6,7] as well as for certain symptoms of shock [8] and eaehexia [9]. TNF has a protective effect in murine salmonellosis [10] and enhances resistance to bacterial infection in mice ill.12]. TNF may also play a benefi-
Abbreviations: TNF, tumor necrosis [actor, PO, prostaglandin; LT, leukotrlen¢; PUFA,polyunsaturated fattyacid;LPS.lipopolysaccharide; IFN-y, interferon g~Jmma. Correspondence: J.E. Kinsella.Collegeo[ Agricultureand Environmental Sciences, Mrak Hall. Universityof California. Davis. CA 9561S, [J.$.A.
cial role in angiogenesis [13], peritonitis [14] and wound healing [15]. When TNF is present at low levels in tissues the beneficial effects may predominate to mediate enhanced host defense against pathogens and modulate normal tissue remodeling. On the other hand, acute systemic release of TNF causes septic shock and tissue injury [16]. Dietary fatty acids may affect the production of TNF, and eicosanoids have been suggested as mediafurs of this effect. Prostaglandin (PG) Ee causes a dose-dependent suppression of TNF release from lipopoh,,sar,'baride (LPS)-stimnlated macrophages [17], whereas leukotriene (LT) B4, enhances TNF production by human monocytes [18,19]. Eicos~moid synthesis is affected by the amount and type of dietary fat and n-3 polyunsaturated fatty acids (PUFA) decrease synthesis of both PG and LT by peritoneal macrophages [20-23]. Decreasing both PGE z and LTB., production of macrophages by dietary n-3 PUFA could either increase TNF production by decreasing the inhibitory
efti:ct of P G E 2 0 l decrease it by decreasing the enhancing effect of LTB 4. Dietary. n - 3 P U F A may el,her enhance or decrease T N F production in diffcrent systems. Peripheral-blood mononuclear cells, from healthy humans and patients w~th active rheumatoid arthritis, produced significantly less T N F and IL-1, upon LPS stimulation, following fisn oil consum,,tion [24,25]. Kupffer cells isolated from rats consumi.-lg either fish oil or safflower oil secreted less T N F and IL-I than Kupffer cells from rats consuming c o r n oil [26] and supernatants of peritoneal exudatc cells isolated from diabetic mice on fish oil diet had reduced IL-I like activity compared to that from mice o . chow or cOCnllUI o[! 0i~-t [27]. In contrast, a previous study from this laboratory showed that murine peritoneal macrophages from mice consuming menhaden oil produce more TNF, following LPS stimulation, than macrophages tram mice consuming corn oil [2811 In order to clarify the disparity between these results, the presem study was condue.ted using diets with more moderate n - 3 and n - 6 fatty acid concentrations than the previous study and control diets for both P U F A content and type of fatty acid ( n - 3 versus n - 6 ) . The present study furthermore concentrates on the role that PGE2 may play in mediating the dietaB, effect.
Animals Female BALB-C mice, weighing 18-20 g, were purchased from Charles River Laboratory (Wilmington, MAt. They were maintained in cages (six mice per cage) in a room with 12 h light/dark cycle and a temperature of 2506. Following 1 week maintenance on Prolab diet (Agway, Syracuse, NY), the mice were placed on a fat free diet (1CN Bioehemicals, Cleveland, O H ) for 1 week. Yhe mice (12 per group) were then fed one of four experimental diets for 5 weeks. Diets a n d study design Aft diets contained 10 wt% fat. The basal linoleie acid (18:201-6)) content of all diets, supplied by safflower oil (75% linoleic acid) (Hollywood Foods, Los Angele:,, CA), was maintained at 1.5 wt%. The n - 3 P U F A were supplied by sardine oil (Nippon Suisan Kaisha, Tokyo, Japan) containing 5.4% 18:4(n-31, 27.1% eieosapentaenoic acid (20:5(n-311, 2.5% docosapentaenoic acid (22: 5(n-3)), 12.0% doeosahexaenoic acid ( 2 2 : 6 ( n - 3 ) ) and minimal amounts of 1 8 : 2 ( n - 6 ) (1.v8%). The filter Oils consisted of equal amounts of high oleic sunflower oil (85% 18:11 (SVO, Eastlake, O H ) and tripalmitin (99% 16:0) (Sigma, St. Louis, MO). Diet A, the high n - 3 diet, contained 1.5 wt% n 3 fn~ty acids and diet B, the low n - 3 diet, 0.15 wt% n - 3
i ABLE i Fatty acid cv,nposiaon vf the ~rper;memat ,gets (mo1%1 Diets: A: 1.5 wl% n-3+1.5 ~lq; n-6; B: 015 wl'7~ ,i-3+15 wt% ,t-6: C: 1.5 wtql n-6. D: 3 ~1~ n-6. n.d.: hal delectable levels of Ihe particular fatty acid. Folly acid ta:o lfi:fl 1[~:1 16:2 16:3 16:4 17:0 Ig:l 18:2tn 6) 18:3OI-31 i.o.:.,~ ~) 20:1 20:4(n-6) 20:4(n-31 20:5(11-3) 21:5(n-3) 22 : I 22:5(n-31 22:61n-31 n-3/n-b ~ PIIFA+ 18:2(n-61 h
A B C D high , .~ low, 3 low n 6 high n-6 2x,.~ a.ss a.lo n.d. 14.45 19,17 15.54 14.33 4.71 0.SO ll.d. n.d. I.~ n.a. n.d. n.d. 0.82 n.d. tl.d. m.d. 2.53 0.1D fl.d. n.d. 2.87 4.03 n.d. n.d. 25.83 51.32 55.62 -'1.73 20.42 21.82 28.7a 43.94 0.48 n.d. n.d. n.d. 2.0~ 0.!7 ,,.a. n.d. 0.41 a.d. n.d. n.d. 0.21 a.d. n.d. a.O. 0.90 ~.d. lad n.d. 13.14 139 n.th n.d. 0.26 n.d. n.d. n.d. 0.53 n.d. ,.d. n.d. 0.07 n.d. n.d. n.d. 5.89 0.62 n.d. ll.d. Ida O.tO 44.14 21.82 28.74 43.94
n-3/n-6: ):lS:Stn-3t; IS:4(n-3); 20:4(n-3); 20:5in-S); 2t :Stn - 3~. 22:5tn-3. 22:6(n-3/18: 2(/r-6h h PUFA+LA: Vla:2(n-6); tS:3{n-3); 18:4(n-3g 20:4(n-3); 20:5(n-3):22:51n 3):22:6(n 3).
fatty acids (Table 1). Diet C, the low n - 6 diet, contained 1.5 wt% n - 6 fatty acids and diet D, the high n - 6 diet, 3 wt% n - 6 fatty acids, pLIFA + 1 8 : 2 ( n - 6 ) content of diets B and C were similar, 21.8 and 28.7 tool%, respectively, as well as P U F A + 1 8 : 2 ( n - 6 ) contents of diets A and D, 44.l and 43.9 real%, respectively (Table 11. The diets were designed to control for both total P U F A content and tile type of P U F A ( n - 3 versus n-6). The remainder of the diet was composed of vitamin free casein (187 g/kg), alphacel (147 g/kg), sucrose (520 g/kg), salt mixture U S P X I V (36 g / k g ) and ICN vitamin mix with supplemental choline chlorides (5 g/kg). Diets were prepared in bulk and daily portions packed in whirl.pak bags, flushed with nitrogen, sealed and stored at 4°C. W a t e r and food were provided ad libitum and fresh diets were provided daily to minimize exposure to air prior to consumption. U n e a t e n food was discarded and the feedcups were washed daily. Isolation and activation o f macrophages The mice were killed by diethy[ ether inhalation and the peritoneal cells collected in phosphate-buffered saline (PBS) without calcium or magnesium containing less than 0.1 ng endotoxin/ml, as determined by the
limulus amebocyte lysate assay (Sigma. St. Louis. MO). The port,ordeal cells were washed twice with PBS nnd suspended in KC 211O0(Hazelton Biologics, Lcnexm KA), a serum ]::dependent medit,~l. Cells were counted on a hemacytometer and differential counts ~ere obtained following staining with Diff-Ouik Stain Set (Baxter, McGaw Park. IL). The peritoneal cell c,mcentration was adjusted to 1.10 b cells/ml and [).5 ml plated per wall ili 24 ~,eil plates (Corning, Coming, NY). After 2 h incubation, at 37~C and 5% CO,, nonadhcrent co! ~ were re,upped by ~piration, ctmnted ( ~ 20% of the total cell number) and discarded. The adherent cells were washed twice with PBS. Cell viability, assessed by Trypan blue exclusion was greater tha;a 95%. The adherent cell cultures were incubated in 0.5 mL KC-20~f~ ".kb c~r ~.it'.~t LF3 (2 ,ag /¢,d) (E. colt 055 : 195. Calbiochem, La Jolla, CA). indomethacin (0.1 ~ M ) (Sigma, St. Louis, MO). interferon-z, (IFN-3,) (100 U / m l ) (Gcnzyme, Cambridge, MA), and PGE a (100 nM) (Calbiochem, La Jolla, CA). After 6 h incubation the supernatants were removed and analyzed for PQ and TNF prodvction.
TNF assay Aliquots of the supernatants were frozen and stored at -70°C until anaWysis of TNF by an enzyme-linked immunosorbent assay were conducted. Th,e hamster anti-murine TNF monoclonal antibody, the rabbit aoti-murine TNF polyelonal antibody and the recombinant mouse TNF-a, used as a standard, were all obtained from Genz3,me Corporation (Cambridge, MA). The anti-rabbit lgG alkaline phosphatase and the paranitrophenyl phosphate, used as a substrate for the alkaline phosphatase, were obtained from Sigma (St. Louis, MO).
Pelleted peritoneal cells (2. 10n) were resuspended ill O.8 ml saline. The fluids were extracted with chlorofofnl/lnelhantd {i : 2. v/v). followed by chloroform/ saline (1:I. v/vk toIIowed by one part chk)roform (twice) [301. l h e pooled chloroform extracts were cvaporated to dl3:ncss under nitrogen and the lipids rcdis~oIved ~n 25 ~1 chh)rotk~rm. The phospholipids were separated from the neutral lipids by thin-layer chromatography a~ing a chloroform/methanol ( 8 : L ,~/v) solvent sV~lem and vist~alized with (llV/e 8-hydroxyl 3,fi-pyrene-trisullbnic acid trisodium salt (Eastman Kodak, Rochester, N'f) in methanol. The phospholipids were recovered from thin-layer chromatography plates by scraping the appropriate bands and re~t,spendcd iv toluene. They were then saponified with 0 5 M KOH in methanol for 8 min at 86°C. Following acidification, with 0.7 M HC~ in methanol, fatty acids were extracteu ~vlt~ equal volumes of hexane (twice), evaporated under nitrogen and methylated with ethereal diazomethane. Fo!lowing evaporation, the fatty. acid methyl esters were resuspended in bexane and analyzed by gas chromatography on a DB23 capillary column (0.25 mm x 30 m) (J&W Chromatography, Folsom, OH) with hydrogen as the carrier gas. Fatty acid composition of the diets was determined by using the method described above without separating phospholipids and neutral Iipids on a TLC plate.
Statistical ana(vsis The results are expressed as means + S.E. The results were evaluatcd by Factorial Analysis of Variance and differences determined significant by Fisher's Proteeted Least Significant Difference at P < 0.05.
MiFe growth and cell coums Eicosanoid determinations Portions (2.00 ttl) of the supernatants were acidified with acetic acid and extrat~ted three times with t ml ethyl acetate [29]. The pooled extract was evaporated to dryness under nitrogen and the PG resusgended in PBS containing 0.1% gelatin. Each sample was analyzed by radioimmunoassay for PGE z and 6-keto POFla. Antiserum was obtained f~om Advanced Magnetics (Cambridge, MA), tritiated PGE.~ and 6-ketoPGFt, from Du Pont (Wilmington, DE) and PGE, and 6-keto-PGFi, standards from Cayman Chemica] (Ann ,,M'bor, M]). The eross-reactivty of the PGE 2 antiserum with PGEs was determined to be 26% at half ma;amum binding (data not shown).
Fatty acid separation and analysis Livers were per/used with ce!d saline (0.9%), containing EDTA (0.1%), removed from the animal and a small portion ( ~ 100 mg) homogenized in 0.8 ml saline.
There was no difference in the body weights or relative weight gains of mice on the different diets over the 5 weeks feeding period. There was no difference in the numbers or relative proportions of cells from the peritoneum of mice on different diets.
Liver and peritoneal cell lipid composition Fatty acid composition of ohosphollpids from livers and resident peritoneal cells differed between diets (Tables II and liD. Livers from mice on the two n-3 diets had significantly higher concentrations of n-3 PUFA and significantly lower concentration of n-6 fatty acids in phospholipids than livers from mice on the two n-6 diets. The n - 3 / n - 6 PUFA ratio of phospholipids in livers from mice on the high n-3 diet was significantly higher (3.44) than the n - 3 / n - 6 PUFA ratio pf livers from mice on the low n-3 ibet (0.88) which was significantly higher than in li,,ers from mice on the two n-6 diets (0.19 and 0.18).
190 TABLE II
7~1elath' acid composition f,no/~l o.¢phospholipldz ertracred from l,icersof mice maimained on ext~rimemal t~lel.~ Mice were fed experimental diets for 5 weeks, livers removed and extracted as described in Methods. Diets: A: 1.5 wl% n -3 + 1,5 ~ t % n -6; B: 0.15 ~ t ~ n 3 + 1.5 wt% ,,-6; C: 1.5 wt% n-6; D: 3 w1% n-6. Data represent mean ± S.E. for 12 mice. Means with the same letter within the ~ m e ,'n~ are not significantly different al P < 0.05, Ftshers Protected Least Significant Difference. n.d.: not detectable levels of the particular I'ally acid.
........
h,~h~o 3
LO3
~. . . .
~. . . . .
16:0 16:1 18:0 18:1 18:2(n 6) 20:1 20;2tn 6)+20:3(n 91 20:2(n-61 20:44n-6) 20:5(,, -3) 22:4(n -6) 22:5(n-6) 22:5( n -3) 22:fi(n 3) n - 3 / n - 6 PUFA t PUFA 2
22.63±0,38 u I 19±0.04 ~ 16.78 ± 0,74 ~ 10.10 ± 0.24 a 11.23±0.96 a~ n.d. a 0.04±0.03 ~ 1.34 ±0.03 a 7.24±0.12 " 9.06 ± 0.29 o n.6. a n.d. ~ I.,16 .L0.03 ~ 18.93±0.21 a 3.44±0.08 a 38,04±0.47 ~
20.82±0.50 b 1.07±0.09 a 15.89 ±0.48 ~ 14.85 ± 0.47 t' 11.65±0.95 ab O.04.[:0.03 a 0.81±0.06 b 2.34±0.07 ~' 16.16±0.27 b 1,11 ± 0.07 b n.d. ~ 0.05±0.03" 0.29 ± 0.05 u 14.92±0.21 0.88_+0.02 b 34.87±0.29 b
18.88 ±0.37 ~ 1.27 ±0,04 ~ 16,81 ±0.41 ° 17.46 ±0,31 c 10.36 ±0.22 a 0.26 _+l~q4 ~ 1,041 ±0.05 e 2.06 ±0.07 : 22,36 ±0.25 ¢ n.d. ¢ 0.39 ±0.04 b 3.70 +0.20 b n.d. b 5.42 ±0.17 c 0.19 ±0.01 ~ 33.93 ~0,29 ~
19.06±0.46 c 0.86 ±0.04 ~ 17.353_0.90 ~ 13.20 ± 0.31 d 12.02±0.30 b 0.14±0.04 ¢ 0.54±0.02 a 1.60 ±C.95 ~ 24.72+0.44 a n.d. ¢ 0.53 ± 0.01 c 4,51 ±0.12 c nd. h 5.46±0.14 c 0.18±0.01 c 36.82±0.57 ~
i n - 3 / n - 6 : 2 2 0 : 5 ( n - 3 ) , 22:5(n-31, 22:6(n-31/'220:3(n-6), 20:4(n-61, 22:4(n-61, 22:5ln-6)). PUFA: 2"20:31n-6), 20:41n -61, 20:51n-31, 22:4(n-61, 22:51n-6), 22:5(n -31, 22:6(n-311.
Peritoneal cells from mice on both n-3 diets also showed elevated concentrations of n-3 PUFA and
pholipids. The n-3/n-6 PUFA ratio in phospholipids of peritoneal cells from mice on the high n-3 diet was
diminished concentrations of n-6
s i g n i f i c a n t l y h i g h e r (1.21 t h a n i n p e r i t o n e a l
fatty acids in phos-
cells from
TABLE 111
The fatly acid composition ( ~ l % ) of phospholiplds ~ , ~ t e d from p e t i t ~ l cells of mice maintained on ~¢perimentat diets M i ~ were fed exl~erimental diets for 5 weeks, peritoneal calls r e m ~ e d and extracted as described in Methods, Diets: A: 1.5 wt% n - 3 + 1.5 wt% n -6; B: 0.15 wl% n -3 ÷ 13 wt% n -6; C: 1,5 wt% n -6; D: 3 va% n -6. Data represent mean ± S.E. for five to eight mice. Means with the same lever within the same haw are not significantly dilffcrent at P < 0.05, Fishers Prolectcd Least Signifieam Difference. n~d.: not detectable levels of the particular fatty acid. ........
A
16: 0 16:1 I8:0 18 : 1 18:21n-61 20:1 20:3(n 6) 20 : 41n -6) 20:5(n-31 22:41n-6) 22:5(n -6) 22:5(n -31 22:61r7-31
36.09 ± 0.83 ~ 1.97±0.05 s 27.19 ± 1.14 aD 14.59 + 0.40 " 6.02±0.29 ab 0.03 ±0.0~ a 1.72±0,23 ~ 6.78 ± 0.61 a 3.I0±0.29 a 0.13±0.10" 0.07 :c 0.07" 2.21 ±0.78 a 5,13 ± 0.38 a L20 ± 0.20 a 19.14 ± 1.22 a
gh n - 3
n-3/n-fPUFA 1 PUF;A2
B low n - 3
C
33.10 ± 1.82 ~b 1.60 ± 0.74 a 24.15 ± 1.60 ~ 15.92 ± 0.97 ~ 5 . l l ±0.54 ~ 0.20±0.10 a 1,66±0.36 a 10.29 :l: t ,38 b 1.22±0.22 b 1.21 ±0.24 ~u 0.04±0.04 a 0.82±0.38 .b 4.88±0.76 a 0.52±0.06 b 20A2~ 2.36 "
32.33 q- 1.52 ~b 1.39±0.69 ~ 22.91 ± 1.63 a~ 16.50± 1.34 ~ 5.31 ±0.51 a 0.15 ± 0.10 " L03.t:0.47 a 13,63 ± 1A7 b 0.56±0,33 b 1.96±0.98 a 1.02±0.39 h n.d. ~ 3.34+0.93 ~ 0.22±0.13 h 21-56± 2.31 a
w n-h
n - 3 / n - 6 : .v.20:51n-3), 22:51n 3), 22:6(n-3)/~20:3n6, 20:41n-6), 22:4(n-61, 22:5{n). z PUFA: ' r 2 0 : ~ n - 6 L 20:4(n-fi), 20:51n-3). 22:41n-6',. 22:51n -61, 22:51n-31, 22:61n 3)1.
D high n - 0
29.26 ± 0.50 b 2.26± 1.01 ~ 19.78 ±0.49 b 16.54+ 1.130 " 6.94:t:0.36 b 0.20±0.08 a 2.15±0.44 ~ 14.03 ± 1.56 b 1.08±0.45 b 2.76 ± 0.KI b 1.17± 0.26 b 0.10±0.116 I, 3.93 :k 1,00 a 0.25 ± 0.09 ~ 25.21 ± IA4 b
i --i
.
.
.
.
.
.
.
LPS LPS ~IM LP$ +IM*PGE2 LP$.IFN-g LP~*IFN-g.IM Fig. I. The effecl of diet on T N F produclion by resident peritoneal macrophages. Macrophages from mice on experimental diels were incubated wilh one or m o ~ of the following: LPS (2 ~ g / m l ) , ]FN-y (ITS) U / m l ) . iadomclhacin (IM) (0, I g M I and PGE 2 (1(~ nM), After ~ h the supernatanls were collected and T N F measured. Dietary groups: closed ba~. diet A (1.5% n - 3 + 1.5( b n - 6 ) ; hatched ba~. diet 19 (0,15% n 3 + 1.5% n -6); open bars, dJ¢l C ( 1.5% tl-O~ checkered o a ~ diet D {3% ~ 6), "¢alue~ are mean ~ S.E. for twelve mice. Bars with the same letters are not significantly ('fffcrcn t ( F < 0.[)5) for each treatment (Fishers's Protected Least Significanl Differc nee).
,so
G LPS
LP$*IM
LPS÷IM.PGS2
LP$*IFN-9
LPS~IFN-g*IU
Fig. 2. The effecl of diel on PGE~ production by resident peritoneal macmphngc~. Macrophagcs from mice on exgnrimental diets were incubated with one or more h i the following: LPS (2 ~ g / m l ) . | F N - 7 (100 U / r a P , indomethacin ( I M ) (0.1 # M ) and P G E 2 (100 aM). After 6 h the s u p e r n a t ~ t s were c o l l a t e d and PGE 2 extracted and measured. Dietary g ~ u p s : closed b a ~ . diet A (1.5% n - 3 + 1.5,% ~z-6); halched bars, diet 19 [{).15% I¢-3 + 1.5% n - 6 ) ; ripen bars. diet C (1.5% n - 6 ~ check¢~d bars. diet D (3% n -6). Values are mean ± S.F~ nor I2 m;ce. B a ~ g'ith the same l e u e r s at© not signff]camly different ( P < O.O5) for each Ir~atmenl (Fishcrs's Prolecled Least Significant Difference),
micc on the low n-3 diet (0.52). The n - 3 / t l 6 PUFA ratio of 0eritorteal ceils from mice on the low n-3 diet (0.52) was. however, not significanfiy different from that of peritoneal cells from mice on the two n - 6 diets (0.22 and 0.25 for the low and the high n-6 diets, respectively). The cg,~Cenl~t~o-~ of pi I F A in liver phospholipids wcrc higher in livers from mice on the high n-6 and the high n-3 diets (36.8 and 38.0 tool%) than in mice on the low n-6 and the low n-3 diets (33.9 and 34.9 tool%) reflecting the P U F A + 18:2(n 6) concentrations in the diets. The FUFA concentrations in the peritoneal cells did not refi=ct the PUFP, + 18:2(n-6) concentrations of the diel as thL3ywere higher in peritoneal cells from mice on the high n-6 diet (25.2 tool%) versus mice on the other diets, which had similar PUFA concentrations (19.1-21.6 mol%) (Table Ill).
LPS buhwed PG and TNF proJ.ction Macrophagcs from mice on the high n-3 diet when stimulated with LPS (2 ~g/mI) secreted 6 to 8-fold more TNF (27.7 ng/ml) than macrophagcs from mice on the other diets which produced slightly but not significantly different amounts of TNF (3.2-4.4 ng/ml) (Fig. 1). In the absence of LPS, the levels of TNF were below the limits of quantitation for all diets except for the high n-3 diet where the levels were 0.64 ng/ml, or 43-fold less than that with LPS, Maerophages from
mice on the high n-3 diet produced the least amount of PGE 2 (49 nM) and 6-keto PGF,. (56 nM) and significantly less than macrophages from mice on the other diets (Figs. 2 and 3). Macrophages from mice on the low n-3 diet produced intermediate amounts of PGE 2 {98.6 nM) ~nd 6-keto PGFI~ (265 nM) and significantly Iess than macrophages from mice on the low n-6 diet which had the highest PG production (134.5 nM PGE~ and 347.3 nM 6-keto-PGFl~).
LPS induced PC- and TNF production with indomethac#z and PGE. To examine the role PG play in regulating TNF production, indomethaein, a cyclooxygenase inhibitor, was added to the cultured mfierophages from mice on the experimental diets, lndomethacin by itself did not stimulate TNF production. The addition of indomcthacin (0.l /xM) to the cultured macrophages seconds prior to stimulation with LPS resulted in increased TNF production by macrophages from mice on all diets (Fig. It. The increase was smallest in macrophages from mice on the high n-3 diet (39%) but greater (3 to 5-fold) in macrophages from mice on the other three diets. Macrophages from mice on the high n-3 diet still secreted 2.3-fold more TNF than maerophages from mice on the other diets, i.e., 38.45 og/ml for maerophages from mice on the high n-3 diet versus 12.9-10.o ng/ml for macrophages from mice on the other diets,
{
LP$
LP$ ~IM
LPS÷IM,PGE2
LPa~IFN.E
LP$~IFN-g+tM
Fig. 3, The effect of diet on 6-keto-POF1. production by residenl poraonea] macrophagos. Macropbages front mice on ¢nperimcntal dicls were incubated with one or mare of zhe following: LPS (2 #ig/ml), IFN~¢ (|00 U/m[), indomctha¢in (]Mt (0.1 ~M), and P G E z (Ifi0riM). After 6 h
the supe[naE~ntswere collectedand 6-EcIo-PGFI. extracledand measured, Dietarygroups: closed bars. diet A (1.5% n-3 + 1.5%n-6); hatched ba,~, di~t8 (O,15% n 3+ 1.5%n-O};open ban, di=t C (1.5% n-6); checkered bars, dlel D (3% n-6). Values arc mean±5,E. of 12 mien, Bars with the s~melettersare not significantlydifferent (P < O.05)for each IreatmcnttFishers's Protected LcaslSignificantDifturencu).
lndomethacin decreased both PGE_, and 6-ketoPGFt, production by macrophages from mice on all diets (Figs. 2 and 3). The effect of PGE, on TNF production was exam ined by providing the cultured macrophages with exogenous PGE z at concentrations similar to the concentrations produced by LPS-stimulated macroplaages l¥om mice on the low n 6 diet and the low ~-3 diet (100 nM). PGE z by itself did not stimulate TNF production. Exogenous PGE, greatI~, decreased the TNF produc tion by macropl~ages from mice on all diets when added to cultures with indomethacin (0.1 ~tM) and LPS (2 ~tg/ml) (Fig. 1). Macrophages from mice on the high n-3 diet produced 11% of the TNF produced when incubated with LPS and indomethacin and 15~} of that. ~reduced when incubated with LPS only. Macrophagcs from mice on the other three d;ets when incubated with exogenous POE 2 secreted I6-26/% of the TNF secreted with LPS and indomethaein and 77-91% of that secreted with LPS only. Macrophages from mice on the high n-3 diet still secreted more TNF than macrophage from mice on the other diets after addition 01 exogenous FGE2. Even though the difference was not great, 4.2 ng/ml for macrophages from mice on the high n-3 diet versus 2.7-3.4 ng/ml for maerophage from mice on the other three diets, it was significant at P
Ig7
Tumor necrosis factor production by murinc resident peritoneal macrophages is enhanced by dietary n-3 polyunsaturated fatty acids Ingibj6rg H a r d a r ~ o t t i r i a n d J o h n E. Kinse!la ~'-' i Lipid~ Research Laborawry. Suwking Hall ('orm'/I U,Uter, it~. Ithaca. ,'~Y ( U .;'A ~ and ( ?,llek~"o f Agrlculture" and Em'ir~ment,d S¢'ie,we~. Mr,d. Hull. U,,i, ,'r,#v ' 4 c'ahfi,r,t,,. D,,, t,. (~.t rU.S A )
IRcceivcd 28 March it)t)ll
Keywords: Tumor necrosisfactor: a-3
Fatty
acid: Pt,tVunsaturatedfatty:mid;Prostaglandin:Dietaryfat
Tumor necrosis factor (TNF) is a macrophage deriveO peptide that has an antitumor attion and modulates immune and inflammatory reactions. Dietary fatty acids may modulate TNF production as dietary n-3 polyunsaturated fatty acids suppress human monocyte TNF production, but enhantx its secretion by routine peritoneal macrughages. Mice were maintained for 5 weeks on diets containing different amourl~s of n-3 and n-6 fatty acids. TNF, PGE z and 6-hero PGF|a production was monitored following in vitro stimulation of resident i~ritoneal macrophages with lipopolysaceharide. Maerophagcs from mice fed the high n - 3 diet produced g-fold more TNF and half the PGE 2 produced hy maeraphages from mice on the other diets. Indomethacin caused an increase in the TNF production by macrophages from mice on all diets but maeraphages from mice on the high n - 3 di¢4 produced mare TNF than maerophages from mice on the other diets. Exogenous PGE z II00 nM) greatly decreased TNF production by macraphages from mice on all diets, hut maerophages from mice on the high n-3 diet secreted 70% more TNF than maerophages ilrom mice fed the other diets, Mdicatlng that PGE z is only partly responsible for the effects observed. The resulis show that feeding n-3 polyunsaturated fatty acids may cause enhanced TNF production by resident peritoneal maerophages and that PGE 2 is partly responsible for the effect.
Tumor necrosis factor (TNF)/cachectin is a macrophage derived cytokine with diverse biological activities [1,2]. It induces hemorrhagic necrosis of certain tumors [3,4] and exerts antiproliferative effects on several tumor cell lines [5]. TNF may be responsible for the blockade of the lipoprotein lipase in adipocytes [6,7] as well as for certain symptoms of shock [8] and eaehexia [9]. TNF has a protective effect in murine salmonellosis [10] and enhances resistance to bacterial infection in mice ill.12]. TNF may also play a benefi-
Abbreviations: TNF, tumor necrosis [actor, PO, prostaglandin; LT, leukotrlen¢; PUFA,polyunsaturated fattyacid;LPS.lipopolysaccharide; IFN-y, interferon g~Jmma. Correspondence: J.E. Kinsella.Collegeo[ Agricultureand Environmental Sciences, Mrak Hall. Universityof California. Davis. CA 9561S, [J.$.A.
cial role in angiogenesis [13], peritonitis [14] and wound healing [15]. When TNF is present at low levels in tissues the beneficial effects may predominate to mediate enhanced host defense against pathogens and modulate normal tissue remodeling. On the other hand, acute systemic release of TNF causes septic shock and tissue injury [16]. Dietary fatty acids may affect the production of TNF, and eicosanoids have been suggested as mediafurs of this effect. Prostaglandin (PG) Ee causes a dose-dependent suppression of TNF release from lipopoh,,sar,'baride (LPS)-stimnlated macrophages [17], whereas leukotriene (LT) B4, enhances TNF production by human monocytes [18,19]. Eicos~moid synthesis is affected by the amount and type of dietary fat and n-3 polyunsaturated fatty acids (PUFA) decrease synthesis of both PG and LT by peritoneal macrophages [20-23]. Decreasing both PGE z and LTB., production of macrophages by dietary n-3 PUFA could either increase TNF production by decreasing the inhibitory
efti:ct of P G E 2 0 l decrease it by decreasing the enhancing effect of LTB 4. Dietary. n - 3 P U F A may el,her enhance or decrease T N F production in diffcrent systems. Peripheral-blood mononuclear cells, from healthy humans and patients w~th active rheumatoid arthritis, produced significantly less T N F and IL-1, upon LPS stimulation, following fisn oil consum,,tion [24,25]. Kupffer cells isolated from rats consumi.-lg either fish oil or safflower oil secreted less T N F and IL-I than Kupffer cells from rats consuming c o r n oil [26] and supernatants of peritoneal exudatc cells isolated from diabetic mice on fish oil diet had reduced IL-I like activity compared to that from mice o . chow or cOCnllUI o[! 0i~-t [27]. In contrast, a previous study from this laboratory showed that murine peritoneal macrophages from mice consuming menhaden oil produce more TNF, following LPS stimulation, than macrophages tram mice consuming corn oil [2811 In order to clarify the disparity between these results, the presem study was condue.ted using diets with more moderate n - 3 and n - 6 fatty acid concentrations than the previous study and control diets for both P U F A content and type of fatty acid ( n - 3 versus n - 6 ) . The present study furthermore concentrates on the role that PGE2 may play in mediating the dietaB, effect.
Animals Female BALB-C mice, weighing 18-20 g, were purchased from Charles River Laboratory (Wilmington, MAt. They were maintained in cages (six mice per cage) in a room with 12 h light/dark cycle and a temperature of 2506. Following 1 week maintenance on Prolab diet (Agway, Syracuse, NY), the mice were placed on a fat free diet (1CN Bioehemicals, Cleveland, O H ) for 1 week. Yhe mice (12 per group) were then fed one of four experimental diets for 5 weeks. Diets a n d study design Aft diets contained 10 wt% fat. The basal linoleie acid (18:201-6)) content of all diets, supplied by safflower oil (75% linoleic acid) (Hollywood Foods, Los Angele:,, CA), was maintained at 1.5 wt%. The n - 3 P U F A were supplied by sardine oil (Nippon Suisan Kaisha, Tokyo, Japan) containing 5.4% 18:4(n-31, 27.1% eieosapentaenoic acid (20:5(n-311, 2.5% docosapentaenoic acid (22: 5(n-3)), 12.0% doeosahexaenoic acid ( 2 2 : 6 ( n - 3 ) ) and minimal amounts of 1 8 : 2 ( n - 6 ) (1.v8%). The filter Oils consisted of equal amounts of high oleic sunflower oil (85% 18:11 (SVO, Eastlake, O H ) and tripalmitin (99% 16:0) (Sigma, St. Louis, MO). Diet A, the high n - 3 diet, contained 1.5 wt% n 3 fn~ty acids and diet B, the low n - 3 diet, 0.15 wt% n - 3
i ABLE i Fatty acid cv,nposiaon vf the ~rper;memat ,gets (mo1%1 Diets: A: 1.5 wl% n-3+1.5 ~lq; n-6; B: 015 wl'7~ ,i-3+15 wt% ,t-6: C: 1.5 wtql n-6. D: 3 ~1~ n-6. n.d.: hal delectable levels of Ihe particular fatty acid. Folly acid ta:o lfi:fl 1[~:1 16:2 16:3 16:4 17:0 Ig:l 18:2tn 6) 18:3OI-31 i.o.:.,~ ~) 20:1 20:4(n-6) 20:4(n-31 20:5(11-3) 21:5(n-3) 22 : I 22:5(n-31 22:61n-31 n-3/n-b ~ PIIFA+ 18:2(n-61 h
A B C D high , .~ low, 3 low n 6 high n-6 2x,.~ a.ss a.lo n.d. 14.45 19,17 15.54 14.33 4.71 0.SO ll.d. n.d. I.~ n.a. n.d. n.d. 0.82 n.d. tl.d. m.d. 2.53 0.1D fl.d. n.d. 2.87 4.03 n.d. n.d. 25.83 51.32 55.62 -'1.73 20.42 21.82 28.7a 43.94 0.48 n.d. n.d. n.d. 2.0~ 0.!7 ,,.a. n.d. 0.41 a.d. n.d. n.d. 0.21 a.d. n.d. a.O. 0.90 ~.d. lad n.d. 13.14 139 n.th n.d. 0.26 n.d. n.d. n.d. 0.53 n.d. ,.d. n.d. 0.07 n.d. n.d. n.d. 5.89 0.62 n.d. ll.d. Ida O.tO 44.14 21.82 28.74 43.94
n-3/n-6: ):lS:Stn-3t; IS:4(n-3); 20:4(n-3); 20:5in-S); 2t :Stn - 3~. 22:5tn-3. 22:6(n-3/18: 2(/r-6h h PUFA+LA: Vla:2(n-6); tS:3{n-3); 18:4(n-3g 20:4(n-3); 20:5(n-3):22:51n 3):22:6(n 3).
fatty acids (Table 1). Diet C, the low n - 6 diet, contained 1.5 wt% n - 6 fatty acids and diet D, the high n - 6 diet, 3 wt% n - 6 fatty acids, pLIFA + 1 8 : 2 ( n - 6 ) content of diets B and C were similar, 21.8 and 28.7 tool%, respectively, as well as P U F A + 1 8 : 2 ( n - 6 ) contents of diets A and D, 44.l and 43.9 real%, respectively (Table 11. The diets were designed to control for both total P U F A content and tile type of P U F A ( n - 3 versus n-6). The remainder of the diet was composed of vitamin free casein (187 g/kg), alphacel (147 g/kg), sucrose (520 g/kg), salt mixture U S P X I V (36 g / k g ) and ICN vitamin mix with supplemental choline chlorides (5 g/kg). Diets were prepared in bulk and daily portions packed in whirl.pak bags, flushed with nitrogen, sealed and stored at 4°C. W a t e r and food were provided ad libitum and fresh diets were provided daily to minimize exposure to air prior to consumption. U n e a t e n food was discarded and the feedcups were washed daily. Isolation and activation o f macrophages The mice were killed by diethy[ ether inhalation and the peritoneal cells collected in phosphate-buffered saline (PBS) without calcium or magnesium containing less than 0.1 ng endotoxin/ml, as determined by the
limulus amebocyte lysate assay (Sigma. St. Louis. MO). The port,ordeal cells were washed twice with PBS nnd suspended in KC 211O0(Hazelton Biologics, Lcnexm KA), a serum ]::dependent medit,~l. Cells were counted on a hemacytometer and differential counts ~ere obtained following staining with Diff-Ouik Stain Set (Baxter, McGaw Park. IL). The peritoneal cell c,mcentration was adjusted to 1.10 b cells/ml and [).5 ml plated per wall ili 24 ~,eil plates (Corning, Coming, NY). After 2 h incubation, at 37~C and 5% CO,, nonadhcrent co! ~ were re,upped by ~piration, ctmnted ( ~ 20% of the total cell number) and discarded. The adherent cells were washed twice with PBS. Cell viability, assessed by Trypan blue exclusion was greater tha;a 95%. The adherent cell cultures were incubated in 0.5 mL KC-20~f~ ".kb c~r ~.it'.~t LF3 (2 ,ag /¢,d) (E. colt 055 : 195. Calbiochem, La Jolla, CA). indomethacin (0.1 ~ M ) (Sigma, St. Louis, MO). interferon-z, (IFN-3,) (100 U / m l ) (Gcnzyme, Cambridge, MA), and PGE a (100 nM) (Calbiochem, La Jolla, CA). After 6 h incubation the supernatants were removed and analyzed for PQ and TNF prodvction.
TNF assay Aliquots of the supernatants were frozen and stored at -70°C until anaWysis of TNF by an enzyme-linked immunosorbent assay were conducted. Th,e hamster anti-murine TNF monoclonal antibody, the rabbit aoti-murine TNF polyelonal antibody and the recombinant mouse TNF-a, used as a standard, were all obtained from Genz3,me Corporation (Cambridge, MA). The anti-rabbit lgG alkaline phosphatase and the paranitrophenyl phosphate, used as a substrate for the alkaline phosphatase, were obtained from Sigma (St. Louis, MO).
Pelleted peritoneal cells (2. 10n) were resuspended ill O.8 ml saline. The fluids were extracted with chlorofofnl/lnelhantd {i : 2. v/v). followed by chloroform/ saline (1:I. v/vk toIIowed by one part chk)roform (twice) [301. l h e pooled chloroform extracts were cvaporated to dl3:ncss under nitrogen and the lipids rcdis~oIved ~n 25 ~1 chh)rotk~rm. The phospholipids were separated from the neutral lipids by thin-layer chromatography a~ing a chloroform/methanol ( 8 : L ,~/v) solvent sV~lem and vist~alized with (llV/e 8-hydroxyl 3,fi-pyrene-trisullbnic acid trisodium salt (Eastman Kodak, Rochester, N'f) in methanol. The phospholipids were recovered from thin-layer chromatography plates by scraping the appropriate bands and re~t,spendcd iv toluene. They were then saponified with 0 5 M KOH in methanol for 8 min at 86°C. Following acidification, with 0.7 M HC~ in methanol, fatty acids were extracteu ~vlt~ equal volumes of hexane (twice), evaporated under nitrogen and methylated with ethereal diazomethane. Fo!lowing evaporation, the fatty. acid methyl esters were resuspended in bexane and analyzed by gas chromatography on a DB23 capillary column (0.25 mm x 30 m) (J&W Chromatography, Folsom, OH) with hydrogen as the carrier gas. Fatty acid composition of the diets was determined by using the method described above without separating phospholipids and neutral Iipids on a TLC plate.
Statistical ana(vsis The results are expressed as means + S.E. The results were evaluatcd by Factorial Analysis of Variance and differences determined significant by Fisher's Proteeted Least Significant Difference at P < 0.05.
MiFe growth and cell coums Eicosanoid determinations Portions (2.00 ttl) of the supernatants were acidified with acetic acid and extrat~ted three times with t ml ethyl acetate [29]. The pooled extract was evaporated to dryness under nitrogen and the PG resusgended in PBS containing 0.1% gelatin. Each sample was analyzed by radioimmunoassay for PGE z and 6-keto POFla. Antiserum was obtained f~om Advanced Magnetics (Cambridge, MA), tritiated PGE.~ and 6-ketoPGFt, from Du Pont (Wilmington, DE) and PGE, and 6-keto-PGFi, standards from Cayman Chemica] (Ann ,,M'bor, M]). The eross-reactivty of the PGE 2 antiserum with PGEs was determined to be 26% at half ma;amum binding (data not shown).
Fatty acid separation and analysis Livers were per/used with ce!d saline (0.9%), containing EDTA (0.1%), removed from the animal and a small portion ( ~ 100 mg) homogenized in 0.8 ml saline.
There was no difference in the body weights or relative weight gains of mice on the different diets over the 5 weeks feeding period. There was no difference in the numbers or relative proportions of cells from the peritoneum of mice on different diets.
Liver and peritoneal cell lipid composition Fatty acid composition of ohosphollpids from livers and resident peritoneal cells differed between diets (Tables II and liD. Livers from mice on the two n-3 diets had significantly higher concentrations of n-3 PUFA and significantly lower concentration of n-6 fatty acids in phospholipids than livers from mice on the two n-6 diets. The n - 3 / n - 6 PUFA ratio of phospholipids in livers from mice on the high n-3 diet was significantly higher (3.44) than the n - 3 / n - 6 PUFA ratio pf livers from mice on the low n-3 ibet (0.88) which was significantly higher than in li,,ers from mice on the two n-6 diets (0.19 and 0.18).
190 TABLE II
7~1elath' acid composition f,no/~l o.¢phospholipldz ertracred from l,icersof mice maimained on ext~rimemal t~lel.~ Mice were fed experimental diets for 5 weeks, livers removed and extracted as described in Methods. Diets: A: 1.5 wl% n -3 + 1,5 ~ t % n -6; B: 0.15 ~ t ~ n 3 + 1.5 wt% ,,-6; C: 1.5 wt% n-6; D: 3 w1% n-6. Data represent mean ± S.E. for 12 mice. Means with the same letter within the ~ m e ,'n~ are not significantly different al P < 0.05, Ftshers Protected Least Significant Difference. n.d.: not detectable levels of the particular I'ally acid.
........
h,~h~o 3
LO3
~. . . .
~. . . . .
16:0 16:1 18:0 18:1 18:2(n 6) 20:1 20;2tn 6)+20:3(n 91 20:2(n-61 20:44n-6) 20:5(,, -3) 22:4(n -6) 22:5(n-6) 22:5( n -3) 22:fi(n 3) n - 3 / n - 6 PUFA t PUFA 2
22.63±0,38 u I 19±0.04 ~ 16.78 ± 0,74 ~ 10.10 ± 0.24 a 11.23±0.96 a~ n.d. a 0.04±0.03 ~ 1.34 ±0.03 a 7.24±0.12 " 9.06 ± 0.29 o n.6. a n.d. ~ I.,16 .L0.03 ~ 18.93±0.21 a 3.44±0.08 a 38,04±0.47 ~
20.82±0.50 b 1.07±0.09 a 15.89 ±0.48 ~ 14.85 ± 0.47 t' 11.65±0.95 ab O.04.[:0.03 a 0.81±0.06 b 2.34±0.07 ~' 16.16±0.27 b 1,11 ± 0.07 b n.d. ~ 0.05±0.03" 0.29 ± 0.05 u 14.92±0.21 0.88_+0.02 b 34.87±0.29 b
18.88 ±0.37 ~ 1.27 ±0,04 ~ 16,81 ±0.41 ° 17.46 ±0,31 c 10.36 ±0.22 a 0.26 _+l~q4 ~ 1,041 ±0.05 e 2.06 ±0.07 : 22,36 ±0.25 ¢ n.d. ¢ 0.39 ±0.04 b 3.70 +0.20 b n.d. b 5.42 ±0.17 c 0.19 ±0.01 ~ 33.93 ~0,29 ~
19.06±0.46 c 0.86 ±0.04 ~ 17.353_0.90 ~ 13.20 ± 0.31 d 12.02±0.30 b 0.14±0.04 ¢ 0.54±0.02 a 1.60 ±C.95 ~ 24.72+0.44 a n.d. ¢ 0.53 ± 0.01 c 4,51 ±0.12 c nd. h 5.46±0.14 c 0.18±0.01 c 36.82±0.57 ~
i n - 3 / n - 6 : 2 2 0 : 5 ( n - 3 ) , 22:5(n-31, 22:6(n-31/'220:3(n-6), 20:4(n-61, 22:4(n-61, 22:5ln-6)). PUFA: 2"20:31n-6), 20:41n -61, 20:51n-31, 22:4(n-61, 22:51n-6), 22:5(n -31, 22:6(n-311.
Peritoneal cells from mice on both n-3 diets also showed elevated concentrations of n-3 PUFA and
pholipids. The n-3/n-6 PUFA ratio in phospholipids of peritoneal cells from mice on the high n-3 diet was
diminished concentrations of n-6
s i g n i f i c a n t l y h i g h e r (1.21 t h a n i n p e r i t o n e a l
fatty acids in phos-
cells from
TABLE 111
The fatly acid composition ( ~ l % ) of phospholiplds ~ , ~ t e d from p e t i t ~ l cells of mice maintained on ~¢perimentat diets M i ~ were fed exl~erimental diets for 5 weeks, peritoneal calls r e m ~ e d and extracted as described in Methods, Diets: A: 1.5 wt% n - 3 + 1.5 wt% n -6; B: 0.15 wl% n -3 ÷ 13 wt% n -6; C: 1,5 wt% n -6; D: 3 va% n -6. Data represent mean ± S.E. for five to eight mice. Means with the same lever within the same haw are not significantly dilffcrent at P < 0.05, Fishers Prolectcd Least Signifieam Difference. n~d.: not detectable levels of the particular fatty acid. ........
A
16: 0 16:1 I8:0 18 : 1 18:21n-61 20:1 20:3(n 6) 20 : 41n -6) 20:5(n-31 22:41n-6) 22:5(n -6) 22:5(n -31 22:61r7-31
36.09 ± 0.83 ~ 1.97±0.05 s 27.19 ± 1.14 aD 14.59 + 0.40 " 6.02±0.29 ab 0.03 ±0.0~ a 1.72±0,23 ~ 6.78 ± 0.61 a 3.I0±0.29 a 0.13±0.10" 0.07 :c 0.07" 2.21 ±0.78 a 5,13 ± 0.38 a L20 ± 0.20 a 19.14 ± 1.22 a
gh n - 3
n-3/n-fPUFA 1 PUF;A2
B low n - 3
C
33.10 ± 1.82 ~b 1.60 ± 0.74 a 24.15 ± 1.60 ~ 15.92 ± 0.97 ~ 5 . l l ±0.54 ~ 0.20±0.10 a 1,66±0.36 a 10.29 :l: t ,38 b 1.22±0.22 b 1.21 ±0.24 ~u 0.04±0.04 a 0.82±0.38 .b 4.88±0.76 a 0.52±0.06 b 20A2~ 2.36 "
32.33 q- 1.52 ~b 1.39±0.69 ~ 22.91 ± 1.63 a~ 16.50± 1.34 ~ 5.31 ±0.51 a 0.15 ± 0.10 " L03.t:0.47 a 13,63 ± 1A7 b 0.56±0,33 b 1.96±0.98 a 1.02±0.39 h n.d. ~ 3.34+0.93 ~ 0.22±0.13 h 21-56± 2.31 a
w n-h
n - 3 / n - 6 : .v.20:51n-3), 22:51n 3), 22:6(n-3)/~20:3n6, 20:41n-6), 22:4(n-61, 22:5{n). z PUFA: ' r 2 0 : ~ n - 6 L 20:4(n-fi), 20:51n-3). 22:41n-6',. 22:51n -61, 22:51n-31, 22:61n 3)1.
D high n - 0
29.26 ± 0.50 b 2.26± 1.01 ~ 19.78 ±0.49 b 16.54+ 1.130 " 6.94:t:0.36 b 0.20±0.08 a 2.15±0.44 ~ 14.03 ± 1.56 b 1.08±0.45 b 2.76 ± 0.KI b 1.17± 0.26 b 0.10±0.116 I, 3.93 :k 1,00 a 0.25 ± 0.09 ~ 25.21 ± IA4 b
i --i
.
.
.
.
.
.
.
LPS LPS ~IM LP$ +IM*PGE2 LP$.IFN-g LP~*IFN-g.IM Fig. I. The effecl of diet on T N F produclion by resident peritoneal macrophages. Macrophages from mice on experimental diels were incubated wilh one or m o ~ of the following: LPS (2 ~ g / m l ) , ]FN-y (ITS) U / m l ) . iadomclhacin (IM) (0, I g M I and PGE 2 (1(~ nM), After ~ h the supernatanls were collected and T N F measured. Dietary groups: closed ba~. diet A (1.5% n - 3 + 1.5( b n - 6 ) ; hatched ba~. diet 19 (0,15% n 3 + 1.5% n -6); open bars, dJ¢l C ( 1.5% tl-O~ checkered o a ~ diet D {3% ~ 6), "¢alue~ are mean ~ S.E. for twelve mice. Bars with the same letters are not significantly ('fffcrcn t ( F < 0.[)5) for each treatment (Fishers's Protected Least Significanl Differc nee).
,so
G LPS
LP$*IM
LPS÷IM.PGS2
LP$*IFN-9
LPS~IFN-g*IU
Fig. 2. The effecl of diel on PGE~ production by resident peritoneal macmphngc~. Macrophagcs from mice on exgnrimental diets were incubated with one or more h i the following: LPS (2 ~ g / m l ) . | F N - 7 (100 U / r a P , indomethacin ( I M ) (0.1 # M ) and P G E 2 (100 aM). After 6 h the s u p e r n a t ~ t s were c o l l a t e d and PGE 2 extracted and measured. Dietary g ~ u p s : closed b a ~ . diet A (1.5% n - 3 + 1.5,% ~z-6); halched bars, diet 19 [{).15% I¢-3 + 1.5% n - 6 ) ; ripen bars. diet C (1.5% n - 6 ~ check¢~d bars. diet D (3% n -6). Values are mean ± S.F~ nor I2 m;ce. B a ~ g'ith the same l e u e r s at© not signff]camly different ( P < O.O5) for each Ir~atmenl (Fishcrs's Prolecled Least Significant Difference),
micc on the low n-3 diet (0.52). The n - 3 / t l 6 PUFA ratio of 0eritorteal ceils from mice on the low n-3 diet (0.52) was. however, not significanfiy different from that of peritoneal cells from mice on the two n - 6 diets (0.22 and 0.25 for the low and the high n-6 diets, respectively). The cg,~Cenl~t~o-~ of pi I F A in liver phospholipids wcrc higher in livers from mice on the high n-6 and the high n-3 diets (36.8 and 38.0 tool%) than in mice on the low n-6 and the low n-3 diets (33.9 and 34.9 tool%) reflecting the P U F A + 18:2(n 6) concentrations in the diets. The FUFA concentrations in the peritoneal cells did not refi=ct the PUFP, + 18:2(n-6) concentrations of the diel as thL3ywere higher in peritoneal cells from mice on the high n-6 diet (25.2 tool%) versus mice on the other diets, which had similar PUFA concentrations (19.1-21.6 mol%) (Table Ill).
LPS buhwed PG and TNF proJ.ction Macrophagcs from mice on the high n-3 diet when stimulated with LPS (2 ~g/mI) secreted 6 to 8-fold more TNF (27.7 ng/ml) than macrophagcs from mice on the other diets which produced slightly but not significantly different amounts of TNF (3.2-4.4 ng/ml) (Fig. 1). In the absence of LPS, the levels of TNF were below the limits of quantitation for all diets except for the high n-3 diet where the levels were 0.64 ng/ml, or 43-fold less than that with LPS, Maerophages from
mice on the high n-3 diet produced the least amount of PGE 2 (49 nM) and 6-keto PGF,. (56 nM) and significantly less than macrophages from mice on the other diets (Figs. 2 and 3). Macrophages from mice on the low n-3 diet produced intermediate amounts of PGE 2 {98.6 nM) ~nd 6-keto PGFI~ (265 nM) and significantly Iess than macrophages from mice on the low n-6 diet which had the highest PG production (134.5 nM PGE~ and 347.3 nM 6-keto-PGFl~).
LPS induced PC- and TNF production with indomethac#z and PGE. To examine the role PG play in regulating TNF production, indomethaein, a cyclooxygenase inhibitor, was added to the cultured mfierophages from mice on the experimental diets, lndomethacin by itself did not stimulate TNF production. The addition of indomcthacin (0.l /xM) to the cultured macrophages seconds prior to stimulation with LPS resulted in increased TNF production by macrophages from mice on all diets (Fig. It. The increase was smallest in macrophages from mice on the high n-3 diet (39%) but greater (3 to 5-fold) in macrophages from mice on the other three diets. Macrophages from mice on the high n-3 diet still secreted 2.3-fold more TNF than maerophages from mice on the other diets, i.e., 38.45 og/ml for maerophages from mice on the high n-3 diet versus 12.9-10.o ng/ml for macrophages from mice on the other diets,
{
LP$
LP$ ~IM
LPS÷IM,PGE2
LPa~IFN.E
LP$~IFN-g+tM
Fig. 3, The effect of diet on 6-keto-POF1. production by residenl poraonea] macrophagos. Macropbages front mice on ¢nperimcntal dicls were incubated with one or mare of zhe following: LPS (2 #ig/ml), IFN~¢ (|00 U/m[), indomctha¢in (]Mt (0.1 ~M), and P G E z (Ifi0riM). After 6 h
the supe[naE~ntswere collectedand 6-EcIo-PGFI. extracledand measured, Dietarygroups: closed bars. diet A (1.5% n-3 + 1.5%n-6); hatched ba,~, di~t8 (O,15% n 3+ 1.5%n-O};open ban, di=t C (1.5% n-6); checkered bars, dlel D (3% n-6). Values arc mean±5,E. of 12 mien, Bars with the s~melettersare not significantlydifferent (P < O.05)for each IreatmcnttFishers's Protected LcaslSignificantDifturencu).
lndomethacin decreased both PGE_, and 6-ketoPGFt, production by macrophages from mice on all diets (Figs. 2 and 3). The effect of PGE, on TNF production was exam ined by providing the cultured macrophages with exogenous PGE z at concentrations similar to the concentrations produced by LPS-stimulated macroplaages l¥om mice on the low n 6 diet and the low ~-3 diet (100 nM). PGE z by itself did not stimulate TNF production. Exogenous PGE, greatI~, decreased the TNF produc tion by macropl~ages from mice on all diets when added to cultures with indomethacin (0.1 ~tM) and LPS (2 ~tg/ml) (Fig. 1). Macrophages from mice on the high n-3 diet produced 11% of the TNF produced when incubated with LPS and indomethacin and 15~} of that. ~reduced when incubated with LPS only. Macrophagcs from mice on the other three d;ets when incubated with exogenous POE 2 secreted I6-26/% of the TNF secreted with LPS and indomethaein and 77-91% of that secreted with LPS only. Macrophages from mice on the high n-3 diet still secreted more TNF than macrophage from mice on the other diets after addition 01 exogenous FGE2. Even though the difference was not great, 4.2 ng/ml for macrophages from mice on the high n-3 diet versus 2.7-3.4 ng/ml for maerophage from mice on the other three diets, it was significant at P
