N-3 Polyunsaturated fatty acids and immune cell function

Advrm. Enzytne Regul.. Vol 37, pp. 197-237, 1991

Copyright0 1997ElsevlerScienceLtd Pnntedtn GreatBritain. All rightsreserved 0065-2571197W32

00

PII:SOO65-2571(96)00004-O

N-3 POLYUNSATURATED ACIDS AND IMMUNE FUNCTION

FATTY CELL

PHILIP C. CALDER School of Biological Sciences,University of Southampton. Bassett Crescent East, Southampton SO16 7PX, U.K. THE

ORIGIN

OF N-3 POLYUNSATURATED

FATTY

ACIDS

All mammals can synthesize fatty acids de nova from acetyl coenzyme A. The end product of the fatty acid synthetase enzyme (E.C. 2.3.1.8.5)is palmitic acid (16:0), which in turn can be elongated to stearic acid (18:O).There is little need for the synthesis of saturated fatty acids in Western man, since the diet normally supplies adequate amounts. However, cell membranes require unsaturated fatty acids to maintain their structure, fluidity and function. Therefore a mechanism for the introduction of double bonds (i.e. desaturation) exists. The introduction of a single double bond between carbon atoms 9 and 10 is catalyzed by the enzyme A9-desaturase(E.C. 1.14.99.5), which is universally present in both plants and animals. This enzyme results in the conversion of stearic acid to oleic acid (18:ln-9). Plants, unlike animals, can insert additional double bonds into oleic acid between the existing double bond at the 9-position and the methyl terminus of the carbon chain; a A12desaturase converts oleic acid into linoleic acid (18:2n-6) while a A15desaturase converts linoleic acid into or-linolenic acid (ALA; 18:3n-3). Since animal tissuesare unable to synthesize linoleic acid and ALA, these fatty acids must be consumed in the diet and so are termed essential fatty acids. Using the pathway outlined in Figure 1, animal cells can convert dietary ALA into eicosapentaenoic acid (EPA; 20:5n-3); by a similar series of reactions dietary linoleic acid is converted via y-linolenic (18:3n-6) and dihomo-y-linolenic (20:3n-6) acids to arachidonic acid (20:4n-6). The conversion of EPA and arachidonic acids to their longer-chain more unsaturated derivatives has been a source of much speculation over the years. Until relatively recently it was thought that a poorly defined A4-desaturasewas involved in this conversion. However, Voss et al. (1) demonstrated that in rat liver the conversion of EPA into docosahexaenoic acid (DHA; 22:6n-3) involves four steps: two elongations to yield 24:5n-3, a A6-desaturation and one round of P-oxidation (Fig. 1). The A6-desaturase (E.C. 1.14.99.25) is the same as that involved in the

198

P. C. CALDER

A15 lmole~cacid a-linolenic acid4 18:21x-6 + (plants) 18:3n-3

I

A12 (plants)

oleic acid lB:ln-9 A

stearic acid 18.0

A6

y-lmolenic acid I8:3n-6 I elongase 20:4n-3 A5

dihomo y-linolenic acid 20~311.6

1 A5

I eicosapentaenoicacid 20~511-3

arachidonic acid 20:4n-6

I

I

elongase

elongase

22:5n-3

22x4~6

I I

I I

elongase

elongase

24:5n-3

24:4n-6

A6

A6

24&t-3

Further desaturation and elongation

24:5n-6

I I b-oxidation

docosahexaenoic acid 22:6n-3

P-oxidation

22:5n-6

FIG. 1. Pathway of formation of PUFAs. A5, A6, A9, Al2 and Al5 indicate desaturase enzymes.

desaturation of linoleic acid and ALA (Fig. 1). The P-oxidation stepsinvolved in this pathway most likely take place in peroxisomes. An analogous pathway for conversion of arachidonic acid to docosapentaenoic acid (22:5n-6) probably also occurs (Fig. 1). Many marine plants, especially the unicellular algae in phytoplankton, also carry out chain elongation and further desaturation of ALA to yield the long-chain n-3 polyunsaturated fatty acids (PUFAs) EPA and DHA. It is the

LIPIDS AND THE IMMUNE

199

SYSTEM

formation of these long chain n-3 PUFAs by marine algae and their transfer through the food chain to fish that accounts for the abundance of the n-3 PUFAs in some marine fish oils (FO). The n-9, n-6 and n-3 families of PUFAs are not metabolically interconvertible in mammals. IMMUNOMODULATORY OF N-3

AND ANTI-INFLAMMATORY POLYUNSATURATED F.ATTY

EFFECTS ACIDS

In vitro Effects of N-3 PUFAs on Functions of Cells of the Immune System Lymphocyte functions. The in vitro effects of fatty acids upon lymphocyte proliferation have been studied since the early 1970’sand have been reviewed in detail elsewhere (2-6). However, apart from an early study of the effect of ALA on the proliferation of human peripheral blood lymphocytes (PBLs) (7). the effects of n-3 PUFAs upon lymphocyte functions were not investigated until relatively recently. These studies have been reviewed elsewhere(4-6) and are summarised in Table 1. A number of studies have shown that ALA, EPA and DHA inhibit the proliferation of lymphocytes isolated from rodent lymphoid tissues (lymph nodes, spleen,thymus) and from human peripheral blood (seeTable 1 for references); in these studies proliferation has been stimulated by a variety of agents including T-cell mitogens (concanavalin A (Con A), phytohaemagglutinin (PHA)), a B-cell mitogen (bacterial lipopolysaccharide (LPS)), a monoclonal antibody directed against CD3 and cytokines (interleukin (IL)-lcr, IL-2). Among the fatty acids texted, EPA appears to be the most inhibitory. Not only do n-3 PUFAs affect the response of lymphocytes to antigen (and to mitogens in experimental conditions), they may also affect the ability of antigen presenting cells to present antigen (29). The proliferation of lymphocytes and the regulation of the function of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, B-cells and macrophages depend upon the production of IL-2. ALA, EPA and DHA all suppressthe production of IL-2 by mitogen-stimulated rat or human lymphocytes (10, 14, 15, 2 1). Triacylglycerols (TAGS) containing n-3 PUFAs have been reported to directly inhibit NK cell activity (27, 28). In addition, ALA inhibits the degranulation of CTLs (26), and therefore presumably CTL activity, although this has not beendirectly tested. Other unsaturated fatty acids which inhibit CTL degranulation (26) inhibit CTL activity (30, 3 1). Monocyte and phugocyte functions. Although it has been known for some time that fatty acids can directly affect the functioning of phagocytic cells (e.g. (32) there are few studies of the effects of n-3 PUFAs on such functions. The proliferation of a monocytic cell line is inhibited by addition of FO to the cultures (33) and n-3 PUFAs have been shown to influence the production of cytokines by monocytic cell lines and human peripheral blood monocytes in vitro (34). Poulos et al. (35) reported that both EPA and DHA are potent

200

P. C. CALDER

TABLE I. EFFECTS OF N-3 PUFAS ON LYMPHOCYTE FUNCTIONS IN VITRO Stimulus

n-3 PUFAs tested

Human blood

PHA, Con A, PPD, anti-CD3, IL-2

Rat lymph node

LPS, Con A

Rat spleen Human synovial fluid Mouse thymus

Con A PHA + IL-2 PHA, IL-lfi, IL-2

ALA, EPA. DHA, n-3 PUFA rich emulsion ALA, EPA, DHA, tTri-ALA, n-3 PUFA-rich emulsion EPA, DHA EPA ALA, EPA

Human blood

Con A, PHA

ALA, EPA, DHA

IO, 14,

Rat lymph node

Con A

ALA, EPA, DHA

15 21

Allogeneic cells

ALA

26

Allogeneic cells

Tri-EPA, Tri-DHA

KLHJ

Tri-EPA

Cell origin

Effect

Ref.

Proliferation*

7716 16-23 13 24 25

IL-2 production

CTL degranulation

Human blood NK cell activity

Human blood

21,28

Antigen presentation

Mouse spleen

29

*In all casesproliferation was measured as thymidine incorporation. tTri-ALA, -EPA and -DHA indicate triacylglycerols with ALA, EPA or DHA at all three sn-positions JKLH indicates keyhole limpet haemocyanin; other abbreviations as in text.

activators of superoxide production by human neutrophils; DHA also enhanced the responses to two known neutrophil agonists, N-formyl+ methionyl-L-leucyl+phenylalanine (fMLP) and phorbol ester. In contrast to these observations, Chen et al. (36) showed that EPA and DHA significantly suppressed phorbol ester-stimulated superoxide generation by human neutrophils. DHA inhibited the interferon (IFN)-pstimulated tumouricidal action of murine peritoneal macrophages (M$) and of a M+ cell line towards a TNF-a-resistant, nitric oxide-sensitive target cell line (37). It appeared that this effect was due to inhibition of IFN-y-dependent signals. More recently, it has been shown that DHA inhibits IL-4- or IFN-y-induced cell surface expression of Ia (i.e. major histocompatability class (MHC) II antigens) on mouse peritoneal M$; DHA was more inhibitory than EPA and other 20-carbon fatty acids (38). DHA acted by inhibiting the increase in Ia mRNA after stimulation of M+ with IFN-y (38). These effects of DHA were not due to changesin the levels and types of eicosanoids produced (37, 38).

LIPIDS AND THE IMMUNE SYSTEM

20 1

Effects of Dietary N-3 PUFAs on ex vivo Functions of’ Cells of the Immune System Lymphocyte proliferation. Studies involving the feeding of diets rich in n-6 PUFAs, such as corn, soybean, safflower or sunflower oils, to laboratory animals have been reviewed several times (2. 4-6). In recent years there has been increased interest in the effects of n-3 PUFA-containing oils (e.g. canola oil, linseed oil (LO), FO) upon lymphocyte proliferation. Many studies have reported that feeding such oils to laboratory animals (rats, mice, rabbits, chickens) suppressesthe response of spleen lymphocytes to mitogenic stimuli including Con A, PHA and pokeweed mitogen (PWM) (seeTable 2 for references). Recently, we reported that feeding rats for lo-12 weeks on a diet containing 20”/0 FO results in markedly suppressed Con A- and PHAstimulated spleen, thymus, lymph node and PBL proliferation ex viva (40,42,48) (Fig. 2). Feeding the FO diet did not affect the proportions of T-, B-, CD4’- or CD8’-cells or M+ in the spleen, thymus, lymph nodes or blood of these rats (40,42, 48). However, FO feeding lowered the proportion of spleenlymphocytes bearing the IL-2 receptor (IL-2R), the proportion of thymic lymphocytes bearing the IL-2R and transferrin receptor and the proportion of lymph node lymphocytes bearing the transferrin receptor following Con A stimulation (40). Spleen lymphocytes from animals fed this diet also showed

n Lowfat El sam0w.3oil u

Con A

Mitogen

FO

PHA

FIG. 2. The effect of dietary lipids upon rat lymphocyte proliferation. Rats were fed for 10 weeks on a low fat diet or on diets containing 20% by weight of safflower oil or FO. The proliferation of mesenteric lymph node lymphocytes in responseto Con A or PHA was measured as thymidine incorporation and is expressed as a stimulation index. Data are mean + SEM from five animals fed on each diet and are taken from Ref. (40)

202

P. C. CALDER

TABLE 2. EFFECTS OF DIETARY N-3 PUFAS UPON LYMPHOCYTE FUNCTIONS TESTED EX VW0 Cell source

Details of diet used

Stimulus

Effect

Ref.

10% LO (3 weeks); 20% LO or FO (IO-12 weeks); 15% canola oil (8 weeks) 20% FO (8 weeks) 7.6% FO (20 weeks) 7.6% FO (20 weeks) 7% LO or FO (3 weeks) 20% FO (IO weeks) 20% FO (IO weeks) 20% FO (IO weeks) Encapsulated FO (up to 18 g/day) (6-24 weeks)

Con A. PHA

3943

Con A Con A, PHA Con A, PHA, LPS Con A, PWM

44,45 46 46 47

Con A, PHA Con A Sub-optimal Con A Con A, PHA, Anti-CD3

40 40 48 9,49-52

Proliferation *

Rat spleen

Mouse spleen Rabbit spleen Rabbit blood Chicken spleen Rat lymph node Rat thymus Rat blood Human blood NK cell activity

Mouse spleen Rat spleen

10% LO or FO (6-10 weeks); 18.6% FO (6 weeks) 20% LO or FO (IO-12 weeks)

Allogeneic cells

53-56

Allogeneic cells

42,43, 57

10% LO or FO (dams fed) 20% FO (10 weeks)

Allogeneic cells

56

Allogeneic cells

57

10% FO (8-10 weeks) 7% LO or FO (9 weeks)

Vaccinia virus

54

Sheep erythrocytes

58

KLH

29

LAK cell activity

Mouse spleen Rat spleen CTL activity

Mouse spleen Chicken spleen

Antigen presentation

Mouse spleen

2% EPA-ethyl ester (4-5 weeks)

*In all casesproliferation was measured as thymidine incorporation.

a 60% lower level of expression of the IL-2R following Con A stimulation (42). Studies of the effects of dietary n-3 PUFAs upon the functions of human PBLs are summarised in Table 2. Meydani et al. (49) reported the results of supplementing the diets of healthy young (22-33 years of age) or older (5168 years of age) women with encapsulated n-3 PUFAs (approximately 2.4 g/ day); the mitogenic response of PBLs to PHA was lowered after 12 weeks of

LIPIDS AND THE IMMUNE SYSTEM

203

supplementation in the older women. More recently, Meydani et al. (51) reported a decreasedresponse of PBLs to Con A and PHA following supplementation of the diet of volunteers on a low fat, low cholesterol diet with rz-3 PUFAs, while Endres er al. (52) found that 18 g of FOlday for 6 weeks resulted in lowered PHA-stimulated proliferation of PBLs 10 weeksafter supplementation had ended (but not at the end of the supplementation period). Meydani et al. (51) observed that consumption of the low fat, low cholesterol, n-3 PUFA-rich diet resulted in a lower proportion of CD4+ and a higher proportion of CD8+ PBLs; the proportion of CD3’ cells (i.e. T-cells) was unaffected. Soyland et al. (59) reported that supplementation of the diet of patients with psoriasis or atopic dermatitis with n-3 PUFA ethyl esters (6 g! day) caused a significant reduction in the percentage of IL-2’ blood lymphocytes following PHA stimulation; the level of expression of the IL-2R on the positive cells was also significantly reduced. Lymphocyte-mediated cytolysis. CTL activity has been reported to be significantly diminished following the feeding of n-3 PUFA-rich diets to mice or chickens (54, 58). Meydani et al. (53) found that feeding young mice a diet containing 10% FO for 6 weekscauseda decreasein spleenNK cell activity compared with feeding chow or corn oil; there were no differences in NK cell activity when these diets werefed to older mice. Feedingmice a diet containing 10%FO suppressedspleen NK cell activity compared with a 10%LO diet (54). In the study of Berger et al. (56) female mice were fed for 5 months on diets containing 10% olive oil, safflower oil, LO or FO and the spleenNK cell activity of the pups wasdetermined before they were weaned;the activity was lower in the FO group than in the safflower or olive oil groups. Recently, Yaqoob et al. (57) showedthat feeding 20% FO to weanling rats for 10 weekssignificantly decreasedspleenNK cell activity compared with feeding a low fat diet or high fat diets containing coconut, safflower or evening primrose oils (Table 3). No studies haveinvestigated the effect of dietary lipids on human NK cell activity, although intravenous injection of a TAG containing EPA into healthy human volunteers resulted in suppressionof peripheral blood NK cell activity 24 hr later (28). Lymphokine-activated killer (LAK) cells are generated by the culture of mitogen-stimulated lymphocytes with exogenous IL-2. The resulting activity is diminished if animals have been previously fed a FO-rich diet (56, 57). Production of reactive oxygen species and nitric oxide. The enzymeswhich result in the synthesis of superoxide, hydrogen peroxide and nitric oxide are regulatedby eicosanoids,cytokines and protein kinase C (PKC). Sincen-3 PUFAs affect the production of eicosanoids and cytokines (see below) and might modulate PKC activity (seebelow), they might affect the production of reactive oxygen speciesand nitric oxide by MQ and so regulate the cytotoxic activities of

204

P. C. CALDER

TABLE 3. THE EFFECT OF DIETARY LIPIDS UPON RAT SPLEEN LYMPHOCYTE NK CELL ACTIVITY % Cytolysis Diet Low fat Coconut oil Safflower oil FO

L:T...

100:1 46.5 2 40.4 + 42.1 + 29.4 f

2.0 2.4 1.5 2.1

5O:l 29.1 f 22.9 f 24.2 k 13.6 f

2.8 1.5 1.3 0.7

25:1 11.1+ 2.1 11.5+0.6 11.2 f 1.1 5.0? 0.3

Rats were fed for 10 weeks on a low fat diet or on diets containing 20% by weight of hydrogenated coconut oil, safflower oil or FO. The NK cell activity of spleen lymphocytes towards YAC-1 murine lymphoma cells was determined by chromium release at lymphocyte to target cell (L:T) ratios of 100:I, 50:1 and 25: 1. Data are mean f SEM from four animals fed on each diet and are taken from Ref. (57).

thesecells.However, investigations of the effectsof diets rich in n-3 PUFAs upon the production of hydrogen peroxide, superoxide and nitric oxide have yielded contradictory results (see Table 4 for references).Studies have reported that production of thesemediators is enhanced,diminished or not affected following FO feeding to laboratory animals (seeTable 4 for references).The reasonsfor such significantly different experimental observations might include the different speciesof origin of the cells studied (mouse,rat, pig, guinea pig, rabbit, man), the anatomical site of origin of the cells (liver, lung, peritoneal cavity, blood), the stateof cellular differentiation (monocyte, M4), the stateof activation of the cell (resident, inflammatory, activated), the stimulus used to elicit mediator production (seeTable 4 for a listing), the nature of the culture conditions used(presence or absenceof serum, serum source,time of culture etc.), the level of FO in the diet, the duration of feeding, the level of antioxidants in the diet and so on. In a recent study, thioglycollate-elicited peritoneal M$ from mice fed 20% FO for 8 weeksproduced more superoxideand hydrogenperoxide in responseto stimulation by PMA than M$ from mice fed a low fat diet (61). Furthermore, M$ from the FO-fed mice produced more nitric oxide in responseto stimulation with LPS than those from mice fed the low fat diet (61). Sincesuperoxide,hydrogenperoxide and nitric oxide are important Mederived cytotoxic agents these observations suggestthat dietary FO could affect the killing of microbial or tumour cells by Me. Nitric oxide appears to regulate lymphocyte functions and so FO-induced modulation of its generation from M+ could affect lymphocyte activity. Phagocytosis by macrophages.There are reports that dietary FO suppresses or does not affect phagocytosis of bacterial cells or particles by MQ of various origins (seeTable 4 for references). Macrophage-mediated cytolysis. Dietary FO has been reported to significantly suppress lysis of target tumour cells by mouse elicited peritoneal

205

LIPIDS AND THE IMMUNE SYSTEM TABLE 4. EFFECTS OF DIETARY N-3 PUFAS UPON PHAGOCYTIC CELL FUNCTIONS TESTED EX VW0 Cell type

Details of diet used

Stimulus

Effect

Ref.

Nitric oxide production

Mouse elicited peritoneal Mo Pig alveolar Mo Rat alveolar M$ Rat resident peritoneal M4 Mouse elicited peritoneal Mo

lOor20%FO (6-l 5 weeks) 10.5% FO (4 weeks) 9% FO ( 12 days) 8 Or loo/ FO (6 weeks) 10% LO or FO (3 weeks)

LPS, IFN-y,

60.61

LPS IFN-1 + LPS LPS

63 64. 65

IFN-y + LPS

66

3% FO (12 weeks)

fMLP, PMA

67

2.16 g EPA/day (7 weeks) 3.6 g EPA +2.4 g DHA/day (6 weeks) 5 g/kg per day FO by gastric tube (1 week) 10% LO; 10 or 20% FO (8-20 weeks) 8% FO (6 weeks)

fMLP, PAF

68

Latex particles

69

Zymosan

70

17% FO (4 weeks)

62

Superoxide production

Guinea pig elicited peritoneal neutrophils Human blood neutrophils Human blood monocytes Rabbit alveolar M$ Mouse elicited peritoneal Mg Rat resident peritoneal M$ Mouse Kupffer

PMA

56,61

PMA

64

Oral Salmonella

71

typhimurium Hydrogen peroxide production

Mouse elicited peritoneal MI+ Mouse elicited peritoneal Me Rat resident peritoneal M@ Mouse elicited peritoneal Mg

10% FO (4 weeks)

Zymosan

72

10% FO (4 weeks)

PMA

72

8% FO (6 weeks)

PMA

64

20% FO (8 weeks)

PMA

61

5 g/kg per day FO by gastric tube (1 week) 10% FO (4 weeks)

Staphylococcus aureus

70

Phagocytosis

Rabbit alveolar M$ Mouse elicited peritoneal MQ Pig alveolar MI+ Mouse Kupffer

10.5% FO (4 weeks) 17% FO (4 weeks)

Sheep erythrocytes, yeast Latex beads Oral Salmonella

None

72

None J

62 71

J.

60,66, 73,74

typhimurium Macrophage-mediated cytoiysis

Mouse elicited peritoneal MQ

10% FO (24 weeks)

Allogeneic cells + IFN-1 + LPS

206

P. C. CALDER

M$ (seeTable 4 for references). The target cell lines used in these studies are sensitive to killing by TNF-a (L929 cells; (60, 74) or nitric oxide (P815 cells; (66, 73). Thus, the suppressedM+mediated cytolysis observed after FO feeding is consistent with the decreasedproduction of nitric oxide (see Table 4) and TNF (seeTables 5 and 6) reported by some studies. MHC expression and antigen presentation. Inclusion of n-3 PUFAs in the diet of mice or rats results in a dimished percentageof peritoneal exudate cells bearing MHC II antigens on their surface (75-77). Huang et al. (77) also reported that the level of MHC II expression on positive cells was suppressed by FO feeding. Recently, it was found that feeding weanling rats a diet containing 20% FO for 12 weeks results in a decreasedlevel of MHC II expression on thioglycollate-elicited peritoneal M$ (78). In accordance with these animal studies, Hughes et al. (79) reported that supplementation of the diet of human volunteers with n-3 PUFAs for 3 weeks results in a decreasedlevel of MHC II (HLA-DP, -DQ and -DR) expression on the surfaceof peripheral blood monocytes. These observations suggest that diets rich in n-3 PUFAs will result in diminished antigen presentation. Indeed, Fujikawa et al. (29) found that feeding mice the ethyl ester of EPA for a period of 4-5 weeksresulted in diminished presentation of antigen (keyhole limpet haemocyanin; KLH) by spleen cells ex vivo.

Dendritic cells are the key antigen presenting cells in vivo. We have recently observed that feeding rats a diet containing 20% FO significantly diminishes MHC II expression on dendritic cells and ex vivo antigen (KLH) presentation by dendritic cells (obtained by cannulation of the thoracic duct) to KLHsensitised spleen lymphocytes (80) (Figure 3). Chemotaxis. Chemotaxis of blood neutrophils and monocytes towards a variety of chemoattractants including leukotriene (LT) B,, platelet activating factor (PAF), fMLP and autologous serum is suppressedfollowing the supplementation of the human diet with n-3 PUFAs (81-84). Effects of N-3 Pufas on Cellular Adhesion In vitro studies. That n-3 PUFAs can affect the expression of adhesion

molecules on the cell surface was shown by the recent study of De Caterina et al. (85) who reported that culture of human adult saphenous vein endothelial cells with DHA (but not EPA) significantly decreased the cytokineinduced expression of vascular cell adhesion molecule 1 (VCAM-I), E-selectin (also known as endothelial leukocyte adhesion molecule 1 or ELAM-I) and CD54 (also known as intercellular adhesion molecule 1 or ICAM-1) in a dose-dependent manner. The effects of DHA were independent of eicosanoid production (85). The adhesion of U937 monocytes and human peripheral blood monocytes to endothelial cells was diminished

207

LIPIDS AND THE IMMUNE SYSTEM TABLE 5. EFFECTS OF DIETARY N-3 PUFAS UPON EX VW0 CYTOKINE PRODUCTION BY RODENT AND PIG MACROPHAGES AND LYMPHOCYTES

species

Cell type

Details of diet used

Mouse

Kupffer Resident peritoneal W Elicited peritoneal M$I

Mouse

Elicited peritoneal MI$

Mouse

Elicited peritoneal M$

Rat

Rat

Resident peritoneal W Resident peritoneal W Elicited peritoneal M$

15% FO (6 weeks) 10% LO or FO (4-6 weeks) 10% LO or 20% FO (4 weeks) 10% LO or FO (3-5 weeks) 10% or 20% FO (415 weeks) 12.5% LO (4 weeks)

Rat Pig

Alveolar Mg Alveolar M@

Effect

Ref

t

90 91-94

t

95,96

TNFproduction

Rat Mouse

Rat

15% FO (6 weeks) 12.5% LO (4 weeks) or 10% FO (8 weeks) 9% FO (12 days) 10.5%LO or FO (4 weeks)

66. 92

1

60.61

t

91

1

65

None

97. 98

T

63 62

IL-1 production

Rat Mouse

Kupffer Resident peritoneal MQ Elicited peritoneal M+ Elicited peritoneal M$r Elicited peritoneal M$

15% FO (6 weeks) 10% FO (4 weeks)

t

90 91

10% FO (15 weeks) 20% FO (8 weeks) 10% FO (8 weeks)

1 None J.

60 61 98

Elicited peritoneal M(I Elicited peritoneal MO

20% FO (8 weeks) 10% FO (8 weeks)

t

61 98

Pig

Alveolar lymphocytes

5

62

Mouse Mouse

Spleen lymphocytes Spleen lymphocytes

10.5% LO or FO (4 weeks) 10% FO (6.5 months) 20% FO (8 weeks)

t None

99 45

Mouse Mouse Rat IL-6 production

Mouse Rat IL-2 production

IL-4 production

Mouse

Spleen lymphocytes

10 or 20% FO (8 weeks-65 months)

None

following incubation of the latter with DHA (85). The binding between monocytes and endothelial cells partially depends upon VCAM-1 expression on the endothelial cells; thus the reduced expression of VCAM-1 caused by DHA appears to have a functional effect. Recently, Kim et al. (86) reported that incubation of LPS-stimulated pig aortic endothelial ceils with

208

P. C. CALDER

TABLE 6. EFFECTS OF SUPPLEMENTATION OF THE HUMAN DIET WITH N-3 PUFAS UPON EX VW0 CYTOKINE PRODUCTION BY PERIPHERAL BLOOD MONONUCLEAR CELLS Details of supplementation used

Cytokine

Encapsulated n-3 PUFAs (2.7 g EPA +1.8 g DHAlday), 6 weeks

IL-la IL-l!3 TNF IL-1p IL-2 IL-6 TNF IL-1p TNF-a IL-1 IL-6 TNF-a IL-1p IL-6 TNF IL-1p IL-2 IFN-1 TNF-a IL-Q3 IL-2 TNF IL-6 IL-2 TNF TNF-a

Encapsulated n-3 PUFAs (2.4 g/day), 12 weeks Encapsulated FO (4 g/day), 7 weeks Encapsulated FO (4.5 g/day), 6-8 weeks Low fat, low cholesterol diet + n-3 PUFAs (I .23 g/day), 24 weeks FO (6 g/day), 24 weeks

FO concentrate (18 g/day), 6 weeks n-3 PUFA ethyl esters (6 g/day), 16 weeks Approx. 14 g/day ALA as LO and LO-based spreads and cooking oil Approx. 14 g/day ALA as LO and LO-based spreads and cooking oil + FO capsules (1.62 g EPA +l.OS g DHA/day)

Effect

Ref. 81

i 49 f f None None

50 100

f None 51 i 101 f f 52 i None None None J.

IL-1p

J

TNF-a

J.

IL-IS

1

59* 102 102

* Subjects were patients with psoriasis or atopic dermatitis.

EPA resulted in diminished binding between these cells and U937 monocytes (they did not investigate the effects of DHA). EPA was shown to reduce the expression of VCAM- 1, ELAM- 1 and ICAM- 1 on the surface of LPS-stimulated human umbilical vein endothelial cells (86). Calder et al. (87) reported that murine thoiglycollate-elicited peritoneal M$ cultured in the presence of EPA or DHA were less adherent to artificial surfaces (the adhesion to one of these surfaces is mediated by leukocyte function associated molecule-l or LFA-1) than those cultured with some other fatty acids; ALA was without effect. Twisk et al. (88) found that culture of murine spleen lymphocytes with ALA did not affect their ability to bind to high endothelial venules of either lymph nodes or Peyer’s patches; the effects of EPA and DHA were not examined. The expression of LFA-1 on the surface

209

LIPIDS AND THE IMMUNE SYSTEM

+

MO

-m-

Low rat

--t

salflower oil

3oooo-

2oooo-

lOOOO-

01 0

I 5000 Dendritic cell number

, 10000

FIG. 3. The effect of dietary lipids upon antigen presentation by rat dendritic cells. Mesenteric lymphadenectomised rats were fed for six weeks on a low fat diet or on diets containing 20?&by weight of safflower oil or FO. Lymphoid dendritic cells were purified from lymph collected by thoracic duct cannulation and were used as antigen presenting cells. The antigen used was KLH and the responder cells were spleen lymphocytes isolated from KLH-sensitized rats fed on standard laboratory chow. The antigen presentation assay was performed at different dendritic cell:responder cell ratios. Data are mean ?r SEM from six animals fed on each diet and are taken from Ref. (80).

of spleen T and B lymphocytes was unaffected by culture of the cells in the presence of ALA (88). Effects of dietary n-3 PUFAs. Feeding rats a diet containing 20% FO for lo-12 weeks caused a 30% reduction in the level of expression of CD18 (the B-chain of LFA-1) on thioglycollate-elicited peritoneal MQ,(78). Feeding rats this diet caused a 20 to 30% decrease in the level of expression of the adhesion molecules CD2 and CD 11a (the a-chain of LFA- 1) on resting spleen lymphocytes; similar effects were observed on both CD4” and CD8’ cells (42). The level of expression of the adhesion molecules CD2, ICAM- 1, LFA-1 on Con A-stimulated spleen lymphocytes was reduced by 25%, 33% and 20%, respectively, if the animals were fed a FO-rich diet (42). In addition, FO feeding reduced the level of expression of CD2, ICAM- and LFA-1 on the surface of lymphocytes obtained from the popliteal lymph nodes of rats undergoing a localised graft versus host (GvH) response (see below) by 20%, 12% and lo%, respectively (89). Following a localised host vs graft (HvG) response the level of expression of CD2 and LFA-1 on popliteal lymph node lymphocytes was reduced by 12% and lo%,

210

P. C. CALDER

respectively, if the animals had been fed on FO (89). In accordance with these observations we have recently observed that lymph node lymphocytes obtained from FO-fed rats adhere less well to M$ and endothelial cell monolayers than lymphocyte from rats fed some other diets (I? Sanderson and P. C. Calder, unpublished observations). Recently, it was shown that supplementation of the human diet with n-3 PUFAs results in significantly lower levels of expression of ICAM- and LFA-1 on peripheral blood monocytes (79). These observations suggest that FO feeding will affect the movement of lymphocytes and monocytes between body compartments and perhaps into sites of inflammatory or autoimmune activity. Effects of Dietary N-3 PUFAs on ex vivo Cytokine Production Macrophage-derivedcytokines. Sincecytokine production by M$ is regulated by eicosanoids and sincedietary lipids affect eicosanoid production (seebelow), it might be expectedthat dietary lipids, especially those containing n-3 PUFAs, will affect cytokine production. A number of studies have reported that feeding rodents n-3 PUFA-containing oils results in enhanced production of TNF ex vivo (see Table 5 for references), although there are reports of decreased production or no effect following FO feeding (seeTable 5 for references). A recent study showed that dietary FO increases IL-6 production by rat peritoneal Mg (98), while an earlier study reported increased IL-l production (91). Again however, there are contradictory reports (seeTable 5). The most likely reason for the variations in experimental observations are the differing protocols used: studies have differed with respect to speciesof origin of the cells studied (mouse, rat, pig), the anatomical site of origin of the cells (liver, lung, peritoneal cavity), the state of activation of the cell (resident, inflammatory, activated), the stimulus used to elicit cytokine production (LPS, calcium ionophore, another cytokine), the nature of the culture conditions used (presence or absenceof serum, serum source, time of culture etc.), the level of FO in the diet, the duration of feeding and the method used to quantitate cytokine concentrations. In a recent study in which all experimental procedures were standardised, it was found that feeding weanling mice a 20% FO diet for 8 weeks resulted in diminished ability of thioglycollate-elicited peritoneal M$ to produce TNF-a and IL-6 in response to LPS ex vivo; IL-la production was also decreasedby FO feeding but not significantly (61) (Figure 4). In agreement with some of the animal experiments (but in contrast to others), Endres et al. (52, Bl), Meydani et al. (49, 51) and others have found that supplementation of the human diet with n-3 PUFAs results in a significantly diminished ability of peripheral blood monocytes to produce TNF, IL-la, IL-lp and IL-6 ex vivo (see Table 6 for references; Figure 5). These studies have been reviewed in detail elsewhere(103, 104). These studies have focussed on the effects of the long chain n-3 PUFAs found in fish oil (i.e. EPA and

211

LIPIDS AND THE IMMUNE SYSTEM 14 12

TT

i

IL-1

IL-6 Cytokine

n Lowfat q Saltlower q FO

oil

TNF-a

FIG. 4. The effect of dietary lipids upon the production of cytokines by murine peritoneal macrophages. Mice were fed for 8 weeks on a low fat diet or on diets containing 200/oby weight of saffIower oil or FO. Thioglycollate-elicited peritoneal macrophages were prepared and cultured with LPS for 8 hr (TNF-a) or 24 hr (IL-la, IL-6). The medium was collected and cytakine concentrations were measured using enzyme-linked immunosorbant assays.IL-6 concentrations are expressedas rig/ml and IL-la and TNF-a concentrations are expressedas ng/lO ml. Data are mean f SEM from three animals fed on each diet and are taken from Ref. (61).

DHA). However, a recent study showed that increasing the level of ALA in the diet of healthy humans also results in a significant reduction in ex viva cytokine production (102). Lymphocyte-derivedcytokines. In contrast to the large number of studies of the effects of FO feeding upon the ex viva production of monocyte/M+derived cytokines (Tables 5 and 6), there have been few studies on lymphocytederived cytokines. Three studies have reported that supplementation of the diet of healthy human volunteers with n-3 PUFAs significantly lowers ex viva IL-2 production by PBLs (seeTable 6 for references;seealso 103 for a review). Gallai ef al. (101) also reported dimished ex vivo IFN-y production. One animal study has reported diminished ex vivo production of IL-2 (by pig alveolar lymphocytes) following LO and FO feeding (62). Fernandes et al. (99) found that feeding autoimmune disease-pronemice a 10%FO diet resulted in enhanced ex vivo IL-2 production by spleen lymphocytes in response to Con A; IL-4 production was unaffected. A recent study showed no significant effect of feeding 20% FO to weanling mice upon ex vivo production of IL-2 or IFN-y by Con A-stimulated spleen lymphocytes (45). Mitogen-stimulated spleen lymphocytes from mice fed FO produced less IL-4 and IL-10 than

P.C. CALDER 0.8

= E

0.6

Baseline

Baseline

3 months

3 months

3 months

BarelIne

3 months

FIG. 5. The effect of dietary supplementation with n-3 PUFAs upon cytokine production by human peripheral blood mononuclear cells. Healthy young (23-33 years of age) and older (5168 years of age) women supplemented their habitual diet with 1.68 g EPA and 0.72 g DHA per day for 3 months. PBLs were isolated and incubated with Con A (IL-6(b), IL-2 (e)) for 48 hr, LPS for 24 hr (IL-1B (a), TNF (c)) or heat-killed Staphylococcus epidemidis for 24 hr (TNF (d)). The medium was collected and cytokine concentrations measured using radioimmunoassays (IL-l& TNF, IL-6) or a bioassay (IL-2). Data are mean It SEM from six subjects in each group and are taken from Ref. (49).

those from mice fed a low fat diet, although this was not statistically significant (45). Effects of Dietary N-3 PUFAs on in vivo Measures of Inflammation and Ceil-mediated Immunity Acute inflammatory responses.Arachidonic acid-derived eicosanoids are involved in mediating inflammatory responses.Since n-3 PUFAs diminish the

LIPIDS AND THE IMMUNE

SYSTEM

213

production of thesemediators (seebelow), they should exert anti-inflammatory activities In fact, Yoshino and Ellis (105) found that administering rats EPA (500 mg/kg per day) and DHA (333 mg/kg per day) by a gastric tube for 50 days did not affect either antigen-induced inflammation of the air pouch or carrageenan-inducedinflammation of the footpad. This was&spite a significant reduction in the production of pro-inflammatory eicosanoids (prostaglandin (PG) E, and LTB,). Such dietary n-3 PUFA-induced changesin the pattern of inflammatory eicosanoid production were also reported in a different model of acute inflammation (intraperitoneal injection of zymosan) in rodents (106). That study also reported that dietary n-3 PUFA inhibited the influx of neutrophils into the peritoneal cavity which accompaniessuch treatment. In contrast to the findings of Yoshino and Ellis (105), Reddy and Lokesh ( 107)reported that feeding rats 10% FO for 10 weeks significantly lowered (by 40%) the inflammatory response to carrageenan injection into the footpad. In accordance with that observation, Nakamura et al. (108) showedthat feedingrats high fat diets containing 2OhEPA or DHA as their ethyl estersresulted in a 50%reduction in footpad swelling in response to carrageenan injection; both n-3 PUFAs were equally effective. In vivo response lo endotoxin. Mascioli et al. (109) showed that two 24 hr intravenous infusions of a 10% lipid emulsion rich in FO into guinea pigs significantly enhancedsurvival to intraperitoneally-injected LPS compared with infusion of a 10%safflower oil emulsion; the total amount of lipid infused was 13 g/animal. Guinea pig survival in the FO group was 10/l 1 at 9 hr postinjection and 7111at 96 hr post-injection. In comparison the survival rate at these times post-injection in the safflower oil group was 2/l 1. The sameworkers later showed that feeding a 14.5% FO diet to guinea pigs for 6 weeks significantly increased survival to an intraperitoneal injection of LPS (0.35 mg/lOOg body weight) compared with animals fed a 15% &Bower oil diet (110). Guinea pig survival 18 hr post-injection was 26/30 in the FD-fed group (compared with 16/30 in the &Bower oil-fed group) while at 96 hr post-injection it was 19130 (compared with 9/30). In accordance with the diminished susceptibility to the lethal effects of endotoxin in experimental animals, Mulrooney and Grimble (111) reported that feeding weanling rats a 10%FO diet for 8 weekssignificantly decreaseda number of responsesto intraperitoneal administration of TNF-a: the rises in liver zinc and plasma C3 concentrations, the fall in plasma albumin concentration and the increasesin liver, kidney and lung protein synthesisrates were all prevented by the FO diet. FO feeding also diminishes the pyrogenic ( 112)and anorexic effects(111, 113) of IL- 1 and TNF-c(. Delayed-type hypersensitivity. The delayed-type hypersensitivity (DTH) reaction is the result of a cell-mediated response to challenge with an antigen to which the individual has already been primed. There are reports that the DTH

214

P. C. CALDER

response is significantly reduced following the feeding of diets rich in FO to rodents (105, 114).Taki et al. (115) reported suppression of the DTH response to sheepred blood cells in mice following tail-vein injections of emulsions of TAGS rich in EPA or DHA. Both Kelley et al. (116) and Meydani et al. (51) reported that supplementation of the human diet with n-3 PUFAs diets diminished the DTH response to sevenrecall antigens. Antibody production. Studies which have investigated the effectsof diets rich in n-3 PUFAs upon antibody production in responseto antigen challenge have produced contradictory results. Prickett et al. (117) reported enhanced production of IgG and IgE to ovalbumin in rats fed 25% FO compared with those fed 25% tallow, while Fritsche et al. (47) showed that chickens fed FO produced a higher level of antibodies to sheepred blood cells than those fed lard or corn oil. In contrast, Kelley et al. (46) found no effect of feeding rabbits LO or FO on production of antibodies to albumin, while Prickett et al. (118) reported that dietary FO decreasedantibody production in rats. Recently,Atkinson and Maisey(119) reported that the production of anti-rat erythrocyte antibodies by mice injected with rat erythrocytes was significantly reduced if the mice were fed a 20% FO diet for 8 or 9 weeks.Virella et al. (120) reported that supplementation of the diet of healthy humans with 6 g FO per day for 6 weeks decreasedthe circulating levels of IgM and IgG following tetanus toxin challenge, but they usedonly a single subjectand a later study with more subjectsfound no significant effect of FO (9). Animal models of inflammatory and autoimmune diseases.Dietary FO has been shown to have significant beneficial clinical, immunological and biochemical effects in a number of animal diseasemodels including autoimmune glomerulonephritis, a model for systemic lupus erythematosus (75, 121-124; see (125) for a review), amyloidosis (126), collagen-induced arthritis (118, 127) and ulcerative colitis (128, 129). These observations suggest that diets enriched in n-3 PUFAs might be of some therapeutic benefit in these diseasesin man. Graft vs. host and host vs. graft responses.The so-called popliteal lymph node (PLN) assayprovides a useful experimental model in rodents for measuring GvH and HvG responseselicited by injection of allogeneic cells into the footpad of the host. The GvH response primarily involves the polyclonal activation, and subsequent proliferation, of host B-cells, although NK cells may also be involved in the host defence. In contrast, the HvG reaction is a T-cell mediated response,in which CTLs of the host recognise MHC antigens on the injected cells. In both casesthe enlargement in PLN size (more than 15-fold in the GvH response and four-fold in the HvG response (89)) is due largely to proliferation of activated host cells; most of these originate within

215

LIPIDS AND THE IMMUNE SYSTEM

the PLN although there is also somerecruitment of cells from the bloodstream. Using this assay, Mertin et al. (130) reported that both the GvH and HvG responses were suppressed following a single administration of a FO concentrate (750 mg/kg body weight) by oesophageal catheter to mice prior to, or immediately after, the inoculation with allogeneic ceils. Hinds and Sanders (131) showed a suppressed HvG response in mice fed a 16% FO diet compared with those fed a standard chow diet. Sanderson et al. (89) reported significantly diminished GvH and HvG responses (by 34% and 20%, respectively) in rats fed 20% FO compared with those fed a low fat diet or diets containing 20% by weight of coconut, olive, safflower or evening primrose oils (Fig. 6). Recently, we have observed that feeding rats a diet containing 20% LO also results in a diminished GvH response and that there is a gradual

Low fat

Safflower Diet

oil

Fish oil

1

-

Low fat

Safflower Diet

oil

Fish oil

FIG. 6. The effect of dietary lipids upon localised GvH and HvG reqonses in the rat. Rats were fed for 10w&s on a low fat diet or on diets containing 2C%by weight of safflower oil or FO. Localii GvH (a) or HvG (b) responseswere elicited by an injection of appropriate abgeneic cells into the footpad. Data are mean + SEM of IO-15 animals fed on each diet and are taken from Ref. (89).

216

P. C. CALDER

reduction in this response as the n-6 to n-3 PUFA ratio of the diet is decreased (43) (Figure 7). Such observations accord with the demonstrations of significantly diminished ex vivo T and B lymphocyte proliferation, NK cell activity and CTL activity following LO and FO feeding (seeearlier). Feeding rats a 20% FO diet resulted in fewer IL-2R+ cells and CD 16+/CD3- cells in the PLN following the GvH response, indicating an inhibition of lymphocyte activation and a decreasein the proportion of NK cells, respectively (89). Animal models of organ transplantation. Graft rejection in transplantation surgery is caused by an immune reaction to the foreign material introduced to the body; T-cells have been implicated in accelerated graft rejection, but antibodies with specificity for the graft donor have also been observed following rejection, implying that both cell-mediated and humoral immunity play a part in the rejection process.Studies investigating the effects of eicosanoids on organ transplantation pre-date those investigating the effects of fatty acids. Some eicosanoids promote graft rejection (see (132, 133) for reviews). Therefore, since n-3 PUFAs affect the levels and types of eicosanoids formed (seebelow) they would be expected to influence graft survival. In addition, n-3 PUFAs exert immunomodulatory effects which might be independent of eicosanoids and so these effects might play a role in enhancing graft survival.

14.8 6.5 0.8 n-6:n-3 PUFA ratio of diet

FIG. 7. The effect of the n-6:n-3 PUFA ratio of the rat diet upon the localised GvH response.Rats were fed for 6 weeks on diets containing 20% by weight of sunflower oil or LO or mixtures of the two. A local&d GvH responsewas elicited by an injection of appropriate allogeneic cells into the footpad. Data are mean + SEM of 12 animals fed on each diet and are taken from Ref. (43).

217

LIPIDS AND THE IMMUNE SYSTEM

Animal studies of DTH, GvH and HvG responses(see above) indicate that dietary n-3 PUFAs can suppress the response to foreign antigens. The immunosuppressive effect of dietary FO has also been evaluated, usually favourably, in studies of heart and kidney transplantation in laboratory animals (114, 134-l 37). In a typical result, it was reported that intravenous fat emulsions rich in n-3 PUFAs prolonged the survival of rat cardiac transplants by up to 60% (137). Use of Dietary N-3 PUFAs to Prevent Graft Rejection in Man

The animal studies described aboveindicate that FO could be usedto prolong the survival of organ transplants. This was recently shown in a study of Homan van der Heide et al. (138). These authors reported that renal transplant patients who received FO (6 g/day for the first post-operative year) in combination with cyclosporin A had better kidney function and fewer rejections over one year compared with patients who received coconut oil and cyclosporin A. The beneficial effects of n-3 PUFA supplementation in renal transplantation in man have been recently confirmed (139, 140). N-3 PUFAs Could Diminish Host Defence The inhibitory actions of n-3 PUFAs upon cell-mediated immunity suggest

that there could be some detrimental effects resulting from the overconsumption of n-3 PUFAs. This is evident from animal experiments. Albina et al. (141) found that the wound healing response was diminished in rats fed for 21 days pre-wounding and 30 days post-wounding on a 17% F0/3% corn oil diet compared with those fed 20% corn oil. In a study investigating resistance to bacteria (oral administration of Salmonella typhimurium), Chang et al. (142) reported that mice fed 20% FO showed lower survival over 15 days (48%) than those fed corn oil (62.5%), coconut oil (87.5%) or a low fat diet (88%); spleensfrom the FO-fed animals had a greater number of bacteria per spleen than those from animals fed the other diets. Similarly, a study of experimental tuberculosis in guinea pigs reported an increased number of bacteria in the spleen of FO-fed animals and it was concluded that this represented persistence of the experimental infection (143). MECHANISMS BY WHICH N-3 POLYUNSATURATED ACIDS MIGHT EXERT IMMUNOMODULATORY

FATTY ACTIONS

Fatty acids as Eicosanoid Precursors The precursors andpathways of eicosanoid synthesis. Eicosanoids are a fam-

ily of oxygenated derivatives of dihomo-ylinolenic, arachidonic and eicosapentaenoic acids. Eicosanoids include PGs and thromboxanes (TXs), which

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P. C. CALDER

together are termed prostanoids, and LTs, lipoxins (LXs), hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs). In most conditions the principal precursor for these compounds is arachidonic acid and the eicosanoids produced from arachidonic acid appear to have more potent biological functions than those produced from dihomo-y-linolenic acid or EPA. The precursor PUFA is released from membrane phosphatidylcholine (PC) by the action of phospholipase A, (E.C. 3.1.1.4) or from membrane phosphatidylinositol-4,Sbisphosphate (PIP,) by the actions of phospholipase C (E.C. 3.1.4.11) and a diacylglycerol (DAG) lipase (E.C. 2.7.1.107) (Fig. 8).

The pathways of eicosanoid synthesis begin with cyclooxygenase (E.C. 1.14.99.1),which yields the PGs and TXs, or with the 5-, 12-or 15-lipoxygenases (E.C. 1.13.11.12), which yield the LTs, HPETEs, HETEs and LXs (Fig. 9). A third pathway which operatesthrough the microsomal cytochrome P-450results in formation of epoxides which are converted to HETEs. The amounts and types of eicosanoids synthesized are determined by the availability of arachidonic acid, by the activities of phospholipase A, and phospholipase C and by the activities of cyclooxygenase and the lipoxygenases. The major biologically active products of the cyclooxygenase pathway are PGA,, PGE,, PGI, (prostacyclin), PGF,, and TXA,, although these are produced in a cell-specific manner. These compounds usually have a short half-life and act locally to the cell from which they are produced. Their production is initiated by particular stimuli (e.g. cytokines, growth factors, endotoxin, zymosan, oxygen free radicals, antigen-antibody complexes,bradykinin, collagen, thrombin) and, once produced, they themselves are able to modify the response to the stimulus. Different prostanoids have different, sometimes opposite, effects; for example, TXA, increases platelet aggregation whereas PGIz inhibits platelet aggregation. PGs are associated with the inflammatory response and cyclooxygenase inhibitors include aspirin and related nonsteroidal anti-inflammatory drugs. It appears that each of the lipoxygenase enzymeshas a particular cellular distribution: Slipoxygenase is found in mast cells, monocytes, M$ and granulocytes, 1Zlipoxygenase is found in platelets and some epithelial cells and 1S-lipoxygenase is found in young myeloid cells and some epithelial cells. The n-3 PUFAs, EPA and DHA, competitively inhibit the oxygenation of arachidonic acid by cyclooxygenase. In addition, EPA (but not DHA) is able to act as a substrate for both cyclooxygenase and 5-lipoxygenase (Fig. 9). Ingestion of FO which contain n-3 PUFAs will result in a decreasein membrane arachidonic acid levels and a concomitant decreasein the capacity to synthesize eicosanoids from arachidonic acid (seebelow); EPA gives rise to the 3-series PGs and TXs and the 5-series LTs (Fig. 9). The eicosanoids produced from EPA do not always have the same biological properties as the analogues produced from arachidonic acid. For example, TXA, is lessactive than TXA,

2

PLAZ

!I-glycerophosphocholine

acid

I-alkyl-glycera

Arachidonic

CoASH

:tiva sting factor

5,

m aeetyl CoA :tyl transferase

sphocholine

PLAZ

@iiiiZZacid)

1-alkyl-2-arachidonyl-glycerophosphocholine

Diacylglycerol

(DAG)

PLC

I-acyl-2-arachidonyl-glycerophospho inositol-4 isphosphate (PIPz)

FIG. 8. The reactions by which arachidonic acid is released from membrane phospholipids.

I-acyl-glycerophosphocholine

I-acyl-t-arachidony

P. C. CALDER

220

Phospholipid Phospholipase A2

Arachidonic acid (20:4n-6)

Cyclooxygenase

Lipoxygenases

PC12

LTAI LTB4 LTC4 LTD4 LTE4. S-HPETE 5-HETE 15HPETE ISHETE I2-HPETE 12-HETE

TXA2 TXBz PGD2 PGE2 PGF2

Eicosapentaenoic acid

A Cyclooxygenase

Lipoxygenases

PG13 TXA3 TXB3 PGD3 PGE3 PGR

LTA5 LTBs LTCs LTDs LTES 5-HEPE 5-PEPE

FIG. 9. Eicosanoid synthesis from arachidonic acid and EPA.

in aggregating platelets and constricting blood vesselsand LTB, is less active than LTB, with regard to chemotactic and aggregatory properties in human neutrophils. In contrast, PGIs is as active as PGIz in inhibiting platelet aggregation and promoting vasodilation. The synthesis of eicosanoids by cells of the immune system. M$ synthesize a range of cyclooxygenaseand lipoxygenase products (see(144, 145)for reviews); the exact profile of eicosanoids formed depends upon the species and anatomical site of origin of the M+ and the nature of the stimulus used. Studies concerning eicosanoid synthesis by lymphocytes have proved controversial (see 146). Many studies indicate that lymphocytes do not synthesize cyclooxygenase or lipoxygenase products (see (146) for references). Nevertheless, lymphocytes contain similar amounts of arachidonic acid in their membrane phospholipids to Mg and mitogen-stimulated lymphocytes release arachidonic acid extracellularly. There is evidence that Me utilize this released arachidonic acid for eicosanoid synthesis. Indeed, in the

LIPIDS AND THE IMMUNE

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221

presence of lymphocytes, M+ produce increased amounts of PGE, in vitro. Thus, it seems that among immunocompetent cells, M$ are the principal, and perhaps the only, source of eicosanoids, but that an interaction between lymphocytes and M$ exists. Effects of eicosanoi& upon cells of the immune system. Not only are immune cells a source of eicosanoids, but they are subject to their regulatory effects; the most well documented effects are those of PGE,. In vivo, PGs are involved in modulating the intensity and duration of inflammatory and immune responses; PGE, has a number of pro-inflammatory effects including fever, erythema, increased vascular permeability, vasodilation and enhancement of pain and oedema caused by other agents such as bradykinin and histamine. In chronic inflammatory conditions increased activity of suppressor T-cells and increased rates of PGE, production are observed, and elevated PGE, production has been observed in patients suffering from infections, whose T-cells show depressed functional responses. It is generally accepted that PGE, is a regulator of immune cell functions; the age and type of target cell and the concentration of PGE, determine the nature of the response. PGs appear to play a role in regulating the differentiation of both T and B lymphocytes; for example PGE, induces immature thymocytes to differentiate into mature T-cells. In addition, the functions of T-cells, B-cells, NK cells and M$ are modulated by eicosanoids; these effects are reviewed elsewhere (145-147) (Fig. 10). T-lymphocytes have receptors for PGE, and PGE, and these compounds suppress T-lymphocyte proliferation, T-cellmediated cytotoxicity, IL-2 production and NK cell activity in vitro (see 145-147 for reviews). B-cells have receptors for the E-series PGs and PGE, can influence antibody production. PGs inhibit production of IL-l and TNF by macrophages (148). Some MQ,enzyme activities are also modulated by PGs, as is expression of MHC II receptors (149). There are conflicting reports about the effects of LTs on lymphocyte proliferation (see (6, 145 147) for reviews), but NK cell activity is enhanced by LTB, (150, 151). LTB, and LTC, enhance IL-1 production by macrophages (148) and LTB4 enhances IFN-y production by lymphocytes (150). Since they influence eicosanoid production (see below), it is clear that n-3 PUFAs can modulate immune cell functions by eicosanoid-mediated effects. Modulation of eicosanoid synthesis by n-3 PUFAs. Culture of M+ or lymphocytes with n-3 PUFAs results in replacement of arachidonic acid in phospholipids by the n-3 PUFA provided (87, 152-154). As a result of this modification, fewer arachidonic acid-derived eicosanoidsare produced by these cells.Dietary lipid modulation also results in significant modification of the fatty acid composition of M+ isolated from the peritoneal cavity of mice (94, 15%

222

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ARACHIDONIC

Lipoxygenase

Cyclooxygenase

PGE2 I

ACID

LTB4

15HETE

Lipoxins

FIG. 10. Summary of the immunomodulatory effects of eicosanoids

(156), rats (92, 157,158) or hamsters(159), of M$ isolated from the lungs of pigs (160), of lymphocytes isolated from rodent lymphoid tissues(157, 161, 162)and of monocytesisolated from human peripheral blood (8 1,82). It is widely reported that feeding laboratory animals n-3 PUFA-containing oils such as LO or FO results in decreased production of arachidonic acid-derived eicosanoids (61,74,93,94, 155, 157, 160) (Fig. 11). FO supplementation of the human diet results in similar changes(8 1,82). The suppressionin the amount of arachidonicacid derived eicosanoids is mirrored by an elevation in the level of EPA-derived eicosanoids (163). As indicated earlier, these latter compounds are often less biologically potent than the analoguessynthesizedfrom arachidonic acid. Thus, significant effectsupon processessuch as platelet aggregation, vasoconstriction, neutrophil function, inflammation and immunity result. A thorough review of the effects of dietary lipids upon eicosanoid production, particularly by M$, may be found elsewhere(164). N-3 PlJFAs and Membrane Fluidity Fatty acids have important roles in membrane structure and there are several ways by which they can potentially influence the functions of membrane proteins. The fluid mosaic model of membrane structure describes biological membranes as dynamic and responsive structures. It is now also recognised that domains exist in membranes, where lipid-protein and lipid-lipid interactions may be highly specific. The composition of phospholipids in cell

223

LIPIDS AND THE IMMUNE SYSTEM loo

n Lowfat E Ssmowcr oil

80 f 60 2 % 4 u 40 20

0 PGE2

Q-keto-PGFla

TXB2

Eicosanoid

FIG. I 1. The effect of dietary lipids upon production of arachidonic acid-derived eicosanoids by murine peritoneal macrophages. Mice were fed for 8 weeks on a low fat diet or on diets containing 20% by weight of safflower oil or FO. Thioglycollate-ehcited peritoneal macrophages were prepared and cultured with LPS for 8 hr. The medium was collected and eicosanoid concentrations measured using enzyme-linked immunosorbant assays.PGE, concentrations are expressed as ng/mi and TXB, and 6-keto-PGF,, concentrations are expressed as ng/lOOml. Data are mean t SEM for three animals fed on each diet and are taken from Ref. (61).

membranes is usually characteristic for the cell type, but may change with the cell cycle, with age,in response to stimuli or to changesin the environment or the diet (165); these changes may have functional consequences.A number of membrane-bound enzymes have been shown to be particularly sensitive to their fatty acid environments (see(165-167) for reviews). The sameis true for a number of receptors, including adrenergic and insulin receptors. The mechanisms by which membrane lipids modulate enzyme activity or receptor function are not fully understood, but suggestions include changes in membrane fluidity (165, 166)and fatty acid-dependent effectson the conformation of the protein complex (167). The stimulation of lymphocytes is accompanied by de ~OVOsynthesis and turnover of membrane phospholipids (168-l 7 1). Within minutes of stimulation remodelling of the membrane phospholipids begins, with saturated fatty acids in phospholipids being substituted by PUFAs (171). As a result, changes in the fatty acid composition of lymphocyte phospholipids are detectable within minutes of stimulation and are quite marked by 4 hr post-stimulation (17 1). Culture of mitogen-stimulated lymphocytes for several days results in a very significant increase in the content of unsaturated

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fatty acids in the membrane phospholipids (154, 172). This change in fatty acid composition is accompanied by an increase in membrane fluidity (154, 172), although this has been a controversial subject for many years (see (154) for references). In support of the proposal that membrane fluidity changes are an inherent component of lymphocyte activation and subsequent proliferation (154, 172), Curtain et al. (173) found that a nonmitogenic lectin (wheatgerm agglutinin) does not affect lymphocyte membrane fluidity, in contrast to the effect of the mitogenic lectins Con A and PHA. That plasma membrane fluidity does influence lymphocyte activity was shown by the studies of Maccecchini and Burger (174) and Huber et al. (175). In contrast to the situation with lymphocytes, stimulation of M$ with LPS appears to cause a decrease in plasma membrane fluidity (176). As described earlier, culture of lymphocytes, M$ or monocytes in the presence of n-3 PUFAs results in significant alterations in the fatty acid composition of the membrane phospholipids. Such changes result in increased lymphocyte plasma membrane fluidity (154). In contrast, even though they elicit significant changesin the fatty acid composition of membrane phospholipids (see above for references), diets rich in n-3 PUFAs do not appear to significantly alter the fluidity of the plasma membranes of lymphocytes (162, 177), M$ (158, 176) or monocytes (82). There are at least two possible reasonsfor this. Firstly, the effect of increasing the PUFA content of membrane phospholipids might be counteracted by altering the cholesterol content of the membrane and/or by altering the proportions of different types of phospholipids present. Secondly, the techniques used yield values for the fluidity of the plasma membrane as a whole and it is possible that changes occur in particular regions of the membrane. N-3 PUFAs and Signal Transduction Apart from influencing the pattern of eicosanoids produced (see above), it is possible that n-3 PUFAs may influence signalling within cells of the immune system in other ways. Many lipids are involved directly in intracellular signalling pathways; for example hydrolysis of membrane phospholipids such as PIP, and PC by phospholipases generates second messengers such as DAG. Other phospholipids have roles in activating or stabilising enzymes involved in intracellular signalling; for example phosphatidylserine (PS) is required for protein kinase C (PKC) activation. Since PIP,, PC, PS and DAG all contain fatty acyl chains attached to the sn-1 and -2 positions of the glycerol moiety, it is conceivable that changing the type of fatty acid present may alter the precise properties of these compounds with regard to their functions in signal transduction. Indeed, PKC is more active in the presence of dioleoylglycerol or diarachidonoyl-glycerol than in the presence of diacylglycerols containing two saturated fatty acids or one

LIPIDS AND THE IMMUNE

SYSTEM

22.5

saturated and one unsaturated fatty acid (178). Bell and Sargent (179) showed that rat spleen PKC was less active in the presence of an n-3 PUFArich PS compared with a PUFA-poor PS, irrespective of the fatty acid composition of the DAG. Recently, Fowler et al. (180) reported that feeding mice for 10 days on a diet containing 1% purified ethyl ester of either EPA or DHA resulted in enrichment of spleen lymphocyte DAG species with the fatty acid fed; the total mass of DAG was elevated in the DHA-fed mice. In addition to the effects of fatty acids on intracellular signalling mechanisms due to changes in the fatty acid composition of the phospholipids which are involved, it has been proposed that unsaturated fatty acids themselves may have a direct effect (see (181) for references). This direct modulatory effect of fatty acids has been most extensively documented in relation to PKC activity which was shown to be enhanced by DHA (182). In contrast, although Speizer et al. (183) reported that EPA and DHA increased brain PKC activity in the absence of PS and DAG, they found that in the presence of both PS and DAG each of these fatty acids caused up to 60% inhibition of PKC activity. Another study has shown that EPA and DHA inhibit rat lymphocyte PKC activity in the presence of calcium, PS and DAG (184); protein kinase A (PKA) (E.C. 2.7.1.37) activity was unaffected by EPA and DHA (184). Recently, rat peritoneal M$ PKC activity was shown to be inhibited by EPA and DHA; again PKA activity was unaffected (185). In accordance with both the direct effects of n-3 PUFAs in vitro (184, 185) and the effects of enriching PS with n-3 PUFAs (179). Van Meter et al. (186) found that feeding mice FO resulted in diminished spleen lymphocyte PKC activity. A change in the concentration of intracellular free calcium is often a key component in the intracellular signalling pathway which follows the stimulation of lymphocytes, M$ and other cells by growth factors, cytokines and antigens. There is now considerable evidence that free fatty acids influence these changes. For example, Richieri and Kleinfeld (187) reported that oleic acid, but not stearic acid, inhibited the target cell- or Con A-stimulated rise in intracellular free calcium concentration in a CTL cell line. Several unsaturated fatty acids including ALA, EPA and DHA inhibit the anti-CD3-induced increasein intracellular free calcium concentration in the JURKAT T-cell line (188, 189); the fatty acids appeared to act by blocking calcium entry into the cells and it was concluded that they act directly upon receptor-operated calcium channels (188). N-3 P UFAs and GeneExpression It is now well documented that fatty acids affect the expression of genes involved in hepatic fatty acid and lipoprotein metabolism (see (190) for a

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review) and the genes involved in adipocyte differentiation and development (see (191) for a review). Dietary n-3 PUFAs have particularly potent effects upon the expression of genes for proteins involved in hepatic peroxisomal proliferation, fatty acid oxidation and lipoprotein assembly (192). They may act in a fashion analogous to that of steroid hormones, possessing intracellular receptors which directly influence transcription (see (193, 194) for reviews). Alternatively, unsaturated fatty acids may act by increasing or decreasing the levels of other mediators (e.g. eicosanoids, cytokines) which affect gene expression. Studies of the effects of fatty acids upon the expression of genes important in immune cell functioning are few. However, a recent study showed that inclusion of FO in the diet of autoimmune disease-prone mice results in elevated levels of mRNA for IL-2, IL-4 and transforming growth factor-l3 and reduced levels of mRNA for c-myc and c-ras in the spleen (99). The same workers showed that dietary FO completely abolished mRNA production for IL- 1l3, IL-6 and TNF-a in the kidneys of these animals (195). Robinson et al. (125) reported that feeding mice a FO-rich diet significantly diminished ex viva IL-l j3 mRNA production by LPS-or PMA-stimulated spleen lymphocytes; the lower IL-l l3mRNA level was not due to accelerated degradation but to impaired synthesis. FO feeding to mice lowered basal TNF-a mRNA and LPS-stimulated TNF-cx and IL-lj3 mRNA levels (60) These studies suggest that n-3 PUFAs might affect immune cell functioning by control at the transcriptional level.

SUMMARY

The amount and type of eicosanoids made can be affected by the type of fat consumed in the diet. It is now apparent that both eicosanoids and n-3 PUFAs are potent modulators of lymphocyte and M+ functions in vitro. Inclusion in the diet of high levels of certain lipids containing n-3 PUFAs markedly affects the functions of cells of the immune system subsequently tested in vitro. Cellular components of both natural and acquired immunity are affected. In vivo tests are perhaps the most appropriate approach for determining the effect of different dietary n-3 PUFAs upon immune function. Several studies indicate that diets rich in n-3 PUFAs are antiinflammatory and immunosuppressive in vivo, although there have been relatively few studies in man. Although some of the effects of n-3 PUFAs may be brought about by modulation of the amount and types of eicosanoids made, it is clear that these fatty acids can also elicit their effects by eicosanoid-independent mechanisms (Fig. 12). Such n-3 PUFA-induced effects may be of use as a therapy for acute and chronic inflammation, for disorders which involve an inappropriately-activated immune response and for the enhancement of graft survival.

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IMMUNE CELL FUNCTION

FIG. 12. Mechanisms by which n-3 PUFAs might exert immunomodulatorv inflammatory effects.

and anti-

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