Effects of dietary lipid manipulation upon rat spleen lymphocyte functions and the expression...

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Section: ORIGINAL RESEARCH

Rats were fed for 12 weeks on a low fat (LF; 2.5% by weight) diet or on one of five high fat (20% by weigh) diets; the latter contained hydrogenated coconut oil (HCO), olive oil (00), safflower oil (SO), evening primrose oil (EPO) or menhaden (fish) oil (MO). Feeding the EPO or MO diets resulted in inhibition of mitogen-stimulated spleen lymphocyte proliferation compared with feeding any of the other diets; in addition, feeding the OO diet resulted in inhibition of proliferation compared with feeding the LF diet. Feeding the 00, EPO or MO diets resulted in inhibition of spleen lymphocyte natural killer (NK) cell activity compared with feeding the LF or HCO diets; feeding the SO diet resulted in inhibition of NK cell activity compared with feeding the HCO diet. There was no effect of dietary lipid manipulation upon the proportion of T-cells or of CD4 (sup +) or CD8 (sup +) cells in the spleen. The level of expression of CD2 on CD4(sup +) and CD8(sup +) spleen lymphocytes was decreased by MO feeding compared with feeding the LF, HCO, SO or EPO diets; 00 feeding decreased the level of expression of CD2 on CD4 (sup +) spleen lymphocytes compared with feeding the LF diet. The level of expression of LFA-1 was decreased on both CD4 (sup +) and CD8(sup +) spleen lymphocytes from animals fed the O0 or MO diets compared with that on lymphocytes from animals fed some of the other diets. Following mitogenic stimulation the level of expression of CD8, CD2, ICAM-1, LFA-1 and the interleukin-2 receptor was lower on spleen lymphocytes from animals fed the MO diet and, for some of the markers, on those from animals fed the O0 or EPO diets. These observations indicate that diets containing MO, O0 and EPO exert immunomodulatory effects by influencing the expression of important molecules upon the lymphocyte surface. This may occur through effects on signal transduction pathways and/or gene expression.

Keywords: lymphocyte proliferation, dietary lipids, fish oil, natural killer cells, lymphocyte subsets, adhesion molecules.

INTRODUCTION

In vitro studies indicate that unsaturated fatty acids inhibit a number of immune cell functions including lymphocyte proliferation [ 1-7], interleukin-2 (IL-2) production [ 5, 6, 8], activation marker expression [ 5], natural killer (NK) cell activity [ 9] and antigen presentation [ 10]. In studies where the effects of a range of fatty acids have been compared, it has often been found that the n-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid is the most potent inhibitor of these functions [ 3-7], although other n-3 PUFAs and n-6 PUFAs are also inhibitory [ 1-7]. Such in vitro studies indicate that the consumption of oils containing unsaturated fatty acids, especially fish oils which contain n-3 PUFAs, may cause immunosuppression. As such, these oils could be of use in the therapy of diseases characterized by an overactive immune system [ 11-15] or in the prolongation of grafts [ 16]. However, the exact nature of the effects of different dietary lipids is unclear. For example, some studies have shown that feeding animals diets rich in n-6 PUFAs results in inhibition of lymphocyte functions [ 17-19], whereas other studies show no effect on [ 20, 21], or even enhancement of [ 22], these functions. Part of the reason for the different findings of such studies is the different experimental protocols used: the studies differ greatly in the amount and source of fat used, the composition of other dietary components, the amount and type of anti-oxidant present, the duration of the feeding, the species, age and sex of the animal studied, and the conditions under which the tests of immune function were made.

Recently, we reported the results of a direct comparison of feeding weanling rats for 10 weeks on diets containing 20% (by weight) of hydrogenated coconut oil (HCO: rich in saturated fatty acids), olive oil (OO: rich in the n-9 monounsaturated fatty acid oleic acid), safflower oil (SO: rich in the n-6 PUFA linoleic acid), evening primrose oil (EPO: rich in linoleic acid and also containing the n-6 PUFA gamma-linolenic acid) or menhaden (fish) oil (MO: rich in n-3 PUFAs); all other components of the diets were identical. Feeding each of these diets suppressed spleen lymphocyte proliferation ex vivo compared with feeding a low fat (LF) diet [ 23], and feeding the OO, EPO and MO diets suppressed the proliferation of lymph node lymphocytes compared with feeding the LF, HCO or SO diets [ 23]. For both cell types the effects of the dietary manipulation were masked when the cells were cultured in fetal calf serum (FCS) rather than autologous serum [ 23]. Furthermore, feeding the HCO, OO or MO diets suppressed the proliferation of lymphocytes in response to low concentrations of mitogen in whole blood culture compared with feeding the LF, SO or EPO diets [ 24]. Feeding the OO, EPO and MO diets resulted in inhibition of spleen NK cell activity compared with feeding the LF diet; feeding the MO diet inhibited NK cell activity compared with feeding HCO, SO or EOP diets [ 25]. Thus, it appears that feeding certain lipids (especially OO, EPO and MO) to very young animals during the rapid phase of growth [ 26].can result in suppression of lymphocyte functions, although it must be noted that the outcome of the ex vivo tests of function employed can be influenced by the cell culture conditions used [ 23]. However, the question of whether it is possible to manipulate lymphocyte functions in older animals has still not been resolved. This may be important clinically since dietary intervention is more likely to be used in older individuals once they develop symptoms of inflammatory and auto-immune conditions. Therefore, in this study, we examined the effect on spleen lymphocyte functions of feeding 8-week-old rats different dietary lipids for 12 weeks. We also examined the effect of these lipids on the expression of a number of important molecules on the surface of both resting and activated spleen lymphocytes.
MATERIALS AND METHODS Animals and Diets

Male Lewis rats (8 weeks old and weighing between 165 and 185 g) were obtained from Harlan-Olac, Bicester, Oxfordshire, UK. The animals were housed in the Department of Biochemistry for a period of 12 weeks prior to sacrifice, during which time they were allowed access, ad libitum, to water and to one of six experimental diets. The diets used were a LF diet (prepared by SDS, Witham, Essex, UK) and five high fat diets (prepared by ICN Biomedicals, High Wycombe, Buckinghamshire, UK). The LF diet contained approximately 2.5% by weight of an unspecified oil which was rich in linoleic, palmitic and oleic acids [ 26]. The high fat diets contained 20% by weight of the lipid under study (HCO, OO, SO, EPO or MO) plus 1% corn oil to prevent essential fatty acid deficiency. All the high fat diets contained identical amounts of protein, starch, sucrose and vitamin E. The energy content of the diets was measured by Knight Energy Services Ltd, St Helens, Merseyside, UK. Details of the composition of these diets have been given elsewhere [ 26].
Chemicals

Chemicals, culture medium and medium supplements were obtained from the sources described elsewhere [ 3, 5, 6, 25]. In addition, mouse immunoglobulin G (IgG), the monoclonal antibody to CD3, biotinylated monoclonal antibodies to CD4 or CD8 and phycoerythrin-conjugated streptavidin were purchased from Serotee, Kidlington, Oxford-shire, UK. Monoclonal antibodies to the alpha-chain of LFA-I (WT-1) and ICAM-1 (IA-29) were gifts from Professor M. Miyasaka, Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Japan; all other monoclonal antibodies were generously supplied by the MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxfordshire, UK.
Lymphocyte Preparation

Spleen lymphocytes were prepared as described previously [ 23, 25].
Lymphocyte Proliferation Assay

Spleen lymphocyte proliferation in response to the T-cell mitogen concanavalin A (Con A; 5 micro g ml(sup -1)) was determined as described elsewhere [ 23]. The culture medium contained 2.5% (v/v) heat-inactivated (56 degrees C, 30 min) autologous serum.
Analysis of Lymphocyte Subpopulations and Receptor Expression

Flow cytometry was used to measure the presence of various markers on the surface of freshly prepared and Con A-stimulated spleen lymphocytes. Where Con A-stimulated cells were used, lymphocytes were cultured for 24 h in the conditions described elsewhere [ 23], except at a density of 5 X 10(sup 6) cells/well and a total culture volume of 2 ml. Following culture, the cells were collected by centrifugation and washed three times in phosphate-buffered saline (PBS) supplemented with 0.1% (w/v) bovine serum albumin and 10 mM-sodium azide (modified PBS).

For single staining, approximately 10(sup 6) cells (resuspended in modified PBS) were incubated for 20 min at 4 degrees C with monoclonal antibodies to the alpha beta T-cell receptor (TcR; R73), CD4 (W3/25), CD8 (MRC OX-8), the alpha-subunit of the IL-2 receptor (IL-2R; MRC OX-39), the transferrin receptor (TfnR; MRC OX-26), CD2 (MRC OX-34), CDlla (LFA-1; WT-I) or CD54 (ICAM-1; IA-29). Incubation with a monoclonal antibody to the human C3b inactivator protein (MRC OX-21) was used as a negative control. Following staining with monoclonal antibodies, the cells were washed twice with modified PBS and incubated with fluorescein isothiocyanate-labelled rabbit anti-mouse IgG (RAM-FITC) for 20 min at 4 degrees C. They were washed twice with modified PBS and then suspended in FACS-Fix (2% (v/v) formaldehyde in PBS) and examined for fluorescence using a Becton Dickinson FACScan fluorescence-activated cell sorter. Fluorescence data were collected on 10(sup 4) viable cells.

For double staining, the above procedure was used to stain for IL-2R, TfnR, CD2, LFA-1 or ICAM-1. After incubation with RAM-FITC, the cells were washed twice with modified PBS and then incubated for 10 rain with 5 micro l of 125 micro g ml(sup -1) mouse IgG. Then 10 micro l of 50 micro g ml (sup -1) biotinylated monoclonal antibody to CD4 or CD8 were added. After incubation at 4 degrees C for 20 min, the cells were washed twice with modified PBS and incubated with 10 micro l of phycoerythrin-conjugated streptavidin for 20 min at 4 degrees C. Finally, the cells were washed twice with modified PBS, suspended in FACS-Fix and examined for fluorescence using a Becton Dickinson FACScan fluorescence-activated sorter. Fluorescence data were collected on 10(sup 4) viable cells.
Spleen NK Cell Activity Assay

Spleen lymphocyte NK cell activity was determined using the chromium release assay described elsewhere [ 25].
Statistical Analysis

Statistical significance was determined using one- or two-way analysis of variance (second factor day), followed by a least squared difference test.

RESULTS
Food and Energy Intake and Weight Gain

The food intake of the LF-fed animals was significantly greater than that of animals fed on the high fat diets (Table 1). There were no differences in food intake between animals fed the high fat diets. The total energy over the 12-week feeding period was not significantly different between animals fed the different diets (see Table 1). The weight gain of the animals fed the HCO, OO, EPO or MO diets was greater than that of the LF-fed animals (see Table 1). As a result, the LF-fed rats had smaller final weights than rats fed on each of the high fat diets, except SO (see Table 1). The weight gains of animals fed the HCO, OO, EPO or MO diets were almost identical (see Table 1).
Surface Markers on Spleen Lymphocytes

There were no differences in either the proportion or mean fluorescence of TcR (sup +), CD4 (sup +) or CD8(sup +) lymphocytes between the different diets (Table 2). However, although the proportion of CD2(sup +) cells was unaffected by dietary lipid manipulation, the mean fluorescence of CD2 + cells was decreased by MO feeding compared with feeding the LF, HCO, SO or EPO diets (see Table 2). Also, feeding the OO diet decreased the mean fluorescence of CD2 + cells compared with feeding the LF diet (see Table 2). In addition, the mean fluorescence of LFA-1 was significantly decreased on spleen lymphocytes prepared from animals fed the OO or MO diets compared with those from animals fed the LF or HCO diets; furthermore, the mean fluorescence of those from the MO-fed animals was lower than that of lymphocytes from animals fed the SO or EPO diets (see Table 2).

The decreased mean fluorescence of CD2 + cells from animals fed the MO diet was observed in both CD4 + and CD8 + populations (Table 3), while the effect of the OO diet was seen in the CD4 + cells only (see Table 3). The decreased mean fluorescence of LFA- 1 on spleen lymphocytes following OO or MO feeding (see Table 2) was seen in both CD4 + and CD8 + cell populations (see Table 3).
Surface Markers on Mitogen-stimulated Spleen Lymphocytes

Following mitogenic stimulation with the T-cell mitogen Con A, the proportion of CD4 cells was lower for spleen lymphocytes from animals fed the OO diet than for those fed the LF, SO, EPO or MO diets (Table 4); however, the mean fluorescence was unaffected (see Table 4). In contrast, there was no effect upon the proportion of CD8 + cells (see Table 4), but the mean fluorescence was lower for cells from animals fed the HCO, SO or MO diets than for those from animals fed the LF diet (see Table 4). There were no effects of dietary manipulation on the proportion of IL-2R [suP +] cells following mitogenic stimulation (see Table 4); however, the mean fluorescence of IL-2R + cells was decreased for those from animals fed the OO, SO, EPO or MO diets compared with those from animals fed the LF or HCO diets (see Table 4). Furthermore, the IL-2R + lymphocytes from rats fed the MO diet were less fluorescent than those from animals fed the OO or SO diets (see Table 4).

There was no effect of dietary lipid manipulation upon the proportion of spleen lymphocytes that were positive for CD2, ICAM-1 or LFA-1 following mitogenic stimulation (Table 5). However, the mean fluorescence of cells positive for each of these markers was significantly affected by dietary lipid manipulation. The mean fluorescence of CD2 + cells was decreased by MO feeding compared with feeding the LF or HCO diets and by EPO feeding compared with feeding the HCO diet (see Table 5). The mean fluorescence of ICAM-1 + cells was lower for the MO and EPO groups than for the cells from the animals fed the LF or HCO diets (see Table 5). The mean fluorescence of the cells from the MO-fed animals was also less than that of the cells from animals fed the OO or SO diets (see Table 5). Finally, the mean fluorescence of LFA-I was lower on cells from animals fed the OO, SO, EPO or MO diets than for those from animals fed the LF diet (see Table 5). In addition, the mean fluorescence of LFA-1 on cells from animals fed the EPO or MO diets was lower than that on those from animals fed the HCO diet (see Table 5).

There were no effects of dietary lipid manipulation on the proportion of mitogen-stimulated spleen lymphocytes staining 'double-positive' for CD2 and CD4 (approximately 50%), CD2 and CD8 (approximately 30%), IL-2R and CD4 (approximately 20%), IL-2R and CD8 (approximately 15%), TfnR and CD4 (approximately 20%), TfnR and CD8 (approximately 20%), LFA-1 and CD4 (approximately 50%), LFA-1 and CD8 (approximately 30%), ICAM-1 and CD4 (approximately 20%) or ICAM-1 and CD8 (approximately 20%). There were, however, a number of significant differences in the mean fluorescence of double-positive cells between lymphocytes from animals fed the different diets (Table 6). The mean fluorescence of mitogen-stimulated spleen lymphocytes staining double-positive for CD2 and CD4, CD2 and CD8, IL-2R and CD4 or IL-2R and CD8 was significantly lower for cells from EPO or MO-fed animals than for those from animals fed the LF or HCO diets (see Table 6). In addition, there were a number of other significant differences in mean fluorescence between cells obtained from animals fed the MO diet and those fed the other diets (see Table 6). There were no significant differences in the mean fluorescence of cells staining double-positive for TfnR and CD8, LFA-1 and CD4, LFA-1 and CD8, ICAM-1 and CD4 or ICAM-1 and CD8, although there was a trend for these to be lower on spleen lymphocytes from animals fed the OO, EPO or MO diets than on those from animals fed the LF or HCO diets (see Table 6).
Spleen Lymphocyte Proliferation

The stimulation index values for spleen lymphocytes cultured in the presence of 2.5% autologous serum and Con A were significantly lower for cells obtained from animals fed the OO, EPO or MO diets than for those from animals fed the LF diet (Table 7). In addition, spleen lymphocytes from animals fed the EPO or MO diets proliferated less well than those from animals fed on the HCO, OO or SO diets (see Table 7).
Spleen NK Cell Activity

Spleen lymphocytes from animals fed the OO, EPO or MO diets showed decreased NK cell activity compared with those from animals fed on the LF or HCO diets at lymphocyte:target cell ratios of 100:1, 50:1 and 25:1 (Table 8); the difference between the NK cell activity of cells from the OO-, EPO- and MO-fed animals and those from animals fed HCO was also observed at a lymphocyte:target cell ratio of 12.5:1 (see Table 8). The NK cell activity of spleen lymphocytes from animals fed the HCO diet was greater than that of cells from animals fed each of the other high fat diets (see Table 8).

DISCUSSION

It has been proposed that the changes in lymphocyte functions caused by fatty acids in vitro are linked to changes in the fatty acid composition of the lymphocyte phospholipids [ 27-29]. Thus, if dietary lipids are to affect lymphocyte functions, they must cause changes in the fatty acid composition of lymphocyte phospholipids. Such changes have been documented following dietary lipid manipulation of weanling rats [ 30, 31]. However, such animals are in a very rapid phase of growth and so the fatty acid composition of phospholipids may be able to be changed more readily than in more mature, slower growing animals. Therefore, we investigated the effect of feeding more mature rats (approximate weight 175 g) a variety of high fat diets, each with a characteristic fatty acid composition, on the function of spleen lymphocytes. We have previously reported the effects of feeding these diets to weanling rats on spleen lymphocyte subsets and proliferation [ 23] and NK cell activity [ 25]. The weanling rats in those studies initially grew at a rate of approximately 6 g/day and grew at rates of between 3.6 and 4.6 g/day over the 10-week feeding period [ 26]. In the current study, the rate of weight gain of the rats was between 2.7 and 3.5 g/day, depending on the diet they were fed. Thus, the rates of weight gain of weanling and more mature rats fed identical diets are different, with the latter showing the lower rate. There were no differences in energy intake between the rats fed the different diets (see Table 1). However, the animals gained different amounts of weight: those fed the HCO, OO, EPO or MO diets gained more weight than those fed the LF or SO diets (see Table 1). The difference in weight gain, despite the similar energy intake, indicates that some high fat diets may result in lower energy expenditure.

We have previously shown that feeding weanling rats the OO, EPO or MO diets results in suppression of spleen lymphocyte proliferation and NK cell activity, examined ex vivo [ 23, 25]. The current study shows that feeding more mature rats these three diets also results in suppression of spleen lymphocyte proliferation compared with feeding the LF diet, while feeding the EPO or MO diets resulted in suppression compared with feeding the HCO, OO or SO diets (see Table 7). Furthermore, feeding the OO, EPO or MO diets also resulted in significant suppression of spleen lymphocyte NK cell activity compared with feeding the LF or HCO diets (see Table 8). The one significant difference between the current study and our previous study with weanling rats is the enhancement of spleen lymphocyte NK cell activity observed after feeding mature rats the HCO diet (see Table 8). The reason for this difference between mature and weanling rats is not known.

We previously showed that the diets used in this study did not affect either the proportions of TcR+, CD4+ or CD8+ lymphocytes in the spleens of weanling rats or the proportions of CD4+, CD8+, IL-2R+ or TfnR+ spleen lymphocytes following mitogenic stimulation [ 23]. The current study confirms these findings with spleen lymphocytes from older rats fed different diets (see Tables 2 and 4), but extends the study of the effects of dietary lipid manipulation upon lymphocyte cell surface molecules to include a variety of adhesion molecules (CD2, ICAM-1, LFA-1) and determinations of the level of expression of surface molecules measured as mean fluorescence. The adhesion molecules investigated are involved in interaction between lymphocytes and the other cells during the immune response and in the movement of lymphocytes between tissue compartments.

The level of expression of TcR, CD4 and CD8 by spleen lymphocytes was unaffected by dietary lipid manipulation (see Table 2). However, although the proportion of spleen lymphocytes expressing CD2 was unaffected by dietary lipid manipulation, the level of expression was significantly decreased following feeding the MO diet compared with feeding the LF, HCO, SO or EPO diets (see Table 2); feeding the OO diet also resulted in a decreased level of expression of CD2 compared with feeding the LF diet (see Table 2). Furthermore, feeding the MO diet significantly decreased the level of expression of LFA-1 compared with feeding the LF, HCO, SO or EPO diets, and feeding the OO diet resulted in a lower level of LFA-1 expression than feeding the LF or HCO diets (see Table 2). The effects of feeding the MO or OO diets were exerted on both CD4 and CD8 lymphocyte subsets (see Table 3).

Although the proportion of cells expressing the IL-2R following Con A stimulation was unaffected by dietary lipid manipulation, the level of expression was significantly affected: feeding the OO, SO, EPO or MO diets resulted in a lower level of expression compared with feeding the LF or HCO diets, while feeding the MO diet also resulted in a decreased level of expression compared with feeding the OO or SO diets (see Table 4). The level of expression of IL-2R correlated well (r=0.963; p < 0.01) with the proliferative response (i.e. SI; see Table 7) of the cells. Just as feeding the MO diet resulted in a lower level of adhesion molecule expression on the surface of freshly prepared spleen lymphocytes, this diet also resulted in a lower level of expression of CD2, ICAM- 1 and LFA-1 on the surface of Con A-stimulated spleen lymphocytes (see Table 5). The effects of the MO diet upon the level of expression of IL-2R and CD2 were observed in both CD4+ and CD8+ lymphocytes (see Table 6). Feeding some of the other high fat diets, in particular the EPO diet, also resulted in a lower level of expression of adhesion molecules by Con A-stimulated lymphocytes (see Table 5).

Thus, fish oil feeding in particular results in a significantly decreased level of expression of two key adhesion molecules, LFA-1 and CD2, on the surface of freshly prepared spleen lymphocytes and a significantly decreased level of expression of three key adhesion molecules, LFA-1, CD2 and ICAM-1, on the surface of mitogen-stimulated spleen lymphocytes. This may help to explain the immunosuppresive effects of fish oil, since decreased levels of expression of these molecules may result in less adhesion molecule-mediated interaction between lymphocytes and accessory cells, thus suppressing lymphocyte activation and subsequent function. Furthermore, decreased levels of adhesion molecule expression may result in a lowered ability of lymphocytes to make their way to sites of inflammatory or auto-immune activity. Such an effect may provide an explanation for the beneficial effects of fish oil feeding observed in disorders such as rheumatoid arthritis [ 11-13], psoriasis [ 14] and multiple sclerosis [ 15]. These effects of fish oil feeding upon adhesion molecule expression may also be useful in the prevention of graft rejection; indeed, a recent study has shown that feeding human kidney transplant recipients fish oil results in improved graft survival and function [ 16]. Interestingly, the other diets which were observed to result in a lowered level of adhesion molecule expression either on fresh or mitogen-stimulated lymphocytes or both (OO and EPO) have also been reported to have a beneficial effect in inflammatory disease [ 32, 33].

The results of the present study suggest that some dietary lipids, in particular fish oil, but also OO and EPO, exert immunomodulatory effects by influencing the expression of important molecules upon the lymphocyte cell surface. These effects may be due to changes in the fatty acid composition of membrane phospholipids. Feeding weanling rats on the diets used in the current study results in marked changes in the fatty acid composition of the phospholipids of spleen lymphocytes [ 31]. That these changes may influence the functioning of the cells is indicated by the observation that FCS can mask the effects of dietary lipid manipulation if it is used in ex vivo lymphocyte cultures rather than autologous serum [ 23]. The fatty acid composition of the phospholipids of spleen lymphocytes isolated from animals fed different dietary lipids is maintained when the cells are cultured in autologous serum [ 34]. In contrast, the diet-induced differences in the fatty acid composition of spleen lymphocyte phospholipids are lost if the cells are cultured in FCS or in serum-free medium [ 34].

How diet-induced changes in the fatty acid composition of lymphocyte phospholipids (or other lipid fractions) might affect the level of expression of cell surface molecules is unclear. They may act upon the pathways which lead to the synthesis of the molecules by affecting signal transduction or gene expression or both. Differential effects of fatty acids upon components of signal transduction pathways [ 35, 36] and upon gene expression [ 37-39] have been reported, with the n-3 PUFAs found in fish oil usually having the most potent effects. Given the potentially important clinical role of dietary lipid manipulation, it will be important to investigate the mechanism of the immunomodulatory action of these lipids further.

In this, and our previous studies [ 23-26, 31], rats have been fed diets containing 20% by weight of a particular lipid. Feeding this level of some lipids, particularly fish oil, clearly results in immunomodulatory effects. It will be important to investigate the effects of lower levels of these lipids, and also mixtures of different lipids, upon the properties and functions of cells of the immune system.

ACKNOWLEDGEMENTS

Peter Sanderson holds an MRC Studentship and Parveen Yaqoob holds a Post-Doctoral Fellowship from the Ministry of Agriculture, Fisheries and Food. We thank Dr David Horrobin and Scotia Pharmaceuticals for the generous gift of evening primrose oil.

TABLE 1. Food and energy intake, weight gain and final weight of rats fed different dietary lipids

Legend for Chart:

A - Diet
B - Food intake (g/day)
C - Energy intake (kcal/day)
D - Initial weight (g)
E - Final weight (g)
F - Weight gain (g)

A B C D
E F

LF 24.4 +/- 0.9[bcdf] 96.9 +/- 3.5 171.7 +/- 4.6
403.3 +/- 5.5[bcef] 231.7 +/- 7.6[bcef]

HCO 18.4 +/- 1.3[a] 92.6 +/- 5.2 169.7 +/- 3.3
452.8 +/- 7.9[a] 283.2 +/- 6.1[ad]

OO 18.0 +/- 0.9[a] 92.7 +/- 4.4 179.5 +/- 2.8
459.5 +/- 15.6[a] 280.0 +/- 15.0[a]

SO 18.3 +/- 0.9[a] 94.9 +/- 4.6 177.0 +/- 1.6
429.3 +/- 13.9[e] 252.3 +/ 13.9[bef]

EPO 18.4 +/- 1.0[a] 93.4 +/- 4.7 181.0 +/- 3.7
470.8 +/- 7.2[ad] 289.8 +/- 7.9[ad]

MO 18.6 +/- 1.0[a] 95.2 +/- 5.3 175.7 +/- 2.4
460.2 +/- 11.3[a] 284.5 +/- 9.5 [ad]

Male Lewis rats were fed for 12 weeks on the diets described in the text. Food intake and weight were monitored throughout the duration of the study. Data are mean +/- SEM for six animals fed each diet. Statistical significance (one-way analysis of variance) for p < 0.05 at least is indicated as follows: a VS LF; b vs HCO; c vs OO; d vs SO; e vs EPO; f vs MO.

TABLE 2. Effect of different dietary lipids upon the expression of lymphocyte surface markers

Legend for Chart:

A - Diet
B - CD4 &P
C - CD4 MF
D - CD8 &P
E - CD8 MF
F - TcR &P
G - TcR MF
H - CD2 %P
I - CD2 MF
J - LFA-1 MF

A B C D
E F G
H I J

LF 55.6 +/- 3.1 664 27.8 +/- 1.6
759 57.7 +/- 2.6 98
72.7 +/- 2.0 409[c][f] 399[c][f]

HCO 55.2 +/- 1.9 621 29.8 +/- 0.8
678 55.5 +/- 6.4 103
72.8 +/- 2.2 384[f] 396[c][f]

OO 49.4 +/ -4.4 615 27.1 +/- 2.2
731 55.3 +/- 4.4 102
66.4 +/- 3.3 362[a] 329[ab]

SO 55.5 +/- 2.0 652 28.3 +/- 1.8
732 59.4 +/- 2.8 105
70.5 +/- 1.4 392[f] 371[f]

EPO 51.3 +/- 1.1 593 31.2 +/- 3.2
697 56.4 +/- 3.7 105
64.8 +/- 2.3 374[f] 377[f]

MO 55.6 +/- 2.8 573 29.0 +/- 2.2
670 54.6 +/- 2.5 96
67.0 +/- 0.8 336[a][b][d][e] 313[a][b][d][e]

Pooled SD -- 49.1 --
52.1 -- 13.5
-- 26.6 35.2

Male Lewis rats were fed for 12 weeks on the diets described in the text. Spleen lymphocytes were prepared and the proportion of cells staining positive (% P) for various surface markers (CD4, CD8, T-cell receptor (TcR), CD2 and LFA- 1) and the extent of staining (mean fluorescence; MF) were determined using flow cytometry and appropriate monoclonal antibodies (see text). Data are mean +/- SEM for at least five animals fed each diet. Statistical significance (one-way analysis of variance for %P and two-way analysis of variance for MF; in the latter case pooled SD values are shown) for p < 0.05 at least is indicated as follows: a vs LF; b vs HCO; c vs 00; d vs SO; e vs EPO; f vs MO.

Since all lymphocytes express LFA-1%P data for this marker are omitted.

TABLE 3. Effect of different dietary lipids upon the expression of lymphocyte surface markers

Legend for Chart:

A - Diet
B - CD2/CD4 %DP
C - CD2/CD4 MF
D - CD2/CD8 %DP
E - CD2/CD8 MF
F - LFA-1/CD4 %DP
G - LFA-1/CD4 MF
H - LFA-1/CD8 %DP
I - LFA-1/CD8 MF

A B C D
E F G
H I

LF 46.3 +/- 2.8 337[bcef] 24.2 +/- 1.3
341[f] 38.3 +/- 1.8 231[c][f]
24.2 +/- 2.4 248[c][f]

HCO 46.5 +/- 2.6 291[a] 25.9 +/- 1.8
338[f] 36.5 +/- 2.2 218[c][f]
20.6 +/- 1.8 259[c][f]

OO 40.5 +/- 3.1 308[a][f] 22.7 +/- 1.7
311 40.1 +/- 3.3 179[a][b]
20.4 +/- 2.4 198[a][b]

SO 46.1 +/-1.8 327[b][f] 23.8 +/- 0.9
325[f] 37.9 +/- 0.9 201
21.0 +/- 2.7 214

EPO 41.3 +/-3.5 303[a][f] 23.6 +/- 1.5
315[f] 33.3 +/-1.8 204
25.3 +/- 3.5 234[f]

MO 43.0 +/- 1.2 274[a][c][d][e] 23.4 +/- 1.4
275[a][b][d][e] 34.7 +/- 1.8 166[a][b]
20.8 +/- 3.3 178[a][b][e]

Pooled SD -- 19.6 --
26.5 -- 22.9
-- 25.9

Male Lewis rats were fed for 12 weeks on the diets described in the text. Spleen lymphocytes were prepared and the proportion of cells staining double-positive (%DP) for various surface markers (CD2 and CD4 or CDS, LFA- 1 and CD4 or CD8) and the extent of staining (mean fluorescence; MF) were determined using flow cytometry and appropriate monoclonal antibodies (see text). Data are mean +/- SEM for at least five animals fed each diet. Statistical significance (one-way analysis of variance for %P and two-way analysis of variance for MF; in the latter case pooled SD values are shown) for p < 0.05 at least is indicated as follows: a vs LF; b vs HCO; c vs OO; d vs SO; e vs EPO: f vs MO.

TABLE 4. Effect of different dietary lipids upon the expression of markers on the surface of stimulated lymphocytes

Legend for Chart:

A - Diet
B - CD4 %P
C - CD4 MF
D - CD8 %P
E - CD8 MF
F - TcR %P
G - TcR MF
H - IL-2R %P
I - IL-2R MF
J - TfnR %P
K - TfnR MF

A B C
D E
F G
H I
J K

LF 53.7 +/- 1.5[c] 551
33.2 +/- 1.7 623[bdf]
62.6 +/- 3.8 72
47.9 +/- 7.9 359[c][d][e][f]
46.0 +/- 3.1 185

HCO 51.3 +/- 1.9 528
34.7 +/- 2.6 542[a]
63.4 +/- 3.2 74
47.3 +/- 5.4 325[c][d][e][f]
47.6 +/- 3.5 256[a][d]

OO 42.4 +/- 3.8[a][d][e][f] 498
32.9 +/- 2.2 585
56.8 +/- 6.2 75
48.1 +/- 7.4 226[a][b][f]
50.9 +/- 4.9 213

SO 55.2 +/- 1.0[c] 551
34.6 +/- 1.8 539[a]
62.8 +/- 6.0 75
46.7 +/- 8.2 234[a][b][f]
47.6 +/- 4.8 198

EPO 57.4 +/- 3.4[c] 503
37.7 +/- 4.8 565
63.1 +/- 6.8 74
45.0 +/- 6.4 189[a][b]
48.6 +/- 3.3 231

MO 58.2 +/- 7.5[c] 472
42.5 +/- 4.6 541[a]
72.4 +/- 4.0 77
46.9 +/- 5.6 134[a][b][c][d]
53.6 +/- 4.4 251[a][d]

Pooled SD -- 40.3
-- 28.2
-- 3.4
-- 43.1
-- 34.4

Male Lewis rats were fed for 12 weeks on the diets described in the text. Spleen lymphocytes were prepared and cultured for 24 h in the presence of the T-cell mitogen Con A. The proportion of cells staining positive (%P) for various surface markers (CD4, CD8, T-cell receptor (TcR), IL-2 receptor (IL-2R) and transferrin receptor (TfnR)) and the extent of staining (mean fluorescence; MF) were determined using flow cytometry and appropriate monoclonal antibodies (see text). Data are mean +/- SEM for at least five animals fed each diet. Statistical significance (one-way analysis of variance for %P and two-way analysis of variance for MF; in the latter case pooled SD values are shown) for p < 0.05 at least is indicated as follows a vs LF; b vs HCO; c vs OO; d vs SO; e vs EPO; f vs MO.

TABLE 5. Effect of different dietary lipids upon the expression of adhesion molecules on the surface of stimulated lymphocytes

Legend for Chart:

A - Diet
B - CD2 %P
C - CD2 MF
D - ICAM-1 %P
E - ICAM-1 MF
F - LFA-1 MF

A B C D
E F

LF 81.8 +/- 2.3 404[e][f] 56.2 +/- 4.4
269[d][e][f] 276[c][d][e][f]

HCO 79.8 +/- 2.6 357[f] 56.3 +/- 1.7
248[e][f] 272[e][f]

OO 74.3 +/- 3.0 334 64.9 +/- 5.2
221[f] 245[a]

SO 83.1 +/- 1.9 335 56.9 +/- 6.7
24l[a][f] 246[a]

EPO 81.8 +/- 5.6 315[b] 54.2 +/- 6.9
203[a][b] 240[a][b]

MO 88.9 +/- 2.8 297[a][b] 56.8 +/- 4.9
179[a][b][c][d] 225[a][b]

Pooled SD -- 31.0 --
27.4 22.0

Male Lewis rats were fed for 12 weeks on the diets described in the text. Spleen lymphocytes were prepared and cultured for 24 h in the presence of the T-cell mitogen Con A. The proportion of cells staining positive (%P) for various adhesion molecules (CD2, ICAM-I, LFA-I) and the extent of staining (mean fluorescence; MF) were determined using flow cytometry and appropriate monoclonal antibodies (see text). Data are mean +/- SEM for at least five animals fed each diet. Statistical significance (one-way analysis of variance for %P and two-way analysis of variance for MF; in the latter case pooled SD values are shown) for p < 0.05 at least is indicated as follows: "vs LF; b vs HCO; c vs OO; d vs SO; e vs EPO; f vs MO.

TABLE 6. Effect of different dietary lipids upon the expression of markers on the surface of stimulated lymphocytes

Legend for Chart:

A - Diet
B - MF CD2/CD4
C - MF CD2/CD8
D - MF IL-2R/CD4
E - MF IL-2R/CD8
F - MF TfnR/CD4
G - MF TfnR/CD8
H - MF LFA-1/CD4
I - MF LF-1/CD8
J - MF ICAM-1/CD4
K - MF ICAM-1/CD8

A B C D
E F G
H I J
K

L 335[e][f] 318[e][f] 240[cdef]
217[c][d][e][f] 150[b][f] 151
161 199 179
167

HCO 306[f] 301[e][f] 211[d][e][f]
195[e][f] 174[a][c][e] 176
167 212 177
157

OO 307[f] 275 172[a][f]
165[a] 156[b][f] 169
137 176 158
148

SO 308[e][f] 277 165[a][b]
155[a] 157[f] 168
158 173 161
147

EPO 286[a] 259[a][b] 154[a][b]
146[a][b] 153[b][f] 183
141 178 155
143

MO 273[a][b][c][d] 245[a][b] 126[a][b][c]
123[a][b] 180[a][c][d][e] 195
151 184 158
151

Pooled SD 22.2 28.2 28.6
31.4 12.4 30.0
19.7 29.9 15.2
14.6

Male Lewis rates were fed for 12 weeks on the diets described in the text. Spleen lymphocytes were prepared and cultured for 24 h in the presence of the T-cell mitogen Con A. The proportion of cells staining double-positive (%DP) for various surface markers (CD2 and CD4 or CD8, LFA-1 and CD4 or CD8, IL-2R and CD4 or CD8, TfnR and CD4 and CD8,1CAM-1 and CD4 and CD8) and the extent of staining (mean fluorescence; MF) were determined using flow cytometry and appropriate monoclonal antibodies (see text). Data are mean values for at least five animals fed each diet. Statistical significance (two-way analysis of variance; pooled SD values are shown) for p < 0.05 at least is indicated as follows:

a vs LF; b vs HCO; c vs OO; d vs SO; e vs EPO; f vs MO.

TABLE 7. Proliferation of spleen lymphocytes from rats fed different lipids

Stimulation
Diet index

LF 181.1 +/- 46.1[c][e][f]
HCO 123.2 +/- 22.7[e][f]
OO 63.5 +/- 20.1[a][e][f]
SO 86.8 +/- 27.9[e][f]
EPO 10.5 +/- 8.2[a][b][c][d]
MO 8.0 +/- 5.1[a][b][c][d]

Male Lewis rats were fed for 12 weeks on the diets described in the text. Spleen lymphocytes were prepared and cultured as described in [ 23]; proliferation was measured as thymidine incorporation over the final 18 h of a 66-h culture period and is expressed as stimulation index (see [ 23]). Data are mean +/- SEM for at least four animals fed each diet. Statistical significance (one-way analysis of variance) for p < 0.05 at least is indicated as follows: a vs LF; b vs HCO; c vs OO; d vs SO; e vs EPO; f vs MO.

TABLE 8. NK cell activity of spleen lymphocytes from rats fed different lipids

Legend for Chart:

A - Diet L:T
B - NK cell activity (% cytolysis), 100:1
C - NK cell activity (% cytolysis), 50:1
D - NK cell activity (% cytolysis), 25:1
E - NK cell activity (% cytolysis), 12.5:1

A B C
D E

LF 39.7 +/- 2.9[c][e][f] 24.7 +/- 1.7[b][c][e][f]
8.7 +/- 1.6[c][e] 3.1 +/- 1.1

HCO 44.9 +/- 2.1[c][d][e][f] 31.8 +/- 2.6[a][c][d][e][f]
11.5 +/- 0.9[c][d][e][f] 5.0 +/- 0.8[c][d][ef]

tO 28.7 +/- 1.7[a][b] 15.6 +/- 1.7[a][b][d]
4.4 +/- 0.9[a][b] 1.7 +/- 0.5[b]

SO 35.5 +/- 3.6[b] 22.5 +/- 2.3[b][c][f]
7.2 +/- 1.2[b] 2.2 +/- 0.7[b]

EPO 29.6 +/- 2.9[a][b] 17.4 +/- 1.9[a][b]
5.1 +/- 1.1[a][b] 2.4 +/- 0.6[b]

MO 29.5 +/- 3.3[a][b] 15.5 +/- 3.0[a][b][d]
5.7 +/- 1.1[b] 2.4 +/- 0.7[b]

Male Lewis rats were fed for 12 weeks on the diets described in the text. Spleen lymphocytes were prepared and natural killer cell activity was determined at different lymphocyte:target cell ratios (L:T) as described in [ 25]. Data are mean +/- SEM for at least four animals fed each diet. Statistical significance (one-way analysis of variance) for p < 0.05 at least is indicated as follows: a vs LF; b vs HCO; c vs OO; d vs SO; e vs EPO; f vs MO.
REFERENCES

[1] Weyman C, Morgan SJ, Belin Jet al. Phytohaemmagglutinin stimulation of human lymphocytes: effect of fatty acids on uridine uptake and phosphoglyceride fatty acid profile. Biochim Biophys Acta 1977; 496: 155-46.

[2] Buttke TM. Inhibition of lymphocyte proliferation by free fatty acids. I. Differential effects on mouse B and T lymphocytes. Immunology 1984; 53: 235-42.

[3] Calder PC, Bond JA, Bevan SJ et al. Effect of fatty acids on the proliferation of concanavalin A-stimulated rat lymph node lymphocytes. Int J Biochem 1991; 23: 579-88.

[4] Calder PC, Beyan SJ, Newsholme EA. The inhibition of T-lymphocyte proliferation by fatty acids is via an eicosanoid-independent mechanism. Immunology 1992; 75: 108-15.

[5] Calder PC, Newsholme EA. Unsaturated fatty acids suppress interleukin-2 production and transferrin receptor expression by concanavalin A-stimulated rat lymphocytes. Mediat Inflamm 1992; 1: 107-15.

[6] Calder PC, Newsholme EA. Polyunsaturated fatty acids suppress human peripheral blood lymphocyte proliferation and interleukin-2 production. Clin Sci 1992; 82: 695-700.

[7] Soyland E, Nenseter MS, Braathen Let al. Very long chain n-3 and n-6 polyunsaturated fatty acids inhibit proliferation of human T lymphocytes in vitro. Eur J Clin Invest 1993; 23:112-21.

[8] Virella G, Kilpatrick JM, Rugeles MT et al. Depression of humoural responses and phagocytic functions in vivo and in vitro by fish oil and eicosapentaenoic acid. Clin Immunol Immunopathol 1989; 52: 277-90.

[9] Yamashita N, Maruyama M, Yamazaki K et al. Effect of eicosapentaenoic and docosahexaenoic acid on natural killer cell activity in human peripheral blood lymphocytes. Clin Immunol Immunopathol 1991; 59: 335-45.

[10] Fujikawa M, Yamashita N, Yamazaki K et al. Eicosapentaenoic acid inhibits antigen-presenting cell function of murine splenocytes. Immunology 1992; 75: 330-35.

[11] Kremer JM, Bigauoette J, Michalek AU et al. Effects of manipulation of dietary fatty acids on clinical manifestations of rheumatoid arthritis. Lancet 1985; i: 184-7.

[12] Kremer JM, Jubiz W, Michalek Aet al. Fish oil fatty acid supplementation in active rheumatoid arthritis. Ann Intern Med 1987; 106: 497-503.

[13] Kremer JM, Lawrence DA, Jubiz Wet al. Dietary fish oil and olive oil supplementation in patients with rheumatoid arthritis. Arthritis Rheum 1990; 33: 810-20.

[14] Bittiner SB, Tucker WFG, Cartwright et al. A double-blind, randomised, placebo-controlled trial of fish oil in psoriasis. Lancet 1988; i: 378-80.

[15] Bates D, Cartlidge NEF, French JM et al. A double-blind controlled trial of long chain n-3 polyunsaturated fatty acids in the treatment of multiple sclerosis. J Neurol Neurosurg Psychol 1989; 52: 18-22.

[16] Homan van der Heide JJ, Bilo HJG, Donker JM et al. Effect of dietary fish oil on renal function and rejection in cyclosporine-treated recipients of renal transplants. N Engl J Med 1993; 329: 769-73.

[17] Levy JA, Ibrahim AB, Shiral I et al. Dietary fat affects immune response, production on antiviral factors and immune complex disease in NZB/NZW mice. Proc Natl Acad Sci USA 1982; 79: 1974-8.

[18] Erickson KL, Adams DA, McNeill CJ. Dietary lipid modulation of immune responsiveness. Lipids 1983; 18: 468-74.

[19] Mark DA, Kim YT, Quimby F. Influence of diet on immune function, prostaglandin biosynthesis and cardiovascular pathology function of MRL/LPR and MRL/N mice. Fed Proc 1983; 42: 1188.

[20] Locniskar M, Nauss Newberne PM. The effect of quality and quantity of dietary fat on the immune system. J Nutr 1983; 113: 951-61.

[21] de Deckere EAM, Verplancke CJ, Blonk CG et al. Effects of type and amount of dietary fat on rabbit and rat lymphocyte proliferation in vitro. J Nutr 1988; 118: 11-18.

[22] Ossman JB, Erickson KL, Canolty NL. Effects of saturation and concentration of dietary fats on lymphocyte transformation in mice. Nutr Rep Int 1980; 22: 279-84.

[23] Yaqoob P, Newsholme EA, Calder PC. The effect of dietary lipid manipulation on rat lymphocyte subsets and proliferation. Immunology 1994; 82: 603-10.

[24] Yaqoob P, Newsholme EA, Calder PC. The effect of dietary lipid manipulation on leucocyte proliferation in whole blood. Nutr Res 1995; 15: 279-87.

[25] Yaqoob P, Newsholme EA, Calder PC. Inhibition of natural killer cell activity by dietary lipids. Immunol Lett 1994; 41: 241-7.

[26] Yaqoob P, Sherrington EJ, Jeffery NM et al. Comparison of the effects of a range of dietary lipids upon serum and tissue lipid composition in the rat. Int J Biochem Cell Biol 1995; 27: 297-310.

[27] Ariel A, Naval J, Gonzalea Bet al. Fatty acid metabolism in human lymphocytes. I. Time course changes in fatty acid composition and membrane fluidity during blastic transformation of peripheral blood lymphocytes. Biochim Biophys Acta 1990; 1044: 332-9.

[28] Calder PC, Yaqoob P, Harvey DJ et al. Incorporation of fatty acids by concanavalin A-stimulated lymphocytes and the effect on fatty acid composition and membrane fluidity. Biochem J 1994; 300: 509-18.

[29] Calder PC, Yaqoob P, Newsholme EA. Triacylglycerol metabolism by lymphocytes and the effect of triacylglycerols on lymphocyte proliferation. Blochem J 1994; 298: 605-11.

[30] Brouard C, Pascaud M. Effects of moderate dietary supplementations with n-3 fatty acids on macrophage and lymphocyte phospholipids and macrophage eicosanoid synthesis in the rat. Blochem Biophys Acta 1990; 1047: 19-28.

[31] Yaqoob P, Harvey DJ, Newsholme EA et al. Dietary lipid manipulation alters lymphocyte phospholipid fatty acid composition but not membrane fluidity. Proc Nutr Soc 1994; 53: 70A.

[32] Belch JJF, Ansell D, Madhok R et al. Effects of altering dietary essential fatty acids on requirements for non-steroidal anti-inflammatory drugs in patients with rheumatoid arthritis: a double-blind placebo controlled study. Ann Rheum Dis 1988; 47: 96-104.

[33] Linos A, Kaklamaris E, Kontomerkos A et al. The effect of olive oil and fish consumption on rheumatoid arthritis--a case control study. Scand J Rheumatol 1991; 20: 419-26.

[34] Yaqoob P, Newsholme EA, Calder PC. Influence of cell culture conditions on diet-induced changes in lymphocyte fatty acid composition. Blochim Biophys Acta 1995; 1255: 333-40.

[35] Sumida C, Graber R, Nunez E. Role of fatty acids in signal transduction: modulators and messengers. Prostaglandins Leukot Essent Fatty Acids 1993; 48:117-22.

[36] May CL, Southworth AJ, Calder PC. Inhibition of lymphocyte protein kinase C by unsaturated fatty acids, Biochem Biophys Res Commun 1993; 195: 823-8.

[37] Fernandes G, Venkatraman J, Khare A et al. Modulation of gene expression in autoimmune disease and ageing by food restriction and dietary lipids. Proc Soc Exp Biol Med 1990; 193: 16-22.

[38] Tebbey PW, Buttke TM. Independent arachidonic acid-mediated gene regulatory pathways in lymphocytes. Biochem Biophys Res Commun 1993; 194: 862-8.

[39] Chandrasekar B, Fernandes G. Decreased pro-inflammatory cytokines and increased antioxidant enzyme gene expression by n-3 lipids in murine lupus nephritis. Biochem Biophys Res Commun 1994; 200: 893-8.

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By PETER SANDERSON BSc, PARVEEN YAQOOB DPHiL AND PHILIP C. CALDER PHD DPHIL, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

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