Fiona A. Wallace

Fatty acids and lymphocyte functions

The immune system acts to protect the host against pathogenic invaders. However, components of the immune system can become dysregulated such that their activities are directed against host tissues, so causing damage. Lymphocytes are involved in both the beneficial and detrimental effects of the immune system. Both the level of fat and the types of fatty acid present in the diet can affect lymphocyte functions. The fatty acid composition of lymphocytes, and other immune cells, is altered according to the fatty acid composition of the diet and this alters the capacity of those cells to produce eicosanoids, such as prostaglandin E2, which are involved in immunoregulation. A high fat diet can impair lymphocyte function. Cell culture and animal feeding studies indicate that oleic, linoleic, conjugated linoleic, gamma-linolenic, dihomo-gamma-linolenic, arachidonic, alpha-linolenic, eicosapentaenoic and docosahexaenoic acids can all influence lymphocyte proliferation, the production of cytokines by lymphocytes, and natural killer cell activity. High intakes of some of these fatty acids are necessary to induce these effects. Among these fatty acids the long chain n-3 fatty acids, especially eicosapentaenoic acid, appear to be the most potent when included in the human diet. Although not all studies agree, it appears that fish oil, which contains eicosapentaenoic acid, down regulates the T-helper 1-type response which is associated with chronic inflammatory disease. There is evidence for beneficial effects of fish oil in such diseases; this evidence is strongest for rheumatoid arthritis. Since n-3 fatty acids also antagonise the production of inflammatory eicosanoid mediators from arachidonic acid, there is potential for benefit in asthma and related diseases. Recent evidence indicates that fish oil may be of benefit in some asthmatics but not others.

Dietary fatty acids influence the production of Th1- but not Th2-type cytokines

C57Bl6 mice were fed for 6 weeks on a low‐fat diet or on high‐fat diets containing coconut oil (rich in saturated fatty acids), safflower oil [rich in n‐6 polyunsaturated fatty acids (PUFAs)], or fish oil (rich in n‐3 PUFAs) as the main fat sources. The fatty acid composition of the spleen lymphocytes was influenced by that of the diet fed. Thymidine incorporation into concanavalin A‐stimulated spleen lymphocytes and interleukin (IL)‐2 production were highest after feeding the coconut oil diet. Interferon (IFN)‐γ production was decreased by safflower oil or fish oil feeding. IL‐4 production was not significantly affected by diet, although production was lowest by lymphocytes from fish oil‐fed mice. The ratio of production of Th1‐ to Th2‐type cytokines (determined as the IFN‐γ/IL‐4 ratio) was lower for lymphocytes from mice fed the safflower oil or fish oil diets. After 4 h of culture, IL‐2 mRNA levels were higher in cells from mice fed coconut oil, and IFN‐γ mRNA levels were higher in cells from mice fed coconut oil or safflower oil. After 8 h of culture, IL‐2, IFN‐γ, and IL‐4 mRNA levels were lowest in cells from mice fed fish oil. The ratio of the relative levels of IFN‐γ mRNA to IL‐4 mRNA was highest in cells from mice fed coconut oil and was lowest in cells of mice fed fish oil. The influence of individual fatty acids on IL‐2 production by murine spleen lymphocytes was examined in vitro. Although all fatty acids decreased IL‐2 production in a concentration‐dependent manner, saturated fatty acids were the least potent and n‐3 PUFAs the most potent inhibitors, withn‐6 PUFAs falling in between in terms of potency. It is concluded that saturated fatty acids have minimal effects on cytokine production. In contrast, PUFAs act to inhibit production of Th1‐type cytokines with little effect on Th2‐type cytokines; n‐3 PUFAs are particularly potent. The effects of fatty acids on cytokine production appear to be exerted at the level of gene expression.

Effect of low-to-moderate amounts of dietary fish oil on neutrophil lipid composition and function

Although essential to host defense, neutrophils are also involved in numerous inflammatory disorders including rheumatoid arthritis. Dietary supplementation with relatively large amounts of fish oil [containing >2.6 g eicosapentaenoic acid (EPA) plus 1.4 g docosahexaenoic acid (DHA) per day] can attenuate neutrophil functions such as chemotaxis and superoxide radical production. In this study, the effects of more moderate supplementation with fish oil on neutrophil lipid composition and function were investigated. The rationale for using lower supplementary doses of fish oil was to avoid adverse gastrointestinal problems, which have been observed at high supplementary concentrations of fish oil. Healthy male volunteers aged <40 yr were randomly assigned to consume one of six dietary supplements daily for 12 wk (n=8 per treatment group). The dietary supplements included four different concentrations of fish oil (the most concentrated fish oil provided 0.58 g EPA plus 1.67 g DHA per day), linseed oil, and a placebo oil. The percentages of EPA and DHA increased (both P<0.05) in neutrophil phospholipids in a dose-dependent manner after 4 wk of supplementation with the three most concentrated fish oil supplements. No further increases in EPA or DHA levels were observed after 4 wk. The percentage of arachidonic acid in neutrophil phospholipids decreased (P<0.05) after 12 wk supplementation with the linseed oil supplement or the two most concentrated fish oil supplements. There were no significant changes in N-formyl-met-leu-phe-induced chemotaxis and superoxide radical production following the dietary supplementations. In conclusion, low-to-moderate amounts of dietary fish oil can be used to manipulate neutrophil fatty acid composition. However, this may not be accompanied by modulation of neutrophil functions such as chemotaxis and superoxide radical production.

Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects

Studies on animal and human subjects have shown that greatly increasing the amount of linseed (also known as flaxseed) oil (rich in the n-3 polyunsaturated fatty acid (PUFA) alpha-linolenic acid (ALNA)) or fish oil (FO; rich in the long-chain n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) in the diet can decrease a number of markers of immune function. The immunological effects of more modest doses of n-3 PUFA in human subjects are unclear, dose-response relationships between n-3 PUFA supply and immune function have not been established and whether ALNA has the same effects as its long-chain derivatives is not known. Therefore, the objective of the present study was to determine the effect of enriching the diet with different doses of FO or with a modest dose of ALNA on a range of functional responses of human monocytes and lymphocytes. In a randomised, placebo-controlled, double-blind, parallel study, forty healthy males aged 18-39 years were randomised to receive placebo or 3.5 g ALNA/d or 0.44, 0.94 or 1.9 g (EPA+DHA)/d in capsules for 12 weeks. The EPA:DHA ratio in the FO used was 1.0:2.5. ALNA supplementation increased the proportion of EPA but not DHA in plasma phospholipids. FO supplementation decreased the proportions of linoleic acid and arachidonic acid and increased the proportions of EPA and DHA in plasma phospholipids. The interventions did not alter circulating mononuclear cell subsets or the production of tumour necrosis factor-alpha, interleukin (IL) 1beta, IL-2, IL-4, IL-10 or interferon-gamma by stimulated mononuclear cells. There was little effect of the interventions on lymphocyte proliferation. The two higher doses of FO resulted in a significant decrease in IL-6 production by stimulated mononuclear cells. It is concluded that, with the exception of IL-6 production, a modest increase in intake of either ALNA or EPA+DHA does not influence the functional activity of mononuclear cells. The threshold of EPA+DHA intake that results in decreased IL-6 production is between 0.44 and 0.94 g/d.

Dietary fish oil reduces intercellular adhesion molecule 1 and scavenger receptor expression on murine macrophages

During atherogenesis, a pathological accumulation of lipids occurs within aortic intimal macrophages through uptake of oxidised low-density lipoprotein (LDL) via scavenger receptors. Here we investigate whether some of the anti-atherosclerotic effects ascribed to dietary fish oil are mediated through effects on macrophage intercellular adhesion molecule 1 (ICAM-1) and scavenger receptor expression. Mice were fed on a low fat diet (containing 25 g/kg corn oil) or on high fat diets containing 200 g/kg coconut oil, safflower oil or fish oil. Thioglycollate-elicited peritoneal macrophages were analysed for fatty acid composition by gas chromatography. Macrophage scavenger receptor A (MSR-A) type I+type II and ICAM-1 expression were measured by flow cytometry and the levels of mRNA coding for MSR-A type I, MSR-A type II and ICAM-1 were measured by reverse-transcription polymerase chain reaction. Feeding mice diets enriched with different fats resulted in significant changes in the fatty acid profile of macrophages, which reflected the fatty acid compositions of the diets. Macrophages from the fish oil fed mice had the lowest expression of ICAM-1 and MSR-A at the level of both mRNA and cell surface expression. The reduced expression of ICAM-1 and MSR-A on macrophages from mice fed on a fish oil-rich diet supports our hypothesis that part of the protective effect of fish oil against atherosclerosis might be due to an altered macrophage phenotype and function ameliorating macrophage-induced plaque formation.

Dietary fats affect macrophage-mediated cytotoxicity towards tumour cells.

In the present study, the effects of feeding mice diets of different fatty acid compositions on the production of TNF‐α and nitric oxide by lipopolysaccharide‐stimulated peritoneal macrophages and on macrophage‐mediated cytotoxicity towards L929 and P815 cells were investigated. C57Bl6 mice were fed on a low‐fat (LF) diet or on high‐fat diets (21% fat by weight), which included coconut oil (CO), olive oil (OO), safflower oil (SO) or fish oil (FO) as the principal fat source. The fatty acid composition of the macrophages was markedly influenced by that of the diet fed. Lipopolysaccharide (LPS)‐stimulated macrophages from FO‐fed mice showed significantly lower production (up to 80%) of PGE2 than those from mice fed on each of the other diets. There was a significant positive linear correlation between the proportion of arachidonic acid in macrophage lipids and the ability of macrophages, to produce PGE2. Lipopolysaccharide‐stimulated TNF‐α production by macrophages decreased with increasing unsaturated fatty acid content of the diet (i.e. FO < SO < OO < CO < LF). Macrophages from FO‐fed mice showed significantly lower production of TNF‐α than those from mice fed on each of the other diets. Nitrite production was highest for LPS‐stimulated macrophages from mice fed on the LF diet. Macrophages from FO‐fed mice showed significantly higher production of nitrite than those from mice fed on the OO and SO diets. Compared with feeding the LF diet, feeding the CO, OO or SO diets significantly decreased macrophage‐ mediated killing of P815 cells (killed by nitric oxide). Fish oil feeding did not alter killing of P815 cells by macrophages, compared with feeding the LF diet; killing of P815 cells was greater after FO feeding than after feeding the other high fat diets. Compared with feeding the LF diet, feeding the OO or SO diets significantly decreased macrophage‐mediated killing of L929 cells (killed by TNF). Coconut oil or FO feeding did not alter killing of L929 cells by macrophages, compared with feeding the LF diet. It is concluded that the type of fat in the diet affects macrophage composition and alters the ability of macrophages to produce cytotoxic and immunoregulatory mediators and to kill target tumour cells.

Dietary glutamine enhances cytokine production by murine macrophages

To examine the effects of dietary glutamine on cytokine production by macrophages, mice were fed for 2 wk on a control diet that included 200.0 g casein/kg providing 19.6 g glutamine/kg or a glutamine-enriched diet that provided 54.8 g glutamine/kg partly at the expense of casein. There were no differences in weight gain between animals fed the two diets. The plasma concentrations of a number of amino acids differed according to the diet fed; this variation largely reflected the variation in the levels of the different amino acids in the diets. Plasma glutamine concentration was not significantly affected by dietary glutamine level. The production of three cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, was greater for lipopolysaccharide-stimulated macrophages from mice fed the glutamine-enriched diet. Thus, increasing the amount of glutamine in the murine diet enhances the ability of macrophages to respond to stimulation, at least in terms of cytokine production. These observations suggest that increasing the availability of glutamine orally could promote immune responses involving macrophage-derived cytokines.

An olive oil-rich diet reduces scavenger receptor mRNA in murine macrophages

During atherogenesis, a pathological accumulation of lipids occurs within aortic intimal macrophages through uptake of oxidised LDL via scavenger receptors. Here we investigated whether some of the anti-atherosclerotic effects ascribed to an olive oil rich-diet are mediated through effects on macrophage scavenger receptors (MSR). Male C57 Bl6 mice aged 6 weeks were fed for 12 weeks on a low-fat diet (containing 25 g corn oil/kg) or on high-fat diets containing 200 g coconut oil, olive oil or safflower oil/kg. Thioglycollate-elicited peritoneal macrophages were analysed for fatty acid composition by GC and the levels of mRNA coding for three MSR (MSRA type I, MSRA type II and CD36) were measured by reverse-transcription polymerase chain reaction. Feeding mice diets enriched with different fats resulted in significant differences in the fatty acid profile of macrophages, which reflected the fatty acid compositions of the diets. These differences were accompanied by a lower level of mRNA for MSRA type I, MSRA type II and CD36 in macrophages from mice fed an olive-oil-enriched diet compared with the mice fed on the low-fat diet. These data suggest that part of the protective effect of olive oil against atherosclerosis might be via reducing macrophage uptake of oxidised LDL. Whether this effect is due to the downregulation of gene transcription directly by unsaturated fatty acids or is the result of the effect of monounsaturated fatty acids or other components of olive oil on LDL composition and oxidation remains to be ascertained.

DIETARY FAT INFLUENCES THE PRODUCTION OF TH1- BUT NOT TH2-DERIVED CYTOKINES

Diets enriched in long-chain n-3 polyunsaturated fatty acids decreased the production of interleukin-2 (IL-2) by murine and human lymphocytes (1,2). IL-2 and interferon-], (IFN-y) are produced by the Thl class of lymphocytes involved in cell-mediated immunity, macrophage and natural killer cell activation, transplant rejection, and inflammation. There is little information about the effects of dietary fatty acids on the production of IFN-y or on the production of cytokines such as IL-4 which are produced by the Th2 class of lymphocytes involved in antibody-dependent responses and allergic reactions. Therefore, in the current study the effect of feeding mice diets enriched in different types of fatty acids upon IL2, IFN-y, and IL-4 production by spleen lymphocytes was investigated. Male C57B1/6 mice (n = 6 per diet) were fed for 6 wk on diets containing 200 g/kg of either coconut oil (CO), olive oil (OO), safflower oil (SO), or fish oil (FO). Spleen lymphocytes were prepared by standard procedures (3) and 2 x t06 cells were cultured for 24 h in the presence of 2.5 Bg/mL concanavalin A, a T-cell mitogen; the final culture volume was 2 mL and the cultures contained 5% (vol/vol) fetal calf serum. The concentrations of the cytokines IL-2, IFN-y, and IL-4 in the culture medium were measured by commercially available ELISA kits (Biosource International, Camarillo, CA). Lymphocyte proliferation was assessed as concanavalin Astimulated [3H]thymidine incorporation over the final 18 h of a 66-h culture period (3). Each of the diets rich in unsaturated fatty acids (OO, SO, FO) decreased lymphocyte proliferation (i.e., thymidine incorporation) and IL-2 production compared with feeding the CO diet (Table 1). IFN-y production was reduced by SO or FO feeding compared with feeding the COor OO-rich diets (Table 1). IL-4 production was not significantly affected by the type of fat in the diet, although production was lowest by cells from FO-fed mice (Table 1). The effects of the unsaturated fatty acid-rich diets on spleen lymphocyte proliferation are in agreement with those published previously in a variety of species (3). Furthermore

Reduction of scavenger receptor expression and function by dietary fish oil is accompanied by a reduction in scavenger receptor mRNA.

During atherosclerosis, macrophages (MO) within plaques take up oxidized low density lipoprotein (LDL) via the scavenger receptor (SR) to become lipid-laden foam cells. Dietary fish oil (FO) provides some protection against the development of atherosclerosis in a variety of species including mice, pigs, nonhuman primates, and man (1,2). We hypothesized that at least part of this beneficial effect might be due to a reduction in SR expression on MO. Thus, in this study the effect of feeding mice high-fat diets with different fatty acid compositions upon class A SR (SRA) expression (mRNA and protein) and function [uptake of acetylated-LDL (Ac-LDL)] was investigated. Male C57B1/6 mice were fed for 12 wk on a low-fat (LF) diet (25 g corn oil/kg) or on diets containing 200 g/kg of either coconut oil (CO), olive oil (OO), safflower oil (SO), or fish oil (FO). Four days prior to sacrifice, the mice were injected intraperitoneally with thioglycolate broth to elicit M ~ migration to the peritoneal cavity. At sacrifice the MO were collected by lavaging the peritoneal cavity. Flow cytometry was used to assess cell surface SRA expression (staining with a fluorescently-labeled monoclonal antibody [clone 2F8; Serotec, Kidlington, United Kingdom]) and function (uptake of fluorescently-labeled Ac-LDL [Biogenesis, Poole, United Kingdom] over 1 h); flow cytometry data are expressed as % of positive cells and median fluorescence intensity (MFI). The 2F8 monoclonal antibody recognises both the type I and type II forms of the SRA (3) and both forms are involved in Ac-LDL uptake by MO. RNA was extracted, and SRA type 1 and type II mRNA levels were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR); mRNA levels are expressed relative to cyclophilin mRNA levels also determined by RT-PCR. FO feeding significantly reduced the percentage of SRApositive MO compared with the other high-fat diets (Table 1). This was mirrored by a trend toward a reduced number of MO able to take up Ac-LDL following FO feeding. The level of