Fatty Acids and Immune Functions
Darshan S. Kelley1
Dietary lipids comprise mainly triglycerides and only small amounts of phospholipids, cholesterol, and other sterols. Chemically triglycerides are the triacylglycerols or glycerol molecules esterified with three fatty acids. Fatty acids without any double bond in their carbon chain are called saturated, and those with one or more double bonds are called unsaturated. Unsaturated fatty acids with more than one double bond are called polyunsaturated fatty acids (PUFAs). Based on the position of the first double bond from the methylene end, unsaturated fatty acids are classified into the n-3, n-6, and the n-9 series, which cannot be interconverted in animals. The first double bond for the n-3 series is between C3 and C4 from the methylene end, for the n-6 series it is between C6 and C7, and for the n-9 series it is between C9 and C10. Examples of the saturated fatty acids include palmitic acid, 16:0, and stearic acid, 18:0; for the n-9 type include palmitoleic acid, 16:1n-9, and oleic acid, 18:1n-9; for the n-6 type, linoleic acid, 18:2n-6 (LA), and arachidonic acid, 20:4n-6 (AA); for the n-3 type, α-linolenic acid, 18:3n-3 (ALA), eicosapentaenoic acid, 20:5n-3
(EPA), and docosahexaenoic acid, 22:6n-3 (DHA). Dietary sources of fatty acids include animal fats, coconut and palm oils for saturated fatty acids; olive and canola oils for oleic acid; sunflower, corn, and soybean oils for LA; organ meats and eggs for AA; flaxseed and pyrilla oils for ALA; marine oils for EPA and DHA. Humans cannot synthesize PUFA of the n-3 and n-6 series, which are termed essential fatty acids (EFAs); they can however, elongate LA to AA and convert ALA to EPA and DHA by a complex pathway. The same desaturases and elongases are involved in the elongation of the n-3 and n-6 PUFA, and these enzymes seem to have a preference for n-3 over the n-6 PUFA.
AA is the major PUFA in most cell membranes, and it is a precursor for a number of compounds termed eicosanoids. AA is converted into prostaglandins, prostacyclins, and thromboxanes through the cyclooxygenase pathway and to leukotrienes and lipoxins through the lipoxygenase pathway. Depending on their concentration and type, prostaglandins and leukotrienes stimulate or inhibit the activity of the immune cells. Other 20-C fatty acids, like dihomo-gamma linolenic acid (DGLA, 20:3n-6) and EPA compete with AA for the lipoxygenase and cyclooxygenase enzymes and reduce the products formed from AA, including eicosanoids of the 2-series and leukotriene B4, which are potent modulators of the immune cells.
Both the total fat intake and the ratios between fatty acids of different classes influence the activity of immune cells. Such information was initially obtained through epidemiological human studies and studies conducted with cultured cells and animal models. These studies showed that EFAs are required for the growth and maintenance of the immune cells, and FFAs (free fatty acids) are produced and secreted during the activation of these cells. A number of intervention studies regarding the effects of the amount and composition of dietary fat on human immune response have been conducted in the last decade, result from these human studies are discussed here.
Amount of Fat Intake and Human Immune Response
Reduction in total fat intake has been found to enhance immune response (IR) in humans as noted in several studies. We noted that the proliferation of peripheral blood mononuclear cells (PBMCs) cultured with T- and B-cell-specific mitogens almost doubled when the level of fat in the diet of healthy men was reduced from 30 to 25 energy percent (en%) for 11 weeks (Kelley et al., 1989). Similar results were found in another study conducted in healthy women whose fat intake was reduced from 40 en% to 25 or 31 en% for 6 weeks (Kelley et al., 1992a). In these studies, the reduction in fat intake also resulted in an increase in the number of circulating T- and B-lymphocytes, while the ratio between the Th-(helper) and Ts-(suppressor) cells did not change. An increase in the proliferation of PBMCs in response to the T-cell mitogen concanavalin A (Con-A) and in the in vitro secretion of interleukin (IL)-1 and tumor necrosis factor (TNF) was also found in a group of elderly men and women when their
fat intake was reduced from 36 to 27 en% for 6 months (Meydani et al., 1993). An increase in PBMC proliferation suggests a faster response in the event of pathogenic attack. Results from two other studies show an increase in the number (Rasmussen et al., 1994) and activity (Barone et al., 1989) of natural killer (NK) cells when the fat intake was reduced by about 10 en%. Other indices of IR tested in these studies, including delayed-type hypersensitivity (DTH) and the secretion of IL-6 were not altered, which suggests that not all indices of IR are equally affected by the reduction in fat intake. This is to be expected because different immune cells have different half lives and respond to different stimuli.
n-6 Pufa and Human IR
The intake of n-6 PUFA was also changed in the two studies dealing with total fat intake mentioned earlier (Kelley et al., 1989, 1992a). In one of the studies, the level of LA was changed from 5 en% to 3 en% for one-half of the subjects and to 13 en% for the remaining half of subjects for 11 weeks (Kelley et al., 1989). The other study was a cross-over type, wherein each subject was fed a diet with 3 and 10 en% LA for 6 weeks (Kelley et al., 1992a), and the stabilization diet contained 5 en% LA. In these two studies, changing the level of LA from 3 to 13 en% had no adverse effect on several indices of IR tested, when the total fat contents of the intervention diets were maintained constant. Inhibition of IR may have happened if the increase in n-6 PUFA was accompanied by an increase in total fat intake. When the increase in LA intake was accompanied with an increase in total fat (22 to 28 en%), it did inhibit NK cell activity in the healthy men (Barone et al., 1989). In this study, inhibition may have resulted from the additive inhibitory effects of total fat and of n-6 PUFA. In general, these studies indicate that moderate levels of LA consumption have no adverse effects on human IR. The plasma and adipose tissue levels of n-6 PUFA presumably reflect their dietary intake, and significant negative correlations were found between NK cell activity and the plasma levels of total PUFA, n-6 PUFA, and LA (Rasmussen et al., 1994) in a group of Danish men with mean age of 71 years. However, no correlation was found between the adipose tissue PUFA and NK cell activity or PBMC proliferation in American men with mean age of 47 years (Berry et al., 1987). The difference in the age of the subjects and the tissue being investigated may account for the difference in these two studies. Other factors that may explain inconsistencies between some of the results from various studies include differences in total fat intake, antioxidant nutrients, duration of feeding, and the index being examined.
In the above studies regarding n-6 PUFA, the level of LA and not that of AA in the diets was changed. LA is rapidly metabolized with only a limited conversion to AA. Studies conducted in animals indicate that dietary AA may be metabolized differently from the endogenously synthesized AA (Whelan et al., 1993), and preliminary human studies indicated harmful effects of dietary
AA (Seyberth et al., 1975). AA and its metabolites have been found to inhibit activity of immune cells in vitro. In a recent study, this author examined the effect of dietary AA on IR and several other health parameters of young men (Kelley et al., 1997). Ten healthy men were fed a basal diet containing 30 en% fat (10:10:10; saturated: monounsaturated: polyunsaturated) and 200 mg/d of AA for 15 days, after which the diet of six men was supplemented with 1.5 g of additional AA from ARASCO Oil (Martek Biosciences Corporation, Columbia, Md.) for 50 days, and the other four men remained on the basal diet. The diets of the two groups were crossed over for the next 50 days. AA supplementation had no adverse effect on a number of indices of IR tested, including DTH response, NK cell activity, lymphocyte proliferation in response to Con-A, phytohemagglutinin (PHA), and pokeweed mitogens, as well as in vitro secretion of IL-1, IL-2, and TNF. However, it significantly increased the number of circulating neutrophils and the secondary response to influenza vaccine. The feeding of the low-fat, nutritionally balanced diet was found in this study to enhance several indices of IR including PBMC proliferation and cytokine production, which was also found in the earlier studies from this laboratory (Kelley et al., 1989, 1992a). The results of this study show that moderate levels of AA fed as natural triglycerides have no adverse effects on human IR, even if AA is a precursor for the inflammatory eicosanoids. It is possible that higher amounts of AA may have adverse effects on human IR; however, for nutritional purposes, the amount of AA fed was adequate.
n-3 Pufa and Human Immune Response
In the last few years, interest in examining the effect of n-3 PUFA on immune status has increased, because these fatty acids have been found (1) to be beneficial in the management of some human autoimmune diseases and (2) to reduce the incidence of certain types of cancer in animal models. Studies examining the effect of n-3 PUFA on human IR have used both plant sources (containing ALA) and marine oils (containing EPA and DHA). Most of these human studies indicate inhibition of human IR by n-3 PUFA. Adding flax seed oil to provide 6 en% ALA for 8 weeks to a basal diet containing 23 en% fat inhibited the PBMC proliferation and DTH response of male soldiers (Kelley et al., 1991). The design of this study does not permit one to distinguish if this inhibition by flaxseed oil was due to increased fat intake or increased ALA intake or both. The feeding of flax seed oil caused small but significant increases in the ALA and EPA levels of the PBMC (Kelley et al., 1993), making it difficult to distinguish if the inhibition was caused by ALA or EPA or both. Several other indices of IR, including serum and salivary immunoglobulin levels, serum levels of complement proteins C3 and C4, and the number of circulating lymphocytes bearing markers for T-, Th-, Ts-, and B-cells were not changed in the study with flax seed oil (Kelley et al., 1991). ALA supplementation has also been reported to inhibit PBMC proliferation in ALA-
deficient patients (Bjerve et al., 1989) and the in vitro secretion of TNF-α and IL-1β in healthy men (Caughey et al., 1996). The latter study with healthy men provided 14 g of ALA for 4 weeks and included a control group that provided only 1.1 g ALA. The total fat contents of the ALA and control diets were comparable (29.4 en%), which indicates that the inhibition of cytokine production was specifically caused by ALA.
Fish oils have been found to inhibit several aspects of neutrophil, monocyte, and lymphocyte functions in several human studies (Endres et al., 1989, 1993; Kramer et al., 1991; Lee et al., 1985; Meydani et al., 1991; Molvig et al., 1991; Payan et al., 1986; Virella et al., 1989). Fish oil intake of 18 g/d, in addition to the fat content of the basal diet, inhibited neutrophil and monocyte functions within 6 weeks of its supplementation, while it failed to inhibit T-cell functions as determined from IL-2 production within this time of intake (Caughey et al., 1996; Endres et al., 1989). The production of IL-2 was, however, significantly inhibited 10 weeks after the discontinuation of fish oil intake. The inhibition of neutrophil functions by fish oils could be overcome within 10 weeks of discontinuation, while it took 20 weeks to overcome the inhibition of lymphocyte and monocyte functions. The differences in the time taken to cause or overcome inhibition are presumably due to different half-lives of various immune cells, as mentioned earlier for the total fat intake. In this study, inhibition of IR may have resulted from both the intake of n-3 PUFA and the added fat. The amount of fish oil has varied from 6 to 20 g/d, and the inhibition of immune parameters was observed within 6 or more weeks of fish oil intake in various studies (Endres et al., 1989, 1993; Kramer et al., 1991; Lee et al., 1985; Meydani et al., 1991; Molvig et al., 1991; Payan et al., 1986; Virella et al., 1989). Since 1 g of fish oils contains on an average 180 mg EPA and 120 mg DHA, the intake of EPA and DHA in these studies ranged from 2 to 6 g/d.
In addition to the above studies with fish oils, the effect of fish intake on human IR has also been examined in several studies (Kelley et al., 1992b; Meydani et al., 1993). Reducing total fat intake from 36 to 27 en%, with a concomitant increase in fish intake (EPA + DHA = 1.23 g/d or 0.54 en%) by men and women over the age of 40 years for 6 months significantly inhibited several indices of IR, including DTH, lymphocyte proliferation, and secretion of IL-1, IL-6, and TNF, when compared with the corresponding values when subjects were fed the high-fat diet (Meydani et al., 1993). However, the switch to low-fat and low-fish intake (EPA + DHA = 0.27 g/d or 0.13 en%) for the same period did not inhibit any of these indices. The lymphocyte proliferation and the secretion of TNF and IL-1 were actually enhanced by the low-fish diet, which was perhaps caused by the reduction in fat intake from 36 to 27 en%, rather than by the low n-3 PUFA intake. In a cross-over study young, healthy men (25–40 years) were fed salmon, 500 g/d (EPA + DHA = 2.1 en%) for 40 days. None of the indices of IR tested were inhibited by the consumption of this amount of salmon (Kelley et al., 1992b). However, the DTH response was
significantly increased, which was perhaps due to the reduction in total fat intake from 29 to 23 en%. Thus, it is important not to view just the changes in n-3 PUFA intake but also in total fat and the ratios between n-6 and n-3 PUFA. The age and antioxidant nutritional status are other important factors that determine the impact of n-3 PUFA on human IR.
Clinical Trials With n-3 PUFA
The immuno-inhibitory effects of n-3 PUFA along with their beneficial effects for cardiovascular health prompted a number of studies with these fatty acids in the management of autoimmune and inflammatory diseases. The overall health of the patients was improved by fish oil supplementation in these studies, although the laboratory tests usually did not show corresponding improvements. The diets of rheumatoid arthritis patients when supplemented with fish oils led to a reduction in swollen and tender joints, morning stiffness, and pain index (Kremer et al., 1995). Fish oil supplementation has also been reported to decrease symptoms of lupus (Walton et al., 1991), psoriasis (Kojima et al., 1989), cystic fibrosis (Lawrence and Sorrell, 1993), ulcerative colitis (Stenson et al., 1992), and inflammatory bowel disease (O'Marn, 1987). Early restenosis after angioplasty decreased (Gapinski et al., 1993) and renal functions in patients maintained on cyclosporin after kidney or liver transplant improved (Badalamenti et al., 1995; Berthoux et al., 1992) with fish oil supplementation. The intake of fish oils in these studies ranged from 4 to 20 g/d for 6 weeks to 6 months. The circulating levels of several cytokines including IL-1, IL-2, IL-4, IL-6, TNF, and interferon-γ decreased with fish oil consumption in patients with advanced colorectal cancer compared with the corresponding values prior to fish oil consumption (Purasiri et al., 1994). It took from 2 to 6 months to lower the levels of various cytokines, which were restored to the original levels within 3 months of fish oil discontinuation. In healthy adults, the maximal increase in oral temperature following typhoid vaccination was attenuated by the prior consumption of fish oils (4.5 g/d) for 6 to 8 weeks (Cooper et al., 1993). Thus, evidence from both in vitro and in vivo studies shows the inhibition of IR by n-3 PUFA. In contrast to the results from these in vivo studies with several patient groups and results from in vitro studies conducted with the cells isolated from healthy subjects after the feeding of n-3 PUFA, where n-3 PUFA was found to inhibit IR, in patients with asthma (Payan et al., 1986) and ALA deficiency (Bjerve et al., 1989), n-3 PUFA supplementation was found to enhance PBMC proliferation and the number of circulating T-lymphocytes, respectively. Interaction with the drugs taken or the effects of EFA deficiency may have resulted in the stimulation of these indices of IR in these patients.
Although the results from clinical trials with fish oils seem encouraging, any benefit from their intake must outweigh the risk associated with the overall suppression of IR. The clinical relevance of such immune suppression is not now known and must be evaluated before the intake of fish oils can be
recommended in the management of such disorders. Because most fish oils are rich in cholesterol, the risk of increased cholesterol intake should also be considered. Moreover, the increased need for antioxidant nutrients should be considered whenever the diet is supplemented with fish oils. The risk/benefit ratios may vary in different individuals and for the same individual under various set of conditions. Thus, fish oil supplementation should not be done without clinical supervision. The safe level of fish oil intake for subjects over 40 years is probably between 0.27 and 1.23 g/d of EPA + DHA, because 1.23 g/d ihibited several indices of IR, while 0.27 g/d did not. From a nutritional point of view, the consumption of fish 2 to 3 times a week or that of ALA up to 5 g/d by the general adult population should not have any adverse effect on IR. Further studies are needed to determine the requirements and the safe levels for EPA and DHA intake in various population groups. Because fish oils contain both EPA and DHA, it is also important to establish the role of these fatty acids individually. The author has just completed a metabolic unit study in which the effect of adding DHA to the diets of healthy men was examined. Results from this study should be available shortly.
Mechanisms by Which Dietary Fat Alters Immune Response
A number of substances and/or mechanisms listed below may be involved in mediating the effects of dietary fat on IR:
- Serum lipoproteins. Both the concentration and composition of dietary fat (fatty acids as well cholesterol) can alter serum lipoprotein profile, which influences the activity of the immune cells. In vitro studies have shown that low-density lipoproteins (LDL) inhibited lymphocyte and neutrophil functions, while high-density lipoproteins (HDL) enhanced neutrophil chemotaxis and phagocytosis. Other studies have indicated that lymphocyte proliferation in vitro was positively correlated with the number of HDL receptors and negatively correlated with the number of LDL receptors. One of the mechanisms by which LDL inhibits lymphocyte and monocyte functions is through apo proteins B and E (apo B and E). Other lipoproteins (very low density, intermediate density and chylomicrons) that are enriched in apo B and apo E also inhibit immune cells. Whether the serum lipoprotein changes caused by dietary fat are large enough to affect IR in vivo needs to be investigated.
- Eicosanoid type and concentration. Fatty acids of the n-3 and n-6 type yield different types of eicosanoids, which have different effects on immune cells. Changing the ratios between n-3 and n-6 PUFA intake alters the type and concentration of eicosanoids produced, since the same enzymes are involved in the metabolism of both types of PUFA. In general, eicosanoids derived from the n-3 PUFA are less potent mediators of inflammation than those derived from
- AA. Furthermore, the effects of these eicosanoids are dose dependent, that is, small concentrations (10-10 – 10-12 M) of prostaglandin-E2 (PGE2) and leukotriene-B4 (LTB4) stimulate some of the immune cells, while concentrations higher than 10-9 M inhibit the same cells.
- Oxidative stress. Increasing the PUFA intake increases oxidative stress, which if not counterbalanced by antioxidant nutrients, can damage cells and inhibit IR. The clearance of oxidized lipoproteins is through the scavenger receptors on the macrophages, and this receptor is not subject to the same feedback inhibition by the intracellular cholesterol as is the receptor for the native LDL. Thus, the antioxidant nutrient status can have profound effects on the IR, and it must be considered when evaluating the effects of dietary fat on IR.
- Membrane fluidity. Dietary fatty acids incorporated into membrane lipids can change membrane fluidity, which increases with an increased content of unsaturated membrane lipids. Changes in membrane fluidity can affect intercellular interaction, receptor expression, nutrient transport and signal transduction. All these factors can affect cell growth.
Any single dietary intervention may involve more than one of the above substances and/or mechanisms. It is also possible that some fatty acids directly affect the cells of the immune system.
Author's Conclusions and Recommendations
Interaction between several factors, including total fat, its composition, and the ratios between various classes of fatty acids; duration of feeding; antioxidant nutrient status; and age and health status of the subjects determines the net effect of dietary fat on IR. Because of this complex interaction, not all individuals respond equally to changes in fat intake. Furthermore, different indices of IR respond differently to changes in fat intake. In general, when other factors are maintained constant, reduction in total fat intake enhances several indices of human IR, and the converse is true when fat intake is increased. If total fat intake is not changed, a moderate increase in the consumption of n-6 PUFA (LA or AA) does not adversely affect several indices of IR tested. A number of studies indicate inhibition of several indices of human IR, with an increased consumption of n-3 PUFA (ALA, EPA, and DHA). Most of these studies lacked a concomitant control group, or the studies did not maintain a constant total fat intake. The inclusion of a control group is important in order to rule out the effects of seasonal changes or the inhibition in IR due to increased fat intake. There has been limited success in the management of autoimmune disorders by supplementing the diets of such patients with n-3 PUFA. However, such practice is not recommended without clinical supervision because of the risk of overall inhibition of IR. Current recommendations by the American Heart Association to reduce total fat intake to 30 en%, with 10 en% from each of the
saturated, monounsaturated, and polyunsaturated fatty acids to improve cardiovascular health, will also improve IR in most individuals who consume diets containing more than 30 en% fat. There are currently no recommendations in the United States regarding the intake of n-3 PUFA. However, daily intakes of 200 to 400 mg of EPA+ DHA or up to 5 g of ALA should have no adverse effects on the IR of healthy adults.
These findings regarding the effects of total fat and n-3 PUFA have significant implications for developing nutritional guidelines for the military. The current fat content of the military rations is well above 30 en percent and reducing it to 30 en percent will improve not only the cardiovascular health but will also enhance several indices of IR. However, this improvement in IR must outweigh the reduction in energy density and palatability of military rations. Enhanced IR may improve general health under a variety of situations, but it can be deleterious under several other conditions like autoimmune disorders or organ transplant. We need to develop guidelines tailored to individual needs, however for the general public including the military, a reduction in total fat with some increase in the intake of n-3 PUFA should prove to be generally healthy. There may also be specific situations under which the incorporation of small amounts of n-3 PUFA into the diet of the military personnel will be useful in the management of autoimmune disorders.
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MELVIN MATHIAS: The decrease in dietary fat seems to be very clear-cut and reproducible with many investigators, in many investigator's hands, but the mechanism of that response—you had a shopping list of possible interactions. From your reading of the literature is there any particular mechanism that might jump out?
All the other parameters could involve eicosanoids, but just a decrease in fat is so clear and profound that we don't have a good—I don't have a good—mechanism. Do you have one?
DARSHAN KELLEY: I have a slide on the various mechanisms; somehow it got buried somewhere. The decrease in fat intake is very difficult to attain without changing the ratios between fatty acids, and also other nutrients. Now, we can maintain the ratios, but in most of the studies when you change the fat,
you are changing the ratios within fatty acids, and also some of the other nutrients.
Even if you are maintaining the ratios, if you decrease the total fat, you are decreasing the antioxidant load. You also would decrease the lipoproteins in the serum, particularly lipoprotein B and E are significantly inhibitory of the immune cells.
So, any of those two mechanisms could easily account for the increase in IR by the decrease in fat intake on the activity of the immune cells, in addition to eicosanoids that you mentioned.
STEVE GAFFIN: A number of studies show that fish oil omega-3 fatty acids are beneficial, effective against endotoxin shock or gram negative sepsis. Your studies show the opposite general effect, that it is actually immunosuppressive. Can you explain why it should be protective in one case and not beneficial in another?
DARSHAN KELLEY: I think probably fish oils have more than one kind of effect. One is that the inhibitory effect may be mediated through the eicosanoid production, decreasing the production of series-2 eicosanoids. But I think in the beneficial effects, we are talking about in sepsis, that could be the increased production of free radicals that could be harmful to the pathogens. Again, a double-edged sword.