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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:185-189.)
© 1995 American Heart Association, Inc.

Articles

Dietary Fish Oil Supplementation Alters Leukocyte Function and Cytokine Production in Healthy Women

Julie M. W. Wallace; Eithne Turley; William S. Gilmore; John J. Strain

From the School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland.

Correspondence to William S. Gilmore, PhD, School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, UK.

	Abstract

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Abstract The effect of low-dose fish oil supplementation on cytokines and white cell function in women was investigated. Thirty-three healthy, nonsmoking women entered the double-blind study. For 4 weeks, 2.4 g of either fish oil (n=16) or fish oil with vitamin E (n=17) was added daily to the subjects' otherwise unchanged diets. Venous blood samples were taken at the onset of the trial, after the supplementation period, and again after a 9-week washout period. Plasma levels of platelet-derived growth factor and myeloperoxidase were measured using immunoassays. The intracellular peroxidase content of white blood cells was measured using a staining technique. Platelet-derived growth factor levels were significantly lowered after supplementation (P.05). Intracellular peroxidase was increased (P.01), and extracellular myeloperoxidase levels were lowered (P.05). Taken together, these results suggest that the anti-inflammatory effect of fish oil may be due at least partly to alterations in white cell function and growth factor levels.

Key Words: atherosclerosis • inflammation • fish oil • cytokines • leukocytes

	Introduction

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Atherosclerosis and its complications such as myocardial infarction and stroke remain the largest cause of premature death in Western societies today. To date, no single cause of vascular disease has been recognized, but a number of factors have been implicated, including smoking, hypertension, obesity, and elevated plasma low-density lipoprotein (LDL) and fibrinogen levels. Combinations of these risk factors lead to an acceleration in the progression of atherosclerosis. A number of these risk factors are linked by their ability to produce chronic inflammatory responses in vascular tissues.¹ Inflammatory response, as assessed by measurement of reactive oxygen species production, is elevated in people at risk of coronary heart disease.^{2 3 4} Furthermore, expression of cytokines and growth factors, which control cardiac growth and development and are produced during inflammation by activated leukocytes, is altered in atherosclerosis.^{5 6}

Epidemiological studies have suggested that increased fish intake is correlated with a reduced risk of coronary heart disease; consequently, many studies have been carried out on the effects of fish and fish oil on blood and vascular composition and function.⁷ The beneficial effects of fish oil are usually attributed to the n3 fatty acids. The anti-inflammatory effects of these fatty acids may be due to a number of factors, not least an alteration in levels of reactive oxygen species, cytokines, and growth factors. In mononuclear blood cells and polymorphonuclear neutrophils (PMNs), fish oil supplementation decreases indices of inflammatory response.^{8 9} A diet supplemented with fish oil has been shown to reduce interleukin-1 and tumor necrosis factor production by stimulated human mononuclear cells.¹⁰ In vitro, addition of fish oil to tissue culture medium causes a reduction in platelet-derived growth factor (PDGF) production by cultured endothelial cells, an effect inhibited by the addition of antioxidants, including vitamin E.¹¹ This implicates oxidative processes in growth factor regulation.

The level of supplementation used in the present study was based on the Cardiff diet and reinfarction trial, the only dietary intervention trial to produce a substantial drop in all-cause mortality.¹² To investigate the possible relation between n3 fatty acid consumption and cytokine levels, we measured the effects of short-term, low-dose fish oil supplementation with and without vitamin E on plasma PDGF levels. To determine the effects of low-dose fish oil on neutrophil activation, we also measured plasma myeloperoxidase (MPO) concentrations. MPO is an enzyme produced mainly by activated PMNs that catalyzes the formation of the reactive oxygen species OCl^- and is a measure of inflammatory activity.

	Methods

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Subjects and Study Design
The study was approved by the Research Ethics Committee of the University of Ulster. Thirty-three healthy female volunteers entered this double-blind study after giving informed consent. Their mean age was 22 years (range, 18 to 28 years). All subjects were nonsmokers and were taking no prescribed medication or nutritional supplements. The subjects were instructed not to change their diets during the experimental period.

For 4 weeks the subjects added 2.4 g of fish oil (three capsules per day) with or without vitamin E to their otherwise unchanged diets. The fish oil, kindly provided by Seven Seas, contained 18% docosahexaenoic acid and 12% eicosapentaenoic acid. The fish oil with vitamin E contained vitamin E at a concentration of 3 mg/g.

Blood Sampling
Venous blood samples (25 mL) were taken before and after the 4-week treatment and again after a 9-week washout period. Aliquots of blood were measured into anticoagulant tubes as follows: 10 mL into heparinized tubes, 9 mL into citrated tubes, 3 mL into EDTA tubes, and 3 mL into serum separator tubes. All blood samples were kept cool until centrifugation. Aliquots of plasma were kept at -70°C until required. A full blood picture was obtained for each subject at each sampling time.

Determination of PDGF Concentration
Plasma was obtained from heparin anticoagulant tubes prechilled to 4°C. PDGF analysis was done with a Quantikine human PDGF-AB immunoassay (R&D Systems). This assay is done with a quantitative "sandwich" enzyme immunoassay technique and is carried out on a microtiter plate coated with PDGF-AA antibody. A 50-µL aliquot of assay diluent was added to each well on a 96-well plate. To this aliquot 200 µL of sample (1:10 dilution of plasma) or standard was added; they were mixed and left at room temperature for 2 hours. The wells were then aspirated and washed, and 200 µL of PDGF-AB conjugate was added to each well for 2 hours. Again this was aspirated, the wells were washed, and 200 µL of substrate solution was added to each well for 20 minutes. After this time 50 µL of stop solution was added to each well, and the absorbance at 450 nm was measured (Bio-Tek Instruments).

Determination of MPO
Plasma was obtained from prechilled heparin anticoagulant tubes. MPO analysis was carried out using a Bioxytech enzyme-linked immunoassay for human MPO. Samples were incubated in the wells of a microtiter plate that had been coated with a monoclonal antibody against MPO. A 100-µL aliquot of sample (1:15 dilution of plasma) or standard was added to each well and incubated at 37°C for 2 hours. The wells were then aspirated and washed, and 100 µL of MPO polyclonal antibody was added to each well. The plate was again incubated at 37°C for 2 hours. The wash procedure was repeated, 100 µL of avidin-coupled alkaline phosphatase solution was added, and the plates were further incubated for 2 hours. The wash procedure was repeated a third time, and 100 µL of p-nitrophenyl phosphate was added to each well. The plate was incubated at 37°C until the absorbance of the highest standard reached approximately 2.5. Finally, 50 µL of stop solution was added to each well, and the absorbance was measured at 405 nm using a multiwell plate reader (Bio-Tek Instruments).

Blood Count
A full blood picture was obtained using the Technicon H*1 hematology analyzer (Bayer Diagnostics). This machine uses the principles of flow cytometry to perform complete blood cell counts and differential counts of the mature white blood cells. It can also be used to measure particular red and white cell morphometric features. One of these is the mean peroxidase index (MPXI), a measure of the peroxidase content of neutrophils. To estimate this, red cells are lysed and the white cells are fixed and then stained for peroxidase. A dark precipitate forms in the primary granules of leukocytes containing peroxidase when a chromagen is added with hydrogen peroxide as the substrate. The cells are then classified by size, and only the neutrophils are assessed to determine MPXI. The staining intensity of the neutrophil is dependent on its peroxidase content.

Statistical Analysis
The results are expressed as mean±SEM. Student's t test for paired observations was used to compare values in the same subject at each sampling time; a value of P.05 was regarded as significant.

	Results

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The fish oil supplements were well tolerated by all study participants, but hiccups and a fishy aftertaste were common. Verbal questioning revealed a satisfactory compliance of all the subjects with the study protocol.

The effect of fish oil supplementation on plasma PDGF concentration is shown in Fig 1. The concentration of plasma PDGF was decreased significantly after 4 weeks of supplementation with either fish oil (P.01, n=14) or fish oil with vitamin E (P.05, n=15). Nine weeks after the end of supplementation PDGF levels had decreased further, but the levels recorded were not significantly different from those recorded after 4 weeks of supplementation. There was no significant difference between the effects of fish oil and fish oil with vitamin E on plasma PDGF concentration.

View larger version (73K): [in this window] [in a new window]   Figure 1. Bar graph showing the effect of 4 weeks of oral supplementation with low-dose fish oil on plasma platelet-derived growth factor (PDGF) concentration. Plasma samples were obtained from subjects at the start of the trial, after the 4-week supplementation period, and after the 9-week washout period. Plasma PDGF concentrations were determined using an enzyme immunoassay system. Data are reported as mean±SEM for the total group (n=29), the fish oil only group (n=14), and the fish oil with vitamin E (Vit.E) group (n=15). Speckled bars indicate presupplementation PDGF concentrations; shaded bars, postsupplementation concentrations; and solid bars, washout concentrations. Results were tested for significant difference from presupplementation concentrations using Student's t test. *P.05, **P.01, ***P.001.

Fig 2 shows the effect of fish oil supplementation on the MPXI. Both supplements caused a significant increase in peroxidase staining after 4 weeks (fish oil, P.01, n=11; fish oil with vitamin E, P.01, n=14). Although levels had not returned to baseline after the 9-week washout period, they were not significantly different from initial measurements. Again, there was no significant difference between the effects of fish oil and fish oil with vitamin E on the MPXI.

View larger version (34K): [in this window] [in a new window]   Figure 2. Bar graph showing the effect of 4 weeks of oral supplementation with low-dose fish oil on the peroxidase staining intensity (MPXI) of neutrophils. Blood samples were obtained from subjects at the start of the trial, after the 4-week supplementation period, and after the 9-week washout period. MPXI is estimated by lysing the red blood cells and fixing and staining the white blood cells for peroxidase content. Staining intensity is dependent on peroxidase content. Data are reported as mean±SEM for the total group (n=25), the fish oil only group (n=11), and the fish oil with vitamin E (Vit.E) group (n=14). Speckled bars indicate presupplementation MPXI staining intensity; shaded bars, postsupplementation intensity; and solid bars, washout intensity. Results were tested for significant difference from presupplementation intensity using Student's t test. **P.01, ***P.001.

The effect of supplementation on plasma MPO concentration is shown in Fig 3. The concentration of MPO was significantly decreased after 4 weeks of supplementation with either fish oil (P.01, n=13) or fish oil with vitamin E (P.05, n=12). After the 9-week washout period, MPO levels increased but did not return to baseline. There was no significant difference between the effects of fish oil and fish oil with vitamin E on plasma MPO concentration.

View larger version (71K): [in this window] [in a new window]   Figure 3. Bar graph showing the effect of 4 weeks of oral supplementation with low-dose fish oil on plasma myeloperoxidase (MPO) concentrations. Plasma samples were obtained from subjects at the start of the trial, after the 4-week supplementation, and after the 9-week washout period. MPO levels were estimated using an enzyme immunoassay system. Data are reported as mean±SEM for the total group (n=25), the fish oil only group (n=13), and the fish oil with vitamin E (Vit.E) group (n=12). Speckled bars indicate presupplementation MPO concentrations; shaded bars, postsupplementation concentrations; and solid bars, washout concentrations. Results were tested for significant difference from presupplementation concentrations using Student's t test. *P.05, **P.01, ***P.001.

The Table shows the data depicted in Figs 1 through 3.

View this table: [in this window] [in a new window]   Table 1. Effect of 4 Weeks of Dietary Supplementation With Low-Dose Fish Oil With or Without Vitamin E on Plasma PDGF and MPO Concentrations and MPXI

	Discussion

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Our results show that low-dose dietary supplementation with n3 fatty acids reduces plasma PDGF and MPO levels while increasing the peroxidase content of neutrophils. This reduction in PDGF concentration and neutrophil activation, as demonstrated by a reduction in MPO release, may contribute to the decreased inflammatory responses reported in patients receiving fish oil supplementation.¹³ Previous studies have focused mainly on intervention trials in which large doses of fish oil were given for varying periods of time. Here we report significant effects after low-dose fish oil supplementation. These results indicate another possible mechanism for the purported antiatherosclerotic effect of fish oil.

The reduction in PDGF concentration after 4 weeks of supplementation is important because of the effect of PDGF on arterial smooth muscle cell proliferation. PDGF is a major serum mitogen for smooth muscle cells; in addition, it mediates stimulation by interleukin-6 of smooth muscle cells.¹⁴ The addition of PDGF antibodies to plasma from patients with coronary heart disease has been shown to lower the growth factor activity by 40%, indicating the potential importance of PDGF in atherosclerosis.¹⁵ In this study the decline in PDGF persisted after a washout period of 9 weeks. Indeed, PDGF concentration declined further during the washout period, although not significantly. The persistent suppression of PDGF for as long as 9 weeks after the cessation of supplementation is in keeping with the results of other studies that show a lasting effect of fish oil on various parameters.^{16 17} In addition, Endres et al¹⁰ report that interleukin-1 and tumor necrosis factor production by mononuclear cells remains suppressed for at least 10 weeks after supplementation.

Supplementation of the diet with n3 fatty acids results in the downregulation of mRNA levels of PDGF-A and PDGF-B in unstimulated human monocytes,¹⁸ an effect not seen on other growth factor genes.¹⁹ Like oxidized LDL in vitro,²⁰ fish oil suppresses PDGF production in vivo, as shown by our results. This points to fish oil's exerting a possible pro-oxidant effect. However, it has been suggested that fish oil can inhibit oxidative modification of LDL,²¹ and vitamin E, the major lipid-soluble chain-breaking antioxidant, had no modulating effect in our study, implying that another mechanism is involved in the suppression of PDGF.

An increase in MPXI after supplementation was observed in both groups. Although peroxidase staining increased significantly after 4 weeks, the values were still within the normal range (-10 to +10). Whole leukocyte preparations were stained for peroxidase. The main producer of this enzyme is the neutrophil, in which peroxidase comprises 5% of the dry weight of the cell. Peroxidase is also found in eosinophils and, in smaller amounts, in monocytes.²² In measuring MPXI, the Technicon H*1 differentiates the cells by size and uses only the neutrophils to calculate the index. This increased peroxidase content of neutrophils could mean that the body has an enhanced potential to initiate a phagocytic response should the need arise. The increased content is also related to our finding of a decreased plasma MPO concentration after supplementation. A reduction in MPO indicates reduction of white cell and, in particular, PMN activation. Taken together, these two results indicate an alteration in peroxidase release rather than in peroxidase production in leukocytes. There was no significant difference between the effects of fish oil and fish oil with vitamin E; thus, the n3 fatty acids and not vitamin E seem to be the major cause of this decrease. The reduction in monocyte and PMN activation after n3 supplementation has been reported previously.^{9 17 23}

After 9 weeks of washout, both MPXI and MPO levels had not returned fully to presupplementation baseline levels. Because a new population of circulating PMN was then being studied, this persistent effect on neutrophil function after dietary enrichment with fish oil could be explained by a recycling of n3 fatty acids from a slow turnover compartment, as previously proposed.¹⁰

Low-dose fish oil exerts a protective effect against mortality,¹² but the precise mechanism is not known. Our findings of reduced PDGF levels and leukocyte function are of interest because studies indicate that activated white blood cells, reactive oxygen species, cytokines, and growth factors play important roles in ischemic heart disease.^{24 25 26} One of the earliest recognized cellular events in the development of the atherosclerotic plaque is the appearance of monocytes on the endothelium,²⁷ and many studies have investigated this initial step in atherosclerosis.^{28 29} These monocytes subsequently migrate into the subendothelial space, differentiate into macrophages, accumulate LDL that has previously been oxidized, and ultimately form foam cells.³⁰ However, the role of the monocyte does not end here because these cells are also producers of cytokines and growth factors, which cause smooth muscle cell proliferation and ultimately lesion formation.³¹ Previous studies on the effects of fish oil on monocyte function have shown reduced chemotaxis³² and a shift in leukotriene production toward the less potent chemotactic mediator leukotriene B₅.^{17 33} Epidemiological studies have reported white blood cell and differential leukocyte counts to be related to the risk of vascular disease, the strongest association being with the neutrophil count.^{34 35} In response to this, investigators have endeavored to determine the reason for the link between neutrophil count and ischemic heart disease. One possible mechanism could be an involvement of reactive oxygen species, released by activated neutrophils in the regulation of cytokine and growth factor levels. It is known that oxidative processes are involved in the regulation of cytokine and growth factor levels in vitro. From the review of recent in vitro studies, we propose the following scheme (see Fig 4): Activated PMNs release MPO,³⁶ an enzymatic protein that catalyzes the formation of OCl^- from hydrogen peroxide and chlorine. OCl^- is a reactive oxygen species that in turn leads to the formation of oxidized LDL.³⁷ Oxidized LDL interacts with endothelial cells, causing them to release granulocyte macrophage–colony stimulating factor,³⁸ which can in turn increase PMN activation.³⁶

View larger version (12K): [in this window] [in a new window]   Figure 4. Diagram of a possible mechanism by which reactive oxygen species affect growth factor levels. The scheme is derived from a number of in vitro studies. MPO indicates myeloperoxidase; H2O2, hydrogen peroxide; Cl2, chlorine; OCl-, hypochlorite anion; LDL, low-density lipoprotein; ox-LDL, oxidized LDL; EC, endothelial cells; GM-CSF, granulocyte macrophage–colony stimulating factor; and PMN, polymorphonuclear neutrophils.

The prolonged effects of n3 fatty acids on leukocyte activation and PDGF production reported in this study have implications for the design of future trials, indicating that even with a low-dose supplement a long washout period should be incorporated into the design of any trial. In addition, the inclusion of a control group would be desirable and should also be part of the design.

In conclusion, it is possible that the beneficial effects on mortality of small amounts of fish oil in the diet might be at least partly mediated by an effect of n3 polyunsaturated fatty acids on neutrophil function and cytokine concentration.

	Acknowledgments

 
This work was supported by a grant from the Northern Ireland Chest, Heart and Stroke Association. The fish oil was kindly provided gratis by Seven Seas Ltd. The authors are grateful to the staff of the hematology laboratory of the Waveney Hospital in Ballymena for determination of the blood counts of subjects.

Received August 2, 1994; accepted November 14, 1994.

	References

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