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The Effect of Eicosapentaenoic Acid Consumption on Human Neutrophil Chemiluminescence 1 Philip J. Thompson*,a, Neil L.A. Missoa, Marion Passarellia a n d Martin J. Phillip,~ aDepartment of Medicine, Universityof Western Australia and bDepartment of Respiraton/Medicine, Sir Charles Gairdner Hospital, Nedlands, W.A. 6009, Australia The effect of eicosapentaenoic acid (EPA) on the inflammatory potential of neutrophils was investigated by supplementing the diets of 12 subjects with 2.16 g of EPA or 12 g of olive oil per day for 4 weeks in a double blind crossover study. Neutrophil function as assessed by iuminol enhanced chemiluminescence responses to plateletactivating factor (PAF) and formyl-methionyl-leucylphenylalanine (FMLP) was significantly reduced after EPA but not after olive oil consumption in the subjects who consumed EPA first. In contrast, EPA had no significant effect on neutrophil chemil-mlnescence in the subjects who consumed olive oil first. Dietary supplementation with EPA inhibits neutrophil responses to inflammatory mediators such as PAF while other fatty acids appear to modify the effects of EPA. Lipids 26, 1223-1226 (1991). Populations such as the Greenland Eskimos and Japanese have a high dietary intake of fish oil compared to most Western diets and are noted to have a much lower 4~revalence of coronary artery disease (1) and chronic inflammatory diseases such as asthma, rheumatoid arthritis and psoriasis (2). These epidemiological observations have led to the hypothesis that the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in oily cold-water fish may offer protection against these diseases. EPA inhibits the uptake of arachidonic acid (AA) into ceil membrane phospholipids and competes with AA for metabolism by the cyclooxygenase and lipoxygenase pathways. DHA is an inhibitor of these pathways (3). Metabolites of AA (thromboxane A2, prostaglandins and leukotrienes) are thought to contribute to the inflammatory process in asthma by recruiting and activating neutrophils, eosinophils and macrophages (4). Platelet-activating factor (PAF), another likely mediator of inflammation in asthma, is derived from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine, and its formation is closely linked with AA metabolism in neutrophils (5). Dietary supplementation with fish oil increases the proportion of EPA to AA in ceil membrane phospholipids (6) and thus could result in a decreased production of PAF and inflammatory eicosanoids as well as the production of less potent analogues such as thromboxane A 3 and LTB 5 (leukotriene B) (3,7). 1Based on a paper presented at the Third InternationalConference on Platelet~ActivatingFactor and StructurallyRelatedAlkylEther Lipids, Tokyo, Japan, May 1989. *To whom correspondence should be addressed at the University Department of Medicine, Queen Elizabeth II Medical Centre, Nediands, W.A. 6009, Australia. Abbreviations: AA, arachidonicacid;CL, chemiluminescence;DHA, docosahexaenoicacid; DMSO, dimethylsulfoxide;EPA, eicosapentaenoic acid;FMLP,formyl-methionyl-leucyl-phenylalanine;HBSSHEPES, Hanks balanced salt solutionbufferedwith N-2-hydroxyethylpiperazine~hr-2-ethanesulfonicacid; 5-HETE, 5-hydroxyeicosatetraenoic acid; LTB, leukotrieneB; PAF, platelet-activatingfactor.
An important function of stimulated neutrophils is the production of reactive oxygen metabolites (8), a process which can be monitored by the technique of luminolenhanced chemiluminescence (CL) (9). In this study the effect of dietary supplementation with EPA on the inflammatory potential of neutrophils was assessed by measuring neutrophil CL responses to in vitro stimulation with PAF and formyl-methionyl-leucyl-phenylalanine (FMLP) and comparing this with the effect of a placebo in the form of olive oil. MATERIALS AND METHODS Twelve healthy volunteers were entered into a double blind balanced crossover study comparing the effects of EPA and olive oil (placebo) on neutrophil chemiluminescence. They were asked to avoid all medication for two weeks prior to entry, and to maintain their normal diets prior to and during the study. The study was of sixteen weeks' duration and divided into 4 four-week intervals. Six subjects (Group 1, 3 males, 3 females aged 24-40 years with a mean age of 33 years) consumed MaxEPA capsules (Lipitac, Reckitt & Colman Products Pty. Ltd., West Ryde, NSW, Australia) in the first four weeks while the other six subjects (Group 2, 3 males, 3 females aged 23-38 years with a mean age of 29.5 years) consumed identical capsules containing olive oil. The dosage was 12 capsules daily, which provided 2.16 g of EPA per day or in the case of olive oil 12 g per day. After 4 weeks all subjects entered a four-week wash-out period when no capsules were taken. In the third four-week period, subjects were crossed over to the alternate capsules, and the study concluded with a four-week wash-out period. At the commencement of the study and after each four-week period (day 0, 28, 56, 85, and 112 of the study), blood was taken for neutrophil isolation. Neutrophils were isolated by centrifugation of heparin anticoagulated whole blood on discontinuous Percoll (Pharmacia LKB, Uppsala, Sweden) gradients (10). Neutrophils were washed in phosphate buffered saline, counted in a haemocytometer and resuspended in Hanks balanced salt solution buffered with N-2-hydroxyethylpiperazine~N'-2-ethanesulfonic acid (HBSS-HEPES) at 1.8 • 106 cells/mL. A 5 mM stock solution of luminol (Boehringer Mannheim, Mannheim, W. Germany) in dimethylsulfoxide (DMSO) was diluted in HBSS to give a 0.5 mM working solution. PAF (1-O-hexadecyl-2-acetyl-sn-glycer~3-phosphocholine, Sigma Chemical Co., St. Louis, MO) was stored as a 1 mM solution in ethanol at - 2 0 ~ On the day of use, 0.1 mL of the stock solution was evaporated under N 2 and resuspended in 1 mL of HBSS containing 0.25% human serum albumin to give a 10 -4 M solution which was further diluted with HBSS to give working solutions of the required concentration. FMLP (Sigma) was stored as a 10 -2 M solution in 50 /zL aliquots of DMSO at LIPIDS, Vol. 26, No. 12 (1991)
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P.J. THOMPSON E T AL. - 7 0 ~ Dilutions to give working solutions of the required concentration were made with HBSS. Chemiluminescence was measured in a LKB 1250 luminometer. The reaction mixture (total vol 0.5 mL) contained 7.5 • 105 neutrophils and 25 ~V[ luminol. The reaction was started by the addition of 50 ~L of PAF or F M L P to give the required final concentration. The CL response was monitored for 10-15 m l n on a digital display/printer unit and chart recorder. CL responses were measured as the m-ximum amplitude in mV, and the data were analyzed by calculating mean values (+_ standard error of mean). Students t-test for paired data with a significance level of p < 0.05 was used when comparing data from the same group of subjects on different days. RESULTS
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The luminol-enhanced CL response of neutrophils to PAF 22 and FMLP was biphasic. An initial rapid burst of chemiB luminescence which peaked at 1-2 rain after the addition 20 of the stimulus was followed by a more gradual rise in 18 CL which reached a maximum at 8-10 mln. In this study 16 the maximum amplitude of the initial peak which is i ~ 14 associated with the production of extracellular reactive E. 9 ~ 12 oxygen metabolites (11) was used as a measure of neutrophil CL responses to PAF and FMLP. O Neutrophil CL dose response curves to PAF and FMLP Q. 8 representing the mean responses of the 12 subjects at the 9 6 commencement of the study are shown in Figure 1. The -I mean msxlmum response to PAF was observed at a con4 2 ~ " centration of 5 • 10 -6 M. The mean FM L P response was maximum at 10 -6 M and was higher than the mean 0 | ........| ........! ........| ........ ! ........! ...... -4 PAF response {14.2 _+ 7.3 vs 10.6 -!-_2.4 mV). Similar PAF 1010 10.9 108 107 106 105 and FMLP concentrations have been used previously in FMLP Concentration (M) the measurement of neutrophil CL responses and have not been shown to affect cellular integrity (12,13). The FIG. I. Dose responses of neutrophU chemiluminescence to P A F {A} FMLP response was more variable at 10 -5 M than at and FMLP (B). Each point represents the mean (+__SENDCL response 10 -6 M, and for this reason the latter concentration was for all 12 subjects on day 0. For each test, P A F or F M L P was added used when comparing the mean responses on different to 7.5 X 105 neutrophils with 25/~M l.minoL, and the maximum amplitude of the CL response was measured in mV. days. At the P A F concentration which produced a m v x i m u m response, neutrophils from Group 1 subjects who con- were so low that meaningful variations with time could sumed E P A from day 0 to day 28 showed a significant not be discerned. For the Group 2 subjects, mean C L decrease in the mean C L response on day 28 compared responses to P A F concentrations of 10 -6, 10 -6 and 10 -7 to day 0 (4.9 _ 2.9 vs 10.9 + 4.6 mV, p < 0.005) (Fig. 2A). M did not change significantly during the course of the study. At the end of the firstwash-out period (day 56), the C L Comparisons of the m e a n C L responses to 10 -e M response had increased to 8.3 _ 3.4 m V which was not significantlydifferentto day 0. There were no significant F M L P were used to assess the effect of E P A consumpchanges in the C L responses measured afterconsumption tion on neutrophil response to this stimulus (Fig. 3A). In of placebo in the form of olive oil (day 85) or at the end Group 1 subjects, the C L response to 10 -6 M F M L P was of the study (day 112) (Fig. 2A). 13.8 _ 2.5 m V on day 0. After E P A consumption this In the Group 2 subjects who consumed placebo capsules decreased to 7.6 ___3.3 m V on day 28 and decreased furfrom day 0 to day 28, the mean CL response to a max- ther to 5.7 +_ 1.5 m V at the end of the firstwash-out on imum PAF concentration was not significantly different day 56 which was significantlyless than day 0 (p < 0.05). on day 28 (6.4 + 1.3 mV) compared to day 0 (10.3 +_ After subsequent placebo {olive oil) consumption, the 4.3 mV) (Fig. 2B). However the mean responses on day mean C L response returned to 12.0 _ 3.6 m V on day 85 85 (8.8 + 2.0 mV) after E PA consumption and on day 112 and to 13.4 +_ 5.2 m V at the end of the study. Neutrophils from Group 2 subjects who consumed olive (6.3 +_ 2.0 mV) were also not significantly different to the response on day 0 or on day 56 (6.9 _ 1.6 mV) prior to oil first gave a mean C L response to 10 -6 M F M L P of E P A consumption. 9.4 _+ 3.0 m V on day 0 {Fig. 3B). This did not change In response to 10 -5 and 10 -6 M PAF, similar trends significantly after placebo consumption (11.8 __ 5.3 m V were observed with a reduction in CL at day 28 in the on day 28, 11.5 _+ 3.9 m V on day 56) and although there Group 1 subjects. With 10 -7 M PAF, the CL responses was a decrease in the response after E P A consumption LIPIDS,Vol. 26, No. 12 (1991)
1225
NEUTROPHIL CHEMILUMINESCENCE AFTER EPA CONSUMPTION A
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FIG. 2. Mean (~SEM) neutrophil chemiluminescence responses to PAF (5 X 10 -6 M) in Group 1 subjects who consumed EPA from day 0 to day 28 (A) and in Group 2 subjects who consumed olive oil capsules from day 0 to day 28 (B). * = p < 0.005 compared to day 0 by paired t-test.
FIG. 3. Mean (+__SEM) neutrophil chemiluminescence responses to FMLP (10-6 M) in Group I subjects who consumed EPA from day 0 to day 28 (A) and in Group 2 subjects who consumed olive oil capsules from day 0 to day 28 (B). * = p < 0.05 compared to day 0 by paired t-test.
(7.0 + 1.8 mV on day 85, 3.9 _+ 1.0 mV on day 112), this did not reach statistical significance. In the Group 1 subjects, CL responses to 10 -5 and 10 -s M FMLP showed similar trends to those described for 10 -6 M FMLP with CL being reduced on day 56 compared to day 0. With 10 -7 M FMLP, the trend for a reduction in CL could not be discerned. For the Group 2 subjects, the CL responses to 10 -5, 10 -7 and 10 -8 M FMLP did not change significantly with time.
the change was not statistically significant. Thus the results from the Group 2 subjects have to be regarded as inconclusive although there is some suggestion that olive oil may inhibit EPA suppression of neutrophil chemiluminescence. Three previous studies have examined the effects of dietary supplementation with EPA and DHA on human neutrophil chemotaxis and LTB4 production {14-16}. Higher doses {3.2-4.0 g} and longer durations {6-10 weeks) were used compared to this study. Neutrophil chemotaxis was reduced by up to 70% {14) and LTB4 production was decreased, with an increase in LTB5 production {15,16). In one of these studies, neutrophil function was not altered by consumption of placebo capsules containing olive off {16}. These studies have all assessed neutrophil function by measuring chemotactic responses to various stimuli (LTB4, FMLP, CSa). The chemiluminescence response investigated in this study monitors the release of reactive oxidant species, an important function of stimulated neutrophils. The chemotactic and chemiluminescence responses may be modified differently by dietary supplementation with EPA. Furthermore the
DISCUSSION
In the group of six subjects who consumed EPA capsules from day 0 to day 28, there was a significant reduction in the neutrophil CL responses to PAF and FMLP immediately after the consumption of EPA. Consumption of placebo capsules containing olive oil from day 56 to day 85 had no significant effect on neutrophil CL responses to PAF or FMLP in this group. In the other group of six subjects, olive oil consumption from day 0 to day 28 did not produce significant changes in CL response. After EPA consumption from day 56 to 85, the CL response to FMLP, but not to PAF, declined although
LIPID& Vol. 26, No. 12 (1991)
1226
P.J. THOMPSON E T AL. effect of EPA on PAF-induced neutrophil responses had not been investigated prior to this study. EPA consumption may have differential effects on neutrophil responses to PAF and other stimuli such as FMLP, LTB4 and C5a. The normal product of lipoxygenase activity in stimulated neutrophils, LTB4, may amplify the chemotactic and chemiluminescence responses to other stimuli such as PAF and FMLP. PAF has been shown to stimulate the production of LTB 4 in neutrophils (17,18), and in support of this data the lipoxygenase inhibitor, nordihydroguaiaretic acid, inhibits the neutrophil CL response to PAF {12}. CL responses to FMLP have also been linked to lipoxygenase activity (12). Other workers have shown that consumption of 3.2 g of EPA per day for 3 weeks (total dose 67.2 g) resulted in a 500% increase in the content of EPA in neutrophil phospholipids (14}. It seems likely that in our study in which the total dose was 60.5 g of EPA {2.16 g per day for 4 weeks) a similar incorporation of EPA into neutrophil phospholipids would have occurred and that this would have caused decreased LTB4 production, and increased LTB 5 production. Consequently, reduced CL responses to PAF and FMLP as observed in this study would be expected to occur. Incorporation of EPA into the phospholipids of neutrophil membranes may alter the structure and/or fluidity of the membrane such that function of surface receptors, such 9as those for PAF and FMLP, may be altered. However, in one previous study, evidence for a change in neutrophil membrane fluidity after dietary supplementation with EPA was lacking {14}. In this crossover study, there was some indication that the effect of EPA persisted for up to four weeks after dietary supplementation ceased. Thus, in Group 1 subjects, the mean CL response to FMLP was less on day 56 than on day 28, and in Group 2 subjects the mean FMLP response was less on day 112 than on day 85. This effect was not observed in the mean responses to PAF. In the design of this study, the duration of the wash-out period was determined on the basis that neutrophils are turned over rapidly, with a blood clearance half-time of approximately 7 hr. The fact that the effects of EPA on FMLP-induced CL responses persisted at the end of a four-week wash-out period suggests that the EPA was turned over and reincorporated into maturing cells or possibly incorporated into stem cells during dietary supplementation. Consumption of placebo capsules which contained olive off did not alter neutrophil CL in this study, and is in agreement with the lack of effect on chemotaxis as reported previously {16}. However prior consumption of olive oil appeared to reduce the effectiveness of EPA in inhibiting neutrophil CL. Oleic acid, the major fatty acid constituent of olive off {approximately 75%), can be metabolized to eicosatrienoic acid which may be converted to LTB 3 by the lipoxygenase pathway (7). Recent studies {7,19} have suggested that LTB 8 may be as potent as LTB 4 as an inflammatory stimulus. Olive oil also contains linoleic acid (5%) which can be metabolized to AA. Thus consumption of placebo capsules by the Group 2 subjects may have increased the amounts of proinflammatory eicosatrienoic acid and AA in their neutrophil membranes thereby reducing the subsequent effectiveness of the fixed dose of EPA. Other fatty acids may also be capable of influencing the effects of EPA, and it LIPIDS, Vol. 26, No. 12 (1991)
has been suggested (20) that the low prevalence of coronary artery disease and inflammatory disorders in Eskimos is due to the combined effects of high dietary EPA and genetically determined high plasma concentrations of dihomo-gamma-linolenic acid. Olive off is probably not an ideal placebo because, as this study has shown, it may be metabolized by similar biochemical pathways to those which metabolize EPA. Indeed the question of whether olive oil affects neutrophil function is worthy of investigation. The only ideal placebo for the study of the effects of EPA would be a marine lipid concentrate from which all the EPA is removed, but this is expensive and difficult to produce. Although the dose (2.16 g} and duration (4 weeks) of EPA supplementation were reduced in this study in comparison with others (3-4 g of EPA for 6-10 weeks) (14-16), the results are in agreement that dietary supplementation with EPA suppresses neutrophil function in vitro, and suggest lower doses may be effective. Although several studies have shown EPA has no effect on clinical symptoms when administered to asthmatic subjects (16,21,22), this may reflect clinical trial design. Alternatively, neutrophils may not play the major role in the inflammatory processes of asthma, and the concentration and duration of EPA supplementation needed to influence other inflammatory cell types and mediators involved in the pathogenesis of asthma (4) have yet to be elucidated. REFERENCES 1. Dyerberg, J., and Bang, H.O. (1978) Lancet 2, 152. 2. Kromann, N., and Green, A. (1980) Acta. Med. ScandL 208, 401-406. 3. Lee, T.H., and Arm, J.P. {1988}Br. Med. J. 297, 1421-1422. 4. Barnes,P.J., Chung,K.F.,and Page, C.P. (1988}PharmacoL Rev. 40, 49-84. 5. Chilton, F.H., and Murphy, R.C. (1986) J. Biol. Chem. 261, 7771-7777. 6. Thorngren,M., and Gustafson, A. (1981}Lancet 2, 1190-1193. 7. Lee, T.H. (1989) Cli~ Exp. Allergy 19 (SuppL 1), 15-23. 8. Babior, B.M. (1984}Blood 64 959-966. 9. Easmon, C.S.F., Cole, P.J., Williams, A.J., and Hastings, M. (1980) Immunology 41, 67-74. 10. Roberts, R.L., Mouness~ N.L., and Gallin, J.I. (1984) J. ImmunoL 132, 2000-2006. 11. Dahlgren, C. (1987) Agents and Actions 21, 104-112. 12. Edinboro,L.E., Van Dyke, K., Peden, D., Castranova, V., and Wierda, D. (1985) Microchem. J. 31, 261-271. 13. Edwards, S.W. (1987)J. Clin. Lab. Immunol. 22, 35-39. 14. Lee,T.H., Hoover,R.L.,Williams,J.D., Sperling,R.I., Ravalese, J., Spur, B.W., Robinson, D.R., Corey,E.J., Lewis, R.A., and Austen, K.F. (1985) N. EngL J. Med. 312, 1217-1224. 15. Payan,D.G., Wong, M.Y.S., Chernov-Rogan,T., Valone, F.H., Pickett, W.C.,Blake, V.A., Gold, W.M., and Goetzl,E.J. (1986) J. Clim Immunol. 6, 402-410. 16. Arm, J.P., Horton, C.E., Mencia-Huerta,J.M., House, F., Eiser, N.M., Clark, T.J.H., Spur, B.W., and Lee, T.H. (1988} Thorax 43, 84-92. 17. Chilton, F.H., O'Flaherty, J.T., Walsh, C.E., Thomas, M.J., Wykle, R.L., De Chatelet, L.R., and Waite, B.M. (1982)s Biol. Chem. 257, 5402-5407. 18. Lin, A.H., Morton, D.R., and Gorman, R.R. {1982}J. Cli~ Invest. 70, 1058-1063. 19. Lee, T.H., Sethi, E.G., Peters, W., Arm, J.P., Horton, C.E., Walport, M.J., and Spur, B.W. {1988} Clin. Sci. 74, 467-475. 20. Horrobin, D.F. {1987}Medical Hypotheses 22, 421-428. 21. Picado,C., Castillo,J.A., Schinca,N., Pujades, M., Ordinas, A., Coronas, A., and Agusti-Vidal, A. (1988} Thorax 43, 93-97. 22. Kitsch,C.M.,Payan,D.G.,Wong,M.Y.S.,Doblman,J.G., Blake, V.A., Petri, M.A., Offenberger,J., Goetzl,E.J., and Gold,W.M. (1988) Clir~ Allergy 18, 177-187. [Received August 24, 1989; Revision accepted April 13, 1990]
The Effect of Eicosapentaenoic Acid Consumption on Human Neutrophil Chemiluminescence 1 Philip J. Thompson*,a, Neil L.A. Missoa, Marion Passarellia a n d Martin J. Phillip,~ aDepartment of Medicine, Universityof Western Australia and bDepartment of Respiraton/Medicine, Sir Charles Gairdner Hospital, Nedlands, W.A. 6009, Australia The effect of eicosapentaenoic acid (EPA) on the inflammatory potential of neutrophils was investigated by supplementing the diets of 12 subjects with 2.16 g of EPA or 12 g of olive oil per day for 4 weeks in a double blind crossover study. Neutrophil function as assessed by iuminol enhanced chemiluminescence responses to plateletactivating factor (PAF) and formyl-methionyl-leucylphenylalanine (FMLP) was significantly reduced after EPA but not after olive oil consumption in the subjects who consumed EPA first. In contrast, EPA had no significant effect on neutrophil chemil-mlnescence in the subjects who consumed olive oil first. Dietary supplementation with EPA inhibits neutrophil responses to inflammatory mediators such as PAF while other fatty acids appear to modify the effects of EPA. Lipids 26, 1223-1226 (1991). Populations such as the Greenland Eskimos and Japanese have a high dietary intake of fish oil compared to most Western diets and are noted to have a much lower 4~revalence of coronary artery disease (1) and chronic inflammatory diseases such as asthma, rheumatoid arthritis and psoriasis (2). These epidemiological observations have led to the hypothesis that the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in oily cold-water fish may offer protection against these diseases. EPA inhibits the uptake of arachidonic acid (AA) into ceil membrane phospholipids and competes with AA for metabolism by the cyclooxygenase and lipoxygenase pathways. DHA is an inhibitor of these pathways (3). Metabolites of AA (thromboxane A2, prostaglandins and leukotrienes) are thought to contribute to the inflammatory process in asthma by recruiting and activating neutrophils, eosinophils and macrophages (4). Platelet-activating factor (PAF), another likely mediator of inflammation in asthma, is derived from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine, and its formation is closely linked with AA metabolism in neutrophils (5). Dietary supplementation with fish oil increases the proportion of EPA to AA in ceil membrane phospholipids (6) and thus could result in a decreased production of PAF and inflammatory eicosanoids as well as the production of less potent analogues such as thromboxane A 3 and LTB 5 (leukotriene B) (3,7). 1Based on a paper presented at the Third InternationalConference on Platelet~ActivatingFactor and StructurallyRelatedAlkylEther Lipids, Tokyo, Japan, May 1989. *To whom correspondence should be addressed at the University Department of Medicine, Queen Elizabeth II Medical Centre, Nediands, W.A. 6009, Australia. Abbreviations: AA, arachidonicacid;CL, chemiluminescence;DHA, docosahexaenoicacid; DMSO, dimethylsulfoxide;EPA, eicosapentaenoic acid;FMLP,formyl-methionyl-leucyl-phenylalanine;HBSSHEPES, Hanks balanced salt solutionbufferedwith N-2-hydroxyethylpiperazine~hr-2-ethanesulfonicacid; 5-HETE, 5-hydroxyeicosatetraenoic acid; LTB, leukotrieneB; PAF, platelet-activatingfactor.
An important function of stimulated neutrophils is the production of reactive oxygen metabolites (8), a process which can be monitored by the technique of luminolenhanced chemiluminescence (CL) (9). In this study the effect of dietary supplementation with EPA on the inflammatory potential of neutrophils was assessed by measuring neutrophil CL responses to in vitro stimulation with PAF and formyl-methionyl-leucyl-phenylalanine (FMLP) and comparing this with the effect of a placebo in the form of olive oil. MATERIALS AND METHODS Twelve healthy volunteers were entered into a double blind balanced crossover study comparing the effects of EPA and olive oil (placebo) on neutrophil chemiluminescence. They were asked to avoid all medication for two weeks prior to entry, and to maintain their normal diets prior to and during the study. The study was of sixteen weeks' duration and divided into 4 four-week intervals. Six subjects (Group 1, 3 males, 3 females aged 24-40 years with a mean age of 33 years) consumed MaxEPA capsules (Lipitac, Reckitt & Colman Products Pty. Ltd., West Ryde, NSW, Australia) in the first four weeks while the other six subjects (Group 2, 3 males, 3 females aged 23-38 years with a mean age of 29.5 years) consumed identical capsules containing olive oil. The dosage was 12 capsules daily, which provided 2.16 g of EPA per day or in the case of olive oil 12 g per day. After 4 weeks all subjects entered a four-week wash-out period when no capsules were taken. In the third four-week period, subjects were crossed over to the alternate capsules, and the study concluded with a four-week wash-out period. At the commencement of the study and after each four-week period (day 0, 28, 56, 85, and 112 of the study), blood was taken for neutrophil isolation. Neutrophils were isolated by centrifugation of heparin anticoagulated whole blood on discontinuous Percoll (Pharmacia LKB, Uppsala, Sweden) gradients (10). Neutrophils were washed in phosphate buffered saline, counted in a haemocytometer and resuspended in Hanks balanced salt solution buffered with N-2-hydroxyethylpiperazine~N'-2-ethanesulfonic acid (HBSS-HEPES) at 1.8 • 106 cells/mL. A 5 mM stock solution of luminol (Boehringer Mannheim, Mannheim, W. Germany) in dimethylsulfoxide (DMSO) was diluted in HBSS to give a 0.5 mM working solution. PAF (1-O-hexadecyl-2-acetyl-sn-glycer~3-phosphocholine, Sigma Chemical Co., St. Louis, MO) was stored as a 1 mM solution in ethanol at - 2 0 ~ On the day of use, 0.1 mL of the stock solution was evaporated under N 2 and resuspended in 1 mL of HBSS containing 0.25% human serum albumin to give a 10 -4 M solution which was further diluted with HBSS to give working solutions of the required concentration. FMLP (Sigma) was stored as a 10 -2 M solution in 50 /zL aliquots of DMSO at LIPIDS, Vol. 26, No. 12 (1991)
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P.J. THOMPSON E T AL. - 7 0 ~ Dilutions to give working solutions of the required concentration were made with HBSS. Chemiluminescence was measured in a LKB 1250 luminometer. The reaction mixture (total vol 0.5 mL) contained 7.5 • 105 neutrophils and 25 ~V[ luminol. The reaction was started by the addition of 50 ~L of PAF or F M L P to give the required final concentration. The CL response was monitored for 10-15 m l n on a digital display/printer unit and chart recorder. CL responses were measured as the m-ximum amplitude in mV, and the data were analyzed by calculating mean values (+_ standard error of mean). Students t-test for paired data with a significance level of p < 0.05 was used when comparing data from the same group of subjects on different days. RESULTS
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-4
PAF Concentration (M)
The luminol-enhanced CL response of neutrophils to PAF 22 and FMLP was biphasic. An initial rapid burst of chemiB luminescence which peaked at 1-2 rain after the addition 20 of the stimulus was followed by a more gradual rise in 18 CL which reached a maximum at 8-10 mln. In this study 16 the maximum amplitude of the initial peak which is i ~ 14 associated with the production of extracellular reactive E. 9 ~ 12 oxygen metabolites (11) was used as a measure of neutrophil CL responses to PAF and FMLP. O Neutrophil CL dose response curves to PAF and FMLP Q. 8 representing the mean responses of the 12 subjects at the 9 6 commencement of the study are shown in Figure 1. The -I mean msxlmum response to PAF was observed at a con4 2 ~ " centration of 5 • 10 -6 M. The mean FM L P response was maximum at 10 -6 M and was higher than the mean 0 | ........| ........! ........| ........ ! ........! ...... -4 PAF response {14.2 _+ 7.3 vs 10.6 -!-_2.4 mV). Similar PAF 1010 10.9 108 107 106 105 and FMLP concentrations have been used previously in FMLP Concentration (M) the measurement of neutrophil CL responses and have not been shown to affect cellular integrity (12,13). The FIG. I. Dose responses of neutrophU chemiluminescence to P A F {A} FMLP response was more variable at 10 -5 M than at and FMLP (B). Each point represents the mean (+__SENDCL response 10 -6 M, and for this reason the latter concentration was for all 12 subjects on day 0. For each test, P A F or F M L P was added used when comparing the mean responses on different to 7.5 X 105 neutrophils with 25/~M l.minoL, and the maximum amplitude of the CL response was measured in mV. days. At the P A F concentration which produced a m v x i m u m response, neutrophils from Group 1 subjects who con- were so low that meaningful variations with time could sumed E P A from day 0 to day 28 showed a significant not be discerned. For the Group 2 subjects, mean C L decrease in the mean C L response on day 28 compared responses to P A F concentrations of 10 -6, 10 -6 and 10 -7 to day 0 (4.9 _ 2.9 vs 10.9 + 4.6 mV, p < 0.005) (Fig. 2A). M did not change significantly during the course of the study. At the end of the firstwash-out period (day 56), the C L Comparisons of the m e a n C L responses to 10 -e M response had increased to 8.3 _ 3.4 m V which was not significantlydifferentto day 0. There were no significant F M L P were used to assess the effect of E P A consumpchanges in the C L responses measured afterconsumption tion on neutrophil response to this stimulus (Fig. 3A). In of placebo in the form of olive oil (day 85) or at the end Group 1 subjects, the C L response to 10 -6 M F M L P was of the study (day 112) (Fig. 2A). 13.8 _ 2.5 m V on day 0. After E P A consumption this In the Group 2 subjects who consumed placebo capsules decreased to 7.6 ___3.3 m V on day 28 and decreased furfrom day 0 to day 28, the mean CL response to a max- ther to 5.7 +_ 1.5 m V at the end of the firstwash-out on imum PAF concentration was not significantly different day 56 which was significantlyless than day 0 (p < 0.05). on day 28 (6.4 + 1.3 mV) compared to day 0 (10.3 +_ After subsequent placebo {olive oil) consumption, the 4.3 mV) (Fig. 2B). However the mean responses on day mean C L response returned to 12.0 _ 3.6 m V on day 85 85 (8.8 + 2.0 mV) after E PA consumption and on day 112 and to 13.4 +_ 5.2 m V at the end of the study. Neutrophils from Group 2 subjects who consumed olive (6.3 +_ 2.0 mV) were also not significantly different to the response on day 0 or on day 56 (6.9 _ 1.6 mV) prior to oil first gave a mean C L response to 10 -6 M F M L P of E P A consumption. 9.4 _+ 3.0 m V on day 0 {Fig. 3B). This did not change In response to 10 -5 and 10 -6 M PAF, similar trends significantly after placebo consumption (11.8 __ 5.3 m V were observed with a reduction in CL at day 28 in the on day 28, 11.5 _+ 3.9 m V on day 56) and although there Group 1 subjects. With 10 -7 M PAF, the CL responses was a decrease in the response after E P A consumption LIPIDS,Vol. 26, No. 12 (1991)
1225
NEUTROPHIL CHEMILUMINESCENCE AFTER EPA CONSUMPTION A
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8 6 4
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FIG. 2. Mean (~SEM) neutrophil chemiluminescence responses to PAF (5 X 10 -6 M) in Group 1 subjects who consumed EPA from day 0 to day 28 (A) and in Group 2 subjects who consumed olive oil capsules from day 0 to day 28 (B). * = p < 0.005 compared to day 0 by paired t-test.
FIG. 3. Mean (+__SEM) neutrophil chemiluminescence responses to FMLP (10-6 M) in Group I subjects who consumed EPA from day 0 to day 28 (A) and in Group 2 subjects who consumed olive oil capsules from day 0 to day 28 (B). * = p < 0.05 compared to day 0 by paired t-test.
(7.0 + 1.8 mV on day 85, 3.9 _+ 1.0 mV on day 112), this did not reach statistical significance. In the Group 1 subjects, CL responses to 10 -5 and 10 -s M FMLP showed similar trends to those described for 10 -6 M FMLP with CL being reduced on day 56 compared to day 0. With 10 -7 M FMLP, the trend for a reduction in CL could not be discerned. For the Group 2 subjects, the CL responses to 10 -5, 10 -7 and 10 -8 M FMLP did not change significantly with time.
the change was not statistically significant. Thus the results from the Group 2 subjects have to be regarded as inconclusive although there is some suggestion that olive oil may inhibit EPA suppression of neutrophil chemiluminescence. Three previous studies have examined the effects of dietary supplementation with EPA and DHA on human neutrophil chemotaxis and LTB4 production {14-16}. Higher doses {3.2-4.0 g} and longer durations {6-10 weeks) were used compared to this study. Neutrophil chemotaxis was reduced by up to 70% {14) and LTB4 production was decreased, with an increase in LTB5 production {15,16). In one of these studies, neutrophil function was not altered by consumption of placebo capsules containing olive off {16}. These studies have all assessed neutrophil function by measuring chemotactic responses to various stimuli (LTB4, FMLP, CSa). The chemiluminescence response investigated in this study monitors the release of reactive oxidant species, an important function of stimulated neutrophils. The chemotactic and chemiluminescence responses may be modified differently by dietary supplementation with EPA. Furthermore the
DISCUSSION
In the group of six subjects who consumed EPA capsules from day 0 to day 28, there was a significant reduction in the neutrophil CL responses to PAF and FMLP immediately after the consumption of EPA. Consumption of placebo capsules containing olive oil from day 56 to day 85 had no significant effect on neutrophil CL responses to PAF or FMLP in this group. In the other group of six subjects, olive oil consumption from day 0 to day 28 did not produce significant changes in CL response. After EPA consumption from day 56 to 85, the CL response to FMLP, but not to PAF, declined although
LIPID& Vol. 26, No. 12 (1991)
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P.J. THOMPSON E T AL. effect of EPA on PAF-induced neutrophil responses had not been investigated prior to this study. EPA consumption may have differential effects on neutrophil responses to PAF and other stimuli such as FMLP, LTB4 and C5a. The normal product of lipoxygenase activity in stimulated neutrophils, LTB4, may amplify the chemotactic and chemiluminescence responses to other stimuli such as PAF and FMLP. PAF has been shown to stimulate the production of LTB 4 in neutrophils (17,18), and in support of this data the lipoxygenase inhibitor, nordihydroguaiaretic acid, inhibits the neutrophil CL response to PAF {12}. CL responses to FMLP have also been linked to lipoxygenase activity (12). Other workers have shown that consumption of 3.2 g of EPA per day for 3 weeks (total dose 67.2 g) resulted in a 500% increase in the content of EPA in neutrophil phospholipids (14}. It seems likely that in our study in which the total dose was 60.5 g of EPA {2.16 g per day for 4 weeks) a similar incorporation of EPA into neutrophil phospholipids would have occurred and that this would have caused decreased LTB4 production, and increased LTB 5 production. Consequently, reduced CL responses to PAF and FMLP as observed in this study would be expected to occur. Incorporation of EPA into the phospholipids of neutrophil membranes may alter the structure and/or fluidity of the membrane such that function of surface receptors, such 9as those for PAF and FMLP, may be altered. However, in one previous study, evidence for a change in neutrophil membrane fluidity after dietary supplementation with EPA was lacking {14}. In this crossover study, there was some indication that the effect of EPA persisted for up to four weeks after dietary supplementation ceased. Thus, in Group 1 subjects, the mean CL response to FMLP was less on day 56 than on day 28, and in Group 2 subjects the mean FMLP response was less on day 112 than on day 85. This effect was not observed in the mean responses to PAF. In the design of this study, the duration of the wash-out period was determined on the basis that neutrophils are turned over rapidly, with a blood clearance half-time of approximately 7 hr. The fact that the effects of EPA on FMLP-induced CL responses persisted at the end of a four-week wash-out period suggests that the EPA was turned over and reincorporated into maturing cells or possibly incorporated into stem cells during dietary supplementation. Consumption of placebo capsules which contained olive off did not alter neutrophil CL in this study, and is in agreement with the lack of effect on chemotaxis as reported previously {16}. However prior consumption of olive oil appeared to reduce the effectiveness of EPA in inhibiting neutrophil CL. Oleic acid, the major fatty acid constituent of olive off {approximately 75%), can be metabolized to eicosatrienoic acid which may be converted to LTB 3 by the lipoxygenase pathway (7). Recent studies {7,19} have suggested that LTB 8 may be as potent as LTB 4 as an inflammatory stimulus. Olive oil also contains linoleic acid (5%) which can be metabolized to AA. Thus consumption of placebo capsules by the Group 2 subjects may have increased the amounts of proinflammatory eicosatrienoic acid and AA in their neutrophil membranes thereby reducing the subsequent effectiveness of the fixed dose of EPA. Other fatty acids may also be capable of influencing the effects of EPA, and it LIPIDS, Vol. 26, No. 12 (1991)
has been suggested (20) that the low prevalence of coronary artery disease and inflammatory disorders in Eskimos is due to the combined effects of high dietary EPA and genetically determined high plasma concentrations of dihomo-gamma-linolenic acid. Olive off is probably not an ideal placebo because, as this study has shown, it may be metabolized by similar biochemical pathways to those which metabolize EPA. Indeed the question of whether olive oil affects neutrophil function is worthy of investigation. The only ideal placebo for the study of the effects of EPA would be a marine lipid concentrate from which all the EPA is removed, but this is expensive and difficult to produce. Although the dose (2.16 g} and duration (4 weeks) of EPA supplementation were reduced in this study in comparison with others (3-4 g of EPA for 6-10 weeks) (14-16), the results are in agreement that dietary supplementation with EPA suppresses neutrophil function in vitro, and suggest lower doses may be effective. Although several studies have shown EPA has no effect on clinical symptoms when administered to asthmatic subjects (16,21,22), this may reflect clinical trial design. Alternatively, neutrophils may not play the major role in the inflammatory processes of asthma, and the concentration and duration of EPA supplementation needed to influence other inflammatory cell types and mediators involved in the pathogenesis of asthma (4) have yet to be elucidated. REFERENCES 1. Dyerberg, J., and Bang, H.O. (1978) Lancet 2, 152. 2. Kromann, N., and Green, A. (1980) Acta. Med. ScandL 208, 401-406. 3. Lee, T.H., and Arm, J.P. {1988}Br. Med. J. 297, 1421-1422. 4. Barnes,P.J., Chung,K.F.,and Page, C.P. (1988}PharmacoL Rev. 40, 49-84. 5. Chilton, F.H., and Murphy, R.C. (1986) J. Biol. Chem. 261, 7771-7777. 6. Thorngren,M., and Gustafson, A. (1981}Lancet 2, 1190-1193. 7. Lee, T.H. (1989) Cli~ Exp. Allergy 19 (SuppL 1), 15-23. 8. Babior, B.M. (1984}Blood 64 959-966. 9. Easmon, C.S.F., Cole, P.J., Williams, A.J., and Hastings, M. (1980) Immunology 41, 67-74. 10. Roberts, R.L., Mouness~ N.L., and Gallin, J.I. (1984) J. ImmunoL 132, 2000-2006. 11. Dahlgren, C. (1987) Agents and Actions 21, 104-112. 12. Edinboro,L.E., Van Dyke, K., Peden, D., Castranova, V., and Wierda, D. (1985) Microchem. J. 31, 261-271. 13. Edwards, S.W. (1987)J. Clin. Lab. Immunol. 22, 35-39. 14. Lee,T.H., Hoover,R.L.,Williams,J.D., Sperling,R.I., Ravalese, J., Spur, B.W., Robinson, D.R., Corey,E.J., Lewis, R.A., and Austen, K.F. (1985) N. EngL J. Med. 312, 1217-1224. 15. Payan,D.G., Wong, M.Y.S., Chernov-Rogan,T., Valone, F.H., Pickett, W.C.,Blake, V.A., Gold, W.M., and Goetzl,E.J. (1986) J. Clim Immunol. 6, 402-410. 16. Arm, J.P., Horton, C.E., Mencia-Huerta,J.M., House, F., Eiser, N.M., Clark, T.J.H., Spur, B.W., and Lee, T.H. (1988} Thorax 43, 84-92. 17. Chilton, F.H., O'Flaherty, J.T., Walsh, C.E., Thomas, M.J., Wykle, R.L., De Chatelet, L.R., and Waite, B.M. (1982)s Biol. Chem. 257, 5402-5407. 18. Lin, A.H., Morton, D.R., and Gorman, R.R. {1982}J. Cli~ Invest. 70, 1058-1063. 19. Lee, T.H., Sethi, E.G., Peters, W., Arm, J.P., Horton, C.E., Walport, M.J., and Spur, B.W. {1988} Clin. Sci. 74, 467-475. 20. Horrobin, D.F. {1987}Medical Hypotheses 22, 421-428. 21. Picado,C., Castillo,J.A., Schinca,N., Pujades, M., Ordinas, A., Coronas, A., and Agusti-Vidal, A. (1988} Thorax 43, 93-97. 22. Kitsch,C.M.,Payan,D.G.,Wong,M.Y.S.,Doblman,J.G., Blake, V.A., Petri, M.A., Offenberger,J., Goetzl,E.J., and Gold,W.M. (1988) Clir~ Allergy 18, 177-187. [Received August 24, 1989; Revision accepted April 13, 1990]
