Scand J Clin Lab Invest 1992; 52: 229-236
Long-term supplementation with n-3 fatty acids, 11: effect on neutrophil and monocyte chemotaxis
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E. B. SCHMIDT, K . VARMING, J . 0. PEDERSEN, H.-H. LERVANG, N. G R U N N E T , C . J E R S I L D , & J . D Y E R B E R G Departments of Clinical Chemistry and Clinical Immunology, Aalborg Hospital, Aalborg, Denmark.
Schmidt EB, Varming K, Pedersen JO, Lervang H.-H, Grunnet N, Jersild C, Dyerberg J. Long-term supplementation with n-3 fatty acids, 11: effect on neutrophil and monocyte chemotaxis. Scand J Clin Lab Invest 1992; 52: 229-236. The effect of a daily supplement with 4 g of n-3 polyunsaturated fatty acids (PUFA) for 9 months to 24 healthy volunteers on neutrophil and monocyte chemotaxis was studied using the under-agarose technique. Autologous serum and n-formyl-methionyl-leucyl-phenylalanine were used as chemoattractants. The effect after 9 months of supplementation with n-3 PUFA was also compared to results after short-term supplementation with n-3 PUFA for 6 weeks. Monocyte chemotaxis was reduced after 9 months of supplementation with n-3 PUFA to the same extent as after 6 weeks supplement. Neutrophil-directed migration towards chemoattractants was reduced after 9 months on fish oil, and this decrease was significantly greater than the decrease obtained after 6 weeks of supplementation. The spontaneous migration of neutrophils was significantly attenuated after 9 months compared to baseline and to 6 weeks. These findings lend support to a role for n-3 PUFA in the management of chronic inflammatory and atherosclerotic vascular diseases.
Key words: atheroslcerosis; n-3 fatty acids; fish oils; inflammation; leukocyte chemotaxis J l r n Dyerberg, MD, Dr med. Sci, Medi-Lab AlS, Adelgade 7, DK-I304 Copenhugen K , Denmurk
Intake of marine n-3 polyunsaturated fatty acids (n-3 PUFA) reduces neutrophil reactivity in healthy humans 11-51 and in patients with various diseases [&14]. Monocyte reactivity may also be inhibited by n-3 PUFA [1,3-5, 151. The modulation of leukocyte reactivity may account for the reported clinical effect of n-3 PUFA in inflammatory disorders, such as rheumatoid arthritis [7, 8, 11, 121 and psoriasis [9, 14, 16).
Previous trials investigating the effect of n-3 PUFA on leukocyte function have been of short duration. It is obviously of interest to ascertain the effect on leukocyte function when n-3 PUFA are given on a long-term basis. We have studied the effect of dietary supplementation with n-3 PUFA for 6 weeks and for 9 months on neutrophil and monocyte chemotaxis in healthy volunteers in order to elucidate whether the short-term effect on leukocyte chemotaxis is 229
230
E. B. Schmidt et al.
maintained, attenuated or amplified during supplementation with n-3 PUFA for longer periods of time.
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MATERIALS AND METHODS
published [I71 made by Jahres Fabrikker A/S, Sandefjord, Norway (EPAX 5S00TG) and delivered by Lube A/S, Hadsund, Denmark. The subjects were given 6 capsules of Pikasol daily, corresponding to 4 g of n-3 PUFA per day for 9 months.
Subjects
Isolation of neutrophils and monocytes
Twenty-four healthy subjects were studied. There were 14 females, with a mean age of 40 years (range 21-53), and 10 males, with a mean age of 39 years (range 3 M 7 ) . All subjects were free of medication. Aspirin and non-steroidal anti-inflammatory drugs were abandoned from 2 weeks before and during the study period. The participants were asked to maintain their usual dietary habits and life-style pattern. Informed consent was obtained and the trial was approved by the local Ethics Committee.
Neutrophils were harvested after HypaqueFicoll discontinuous gradient centrifugation [ 181 and washed three times in tissue culture medium (RPMI-1640). The purity of neutrophils was more than Y5%, and cell viability exceeded 98%, as evaluated by the trypan blue exclusion test. Cell counts were adjusted to I X I O x neutrophils ml-' RPMI-1640. Monocytes were purified from the crude mononuclear cell population by further centrifugation on a triple layer Percoll gradient [ 191. Monocytes were purified to more than 80%, the rest of cells being lymphocytes. The viability of monocytes after the purification procedure was above 88%. Cell counts were adjusted to 5x10' monocytes ml-' RPMI-1640.
Blood sampling Blood was drawn from an antecubital vein after an overnight fast and after 10 min of rest in the supine position. Blood was sampled before, after 6 weeks and after 9 months of supplementation with n-3 PUFA and again at least 3 months after the supplement wasstopped. E D T A in a final concentration of 0.003 mol I-' was used for anti-coagulation of the blood. Unfortunately, chemotaxis could not be determined in samples from 12 of the subjects before n-3 supplementation due to infection in the culture medium. Instead results obtained 3 months after the supplement was stopped were used as baseline values. This was felt justified, as chemotaxis has been shown to return to baseline levels 6 weeks after termination of supplementation with n-3 PUFA [l]. Furthermore, there was no difference between pre- and post-treatment results in the 12 participants with all test results available. In these subjects the mean of initial and post-supplement values was used as a baseline result. Consequently, no values are given for chemotaxis determined 3 months after the intake of n-3 PUFA had stopped.
Oil supplement The source of n-3 PUFA was PikasolK (a fish oil triglyceride with characteristics as previously
Chemotactic assays Neutrophil and monocyte chemotaxis were investigated using the under-agarose technique [4]. Ten microlitres of cell suspension ( 1 X 10" neutrophils and SX lo5 monocytes) were transferred to each of the wells in the middle row (Fig. 1). Ten microlitres of chemoattractant were placed in one row of wells, while 10 yl of RPMI-1640 were placed in the opposite wells as controls. The chemoattractants used were autologous serum and n-formyl-methionyl-leucylphenylalanine (FMLP) in a concentration of 1 x 10-xmol I-' RPMI-1640. This concentration of FMLP elicits a submaximal chemotactic response and ensures that no inhibition of front cell migration takes place, since the maximal concentration of chemoattractant at the edge of the cell well never exceeds 10%, of the original concentration. Cell migration was determined using a microscope equipped with a scale for measurement of migrated distances and a grid for counting the number of cells migrating into a predefined area of 250 pmx250 ym (Fig. 1). Each sample was investigated in triplicate and mean values of the following measurements were recorded.
Fish oil und leukocyte chemotaxis
0
231
migrated into the predefined area with its base at B (spontaneous migration) for neutrophils and B/2 for monocytes (Fig. 1).
CHEYO-
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ATTRACTANT
Assay variation Intra-assay (well-to-well) coefficient of variation was below 0.10 for neutrophils and below 0.23 for monocytes. Interassay (day-to-day) coefficient of variation was below 0.17 for neutrophils and below 0.24 for monocytes. Statistics Pratt’s test was used for comparison of chemotaxis. A p-value below 0.05 (two-tailed) was considered as significant.
RESULTS The fish oil concentrate was well tolerated and found acceptable for continued use. No dropouts were recorded. Determination of platelet fatty acid composition revealed an excellent compliance of the subjects to the fish oil supplement, as documented by an increase in both eicosapentaenoic acid and docosahexaenoic acid in each subject after the supplement [17]. Neutrophil chemotaxis FIG 1. The assay for determination of leukocyte migration is shown schematically. (A) Active migration by the front leading leukocytes towards the chernoattractant. (B) Spontaneous migration of leukocytes towards control medium. (C) Location of leukocytes after incubation (shaded area). (D) Cell density (number of leukocytes) in this 250x250 pm
area. Directed migration. The distance migrated by the leading front of cells towards the chemoattractant. Spontuneous migration. The distance rnigrated by the leading front of neutrophils towards control. Because of the long incubation period, monocytes moving towards control will be reached by the chemoattractant diffusing into the agarose. Consequently, spontaneous migration could not be determined for monocytes. Cell density. The number of cells that had
The spontaneous migration of neutrophils was unchanged after 6 weeks, but significantly reduced after 9 months of supplementation with n-3 PUFAs (Figs 2 and 3). Directed migration was reduced after 6 weeks on fish oil towards autologous serum, but not when FMLP was used as chemoattractant. Long-term supplementation with n-3 PUFAs for 9 months significantly inhibited neutrophil directed migration towards both chemoattractants compared t o results before supplementation and compared to results after 6 weeks of supplementation. Neutrophil chemotactic responsiveness (expressed as directed-spontaneous migration) was significantly decreased towards autologous serum after 6 weeks and after 9 months of supplement, with no difference between the two periods (Fig. 2). When FMLP was used as chemoattractant no changes in neutrophil chemotactic responsiveness could be demonstrated (Fig. 3).
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Neutrophil migration Directed
Sponteneous
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Irm 1
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Scand J Clin Lab Invest Downloaded from informahealthcare.com by Hochschulbibliothek Darmstadt on 11/24/14 For personal use only.
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FIG2. Neutrophil chemotaxis towards autologous serum before, alter 6 weeks, and after 9 months of supplementation with 4 g of n-3 PUFA daily. Results are given as medians with 25-75 pcrcentilc rangcs. p-values are indicated in the boxes.
Cell density was not significantly changed after ingestion of n-3 PUFA for 9 months (Figs 2 and 3). There was a significant increase in cell density between 6 weeks and 9 months of n-3 PUFA supplementation when autologous serum was used as chemoattractant. This finding must be interpreted bearing in mind the simultaneously large reduction seen in directed migration, since cell density inherently tends t o
increase when directed migration decreases in this assay (Fig. 1). Monocyte chemotaxis Monocyte-directed migration was significantly reduced after 6 weeks of n-3 PUFA ingestion towards both chemoattractants (Fig. 4). The decrease was maintained, and no
Fish oil and leukocyte chemotaxis
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Scand J Clin Lab Invest Downloaded from informahealthcare.com by Hochschulbibliothek Darmstadt on 11/24/14 For personal use only.
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FIG3. Neutrophil chemotaxis towards FMLP before, after 6 weeks and after 9 months of supplementation with 4 g of n-3 PUFA daily. Results are given as medians with 25-75 percentile ranges. p-values are indicated in the boxes.
additional effect was observed after 9 months of supplementation. Cell density was unaltered by long-term supplementation with n-3 PUFA. Cell density, however, temporarily decreased after 6 weeks intake of n-3 PUFA,when autologous serum was used as chemoattractant.
DISCUSSION In the present study neutrophil chemotaxis was
reduced after 6 weeks of supplementation with n-3 PUFA. This confirms the previously reported short-term effect of n-3 PUFA on neutrophil chemotaxis [I, 3, 4, 5, 7, 10, 131. Supplementation with n-3 PUFA for 9 months further decreased neutrophil chemotaxis, which is a new observation. Supplementation with n-3 PUFA could thus be beneficial in conditions with increased neutrophil reactivity. This is supported by observations of a reduction in neutrophil function simultaneous with a clinical
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E . B. Schmidt et al Directed migration
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Fic 4. Monocyte chemotaxis before, after h weeks, and after 9 months o f supplemcntation with 4 g o f n-3 PUFA daily. Results are given as medians with 25-75 percentile ranges. p values are indicated in thc boxes.
effect exerted by n-3 PUFA in patients with .inflammatory disorders [7, 8, 9, 11, 12, 14). Furthermore, inflammatory diseases-as well as coronary heart disease-are rare in Greenland Eskimos [20], a population with a very high intake of n-3 PUFA. In the present study monocyte chemotaxis was significantly reduced after 6 weeks and 9 months of supplementation with 4 g of n-3 PUFA. We have previously reported that monocyte chemotaxis was reduced after supplementation with n-3 PUFA for 6 weeks [4, 51. Furthermore, ingestion of n-3 PUFA has been reported to reduce mononuclear generation of pro-inflammatory leukotriene (LT) B4, plateletactivating factor, interleukin-1 and tumour necrosis factor [ l , 3 , 6, 8, 151. Monocytes are known to take part in chronic inflammatory
processes and to be important cells in atherogenesis [21]. A reduction in monocyte reactivity may thus contribute to t h e apparent beneficial effect of n-3 PUFA in coronary heart disease [20,22.23]. In interpreting the present results it should he born in mind that n o control group was included (discussed in [17]). Also, a complete set of measurements (pre-supplement, 6 weeks, 9 months and 3 months post-supplement) was only available in 12 of the subjects. However, as discussed above this probably did not affect results to any significant degree. Dietary n-3 PUFA are incorporated into leukocyte membrane phospholipids, released after appropriate stimulation and converted to LTB5, with much weaker effects than LTB4 on leukocyte adhesion, chemotaxis, aggregation
-
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Fish oil and leukocyte chemotaxis and degranulation (24, 251. Furthermore, the liberation of arachidonic acid from leukocyte membranes may be decreased, and its conversion to LTB4 through the 5-lipo-oxygenase pathway in leukocytes inhibited by n-3 PUFA [ I , 24, 251. Finally, n-3 PUFAs affect the physical properties of cell membranes [23], and thereby could influence the spontaneous and stimulated migration of leukocytes. We did not measure incorporation of n-3 PUFA into leukocyte membranes. However, long-chained n-3 PUFA were incorporated into platelet membranes, and although alterations in membrane fatty acid composition induced by dietary changes d o not necessarily occur in parallel in platelets and leukocytes [26], there is no reason to doubt that n-3 PUFA were actually incorporated into leukocytes also. The levels of arachidonic acid decreased in platelets during fish oil supplementation. Probably, arachidonic acid levels also decreased in leukocytes with a simultaneous reduction of formation of LTB4. W e have previously reported that spontaneous neutrophil migration is unaltered by dietary supplementation with n-3 PUFA for 6 weeks in healthy volunteers [4,51 and in diabetics [27]. In the present study, neutrophil spontaneous migration was unchanged after 6 weeks, but significantly decreased after 9 months of supplementation with n-3 PUFA. Chemotactic responsiveness was equally reduced after 6 weeks and 9 months intake of fish oil. Thus, while short-term supplementation with n-3 PUFA reduces neutrophil chemotactic responsiveness, spontaneous neutrophil migration may only be decreased after intake of n-3 PUFA for prolonged periods. In contrast, the effect of n-3 PUFA on monocyte chemotaxis was fully expressed after 6 weeks supplement. Regardless of the mechanisms involved, it has been established that dietary intake of n-3 PUFA reduces neutrophil and monocyte function. The present study has extended the knowledge on this issue by showing that leukocyte chemotaxis is still reduced, and for neutrophils even further reduced, after supplementation with n-3 PUFA for a longer period.
ACKNOWLEDGMENTS The study was supported by a grant from The
235
Northern Jutland County Fund for Medical Research.
REFERENCES 1 Lee TH. Hoover RL, Williams JD, Sperling RI, Ravalese 111 J , Spur BW, Robinson DR, Corey
EJ, Lewis RA, Austen KF. Effects of dietary enrichment with eicosapentaenoic acid and docosahexaneoic acids on in virro neutrophil and monocyte leukotriene generation and neutrophil function. N Engl J Med 1985; 312: 1217-24. 2 Fisher M, Upchurch KS, Levine PH, Johnson MH, Vaudreuil CH, Nayale A, Hoogasian JJ. Effects of dietary fish oil supplementation on polymorphonuclear leukocyte inflammatory potential. Inflammation 1986; 10: 387-92. 3 Endres S, Ghorhani R, Kelley VE, Georgilis K, Lonnemann G , van der Meer JWM, Cannon JG, Rogers TS, Klempner MS, Weher PC, Schaefer EJ, Wolff SM, Dinnarello CA. The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-I and tumor necrosis factor by mononuclear cells. N Engl J Med 1989; 320: 265-71. 4 Schmidt EB, Pedersen JO, Ekelund S, Grunnet N , Jersild C, Dyerberg J. Cod liver oil inhibits neutrophil and monocyte chemotaxis in healthy males. Atherosclerosis 1989; 77: 53-7. 5 Schmidt EB, Pedersen JO, Varming K , Ernst E, Jersild C, Grunnet N, Dyerherg J. n-3 fatty acids and leukocyte chemotaxis. Effects in hyperlipidaemia and dose-response studies in healthy men. Arteriosclerosis 1991; 1 I: 429-35. 6 Payan DG, Wong MYS, Chernov-Rogan T, Valone FH, Pickett WC, Blake VA, Gold WM, Goetzl EJ. Alterations in human leukocyte function induced by ingestion of eicosapentaenoic acid. J Clin Immunol 1986; 6: 402- 10. 7 Kremer JM, Jubiz W, Michalek A, Rynes RI, Bartholomew LE, Bigaouette J , Timchalk M, Beeler D, Lininger L. Fish-oil fatty acid supplementation in active rheumatoid arthritis. Ann Intern Med 1987; 106: 497-503. 8 Sperling RI, Weinhlatt M, Robin J-L, Ravalese I I I J , Hoover RL, House F, Cohlyn JS, Fraser PA, Spur BW. Robinson DR, Lewis RA, Austen KF. Effects of dietary supplementation with marine fish oil, leukocyte lipid mediator generation and function in rheumatoid arthritis. Arthritis Rheum 1987; 30: 988-97. Y Maurice PDL, Allen BR, Barkley ASJ, Cockbill SR, Stammers J , Bather PC. The effects of dietary supplementation with fish oil in patients with psoriasis. Br J Dermatol 1Y87; 117: 599-606. 10 Arm JP, Horton CE. Mencia-Huerta J-M, House F, Eiser NM, Clark TJH, Spur BW, Lee TH. Effect of dietary supplementation with fish oil lipids on mild asthma. Thorax 1988; 43: 84-92. II Clcland LG, French JK, Betts H, Murphy GA, Elliott MJ. Clinical and biochemical effects of dictary fish oil supplements in rheumatoid arthritis. J Rheumatol 1988; 15: 1471-5.
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12 Magaro M, Altomontc L, Zoli A, Mirone L, De Sole P, Di Mario G , Lippa S, Oradei A. Influence of diet with different lipid composition on neutrophil chemiluminiscence and disease activity in patients with rheumatoid arthritis. Ann Rheum Dis 1988; 47: 793-6. 13 Mehta JL. Lopez LM, Lawson D, Wargowich TJ, Williams LL. Dietary supplementation with omega-3 polyunsaturated fatty acids in patients with stahle coronary heart disease. Am J Med 1988; 84: 45-52. 14 Kragballe K, Fogh K. A low-fat diet supplemented with dietary fish oil (Max-EPA) results in improvement of psoriasis and in formation of leukotriene B,. Acta Derm Venereol (Stoch) 1989; 69: 23-8. 15 Sperling RI, Robin J-L, Kylander KA, Lee TH, Lewis RA, Austen KF. The effects of n-3 polyunsaturated fatty acids on the generation of platelet-activating factor-acether by human monocytes. J lmmunol 1987; 130: 4186-91. 16 Bittiner SB, Tucker WFG, Cartwright I, Bleehen SS. A double-blind, randomised, placebo-controlled trial of fish oil in psoriasis. Lancet 1988; i : 378-80. 17 Schmidt EB. Lervang H-H. Varming K. Madscn P, Dyerberg J . Long-term supplcmcntiition with n-3 fatty acids, I: effect on blood lipids, hacmostasis and blood pressure. Scand J Clin Lab Invest 1992; 52: 221-8. 18 Ferrante A, Thong YH. A rapid one-step procedure for purification of mononuclear and polymorphonuclcar leukocytes from human blood using a modification of the Hypaque/Ficoll technique. J Immunol Meth 1978; 24: 389-93. 19 Al-Sumidaie AM, Jones DL, Young HL. Characterisation of the under agarose method for quantifying migration of highly purified human
20 21 22
23 24
25 26
27
monocytes. J Immunol Methods 1984; 75: 129- 40. Kromann N , Green A. Epidemiologic studies in the Upernavik district, Greenland. Acta Med Scand 1980;208: 401-6. Ross R. The pathogenesis of atherosclcrosis--an update. N Engl J Med 1986; 314: 4XX-500. Burr ML, Fehily AM, Gilbert JF. Rogers S . Holliday RM, Sweetnam PM, Elwood PC. Deadman NM. Effects of changes in fat, fish. and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART). Lancet 1989; ii: 757-61. Leaf A, Weher PC. Cardiovascular effects of n-3 fatty acids. N Engl J Med 1988; 318: 549-57. Lee TH, Austen KF. Arachidonic acid metabolism by the 5-lipoxygenase pathway, and the effects of alternative dietary fatty acids. Adv lmmunol 1986; 39: 145-75. Schmidt EB, Dyerberg J . n-3 fatty acids and leukocytes. J Intern Med 1989; 225 (Suppl. I): 151-8. Hafstrom I, Ringertz B, Gyllenhammer H, Palmblad J , Harms-Ringdahl M. Effects of Fasting on disease activity, neutrophil functicin, fatty acid composition, and leukotriene biosynthesis in patients with rheumatoid arthritis. Arthritis Rheum 1988; 31: 585-92. Schmidt EB, Sarensen PJ. Pedersen JO, Jersild C, Ditzel J, Grunnet N, Dyerberg J . The efkct of n-3 polyunsaturated fatty acids on lipids, haemostasis, neutrophil and monocyte chemotaxis in insulin-dependent diahetes mcllitus. J Intern Meti 1989; 225 (SUPPI.I): 201-6.
Received 5 April 199I Accepted 3 November 1991
Long-term supplementation with n-3 fatty acids, 11: effect on neutrophil and monocyte chemotaxis
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Hochschulbibliothek Darmstadt on 11/24/14 For personal use only.
E. B. SCHMIDT, K . VARMING, J . 0. PEDERSEN, H.-H. LERVANG, N. G R U N N E T , C . J E R S I L D , & J . D Y E R B E R G Departments of Clinical Chemistry and Clinical Immunology, Aalborg Hospital, Aalborg, Denmark.
Schmidt EB, Varming K, Pedersen JO, Lervang H.-H, Grunnet N, Jersild C, Dyerberg J. Long-term supplementation with n-3 fatty acids, 11: effect on neutrophil and monocyte chemotaxis. Scand J Clin Lab Invest 1992; 52: 229-236. The effect of a daily supplement with 4 g of n-3 polyunsaturated fatty acids (PUFA) for 9 months to 24 healthy volunteers on neutrophil and monocyte chemotaxis was studied using the under-agarose technique. Autologous serum and n-formyl-methionyl-leucyl-phenylalanine were used as chemoattractants. The effect after 9 months of supplementation with n-3 PUFA was also compared to results after short-term supplementation with n-3 PUFA for 6 weeks. Monocyte chemotaxis was reduced after 9 months of supplementation with n-3 PUFA to the same extent as after 6 weeks supplement. Neutrophil-directed migration towards chemoattractants was reduced after 9 months on fish oil, and this decrease was significantly greater than the decrease obtained after 6 weeks of supplementation. The spontaneous migration of neutrophils was significantly attenuated after 9 months compared to baseline and to 6 weeks. These findings lend support to a role for n-3 PUFA in the management of chronic inflammatory and atherosclerotic vascular diseases.
Key words: atheroslcerosis; n-3 fatty acids; fish oils; inflammation; leukocyte chemotaxis J l r n Dyerberg, MD, Dr med. Sci, Medi-Lab AlS, Adelgade 7, DK-I304 Copenhugen K , Denmurk
Intake of marine n-3 polyunsaturated fatty acids (n-3 PUFA) reduces neutrophil reactivity in healthy humans 11-51 and in patients with various diseases [&14]. Monocyte reactivity may also be inhibited by n-3 PUFA [1,3-5, 151. The modulation of leukocyte reactivity may account for the reported clinical effect of n-3 PUFA in inflammatory disorders, such as rheumatoid arthritis [7, 8, 11, 121 and psoriasis [9, 14, 16).
Previous trials investigating the effect of n-3 PUFA on leukocyte function have been of short duration. It is obviously of interest to ascertain the effect on leukocyte function when n-3 PUFA are given on a long-term basis. We have studied the effect of dietary supplementation with n-3 PUFA for 6 weeks and for 9 months on neutrophil and monocyte chemotaxis in healthy volunteers in order to elucidate whether the short-term effect on leukocyte chemotaxis is 229
230
E. B. Schmidt et al.
maintained, attenuated or amplified during supplementation with n-3 PUFA for longer periods of time.
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MATERIALS AND METHODS
published [I71 made by Jahres Fabrikker A/S, Sandefjord, Norway (EPAX 5S00TG) and delivered by Lube A/S, Hadsund, Denmark. The subjects were given 6 capsules of Pikasol daily, corresponding to 4 g of n-3 PUFA per day for 9 months.
Subjects
Isolation of neutrophils and monocytes
Twenty-four healthy subjects were studied. There were 14 females, with a mean age of 40 years (range 21-53), and 10 males, with a mean age of 39 years (range 3 M 7 ) . All subjects were free of medication. Aspirin and non-steroidal anti-inflammatory drugs were abandoned from 2 weeks before and during the study period. The participants were asked to maintain their usual dietary habits and life-style pattern. Informed consent was obtained and the trial was approved by the local Ethics Committee.
Neutrophils were harvested after HypaqueFicoll discontinuous gradient centrifugation [ 181 and washed three times in tissue culture medium (RPMI-1640). The purity of neutrophils was more than Y5%, and cell viability exceeded 98%, as evaluated by the trypan blue exclusion test. Cell counts were adjusted to I X I O x neutrophils ml-' RPMI-1640. Monocytes were purified from the crude mononuclear cell population by further centrifugation on a triple layer Percoll gradient [ 191. Monocytes were purified to more than 80%, the rest of cells being lymphocytes. The viability of monocytes after the purification procedure was above 88%. Cell counts were adjusted to 5x10' monocytes ml-' RPMI-1640.
Blood sampling Blood was drawn from an antecubital vein after an overnight fast and after 10 min of rest in the supine position. Blood was sampled before, after 6 weeks and after 9 months of supplementation with n-3 PUFA and again at least 3 months after the supplement wasstopped. E D T A in a final concentration of 0.003 mol I-' was used for anti-coagulation of the blood. Unfortunately, chemotaxis could not be determined in samples from 12 of the subjects before n-3 supplementation due to infection in the culture medium. Instead results obtained 3 months after the supplement was stopped were used as baseline values. This was felt justified, as chemotaxis has been shown to return to baseline levels 6 weeks after termination of supplementation with n-3 PUFA [l]. Furthermore, there was no difference between pre- and post-treatment results in the 12 participants with all test results available. In these subjects the mean of initial and post-supplement values was used as a baseline result. Consequently, no values are given for chemotaxis determined 3 months after the intake of n-3 PUFA had stopped.
Oil supplement The source of n-3 PUFA was PikasolK (a fish oil triglyceride with characteristics as previously
Chemotactic assays Neutrophil and monocyte chemotaxis were investigated using the under-agarose technique [4]. Ten microlitres of cell suspension ( 1 X 10" neutrophils and SX lo5 monocytes) were transferred to each of the wells in the middle row (Fig. 1). Ten microlitres of chemoattractant were placed in one row of wells, while 10 yl of RPMI-1640 were placed in the opposite wells as controls. The chemoattractants used were autologous serum and n-formyl-methionyl-leucylphenylalanine (FMLP) in a concentration of 1 x 10-xmol I-' RPMI-1640. This concentration of FMLP elicits a submaximal chemotactic response and ensures that no inhibition of front cell migration takes place, since the maximal concentration of chemoattractant at the edge of the cell well never exceeds 10%, of the original concentration. Cell migration was determined using a microscope equipped with a scale for measurement of migrated distances and a grid for counting the number of cells migrating into a predefined area of 250 pmx250 ym (Fig. 1). Each sample was investigated in triplicate and mean values of the following measurements were recorded.
Fish oil und leukocyte chemotaxis
0
231
migrated into the predefined area with its base at B (spontaneous migration) for neutrophils and B/2 for monocytes (Fig. 1).
CHEYO-
Scand J Clin Lab Invest Downloaded from informahealthcare.com by Hochschulbibliothek Darmstadt on 11/24/14 For personal use only.
ATTRACTANT
Assay variation Intra-assay (well-to-well) coefficient of variation was below 0.10 for neutrophils and below 0.23 for monocytes. Interassay (day-to-day) coefficient of variation was below 0.17 for neutrophils and below 0.24 for monocytes. Statistics Pratt’s test was used for comparison of chemotaxis. A p-value below 0.05 (two-tailed) was considered as significant.
RESULTS The fish oil concentrate was well tolerated and found acceptable for continued use. No dropouts were recorded. Determination of platelet fatty acid composition revealed an excellent compliance of the subjects to the fish oil supplement, as documented by an increase in both eicosapentaenoic acid and docosahexaenoic acid in each subject after the supplement [17]. Neutrophil chemotaxis FIG 1. The assay for determination of leukocyte migration is shown schematically. (A) Active migration by the front leading leukocytes towards the chernoattractant. (B) Spontaneous migration of leukocytes towards control medium. (C) Location of leukocytes after incubation (shaded area). (D) Cell density (number of leukocytes) in this 250x250 pm
area. Directed migration. The distance migrated by the leading front of cells towards the chemoattractant. Spontuneous migration. The distance rnigrated by the leading front of neutrophils towards control. Because of the long incubation period, monocytes moving towards control will be reached by the chemoattractant diffusing into the agarose. Consequently, spontaneous migration could not be determined for monocytes. Cell density. The number of cells that had
The spontaneous migration of neutrophils was unchanged after 6 weeks, but significantly reduced after 9 months of supplementation with n-3 PUFAs (Figs 2 and 3). Directed migration was reduced after 6 weeks on fish oil towards autologous serum, but not when FMLP was used as chemoattractant. Long-term supplementation with n-3 PUFAs for 9 months significantly inhibited neutrophil directed migration towards both chemoattractants compared t o results before supplementation and compared to results after 6 weeks of supplementation. Neutrophil chemotactic responsiveness (expressed as directed-spontaneous migration) was significantly decreased towards autologous serum after 6 weeks and after 9 months of supplement, with no difference between the two periods (Fig. 2). When FMLP was used as chemoattractant no changes in neutrophil chemotactic responsiveness could be demonstrated (Fig. 3).
232
E. B. Schmidt et al.
Neutrophil migration Directed
Sponteneous
(
Irm 1
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Density cells/area
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FIG2. Neutrophil chemotaxis towards autologous serum before, alter 6 weeks, and after 9 months of supplementation with 4 g of n-3 PUFA daily. Results are given as medians with 25-75 pcrcentilc rangcs. p-values are indicated in the boxes.
Cell density was not significantly changed after ingestion of n-3 PUFA for 9 months (Figs 2 and 3). There was a significant increase in cell density between 6 weeks and 9 months of n-3 PUFA supplementation when autologous serum was used as chemoattractant. This finding must be interpreted bearing in mind the simultaneously large reduction seen in directed migration, since cell density inherently tends t o
increase when directed migration decreases in this assay (Fig. 1). Monocyte chemotaxis Monocyte-directed migration was significantly reduced after 6 weeks of n-3 PUFA ingestion towards both chemoattractants (Fig. 4). The decrease was maintained, and no
Fish oil and leukocyte chemotaxis
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FIG3. Neutrophil chemotaxis towards FMLP before, after 6 weeks and after 9 months of supplementation with 4 g of n-3 PUFA daily. Results are given as medians with 25-75 percentile ranges. p-values are indicated in the boxes.
additional effect was observed after 9 months of supplementation. Cell density was unaltered by long-term supplementation with n-3 PUFA. Cell density, however, temporarily decreased after 6 weeks intake of n-3 PUFA,when autologous serum was used as chemoattractant.
DISCUSSION In the present study neutrophil chemotaxis was
reduced after 6 weeks of supplementation with n-3 PUFA. This confirms the previously reported short-term effect of n-3 PUFA on neutrophil chemotaxis [I, 3, 4, 5, 7, 10, 131. Supplementation with n-3 PUFA for 9 months further decreased neutrophil chemotaxis, which is a new observation. Supplementation with n-3 PUFA could thus be beneficial in conditions with increased neutrophil reactivity. This is supported by observations of a reduction in neutrophil function simultaneous with a clinical
234
E . B. Schmidt et al Directed migration
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Fic 4. Monocyte chemotaxis before, after h weeks, and after 9 months o f supplemcntation with 4 g o f n-3 PUFA daily. Results are given as medians with 25-75 percentile ranges. p values are indicated in thc boxes.
effect exerted by n-3 PUFA in patients with .inflammatory disorders [7, 8, 9, 11, 12, 14). Furthermore, inflammatory diseases-as well as coronary heart disease-are rare in Greenland Eskimos [20], a population with a very high intake of n-3 PUFA. In the present study monocyte chemotaxis was significantly reduced after 6 weeks and 9 months of supplementation with 4 g of n-3 PUFA. We have previously reported that monocyte chemotaxis was reduced after supplementation with n-3 PUFA for 6 weeks [4, 51. Furthermore, ingestion of n-3 PUFA has been reported to reduce mononuclear generation of pro-inflammatory leukotriene (LT) B4, plateletactivating factor, interleukin-1 and tumour necrosis factor [ l , 3 , 6, 8, 151. Monocytes are known to take part in chronic inflammatory
processes and to be important cells in atherogenesis [21]. A reduction in monocyte reactivity may thus contribute to t h e apparent beneficial effect of n-3 PUFA in coronary heart disease [20,22.23]. In interpreting the present results it should he born in mind that n o control group was included (discussed in [17]). Also, a complete set of measurements (pre-supplement, 6 weeks, 9 months and 3 months post-supplement) was only available in 12 of the subjects. However, as discussed above this probably did not affect results to any significant degree. Dietary n-3 PUFA are incorporated into leukocyte membrane phospholipids, released after appropriate stimulation and converted to LTB5, with much weaker effects than LTB4 on leukocyte adhesion, chemotaxis, aggregation
-
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Fish oil and leukocyte chemotaxis and degranulation (24, 251. Furthermore, the liberation of arachidonic acid from leukocyte membranes may be decreased, and its conversion to LTB4 through the 5-lipo-oxygenase pathway in leukocytes inhibited by n-3 PUFA [ I , 24, 251. Finally, n-3 PUFAs affect the physical properties of cell membranes [23], and thereby could influence the spontaneous and stimulated migration of leukocytes. We did not measure incorporation of n-3 PUFA into leukocyte membranes. However, long-chained n-3 PUFA were incorporated into platelet membranes, and although alterations in membrane fatty acid composition induced by dietary changes d o not necessarily occur in parallel in platelets and leukocytes [26], there is no reason to doubt that n-3 PUFA were actually incorporated into leukocytes also. The levels of arachidonic acid decreased in platelets during fish oil supplementation. Probably, arachidonic acid levels also decreased in leukocytes with a simultaneous reduction of formation of LTB4. W e have previously reported that spontaneous neutrophil migration is unaltered by dietary supplementation with n-3 PUFA for 6 weeks in healthy volunteers [4,51 and in diabetics [27]. In the present study, neutrophil spontaneous migration was unchanged after 6 weeks, but significantly decreased after 9 months of supplementation with n-3 PUFA. Chemotactic responsiveness was equally reduced after 6 weeks and 9 months intake of fish oil. Thus, while short-term supplementation with n-3 PUFA reduces neutrophil chemotactic responsiveness, spontaneous neutrophil migration may only be decreased after intake of n-3 PUFA for prolonged periods. In contrast, the effect of n-3 PUFA on monocyte chemotaxis was fully expressed after 6 weeks supplement. Regardless of the mechanisms involved, it has been established that dietary intake of n-3 PUFA reduces neutrophil and monocyte function. The present study has extended the knowledge on this issue by showing that leukocyte chemotaxis is still reduced, and for neutrophils even further reduced, after supplementation with n-3 PUFA for a longer period.
ACKNOWLEDGMENTS The study was supported by a grant from The
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Northern Jutland County Fund for Medical Research.
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Received 5 April 199I Accepted 3 November 1991
