Atherosclerosis, 53 II (1989) 53-57 Elsevier Scientific Publishers Ireland, Ltd. A TH 04305 Cod liver oil inhibits neutrophil and monocyte chemotaxis in healthy males E.B. Schmidt I, J.O. Pedersen 2, S. Ekelund ‘, N. Grunnet 2, C. Jersild 2 and J. Dyerberg 1 ’ Department of Clinical Chemistry, and 2 Deportment of Clinical Immunology, Aalborg Hospital, DK-9000 Aalborg (Denmark) (Received 29 A ugust, 1988) (Revised, received 23 December, 1988) (A ccepted 4 January, 1989) Summary Epidemiological evidence suggests a reduced rate of chronic inflammatory diseases and ischaemic heart disease in populations with a high consumption of fish. This has been ascribed to the high content in sea food of polyunsaturated fatty acids (PUFAs), belonging to the n - 3 family. We have studied neutrophil and monocyte chemotaxis in 12 healthy males before and after 6 weeks supplementation with cod liver oil, corresponding to 5.3 g n - 3 PUFAs daily. Neutrophil and monocyte chemotaxis were investigated using the under agarose technique with N-formyl-methionyl-leucyl-phenylalanine (N-FMLP) and autologous serum as chemoattractants. Neutrophil chemotaxis towards both chemoattractants and monocyte chemotaxis towards N-FMLP were significantly reduced after supplementation with cod liver oil. Key words: Cod liver oil; n - 3 fatty acids; Neutrophil chemotaxis; diseases; Atherosclerosis Introduction Recent studies have suggested that monocytes and macrophages play an important role in the early stages of atherosclerosis [1,2]. Polymorphonuclear leucocytes (PMNL) have also been implicated in ischaemic heart disease (IHD), particularly in acute myocardial ischaemic events [3-51. Epidemiological evidence has indicated a reduced risk of IHD in populations with a high Correspondence cal Chemistry, to: Dr. E.B. Schmidt, D epartment of Clini- A alborg A alborg, Denmark. 0021-9150/89/$03.50 Hospital, Section North, D K-9000 Monocyte chemotaxis; Inflammatory intake of fish [6,7]. This has been attributed to the high content in sea food of polyunsaturated fatty acids (PUFAs) belonging to the n - 3 family, especially eicosapentaenoic acid (EPA; 20 : 5n 3). In contrast, PUFAs in the traditional western diet nearly exclusively belong to the n - 6 family, represented by linoleic acid and arachidonic acid. Chronic inflammatory diseases seem to be rare in Greenland Eskimos [6]. Interestingly, administration of n - 3 PUFAs to human volunteers has recently been shown to inhibit important functions of leukocytes [5,8,9]. In the present study we have found that supplementation with cod liver oil reduce neutrophil chemotaxis in healthy males. A novel finding is that monocyte chemotaxis also was significantly 0 1989 Elsevier Scientific Publishers Ireland, Ltd. 54 reduced after liver oil. dietary supplementation with cod Material 12 healthy males, all belonging to the hospital staff, were investigated. The age of the volunteers ranged from 29 to 49 years (median 35 years). All were non-smokers free from any medication. Nonsteroidal anti-inflammatory drugs were not taken in the last 2 weeks prior to or during the study. The participants were given their usual unrestricted diet supplemented with 30 ml cod liver oil daily for 6 weeks from P. Moeller, Oslo, Norway, corresponding to an extra daily intake of 5.3 g n - 3 PUFAs (2.5 g EPA). The trial was approved by the local Ethical Committee. Methods Blood was drawn from an antecubital vein in the morning after an overnight fast before and after supplementation with cod liver oil. Alcohol was not allowed for the last 2 days prior to blood sampling. Neutrophil chemotaxis and migration was performed as previously described [lo]. Briefly, PMNL were purified from EDTA-stabilized blood Hypaque/Ficoll discontinuous gradient using centrifugation. Neutrophils were harvested and washed 3 times in culture medium RPM1 1640. Cell counts were adjusted to 1 X lo8 neutrophils/ ml medium. Migration experiments were performed using the under agarose method [ll]. The gels were made from 0.75480,w/v, agarose moulded on gelatinized microscope object slides. The buffer system was Hepes at a final concentration of 0.05 mol/l. pH was adjusted to 7.1 using 1 mol/l NaOH. Gelatin was added to the agarose gel at a final concentration of 0.2548, w/v, as a source of protein [12] to avoid possible influence by allologous plasma proteins in the assay. Six chemotactic systems each comprizing 3 wells (diameter 2.5 mm) were punched out in the agar gel opposite each other at a mutual distance of 2.5 mm. 10 ~1 of cell suspension (1 X lo6 cells) were transferred to each well in the middle row. The chemoattractants used were: (1) N-FMLP at a concentration of lo-’ mol/l RPM1 1640. (2) Autologous serum. By direct application to the agarose gel, serum complement is activated and the chemotactic factor C5a is formed (unpublished results). 10 ~1 of chemoattractant were transferred to 1 well and 10 ~1 of culture medium RPM1 1640 was used as control in the opposite well. The agarose plates were incubated at 37 o C for 120 n-tin in humid atmospheric air. Migration was stopped by floating the agarose plates in 2.5% glutaraldehyde in phosphate buffered saline. After fixation overnight the agarose gels were removed. Slides were stained with Wright’s stain and provided with a coverslip. Monocytes were purified from the mononuclear ring obtained as described above by transferring the crude mononuclear cells to a triple layer discontinuous gradient of Percoll [13]. After centrifugation for 90 min, cells were harvested and washed 3 times in cold RPM1 1640. Cell count was adjusted to 1 X 10’ monocytes/ml RPM1 1640 using esterase staining to identify them. Migration experiments were carried out as described for neutrophils with the modifications that distances between wells were 4.0 mm and incubation time was 20 h. Results were read using an ordinary light microscope provided with a fine calibrated scale for measuring migration distances and a grid for counting cells migrated into a defined plane of were 250 X 250 pm. The following measurements recorded: (A) Directed migration: The distance migrated in pm by the leading front of cells towards the chemoattractant. (B) Spontaneous migration: The distance migrated in pm by the leading front of cells towards control culture medium. (D) Cell density: The number of cells, which migrated into the grid, located with the base at a distance equal to B. For monocytes, B was not considered as spontaneous migration, because after 20 h of incubation the chemoattractant is widely dispersed in the gel, so migration is no longer spontaneous. Cell density was determined with the base of the grid placed at a distance of B/2 for monocytes. Statistics. Results before and after cod liver oil supplementation are compared using Pratt’s test [14]. 55 Results CELL COUNT/AREA 130, Neutrophil migration The median distances migrated by PMNL are shown in Table 1. Both the migrations towards autologous serum and N-FMLP were significantly decreased after ingestion of cod liver oil. Also cell density was significantly lowered after the oil supplement. The spontaneous migrations were unaltered. 120_ llO_ loo_ go_ 80_ 70_ Monocyte migration Two of the 12 persons studied had monocyte chemotaxis, which for technical reasons could not be evaluated. From Table 2 it is seen that the median distances migrated by monocytes were unaffected by cod liver oil. On the other hand, cell density was lowered after oil supplementation. This decrease was statistically significant for 60_ 50_ 40_ 30_ 20_ IO_ TABLE 1 NEUTROPHIL MIGRATION TOWARDS N-FMLP (f), AND AUTOLOGOUS SERUM (s) BEFORE AND AFTER COD LIVER OIL SUPPLEMENTATION Values are medians (interquartile range), n = 12. (A) (f) (s) (B) (D) (f) (s) Before After P 2 135 (2000-2 328) 1568 (1420-l 703) 1630 (1310-l 937) 1377 (858-l 450) < 0.01 < 0.05 892 (767- 993) 797 (665- 927) > 0.1 183 148 (145(95- 207) 192) 123 88 (102(60- 138) 96) < 0.01 < 0.01 A = directed migration in pm; B = spontaneous migration in pm. D = cell density in cells/area. TABLE 2 MONOCYTE MIGRATION TOWARDS N-FMLP (f), AND AUTOLOGOUS SERUM (s) BEFORE AND AFTER COD LIVER OIL SUPPLEMENTATION Values are medians (interquartile range), n = 10. (A) (f) (s) (D) (f) (s) Before After P 1263 (1080-1466) 1467 (1040-1753) 1290 (1133-1407) 1384 (1213-1746) z 0.1 10.1 55 127 (39(67- 107) 146) 30 69 (16(53- 33) 88) < 0.01 > 0.1 A = directed migration in Pm; D = cell density in cells/area. BEFORE AFTER Fig. 1. Monocyte migration towards N-FMLP (cell density) before and after cod liver oil supplementation in each participant. migration towards N-FMLP (P -C0.01). Overall monocyte chemotaxis therefore was reduced after supplementation with cod liver oil. Fig. 1 demonstrates that the decrease in monocyte cell density after cod liver oil, was the result of decrements in all participants. Discussion In the present study the chemotactic responsiveness of PMNL was markedly reduced after supplementation with n - 3 PUFAs, which is in agreement with most other reports [8,15-181. Sperling et al. [19], however, found that PMNL chemotaxis increased after daily supplementation with 6 g n - 3 PUFAs for 6 weeks in patients with rheumatoid arthritis, and no changes were seen in PMNL chemotaxis in 8 healthy persons after dietary addition of 10 g EPA daily for 4 weeks [20]. Dietary supplementation with n - 3 PUFAs leads to formation of leukotriene B, (LTB,) at the expense of proinflammatory leukotriene B4 (LTB,) 56 from PMNL and monocytes. While LTB, has very potent effects on chemotaxis, aggregation and degranulation of leukocytes, LTB, is much less active [21,22]. Thus there is a sound basis for considering n - 3 PUFAs in the management of inflammatory disorders. Supplementation with fish oil of diets for patients with rheumatoid arthritis, psoriasis and ulcerative colitis indeed has given promising results [23-251. Furthermore, fish oil feeding in animals has been shown to reduce myocardial infarct size [26,27] and to reduce the incidence of serious ventricular arrhythmias after coronary artery ligation and during reperfusion [28]. Although the mechanisms are unknown, the important role of PMNL in acute myocardial ischaemia [3-51 indicates that suppression of neutrophil activity by fish oil is likely to have contributed. A new observation is that monocyte chemotaxis was markedly reduced after intake of n - 3 PUFAs. In the only study on this issue so far reported no effect on chemotactic responsiveness of mononuclear cells was observed in 6 patients given 4 g EPA for 8 weeks [16]. This study was conducted in patients with asthma and was methodologically different and not comparable to the present. Further studies are thus clearly indicated. Interestingly, short term supplementation with n - 3 PUFAs has been reported to reduce monocyte production of LTB, [8], platelet activating factor [29] and of interleukin-1 and tumor necrosis factor [30]. The apparent attenuation of monocyte reactivity after intake of n - 3 PUFAs may be of interest to the development of atherosclerosis, as monocytes and macrophages have been increasingly incriminated in this process [1,2]. This is further stressed by the observation of a reduction in atherosclerosis and in macrophage accumulation in aortic intimas after fish oil supplementation in rhesus monkeys on a high cholesterol diet f311. In conclusion, we have found a reduction in PMNL and monocyte chemotactic responsiveness in healthy males after supplementation with cod liver oil. This adds further evidence to an anti-inflammatory effect of n - 3 PUFAs with possible implication for their use in inflammatory disease states and IHD. Acknowledgements This study was supported by grants from the Northern Jutland County Fund for Medical Research, Aalborg Municipal Fund for Medical Research and Aalborg Frivillige Bloddonores Fund for Medical Research. References 1 Ross, R., The pathogenesis of atherosclerosis - an update, N. Engl. J. Med., 314 (1986) 488. 2 Mitchinson, M.J. and Ball, R.Y., Macrophages and atherogenesis, Lancet, ii (1987) 146. D.E., Bagge, U., Matrai, A. and 3 Ernst, E., Hammerschmidt, Dormandy, J.A., Leukocytes and the risk of ischemic diseases, J. Am. Med. Assoc., 257 (1987) 2318. 4 Lucchesi, B.R. and Mullane, K.M., Leukocytes and ischemia-induced myocardial injury, Annu. Rev. Pharmacol. Toxicol., 26 (1986) 201. 5 Moncada, S. and Salmon, J.A., Leucocytes and tissue injury: the use of eicosapentaenoic acid in the control of white cell activation, Wiener Klin. Wochenschr., 98 (1986) 104. 6 Kromann, N. and Green, A., Epidemiological studies in the Upernavik district, Greenland: incidence of some chronic diseases 1950-1974, Acta Med. Stand., 208 (1980) 401. 7 Leaf, A. and Weber, P.C., Cardiovascular effects of n -3 fatty acids, N. Engl. J. Med., 318 (1988) 549. 8 Lee, T.H., Hoover, R.L., Williams, J.D.. Sperling, RI., Ravalese 111, J., Spur, B.W., Robinson, D.R., Corey, E.J., Lewis, R.A. and Austen, K.F., Effects of dietary enrichment with eicosapentaenoic acid and docosahexaenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function, N. Engl. J. Med., 312 (1985) 1217. 9 Strasser, T., Fischer, S. and Weber, P.C., Leukotriene B, is formed in human neutrophils after dietary supplementation with icosapentaenoic acid, Proc. Natl. Acad. Sci. USA, 82 (1985) 1540. 10 Pedersen, J.O., Knudsen, F. and Jersild, C.. Acute effect of hemodialysis on neutrophil migration: impact on humoral and cellular function, Kidney Int., 33 (1988) 86. 11 Nelson, R.D., Quie, P.G. and Simmons, R.L., Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leucocytes and monocytes. J. Immunol., 115 (1975) 1650. 12 Chenoweth, D.E., Rowe, J.G. and Hugli, T.E., A modified method for chemotaxis under agarose, J. Immunol. Methods, 25 (1978) 337. 13 Al-Sumidaie, Jones, D.L. and Young, H.L., Characterisation of the under-agarose method for quantifying migration of highly purified human monocytes, J. Immunol. Methods, 75 (1984) 129. 14 Rahe, A.J., Tables of critical values for the Pratt matched pair signed rank statistic, J. Am. Stat. Assoc., 69 (1974) 368. 57 15 Terano, 16 17 18 19 20 21 22 23 T., Hirai, A., Tamura, Y., Kumagai, A. and Yoshida, S., Effect of dietary supplementation of highly purified eicosapentaenoic acid and docosahexaenoic acid on arachidonic acid metabolism in leucocytes and leukocyte function in healthy volunteers, Adv. Prostaglandin Thromboxane Leukotriene Res., 17 (1987) 880. Payan, D.G., Wong, M.Y.S., Chemov-Rogan, T., Valone, F.M., Pickett, W.C., Blake, V.A., Gould, W.M. and Goetzl, E.J., Alterations in human leukocyte function induced by ingestion of eicosapentaenoic acid, J. Clin. Immunol., 6 (1986) 402. 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., Effect of dietary supplementation with fish oil lipids on mild asthma, Thorax, 43 (1988) 84, Mehta, J.L., Lopez, L.M., Lawson, D., Wargovich, T.J. and Williams, L.L., Dietary supplementation with omega-3 polyunsaturated fatty acids in patients with stable coronary heart disease, Am. J. Med., 84 (1988) 45. Sperling, RI., Weinblatt, M., Robin, J-L., Ravalese, III J., Hoover, R.L., House, F., Coblyn, J.S., Fraser, P.A., Spur, B.W., Robinson, D.R., Lewis, R.A. and Austen, K.F., Effects of dietary supplementation with marine fish oil on leukocyte lipid mediator generation and function in rheumatoid arthritis, Arthritis Rheum., 30 (1987) 988. Knapp, H.R. and FitzGerald, G.A., Effects of dietary fish oil on human leukocyte function, Clin. Res., 34 (1986) 390 (Abstract). Terano, T., Salmon, J.A. and Moncada, S., Biosynthesis and biological activity of leukotriene B,, Prostaglandins, 27 (1984) 217. Lee, T.H. and Austen, K.F., Arachidonic acid metabolism by the 5-lipoxygenase pathway, and the effects of altemative dietary fatty acids, Adv. Immunol., 39 (1986) 145. Kremer, J.M., Jubiz, W., Michalek, A., Rynes, RI., 24 25 26 27 28 29 30 31 Bartholomew, L.E., Bigaouette, J., Timachalk, M., Beeler, D. and Lininger, L., Fish-oil fatty acid supplementation in active rheumatoid arthritis, Ann. Intern. Med., 106 (1987) 497. Bittiner, S.B., Tucher, W.F.G., Cartwright, I. and Bleehen, S.S., A double-blind, randomised, placebo-controlled trial of fish oil in psoriasis, Lancet, ii (1988) 378. McCall, T., O’Leary, D., Bloomfield, J. and G’Morain, C.A., Eicosapentaenoic acid (EPA), Gastroenterology, 94 (1988) 294 (abstract). Culp, B.R., Lands, W.E.M., Lucchesi, B.R., Pitt, B. and Romson, J., The effect of dietary supplementation of fish oil on experimental myocardial infarction, Prostaglandins, 20 (1980) 1021. Hock, C.E., Holahan, M.A. and Reibel, D.K., Effect of dietary fish oil on myocardial phospholipids and myocardial ischemic damage, Am. J. Physiol., 252 (1987) 554. McLennan, P.L., Abeywardena, M.Y. and Chamock, J.S., Dietary fish oil prevents ventricular fibrillation following coronary artery occlusion and reperfusion, Am. Heart J., 116 (1988) 709. Sperling, RI., Robin, J-L., Kylander, K.A., Lee, T.H., Lewis, R.A. and Austen, K.F., The effects of n - 3 polyunsaturated fatty acids on the generation of platelet-activating factor-acether by human monocytes, J. Immunol., 139 (1987) 4186. Endres, S., Ghorbani, R., Kelley, V.E., Lonnemann, G., van der Meer, J.W.M., Cannon, J.G. and Dinarello, C.A., Dietary n - 3 polyunsaturated fatty acids suppress the synthesis of interleukin-1 and tumor necrosis factor in human, Eur. J. Clin. Invest., 18 (1988) A54 (abstract). Davis, H.R., Bridenstine, R.T., Vesselinovitch, D. and Wissler, R.W., Fish oil inhibits development of atherosclerosis in rhesus monkeys, Arteriosclerosis, 7 (1987) 441.