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Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland
2To whom correspondence should be addressed. E-mail: ih{at}hi.is.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • fish oil • cytokines • tumor necrosis factor • interleukin 10 • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Tumor necrosis factor-
(TNF-)3 is a proinflammatory cytokine that is important in the primary immune response against bacterial and viral infections. It is produced by activated monocytes, macrophages and lymphocytes (1
) and leads to proliferation of activated T- and B-cells (2
), up-regulation of major histocompatibility complex expression (3
) and expression of adhesion molecules on endothelial cells (4
).
Interleukin (IL)-10, on the other hand, is an anti-inflammatory cytokine. It is synthesized by monocytes, macrophages and activated T- and B-cells (5
). It was initially identified as a product of T-cells involved in T helper (Th) 2 type immune response and inhibits T-cell proliferation, development and function (6
). It has now been shown to inhibit secretion of both Th1 and Th2 type cytokines (6
,7
). IL-10 protects mice from death during septic peritonitis (8
) and down-regulates lipopolysaccharide (LPS)-induced mRNA expression of proinflammatory cytokines from monocytes/macrophages (9
). It is thought to be produced later in the inflammatory response than TNF (7
,8
).
A number of studies have demonstrated enhanced TNF secretion by LPS-stimulated murine resident peritoneal macrophages from mice fed fish oil (10
–13
). The one study that has examined the effect of dietary fish oil on IL-10 secretion by resident peritoneal macrophages showed no effect of fish oil on IL-10 secretion but it also did not show a significant effect of fish oil on TNF secretion (14
).
Fish oil is currently believed to have anti-inflammatory properties and this effect is now well established in murine T-cells. The increased TNF secretion by peritoneal macrophages from mice fed fish oil indicates a proinflammatory effect of fish oil on these cells. The aim of this study was to determine whether dietary fish oil also has a proinflammatory effect due to decreasing the secretion of the anti-inflammatory cytokine, IL-10, by murine peritoneal macrophages and to investigate whether the effects of fish oil on TNF and IL-10 are interrelated or mediated by eicosanoids.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experimental procedures using laboratory animals complied with the NIH guidelines. Female BalbC mice (18–20g) (Bomholtgaard Copenhagen, Denmark) were assigned randomly to one of two experimental diets. Mice were housed five per cage at 25°C with a 12-h light:dark cycle.
Experimental diets were designed according to AIN-93 guidelines (15
) with modification in fat content. They were based on a nutritionally complete diet made for the addition of 200 g/kg of fat (ICN Pharmaceuticals, Aurora, OH) containing (per kg): 230 g casein, 354.6 g cornstarch, 57.4 g fiber, 3.4 g L-cystine, 40.2 g mineral mix (AIN-93G), 11.5 g vitamin mix (AIN-93), 100 g sucrose and 2.9 g choline bitartrate. The fish oil diet contained 180 g/kg menhaden fish oil and 20 g/kg corn oil (ICN Pharmaceuticals, Asse-Relegem, Belgium). The corn oil diet contained 200 g/kg corn oil. Relative amounts of selected fatty acids in the diets are shown in Table 1
. BHT (1.2 mmol/L; ICN Biomedicals) was added to the oils to prevent their deterioration (16
). Diets were prepared in bulk and daily portions packed in zip-lock bags, flushed with nitrogen, sealed and stored at -20°C. Water and food were consumed ad libitum. The mice were fed the experimental diets for 4 wk.
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Livers were perfused with saline (9 g/L NaCl, 1 g/L EDTA), and the lipids extracted with the method of Bligh and Dyer (17
). Phospholipids were separated from the neutral lipids by TLC using chloroform/methanol (8:1, v/v) and methylated in boron-trifluoride-methanol complex (Merck, Darmstadt, Germany) at 110°C for 45 min. The methyl esters were extracted into hexane (Merck) and after evaporation resuspended in isooctane (Merck) and injected into a gas chromatograph (Hewlett-Packard 5890 series II, Palo Alto, CA) equipped with a CP-Wax 52CB column (25 m x 0.25 mm x 0.2 µm) and a flame ionization detector, with hydrogen as the carrier gas.
Isolation and activation of macrophages.
Mice were anesthetized with isoflurane (Abbot Scandinavia AB, Solna, Sweden) and blood collected by axillary bleeding in acid citrate dextrose (Greiner Bio-One, Kremsmunster, Austria) anticoagulant to minimize the effect of the anticoagulant on cytokine secretion and measurement (18
). Plasma was prepared and heat inactivated at 56°C for 40 min to be used as autologous plasma. Peritoneal cells were collected in cold PBS without calcium or magnesium. After washing twice, the cells were suspended in Dulbecco’s minimum essential medium (DMEM, Gibco BRL, Life Technologies, Paisley, UK) with GlutaMAX–I, penicillin (1000 U/L) and streptomycin (100 mg/L). They were counted on a hemocytometer and 2.5 x 105 cells/well incubated in a 48-well flat-bottomed plate at 37°C with 50 mL/L CO2. After culturing for 2 h, nonadherent cells were discarded and the adherent cells washed twice with PBS and incubated in 0.25 mL DMEM with 50 mL/L autologous plasma. Adherent macrophages were stimulated with LPS (1 mg/L) (E. coli 055:195, Difco, Detroit, MI). TNF and IL-10 were neutralized using neutralizing antibodies that have been shown to be effective in neutralizing mouse TNF- and IL-10 bioactivity, respectively (R&D Systems, Minneapolis, MN). The suitable concentration for the neutralizing antibodies was assessed using duo set ELISA kits for the cytokines. Indomethacin (IM) (Sigma, St. Louis, MO) was used to block prostaglandin (PG) production (0.1 µmol/L). The concentration of IM used has previously been shown to prevent PG secretion by peritoneal macrophages under similar conditions (19
). Macrophages isolated from mice fed a standard nonpurified diet (Special Diets Services, Witham, UK) were stimulated with LPS and cultured with or without 10 nmol/L to 10 µmol/L MK886 (Affinity Research Products, Exeter, UK), a 5-lipoxygenase activating protein (FLAP) inhibitor. A concentration of 20 nmol/L has been shown to be effective in inhibiting leukotriene (LT)B4 synthesis by rat neutrophils after stimulation with the Ca2+ ionophore, A23187 (20
).
Cytokine measurements.
After incubation, the culture plates were centrifuged and cell culture supernatants collected and stored at -70°C. TNF- and IL-10 were measured in the supernatants using Duo Set ELISA kits (R&D Systems).
Statistical analysis.
Differences between dietary groups were analyzed using an unpaired t test. Differences between treatments within dietary groups were analyzed using two-way ANOVA, followed by Bonferroni’s post-hoc test. Differences were determined to be significant when P < 0.01.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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There were no differences in the body weights or relative weight gains of mice fed the different diets. Cell numbers from the peritoneum did not differ between groups.
Liver phospholipid fatty acid composition.
Hepatic phospholipids from mice consuming the fish oil diet had a significantly higher (n-3) polyunsaturated fatty acid (PUFA) content and significantly lower (n-6) PUFA content, than hepatic phospholipids from mice fed the corn oil diet (Table 2
). These differences in fatty acid composition of hepatic phospholipids are similar to what has been shown previously (19
) and demonstrate the effectiveness of the diets in changing tissue fatty acid composition.
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When stimulated with LPS (1 mg/L), macrophages from mice fed the fish oil diet secreted twice the amount of TNF (178 ± 25 ng/L) as macrophages from mice fed the corn oil diet (86 ± 10 ng/L), confirming previous results by others (10
–13
). However, macrophages from mice fed the fish oil diet secreted only one third of the IL-10 (93 ± 10 ng/L) that macrophages from mice fed the corn oil diet (262 ± 30 ng/L) secreted.
Time courses for TNF and IL-10 secretion.
Because IL-10 has been shown to suppress TNF secretion by LPS-stimulated human monocytes (7
), the decrease in IL-10 secretion by macrophages from mice fed the fish oil diet could be responsible for the increase in TNF secretion. We therefore determined the time courses for LPS-induced TNF and IL-10 secretion by macrophages from mice fed corn oil and fish oil diets (Fig. 1
). TNF was significantly increased in the medium 3 h after incubation with LPS and peaked at 6–12 h, somewhat later than has been shown previously (21
). The time course for IL-10 secretion in the present study was similar to that for TNF, with IL-10 increasing in the medium at 3 h and peaking at 6–12 h. The early secretion of IL-10 is similar to what has been shown previously; however, IL-10 secretion peaked at 6–12 h in the present study compared with 24 h in the previous study (22
).
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Effect of neutralizing antibody against IL-10 on TNF secretion by macrophages.
To further elucidate whether decreased IL-10 secretion by macrophages from mice fed the fish oil diet could be responsible for the increased TNF secretion by these cells, we used neutralizing antibody against IL-10. The IL-10 concentration measured in samples with added neutralizing antibody against IL-10, using an ELISA assay, was <5% of the IL-10 measured in samples without antibody. The neutralizing antibody against IL-10 had no effect on TNF secretion by macrophages from mice fed the corn oil diet and slightly increased TNF secretion by macrophages from mice fed the fish oil diet (Fig. 2A
), indicating that the decreased IL-10 secretion by macrophages from mice fed the fish oil diet was not responsible for the effect of fish oil on TNF secretion. Although it was not likely that the effect of fish oil on IL-10 secretion was dependent on its effect on TNF, we also used neutralizing antibody against TNF and measured its effect on IL-10 secretion. The TNF concentration in samples with added neutralizing antibody against TNF was <5% of the TNF measured in samples without the antibody. As expected, the neutralizing anti-TNF antibody had no effect on IL-10 secretion by macrophages from mice fed either of the two experimental diets (Fig. 2
B). These results indicate that the effects of fish oil on TNF and IL-10 secretion by resident peritoneal macrophages are independent of the effect on the other cytokine.
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PGE2 decreases TNF secretion by elicited peritoneal macrophages (23
) but enhances IL-10 secretion (24
). Dietary fish oil decreases PGE2 production by murine peritoneal macrophages (10
) and the decreased PGE2 production is responsible in part for the effect of fish oil on TNF secretion (19
). We therefore determined whether decreased IL-10 secretion by macrophages from mice fed the fish oil diet could also be mediated by the decrease in PGE2 production. As expected, blocking PG production with IM increased LPS-induced TNF secretion by macrophages from mice fed the corn oil diet, but did not affect TNF secretion by macrophages from mice fed the fish oil diet (Fig. 2
A), eliminating the difference in TNF secretion by macrophages from mice fed the two experimental diets. In contrast, IM had no effect on IL-10 secretion by macrophages from mice fed the corn oil and slightly decreased IL-10 secretion by macrophages from mice fed the fish oil diet (Fig. 2B)
; thus, the difference in IL-10 secretion by macrophages from mice fed the different diets remained after inhibition of PG production. Thus, decreased IL-10 secretion by macrophages from mice fed the fish oil diet is not likely to be mediated by decreased PGE2 production.
Effect of blocking LT production on IL-10 secretion.
LTB4 enhances IL-10 secretion by spleen cells after concanavalin A stimulation (25
). Resident peritoneal macrophages do not produce much LTB4 after LPS stimulation but they do produce some (26
). We therefore investigated whether blocking LTB4 production affected IL-10 secretion by the resident peritoneal macrophages. Blocking 5-lipoxygenase (LO) with the FLAP inhibitor, MK886, had little effect on IL-10 secretion (172 ± 41 ng/L without MK886, 152 ± 21 with 10 nmol/L and 146 ± 12 with 20 nmol/L) by macrophages from mice fed a nonpurified diet, rendering it unlikely that the effect of fish oil on IL-10 secretion is mediated by its effect on LTB production. Using a very high concentration (10 µmol/L) of MK886 reduced IL-10 secretion to 117 ± 13 ng/L, but at this concentration it may be affecting cellular mechanisms in addition to the 5-LO (27
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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One other study has investigated the effect of dietary fish oil on IL-10 secretion by resident peritoneal macrophages and showed no effect compared with safflower oil (14
). Neither was there a significant effect of dietary fish oil on TNF secretion (14
), although results from the present study and other similar studies show an increase in TNF production by macrophages from mice fed a fish oil diet (10
–13
). The conditions used in the study by Wallace et al. (14
) are similar to the conditions used in the present study except for the strain of mice (C57Bl/6) and the use of fetal calf serum (FCS) in the cell cultures. The BalbC mouse strain is thought to be more Th2 prone than the C57Bl/6 mouse strain (28
). However, studies using both C57Bl/6 (11
) and BalbC mice (10
) show increased TNF production by macrophages from mice fed fish oil. Similarly, although the effects of dietary lipid manipulation on fatty acid composition are reversed when lymphocytes are cultured in FCS (29
), increased TNF production by resident peritoneal macrophages from mice fed a fish oil diet has been shown in studies using FCS (13
). It is therefore not likely that the strain of mice or the use of FCS in the study by Wallace et al. (14
) is responsible for the lack of effect of fish oil on IL-10 secretion.
The results from the present study show similar time courses for TNF and IL-10 secretion after LPS stimulation and are consistent with other studies (21
,22
). The effect of fish oil on IL-10 secretion was evident at an earlier time point than its effect on TNF secretion. However, the effects of fish oil on IL-10 and TNF secretion seem to be independent of the effect on the other as demonstrated using neutralizing antibodies against each cytokine. Furthermore, the effect of fish oil on IL-10 secretion is probably mediated by some mechanism other than decreasing PG or LT production, although the effect of fish oil on TNF secretion is mediated in part by decreased PG production.
Regulation of LPS-induced IL-10 production by macrophages is not fully understood but is currently under rigorous investigation. The effect of fish oil on IL-10 secretion could be mediated through the different signaling molecules or pathways that are involved in regulation of IL-10 production. Further studies are required to elucidate the mechanism by which dietary fish oil affects secretion of IL-10 by murine resident peritoneal macrophages.
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FOOTNOTES |
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3 Abbreviations used: DMEM, Dulbecco’s minimum essential medium; FCS, fetal calf serum; FLAP, 5-lipoxygenase activating protein; IL, interleukin; IM, indomethacin; LO, lipoxygenase; LPS, lipopolysaccharide; LT, leukotriene; PG, prostaglandin; PUFA, polyunsaturated fatty acids; Th, T helper; TNF-, tumor necrosis factor-.
Manuscript received 15 July 2002. Initial review completed 10 August 2002. Revision accepted 16 September 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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