Abstract
Fish oils are used as therapeutic agents in chronic inflammatory diseases. The omega-3 fatty acids (FA) found in these oils are mainly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. The anti-inflammatory properties of fish oils are attributed to both omega-3 fatty acids. However, it is unknown whether such effects are due to either EPA or DHA. In this study, the effects of EPA and DHA on rat neutrophil function in vitro were compared. Both EPA and DHA increased the production of H2O2 when cells were stimulated or not with lipopolysaccharides (LPS). However, EPA was more potent than DHA in triggering an increase in superoxide release by cells in the basal condition or when stimulated with phorbol myristate acetate (PMA) or zymosan. Only DHA increased the phagocytic capacity and fungicidal activity of neutrophils. Both FA increased the release of tumor necrosis factor-α (TNF-α) in nonstimulated cells, but only EPA increased the production of cytokine-inducing neutrophil chemoattractant-2 (CINC-2) in the absence or presence of LPS, whereas production of interleukin-1 beta (IL-1β) was only increased by DHA in the presence of LPS. In addition, there was no alteration in the production of nitric oxide. In conclusion, we show herein that EPA and DHA can differently modulate aspects of the neutrophil response, which may be relevant for the development of therapies rich in one or other FA depending on the effect required.
Similar content being viewed by others
Abbreviations
- ANOVA:
-
Analysis of variance
- CINC-2:
-
Cytokine-inducing neutrophil chemoattractant-2
- DHA:
-
Docosahexaenoic acid
- DPI:
-
Diphenyliodonium
- ELISA:
-
Enzyme-linked immunosorbent assay
- EPA:
-
Eicosapentaenoic acid
- FA:
-
Fatty acid
- FBS:
-
Fetal bovine serum
- fMLP:
-
N-formyl-methionyl-leucyl-phenylalanine
- HEPES:
-
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- HRP:
-
Horseradish peroxidase
- IL-1β:
-
Interleukin-1 beta
- LPS:
-
Lipopolysaccharides
- NADPH:
-
Reduced nicotinamide adenine dinucleotide phosphate
- NLR:
-
Nucleotide-binding protein oligomerization domain
- PBS:
-
Phosphate-buffered saline
- PMA:
-
Phorbol myristate acetate
- PUFA:
-
Polyunsaturated fatty acid
- RNS:
-
Reactive nitrogen species
- ROS:
-
Reactive oxygen species
- RPMI:
-
Roswell park memorial institute
- TLR:
-
Toll-like receptor
- TNF-α:
-
Tumor necrosis factor-α
References
Segal AW (2005) How neutrophils kill microbes. Annu Rev Immunol 23:197–223
Chen GY, Nunez G (2010) Sterile inflammation: sensing and reacting to damage. Nat Rev Immunol 10:826–837
Barton GM (2008) A calculated response: control of inflammation by the innate immune system. J Clin Invest 118:413–420
Ekman AK, Cardell LO (2010) The expression and function of nod-like receptors in neutrophils. Immunology 130:55–63
Scapini P, Lapinet-Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA (2000) The neutrophil as a cellular source of chemokines. Immunol Rev 177:195–203
Cassatella MA (1995) The production of cytokines by polymorphonuclear neutrophils. Immunol Today 16:21–26
Rudkowska I (2010) Fish oils for cardiovascular disease: impact on diabetes. Maturitas 67:25–28
Wang C, Harris WS, Chung M, Lichtenstein AH, Balk EM, Kupelnick B, Jordan HS, Lau J (2006) n-3 Fatty acids from fish or fish-oil supplements, but not alpha-linolenic acid, benefit cardiovascular disease outcomes in primary- and secondary-prevention studies: a systematic review. Am J Clin Nutr 84:5–17
Kris-Etherton PM, Harris WS, Appel LJ (2002) Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 106:2747–2757
Calder PC (1998) Dietary fatty acids and lymphocyte functions. Proc Nutr Soc 57:487–502
Calder PC (1998) Immunoregulatory and anti-inflammatory effects of n-3 polyunsaturated fatty acids. Braz J Med Biol Res 31:467–490
Mori TA, Beilin LJ (2004) Omega-3 fatty acids and inflammation. Curr Atheroscler Rep 6:461–467
De Caterina R, Madonna R, Massaro M (2004) Effects of omega-3 fatty acids on cytokines and adhesion molecules. Curr Atheroscler Rep 6:485–491
Byrne J, McGuinness J, Chen G, Hill AD, Redmond MJ (2011) Intravenous omega-3, a technique to prevent an excessive innate immune response to cardiac surgery in a rodent gut ischemia model. J Thorac Cardiovasc Surg 141:803–807
Rees D, Miles EA, Banerjee T, Wells SJ, Roynette CE, Wahle KW, Calder PC (2006) Dose-related effects of eicosapentaenoic acid on innate immune function in healthy humans: a comparison of young and older men. Am J Clin Nutr 83:331–342
Mazza M, Pomponi M, Janiri L, Bria P, Mazza S (2007) Omega-3 fatty acids and antioxidants in neurological and psychiatric diseases: an overview. Prog Neuropsychopharmacol Biol Psychiatry 31:12–26
Pisani LF, Lecchi C, Invernizzi G, Sartorelli P, Savoini G, Ceciliani F (2009) In vitro modulatory effect of omega-3 polyunsaturated fatty acid (EPA and DHA) on phagocytosis and ROS production of goat neutrophils. Vet Immunol Immunopathol 131:79–85
Nair SS, Leitch JW, Falconer J, Garg ML (1997) Prevention of cardiac arrhythmia by dietary (n-3) polyunsaturated fatty acids and their mechanism of action. J Nutr 127:383–393
Calder PC (2010) Omega-3 fatty acids and inflammatory processes. Nutrients 2:355–374
de Lima TM, Amarante-Mendes GP, Curi R (2007) Docosahexaenoic acid enhances the toxic effect of imatinib on Bcr-Abl expressing HL-60 cells. Toxicol In Vitro 21:1678–1685
Vinolo MA, Hatanaka E, Lambertucci RH, Newsholme P, Curi R (2009) Effects of short chain fatty acids on effector mechanisms of neutrophils. Cell Biochem Funct 27:48–55
Vinolo MA, Rodrigues HG, Hatanaka E, Sato FT, Sampaio SC, Curi R (2011) Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils. J Nutr Biochem 22:849–855
Rodrigues HG, Vinolo MA, Magdalon J, Fujiwara H, Cavalcanti DM, Farsky SH, Calder PC, Hatanaka E, Curi R (2010) Dietary free oleic and linoleic acid enhances neutrophil function and modulates the inflammatory response in rats. Lipids 45:809–819
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279
Martins de Lima T, Cury-Boaventura MF, Giannocco G, Nunes MT, Curi R (2006) Comparative toxicity of fatty acids on a macrophage cell line (J774). Clin Sci (Lond) 111:307–317
Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP (1997) A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 253:162–168
Hatanaka E, Levada-Pires AC, Pithon-Curi TC, Curi R (2006) Systematic study on ROS production induced by oleic, linoleic, and gamma-linolenic acids in human and rat neutrophils. Free Radic Biol Med 41:1124–1132
Rosenfeld G (1947) Método rápido de coloração de esfregaços de sangue: Noções práticas sobre corantes pancrômicos e estudo de diversos fatores. 20:315–328
Fock RA, Vinolo MA, de Moura Sa Rocha V, de Sa Rocha LC, Borelli P (2007) Protein-energy malnutrition decreases the expression of TLR-4/md-2 and CD14 receptors in peritoneal macrophages and reduces the synthesis of TNF-alpha in response to lipopolysaccharide (LPS) in mice. Cytokine 40:105–114
Rogero MM, Borelli P, Fock RA, de Oliveira Pires IS, Tirapegui J (2008) Glutamine in vitro supplementation partly reverses impaired macrophage function resulting from early weaning in mice. Nutrition 24:589–598
Rogero MM, Borelli P, Vinolo MA, Fock RA, de Oliveira Pires IS, Tirapegui J (2008) Dietary glutamine supplementation affects macrophage function, hematopoiesis and nutritional status in early weaned mice. Clin Nutr 27:386–397
Corazzini R (1993) Avaliação morfo-fisiológica de macrófagos peritoneais de camundongos submetidos ao choque térmico. In Tese de Mestrado em patologia Experimental e Comparada, Faculdade de Medicina Veterinária e Zootecnia 156
Ding AH, Nathan CF, Stuehr DJ (1988) Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol 141:2407–2412
Slusser WN, Wade GN (1981) Testicular effects on food intake, body weight, and body composition in male hamsters. Physiol Behav 27:637–640
Gyllenhammar H (1987) Lucigenin chemiluminescence in the assessment of neutrophil superoxide production. J Immunol Methods 97:209–213
Lecchi C, Invernizzi G, Agazzi A, Ferroni M, Pisani LF, Savoini G, Ceciliani F (2011) In vitro modulation of caprine monocyte immune functions by omega-3 polyunsaturated fatty acids. Vet J 189:353–355
Calder PC, Bond JA, Harvey DJ, Gordon S, Newsholme EA (1990) Uptake and incorporation of saturated and unsaturated fatty acids into macrophage lipids and their effect upon macrophage adhesion and phagocytosis. Biochem J 269:807–814
Kew S, Gibbons ES, Thies F, McNeill GP, Quinlan PT, Calder PC (2003) The effect of feeding structured triacylglycerols enriched in eicosapentaenoic or docosahexaenoic acids on murine splenocyte fatty acid composition and leucocyte phagocytosis. Br J Nutr 90:1071–1080
Schroit AJ, Gallily R (1979) Macrophage fatty acid composition and phagocytosis: effect of unsaturation on cellular phagocytic activity. Immunology 36:199–205
Guzik TJ, Korbut R, Adamek-Guzik T (2003) Nitric oxide and superoxide in inflammation and immune regulation. J Physiol Pharmacol 54:469–487
Wheeler MA, Smith SD, Garcia-Cardena G, Nathan CF, Weiss RM, Sessa WC (1997) Bacterial infection induces nitric oxide synthase in human neutrophils. J Clin Invest 99:110–116
de Lima TM, de Sa Lima L, Scavone C, Curi R (2006) Fatty acid control of nitric oxide production by macrophages. FEBS Lett 580:3287–3295
Komatsu W, Ishihara K, Murata M, Saito H, Shinohara K (2003) Docosahexaenoic acid suppresses nitric oxide production and inducible nitric oxide synthase expression in interferon-gamma plus lipopolysaccharide-stimulated murine macrophages by inhibiting the oxidative stress. Free Radic Biol Med 34:1006–1016
Batot G, Martel C, Capdeville N, Wientjes F, Morel F (1995) Characterization of neutrophil NADPH oxidase activity reconstituted in a cell-free assay using specific monoclonal antibodies raised against cytochrome b558. Eur J Biochem 234:208–215
Remijsen QF, Fontayne A, Verdonck F, Clynen E, Schoofs L, Willems J (2006) The antimicrobial peptide parabutoporin competes with p47(phox) as a PKC-substrate and inhibits NADPH oxidase in human neutrophils. FEBS Lett 580:6206–6210
Winterbourn CC (2008) Reconciling the chemistry and biology of reactive oxygen species. Nat Chem Biol 4:278–286
Poulos A, Robinson BS, Ferrante A, Harvey DP, Hardy SJ, Murray AW (1991) Effect of 22–32 carbon n-3 polyunsaturated fatty acids on superoxide production in human neutrophils: synergism of docosahexaenoic acid with f-met-leu-phe and phorbol ester. Immunology 73:102–108
El-Benna J, Dang PM, Perianin A (2010) Peptide-based inhibitors of the phagocyte NADPH oxidase. Biochem Pharmacol 80:778–785
Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140:805–820
Lo CJ, Chiu KC, Fu M, Lo R, Helton S (1999) Fish oil decreases macrophage tumor necrosis factor gene transcription by altering the NF kappa B activity. J Surg Res 82:216–221
Babcock TA, Novak T, Ong E, Jho DH, Helton WS, Espat NJ (2002) Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-alpha production by omega-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10. J Surg Res 107:135–139
Novak TE, Babcock TA, Jho DH, Helton WS, Espat NJ (2003) NF-kappa B inhibition by omega-3 fatty acids modulates LPS-stimulated macrophage TNF-alpha transcription. Am J Physiol Lung Cell Mol Physiol 284:L84–L89
Zhao Y, Joshi-Barve S, Barve S, Chen LH (2004) Eicosapentaenoic acid prevents LPS-induced TNF-alpha expression by preventing NF-kappaB activation. J Am Coll Nutr 23:71–78
Billiar TR, Bankey PE, Svingen BA, Curran RD, West MA, Holman RT, Simmons RL, Cerra FB (1988) Fatty acid intake and kupffer cell function: fish oil alters eicosanoid and monokine production to endotoxin stimulation. Surgery 104:343–349
Renier G, Skamene E, DeSanctis J, Radzioch D (1993) Dietary n-3 polyunsaturated fatty acids prevent the development of atherosclerotic lesions in mice. Modulation of macrophage secretory activities. Arterioscler Thromb 13:1515–1524
Yaqoob P, Calder P (1995) Effects of dietary lipid manipulation upon inflammatory mediator production by murine macrophages. Cell Immunol 163:120–128
Kelley DS, Taylor PC, Nelson GJ, Schmidt PC, Ferretti A, Erickson KL, Yu R, Chandra RK, Mackey BE (1999) Docosahexaenoic acid ingestion inhibits natural killer cell activity and production of inflammatory mediators in young healthy men. Lipids 34:317–324
Holm T, Berge RK, Andreassen AK, Ueland T, Kjekshus J, Simonsen S, Froland S, Gullestad L, Aukrust P (2001) Omega-3 fatty acids enhance tumor necrosis factor-alpha levels in heart transplant recipients. Transplantation 72:706–711
Chang HR, Arsenijevic D, Pechere JC, Piguet PF, Mensi N, Girardier L, Dulloo AG (1992) Dietary supplementation with fish oil enhances in vivo synthesis of tumor necrosis factor. Immunol Lett 34:13–17
Skuladottir IH, Petursdottir DH, Hardardottir I (2007) The effects of omega-3 polyunsaturated fatty acids on TNF-alpha and IL-10 secretion by murine peritoneal cells in vitro. Lipids 42:699–706
Blok WL, Vogels MT, Curfs JH, Eling WM, Buurman WA, van der Meer JW (1992) Dietary fish-oil supplementation in experimental gram-negative infection and in cerebral malaria in mice. J Infect Dis 165:898–903
Bonatto SJ, Folador A, Aikawa J, Yamazaki RK, Pizatto N, Oliveira HH, Vecchi R, Curi R, Calder PC, Fernandes LC (2004) Lifelong exposure to dietary fish oil alters macrophage responses in Walker 256 tumor-bearing rats. Cell Immunol 231:56–62
Hardardottir I, Kinsella JE (1991) Tumor necrosis factor production by murine resident peritoneal macrophages is enhanced by dietary n-3 polyunsaturated fatty acids. Biochim Biophys Acta 1095:187–195
Petursdottir DH, Olafsdottir I, Hardardottir I (2002) Dietary fish oil increases tumor necrosis factor secretion but decreases interleukin-10 secretion by murine peritoneal macrophages. J Nutr 132:3740–3743
Hardardottir I, Kinsella JE (1992) Increasing the dietary (n-3) to (n-6) polyunsaturated fatty acid ratio increases tumor necrosis factor production by murine resident peritoneal macrophages without an effect on elicited peritoneal macrophages. J Nutr 122:1942–1951
Lokesh BR, Sayers TJ, Kinsella JE (1990) Interleukin-1 and tumor necrosis factor synthesis by mouse peritoneal macrophages is enhanced by dietary n-3 polyunsaturated fatty acids. Immunol Lett 23:281–285
Nguyen HX, Tidball JG (2003) Interactions between neutrophils and macrophages promote macrophage killing of rat muscle cells in vitro. J Physiol 547:125–132
Acknowledgments
This study is supported by FAPESP, CNPq, and CAPES. The authors acknowledge the technical support of José Roberto Mendonça.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Paschoal, V.A., Vinolo, M.A.R., Crisma, A.R. et al. Eicosapentaenoic (EPA) and Docosahexaenoic (DHA) Acid Differentially Modulate Rat Neutrophil Function In Vitro. Lipids 48, 93–103 (2013). https://doi.org/10.1007/s11745-012-3726-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11745-012-3726-6