Biological Trace Element Research

, Volume 189, Issue 2, pp 519–528 | Cite as

Modulatory Effect of the Supplemented Copper Ion on In Vitro Activity of Bovine Lactoferrin to Murine Splenocytes and RAW264.7 Macrophages

  • Hui-Juan Zhao
  • Xin-Huai ZhaoEmail author


Bovine lactoferrin (LF) was supplemented with Cu2+ at three contents of 0.16, 0.32, and 0.64 mg/g LF, respectively. After then, LF and Cu-supplemented LF products were assessed for immuno-modulation in murine splenocytes and RAW264.7 macrophages, using dose levels of 10−40 μg/mL and four evaluation reflectors including stimulation index of splenocytes, T lymphocyte subpopulations, macrophage phagocytosis, and cytokine secretion. The results indicated that LF and Cu-supplemented LF products had suppression on splenocytes as well as concanavalin A (ConA)- or lipopolysaccharide-stimulated splenocytes; however, using lower Cu-supplementation content (i.e., 0.16 mg/g LF) and lower dose level (10 μg/mL) alleviated this suppression significantly (P < 0.05). Compared to LF, Cu-supplemented LF product of lower Cu-supplementation content at lower dose level yielded slightly enhanced macrophage stimulation, increased CD4+/CD8+ ratio of T lymphocyte subpopulations in ConA-stimulated splenocytes, and significant secretion enhancement for interleukin-2 (IL-2), IL-4, interferon-γ (in splenocytes), IL-1β, and tumor necrosis factor-α (in macrophages) (P < 0.05). Furthermore, Cu-supplemented LF product of higher Cu-supplementation content (i.e., 0.64 mg/g LF) at higher dose level mostly showed opposite effects in the cells, in comparison with its counterpart at lower dose level. It is concluded that Cu-supplementation of LF can alleviate or increase LF’s effects on the two immune cells, and moreover, Cu content of supplemented LF is a key factor that modulates these effects.


Lactoferrin Copper Murine splenocytes RAW264.7 macrophages Immuno-modulation 



The authors thank the anonymous referees for their valuable advice.


This study was funded by the National High Technology Research and Development Program (“863” Program) of China (Project No. 2013AA102205).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Naidu AS (2000) Lactoferrin: natural, multifunctional, anti-microbial. CRC Press LLC, FloridaGoogle Scholar
  2. 2.
    García-Montoya IA, Cendón TS, Arévalo-Gallegos S, Rascón-Cruz Q (2012) Lactoferrin a multiple bioactive protein: an overview. Biochim Biophys Acta Gen Subj 1820(3):226–236. Google Scholar
  3. 3.
    Sanchez L, Calvo M, Brock JH (1992) Biological role of lactoferrin. Arch Dis Child 67(5):657–661. Google Scholar
  4. 4.
    Huma B, Trang T, Nidhi B, Lisbeth G, Bhesh B (2014) Evaluation of different methods for determination of the iron saturation level in bovine lactoferrin. Food Chem 152(2):121–127. Google Scholar
  5. 5.
    Brisson G, Britten M, Pouliot Y (2007) Heat-induced aggregation of bovine lactoferrin at neutral pH: effect of iron saturation. Int Dairy J 17(6):617–624. Google Scholar
  6. 6.
    Zimecki M, Wlaszczyk A, Wojciechowski R, Dawiskiba J, Kruzel M (2001) Lactoferrin regulates the immune responses in post-surgical patients. Arch Immunol Ther Exp 49(4):325–333Google Scholar
  7. 7.
    Legrand D (2012) Lactoferrin, a key molecule in immune and inflammatory processes. Biochem Cell Biol 90(3):252–268. Google Scholar
  8. 8.
    Drago-Serrano ME, Campos-Rodríguez R, Carrero JC, de la Garza M (2017) Lactoferrin: balancing ups and downs of inflammation due to microbial infections. Int J Mol Sci 18(3):501. Google Scholar
  9. 9.
    Yoo YC, Watanabe R, Koike Y, Mitobe M, Shimazaki K, Watanabe S, Azuma I (1997) Apoptosis in human leukemic cells induced by lactoferricin, a bovine milk protein-derived peptide: involvement of reactive oxygen species. Biochem Biophys Res Commun 237(3):624–628. Google Scholar
  10. 10.
    Haversen L, Ohlsson BG, Hahn-Zoric M, Hanson LA, Mattsby-Baltzer I (2002) Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell Immunol 220(2):83–95. Google Scholar
  11. 11.
    Rybarczyk J, Kieckens E, Vanrompay D, Cox E (2016) In vitro and in vivo studies on the antimicrobial effect of lactoferrin against Escherichia coli O157:H7. Vet Microbiol 202:23–28. Google Scholar
  12. 12.
    Failla ML (2003) Trace elements and host defense: recent advances and continuing challenges. J Nutr 133(1):1443S–1447S. Google Scholar
  13. 13.
    Tsuji PA, Canter JA, Rosso LE (2016) Trace minerals and trace elements. In: Caballero B, Finglas PM, Toldrá F (eds) Encyclopedia of food and health, 1st edn. Elsevier, Oxford, pp 331–338Google Scholar
  14. 14.
    Goldhaber SB (2003) Trace element risk assessment: essentiality vs. toxicity. Regul Toxicol Pharmacol 38(2):232–242. Google Scholar
  15. 15.
    Ahmed F, Khan MR, Shaheen N, Ahmed K, Hasan A, Chowdhury IA, Chowdhury R (2018) Anaemia and iron deficiency in rural Bangladeshi pregnant women living in areas of high and low iron in groundwater. Nutr Diet 51–52:46–52. Google Scholar
  16. 16.
    Feske S, Wulff H, Skolnik EY (2015) Ion channels in innate and adaptive immunity. Annu Rev Immunol 33(1):291–353. Google Scholar
  17. 17.
    Hood MI, Skaar EP (2012) Nutritional immunity: transition metals at the pathogen-host interface. Nat Rev Microbiol 10(8):525–537. Google Scholar
  18. 18.
    The Commission of the European Communities (2006) Commission directive 2006/141/EC on infant formulae and follow-on formulae and amending directive 1999/21/EC. Brussels, BE, EU, pp 1–33Google Scholar
  19. 19.
    Ainscough EW, Brodie AM, Plowman JE (1979) The chromium, manganese, cobalt and copper complexes of human lactoferrin. Inorg Chim Acta 33(37):145–153. Google Scholar
  20. 20.
    Harrington JP, Stuart J, Jones A (1987) Unfolding of iron and copper complexes of human lactoferrin and transferrin. Int J BioChemiPhysics 19(10):1001–1008. Google Scholar
  21. 21.
    Goldoni P, Sinibaldi L, Valenti P, Orsi N (2000) Metal complexes of lactoferrin and their effect on the intracellular multiplication of Legionella pneumophila. Biometals 13(1):15–22. Google Scholar
  22. 22.
    Marchetti M, Superti F, Ammendolia MG, Rossi P, Valenti P, Seganti L (1999) Inhibition of poliovirus type 1 infection by iron-, manganese and zinc saturated lactoferrin. Med Microbiol Immun 187(4):199–204. Google Scholar
  23. 23.
    Anand N, Kanwar RK, Dubey ML, Vahishta RK, Sehgal R, Verma AK, Kanwar JR (2015) Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite-host interaction. Drug Des Devel Ther 9:3821–3835. Google Scholar
  24. 24.
    Murphy K, Weaver C (2017) Janeway’s immunobiology. Taylor & Francis Group LLC, New YorkGoogle Scholar
  25. 25.
    Hao LX, Zhao XH (2017) In vitro immune potentials of a water-soluble polysaccharide extract from Dioscorea opposita planted in Henan Province, China. Pak J Pharm Sci 30(4):1383–1388Google Scholar
  26. 26.
    Wang MC, Jiang CX, Ma LP, Zhang ZJ, Cao L, Liu J, Zeng XX (2013) Preparation, preliminary characterization and immunostimulatory activity of polysaccharide fractions from the peduncles of Hovenia dulcis. Food Chem 138(1):41–47. Google Scholar
  27. 27.
    Shi J, Zhao XH (2017) In vitro immuno-modulatory ability of tryptic caseinate hydrolysate affected by prior caseinate glycation using the Maillard reaction or transglutaminase. Food Agric Immunol 28(6):1029–1045. Google Scholar
  28. 28.
    Liu X, Zhao XH (2017) Immune potentials of the Mucor-fermented Mao-tofu and especially its soluble extracts for the normal mice. Food Agric Immunol 28(5):859–875. Google Scholar
  29. 29.
    Ripolles D, Harouna S, Parrón JA, Calvo M, Pérez MD, Carramiñana JJ, Sánchez L (2015) Antibacterial activity of bovine milk lactoferrin and its hydrolysates prepared with pepsin, chymosin and microbial rennet against foodborne pathogen Listeria monocytogenes. Int Dairy J 45:15–22. Google Scholar
  30. 30.
    Duarte DC, Nicolau A, Teixeira JA, Rodrigues LR (2011) The effect of bovine milk lactoferrin on human breast cancer cell lines. J Dairy Sci 94(1):66–76. Google Scholar
  31. 31.
    Wong CW, Seow HF, Husband AJ, Regester GO, Watson DL (1997) Effects of purified bovine whey factors on cellular immune functions in ruminants. Vet Immunol Immunopathol 56(1–2):85–96. Google Scholar
  32. 32.
    Miyauchi H, Kaino A, Shinoda I, Fukuwatari Y, Hayasawa H (1997) Immunomodulatory effect of bovine lactoferrin pepsin hydrolysate on murine splenocytes and Peyer’s patch cells. J Dairy Sci 80(10):2330–2339. Google Scholar
  33. 33.
    Richie ER, Hilliard JK, Gilmore R, Gillespie DJ (1987) Human milk-derived lactoferrin inhibits mitogen and alloantigen induced human lymphocyte proliferation. J Reprod Immunol 12(2):137–148. Google Scholar
  34. 34.
    Percival SS (1998) Copper and immunity. Am J Clin Nutr 67(5 suppl):1064S–1068S. Google Scholar
  35. 35.
    Veldhuis NA, Valova VA, Gaeth AP, Palstra N, Hannan KM, Michell BJ, Kelly LE, Jennings I, Kemp BE, Pearson RB, Robinson PJ, Camakaris J (2009) Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase. Int J Biochem Cell Biol 41(12):2403–2412. Google Scholar
  36. 36.
    Steinborn C, Diegel C, Garcia-Käufer M, Gründemann C, Huber R (2017) Immunomodulatory effects of metal salts at sub-toxic concentrations. J Appl Toxicol 37(5):563–572. Google Scholar
  37. 37.
    Cross ML, Gill HS (1999) Modulation of immune function by a modified bovine whey protein concentrate. Immunol Cell Biol 77(4):345–350. Google Scholar
  38. 38.
    Gordon S (2016) Phagocytosis: an immunobiologic process. Immunity 44(3):463–475. Google Scholar
  39. 39.
    Schepetkin IA, Quinn MT (2006) Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. Int Immunopharmacol 6(3):317–333. Google Scholar
  40. 40.
    Wagner D, Maser J, Lai B, Cai ZH, Barry CE, Bentrup KHZ, Russell DG, Bermudez LE (2005) Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell’s endosomal system. J Immunol 174(3):1491–1500. Google Scholar
  41. 41.
    White C, Lee J, Kambe T, Fritsche K, Petris MJ (2009) A role for the ATP7A copper-transporting ATPase in macrophage bactericidal activity. J Biol Chem 284(49):33949–33956. Google Scholar
  42. 42.
    Bertinato J (2016) Copper: physiology. In: Caballero B, Finglas PM, Toldrá F (eds) Encyclopedia of food and health, 1st edn. Elsevier, Oxford, pp 321–326Google Scholar
  43. 43.
    Stafford SL, Bokil NJ, Achard MES, Kapetanovic R, Schembri MA, McEwan AG, Sweet MJ (2013) Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper. Biosci Rep 33(4):541–554. Google Scholar
  44. 44.
    Murphy KM, Reiner SL (2002) The lineage decisions of helper T cells. Nat Rev Immunol 2(12):933–944. Google Scholar
  45. 45.
    Willey S, Aasachapman MMI (2008) Humoral immunity to HIV-1: neutralisation and antibody effector functions. Trends Microbiol 16(12):596–604. Google Scholar
  46. 46.
    He LX, Ren JW, Liu R, Chen QH, Zhao J, Wu X, Zhang ZF, Wang JB, Pettinatoc G, Li Y (2017) Ginseng (Panax ginseng Meyer) oligopeptides regulate innate and adaptive immune responses in mice via increased macrophage phagocytosis capacity, NK cell activity and Th cells secretion. Food Funct 8(10):3523–3532. Google Scholar
  47. 47.
    Saint-Sauveura D, Gauthiera SF, Boutinb Y, Montoni A (2008) Immunomodulating properties of a whey protein isolate, its enzymatic digest and peptide fractions. Int Dairy J 18(3):260–270. Google Scholar
  48. 48.
    Constant SL, Bottomly K (1997) Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Ann Rev Immunol 15(15):297–322 Google Scholar
  49. 49.
    Sakaguchi S (2002) Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101(5):455–458. Google Scholar
  50. 50.
    Sá-Nunes A, Faccioli LH, Sforcin JM (2003) Propolis: lymphocyte proliferation and IFN-γ production. J Ethnopharmacol 87(1):93–97. Google Scholar
  51. 51.
    Sfeir RM, Dubarry M, Boyaka PN, Rautureau M, Tomé D (2004) The mode of oral bovine lactoferrin administration influences mucosal and systemic immune responses in mice. J Nutr 134(2):403–409. Google Scholar
  52. 52.
    Wang M, Yang XB, Zhao JW, Lu CJ, Zhu W (2017) Structural characterization and macrophage immunomodulatory activity of a novel polysaccharide from Smilax glabra Roxb. Carbohydr Polym 156:390–402. Google Scholar
  53. 53.
    Wu WJ, Sun CZ, Wang G, Pan Q, Lai FR, Li XF, Tang YQ, Wu H (2017) Immunomodulatory activities of non-prolamin proteins in wheat germ and gluten. J Cereal Sci 76:206–214. Google Scholar
  54. 54.
    Crouch SP, Slater KJ, Fletcher J (1992) Regulation of cytokine release from mononuclear cells by the iron-binding protein lactoferrin. Blood 80(1):235–240Google Scholar
  55. 55.
    Choe YH, Lee SW (2000) Effect of lactoferrin on the production of tumor necrosis factor-α and nitric oxide. J Cell Biochem 76(1):30–36Google Scholar
  56. 56.
    Miyauchi H, Hashimoto S, Nakajima M, Shinoda I, Fukuwatari Y, Hayasawa H (1998) Bovine lactoferrin stimulates the phagocytic activity of human neutrophils: identification of its active domain. Cell Immunol 187(1):34–37. Google Scholar
  57. 57.
    Salgueiro MJ, Zubillaga M, Lysionek A, Sarabia MI, Caro R, De Paoli T, Hager A, Weill R, Boccio J (2000) Zinc as an essential micronutrient: a review. Nutr Res 20(5):737–755. Google Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Dairy Science, Ministry of EducationNortheast Agricultural UniversityHarbinPeople’s Republic of China

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