Skip to main content
Download PDF
  • Short Communication
  • Published:

The effect of carbohydrate moiety structure on the immunoregulatory activity of lactoferrin in vitro

Cellular & Molecular Biology Letters volume 19, pages 284–296 (2014)Cite this article

Abstract

The aim of this study was to evaluate the immunoregulatory effects of recombinant human lactoferrin (rhLF) in two in vitro models: (1) the secondary humoral immune response to sheep erythrocytes (SRBC); and (2) the mixed lymphocyte reaction (MLR). We compared the non-sialylated glycoform of rhLF as expressed by glycoengineered Pichia pastoris with one that was further chemically sialylated. In an earlier study, we showed that sialylated rhLF could reverse methotrexate-induced suppression of the secondary immune response of mouse splenocytes to SRBC, and that the phenomenon is dependent on the interaction of lactoferrin (LF) with sialoadhesin (CD169). We found that the immunorestorative activity of sialylated rhLF is also dependent on its interaction with the CD22 antigen, a member of the immunoglobulin superfamily that is expressed by B lymphocytes. We also demonstrated that only sialylated rhLF was able to inhibit the MLR reaction. MLR was inhibited by bovine lactoferrin (bLF), a glycoform that has a more complex glycan structure. Desialylated bLF and lactoferricin, a bLF-derived peptide devoid of carbohydrates, did not express such inhibitory activity. We showed that the interaction of LF with sialic acid receptors is essential for at least some of the immunoregulatory activity of this glycoprotein.

Abbreviations

AFC:

antibody-forming cells

bLF:

bovine milk lactoferrin

ConA:

concanavalin A

DegbLF:

deglycosylated bovine lactoferrin

FCS:

fetal calf serum

hLF:

human lactoferrin

LF:

lactoferrin

LPS:

lipopolysaccharide

MLR:

mixed lymphocyte reaction

MTX:

methotrexate

PBMC:

peripheral blood mononuclear cell

rhLF:

recombinant human lactoferrin

rhLF A:

non-sialylated recombinant human lactoferrin

rhLF B:

sialylated recombinant human lactoferrin

SRBC:

sheep red blood cells

References

  1. Legrand, D., Pierce, A., Elass, E., Carpentier, M., Mariller, C. and Mazurier, J. Lactoferrin structure and functions. Adv. Exp. Med. Biol. 606 (2008) 163–194.

    Article  CAS  PubMed  Google Scholar 

  2. Vogel, H.J. Lactoferrin, a bird’s eye view. Biochem. Cell Biol. 90 (2012) 233–244.

    Article  CAS  PubMed  Google Scholar 

  3. Kruzel, M.L., Actor, J.K., Boldogh, I. and Zimecki, M. Lactoferrin in health and disease. Postepy Hig. Med. Dosw. 61 (2007) 261–267.

    Google Scholar 

  4. Kruzel, M.L., Actor, J.K., Radak, Z., Bacsi, A., Saavedra-Molina, A. and Boldogh, I. Lactoferrin decreases LPS-induced mitochondrial dysfunction in cultured cells and in animal endotoxemia model. Innate Immun. 16 (2010) 67–79.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Jonasch, E., Stadler, W.M., Bukowski, R.M., Hayes, T.G., Varadhachary, A., Malik, R., Figlin, R.A. and Srinivas, S. Phase 2 trial of talactoferrin in previously treated patients with metastatic renal cell carcinoma. Cancer 113 (2008) 72–77.

    Article  CAS  PubMed  Google Scholar 

  6. Baldi, A., Ioannis, P., Chiara, P., Eleonora, F., Roubini, C. and Vittorio, D. Biological effects of milk proteins and their peptides with emphasis on those related to the gastrointestinal ecosystem. J. Dairy Res. 72 (2005) 66–72.

    Article  CAS  PubMed  Google Scholar 

  7. Weinberg, E.D. Iron, infection, and neoplasia. Clin. Physiol. Biochem. 4 (1986) 50–60.

    CAS  PubMed  Google Scholar 

  8. Hendrixson, D.R., Qiu, J., Shewry, S.C., Fink, D.L., Petty, S., Baker, E.N., Plaut, A.G. and St Geme, J.W., 3rd. Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites. Mol. Microbiol. 47 (2003) 607–617.

    Article  CAS  PubMed  Google Scholar 

  9. Appelmelk, B.J., An, Y.Q., Geerts, M., Thijs, B.G., de Boer, H.A., MacLaren, D.M., de Graaff, J. and Nuijens, J.H. Lactoferrin is a lipid A-binding protein. Infect. Immun. 62 (1994) 2628–2632.

    CAS  PubMed Central  PubMed  Google Scholar 

  10. Zimecki, M., Mazurier, J., Machnicki, M., Wieczorek, Z., Montreuil, J. and Spik, G. Immunostimulatory activity of lactotransferrin and maturation of CD4-CD8-murine thymocytes. Immunol. Lett. 30 (1991) 119–123.

    Article  CAS  PubMed  Google Scholar 

  11. Zimecki, M., Mazurier, J., Spik, G. and Kapp, J.A. Human lactoferrin induces phenotypic and functional changes in murine splenic B cells. Immunology 86 (1995) 122–127.

    CAS  PubMed Central  PubMed  Google Scholar 

  12. Artym, J. and Zimecki, M. The effects of lactoferrin on myelopoiesis: can we resolve the controversy? Postepy Hig. Med. Dosw. 61 (2007) 129–150.

    Google Scholar 

  13. Legrand, D., Elass, E., Carpentier, M. and Mazurier, J. Interactions of lactoferrin with cells involved in immune function. Biochem. Cell Biol. 84 (2006) 282–290.

    Article  CAS  PubMed  Google Scholar 

  14. Fischer, R., Debbabi, H., Dubarry, M., Boyaka, P. and Tome, D. Regulation of physiological and pathological Th1 and Th2 responses by lactoferrin. Biochem. Cell Biol. 84 (2006) 303–311.

    Article  CAS  PubMed  Google Scholar 

  15. Zimecki, M., Stepniak, D., Szynol, A. and Kruzel, M.L. Lactoferrin regulates proliferative response of human peripheral blood mononuclear cells to phytohemagglutinin and mixed lymphocyte reaction. Arch. Immunol. Ther. Exp. (Warsz.). 49 (2001) 147–154.

    CAS  PubMed  Google Scholar 

  16. Suzuki, Y.A., Lopez, V. and Lonnerdal, B. Mammalian lactoferrin receptors: structure and function. Cell. Mol. Life Sci. 62 (2005) 2560–2575.

    Article  CAS  PubMed  Google Scholar 

  17. Curran, C.S., Demick, K.P. and Mansfield, J.M. Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways. Cell. Immunol. 242 (2006) 23–30.

    Article  CAS  PubMed  Google Scholar 

  18. Ando, K., Hasegawa, K., Shindo, K., Furusawa, T., Fujino, T., Kikugawa, K., Nakano, H., Takeuchi, O., Akira, S., Akiyama, T., Gohda, J., Inoue, J. and Hayakawa, M. Human lactoferrin activates NF-kappaB through the Toll-like receptor 4 pathway while it interferes with the lipopolysaccharide-stimulated TLR4 signaling. FEBS J. 277 (2010) 2051–2066.

    Article  CAS  PubMed  Google Scholar 

  19. Chien, Y.J., Chen, W.J., Hsu, W.L. and Chiou, S.S. Bovine lactoferrin inhibits Japanese encephalitis virus by binding to heparan sulfate and receptor for low density lipoprotein. Virology 379 (2008) 143–151.

    Article  CAS  PubMed  Google Scholar 

  20. van Berkel, P.H., Geerts, M.E., van Veen, H.A., Mericskay, M., de Boer, H.A. and Nuijens, J.H. N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA. Biochem. J. 328(Pt 1) (1997) 145–151.

    PubMed Central  PubMed  Google Scholar 

  21. Elass-Rochard, E., Legrand, D., Salmon, V., Roseanu, A., Trif, M., Tobias, P.S., Mazurier, J. and Spik, G. Lactoferrin inhibits the endotoxin interaction with CD14 by competition with the lipopolysaccharide-binding protein. Infect. Immun. 66 (1998) 486–491.

    CAS  PubMed Central  PubMed  Google Scholar 

  22. Legrand, D., Vigie, K., Said, E.A., Elass, E., Masson, M., Slomianny, M.C., Carpentier, M., Briand, J.P., Mazurier, J. and Hovanessian, A.G. Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells. Eur. J. Biochem. 271 (2004) 303–317.

    Article  CAS  PubMed  Google Scholar 

  23. Shin, K., Wakabayashi, H., Yamauchi, K., Yaeshima, T. and Iwatsuki, K. Recombinant human intelectin binds bovine lactoferrin and its peptides. Biol. Pharm. Bull. 31 (2008) 1605–1608.

    Article  CAS  PubMed  Google Scholar 

  24. Kerrigan, A.M. and Brown, G.D. C-type lectins and phagocytosis. Immunobiology 214 (2009) 562–575.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Paulson, J.C., Macauley, M.S. and Kawasaki, N. Siglecs as sensors of self in innate and adaptive immune responses. Ann. N.Y. Acad. Sci. 1253 (2012) 37–48.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Kocieba, M., Zimecki, M., Kruzel, M. and Actor, J. The adjuvant activity of lactoferrin in the generation of DTH to ovalbumin can be inhibited by bovine serum albumin bearing alpha-D-mannopyranosyl residues. Cell. Mol. Biol. Lett. 7 (2002) 1131–1136.

    CAS  PubMed  Google Scholar 

  27. Groot, F., Geijtenbeek, T.B., Sanders, R.W., Baldwin, C.E., Sanchez-Hernandez, M., Floris, R., van Kooyk, Y., de Jong, E.C. and Berkhout, B. Lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type 1 transmission by blocking the DC-SIGN-gp120 interaction. J. Virol. 79 (2005) 3009–3015.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Choi, B.K., Actor, J.K., Rios, S., d’Anjou, M., Stadheim, T.A., Warburton, S., Giaccone, E., Cukan, M., Li, H., Kull, A., Sharkey, N., Gollnick, P., Kocieba, M., Artym, J., Zimecki, M., Kruzel, M.L. and Wildt, S. Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response. Glycoconj. J. 25 (2008) 581–593.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Baveye, S., Elass, E., Fernig, D.G., Blanquart, C., Mazurier, J. and Legrand, D. Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by the CD14-lipopolysaccharide complex. Infect. Immun. 68 (2000) 6519–6525.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Hwang, S.A., Wilk, K., Kruzel, M.L. and Actor, J.K. A novel recombinant human lactoferrin augments the BCG vaccine and protects alveolar integrity upon infection with Mycobacterium tuberculosis in mice. Vaccine 27 (2009) 3026–3034.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Artym, J., Zimecki, M. and Kruzel, M.L. Effect of lactoferrin on the methotrexate-induced suppression of the cellular and humoral immune response in mice. Anticancer Res. 24 (2004) 3831–3836.

    CAS  PubMed  Google Scholar 

  32. O’Neill, A.S., van den Berg, T.K. and Mullen, G.E. Sialoadhesin — a macrophage-restricted marker of immunoregulation and inflammation. Immunology 138 (2013) 198–207.

    Article  PubMed Central  PubMed  Google Scholar 

  33. Sunshine, G.H., Katz, D.R. and Czitrom, A.A. Heterogeneity of stimulator cells in the murine mixed leukocyte response. Eur. J. Immunol. 12 (1982) 9–15.

    Article  CAS  PubMed  Google Scholar 

  34. Unanue, E.R. Antigen-presenting function of the macrophage. Annu. Rev. Immunol. 2 (1984) 395–428.

    Article  CAS  PubMed  Google Scholar 

  35. Nitschke, L. CD22 and Siglec-G: B-cell inhibitory receptors with distinct functions. Immunol. Rev. 230 (2009) 128–143.

    Article  CAS  PubMed  Google Scholar 

  36. Lisowska, E., Duk, M. and Wu, A.M. Preparation of biotinylated lectins and application in microtiter plate assays and Western blotting. In: A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis, BioMethods 7 (1996) 115–129.

    Article  CAS  Google Scholar 

  37. Endo, Y. and Kobata, A. Partial purification and characterization of an endoalpha-N-acetylgalactosaminidase from the culture of medium of Diplococcus pneumoniae. J. Biochem. 80 (1976) 1–8.

    CAS  PubMed  Google Scholar 

  38. Drzeniek, Z., Krotkiewski, H., Syper, D. and Lisowska, E. Reactivity of glycosidase-treated, blood-group M and N glycopeptides with lectins. Carbohydr. Res. 120 (1983) 315–321.

    Article  CAS  PubMed  Google Scholar 

  39. Mishell, R.I. and Dutton, R.W. Immunization of dissociated spleen cell cultures from normal mice. J. Exp. Med. 126 (1967) 423–442.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Hansen, M.B., Nielsen, S.E. and Berg, K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods. 119 (1989) 203–210.

    Article  CAS  PubMed  Google Scholar 

  41. Vogel, H.J., Schibli, D.J., Jing, W., Lohmeier-Vogel, E.M., Epand, R.F. and Epand, R.M. Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides. Biochem. Cell Biol. 80 (2002) 49–63.

    Article  CAS  PubMed  Google Scholar 

  42. Sato, S., Tuscano, J.M., Inaoki, M. and Tedder, T.F. CD22 negatively and positively regulates signal transduction through the B lymphocyte antigen receptor. Semin. Immunol. 10 (1998) 287–297.

    Article  CAS  PubMed  Google Scholar 

  43. Rosenthal, G.J., Weigand, G.W., Germolec, D.R., Blank, J.A. and Luster, M.I. Suppression of B cell function by methotrexate and trimetrexate. Evidence for inhibition of purine biosynthesis as a major mechanism of action. J. Immunol. 141 (1988) 410–416.

    CAS  PubMed  Google Scholar 

  44. Genestier, L., Paillot, R., Fournel, S., Ferraro, C., Miossec, P. and Revillard, J.P. Immunosuppressive properties of methotrexate: apoptosis and clonal deletion of activated peripheral T cells. J. Clin. Invest. 102 (1998) 322–328.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Danzer, C.P., Collins, B.E., Blixt, O., Paulson, J.C. and Nitschke, L. Transitional and marginal zone B cells have a high proportion of unmasked CD22: implications for BCR signaling. Int. Immunol. 15 (2003) 1137–1147.

    Article  CAS  PubMed  Google Scholar 

  46. Kawasaki, Y., Sato, K., Shinmoto, H. and Dosako, S. Role of basic residues of human lactoferrin in the interaction with B lymphocytes. Biosci. Biotechnol. Biochem. 64 (2000) 314–318.

    Article  CAS  PubMed  Google Scholar 

  47. Artym, J., Zimecki, M., Paprocka, M. and Kruzel, M.L. Orally administered lactoferrin restores humoral immune response in immunocompromised mice. Immunol. Lett. 89 (2003) 9–15.

    Article  CAS  PubMed  Google Scholar 

  48. Actor, J.K., Hwang, S.A., Olsen, M., Zimecki, M., Hunter, R.L., Jr. and Kruzel, M.L. Lactoferrin immunomodulation of DTH response in mice. Int. Immunopharmacol. 2 (2002) 475–486.

    Article  CAS  PubMed  Google Scholar 

  49. Boswell, H.S., Nerenberg, M.I., Scher, I. and Singer, A. Role of accessory cells in B cell activation. III. Cellular analysis of primary immune response deficits in CBA/N mice: presence of an accessory cell-B cell interaction defect. J. Exp. Med. 152 (1980) 1194–1309.

    Article  CAS  PubMed  Google Scholar 

  50. Nakae, S., Asano, M., Horai, R. and Iwakura, Y. Interleukin-1 beta, but not interleukin-1 alpha, is required for T-cell-dependent antibody production. Immunology 104 (2001) 402–409.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  51. Zucali, J.R., Broxmeyer, H.E., Levy, D. and Morse, C. Lactoferrin decreases monocyte-induced fibroblast production of myeloid colony-stimulating activity by suppressing monocyte release of interleukin-1. Blood 74 (1989) 1531–1536.

    CAS  PubMed  Google Scholar 

  52. Leutwyler, C., Schalch, L. and Jungi, T.W. Evidence for interleukin-1 beta being a necessary but not sufficient co-stimulatory signal in monocytedependent anti-CD3-mediated T-cell triggering. Immunol. Lett. 38 (1993) 33–39.

    Article  CAS  PubMed  Google Scholar 

  53. Zimecki, M., Kocieba, M. and Kruzel, M. Immunoregulatory activities of lactoferrin in the delayed type hypersensitivity in mice are mediated by a receptor with affinity to mannose. Immunobiology 205 (2002) 120–131.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Experimental Therapy, Institute of Immunology and Experimental Therapy, Weigla 12, 53-114, Wrocław, Poland

    Michał Zimecki, Jolanta Artym & Maja Kocięba

  2. Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Weigla 12, 53-114, Wrocław, Poland

    Maria Duk

  3. Health Science Center, University of Texas, Houston, Texas, USA

    Marian L. Kruzel

Authors
  1. Michał Zimecki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jolanta Artym
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maja Kocięba
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria Duk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marian L. Kruzel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michał Zimecki.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zimecki, M., Artym, J., Kocięba, M. et al. The effect of carbohydrate moiety structure on the immunoregulatory activity of lactoferrin in vitro. Cell Mol Biol Lett 19, 284–296 (2014). https://doi.org/10.2478/s11658-014-0196-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-014-0196-2

Keywords

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us