Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

Optimization of Dendritic cells purification from mouse spleen A recommended method for purification of Dendritic cells from reproductive organs

Authors
Amir Hassan Zarnani 1
Seyyed Mohammad Moazzeni 2
Fazel Shokri 3
Mojdeh Salehnia 4
Ali Ahmad Bayat 5
Mahmood Jeddi-Tehrani 5
1 Immunology Department, Faculty of Medical Science, Tarbiat Modarres University, Tehran, Iran; Nanobiotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran
2 Immunology Department, Faculty of Medical Science, Tarbiat Modarres University, Tehran, Iran
3 Immunology Department, Faculty of Medical Science, Tehran University of Medical Science, Tehran, Iran
4 Anatomy Department, Faculty of Medical Sciences, Tarbiyat Modares University, Tehran, Iran
5 Nanobiotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran
Abstract
Dendritic cells (DC) are the principal antigen-presenting cells (APC) responsible for induction of primary immune responses by T lymphocytes. Although DCs are present in most lymphoid tissues, they occur in very low frequency accounting for 0.5% or less of nucleated cells in peripheral lymphoid organs. In the present study, we report the purification of DCs from mouse spleen with high yield and purity using a three-step purification technique including: collagenase digestion of tissue, selection of low-density cells using Optiprep density gradient medium and plastic adherence. By using techniques outlined above, we obtained 5-7×107 DC/spleen with purity³ of 97%. Such large numbers of purified DCs enables us to further document their different characteristics including morphology, immunophenotype and to evaluation of their role in immune system. Finally, since DCs have been reported to be present in all reproductive organs, we suggest that this protocol be used for isolation and purification of DCs from those organs for further in vitro studies.
Keywords
Dendritic cell
Density gradient centrifugation
Purification
spleen
  1. Steinman R.M., Cohn Z.A. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med. 1973;137(5):1142-62.
  2. Banchereau J., Briere F., Caux C., Davoust J., Lebecque S., Liu Y.J., Pulendran B., Palucka K.Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767-811.
  3. Reid S.D., Penna G., Adorini L. The Control of T cell response by dendritic cell Subset. Curr Opin Immunol. 2000;12(1):114-21.
  4. Moser M., Murphy K.M. Dendritic cell regulation of Th1/Th2 development. Nat Immunol. 2000; 1(3):199-205.
  5. Lotze M.T., Thomson A.W (eds). Dendritic Cells. Academic Press. Chapters. 23-30:2001.
  6. Kammerer U., Schoppet M., McLellan A.D., Kapp M., Huppertz H.I., Kampgen E., Dietl J. Human decidua contains potent immunostimulatory CD83(+) dendritic cells.Am J Pathol. 2000;157(1):159-69.
  7. Sutton L., Mason D.Y., Redman C.W. HLA-DR positive cells in the human placenta. Immunol. 1983;49(1):103-12.
  8. Abraham S., Indrasingh I., Vettivel S., Chandi G. Gross morphology and ultrastructure of dendritic cells in the normal human decidua.Clin Anat. 2000;13(3):177-80.
  9. Parr M.B., Parr E.L. Langerhans cells and T lymphocyte subsets in the murine vagina and cervix. Biol Reprod. 1991;44(3):491-8.
  10. Parr M.B., Kepple L., Parr E.L.Antigen recognition in the female reproductive tract. II. Endocytosis of horseradish peroxidase by Langerhans cells in murine vaginal epithelium.Biol Reprod. 1991;45(2):261-5.
  11. Figueroa C.D., Caorsi I. Ultrastructural and morphometric study of the Langerhans cell in the normal human exocervix. J Anat. 1980; 131 (Pt4): 669-82.
  12. Coligan J.E., Kruisbeek A.M., Margulies D.H., In Shevach E.M., Strober W. (Eds). Current protocoles in immunology. John Willy sons Inc. 1994-1998 Units 3.7 and 5.8.
  13. Vremec D., Zorbas M., Scollay R., Saunders D.J., Ardavin C.F., Wu L., Shortman K. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med. 1992;176(1):47-58.
  14. Pulendran B., Lingappa J., Kennedy M.K., Smith J., Teepe M., Rudensky A., Maliszewski C.R.,Maraskovsky E. Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J Immunol. 1997;159(5):2222-31.
  15. Fukao T., Koyasu S. Expression of functional IL-2 receptors on mature splenic dendritic cells. Eur J Immunol. 2000;30(5):1453-7.
  16. Crowley M.T., Inaba K., Witmer-Pack M.D., Gezelter S., Steinman R.M. Use of the fluorescence activated cell sorter to enrich dendritic cells from mouse spleen. J Immunol Methods. 1990;133(1):55-66.
  17. Stoll S., Jonuleit H., Schmitt E., Muller G., Yamauchi H., Kurimoto M., Knop J., Enk A.H. Production of functional IL-18 by different subtypes of murine and human dendritic cells (DC): DC-derived IL-18enhances IL-12-dependent Th1 development. Eur J Immunol. 1998;28(10):3231-9.
  18. Brocker T. Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells. J Exp Med. 1997;186(8):1223-32.
  19. Maldonado-Lopez R., De Smedt T., Michel P., Godfroid J., Pajak B., Heirman C., Thielemans K., LeoO., Urbain J., Moser M. CD8alpha+ and CD8alpha- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J Exp Med. 1999;189(3):587-92.
  20. Crowley M., Inaba K., Steinman R.M. Dendritic cells are the principal cells in mouse spleen bearing immunogenic fragments of foreign proteins. J Exp Med. 1990;172(1):383-6.
  21. Steinman R.M., Cohn Z.A. Identification of a novel cell type in peripheral lymphoid organs of mice. II.Functional properties in vitro. J Exp Med. 1974;139(2):380-97.
  22. Steinman R.M., Kaplan G., Witmer M.D., Cohn Z.A. Identification of a novel cell type in peripheral lymphoid organs of mice. V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro. J Exp Med. 1979;149(1):1-16.
  23. Gabrilovich D.I., Patterson S., Harvey J.J., Woods G.M., Elsley W., Knight S.C. Murine retrovirus induces defects in the function of dendritic cells at early stages of infection. Cell Immunol. 1994;158(1):167-81.
  24. Stoll S., Jonuleit H., Schmitt E., Muller G., Yamauchi H., Kurimoto M., Knop J., Enk A.H.Production of functional IL-18 by different subtypes of murine and human dendritic cells (DC): DC-derived IL-18 enhances IL-12-dependent Th1 development. Eur J Immunol. 1998;28(10):3231-9.
  25. Rescigno M., Granucci F., Citterio S., Foti M., Ricciardi-Castagnoli P. Coordinated events during bacteria-induced DC maturation. Immunol Today. 1999;20(5):200-3.
  26. Hartmann G., Weiner G.J., Krieg A.M. CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc Natl Acad Sci USA. 1999;96(16):9305-10.
  27. Banchereau J., Steinman R.M. Dendritic cells and the control of immunity. Nature. 1998;392 (6673):245-52.
  28. Ni K., O’Neill H.C. The role of dendritic cells in T cell activation. Immunol Cell Biol. 1997;75 (3):223-30.
  29. Steinman R.M., Pack M., Inaba K. Dendritic cells in the T-cell areas of lymphoid organs. Immunol Rev. 1997;156:25-37.
  30. Vremec D., Shortman K. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J Immunol. 1997;159 (2):565-73.
  31. Weir D.M. (Eds). Handbook of experimental immunology. Blakwell pub. 4th Edition. 1986;vol.
  32. Boyum A., Lovhaug D., Tresland L., Nordlie E.M. Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality. Scand J Immunol. 1991; 34(6): 697-712.
  33. Ria F., Penna G., Adorini L. Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells. Eur J Immunol. 1998;28 (6):2003-16.
  34. Patel D. (Eds). Separating cells. Bios scientific pub., 2001;chapter 2.
  35. McLellan A.D., Starling G.C., Hart D.N.Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation.J Immunol Methods. 1995;184(1):81-9.
  36. Metlay J.P., Witmer-Pack M.D., Agger R., Crowley M.T., Lawless D., Steinman R.M. The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J Exp Med. 1990;171 (5):1753-71.
Journal of Reproduction & Infertility
Volume 4, Issue 1
Winter 2003
Pages 17-29