Application of Single-Cell Cultures of Mouse Splenocytes as an Assay System to Analyze the Immunomodulatory Properties of Bacterial Components

  • Ludwig Deml
  • Michael Aigner
  • Alexander Eckhardt
  • Jochen Decker
  • Norbert Lehn
  • Wulf Schneider-Brachert
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 94)


Recently, various bacterial components have been suggested as initiating and modulating immune activation, thereby substantially affecting the complex and dynamic host/pathogen interactions. Herein, we present a valuable and simple methodology for determining the capacity of bacteria as well as defined bacterial structures to stimulate cellular effectors of the innate and cognate immune system. This assay format is based on the exposure of freshly prepared single-cell cultures of splenic cells derived from naive mice with the immunogen of interest. Herein, the determination of exclusive panels of cytokines by the ELISA, ELISpot, and FACS technology will serve as an indicator for the activation of defined arms of the immune system. An increased knowledge about microbial components with immunomodulatory properties will substantially contribute to a more detailed understanding of the dynamic interplay between the host and potential pathogens and, based on this knowledge, to the development of novel substances for the prevention and therapy of microbial infections.


  1. 1.
    Janeway, C. A. Jr. (1989) PatThe role of CD4 in T-cell activation: accessory molecule or co-receptor? Immunol. Today 10, 234–238.PubMedCrossRefGoogle Scholar
  2. 2.
    Janeway, C. A. Jr. (1992) The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol. Today 13, 11–16.PubMedCrossRefGoogle Scholar
  3. 3.
    Medzhitov, R. and Janeway, C. A. Jr. (2000) Innate immune recognition: mechanisms and pathways. Immunol. Rev. 173, 89–97.PubMedCrossRefGoogle Scholar
  4. 4.
    Gronowski, A. M., Hilbert, D. M., Sheehan, K. C., Garotta, G., and Schreiber, R. D. (1999) Baculovirus stimulates antiviral effects in mammalian cells J. Virol. 73, 9944–9951.PubMedGoogle Scholar
  5. 5.
    Hayashi, F., Smith, K. D., Ozinsky, A., et al. (2001) The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099–1103.PubMedCrossRefGoogle Scholar
  6. 6.
    Smith, K. D. and Ozinsky, A. (2002) Toll-like receptor-5 and the innate immune response to bacterial flagellin. Curr. Top. Microbiol. Immunol. 270, 93–108.PubMedGoogle Scholar
  7. 7.
    Borges, M. M., Campos-Neto, A., Sleath, P., et al. (2001) Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein. Infect. Immun. 69, 5270–5277.PubMedCrossRefGoogle Scholar
  8. 8.
    Rock, F. L, Hardiman, G., Timans, J. C., Kastelein, R. A, and Bazan, J. F. (1998) A family of human receptors structurally related to Drosophila Toll. Proc. Natl. Acad. Sci. USA 95, 588–593.PubMedCrossRefGoogle Scholar
  9. 9.
    Aderem, A. and Ulevitch, R. J. (2000) Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787.PubMedCrossRefGoogle Scholar
  10. 10.
    Hartmann, G., Weiner, G. J., and Krieg, A. M. (1999) CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc. Natl. Acad. Sci. USA 96, 9305–9310.PubMedCrossRefGoogle Scholar
  11. 11.
    Dundon, W. G., de Bernard, M., and Montecucco, C. (2001) Virulence factors of Helicobacter pylori. Int. J. Med. Microbiol. 290, 647–658.PubMedGoogle Scholar
  12. 12.
    Atherton, J. C. (1998) H. pylori virulence factors. Br. Med. Bull. 54, 105–120.PubMedGoogle Scholar
  13. 13.
    Bodger, K. and Crabtree, J. E. (1998) Helicobacter pylori and gastric inflammation. Br. Med. Bull. 54, 139–150.PubMedGoogle Scholar
  14. 14.
    Shimoyama, T. and Crabtree, J. E. (1998) Bacterial factors and immune pathogenesis in Helicobacter pylori infection. Gut 43(suppl 1), S2–5.Google Scholar
  15. 15.
    Zevering, Y., Jacob, L., and Meyer, T. F. (1999) Naturally acquired human immune responses against Helicobacter pylori and implications for vaccine development. Gut 45, 465.PubMedCrossRefGoogle Scholar
  16. 16.
    Suerbaum, S. and Michetti, P. (2002) Helicobacter pylori infection. N. Engl. J. Med. 347, 1175–1186.PubMedCrossRefGoogle Scholar
  17. 17.
    Wunderlich, M. and Glockshuber, R. (1993) In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA). J. Biol. Chem. 268, 24547–24550.PubMedGoogle Scholar
  18. 18.
    Deml, L., Wolf, H., and Wagner, R. (1999) High level expression of hepatitis B virus surface antigen in stably transfected Drosophila Schneider-2 cells J. Virol. Methods 79, 191–203.PubMedCrossRefGoogle Scholar
  19. 19.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. New York, Cold Spring Harbor Laboratory.Google Scholar
  20. 20.
    Deml, L. and Wagner, R. (1998) Stable transfected Drosophila Schneider-2 cells as a novel tool to produce recombinant antigens for diagnostic, therapeutic and preventive purposes, in U Reischl (ed), Methods in Molecular Medicine. Molecular Diagnosis of Infectious Diseases. Humana, Totowa, NJ, pp. 185–200.Google Scholar
  21. 21.
    White, B. A. (1993) Methods in Molecular Biology: PCR Protocols, Current Methods and Applications. Humana, Totowa, NJ.Google Scholar
  22. 22.
    Inoue, H. and Okayama, H. (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23–28.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Ludwig Deml
    • 1
  • Michael Aigner
    • 1
  • Alexander Eckhardt
    • 1
  • Jochen Decker
    • 2
  • Norbert Lehn
    • 1
  • Wulf Schneider-Brachert
    • 1
  1. 1.Institute for Medical Microbiology and HygieneUniversity of RegensburgRegensburgGermany
  2. 2.Accenture GmbHMunichGermany

Personalised recommendations