Skip to main content
SpringerLink
Log in

IL-10 Detection in Murine B Cells: Pros and Cons of the Different Techniques

  • Protocol
  • First Online:
Regulatory B Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1190))

Abstract

With the recently increased understanding of the roles of B cells in immune response and autoimmune pathogenesis, various techniques have been used for the detection of IL-10 production in B cells. In this chapter, we describe several commonly used methods for the effective detection of IL-10 in B cells at both mRNA and protein levels, including quantitative PCR analysis, intracellular staining of IL-10 by flow cytometry, ELISA assay for secreted IL-10 detection, and ELISPOT assay of enumerating IL-10-producing B cells. Moreover, we provide a detailed protocol for the detection of IL-10-producing B cells in situ by immunofluorescence microscopy. Together, the application of these described methods for the detection of IL-10 will facilitate the characterization of B cell subsets with regulatory functions and enhance our current understanding on the critical roles of B cells in immune response and autoimmune development.

Xiang Lin and Jun Deng have contributed equally to this chapter.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Shlomchik MJ, Craft JE, Mamula MJ (2001) From T to B and back again: positive feedback in systemic autoimmune disease. Nat Rev Immunol 1:147–153

    Article  CAS  PubMed  Google Scholar 

  2. Harris DP, Haynes L, Sayles PC, Duso DK, Eaton SM, Lepak NM, Johnson LL, Swain SL, Lund FE (2000) Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat Immunol 1:475–482

    Article  CAS  PubMed  Google Scholar 

  3. Mauri C, Bosma A (2012) Immune regulatory function of B cells. Annu Rev Immunol 30:221–241

    Article  CAS  PubMed  Google Scholar 

  4. Barr TA, Shen P, Brown S, Lampropoulou V, Roch T, Lawrie S, Fan B, O'Connor RA, Anderton SM, Bar-Or A, Fillatreau S, Gray D (2012) B cell depletion therapy ameliorates autoimmune disease through ablation of IL-6-producing B cells. J Exp Med 209:1001–1010

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Bermejo DA, Jackson SW, Gorosito-Serran M, Acosta-Rodriguez EV, Amezcua-Vesely MC, Sather BD, Singh AK, Khim S, Mucci J, Liggitt D, Campetella O, Oukka M, Gruppi A, Rawlings DJ (2013) Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORgammat and Ahr that leads to IL-17 production by activated B cells. Nat Immunol 14:514–522

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM (2002) B cells regulate autoimmunity by provision of IL-10. Nat Immunol 3:944–950

    Article  CAS  PubMed  Google Scholar 

  7. Matsushita T, Yanaba K, Bouaziz JD, Fujimoto M, Tedder TF (2008) Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J Clin Invest 118:3420–3430

    CAS  PubMed Central  PubMed  Google Scholar 

  8. Mizoguchi A, Mizoguchi E, Takedatsu H, Blumberg RS, Bhan AK (2002) Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity 16:219–230

    Article  CAS  PubMed  Google Scholar 

  9. Yanaba K, Bouaziz JD, Haas KM, Poe JC, Fujimoto M, Tedder TF (2008) A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses. Immunity 28:639–650

    Article  CAS  PubMed  Google Scholar 

  10. Evans JG, Chavez-Rueda KA, Eddaoudi A, Meyer-Bahlburg A, Rawlings DJ, Ehrenstein MR, Mauri C (2007) Novel suppressive function of transitional 2 B cells in experimental arthritis. J Immunol 178:7868–7878

    Article  CAS  PubMed  Google Scholar 

  11. Blair PA, Norena LY, Flores-Borja F, Rawlings DJ, Isenberg DA, Ehrenstein MR, Mauri C (2010) CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients. Immunity 32:129–140

    Article  CAS  PubMed  Google Scholar 

  12. Kessel A, Haj T, Peri R, Snir A, Melamed D, Sabo E, Toubi E (2012) Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells. Autoimmun Rev 11:670–677

    Article  CAS  PubMed  Google Scholar 

  13. Kalampokis I, Yoshizaki A, Tedder TF (2013) IL-10-producing regulatory B cells (B10 cells) in autoimmune disease. Arthritis Res Ther 15(Suppl 1): S1

    Google Scholar 

  14. Yang M, Rui K, Wang S, Lu L (2013) Regulatory B cells in autoimmune diseases. Cell Mol Immunol 10:122–132

    Article  CAS  PubMed  Google Scholar 

  15. Mauri C, Jury EC (2010) Could the expression of CD86 and FcgammaRIIB on B cells be functionally related and involved in driving rheumatoid arthritis? Arthritis Res Ther 12:133

    Article  PubMed Central  PubMed  Google Scholar 

  16. Schioppa T, Moore R, Thompson RG, Rosser EC, Kulbe H, Nedospasov S, Mauri C, Coussens LM, Balkwill FR (2011) B regulatory cells and the tumor-promoting actions of TNF-alpha during squamous carcinogenesis. Proc Natl Acad Sci U S A 108:10662–10667

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Le Huu D, Matsushita T, Jin G, Hamaguchi Y, Hasegawa M, Takehara K, Tedder TF, Fujimoto M (2013) Donor-derived regulatory B cells are important for suppression of murine sclerodermatous chronic graft-versus-host disease. Blood 121:3274–3283

    Article  PubMed  Google Scholar 

  18. Yanaba K, Yoshizaki A, Asano Y, Kadono T, Tedder TF, Sato S (2011) IL-10-producing regulatory B10 cells inhibit intestinal injury in a mouse model. Am J Pathol 178:735–743

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Tedder TF, Matsushita T (2010) Regulatory B cells that produce IL-10: a breath of fresh air in allergic airway disease. J Allergy Clin Immunol 125:1125–1127

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Yang M, Sun L, Wang S, Ko KH, Xu H, Zheng BJ, Cao X, Lu L (2010) Novel function of B cell-activating factor in the induction of IL-10-producing regulatory B cells. J Immunol 184:3321–3325

    Article  CAS  PubMed  Google Scholar 

  21. Yang M, Deng J, Liu Y, Ko KH, Wang X, Jiao Z, Wang S, Hua Z, Sun L, Srivastava G, Lau CS, Cao X, Lu L (2012) IL-10-producing regulatory B10 cells ameliorate collagen-induced arthritis via suppressing Th17 cell generation. Am J Pathol 180:2375–2385

    Article  CAS  PubMed  Google Scholar 

  22. Yoshizaki A, Miyagaki T, DiLillo DJ, Matsushita T, Horikawa M, Kountikov EI, Spolski R, Poe JC, Leonard WJ, Tedder TF (2012) Regulatory B cells control T-cell autoimmunity through IL-21-dependent cognate interactions. Nature 491:264–268

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

    Authors and Affiliations

    1. Department of Pathology, Queen Mary Hospital, The University of Hong Kong, Room 234, University Pathology Building, Pokfulam Road, Hong Kong, Hong Kong

      Xiang Lin, Jun Deng & Liwei Lu

    Authors
    1. Xiang Lin
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Jun Deng
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Liwei Lu
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Liwei Lu .

    Editor information

    Editors and Affiliations

    1. Department of Medical and Biological Sciences, University of Udine, Udine, Italy

      Gaetano Vitale

    2. Department of Medical and Biological Sciences, University of Udine, Udine, Italy

      Francesca Mion

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2014 Springer Science+Business Media New York

    About this protocol

    Cite this protocol

    Lin, X., Deng, J., Lu, L. (2014). IL-10 Detection in Murine B Cells: Pros and Cons of the Different Techniques. In: Vitale, G., Mion, F. (eds) Regulatory B Cells. Methods in Molecular Biology, vol 1190. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1161-5_5

    Download citation

    • DOI: https://doi.org/10.1007/978-1-4939-1161-5_5

    • Published:

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-1160-8

    • Online ISBN: 978-1-4939-1161-5

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation