Skip to main content
SpringerLink
Log in

Functional Analysis of C5a Effector Responses In Vitro and In Vivo

  • Protocol
  • First Online:
The Complement System

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1100))

Abstract

C5a is a powerful proinflammatory and immunomodulatory mediator as evidenced by its involvement in infectious, allergic, and autoimmune diseases as well as in cancer. C5a exerts most of its biologic functions through binding and activation of the G protein-coupled C5a receptor (C5aR). C5aR activation promotes complex signaling pathways eventually resulting in increased intracellular calcium (Ca2+)i concentration. One of the prototypic effector functions associated with C5aR activation is the reconfiguration of the cell cytoskeleton resulting in increased cell motility and ligand-specific cell migration. Here, we describe three assays to assess C5a-mediated effector functions: (1) increase in (Ca2+)i in bone marrow-derived neutrophils using the intracellular fluorescent Ca2+-sensitive indicator Fluo-4 AM and flow cytometry-based methods; (2) in vitro migration of murine, bone marrow-derived neutrophils using a modified Boyden chamber; and (3) in vivo migration of neutrophils from the circulation into the peritoneal cavity. To control for the specificity of the C5a-mediated effects, C5aR-deficient mice, neutralizing C5aR-specific antibodies and potent pharmacologic C5aR inhibitors are available.

Christian M. Karsten and Yves Laumonnier contributed equally to this work.

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 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Klos A, Tenner AJ, Johswich KO et al (2009) The role of the anaphylatoxins in health and disease. Mol Immunol 46:2753–2766

    Article  PubMed  CAS  Google Scholar 

  2. Strey CW, Markiewski M, Mastellos D et al (2003) The proinflammatory mediators C3a and C5a are essential for liver regeneration. J Exp Med 198:913–923

    Article  PubMed  CAS  Google Scholar 

  3. Mastellos D, Papadimitriou JC, Franchini S et al (2001) A novel role of complement: mice deficient in the fifth component of complement (C5) exhibit impaired liver regeneration. J Immunol 166:2479–2486

    PubMed  CAS  Google Scholar 

  4. Hillebrandt S, Wasmuth HE, Weiskirchen R et al (2005) Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans. Nat Genet 37:835–843

    Article  PubMed  CAS  Google Scholar 

  5. Addis-Lieser E, Köhl J, Chiaramonte MG (2005) Opposing regulatory roles of complement factor 5 in the development of bleomycin-induced pulmonary fibrosis. J Immunol 175:1894–1902

    PubMed  CAS  Google Scholar 

  6. Benard M, Gonzalez BJ, Schouft MT et al (2004) Characterization of C3a and C5a receptors in rat cerebellar granule neurons during maturation: neuroprotective effect of C5a against apoptotic cell death. J Biol Chem 279:43487–96

    Article  PubMed  CAS  Google Scholar 

  7. Gerard NP, Gerard C (1991) The chemotactic receptor for human C5a anaphylatoxin. Nature 349:614–617

    Article  PubMed  CAS  Google Scholar 

  8. Boulay F, Mery L, Tardif M et al (1991) Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells. Biochemistry 30:2993–2999

    Article  PubMed  CAS  Google Scholar 

  9. Cain SA, Monk PN (2002) The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74). J Biol Chem 277:7165–7169

    Article  PubMed  CAS  Google Scholar 

  10. Okinaga S, Slattery D, Humbles A et al (2003) C5L2, a nonsignaling C5A binding protein. Biochemistry 42:9406–9415

    Article  PubMed  CAS  Google Scholar 

  11. Crass T, Raffetseder U, Martin U et al (1996) Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U-937 cells. Eur J Immunol 26:1944–1950

    Article  PubMed  CAS  Google Scholar 

  12. Ames RS, Li Y, Sarau HM et al (1996) Molecular cloning and characterization of the human anaphylatoxin C3a receptor. J Biol Chem 271:20231–20234

    Article  PubMed  CAS  Google Scholar 

  13. Chenoweth DE, Goodman MG (1983) The C5a receptor of neutrophils and macrophages. Agents Actions Suppl 12:252–273

    PubMed  CAS  Google Scholar 

  14. Chenoweth DE, Hugli TE (1978) Demonstration of specific C5a receptor on intact human polymorphonuclear leukocytes. Proc Natl Acad Sci U S A 75:3943–3947

    Article  PubMed  CAS  Google Scholar 

  15. Gerard NP, Hodges MK, Drazen JM et al (1989) Characterization of a receptor for C5a anaphylatoxin on human eosinophils. J Biol Chem 264:1760–1766

    PubMed  CAS  Google Scholar 

  16. Werfel T, Oppermann M, Begemann G et al (1997) C5a receptors are detectable on mast cells in normal human skin and in psoriatic plaques but not in weal and flare reactions or in uticaria pigmentosa by immunohistochemistry. Arch Dermatol Res 289:83–86

    Article  PubMed  CAS  Google Scholar 

  17. Köhl J, Baelder R, Lewkowich IP et al (2006) A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma. J Clin Invest 116:783–796

    Article  PubMed  Google Scholar 

  18. Zhang X, Lewkowich IP, Köhl G et al (2009) A protective role for C5a in the development of allergic asthma associated with altered levels of B7-H1 and B7-DC on plasmacytoid dendritic cells. J Immunol 182:5123–5130

    Article  PubMed  CAS  Google Scholar 

  19. Ottonello L, Corcione A, Tortolina G et al (1999) rC5a directs the in vitro migration of human memory and naive tonsillar B lymphocytes: implications for B cell trafficking in secondary lymphoid tissues. J Immunol 162: 6510–6517

    PubMed  CAS  Google Scholar 

  20. Lalli PN, Strainic MG, Yang M et al (2008) Locally produced C5a binds to T cell-expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis 153. Blood 112:1759–1766

    Article  PubMed  CAS  Google Scholar 

  21. Strainic MG, Liu J, Huang D et al (2008) Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells. Immunity 28:425–435

    Article  PubMed  CAS  Google Scholar 

  22. Fusakio ME, Mohammed JP, Laumonnier Y et al (2011) C5a regulates NKT and NK cell functions in sepsis. J Immunol 187: 5805–5812

    Article  PubMed  CAS  Google Scholar 

  23. Han G, Geng S, Li Y et al (2011) GammadeltaT-cell function in sepsis is modulated by C5a receptor signalling. Immunology 133: 340–349

    Article  PubMed  CAS  Google Scholar 

  24. Monk PN, Scola AM, Madala P et al (2007) Function, structure and therapeutic potential of complement C5a receptors. Br J Pharmacol 152:429–448

    Google Scholar 

  25. Skokowa J, Ali SR, Felda O et al (2005) Macrophages induce the inflammatory response in the pulmonary Arthus reaction through G alpha i2 activation that controls C5aR and Fc receptor cooperation. J Immunol 174:3041–3050

    PubMed  CAS  Google Scholar 

  26. Amatruda TT, Gerard NP, Gerard C et al (1993) Specific interactions of chemoattractant factor receptors with G- proteins. J Biol Chem 268:10139–10144

    PubMed  CAS  Google Scholar 

  27. Monk PN, Partridge LJ (1993) Characterization of a complement-fragment-C5a-stimulated calcium- influx mechanism in U937 monocytic cells. Biochem J 295:679–684

    PubMed  CAS  Google Scholar 

  28. Hawlisch H, Belkaid Y, Baelder R et al (2005) C5a negatively regulates toll-like receptor 4-induced immune responses. Immunity 22:415–426

    Article  PubMed  CAS  Google Scholar 

  29. Fernandez HN, Henson PM, Otani A et al (1978) Chemotactic response to human C3a and C5a anaphylatoxins. I. Evaluation of C3a and C5a leukotaxis in vitro and under stimulated in vivo conditions. J Immunol 120:109–115

    PubMed  CAS  Google Scholar 

  30. Markiewski MM, Deangelis RA, Benencia F et al (2008) Modulation of the antitumor immune response by complement. Nat Immunol 9:1225–1235

    Article  PubMed  CAS  Google Scholar 

  31. Köhl J (1997) In: Dodds AW, Sim RB (eds) Complement: a practical approach. Oxford University Press, Oxford, pp 135–163

    Google Scholar 

  32. Gerard C, Bao L, Orozco O et al (1992) Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor. J Immunol 149:2600–2606

    PubMed  CAS  Google Scholar 

  33. Daley JM, Thomay AA, Connolly MD et al (2008) Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol 83:64–70

    Article  PubMed  CAS  Google Scholar 

  34. Zhang X, Majlessi L, Deriaud E et al (2009) Coactivation of Syk kinase and MyD88 adaptor protein pathways by bacteria promotes regulatory properties of neutrophils. Immunity 31:761–771

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute for Systemic Inflammation Research, University of Lübeck, Lübeck, Germany

    Christian M. Karsten & Yves Laumonnier

  2. Division of Cellular and Molecular Immunology, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Jörg Köhl

Authors
  1. Christian M. Karsten
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yves Laumonnier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jörg Köhl
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Division of Infectious Diseases, Brigham and Women’s Hospital, Harvard University Harvard Medical School, Boston, Massachusetts, USA

    Mihaela Gadjeva

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Karsten, C.M., Laumonnier, Y., Köhl, J. (2014). Functional Analysis of C5a Effector Responses In Vitro and In Vivo. In: Gadjeva, M. (eds) The Complement System. Methods in Molecular Biology, vol 1100. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-724-2_23

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-724-2_23

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-723-5

  • Online ISBN: 978-1-62703-724-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation