Principle

Measurement of in vivo cytokine production is problematic owing to the rapid utilization, catabolization, and excretion of cytokine quickly after production and secretion. The In Vivo Cytokine Capture Assay (IVCCA) is an adaptation of the standard cytokine ELISA which facilitates measurement of cytokines in serum by increasing their in vivo half lives dramatically. The IVCCA increases the sensitivity of measurement of in vivo cytokine production 30- to 1000- fold over levels measurable using standard ELISA approaches for serum cytokine measurement.

To increase in vivo cytokine half-life, mice are injected with a small dose of (sterile, low endotoxin) biotinylated neutralizing anti-cytokine antibody; this “in vivo capture” antibody binds the cytokine and inhibits cytokine utilization, degradation, and excretion. Complexes of cytokine/biotin-anti-cytokine antibody accumulate in the blood and are measured in collected serum by ELISA, using microplate wells coated with an antibody reactive with a second epitope of the same cytokine (two-site, sandwich ELISA). IVCCAs have been developed for mouse IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α; kinetics of in vivo activation, in vivo capture, and serum harvest are described in detail in the primary reference (Finkelman, F., et al. 2003. Current Protocols in Immunology, Unit 6.28). More than 25 publications have cited the use of the IVCCA for in vivo cytokine measurement.

Reagent Table

Protocol

For step-by-step methodology of this application, please refer to this detailed reference: Finkelman, F., S. Morris, T. Orekhova, and D. Sehy. 2003. The In Vivo Cytokine Capture Assay for measurement of cytokine production in the mouse. In Current Protocols in Immunology. Unit 6.28. J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, and W. Strober, eds. John Wiley and Sons, New York.

References

1. Marat V. Khodoun, Tatyana Orekhova, Crystal Potter, Suzanne Morris, and Fred D. Finkelman. Basophils Initiate IL-4 Production during a Memory T-dependent Response. J. Exp. Med., Oct 2004; 200: 857 - 870.
   
   
2. Ram Raj Singh, Vijay Saxena, Song Zang, Lily Li, Fred D. Finkelman, David P. Witte, and Chaim O. Jacob. Differential Contribution of IL-4 and STAT6 vs STAT4 to the Development of Lupus Nephritis. J. Immunol., May 2003; 170: 4818 - 4825.
   
   
3. Allen Smith, Kathleen B. Madden, Karla J. Au Yeung, Aiping Zhao, Justin Elfrey, Fred Finkelman, Orville Levander, Terez Shea-Donohue, and Joseph F. Urban, Jr. Deficiencies in Selenium and/or Vitamin E Lower the Resistance of Mice to Heligmosomoides polygyrus Infections. J. Nutr., Apr 2005; 135: 830 - 836.
   
   
4. Roman Puliaev, Phuong Nguyen, Fred D. Finkelman, and Charles S. Via Differential Requirement for IFN-gamma in CTL Maturation in Acute Murine Graft-versus-Host Disease. J. Immunol., Jul 2004; 173: 910 - 919.
   
   
5. Fred D. Finkelman, Mingyan Yang, Tatyana Orekhova, Erin Clyne, Jonathan Bernstein, Michael Whitekus, David Diaz-Sanchez, and Suzanne C. Morris Diesel Exhaust Particles Suppress In Vivo IFN-gamma Production by Inhibiting Cytokine Effects on NK and NKT Cells. J. Immunol., Mar 2004; 172: 3808 - 3813.
   
   
6. Booki Min, Melanie Prout, Jane Hu-Li, Jinfang Zhu, Dragana Jankovic, Ellen S. Morgan, Joseph F. Urban, Jr., Ann M. Dvorak, Fred D. Finkelman, Graham LeGros, and William E. Paul. Basophils Produce IL-4 and Accumulate in Tissues after Infection with a Th2-inducing Parasite. J. Exp. Med., Aug 2004; 200: 507 - 517.
   
   
7. Dmitri Berenzon, Robert J. Schwenk, Lisa Letellier, Mimi Guebre-Xabier, Jackie Williams, and Urszula Krzych. Protracted Protection to Plasmodium berghei Malaria Is Linked to Functionally and Phenotypically Heterogeneous Liver Memory CD8+ T Cells. J. Immunol., Aug 2003; 171: 2024 - 2034.
   
   
8. Lucy A. Gildea, Reta Gibbons, Fred D. Finkelman, and George S. Deepe, Jr. Overexpression of Interleukin-4 in Lungs of Mice Impairs Elimination of Histoplasma capsulatum. Infect. Immun., Jul 2003; 71: 3787 - 3793.
   
   
9. Matthew L. deSchoolmeester, Matthew C. Little, Barrett J. Rollins, and Kathryn J. Else. Absence of CC Chemokine Ligand 2 Results in an Altered Th1/Th2 Cytokine Balance and Failure to Expel Trichuris muris Infection. J. Immunol., May 2003; 170: 4693 - 4700.
   
   
10. Ram Raj Singh , Vijay Saxena, Song Zang, Lily Li, Fred D. Finkelman, David P. Witte and Chaim O. Jacob. Differential Contribution of IL-4 and STAT6 vs STAT4 to the Development of Lupus Nephritis. J. Immunol. 2003. 170: 4818-4825.
    
    
11. Dengping Yin, Nadav Dujovny, Lianli Ma, Anncy Varghese, JiKun Shen, D. Keith Bishop, and Anita S. Chong. IFN-gamma Production Is Specifically Regulated by IL-10 in Mice Made Tolerant with Anti-CD40 Ligand Antibody and Intact Active Bone J. Immunol., Jan 2003; 170: 853 - 860.
    
    
12. Alison L. Woo, Lucy A. Gildea, Leslie M. Tack, Marian L. Miller, Zachary Spicer, David E. Millhorn, Fred D. Finkelman, Daniel J. Hassett, and Gary E. Shull. In Vivo Evidence for Interferon-gamma-mediated Homeostatic Mechanisms in Small Intestine of the NHE3 Na+/H+ Exchanger Knockout Model of Congenital Diarrhea J. Biol. Chem., Dec 2002; 277: 49036 - 49046.
    
    
13. Fred D. Finkelman, Suzanne C. Morris, Tatyana Orekhova, Masaaki Mori, Debra Donaldson, Steven L. Reiner, Nancy L. Reilly, Lisa Schopf, and Joseph F. Urban, Jr. Stat6 Regulation of In Vivo IL-4 Responses. J. Immunol., Mar 2000; 164: 2303 - 2310.
    
    
14. Ann E. Kelly-Welch, Helen Y. Wang, Ling-Mei Wang, Jacalyn H. Pierce, Gilbert Jay, Fred Finkelman, and Achsah D. Keegan. Transgenic Expression of Insulin Receptor Substrate 2 in Murine B Cells Alters the Cell Density-Dependence of IgE Production In Vitro and Enhances IgE Production In Vivo. J. Immunol., Mar 2004; 172: 2803 - 2810.
    
    
15. Matthew L. deSchoolmeester, Matthew C. Little, Barrett J. Rollins, and Kathryn J. Else. Absence of CC Chemokine Ligand 2 Results in an Altered Th1/Th2 Cytokine Balance and Failure to Expel Trichuris muris Infection. J. Immunol., May 2003; 170: 4693 - 4700.
    
    
16. Monica G. Chiaramonte, Margaret Mentink-Kane, Bruce A. Jacobson, Allen W. Cheever, Matthew J. Whitters, Mary E.P. Goad, Anthony Wong, Mary Collins, Debra D. Donaldson, Michael J. Grusby, and Thomas A. Wynn. Regulation and Function of the Interleukin 13 Receptor 2 During a T Helper Cell Type 2–dominant Immune Response. J. Exp. Med., Mar 2003; 197: 687 - 701.
    
    
17. Charles S. Via, Andrei Shustov, Violeta Rus, Thomas Lang, Phuong Nguyen, and Fred D. Finkelman. In Vivo Neutralization of TNF-alpha Promotes Humoral Autoimmunity by Preventing the Induction of CTL. J. Immunol., Dec 2001; 167: 6821 - 6826.
    
    
18. Joseph F. Urban, Jr., Nancy Noben-Trauth, Lisa Schopf, Kathleen B. Madden, and Fred D. Finkelman. Cutting Edge: IL-4 Receptor Expression by Non-Bone Marrow-Derived Cells Is Required to Expel Gastrointestinal Nematode Parasites. J. Immunol., Dec 2001; 167: 6078 - 6081.
    
    
19. Joseph F. Urban, Jr., Lisa Schopf, Suzanne C. Morris, Tatyana Orekhova, Kathleen B. Madden, Catherine J. Betts, H. Ray Gamble, Colleen Byrd, Deborah Donaldson, Kathryn Else, and Fred D. Finkelman. Stat6 Signaling Promotes Protective Immunity Against Trichinella spiralis Through a Mast Cell- and T Cell-Dependent Mechanism J. Immunol., Feb 2000; 164: 2046 - 2052.
    
    
20. Fred D. Finkelman and Suzanne C. Morris. Development of an assay to measure in vivo cytokine production in the mouse. Int. Immunol., Nov 1999; 11: 1811 - 1818.
    
    
21. Suzanne C. Morris, Robert L. Coffman, and Fred D. Finkelman. In Vivo IL-4 Responses to Anti-IgD Antibody Are MHC Class II Dependent and β2-Microglobulin Independent and Develop Normally in the Absence of IL-4 Priming of T Cells. J. Immunol., Apr 1998; 160: 3299 - 3304.
    
    
22. Qian Liu, Cristin Arseculeratne, Zhugong Liu, Jeannette Whitmire, Michael J. Grusby, Fred D. Finkelman, Thomas N. Darling, Allen W. Cheever, James Swearengen, Joseph F. Urban, and William C. Gause. Simultaneous Deficiency in CD28 and STAT6 Results in Chronic Ectoparasite-Induced Inflammatory Skin Disease. Infect. Immun., Jul 2004; 72: 3706 - 3715.
    
    
23. Sherry Thornton, Gregory P. Boivin, Kwang N. Kim, Fred D. Finkelman, and Raphael Hirsch. Heterogeneous Effects of IL-2 on Collagen-Induced Arthritis. J. Immunol., Aug 2000; 165: 1557 - 1563.