Measurement of Soluble and Membrane‐Bound Interleukin 1 Using a Fibroblast Bioassay

Charles A. Dinarello1, Kathrin Muegge2, Scott K. Durum2

1 University of Colorado Health Sciences Center, Denver, Colorado, 2 National Cancer Institute, Frederick, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.2
DOI:  10.1002/0471142735.im0602s37
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Interleukin 1 (IL‐1) is both a major mediator of inflammation and an important signal for activation and differentiation of lymphoid cells. IL‐1 is produced by a large variety of cell types, but in most immunologic systems, activated macrophages are its major cellular source. This unit describes a bioassay for testing biological fluids based on the ability of IL‐1 to induce IL‐8 or IL‐6 in fibroblasts. Also listed are sources of commercially available kits (ELISAs, immunoassays) to measure IL‐1, and antibodies useful in setting up these assays.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: IL‐1 Induction of IL‐8 by Human Fibroblasts
  • Support Protocol 1: Preparation of Fixed Membrane IL‐1α Samples
  • Commentary
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: IL‐1 Induction of IL‐8 by Human Fibroblasts

  Materials
  • Fibroblasts (e.g., MRC‐5; ATCC #CCL‐171; see above)
  • Complete RPMI‐5 ( appendix 2A)
  • Trypsin solution (unit 6.9)
  • Hanks' balanced salt solution (HBSS; appendix 2A) without Ca2+ and Mg2+
  • Sample to be tested for IL‐1 activity
  • 150‐cm2 tissue culture flask
  • 24‐well flat‐bottom tissue culture plate
  • Additional reagents and equipment for counting cells ( appendix 3A), trypan blue exclusion ( appendix 3B), and quantitating IL‐8 by ELISA (unit 6.12)

Support Protocol 1: Preparation of Fixed Membrane IL‐1α Samples

  Materials
  • Cells to be tested for membrane IL‐1α (pro‐IL‐1α)
  • Hanks' balanced salt solution (HBSS; appendix 2A) with and without 1% (w/v) paraformaldehyde, room temperature
  • Serum‐free complete RPMI ( appendix 2A)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Arend, W.P. 1993. Interleukin‐1 receptor antagonists. Adv. Immunol. 54:167‐227.
   Arend, W.P., Malyak, M., Guthridge, C.J., and Gabay, C. 1998. Interleukin‐1 receptor antagonist: role in biology. Annu. Rev. Immunol. 16:27‐55.
   Bendtzen, K., Buschard, K., Diamant, M., Horn, T., and Svenson, M. 1988. Possible role of IL‐1, TNF‐α, and IL‐6 in insulin‐dependent diabetes mellitus and autoimmune thyroid disease. Lymphokine. Res. 8:335‐340.
   Bendtzen, K., Svenson, M., Jonsson, V., and Hippe, E. 1990. Autoantibodies to cytokines—friends or foes? Immunol. Today 11:167‐169.
   Borth, W., Urbanski, A., Prohaska, R., Susanj, M., and Luger, T.A. 1990. Binding of recombinant interleukin‐1 beta to the third complement component and alpha 2‐macroglobulin after activation of serum by immune complexes. Blood 75:2388‐2395.
   Cominelli, F., Nast, C.C., Clark, B.D., Schindler, R., Llerena, R., Eysselein, V.E., Thompson, R.C., and Dinarello, C.A. 1990. Interleukin‐1 gene expression, synthesis and effect of specific IL‐1 receptor blockade in rabbit immune complex colitis. J. Clin. Invest. 86:972‐980.
   Dinarello, C.A. 1996. Biological basis for interleukin‐1 in disease. Blood 87:2095‐2147.
   Dinarello, C.A. 1999. IL‐18: A Th1‐inducing, proinflammatory cytokine and new member of the IL‐1 family. J. Allergy Clin. Immunol. 103:11‐24.
   Dinarello, C.A. and Cannon, J.G. 1993. Cytokine measurements in septic shock. Ann. Intern. Med. 119:853‐854.
   Giri, J.G., Lomedico, P.T., and Mizel, S.B. 1985. Studies on the synthesis and secretion of interleukin 1. I. A 33,000 molecular weight precursor for interleukin 1. J. Immunol. 134:343‐349.
   Granowitz, E.V., Clark, B.D., Vannier, E., Callahan, M.V., and Dinarello, C.A. 1992a. Effect of interleukin‐1 (IL‐1) blockade on cytokine synthesis: I. IL‐1 receptor antagonist inhibits IL‐1‐induced cytokine synthesis and blocks thebinding of IL‐1 to its type II receptor on human monocytes. Blood 79:2356‐2363.
   Granowitz, E.V., Vannier, E., Puotsiaka, D.D., and Dinarello, C.A. 1992b. Effect of interleukin‐1 (IL‐1) blockade on cytokine synthesis: II. IL‐1 receptor antagonist inhibits lipopolysaccharide‐induced cytokine synthesis by human monocytes. Blood 79:2364‐2369.
   Kaplanski, G., Farnarier, C., Kaplanski, S., Porat, R., Shapiro, L., Bongrand, P., and Dinarello, C.A. 1994. Interleukin‐1 induces interleukin‐8 from endothelial cells by a juxacrine mechanism. Blood 84:4242‐4248.
   Kingston, R.E. 1999. Guanidine methods for total RNA preparation. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 4.2.1‐4.2.9. John Wiley & Sons, New York.
   Kurt‐Jones, E.A., Beller, D.I., Mizel, S.B., and Unanue, E.R. 1985. Identification of a membrane‐associated interleukin 1 in macrophages. Proc. Natl. Acad. Sci. U.S.A. 82:1204‐1208.
   Nakamura, K., Okamura, H., Wada, M., Nagata, K., and Tamura, T. 1989. Endotoxin‐induced serum factor that stimulates gamma inteferon production. Infect. Immun. 57:590‐595.
   Okamura, H., Tsutsui, H., Komatsu, T., Yutsudo, M., Hakura, A., Tanimoto, T., Torigoe, K., Okura, T., Nukada, Y., Hattori, K., Akita, K., Namba, M., Tanabe, F., Konishi, K., Fukuda, S., and Kurimoto, M. 1995. Cloning of a new cytokine that induces interferon‐γ. Nature 378:88‐91.
   Ruchatz, H., Leung, B.P., Wei, X.Q., McInnes, I.B., and Liew, F.Y. 1998. Soluble IL‐15 receptor alpha‐chain administration prevents murine collagen‐induced arthritis: a role for IL‐15 in development of antigen‐induced immunopathology. J. Immunol. 160:5654‐5660.
   Satoh, H., Chizzonite, R., Ostrowski, C., Ni‐Wu, G., Kim, H., Fayer, B., Mae, N., Nadeau, R., and Liberato, D.J. 1994. Characterization of anti‐IL‐1α autoantibodies in the sera from healthy humans. Immunpharm. 27:107‐118.
   Shapiro, L., Panayotatos, N., Meydani, S.N., Wu, D., and Dinarello, CA. 1994. Ciliary neurotrophic factor combined with soluble receptor inhibits synthesis of pro‐inflammatory cytokines and prostaglandin‐E2 in vitro. Exp. Cell Res. 215:51‐56.
   Sims, J.E., Giri, J.G., and Dower, S.K. 1994. The two interleukin‐1 receptors play different roles in IL‐1 activities. Clin. Immunol. Immunopathol. 72:9‐14.
   Stevenson, F.T., Bursten, S.L., Fanton, C., Locksley, R.M., and Lovett, D.H. 1993. The 31‐kDa precursor of interleukin‐1α is myristoylated on specific lysines within the 16‐kDa N‐terminal propiece. Proc. Natl. Acad. Sci. U.S.A. 90:7245‐7249.
   Svenson, M., Hensen, M.B., and Bendtzen, K. 1990. Distribution and characterization of autoantibodies to interleukin‐1α in normal human sera. Scand. J. Immunol. 32:695‐701.
   Svenson, M., Hensen, M.B., Kayser, L., Rasmussen, A.K., Reimert, C.M., and Bendtzen, K. 1992. Effects of human anti‐IL‐1α autoantibodies on receptor binding and biological activities of IL‐1. Cytokine 4:125‐133.
   Svenson, M., Hensen, M.B., and Bendtzen, K. 1993. Binding of cytokines to pharmaceutically prepared human immunoglobulin. J. Clin. Invest. 92:2533‐2539.
   Takacs, L., Kovacs, E.J., Smith, M.R., Young, H.A., Tartakovsky, B., and Durum, S.K. 1988. Direct visualization of IL‐1 mRNA in tissue sections by in situ hybridization. J. Immunol. 141:3081‐3095.
Key References
   Dinarello, C.A. and Savage, N. 1989. Interleukin‐1 and its receptor. Crit. Rev. Immunol. 9:1‐20.
   Muegge, K. and Durum, S.K. 1989. The role of interleukin‐1 in the immune response. In Interleukin‐1, Inflammation and Disease (R. Bomford and B. Henderson, eds.) pp. 107‐122. Elsevier Science Publishing, New York.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library