Measurement of Mouse and Human Interleukin 9

Jean‐Christophe Renauld1, Jacques Van Snick1

1 Ludwig Institute for Cancer Research and Experimental Medicine Unit, Catholic University of Louvain, Brussels, Belgium
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.13
DOI:  10.1002/0471142735.im0613s51
Online Posting Date:  November, 2002
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Abstract

This unit describes two proliferation assays to detect or quantitate human and murine interleukin 9 (IL‐9). The first is based on the ability of IL‐9 to stimulate the proliferation of the TS1h9RA3 cell line, a murine IL‐9‐dependent cell line transfected with the human IL‐9 receptor. An alternate protocol is based on the ability of IL‐9 to stimulate the proliferation of the human megakaryoblastic leukemia cell line, M‐O7e. M‐O7e cells depend on either human IL‐3 or granulocyte/macrophage colony‐stimulating factor (GM‐CSF) for growth, although other cytokines including IL‐2, ‐4, ‐6, and ‐9 and steel factor are also weakly mitogenic (relative to IL‐3 and GM‐CSF). Thus, although M‐O7e cells can readily be used to quantitate levels of IL‐9 in the absence of other cytokines, analysis in the presence of complex mixtures of cytokines (e.g., natural sources) requires the use of specific antibodies against IL‐9 and the other cytokines, as described in this unit.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of IL‐9 Activity Using the TS1h9RA3 Cell Proliferation Assay
  • Alternate Protocol 1: Measurement of IL‐9 Activity Using the M‐O7e Proliferation Assay
  • Alternate Protocol 2: Detection of IL‐9 in Mixed Cytokine Samples
  • Support Protocol 1: Maintenance of TS1h9RA3 Cells
  • Support Protocol 2: Maintenance of M‐O7e Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of IL‐9 Activity Using the TS1h9RA3 Cell Proliferation Assay

  Materials
  • Complete DMEM‐10 medium ( appendix 2A)
  • recipe1000 U/ml human or mouse IL‐9 reference standard (see recipe)
  • Unknown samples containing IL‐9 (must not contain any murine IL‐4 or human or mouse LIF)
  • TS1 cell culture (3‐ to 4‐day‐old; see protocol 4)
  • PBS, pH 7.4 ( appendix 2A)
  • 50 mM citrate buffer, pH 5.0
  • 0.25% Triton X‐100
  • 3.75 mM p‐nitrophenyl N‐acetyl‐β‐D‐glucosaminide (Sigma)
  • Glycine buffer: 0.1 M glycine, pH 10.4 and 10 mM EDTA, store at room temperature
  • Multichannel pipettor and tips
  • 96‐well flat‐bottom microtiter plates with lids (e.g., Nunc)
  • 15‐ml conical centrifuge tube
  • Jouan GR422 centrifuge (or equivalent)
  • Additional reagents and equipment for counting viable cells ( appendix 3B), labeling cells and determining [3H]thymidine incorporation ( appendix 3B), and quantitation of interleukin activity (unit 6.3)

Alternate Protocol 1: Measurement of IL‐9 Activity Using the M‐O7e Proliferation Assay

  • Unknown samples containing IL‐9 (but no other cytokines)
  • M‐O7e cell culture (3‐ to 4‐day old; see protocol 5)
  • Semi‐automated cell‐harvesting apparatus
  • Additional reagents and equipment for counting viable cells ( appendix 3B), labeling cells and determining [3H]thymidine incorporation ( appendix 3B), and quantitation of interleukin activity (unit 6.3)

Alternate Protocol 2: Detection of IL‐9 in Mixed Cytokine Samples

  • TS1h9RA3 cells
  • Complete RPMI‐10 medium ( appendix 2A)

Support Protocol 1: Maintenance of TS1h9RA3 Cells

  • M‐O7e cells (Dr. Luigi Pegoraro, University of Perugia, Italy)
  • Complete RPMI‐10 medium ( appendix 2A)
  • 100,000 U/ml human GM‐CSF stock (Table 97.80.4711) or 8000 U/ml human IL‐3 stock (Table 97.80.4711 and unit 6.4)
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Figures

Videos

Literature Cited

Literature Cited
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   Modi, W.S., Pollock, D.D., Mock, B.A., Banner, C., Renauld, J.‐C., and Van Snick, J. 1991. Regional localization of the human glutaminase (GLS) and interleukin‐9 (IL9) genes by in situ hybridization. Cytogenet. Cell. Genet. 57:114‐116.
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   Renauld, J.‐C., van der Lugt, N., Vink, A., van Roon, M., Godfraind, C., Warnier, G., Merz, H., Feller, A., Bems, A., and Van Snick, J. 1994. Thymic lymphomas in interleukin 9 transgenic mice. Oncogene. 9:1327‐1332.
   Richard, M., Grencis, R.K., Humphreys, N.E., Renauld, J.‐C., and Van Snick, J. 2000. Anti‐interleukin‐9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris‐infected mice. Proc. Natl. Acad. Sci. U.S.A. 97:767‐772.
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   Townsend, J.M., Fallon, G.P., Matthews, J.D., Smith, P., Jolin, E.H., and McKenzie, N.A. 2000. IL‐9‐deficient mice establish fundamental roles for IL‐9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development. Immunity. 13:573‐583.
   Uyttenhove, C., Simpson, R.J., and Vans Snick, J. 1988. Functional and structural characterization of P40, a mouse glycoprotein with T‐cell growth factor activity. Proc. Natl. Acad. Sci. U.S.A. 85:6934‐6938.
   Van Damme, J., Uyttenhove, C., Houssiau, F., Put, W., Proost, P., Van Snick, J. 1992. Human growth factor for murine interleukin (IL)‐9 responsive T cell lines: Co‐induction with IL‐6 in fibroblasts and identification as LIF/HILDA. Eur. J. Immunol. 22:2801‐2808.
   Van Snick, J., Goethals, A., Renauld, J.‐C., van Roost, E., Uyttenhove, C., Rubira, M.R., Moritz, R.L., and Simpson, R.J. 1989. Cloning and characterization of a cDNA for a new mouse T cell growth factor (P40). J. Exp. Med. 169:363‐368.
   Vink, A., Warnier, G., Brombacher, F., and Renauld, J.‐C. 1999. IL‐9‐induced in vivo expansion of the B‐1 lymphocyte population. J. Exp. Med. 189:1413‐1423.
   Yang, Y.‐C., Ricciardi, S., Ciarletta, A., Calvetti, J., Kelleher, K., and Clark, S.C. 1989. Expression cloning of a cDNA encoding a novel human hematopoietic growth factor: Human homologue of murine T cell growth factor P40. Blood. 74:1880‐1884.
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