Quantitative Assessment of Macrophage Functions in Repair and Fibrosis

Thomas A. Wynn1, Luke Barron1, Robert W. Thompson1, Satish K. Madala1, Mark S. Wilson1, Allen W. Cheever1, Thirumalai Ramalingam1

1 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.22
DOI:  10.1002/0471142735.im1422s93
Online Posting Date:  April, 2011
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Abstract

Macrophages play key roles in wound repair and fibrosis by regulating extracellular matrix turnover. Macrophages can process matrix components themselves, but also recruit and alter the functions of other cell types that directly build or degrade extracellular matrix. Classically activated macrophages (CAM, also called M1) tend to promote tissue injury while alternatively activated macrophages (AAM, also called M2) are often linked with the mechanisms of wound repair and fibrosis. However, rather than promoting collagen deposition, recent studies suggest that arginase‐1‐expressing AAM suppress chronic inflammation and fibrosis by inhibiting antigen‐specific T cell responses. This unit describes methods to measure arginase activity in macrophages and whole tissues as well as assays to quantify the T cell suppressive activity of AAMs. Modified hydroxyproline and soluble collagen assays that can be used to quantify collagen levels in tissues and brochoalveolar lavage fluid are also described. The protocols in this unit should provide the investigator with all the necessary information required to measure arginase activity and to correlate the observed activity with the progression and resolution of fibrosis. Curr. Protoc. Immunol. 93:14.22.1‐14.22.12. © 2011 by John Wiley & Sons, Inc.

Keywords: urea; collagen; hydroxyproline; immunosuppression; remodeling; fibrosis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Quantifying Arginase Activity in Cells and Whole Tissues
  • Basic Protocol 2: Quantification of Tissue Fibrosis by Measuring Hydroxyproline in Mouse Tissue
  • Alternate Protocol 1: Measurement of Soluble Collagen from Cell Culture or Lavage Fluid
  • Basic Protocol 3: Assay for T Cell Suppressive Activity of M2 Macrophages
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Quantifying Arginase Activity in Cells and Whole Tissues

  Materials
  • Cultured cells of interest (macrophages derived from bone marrow or thioglycollate derived; or primary lung fibroblasts)
  • Iscove's modified DMEM supplemented with 10% fetal bovine serum, penicillin/streptomycin, and L‐arginine
  • 1× PBS ( appendix 2A)
  • Lysis buffer (see recipe)
  • Arginase activation solution (see recipe)
  • Arginase substrate solution (see recipe)
  • Urea standard solution (see recipe)
  • Quantichrom urea assay kit (Bioassay Systems, cat. no. DIUR‐500)
  • Whole tissue samples (∼100‐mg samples)
  • 24‐well tissue culture plates
  • Platform rocker
  • 96‐well PCR reaction plate
  • Thermal cycler
  • Standard ELISA and PCR plates
  • Spectrophotometer

Basic Protocol 2: Quantification of Tissue Fibrosis by Measuring Hydroxyproline in Mouse Tissue

  Materials
  • Mouse liver (see unit 3.2)
  • 6 N HCl ( appendix 2A)
  • Dowex/Norit A mixture (see recipe)
  • 1% phenolphthalein (see recipe)
  • 10 N NaOH ( appendix 2A)
  • Isopropanol
  • Solution A (see recipe)
  • Solution B (see recipe)
  • Hydroxyproline (Calbiochem)
  • 110°C incubator
  • Centrifuge
  • Glass wool filters
  • 16‐ml tubes (Kimax; VWR)
  • 60°C water bath
  • Spectrophotometer
  • 96‐well plates or cuvettes

Alternate Protocol 1: Measurement of Soluble Collagen from Cell Culture or Lavage Fluid

  Materials
  • Cell lines of interest (e.g., fibroblasts or thioglycollate‐induced peritoneal lavage cells (see unit 16.1)
  • DMEM containing 1% fetal bovine serum
  • PBS ( appendix 2A), cold
  • Collagen (Sigma)
  • Collagen‐binding dye reagent (see recipe)
  • 100% ethanol
  • 0.5 M NaOH ( appendix 2A)
  • U‐ or V‐bottomed 96‐well plates
  • 37°C incubator
  • Centrifuge
  • Spectrophotometer
CAUTION: Picric acid is very toxic and is explosive when stored for extended periods of time; therefore, it should always be used in a hood and handled according to manufacturer's instructions.

Basic Protocol 3: Assay for T Cell Suppressive Activity of M2 Macrophages

  Materials
  • 8‐week‐old mice
  • 3% thioglycollated broth (TG broth; BD/Difco), autoclaved (preferably aged in the dark)
  • IL‐4, IL‐13, and GM‐CSF (10 µg/ml stocks; Peprotech)
  • OVA 323‐339 peptide (1 mg/ml stock)
  • OT‐II transgenic mice (or similar T cell receptor transgenic mouse)
  • CD4 magnetic beads (Miltenyi Biotech), optional
  • 50 µM CFSE stock (Invitrogen)
  • 24‐well plates, tissue culture–treated
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Figures

Videos

Literature Cited

Literature Cited
   Bronte, V. and Zanovello, P. 2005. Regulation of immune responses by L‐arginine metabolism. Nat. Rev. Immunol. 5:641‐654.
   Chiaramonte, M.G., Donaldson, D.D., Cheever, A.W., and Wynn, T.A. 1999. An IL‐13 inhibitor blocks the development of hepatic fibrosis during a T‐helper type 2‐dominated inflammatory response. J. Clin. Invest. 104:777‐785.
   El Kasmi, K.C., Qualls, J.E., Pesce, J.T., Smith, A.M., Thompson, R.W., Henao‐Tamayo, M., Basaraba, R.J., König, T., Schleicher, U., Koo, M.S., Kaplan, G., Fitzgerald, K.A., Tuomanen, E.I., Orme, I.M., Kanneganti, T.D., Bogdan, C., Wynn, T.A., and Murray, P.J. 2008. Toll‐like receptor‐induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens. Nat. Immunol. 9:1399‐1406.
   Gordon, M.K. and Hahn, R.A. 2010. Collagens. Cell Tissue Res. 339:247‐257.
   Gordon, S. 1999. Macrophages and the immune response. In Fundamental Immunology (W.E. Paul, ed.) pp. 533‐544. Lippincott‐Raven Publishers, Philadelphia.
   Gordon, S. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3:23‐35.
   Hesse, M., Modolell, M., La Flamme, A.C., Schito, M., Fuentes, J.M., Cheever, A.W., Pearce, E.J., and Wynn, T.A. 2001. Differential regulation of nitric oxide synthase‐2 and arginase‐1 by type 1/type 2 cytokines in vivo: Granulomatous pathology is shaped by the pattern of L‐arginine metabolism. J. Immunol. 167:6533‐6544.
   Modolell, M., Corraliza, I.M., Link, F., Soler, G., and Eichmann, K. 1995. Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow‐derived macrophages by TH1 and TH2 cytokines. Eur. J. Immunol. 25:1101‐1104.
   Mosser, D.M. and Edwards, J.P. 2008. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8:958‐969.
   Pesce, J., Kaviratne, M., Ramalingam, T.R., Thompson, R.W., Urban, J.F. Jr., Cheever, A.W., Young, D.A., Collins, M., Grusby, M.J., and Wynn, T.A. 2006. The IL‐21 receptor augments Th2 effector function and alternative macrophage activation. J. Clin. Invest. 116:2044‐2055.
   Pesce, J.T., Ramalingam, T.R., Mentink‐Kane, M.M., Wilson, M.S., El Kasmi, K.C., Smith, A.M., Thompson, R.W., Cheever, A.W., Murray, P.J., and Wynn, T.A. 2009. Arginase‐1‐expressing macrophages suppress Th2 cytokine‐driven inflammation and fibrosis. PLoS Pathog. 5:e100371.
   Rutschman, R., Lang, R., Hesse, M., Ihle, J.N., Wynn, T.A., and Murray, P.J. 2001. Cutting edge: Stat6‐Dependent substrate depletion regulates nitric oxide production. J. Immunol. 166:2173‐2177.
   Thompson, R.W., Pesce, J.T., Ramalingam, T., Wilson, M.S., White, S., Cheever, A.W., Ricklefs, S.M., Porcella, S.F., Li, L., Ellies, L.G., and Wynn, T.A. 2008. Cationic amino acid transporter‐2 regulates immunity by modulating arginase activity. PLoS Pathog. 4:e100023.
   Wilson, M.S. and Wynn, T.A. 2009. Pulmonary fibrosis: Pathogenesis, etiology and regulation. Mucosal. Immunol. 2:103‐121.
   Wynn, T.A. 2004. Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat. Rev. Immunol. 4:583‐594.
   Wynn, T.A. 2008. Cellular and molecular mechanisms of fibrosis. J. Pathol. 214:199‐210.
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