Measuring Matrix Metalloproteinase Activity in Macrophages and Polymorphonuclear Leukocytes

Kai Kessenbrock1, Markus Brown1, Zena Werb1

1 Department of Anatomy and Biomedical Sciences Program, University of California, San Francisco, San Francisco, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.24
DOI:  10.1002/0471142735.im1424s93
Online Posting Date:  April, 2011
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Abstract

Macrophages and polymorphonuclear cells (PMNs) represent an essential part of the innate immune system. These cells mediate a wide spectrum of immunological functions including bacterial defense, immune modulation, and inflammation; they are necessary for tissue homeostasis and also contribute to pathologies such as malignancy, autoimmunity, and chronic inflammation. Both macrophages and PMNs express a set of matrix metalloproteinases (MMPs), zinc‐dependent endopeptidases that are involved in a variety of biological functions such as the turnover of extracellular matrix (ECM) components, angiogenesis, and the regulation of inflammation. Given the link between unregulated MMP function and diseases such as chronic inflammation or cancer, it is not surprising that these enzymes have been implicated as key effectors in clinical studies. Thus, it is important to widen our knowledge about the role of these enzymes in macrophage and PMN biology. Here, we briefly discuss the general role of inflammatory cell–derived MMPs and describe methods to analyze their activity in macrophages and PMN. Curr. Protoc. Immunol. 93:14.24.1‐14.24.11. © 2011 by John Wiley & Sons, Inc.

Keywords: MMP; macrophage; neutrophil; inflammation; cancer

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring Matrix Metalloproteinase Using Substrate Gel Zymography
  • Alternate Protocol 1: Measuring MMP Activity Using a Fluorogenic Substrate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measuring Matrix Metalloproteinase Using Substrate Gel Zymography

  Materials
  • Sample for analysis, either:
    • Cells [cell lines including RAW264.7 cells (murine macrophage–like) or HL‐60 cells (human neutrophil–like) or primary cells such as mouse bone marrow–derived macrophages (BMDM) or neutrophils]
    • Macrophage‐ or PMN‐conditioned medium (see unit 14.2 for macrophage activation; see unit 3.20 for isolation of PMNs): medium can be mixed 3:1 (v/v) with Laemmli sample buffer and stored at −70°C (use directly in step 14, below)
  • Phosphate‐buffered saline (PBS), pH 7.2 ( appendix 2A), ice cold
  • 4× Laemmli sample buffer (without bromphenol blue; see recipe)
  • Protein assay kit (e.g., BioRad)
  • Gelatin (Sigma, Type A from porcine skin)
  • Casein (Sigma, α‐casein)
  • Sodium azide (NaN 3)
  • 30% acrylamide:bisacrylamide (BioRad; also see unit 8.4)
  • 4× lower gel buffer (see recipe)
  • Substrate gel incubation buffer: 50 mM Tris⋅Cl, pH 7.8, containing 150 mM NaCl and 5 mM CaCl 2
  • MMP substrate (Table 14.24.1)
  • Sodium azide (NaN 3)
  • 10% (w/v) ammonium persulfate (prepare fresh)
  • N,N,N′,N′‐tetramethylethylenediamine (TEMED; BioRad)
  • Butanol‐saturated H 2O (optional)
  • 4× upper gel buffer (see recipe)
  • Prestained molecular weight markers (BRL)
  • 1× running buffer (see recipe)
  • 2.5% (v/v) Triton X‐100 in 50 mM Tris⋅Cl, pH 7.6 (see appendix 2A for Tris⋅Cl)
  • Coomassie Blue stain (see recipe)
  • Destaining solution: 45% (v/v) methanol or isopropanol/10% (v/v) acetic acid/45% (v/v) H 2O
  • Cell scraper
  • 1‐ml syringe with 26‐G needle
  • Centrifuge
  • Gel casting apparatus for preparing 10 × 8–cm gels including mold, combs, 1‐mm‐thickness spacers
  • Hoefer electrophoresis apparatus (Hoefer Scientific instruments)
  • Hamilton syringes
  • Light box
  • Lucite slab overlaid with a single wet‐stretched sheet of cellulose nitrate held in place with a Lucite frame and clamps
  • Densitometer
  • Additional reagents and equipment for preparation of activated macrophages (unit 14.20) and polyacrylamide gel electrophoresis (unit 8.4)
    Table 4.4.1   MaterialsMMP Substrates for Zymography

    Zymography substrate Macrophage and PMN MMPs
    Gelatin, fibrillar collagen MMP‐1, ‐8, ‐13, ‐14
    Gelatin MMP‐2, ‐9
    Casein MMP‐3, ‐10, ‐11
    Elastin MMP‐12

Alternate Protocol 1: Measuring MMP Activity Using a Fluorogenic Substrate

  Materials
  • Recombinant mouse MMP‐3 catalytic domain (CD), produced by refolding from inclusion bodies expressed in E. coli or commercially available from multiple sources (e.g., EMD Biosciences, cat. no. 444217)
  • 100× MMP activation buffer: 0.1 M 4‐aminophenylmercuric acetate (APMA; Sigma, cat. no. A9563)
  • 1000× (20 mM) fluorogenic MMP substrate Dnp‐Pro‐β‐cyclohexyl‐Ala‐Gly‐Cys(Me)‐His‐Ala‐Lys(Nma)‐NH2 (Enzo Life Sciences, cat. no. P‐128) in dimethylsulfoxide (DMSO)
  • 1× TCNB reaction buffer (see recipe)
  • 10× stop solution: 100 mM EDTA/3 mM NaN 3 dissolved in H 2O
  • Eppendorf Thermomixer R dry block heating and cooling shaker
  • Microfluor black flat‐bottom 96‐well plates (Nunc, cat. no. 7605)
  • Spectramax Gemini XS microplate spectrofluorometer (Molecular Devices)
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Figures

Videos

Literature Cited

Literature Cited
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   Bergers, G., Brekken, R., McMahon, G., Vu, T.H., Itoh, T., Tamaki, K., Tanzawa, K., Thorpe, P., Itohara, S., Werb, Z., and Hanahan, D. 2000. Matrix metalloproteinase‐9 triggers the angiogenic switch during carcinogenesis. Nat. Cell Biol. 2:737‐744.
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   Ridnour, L.A., Windhausen, A.N., Isenberg, J.S., Yeung, N., Thomas, D.D., Vitek, M.P., Roberts, D.D., and Wink, D.A. 2007. Nitric oxide regulates matrix metalloproteinase‐9 activity by guanylyl‐cyclase‐dependent and‐ independent pathways. Proc. Natl. Acad. Sci. U.S.A. 104:16898‐16903.
   Sternlicht, M.D. and Werb, Z. 2001. How matrix metalloproteinases regulate cell behavior. Annu. Rev. Cell Dev. Biol. 17:463‐516.
   Taylor, P.R., Martinez‐Pomares, L., Stacey, M., Lin, H.H., Brown, G.D., and Gordon, S. 2005. Macrophage receptors and immune recognition. Annu. Rev. Immunol. 23:901‐944.
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