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


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

PDF or HTML at Wiley Online Library

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
PDF or HTML at Wiley Online Library


Basic Protocol 1: Measuring Matrix Metalloproteinase Using Substrate Gel Zymography

  • 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

  • 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)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Ardi, V.C., Kupriyanova, T.A., Deryugina, E.I., and Quigley, J.P. 2007. Human neutrophils uniquely release TIMP‐free MMP‐9 to provide a potent catalytic stimulator of angiogenesis. Proc. Natl. Acad. Sci. U.S.A. 104:20262‐20267.
   Bar‐Or, A., Nuttall, R.K., Duddy, M., Alter, A., Kim, H.J., Ifergan, I., Pennington, C.J., Bourgoin, P., Edwards, D.R., and Yong, V.W. 2003. Analyses of all matrix metalloproteinase members in leukocytes emphasize monocytes as major inflammatory mediators in multiple sclerosis. Brain 126:2738‐2749.
   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.
   Bonfield, T.L., Swaisgood, C.M., Barna, B.P., Farver, C.F., Kavuru, M.S., and Thomassen, M.J. 2006. Elevated gelatinase activity in pulmonary alveolar proteinosis: Role of macrophage‐colony stimulating factor. J. Leukoc. Biol. 79:133‐139.
   Busiek, D.F., Baragi, V., Nehring, L.C., Parks, W.C., and Welgus, H.G. 1995. Matrilysin expression by human mononuclear phagocytes and its regulation by cytokines and hormones. J. Immunol. 154:6484‐6491.
   de Visser, K.E., Eichten, A., and Coussens, L.M. 2006. Paradoxical roles of the immune system during cancer development. Nat. Rev. Cancer 6:24‐37.
   Fisher, S. and Werb, Z. 1995. The catabolism of extracellular matrix components. In Extracellular Matrix: A Practical Approach (M.A. Haralson and J.R. Hassell, eds.) pp. 261‐287. Oxford University Press, Oxford.
   Gutierrez‐Fernandez, A., Inada, M., Balbín, M., Fueyo, A., Pitiot, A.S., Astudillo, A., Hirose, K., Hirata, M., Shapiro, S.D., Noël, A., Werb, Z., Krane, S.M., López‐Otín, C., and Puente, X.S. 2007. Increased inflammation delays wound healing in mice deficient in collagenase‐2 (MMP‐8). FASEB J. 21:2580‐2591.
   Hattori, S., Fujisaki, H., Kiriyama, T., Yokoyama, T., and Irie, S. 2002. Real‐time zymography and reverse zymography: A method for detecting activities of matrix metalloproteinases and their inhibitors using FITC‐labeled collagen and casein as substrates. Anal. Biochem. 301:27‐34.
   Houghton, A.M., Hartzell, W.O., Robbins, C.S., Gomis‐Rüth, F.X., and Shapiro, S.D. 2009. Macrophage elastase kills bacteria within murine macrophages. Nature 460:637‐641.
   Kahnert, A., Seiler, P., Stein, M., Bandermann, S., Hahnke, K., Mollenkopf, H., and Kaufmann, S.H. 2006. Alternative activation deprives macrophages of a coordinated defense program to Mycobacterium tuberculosis. Eur. J. Immunol. 36:631‐647.
   Kessenbrock, K., Plaks, V., and Werb, Z. 2010. Ratrix metalloproteinases: Regulators of the tumor microenvironment. Cell 141:52‐67.
   Liu, Z., Zhou, X., Shapiro, S.D., Shipley, J.M., Twining, S.S., Diaz, L.A., Senior, R.M., and Werb, Z. 2000. The serpin alpha1‐proteinase inhibitor is a critical substrate for gelatinase B/MMP‐9 in vivo. Cell 102:647‐655.
   Matias‐Roman, S., Gálvez, B.G., Genís, L., Yáñez‐Mó, M., de la Rosa, G., Sánchez‐Mateos, P., Sánchez‐Madrid, F., and Arroyo, A.G. 2005. Membrane type 1‐matrix metalloproteinase is involved in migration of human monocytes and is regulated through their interaction with fibronectin or endothelium. Blood 105:3956‐3964.
   Mostafa Mtairag, E., Chollet‐Martin, S., Oudghiri, M., Laquay, N., Jacob, M.P., Michel, J.B., and Feldman, L.J. 2001. Effects of interleukin‐10 on monocyte/endothelial cell adhesion and MMP‐9/TIMP‐1 secretion. Cardiovasc. Res. 49:882‐890.
   Ortega, N., Wang, K., Ferrara, N., Werb, Z., and Vu, T.H. 2010. Complementary interplay between matrix metalloproteinase‐9, vascular endothelial growth factor and osteoclast function drives endochondral bone formation. Dis. Model Mech. 3:224‐235.
   Parks, W.C., Wilson, C.L., and Lopez‐Boado, Y.S. 2004. Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat. Rev. Immunol. 4:617‐629.
   Redondo‐Munoz, J., Ugarte‐Berzal, E., Terol, M.J., Van den Steen, P.E., Hernández del Cerro, M., Roderfeld, M., Roeb, E., Opdenakker, G., García‐Marco, J.A., and García‐Pardo, A. 2010. Matrix metalloproteinase‐9 promotes chronic lymphocytic leukemia b cell survival through its hemopexin domain. Cancer Cell 17:160‐172.
   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.
PDF or HTML at Wiley Online Library