Measuring Matrix Metalloproteinase Activity in Macrophages and Polymorphonuclear Leukocytes

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.

INTRODUCTION

Macrophages and polymorphonuclear cells (PMN) are innate immune cells with crucial functions in host defense, inflammation, and tissue homeostasis, but they also play important roles in noninfectious diseases such as cancer. During inflammation, tissue macrophages are mainly derived from monocytes as they extravasate from the blood stream into the tissue and differentiate into macrophages. One of the main features of macrophages is their high phagocytic capacity, which is of importance for their regulation of immune responses and tissue homeostasis. PMNs make up the largest proportion of all peripheral blood leukocytes. They are the first cell type to infiltrate inflamed or infected tissue, where they release intracellular granules filled with effector proteins and proteinases upon cellular activation.

Both macrophages and PMNs express a set of matrix metalloproteinases (MMPs), zinc-and calcium-dependent proteolytic enzymes that are involved in many of the key functions of the innate immune system. As their name implies, MMPs are important for the turnover of extracellular matrix (ECM; Sternlicht and Werb, 2001). However, MMPs also specifically regulate immune responses by modulating a variety chemokines and cytokines (Parks et al., 2004). Therefore, macrophage-derived MMPs help to orchestrate the course of an immune or inflammatory response. Moreover, tumor-associated macrophages and PMN potently affect cancer progression (de Visser et al., 2006). In the tumor microenvironment, these myeloid cells are one of the major sources of MMPs, which influence tumor progression in multiple ways (Kessenbrock et al., 2010).

MMPs can be analyzed by a broad range of methodologies including real-time PCR, immunocytochemistry, ELISA methods, in situ zymography, or direct substrate analysis and proteomics (Fisher and Werb, 1995). In this unit, we present a limited number of protocols for analysis of MMPs in inflammatory cells that are based on zymography (Basic Protocol) and fluorogenic substrate peptides to quantify MMP activity (Alternate Protocol).

Macrophages and PMNs may be stimulated prior to the analysis of MMP activity. As detailed above, the pathway of activation will significantly alter the expression profile and the presence of certain MMPs in the cellular fraction and the supernatant. A general priming agent for macrophages is IFN-γ, which can be followed by secondary stimuli such as LPS, immune complexes, or IL-4. For more detailed information on macrophage activation protocols, see UNIT 14.2. PMNs are usually primed with TNF-α. Secondary stimulation of PMNs can be achieved by immune complexes.

BASIC PROTOCOL

MEASURING MATRIX METALLOPROTEINASE USING SUBSTRATE GEL ZYMOGRAPHY

Either cultured cells or macrophage- or PMN-conditioned medium may be used as the analytical sample in this protocol. One should use conditioned medium if the biological process under investigation involves secreted, extracellular proteinases. In contrast, cell lysates should be used when investigating the function of intracellular or membrane-bound proteinases.

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 (NaN₃)

• 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 CaCl₂

• MMP substrate (Table 14.24.1)

• Sodium azide (NaN₃)

• 10% (w/v) ammonium persulfate (prepare fresh)

• N,N,N′,N′-tetramethylethylenediamine (TEMED; BioRad)

• Butanol-saturated H₂O (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) H₂O

• 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 14.24.1.

MMP 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

Prepare samples from cultured cells

If using conditioned medium as the sample, skip steps 1 to 6, prepare gel as in steps 7 to 13, and introduce the sample directly at step 14.

• 1Prepare activated macrophages (see UNIT 14.20). Rinse cultured cells three times with ice-cold PBS, then lyse by adding as small a volume of 4× Laemmli sample buffer as possible.
• 2Scrape the cells from the dish using a cell scraper.
• 3Produce a cell suspension by using a 1-ml syringe fitted with a 26-G needle to aspirate the cells up and down in 4× Laemmli sample buffer, approximately 20 times.
• 4Centrifuge 10 min at 12,000 to 14,000 × g, 4°C, to remove any particulate material.
• 5Determine the protein concentration of the supernatant, using, e.g., the BioRad protein assay.
• 6Freeze the sample at −70°C.

  IMPORTANT NOTE:Since proteinases can autoactivate or be cleaved by other proteinases in the sample, initial analyses should be performed with fresh samples.

Prepare gels

• 7Dissolve gelatin or casein in water at 3 mg/ml (3× stock) with 20 mg/ml NaN₃.

  Proteins may be dissolved in water, or in lower gel buffer (see recipe) if necessary.

• 8Prepare 30% acrylamide:bisacrylamide by dissolving in double-distilled water to a final volume of 100 ml. Filter and store at 4°C in a dark bottle.

  Detailed protocols for the preparation of polyacrylamide gels are provide in UNIT 8.4.

• 9Prepare a substrate gel incubation buffer that will provide the conditions necessary for the enzyme.

  MMPs usually are assayed in 50 mM Tris·Cl, pH 7.8, containing 150 mM NaCl and 5 mM CaCl₂ (referred to as substrate gel incubation buffer in this unit).

• 10Prepare 14 ml of 10% acrylamide containing 1 mg/ml of the substrates listed in Table 14.24.1 by mixing:

  • 4.66 ml of 30% acrylamide:bisacrylamide

  • 3.50 ml of 4× lower gel buffer

  • 4.66 ml of 3 mg/ml substrate suspension (in double-distilled water; Table 14.24.1) containing 20 mg/ml NaN₃

  • 1.04 ml H₂O.

  Degas for 15 min at ambient temperature. Add 50 μl of 10% ammonium persulfate (freshly prepared) and 7 μl of TEMED.

  10% polyacrylamide gels give the best resolution of proteins in the range of 30 to 100 kD.

  By adding an inhibitor to the substrate gel incubation buffer, it is possible to determine the matrix metalloproteinase class or the specific matrix metalloproteinase itself. When the appropriate inhibitors are used, no enzymatic activity is observed in the molecular weight region that was formerly cleared of substrate. MMP inhibitors can also be determined by zymography by adding a limited amount of an MMP to the incubation buffer. This degrades the gelatin substrate to a certain extent so that it stains more lightly. Bands containing MMPs degrade the substrate further, while bands containing MMP inhibitors suppress the general clearing and appear as dark bands. For further information on reverse zymography also see UNIT 10.8.

• 11Pour the acrylamide solution carefully into the mold, avoiding bubbles; overlay with H₂O or butanol-saturated H₂O and allow to polymerize for 120 min.

  After polymerization, the gels can be stored in self-sealing plastic bags with extra 1× lower gel buffer at 4°C for up to 2 weeks.

• 12Prepare 10 ml Laemmli stacking gel (4% acrylamide) by mixing the following:

  • 1.5 ml 30% acrylamide:bisacrylamide

  • 6.1 ml H₂O

  • 2.5 ml 4× upper gel buffer

  • 100 μl 10% ammonium persulfate (freshly prepared)

  • 5μl TEMED.

  This is enough for five mini-gels or two to three regular gel stackers.

• 13Remove H₂O/butanol-saturated H₂O from polymerized gel and pour Laemmli stacking gel into the mold around a 10-, 15-, or 20-well comb, using a Pasteur pipet to fill to the top of the mold. Allow to polymerize for 40 to 90 min at room temperature.
• 14Mix samples 3:1 (v/v) with 4× sample buffer (do not boil!) and load by underlaying with a Hamilton syringe. When feasible, run the gels at 4°C to reduce interaction of the enzyme with the substrate and to achieve more accurate estimates of molecular weight. Run in the running buffer described in Reagents and Solutions at 15 mA/gel while stacking and at 20 mA/gel during the resolving phase. To monitor the progress of the electrophoresis, use prestained molecular weight markers (BRL) as standards. For low-molecular-weight enzymes, terminate electrophoresis when the dye front reaches the bottom of the gel. For enzymes of 40 to 100 kD, terminate electrophoresis when the 25-kD marker reaches the edge of the gel.
• 15Remove the SDS and allow the proteins to renature by soaking in 2.5% Triton X-100 (in 50 mM Tris·Cl, pH 7.6) with gentle shaking for 60 min at ambient temperature, and then repeating with one additional change of solution. Rinse the gel three times in the incubation buffer of choice (i.e., one that will permit proteinase activity; in this case, substrate gel incubation buffer as described above) and incubate in this buffer at 37°C for 2 to 24 hr with gentle shaking.

  Incubation time depends on the degree of proteinase activity in the samples tested and needs to be determined empirically.

• 16Stain the gel with Coomassie Blue (minimum 30 min) with shaking, then destain in 45% methanol (or isopropyl alcohol)/10% acetic acid. Optimize the contrast between cleared regions and background by varying the staining and destaining conditions.

  The areas containing enzyme are pale or clear against blue background (see Fig. 14.24.1)

• 17Transilluminate the wet gel against a light box and photograph it.
• 18Air-dry the gel on a Lucite slab overlaid with a single wet-stretched sheet of cellulose nitrate held in place with a Lucite frame and clamps.
• 19Scan the gel using a densitometer and/or store in a notebook.

Figure 14.24.1.

Monitoring MMP activity. (A) Zymography using a polyacrylamide gel containing gelatin with three samples from lysed human tumor cells identifying the gelatinases MMP-2 and -9 (Bergers et al., 2000). Pro-MMPs show catalytic activity, since SDS chemically activates the enzymes. MMPs can be identified according to their molecular weight. While MMP-9 is present in both its pro-form (~92 kD) as well as its activated form (~82 kD), MMP-2 is only found in the nonactivated pro-form (~68 kD). (B) Fluorogenic substrate assay. MMP-3-mediated hydrolysis of MMP substrate Dnp-Pro-β-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2 was measured using a spectrophotometer at 455 nm after excitation with 360 nm wavelength. Rapid increase in fluorescence arbitrary units (AU) is observed over time in the presence of 150 nM recombinant catalytic domain of MMP-3 (MMP-3 CD; triangles). Only background fluorescence in found in the absence of enzyme (control; squares).

ALTERNATE PROTOCOL

MEASURING MMP ACTIVITY USING A FLUOROGENIC SUBSTRATE

Synthetic fluorogenic substrates to monitor MMP activity

To characterize MMP-dependent myeloid cell functions thoroughly, it is important to determine MMP activity. The use of fluorogenic substrates allows for quantitative measurement of the proteolytic activity of MMPs over time, in vitro. As an example, this section describes an assay to measure the proteolytic activity of MMP-3, one of the MMPs expressed by macrophages, using the substrate Dnp-Pro-β-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2, which is cleaved by MMP-1, -3, -7, -8, -9, -11, -12, -13, -14, and ADAM9 (Dnp = 2,4-dinitrophenyl; Nma = N-Me-2-aminobenzoy). This substrate is cleaved by several MMPs between the Gly-Cys(met) with different k_cat/k_m. For MMP-3, k_cat/k_m is 5 × 10² M⁻¹ sec⁻¹.

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 NaN₃ dissolved in H₂O

• 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)

1. For duplicate measurement, prepare MMP activation solution consisting of 20 μg/ml recombinant MMP-3 in 20 μl of 1× MMP activation buffer. Incubate 1 hr at 37°C in Thermomixer.

2. Transfer 10 μl of the MMP activation solution into a 96-well flat-bottom plate and add 65 μl of 1× (20 μM) fluorogenic MMP substrate solution (diluted with 1× reaction buffer). Incubate 1 hr at 37°C.

3. Optional: Add 7.5 μl of 10× stop solution to stop enzymatic activity.

4. Place the 96-well plate into the microplate spectrofluorometer and measure the fluorescence arbitrary units at 455 nm after excitation of the solution with 360 nm light (Fig. 14.24.1B).

   MMP activity can be determined by end point analysis or measured over time to analyze enzyme kinetics.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see APPENDIX 5.

Coomassie Blue stain

• Dissolve 5 g Coomassie Blue in 600 ml water. Filter, then add 300 ml isopropyl alcohol and 100 ml glacial acetic acid. Store up to 3 months at 4°C.

Laemmli sample buffer (without bromphenol blue), 4×

• 1 g SDS (BioRad)

• 2.5 ml glycerol (BioRad)

• 5 ml 4× upper gel buffer (see recipe)

• H₂O to 10 ml

• Store up to 3 months at −20°C

Lower gel buffer, 4×

• 18.15 g Tris base (Sigma)

• 0.4 g SDS (BioRad)

• Add 50 ml double-distilled H₂O and adjust pH to 8.8 with HCl

• Make up to 100 ml final with double-distilled H₂O

• Store up to 2 months at room temperature

Running buffer, 1×

• 0.025 M Tris base (Sigma)

• 0.19 M glycine (BioRad)

• 0.1% SDS (BioRad)

• pH will be 8.3; do not adjust

• Prepare fresh from 10× stocks (see below)

  Prepare solution using 10× Tris stock (30.275 g Tris base in 1 liter) and 10× glycine-SDS stock (142.25 g glycine and 10 g SDS in 1 liter).

TCNB reaction buffer, 1×

• 50 mM Tris base (Sigma)

• 10 mM CaCl₂ (Sigma)

• 0.15 M NaCl (Sigma)

• 0.05% Brij-35 (BioRad)

• Adjust to pH 7.5 with HCl

• Store up to 1 month at 4°C

  Prepare solution using the following stocks: 5 M NaCl, 2.5 M CaCl₂, 1 M Tris·Cl, pH 7.6 (APPENDIX 2A), 10% Brij-35 (BioRad).

Upper gel buffer, 4×

• 3 g Tris base (Sigma)

• 0.2 g SDS (BioRad)

• Add 20 ml double-distilled H₂O and adjust pH to 6.8 with HCl

• Make up to 50 ml final with double-distilled H₂O

• Store up to 2 months at room temperature

COMMENTARY

Background Information

MMPs consist of several subunits, such as the pre- and pro-domains, the catalytic domain, the hemopexin domain, and, in the case of membrane-anchored MMPs, a glycosylphosphatidylinositol (GPI) or transmembrane domain. The enzymes are synthesized as inactive zymogens that require removal of the N-terminal pro-domain to be converted into active proteases, which usually takes place after secretion of the MMPs into the extracellular space.

While the best characterized properties of MMPs are mediated by their catalytic activity, MMPs also act independently of their proteolytic activity; for example, MMP-12 encodes a bacteriocidal peptide in its hemopexin domain (Houghton et al., 2009), and MMP-9 activates integrin-mediated signaling pathways solely via its hemopexin domain even in the absence of protease function (Redondo-Munoz et al., 2010).

Matrix metalloproteinases of macrophages and polymorphonuclear neutrophils

While macrophages have been reported to express almost the entire spectrum of MMPs, the granules of PMNs are restricted to MMP-8 and -9. In both macrophages and PMNs, MMPs mediate important functions such as maintenance of tissue homeostasis and modulation of inflammatory responses, as well as anti-microbial functions (Bar-Or et al., 2003).

The majority of macrophages are derived from monocytes that infiltrate peripheral tissues. MMPs are critically involved in the differentiation of monocytes into macrophages, since they contribute to monocyte extravasation and migration through the interstitial matrix. During this process, monocytes increase MMP-9 and -14 activity at their leading edge and in lamellopodia (Matias-Roman et al., 2005). Upon attachment to fibronectin and TNF-α-activated endothelial cells, these MMPs regulate monocyte migration via exposure of cryptic adhesion sites on vascular endothelia or regulation of β1- and β2-integrin receptor function. Importantly, freshly isolated monocytes express MT1-MMP (MMP-14) and MMP-2. However, during the process of differentiation into macrophages, monocytes are triggered to up-regulate several MMPs, including MMP-7, -9, and -12 (Parks et al., 2004).

Macrophages are highly efficient sentinel cells that respond to immunogenic stimuli by engaging a broad range of surface receptors (Taylor et al., 2005). Activation of these receptors gives rise to three different types of macrophages: macrophages that are instructed to kill microbes (classical activation, “effector” M1 type macrophages); macrophages that are instructed to promote wound healing (alternative activation, “repair” M2 type macrophages); or macrophages that are instructed to secrete anti-inflammatory cytokines that terminate inflammation (alternative activation, “regulatory” M2 type macrophages). These populations of macrophages are unique in their physiology and express certain sets of MMPs.

Effector macrophages are generally induced by a combination of two signals. The priming signal, a cytokine such as interferon γ (IFN-γ), is usually provided by immune cells including natural killer cells (NK) or Type-1 helper cells (Th1). The secondary stimulus is usually provided by toll-like receptor activation. Classically activated macrophages play an important defensive role in several diseases. They are characterized by their production of nitric oxide (NO), tumor necrosis factor (TNF), IL-6, MMP-1, and MMP-3, and typically show a significant down-regulation of MMP-7, -9, -14, -21, and -25 (Bar-Or et al., 2003). Notably, NO production by macrophages directly influences MMP function via chemical modification and expression regulation. MMP-9 activity was shown to be up-regulated in the presence of exogenous NO and to mediate biological effects such as increased vascular cell migration during wound healing (Ridnour et al., 2007). Several MMPs can mediate anti-bacterial functions of macrophages. For instance, an intracellular pool of MMP-12 is translocated to phagolysosomes after ingestion of bacterial pathogens (Houghton et al., 2009). The microbicidal effect of MMP-12 is due to a unique peptide sequence located at the carboxy-terminal hemopexin domain, which does not require proteolytic activity of MMP-12.

Alternative activation of macrophages can be achieved by exposure to IL-4 and IL-13. They differ from classically activated macrophages in that they show increased expression of mannose receptor and alternated arginine metabolism, which is necessary for their promotion of wound healing and the regulation of humoral immunity. MMP-12 expression significantly increases in vitro in IL-4-activated macrophages, and can serve as an in vivo marker for alternatively activated macrophages during late stages of tuberculosis (Kahnert et al., 2006). Furthermore, alternative activation of macrophages with macrophage colony stimulating factor (M-CSF) triggers MMP-2 and MMP-9 expression. This up-regulation is a prominent feature observed in lung tissue from patients suffering from the autoimmune disease pulmonary alveolar proteinosis (PAP; Bonfield et al., 2006). In a manner similar to what is observed with effector macrophages, expression of MMP-7 is inhibited by IL-4 receptor activation (Busiek et al., 1995).

The population of regulatory macrophages is mainly characterized by the production of IL-10, a key cytokine with anti-inflammatory function. There are several pathways that lead to the generation of regulatory macrophages, which all seem to require a “two-signal” activation. In contrast to classical activation of macrophages, the priming (e.g., IL-10, prostaglandins, immune complexes, adenine nucleotides, apoptotic cells, glucocorticoids) and stimulation signals (e.g., TLR ligands) usually occur simultaneously. These stimulants have effects on MMP expression and activity in macrophages. For instance, MMP-9 activity significantly down-regulated in monocytes/macrophages upon IL-10 activation (Mostafa Mtairag et al., 2001).

The major MMPs expressed by PMNs are MMP-8 and MMP-9. Upon activation, PMNs release their MMP-containing granules into the extracellular spaces, where these MMPs potently alter the microenvironment. For instance, MMP-9 degrades the important serine protease inhibitor, α1-proteinase inhibitor (α1-PI), and thereby prolongs the proteolytic activity of neutrophil elastase during skin blister formation (Liu et al., 2000). Interestingly MMP-9 delivered by PMN may be especially active, since PMN-derived proMMP-9 is not bound by TIMP-1 and can therefore be more easily activated (Ardi et al., 2007). Hence, MMP-9 from PMNs might be more potent in regard to its proteolytic activity, and thus have enhanced capacity.

Much of the current knowledge about MMP function has been learned through the availability of mice that are null mutants for various MMPs. Indeed, the dominant phenotypes for several MMPs are due to the loss of function in PMNs as well as in macrophages and their relatives such as osteoclasts. For example the loss of MMP-8 results in excess inflammation, leading to increased cancer incidence (Gutierrez-Fernandez et al., 2007), and the loss of MMP-9 alters endochondral bone formation because the osteoclasts are unable to degrade the hypertrophic cartilage matrix and mount an effective angiogenic response (Ortega et al., 2010).

Isolation of murine macrophages or PMNs

Most methods to monitor MMP activity are in vitro assays. The first step in analyzing inflammatory cell-derived MMPs is therefore to harvest these cells. Due to their wide functional spectrum with roles in immune defense, immunomodulation, and tissue homeostasis, it is inevitable for macrophages to be widely distributed throughout the whole body. They dramatically change their physiology and expression profile depending on the situation, as well as the tissue that they reside in. Hence, the standardized isolation and characterization represents a critical step in the study of macrophage biology. In UNIT 14.1, Zhang and colleagues provide helpful procedures as how to purify murine macrophages.

In contrast, PMNs are predominantly located in the bone marrow or circulating in the blood stream, which makes them easier to purify. PMNs can also easily be isolated from an inflammatory environment, for example, by peritoneal lavage after induced peritonitis. More details about the isolation of PMNs are given in UNIT 3.20.

Studying MMP activity

The multigene family of MMPs has been traditionally categorized according to substrate specificity, i.e., collagenases, gelatinases, stromelysins, matrilysins, and elastases. Although a classification based on structural aspects is in many ways more useful and appropriate, it is helpful to detect their enzymatic activity by methods such as the zymography technique (Table 14.24.1). Zymography makes use of the proteins’ electrophoretic mobility to separate them and visualize their proteolytic activity. Fortunately, zymography not only shows all the MMPs that can degrade a given substrate class, but also gives information on the estimated molecular weight of an MMP and its various forms. It differs from conventional SDS-polyacrylamide gel electrophoresis in that the gels have the putative substrate incorporated, the samples are mixed in a higher concentration of SDS (without reducing agents), and the samples are not boiled. The elegance of the technique lies in the circumvention of two problems frequently encountered when characterizing MMPs: first, many proenzymes are active when assayed in this manner, thus eliminating the requirement for activation; and second, electrophoresis separates the MMPs from noncovalent proteinase-inhibitor complexes, which would otherwise significantly reduce the activity of MMPs.

It is important to note that MMPs may differ between species in terms of substrate specificity and apparent molecular weights. For example, MMP-9 migrates at apparent molecular weights of 105 and 95 kD for the mouse proenzyme and active enzyme, respectively, while the human counterparts migrate at 92 and 82 kD. Moreover, full-length active enzymes may have different substrate specificities or activities compared to catalytic-domain fragments, which are often generated by autoproteolysis. In this context, human MMP-12 is less elastinolytic than the mouse enzyme, and the 21-kD catalytic domain is much more elastinolytic than the full-length enzyme. Thus, it is important to use species-specific positive controls. For mouse, the best controls are samples isolated from MMP null animals.

Critical Parameters and Troubleshooting

Zymography

The sensitivity is too low

By decreasing the substrate concentration, the sensitivity of the assay can be increased. However, less substrate in the gel will also increase the apparent staining of sample proteins and can therefore cause detection problems. A useful tool under this circumstance is the use of overlay assays, i.e., SDS-PAGE gels are overlaid with membranes impregnated with flurogenic peptides, such as Glutaryl-Ala-Ala-Ala-AFC for the fluorescent detection of elastolytic enzymes. An alternative is the use of the real-time zymography technique. Digestion of a fluorescent protein is directly visualized in the developing buffer using a UV transilluminator with areas of digestion visible as black, nonfluorescent bands on a fluorescent background. Fluorescent substrates include FITC-gelatin and FITC-casein, which have been used for analyzing MMP-2, -3, and -9 (Hattori et al., 2002).

Digestion zones are smeared

Smears indicate that proteolytic activity was too high. If these are observed, either use shorter incubation time, or less enzyme or supernatant, for the assay.

No visible digestion zones

Use appropriate amounts of MMPs that are known to digest the respective substrate as positive controls. This makes it possible to determine whether the sample’s enzyme concentration is too low or whether the experimental setup contains flaws, such as wrong buffer concentrations. Check that the buffer did not contain EDTA, and that the renaturing solution contains the right amount of Triton X-100. Make sure that the sample was not boiled.

Fluorogenic substrates

No increase of fluorescence after addition of enzyme

The activity of MMPs depends on a variety of steps and conditions. MMPs are generally expressed as inactive zymogens, which require proteolytic removal of the pro-peptide to gain enzymatic activity. As described above, this step can be replaced by chemical activation, for example, using APMA. Moreover, the buffer conditions are crucial for MMP activity. For instance, sufficient Ca²⁺ (~10 mM) should be included in the buffer. Furthermore, there is a set of physiological inhibitors of MMP activity, such as the tissue inhibitors of metalloproteinases (TIMPs). Especially when working with samples from macrophages or PMNs, the presence of inhibitors has to be considered, as these may interfere with the hydrolysis of the substrate.

Background fluorescence values are too high

The preparation of fresh substrate solution can be a critical point, as fluorogenic substrates can lose their functionality over time. Fluorogenic substrates are usually light sensitive. Therefore, incubations should be carried out in the dark. The appropriate controls, such as substrate solution without added enzyme, and reaction buffer alone, should always be included in the assay design.

Time Considerations

Sample preparation takes 5 to 15 min, depending on whether conditioned media or cell suspensions are used. The preparation and loading of the gel requires 3 to 4 hr. Gels take 1 to 1.5 hr to run. The renaturing and washing of the gels takes 1.5 hr. The proteolytic digestion of the substrate varies between 2 and 24 hr, depending on the sensitivity required. Although there are variations, staining usually takes 30 min and destaining takes 2 hr.