Materials and Methods

Isolation of bovine granulocytes and production of conditioned media—Samples of whole blood were obtained from 20 healthy cows (450 mL each) by jugular venipuncture into 450-mL containers containing an anticoagulant (citrate dextrose solution; anticoagulant-to-blood ratio of 1:9). Blood samples were centrifuged at 1,000 X g for 20 minutes, and the plasma was removed. The remaining buffy coat that contained residual RBCs was treated with sterile distilled water for 30 seconds to lyse the RBCs.³⁵ Tonicity was restored by addition of 1 volume of saline (2.7% NaCl) solution containing 10mM phosphate buffer and 2% bovine serum albumin and 2 volumes of distilled water.³⁵ The suspension was diluted in 50-mL tubes with PBS solution and recentrifuged at 400 X g for 8 minutes. Cell pellets were washed in PBS solution, and any remaining RBCs were removed by an additional cycle of hypotonic lysis.³⁵

White blood cells were counted, and a differential WBC count was performed to determine the percentage of granulocytes. On average, the isolation procedure resulted in approximately 2 × 10⁹ viable cells, of which approximately 40% were granulocytes. Cell pellets were resuspended in 200 mL of Dulbecco modified Eagle medium^a without fetal bovine serum and stimulated with phorbol 12-myristate 13-acetate (20 ng/mL).^b Cells were incubated at 37°C in a water bath for 30 minutes and periodically stirred. Afterward, the cells were centrifuged, and the conditioned medium was harvested and buffered with 20mM Tris-HCl buffer (pH, 7.5) and stored frozen (–70°C) until protein purification was performed. Because bovine mononuclear cells and alveolar macrophages cultured in vitro require induction of MMP-9 gene expression, which happens after several hours of incubation,³⁶ the potential contribution of monocytes and lymphocytes in 30-minute incubations was ignored. For preparative purification of MMP-9 molecular species, several aliquots of conditioned medium (up to 4,000 mL) were combined.

Purification of molecular species of MMP-9 released by granulocytes—Purification of MMP-9 was based on a method described elsewhere,^12,17 with addition of a gel filtration step. Serum-free conditioned medium^a from phorbol myristate acetate–stimulated bovine granulocytes buffered with Tris-HCl buffer (pH, 7.5) was applied to a reactive red-120 agarose column^b (1 mL of resin/100 mL of conditioned medium) equilibrated with 20mM Tris-HCl buffer (pH, 7.5) that contained 150mM NaCl and 2mM CaCl₂. After the column was washed with the same buffer, bound proteins were eluted with 20mM Tris-HCl buffer (pH, 7.5) that contained 2M NaCl and 2mM CaCl₂. The material eluted from the column was dialyzed against 20mM Tris-HCl buffer (pH, 7.5) that contained 0.5M NaCl, 2mM CaCl₂, and a nonionic surfactant^c and applied on a gelatin-agarose^b column (1 to 3 mL).

After washing, the gelatin-agarose column was eluted with 20 volumes of a 0% to 10% DMSO gradient. Elution was monitored by UV absorption at a wavelength of 280 nm, and protein-containing peaks were collected, concentrated,^d and analyzed by gradient zymography, western blot, and protein staining.¹⁷ Total yield of DMSO-eluted material was approximately 100 μg of protein/L of conditioned medium. For final purification, concentrated protein peaks were subjected to gel filtration on a 1 × 95-cm column of size exclusion chromatography sorbent^e equilibrated with 20mM Tris-HCl buffer that contained 200mM NaCl, 2mM CaCl₂, and nonionic surfactant.^e Elution was monitored by UV absorption, and proteins eluting as peaks were collected, concentrated, and stored in 5mM Tris-HCl buffer (pH, 7.5), 0.5mM CaCl₂, and nonionic surfactant^e at −80°C.

Activation of MMP-9—Bovine MMP-9 molecular species were activated as described elsewhere¹⁷ with activated stromelysin^f in stromelysin-to-MMP-9 molar ratios of 1:15 to 1:30 for 3 to 8 hours at 37°C. Stromelysin activation was achieved by incubation with trypsin treated with tosyl phenylalanyl chloromethyl ketone^g at a trypsin-to-stromelysin molar ratio of 1:15 for 20 to 40 minutes at room temperature (23°C). Trypsin activation was stopped by addition of a 4-fold molar excess of soybean trypsin inhibitor.^b The activation process (proforms no longer detected but N-terminal truncated active species detected) was monitored by means of SDS-PAGE, western plot silver staining, and zymography.

Assays of enzymatic activity of activated MMP-9—Enzymatic activity of stromelysin-activated bovine MMP-9 was measured by means of 2 methods, with gelatin fluorescein conjugate^h as a high–molecular-weight substrate or synthetic fluorogenic peptide^i,j as a low–molecular-weight substrate. Both assays were performed in 50mM Tris-HCl buffer (pH, 7.6) containing 150mM NaCl and 5mM CaCl₂.

The gelatin conjugate assay, which involves gelatin that is so heavily labeled with fluorescein that fluorescence is quenched until cleavage occurs, was performed (MMP-9 concentration range, 0.188 to 3nM; substrate concentration range, 7.5 to 65 μg/mL) between 0 and 90 minutes at room temperature, whereby enzymatic activity (as measured by increasing fluorescence) progressed in a linear fashion. Fluorescence was measured with a microplate reader set for excitation at 495 nm and emission detection at 530 nm. For each experimental point, values for spontaneous degradation of the gelatin fluorescein conjugate (sample containing no enzyme) were subtracted.

The fluorogenic peptide substrate assay was performed with a microplate reader set for excitation at 328 nm and emission detection at 393 nm. Within the ranges of MMP-9 concentration (0.188 to 3nM), substrate concentration (5 to 25μM), and time (0 to 90 minutes), the enzymatic activity yielded linear increases in fluorescence. Fluorogenic coumarin–labeled peptide was stored at −20°C as 1mM stock in DMSO solution. A calibration curve consisting of a reference compound was used for determination of concentration of product of reaction of MMP-9 with the fluorogenic substrate. For each experimental point, values for spontaneous degradation of fluorogenic substrate were subtracted.

Assay of inhibitors of MMP-9 activity—The following stock solutions of MMP inhibitors were used: tetracycline, minocycline, and doxycycline^b (10mM in ethanol), chlorhexidine^k (30mM in DMSO), CGS 27023A^l (1mM in 1:1 acetonitrile and methanol), HMR 1035^m (1mM in water), and gelatinase-specific inhibitor SB-3CTⁿ (10mM in methanol). Reactions were initiated by addition of gelatin fluorescein conjugate or peptide substrate to activated enzyme (3nM) that had been incubated with plain buffer or individual inhibitors at room temperature, and the reaction progression was monitored. For inhibition assays involving peptide substrate, substrate concentrations of 5μM and 15μM were used in each experiment to determine the IC₅₀, K_M, and K_I. For the assay of each inhibitor, incubations were set up in triplicate.

Data analysis—Inhibition curves and calculations of IC₅₀, K_I, and K_M for reactions involving gelatin fluorescein conjugate^o and fluorogenic peptide substrate^p were performed by use of commercial software. Values for IC₅₀ were estimated by means of nonlinear regression analysis.

Results

Purification and activation of MMP-9 monomer and dimer—The combination of gelatin-agarose affinity and gel-filtration columns allowed separation of MMP-9 monomer and dimer on the basis of differences in their affinity to gelatin and in molecule sizes. Activation of bovine neutrophil MMP-9 monomer and dimer by tolyl sulfonyl phenylalanyl chloromethyl ketonetrypsin–activated stromelysin was verified by silver staining, zymography, and western blotting of purified, activated proteins. Characteristic shift in the migration pattern of MMP-9 after activation was detected prior to use of specific aliquots of MMP-9 in inhibition assays.

Calibration curves—Degradation of gelatin fluorescein conjugate by bovine MMP-9 progressed in a linear manner with respect to concentration of MMP-9 and time of incubation when the concentration of the conjugate was 12.5 μg/mL (Figure 1). The relationship between conjugate concentration and relative fluorescence intensity was also linear for the range of substrate concentrations evaluated (7.5 to 65 μg/mL).

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Calibration curves for enzymatic activities of bovine MMP-9 monomer (black circles) and dimer (white circles) at various concentrations of MMP-9 (A), various time points (B), and various concentrations of gelatin fluorescein conjugate (C). Similar results were obtained when fluorogenic peptide was used as a substrate in identical reaction conditions.

Citation: American Journal of Veterinary Research 70, 5; 10.2460/ajvr.70.5.633

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The enzymatic activity of MMP-9 when fluorogenic peptide substrate was used was also linear throughout the range of substrate concentrations evaluated (5 to 25μM) and as time increased (data not shown). When the enzymatic activities of activated MMP-9 monomer and dimer were compared with respect to the same properties, activities appeared similar (data not shown). When the enzyme was not activated prior to exposure to either substrate, the production of fluorescent product was no different than that produced in the absence of enzyme.

Assays of inhibitors—Curves representing the inhibition of bovine MMP-9 by various inhibitors were constructed (Figure 2). When gelatin fluorescein conjugate was used for quantitation of enzymatic activity, there were substantial differences among classes of inhibitors with respect to values of IC₅₀ (Table 1). The tetracyclines (tetracycline, doxycycline, and minocycline) inhibited MMP-9 activity at micromolar concentrations, whereas the hydroxamic acids (HMR 1035 and CGS 27023A) were inhibited MMP-9 activity at nanomolar concentrations. The inhibitor with the least potential for MMP-9 inhibition (greatest IC₅₀) was chlorhexidine.

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Mean ± SD relative fluorescence intensity indicating the activity of MMP-9 when incubated with various concentrations of CGS 27023A (A) and tetracycline (B) with gelatin fluorescein conjugate as the substrate and of HMR 1035 (C) and SB-3CT (gelatinase-specific inhibitor; D) with fluorogenic peptide as the substrate. For all reactions, concentrations of MMP-9 (3nM) were identical in each reaction well. The concentration of inhibitor varied by class of compound and was tested with 12.5 μg of substrate/mL.

Citation: American Journal of Veterinary Research 70, 5; 10.2460/ajvr.70.5.633

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When fluorogenic peptide substrate was used for quantitation of enzymatic activity, similar differences were detected in potency of the various MMP-9 inhibitors. The hydroxamic acids inhibited enzymatic activity of MMP-9 in low nanomolar concentrations (Table 2). The gelatinase-specific inhibitor (SB-3CT) strongly inhibited enzymatic activity of MMP-9, although it was a less potent inhibitor than the hydroxamic acids (Figure 2). There were few or no differences in abilities of the various inhibitors to inhibit enzymatic activities of monomeric versus dimeric forms of MMP-9, regardless of the type of substrate used for quantitation.

Discussion

In the study reported here, the analytic sensitivity of bovine MMP-9 to various compounds that reportedly inhibit MMPs was evaluated. For practical purposes, the compounds were chosen on the basis of their clinical use in animals (including cattle) for various disorders. We evaluated 2 hydroxamic acid MMP inhibitors of low molecular weight (HMR 1035 and CGS 27023A) because our experience indicated that these compounds inhibit bovine MMP-9 in in vitro zymographic assays, they are not antimicrobial agents and consequently will not alter susceptibility of microbes to available antimicrobials, and they are highly bioavailable when administered orally in dogs, rabbits, and humans.^29,30 We also evaluated the inhibitory effects of tetracyclines (tetracycline, doxycycline, and minocycline) because they reportedly inhibit MMPs in various inflammatory conditions.^31,32 Chlorhexidine, a disinfectant commonly used in human and veterinary medicine, was chosen for evaluation on the basis of a report³³ that it inhibits gelatinases and collagenases. Finally, the activity of a gelatinase-specific inhibitor, 3B-SCT, was chosen because it is an irreversible inhibitor and is specific for MMP-2 and MMP-9.³⁴ The activity of purified bovine MMP-9 in the presence of the aforementioned inhibitors was measured by use of the macromolecular substrate of this protease (gelatin) and a synthetic peptide substrate.

The results of the present study may be valuable for evaluating pharmacokinetics, bioavailability, and usefulness of the MMP inhibitors as a means of reducing or modifying the impact of protease-mediated inflammation. Inhibition of proteolytic activity associated with neutrophils and, specifically, enzymatic activity of MMP-9 in vivo is a promising approach for the evaluation and regulation of inflammation attributable to neutrophilic infiltrates. An important prerequisite for in vivo testing is measurement of the kinetics of inhibition of purified native (not partially denatured, as in zymography) bovine neutrophil MMP-9 by relevant inhibitors that are easily administered, are inexpensive, do not alter the microbial flora, and have limited potential for tissue residues. Evaluation of the kinetics of inhibition of human MMP-9 by some inhibitors has been performed,^34,37,38 but the resulting data may not be directly applicable to the bovine enzyme because of species-related differences in enzyme properties and because comparisons between the activity of recombinant human MMP-9 and native bovine neutrophil MMP-9 may not be fully appropriate.

Matrix metalloproteinase 9 is able to cleave several substrates with relevance to the modulation of inflammation.^13–16 In the present study, we used 2 widely used model substrates. Gelatin, or denatured type I collagen, is the macromolecular substrate most commonly used for evaluation of the enzymatic activity of MMP-9. With gelatin as a substrate, we were able to estimate the IC₅₀s of various MMP inhibitors, which is a critical variable in the assessment of inhibitor potency. Lack of reliable data on the number of cleavage sites in the gelatin molecule hindered determination of the K_I when gelatin was used as a substrate. The fluorogenic peptide substrate is also commonly used in the evaluation of MMP enzyme kinetics and inhibition by low–molecular-weight inhibitors.^34,37,38 This peptide has the advantage of having only 1 cleavage site, thus allowing determination of K_I and comparisons with findings of other studies.

The ability of the chosen compounds to inhibit MMP-9 varied by chemical class. The potencies of the various inhibitors evaluated, in order of increasing potency, were as follows: chlorhexidine, tetracyclines, gelatinase-specific inhibitor, and hydroxamic acid inhibitors. The antibiotics tetracycline, doxycycline, and minocycline reportedly inhibit the activity of MMP.^39–42 When fluorogenic peptide is used as a substrate, the concentrations of tetracycline and doxycycline that reportedly reduce the activity of human MMP-2 and MMP-8 by 50% (IC₅₀) are 220μM and 24μM, respectively.^39–42 In the study reported here, regardless of the substrate used, values of IC₅₀ for the tetracyclines were similar to these human data. The lack of a difference in values attributable to the nature of the substrate used suggested that the tetracyclines inhibit MMP-9 via an active site of the MMP-9 molecule, independently of other interactions between substrate and MMP-9 (eg, gelatin-binding modules or hemopexin-like C-terminal domain).^43,44

Chlorhexidine rapidly forms insoluble complexes with cations.³³ The addition of calcium ions to the incubation medium containing gelatinases, collagenases, and chlorhexidine in another study³³ revealed the ability of calcium to reduce the inhibition of bovine MMP-9, suggesting that chlorhexidine acts via chelation of calcium from the medium and destabilization of the active site of MMP-9. The results reported here agree with the hypothesis that chelation of cations is a major action of chlorhexidine. In the other study,³³ addition of a range of chlorhexidine concentrations (0.001% to 0.5%) to collagenase (MMP-8)-containing buffer led to clouding of the reaction mixture, suggesting formation of insoluble chlorhexidine-calcium salts. In the study reported here, the concentration of chlorhexidine-inhibited enzymatic activity of MMP-9 in the presence of gelatin fluorescein conjugate was approximately 140μM (0.008%), whereas that required to inhibit the activity of MMP-9 in the presence of fluorogenic peptide was 0.6 to 1.6mM (0.03% to 0.08%). Such concentrations are reportedly high enough to prevent specific inhibitory action.³³

Results regarding the inhibition of bovine MMP-9 by hydroxamic acids indicated that this class of inhibitors was highly potent and effectively reduced enzymatic activity in the low nanomolar range. Findings generally corresponded with those regarding human MMPs,^29,30 with bovine MMP-9 approximately 10 times as sensitive to inhibition by CGS 27023A as is recombinant human MMP-9 when fluorogenic peptides are used in vitro.²⁹ It is also important to mention that the IC₅₀s obtained for the hydroxamic acids in our study were 50- to 100-fold lower than the corresponding values of tetracyclines and chlorohexidine when peptide was used as a substrate and nearly 1,000-fold lower when gelatin fluorescein conjugate was used as a substrate.

Results for the hydroxamate inhibitors differed from those of the tetracyclines and chlorhexidine in that the IC₅₀s obtained with the peptide substrate were more than 10-fold higher than the IC₅₀s for the gelatin substrate. This suggested that binding of the enzyme to gelatin (gelatin-binding modules) or other molecular interactions of MMP-9 with a high–molecular-weight substrate are important aspects of enzyme inhibition. A high inhibitory potency of hydroxamates with respect to bovine MMP-9 is important because these drugs are highly bioavailable orally in monogastric species. In humans, dogs, and lagomorphs, systemic availability exceeds 50% (nearly 100% in dogs) after oral administration,^29,30 suggesting these compounds may be useful in the inhibition of bovine MMP-9.

The MMP inhibitors HMR 1035 and CGS 27023A are broad-spectrum inhibitors because they interact with the zinc atom of the active site, resulting in inhibition of several MMPs and other metalloenzymes.^29,30 Lack of selectivity toward a specific enzyme limits their in vivo use in humans because of adverse reactions associated with widespread inhibition of proteases. There is interest in developing inhibitors with selectivity for individual proteases. The gelatinase-specific inhibitors (SB-3CT in the present study) have the advantage that their inhibitory potential is limited primarily to gelatinases (MMP-2 and MMP-9).^34,37 Gelatinase-specific SB-3CT, in contrast to nonselective MMP inhibitors, has been evaluated in animals with cancer, with promising results.⁴⁵ Studies^34,37 involving gelatinase-specific inhibitors revealed that they function to inhibit enzymatic activity of human MMP-9, with K_Is ranging from 100 to 600nM; results for bovine MMP-9 in the present study indicated that bovine MMP-9 is apparently more sensitive to inhibition (IC₅₀, 185 to 290nM; K_I, 60 to 80nM) by this type of inhibitor.

The MMPs, specifically neutrophil MMP-9, are important in the early stages of inflammation associated with respiratory disease in cattle.^1–5 Because these proteases are stored preformed in neutrophil granules, inhibition of proteolytic activity as moderated by bovine polymorphonuclear neutrophilic leukocytes may be beneficial clinically through modulation of acute phase inflammation associated with microbial pneumonia. Because bovine neutrophils factor early in the lung response to microbial-induced inflammation, their contribution should be substantial. As inflammation becomes sustained, contributions from other cell types (such as macrophages) may contribute to clearance of debris or exacerbation of lung injury after sufficient time for MMP-9 gene expression. The results reported here narrow the list of low–molecular-weight compounds with properties suitable for additional evaluation. The tetracyclines and chlorhexidine do not appear to be clinically useful in this regard, whereas hydroxamic acid–derived inhibitors and the gelatinase-specific inhibitors appear promising.