ARCHIVES
OF BIOCHEMISTRY
Vol. 292, No. 2, February
AND
BIOPHYSICS
1, pp. 555-562, 1992
Different Effects of Hypochlorous Acid on Human Neutrophil Metalloproteinases: Activation of Collagenase and Inactivation of Collagenase and Gelatinase Jiirgen Michaelis,’ Department
Mamgret C. Vissers, and Christine
of Pathology, Christchurch
School of Medicine,
C. Winterbourn
Christchurch,
New Zealand
Received June 13,1991, and in revised form October 7,199l
Human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) produce the reactive oxidant hypochlorous acid (HOCl) and release the matrix metalloproteinases collagenase and gelatinase from secretory granules. We have investigated the stoichiometry of activation and inactivation of the two metalloproteinases with HOCl. HOC1 activated purified neutrophil procollagenase at ratios between 10 and 40 mol of HOCl/mol enzyme, but caused inac:tivation at higher ratios. Maximum activation was about the same as that achieved by p-aminophenyl-mercuric acetate. However, less than a third of the total collagenase released from PMA-stimulated neutrophils was activated by coreleased HOC1 and most of the activity was destroyed after 1 h of stimulation. These results indicate that the HOCl/enzyme ratio must fall within a narrow range for activation to occur. In contrast to collagenase, purified progelatinase underwent negligible activation (2.5 + 1.2%) at HOCl/enzyme molar ratios < 30 and ‘was destroyed at higher ratios. Likewise no active gelatinase could be detected in supernatant from PMA-st.imulated cells and almost all of the proenzyme was destroyed by HOC1 after 60 min stimulation. Our results; illustrate that only collagenase can be activated by HOC1 in vitro and that gelatinase is much more sensitive to inactivation. Since a precise HOCl/enzyme ratio is required for collagenase activation it is doubtful whether effective enzyme regulation by HOC1 could occur in vivo where various HOCL scavengers are present. 0 1992 Academic Prees, Inc.
Human neutrophils contain the two metalloproteinases collagenase, which degrades native collagens types I, II, ’ TO whom correspondence should be addressed at present address: Peptide Technology Ltd., CSIRO Division of Biomolecular Engineering, P.O. Box 184, North Ryde, Sydney 2113, Australia. 0003.9861/92 $3.00 Copyright 0 1992 by Academic Pxess, All rights of reproduction in any form
and III (1, 2), and gelatinase, which has a greater specificity for native types IV and V collagen and for denatured collagens (3, 4). Both enzymes are thought to play a key role in extracellular matrix degradation by neutrophils (5). Collagenase and gelatinase are present as latent proenzymes and are released as such by stimulated neutrophils. The mechanism by which they are activated is unknown. In vitro studies have shown that both enzymes can be activated by incubation with mercurial compounds (4, 6, 7) or with gold compounds (8). Since this is an artificial reaction not related to normal conditions in vivo, activation of metalloproteinases by limited proteolysis by neutrophil serine proteases such as cathepsin G for collagenase (9) and elastase for gelatinase (10) has been proposed. These observations have been documented with sequence data of the activation site within neutrophil collagenase (9). Another pathway that could be physiologically important is activation by hypochlorous acid (HOC1)2 produced by neutrophils. Neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA) or the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) produce superoxide (0;) and hydrogen peroxide (H,O,) which is consumed by myeloperoxidase coreleased from azurophi1 granules to form HOC1 (11): myeloperoxidase
Hz02 + Cl- + H+ -
HOC1 + HzO.
* Abbreviations used: HOCl, hypochlorous acid; PMA, phorbol 12myristate 13-acetate; fMLP, fMet-Leu-Phe; APMA, p-aminophenyl mercuric acid, PMSF, phenylmethylsulfonyl fluoride; TPCK, L-l-p-tosylamino-2-phenylethyl chloromethyl ketone; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide. 555
Inc. reserved.
556
MICHAELIS,
VISSERS,
Weiss and colleagues first reported activation of neutrophil collagenase (12) and gelatinase (13) by HOCl. Collagenase activation has also been described by others (14) but there appear to be some discrepancies with these findings, since several groups have isolated and purified latent collagenase and gelatinase from PMA-stimulated neutrophils (4, 7). Furthermore, HOC1 is highly reactive with a wide range of biological molecules (15) including proteins (16) and Vissers and Winterbourn (17) have demonstrated that neutrophil proteases are inactivated by a myeloperoxidase-dependent oxidation pathway. In the studies on oxidant activation, the stoichiometric requirement of HOC1 has not been investigated. Similarly, the effect of HOC1 on the protein structure has not been determined. We have, therefore, examined the effect of HOC1 on purified collagenase and gelatinase and in cell supernatants from stimulated neutrophils. We demonstrate the exact stoichiometry necessary for collagenase activation and for inactivation of both collagenase and gelatinase. EXPERIMENTAL
AND
WINTERBOURN
Purification
of Neutrophil
Metalloproteinases
Collagenase was purified from homogenized neutrophils as described previously (6). Gelatinase was purified from homogenized nzutrophils in the presence of PMSF and benzamidine, by successive co!umn chromatography steps using DEAE-Sepharose, Zn2+ chelate-Sepharose (22, 23), Cibacron blue-Sepharose (24), and gelatin-Sepharose (4).
Reaction of Collagenase and Gelatinase with HOC1 Purified collagenase and gelatinase in PBS-Ca were exposed to HOC1 at known molar ratios for 15 min at 37°C unless otherwise stated. The molarity of HOC1 was assessed by its reaction with monochlorodimedon in PBS, monitoring the decrease in monochlorodimedon absorption at 290 nm (Ezw = 19,000 M-’ cm-‘) (25). The effects of HOC1 were related to the molar ratio rather than HOC1 concentration because HOC1 reacts rapidly with biomolecules and at the concentrations used in this study would all be consumed within minutes (26).
Activation
of Collagenase and Gelatinase by APMA
The latent metalloproteinases in neutrophil supernatants or as purified enzymes were activated by incubating at 37°C for 30 min with 0.5 mM APMA (collagenase) or for 1 h with 1 mM APMA (gelatinase). APMA was dissolved at 10 mM in 50 mM NaOH and the pH adjusted to 7.4 pH 7.0, and 2 mM CaCl, prior to use. with 1000 mM Tris/HCl,
PROCEDURES
Materials Ficoll-Hypaque was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Phorbol12-myristate 13-acetate, N-formyl-Met-Leu-Phe, cytochalasin B, p-aminophenyl mercuric acetate (APMA), phenylmethylsulfonyl fluoride (PMSF), monochlorodimedon, type I calf skin collagen, and goat anti-rabbit antibody alkaline phosphatase conjugate were from Sigma Chemical Co., St. Louis, Missouri. Hypochlorous acid was from Reckitt & Colman, Auckland, New Zealand. TPCK-trypsin was purchased from Worthington, Freehold, New Jersey; al-antitrypsin was purified from human plasma by thiol-disulfide interchange (18). All other chemicals were reagent grade. The Centricon 30 microconcentrator was purchased from Amicon, Danvers, Massachusetts; and the Microsep microconcentrator 100 from Filtron Technology, Northborough, Massachusetts. Polyclonal antibodies against purified gelatinase were raised in a rabbit and purified by ammonium sulfate precipitation and ionexchange chromatography on DEAE-Sepharose as described previously (19, 20).
Methods Human neutrophils were prepared from the peripheral blood of healthy donors by centrifugation through Ficoll-Hypaque, dextran sedimentation, and hypotonic lysis of contaminating red cells (21). The cells were suspended at 107/ml in 10 mM sodium phosphate buffer, pH 7.4, and 150 mM NaCl (PBS), containing 1 mM CaCl, (PBS-Ca), 0.5 mM MgCl,, and 1 mg/ml glucose. For stimulation with PMA (0.1 pg/ml) 2 pl PMA (stock 500 pg/ml in dimethyl sulfoxide [Me,SO]) was added to 10 ml of cell suspension. Alternatively the cells were incubated for 2 min with 5 rg/ml cytochalasin B prior to stimulation with lo-’ M fMLP (stock solution in PBS). When appropriate the neutrophils were stimulated in the presence of 1 mM phenylmethylsulfonyl fluoride (added 2 min after addition of the stimulus), 1 mM methionine, or 3% (w/v) human serum albumin (added before the stimulus). After 10. 30, and 60 min at 37°C the neutrobhils were pelleted by ce&ifugation (ll!lOOg) for 5 min and the supernatant was removed. PMSF (2 mM), a,-antitrypsin (5 pg/ ml), and NaN, (1 mM) were added to prevent further proteolysis or inactivation of the metalloenzymes and the supernatant was stored at -80°C.
Activation
of Gelatinase by Trypsin
Neutrophil supernatant was dialyzed against PBS-Ca by spinning twice through a Centricon 30 microconcentrator (M, 30000 cutoff membrane) to eliminate PMSF from the sample and was finally concentrated 30-fold. TPCK-trypsin (2 @g/30 pl neutrophil supernatant) was added and the solution was incubated for 10 min at 37°C. Purified gelatinase (2 fig in 20 /Al PBS-Ca) was activated by 0.5 pg TPCK-trypsin at 37°C for 10 min. The reaction was terminated by the addition of a lo-fold excess of soya bean trypsin inhibitor. Conversion to the cleaved, active form was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.
Enzyme Assays Collagenuse. Collagenase activity was assessed by specific cleavage of soluble type I calf skin collagen. Collagen (70 pg) was incubated at 25°C with either (i) 0.2 pg purified collagenase in 62 ~1 buffer A (100 mM Tris/HCl, pH 7.5, 100 mM NaCl, 2 mM CaCl,, 0.05 mM ZnCl,, 0.1 mM NaN,) for 200 min or (ii) 18 ~1 cell-free medium from stimulated neutrophils in 32 ~1 buffer A for 18 h. Appearance of OI*and /3* collagen fragments was monitored by SDS-PAGE. Gelatinme. Degradation of radiolabeled gelatin was measured essentially as described earlier (4). Type I collagen from rat skin was labeled with [l-‘4C]acetic anhydride (27) and diluted with unlabeled collagen to approximately 200,000 cpm/mg. [“C]Collagen was denatured at 90°C for 15 min and incubated for 200 min at 37°C with either 20 ~1 cell supernatant or 2 pg purified gelatinase in a final volume of 150 ~1. The reaction was stopped by adding trichloroacetic acid to a final concentration of 15% (w/v) and cooling to 4°C. Solubilized radioactivity was determined after centrifugation at SOOOgfor 5 min. Maximum radioactivity was measured in a sample that was not pelleted.
Electrophoresis
and Immunoblotting
SDS-PAGE was performed in a continuous slab gel system (28). Gels were stained with picrate-Coomassie blue (29). Western blots were performed using rabbit anti-neutrophil gelatinase antibody as the first antibody and goat anti-rabbit antibody alkaline phosphatase conjugate as the second antibody (6).
EFFECTS
OF HYPOCHLOROUS
ACID
ON NEUTROPHIL
557
METALLOPROTEINASES
Protein Determination Protein was assayed using the method previously described (30). To account for the high carbohydrate portion of neutrophil collagenase a M. 53000 (9) for the unglycosylated enzyme was used to calculate the molarity. M, 95000 was used for purified neutrophil gelatinase, as this was the predominant form visible on SDS-PAGE under nonreducing conditions (data not shown).
RESULTS
Effect of HOC1 on Collagenase Released from Stimulated Neutrophils To determine the eff’ects of oxidants released from stimulated neutrophils on collagenase, we used cells stimulated with either .PMA or fMLP/cytochalasin B. Both stimuli release collagenase from specific granules and gelatinase from C-particles. However, the amount of HOC1 produced varies considerably. PMA promotes a large and extended release of HzOz which is almost completely converted to HOC1 (31, 32). Stimulation with fMLP/cytochalasin B induces only a brief oxidative burst with little HOC1 production but, in contrast to PMA, releases azurophil granule enzymes, including proteases like cathepsin G and elastase (10, 33). Methionine, which is known for its high HOCl-scavenging ability, was used to define the action of HOC1 (15). Collagenase activity was measured in supernatants from neutrophils stimulated with PMA for 10, 30, and 60 min in the presence and absence of methionine (Fig. 1). The amount of enzyme active and total collagenase present (after procollagenase activation by APMA) was measured by the appearance of the characteristic LY~and PA collagen cleavage products. Active collagenase was detected in the neutrophil supernatant at each time point in samples without metlnionine (lanes 1, 3, 5). Visual examination of the gel in’dicated that this corresponds to less than a third of the maximal activity seen in the presence of APMA and methionine (lanes 8, 10, 12). When cells were stimulated with PMA in the presence of methionine no active collagenase was seen (lanes 7, 9, 11). When APMA was added to these supernatants, maximal cleavage of collagen was observed (lanes 8,10,12). These results are consistent with the conclusion of Weiss et al. (12) that neutrophils can partially activate their released collagenase with coreleased HOCl. In our experiments total collagenase activity in the supernatant without methionine decreased with incubation time to 60 min (lanes 2, 4, 6). It was also higher at all time points when methionine was present (lanes 8, 10, 12). This implies that in addition to being activated by HOCl, collagenase also undergoes progressive inactivation when continually exposed to HOC1 produced by neutrophils. Supernatants from resting cells showed no collagenolytic activity after activation by APMA (Fig. 1, lane c). When neutrophils were stimulated with fMLP/cytochalasin B for 10 min (not shown) a similar level of active collagenase was seen as with PMA. This activity and the
Time (min)...
10
30
50
lo
30
50
FIG. 1. Collagenase activity in neutrophil supernatant. Neutrophils (lo7 cells/ml) were stimulated with PMA for 10, 30, and 60 min without (lanes l-6) and with (lanes 7-12) 1 mM methionine. Supernatants (18 ~1) were incubated with collagen either before (odd lane numbers) or after (even lane numbers) collagenase activation by APMA. Supernatant was incubated with 70 ng type I collagen for 18 h at 25°C and the reaction products were separated by SDS/S% PAGE. The o* and @’ bands indicate the cleavage products of collagen. Lane c, supernatant from resting cells (30 min) after APMA activation.
total activity observed after activation by APMA decreased by about a half after 1 h. In contrast to what was seen with PMA, this activation and inactivation was not due to HOC1 because it was unaffected by the addition of methionine. Activation was most likely caused by proteases released from azurophil granules, since in the presence of the serine proteinase inhibitor PMSF, the released collagenase was entirely latent (not shown). These data are in agreement with Knauper et al. (9), who demonstrated activation of purified collagenase by cathepsin G. Effect of HOC1 on Gelatinase Released from Stimulated Neutrophils Neutrophils were stimulated with either PMA or cytochalasin B/fMLP. Gelatinase activity was measured as the l,lO-phenanthroline inhibitable degradation of 14Clabeled gelatin and was expressed as the percentage of the total gelatinase activity measured in a Triton-lysed cell extract in the presence of APMA. The results from four independent experiments gave a maximum activity of 2.36 1- 0.22 mg gelatin degraded/lo7 cells/h at 37°C (PMA stimulation). This value represented 89% of the total gelatinase in neutrophils and was estimated in the presence of methionine and after progelatinase activation by APMA. The same supernatants from PMA-stimulated cells as used for the collagenase assay in Fig. 1 contained very little active gelatinase (Fig. 2A). At all time points this corresponded to only 1.0 + 0.5% of the total gelatinase present in the neutrophils. No differences were seen when methionine was present (Fig. 2B). Latent gelatinase that could be activated by APMA was released by PMA-stimulated cells. At 10 min this represented only 35% of the total gelatinase in neutrophils, and this decreased to 6% after 60 min (Fig. 2A). However, when methionine was present, 85-90% of the total gelatinase activity was mea-
558
MICHAELIS,
1ooy
VISSERS,
E
A r
(ii, c,
nnn 30
10 Time
1 ,ji 30
10
60
Time
(min)
C
60
(min)
D
40 20 0
10
30 Time
60
10
30 Time
(min)
60
(min)
FIG. 2. Gelatinase activity in neutrophil supernatant. Neutrophils (lo7 cells/ml) were stimulated with PMA or fMLP/cytochalasin B for 10,30, and 60 min at 37°C either with or without the addition of 1 mM methionine. Samples (20 ~1) were incubated with 250 pg i4C-labeled gelatin for 200 min at 37°C. Total gelatinolytic activity in neutrophils was determined after cell lysis with 0.1% (v/v) Triton X-100 and activation by APMA, and corresponds to the degradation of 176 pg gelatin. The data shown are the mean values of a triplicate assay of the same supernatants as those in Fig. 1, the standard deviation being
OF BIOCHEMISTRY
Vol. 292, No. 2, February
AND
BIOPHYSICS
1, pp. 555-562, 1992
Different Effects of Hypochlorous Acid on Human Neutrophil Metalloproteinases: Activation of Collagenase and Inactivation of Collagenase and Gelatinase Jiirgen Michaelis,’ Department
Mamgret C. Vissers, and Christine
of Pathology, Christchurch
School of Medicine,
C. Winterbourn
Christchurch,
New Zealand
Received June 13,1991, and in revised form October 7,199l
Human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) produce the reactive oxidant hypochlorous acid (HOCl) and release the matrix metalloproteinases collagenase and gelatinase from secretory granules. We have investigated the stoichiometry of activation and inactivation of the two metalloproteinases with HOCl. HOC1 activated purified neutrophil procollagenase at ratios between 10 and 40 mol of HOCl/mol enzyme, but caused inac:tivation at higher ratios. Maximum activation was about the same as that achieved by p-aminophenyl-mercuric acetate. However, less than a third of the total collagenase released from PMA-stimulated neutrophils was activated by coreleased HOC1 and most of the activity was destroyed after 1 h of stimulation. These results indicate that the HOCl/enzyme ratio must fall within a narrow range for activation to occur. In contrast to collagenase, purified progelatinase underwent negligible activation (2.5 + 1.2%) at HOCl/enzyme molar ratios < 30 and ‘was destroyed at higher ratios. Likewise no active gelatinase could be detected in supernatant from PMA-st.imulated cells and almost all of the proenzyme was destroyed by HOC1 after 60 min stimulation. Our results; illustrate that only collagenase can be activated by HOC1 in vitro and that gelatinase is much more sensitive to inactivation. Since a precise HOCl/enzyme ratio is required for collagenase activation it is doubtful whether effective enzyme regulation by HOC1 could occur in vivo where various HOCL scavengers are present. 0 1992 Academic Prees, Inc.
Human neutrophils contain the two metalloproteinases collagenase, which degrades native collagens types I, II, ’ TO whom correspondence should be addressed at present address: Peptide Technology Ltd., CSIRO Division of Biomolecular Engineering, P.O. Box 184, North Ryde, Sydney 2113, Australia. 0003.9861/92 $3.00 Copyright 0 1992 by Academic Pxess, All rights of reproduction in any form
and III (1, 2), and gelatinase, which has a greater specificity for native types IV and V collagen and for denatured collagens (3, 4). Both enzymes are thought to play a key role in extracellular matrix degradation by neutrophils (5). Collagenase and gelatinase are present as latent proenzymes and are released as such by stimulated neutrophils. The mechanism by which they are activated is unknown. In vitro studies have shown that both enzymes can be activated by incubation with mercurial compounds (4, 6, 7) or with gold compounds (8). Since this is an artificial reaction not related to normal conditions in vivo, activation of metalloproteinases by limited proteolysis by neutrophil serine proteases such as cathepsin G for collagenase (9) and elastase for gelatinase (10) has been proposed. These observations have been documented with sequence data of the activation site within neutrophil collagenase (9). Another pathway that could be physiologically important is activation by hypochlorous acid (HOC1)2 produced by neutrophils. Neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA) or the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) produce superoxide (0;) and hydrogen peroxide (H,O,) which is consumed by myeloperoxidase coreleased from azurophi1 granules to form HOC1 (11): myeloperoxidase
Hz02 + Cl- + H+ -
HOC1 + HzO.
* Abbreviations used: HOCl, hypochlorous acid; PMA, phorbol 12myristate 13-acetate; fMLP, fMet-Leu-Phe; APMA, p-aminophenyl mercuric acid, PMSF, phenylmethylsulfonyl fluoride; TPCK, L-l-p-tosylamino-2-phenylethyl chloromethyl ketone; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide. 555
Inc. reserved.
556
MICHAELIS,
VISSERS,
Weiss and colleagues first reported activation of neutrophil collagenase (12) and gelatinase (13) by HOCl. Collagenase activation has also been described by others (14) but there appear to be some discrepancies with these findings, since several groups have isolated and purified latent collagenase and gelatinase from PMA-stimulated neutrophils (4, 7). Furthermore, HOC1 is highly reactive with a wide range of biological molecules (15) including proteins (16) and Vissers and Winterbourn (17) have demonstrated that neutrophil proteases are inactivated by a myeloperoxidase-dependent oxidation pathway. In the studies on oxidant activation, the stoichiometric requirement of HOC1 has not been investigated. Similarly, the effect of HOC1 on the protein structure has not been determined. We have, therefore, examined the effect of HOC1 on purified collagenase and gelatinase and in cell supernatants from stimulated neutrophils. We demonstrate the exact stoichiometry necessary for collagenase activation and for inactivation of both collagenase and gelatinase. EXPERIMENTAL
AND
WINTERBOURN
Purification
of Neutrophil
Metalloproteinases
Collagenase was purified from homogenized neutrophils as described previously (6). Gelatinase was purified from homogenized nzutrophils in the presence of PMSF and benzamidine, by successive co!umn chromatography steps using DEAE-Sepharose, Zn2+ chelate-Sepharose (22, 23), Cibacron blue-Sepharose (24), and gelatin-Sepharose (4).
Reaction of Collagenase and Gelatinase with HOC1 Purified collagenase and gelatinase in PBS-Ca were exposed to HOC1 at known molar ratios for 15 min at 37°C unless otherwise stated. The molarity of HOC1 was assessed by its reaction with monochlorodimedon in PBS, monitoring the decrease in monochlorodimedon absorption at 290 nm (Ezw = 19,000 M-’ cm-‘) (25). The effects of HOC1 were related to the molar ratio rather than HOC1 concentration because HOC1 reacts rapidly with biomolecules and at the concentrations used in this study would all be consumed within minutes (26).
Activation
of Collagenase and Gelatinase by APMA
The latent metalloproteinases in neutrophil supernatants or as purified enzymes were activated by incubating at 37°C for 30 min with 0.5 mM APMA (collagenase) or for 1 h with 1 mM APMA (gelatinase). APMA was dissolved at 10 mM in 50 mM NaOH and the pH adjusted to 7.4 pH 7.0, and 2 mM CaCl, prior to use. with 1000 mM Tris/HCl,
PROCEDURES
Materials Ficoll-Hypaque was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Phorbol12-myristate 13-acetate, N-formyl-Met-Leu-Phe, cytochalasin B, p-aminophenyl mercuric acetate (APMA), phenylmethylsulfonyl fluoride (PMSF), monochlorodimedon, type I calf skin collagen, and goat anti-rabbit antibody alkaline phosphatase conjugate were from Sigma Chemical Co., St. Louis, Missouri. Hypochlorous acid was from Reckitt & Colman, Auckland, New Zealand. TPCK-trypsin was purchased from Worthington, Freehold, New Jersey; al-antitrypsin was purified from human plasma by thiol-disulfide interchange (18). All other chemicals were reagent grade. The Centricon 30 microconcentrator was purchased from Amicon, Danvers, Massachusetts; and the Microsep microconcentrator 100 from Filtron Technology, Northborough, Massachusetts. Polyclonal antibodies against purified gelatinase were raised in a rabbit and purified by ammonium sulfate precipitation and ionexchange chromatography on DEAE-Sepharose as described previously (19, 20).
Methods Human neutrophils were prepared from the peripheral blood of healthy donors by centrifugation through Ficoll-Hypaque, dextran sedimentation, and hypotonic lysis of contaminating red cells (21). The cells were suspended at 107/ml in 10 mM sodium phosphate buffer, pH 7.4, and 150 mM NaCl (PBS), containing 1 mM CaCl, (PBS-Ca), 0.5 mM MgCl,, and 1 mg/ml glucose. For stimulation with PMA (0.1 pg/ml) 2 pl PMA (stock 500 pg/ml in dimethyl sulfoxide [Me,SO]) was added to 10 ml of cell suspension. Alternatively the cells were incubated for 2 min with 5 rg/ml cytochalasin B prior to stimulation with lo-’ M fMLP (stock solution in PBS). When appropriate the neutrophils were stimulated in the presence of 1 mM phenylmethylsulfonyl fluoride (added 2 min after addition of the stimulus), 1 mM methionine, or 3% (w/v) human serum albumin (added before the stimulus). After 10. 30, and 60 min at 37°C the neutrobhils were pelleted by ce&ifugation (ll!lOOg) for 5 min and the supernatant was removed. PMSF (2 mM), a,-antitrypsin (5 pg/ ml), and NaN, (1 mM) were added to prevent further proteolysis or inactivation of the metalloenzymes and the supernatant was stored at -80°C.
Activation
of Gelatinase by Trypsin
Neutrophil supernatant was dialyzed against PBS-Ca by spinning twice through a Centricon 30 microconcentrator (M, 30000 cutoff membrane) to eliminate PMSF from the sample and was finally concentrated 30-fold. TPCK-trypsin (2 @g/30 pl neutrophil supernatant) was added and the solution was incubated for 10 min at 37°C. Purified gelatinase (2 fig in 20 /Al PBS-Ca) was activated by 0.5 pg TPCK-trypsin at 37°C for 10 min. The reaction was terminated by the addition of a lo-fold excess of soya bean trypsin inhibitor. Conversion to the cleaved, active form was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.
Enzyme Assays Collagenuse. Collagenase activity was assessed by specific cleavage of soluble type I calf skin collagen. Collagen (70 pg) was incubated at 25°C with either (i) 0.2 pg purified collagenase in 62 ~1 buffer A (100 mM Tris/HCl, pH 7.5, 100 mM NaCl, 2 mM CaCl,, 0.05 mM ZnCl,, 0.1 mM NaN,) for 200 min or (ii) 18 ~1 cell-free medium from stimulated neutrophils in 32 ~1 buffer A for 18 h. Appearance of OI*and /3* collagen fragments was monitored by SDS-PAGE. Gelatinme. Degradation of radiolabeled gelatin was measured essentially as described earlier (4). Type I collagen from rat skin was labeled with [l-‘4C]acetic anhydride (27) and diluted with unlabeled collagen to approximately 200,000 cpm/mg. [“C]Collagen was denatured at 90°C for 15 min and incubated for 200 min at 37°C with either 20 ~1 cell supernatant or 2 pg purified gelatinase in a final volume of 150 ~1. The reaction was stopped by adding trichloroacetic acid to a final concentration of 15% (w/v) and cooling to 4°C. Solubilized radioactivity was determined after centrifugation at SOOOgfor 5 min. Maximum radioactivity was measured in a sample that was not pelleted.
Electrophoresis
and Immunoblotting
SDS-PAGE was performed in a continuous slab gel system (28). Gels were stained with picrate-Coomassie blue (29). Western blots were performed using rabbit anti-neutrophil gelatinase antibody as the first antibody and goat anti-rabbit antibody alkaline phosphatase conjugate as the second antibody (6).
EFFECTS
OF HYPOCHLOROUS
ACID
ON NEUTROPHIL
557
METALLOPROTEINASES
Protein Determination Protein was assayed using the method previously described (30). To account for the high carbohydrate portion of neutrophil collagenase a M. 53000 (9) for the unglycosylated enzyme was used to calculate the molarity. M, 95000 was used for purified neutrophil gelatinase, as this was the predominant form visible on SDS-PAGE under nonreducing conditions (data not shown).
RESULTS
Effect of HOC1 on Collagenase Released from Stimulated Neutrophils To determine the eff’ects of oxidants released from stimulated neutrophils on collagenase, we used cells stimulated with either .PMA or fMLP/cytochalasin B. Both stimuli release collagenase from specific granules and gelatinase from C-particles. However, the amount of HOC1 produced varies considerably. PMA promotes a large and extended release of HzOz which is almost completely converted to HOC1 (31, 32). Stimulation with fMLP/cytochalasin B induces only a brief oxidative burst with little HOC1 production but, in contrast to PMA, releases azurophil granule enzymes, including proteases like cathepsin G and elastase (10, 33). Methionine, which is known for its high HOCl-scavenging ability, was used to define the action of HOC1 (15). Collagenase activity was measured in supernatants from neutrophils stimulated with PMA for 10, 30, and 60 min in the presence and absence of methionine (Fig. 1). The amount of enzyme active and total collagenase present (after procollagenase activation by APMA) was measured by the appearance of the characteristic LY~and PA collagen cleavage products. Active collagenase was detected in the neutrophil supernatant at each time point in samples without metlnionine (lanes 1, 3, 5). Visual examination of the gel in’dicated that this corresponds to less than a third of the maximal activity seen in the presence of APMA and methionine (lanes 8, 10, 12). When cells were stimulated with PMA in the presence of methionine no active collagenase was seen (lanes 7, 9, 11). When APMA was added to these supernatants, maximal cleavage of collagen was observed (lanes 8,10,12). These results are consistent with the conclusion of Weiss et al. (12) that neutrophils can partially activate their released collagenase with coreleased HOCl. In our experiments total collagenase activity in the supernatant without methionine decreased with incubation time to 60 min (lanes 2, 4, 6). It was also higher at all time points when methionine was present (lanes 8, 10, 12). This implies that in addition to being activated by HOCl, collagenase also undergoes progressive inactivation when continually exposed to HOC1 produced by neutrophils. Supernatants from resting cells showed no collagenolytic activity after activation by APMA (Fig. 1, lane c). When neutrophils were stimulated with fMLP/cytochalasin B for 10 min (not shown) a similar level of active collagenase was seen as with PMA. This activity and the
Time (min)...
10
30
50
lo
30
50
FIG. 1. Collagenase activity in neutrophil supernatant. Neutrophils (lo7 cells/ml) were stimulated with PMA for 10, 30, and 60 min without (lanes l-6) and with (lanes 7-12) 1 mM methionine. Supernatants (18 ~1) were incubated with collagen either before (odd lane numbers) or after (even lane numbers) collagenase activation by APMA. Supernatant was incubated with 70 ng type I collagen for 18 h at 25°C and the reaction products were separated by SDS/S% PAGE. The o* and @’ bands indicate the cleavage products of collagen. Lane c, supernatant from resting cells (30 min) after APMA activation.
total activity observed after activation by APMA decreased by about a half after 1 h. In contrast to what was seen with PMA, this activation and inactivation was not due to HOC1 because it was unaffected by the addition of methionine. Activation was most likely caused by proteases released from azurophil granules, since in the presence of the serine proteinase inhibitor PMSF, the released collagenase was entirely latent (not shown). These data are in agreement with Knauper et al. (9), who demonstrated activation of purified collagenase by cathepsin G. Effect of HOC1 on Gelatinase Released from Stimulated Neutrophils Neutrophils were stimulated with either PMA or cytochalasin B/fMLP. Gelatinase activity was measured as the l,lO-phenanthroline inhibitable degradation of 14Clabeled gelatin and was expressed as the percentage of the total gelatinase activity measured in a Triton-lysed cell extract in the presence of APMA. The results from four independent experiments gave a maximum activity of 2.36 1- 0.22 mg gelatin degraded/lo7 cells/h at 37°C (PMA stimulation). This value represented 89% of the total gelatinase in neutrophils and was estimated in the presence of methionine and after progelatinase activation by APMA. The same supernatants from PMA-stimulated cells as used for the collagenase assay in Fig. 1 contained very little active gelatinase (Fig. 2A). At all time points this corresponded to only 1.0 + 0.5% of the total gelatinase present in the neutrophils. No differences were seen when methionine was present (Fig. 2B). Latent gelatinase that could be activated by APMA was released by PMA-stimulated cells. At 10 min this represented only 35% of the total gelatinase in neutrophils, and this decreased to 6% after 60 min (Fig. 2A). However, when methionine was present, 85-90% of the total gelatinase activity was mea-
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FIG. 2. Gelatinase activity in neutrophil supernatant. Neutrophils (lo7 cells/ml) were stimulated with PMA or fMLP/cytochalasin B for 10,30, and 60 min at 37°C either with or without the addition of 1 mM methionine. Samples (20 ~1) were incubated with 250 pg i4C-labeled gelatin for 200 min at 37°C. Total gelatinolytic activity in neutrophils was determined after cell lysis with 0.1% (v/v) Triton X-100 and activation by APMA, and corresponds to the degradation of 176 pg gelatin. The data shown are the mean values of a triplicate assay of the same supernatants as those in Fig. 1, the standard deviation being
