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2, 1992
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ADDITIVE
ENHANCEMENT OF NEUTROPHIL COLLAGENASE BY HOC1 AND CATHEPSIN G
560-567
ACTIVITY
W. Winn Chatham, Warren D. Blackburn, Jr., and Louis W. Heck Division of Clinical Immunology and Rheumatology Department of Medicine, The University of Alabama at Birmingham and Department of Veteran’s Affairs, Birmingham, Alabama Received
March
9,
1992
Summary: Using preparations of latent collagenase derived from neutrophil specific granule extracts, the relative effects of cathepsin G and HOC1 on activation of neutrophil collagenase were determined using a quantitative collagenase assay. Enhancement of collagenase activity by cathepsin G and physiologically relevant concentrations of HOC1 were comparable, but HOC1 mediated collagenase activation was reversible in the presence of HOC1 scavenger. Collagenase activity in preparations treated with cathepsin G and HOC1 simultaneously or sequentially was significantly greater than activity in preparations treated with HOC1 or cathepsin G alone. The results indicated additive, yet independant enhancing effects of HOC1 and cathepsin G on activity of neutrophil collagenase. c 1992Academic Press,1°C.
Human
neutrophil
collagenase is a metalloprotease
specific granules of neutrophils released extracellularly
(1). Depending
may be predominantly
activated (2). Ligands that induce significant
stored in latent form within
upon the PMN
activating ligand, HNC
latent or a significant generation
portion
may be
of activated HNC also induce
significant generation of HOC1 and several studies indicate that latent HNC can be activated by this compound including
(3,4,5).
Serine proteases with trypsin or chymotrypsin-like
the PMN azurophilic
granule constituent
activity,
cathepsin G, are also capable of
activating HNC (4). Results from at least one study suggest that cathepsin G contributes to HNC activation during PMN stimulation with phorbol esters (6); however, the respective roles of HOC1 and cathepsin G in facilitating
HNC activation during PMN stimulation by
physiologic ligands in vivo remains uncertain. Abbreviations collagenase; Sue-AAPPpNa,
used: PMN, polymorphonuclear leukocyte; HNC, human neutrophil SAIgG, surface associated IgG; DFP, diisopropyl fluorophosphate; succinyl-(alanyl)2-prolyl-phenylalanine-p-nitroanilide.
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of neutrophils with SAIgG , a model for PMN interactions with immune
complexes, results in release of activated HNC in association with significant generation of HOC1 and extracellular release of cathepsin G (1,5,7). Although these observations suggest that both HOC1 and cathepsin G potentially
contribute to activation of HNC in vivo, the
relative effects of these PMN products on HNC activation have not been determined using quantitative
measures of collagenase activity. Using a quantitative
preparations
of latent HNC that are free of cathepsin G activity, we determined
of HOC1 and cathepsin G, alone or in combination,
MATERIALS
collagenase assay and the effects
on HNC activation.
AND METHODS
Reapents and Materials. CM cellulose and DE cellulose were obtained from Pharmacia (Piscataway, N.J.). Percoll, DFP , L-methionine, mersalyl, and other chemical reagents were obtained from Sigma (St. Louis, MO). Sue-AAPPpNa was obtained from Cal Biochem (La Jolla, Ca.). Clostridial collagenase was obtained from Worthington Biochemical (Freehold, N.J.). Preparation of latent HNC from neutroDhi1 soecific granules. Neutrophils were prepared from blood of normal donors using established methods (1). For each preparation, 3 x lo8 PMN were suspended in buffer containing 15 mM CaCl,, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl, and 10 mM Pipes, pH 7.4, and sonicated in the presence of 1.0 mM NaN,, 5 mM Lmethionine, and 1.0 mM DFP. Specific granules in the cell lysate were then isolated on Percoll gradients as previously described (8). Following separation from the Percoll by ultracentrifugation (35,000 g x 2 hr), the granules were suspended in 3.0 ml of HBSS with 1.0 mM DFP, to which 0.1% Triton X-100 was added. The granule contents (average protein content = 600 pg/ml) were dialyzed against 50 mM sodium acetate, pH 6.0 with 15 mM CaCl,, then applied over a 1.5 x 3 cm column of CM cellulose equilibrated with the same buffer. The exclusion fraction from the column was collected and dialyzed against 10 mM Tris with 1 mM CaCl,, pH 8.0, then applied over a 1.5 by 3 cm DE cellulose column equilibrated in the same buffer. Following three column washes, collagenase adherant to the column was eluted with 50 mM NaCl in 10 mM Tris/l mM CaCl, and the eluted mixture (mean protein concentration = 12 pg/ml) was dialyzed into HBSS. Specific collagenase activity in mersalyl (1 mM) treated aliquots of the final HNC preparation averaged 0.024 units’/pg (a 140-fold enhancement relative to the specific granule extract) and was consistently three to four times higher than that in aliquots not treated with mersalyl. No cathepsin G activity could be detected in the final HNC preparation during a 24 hour incubation with Sue-AAPPpNa . Preuaration of Catheosin G. Neutrophil cathepsin G was purified from human neutrophils as previously prescribed (9). The cathepsin G preparation had no evidence of collagenase activity and 1.0 ug released 83.4 pmole/min of p-nitrophenol from Sue-AAPPpNa (37°C). Activation of latent HNC by HOC1 and/or CatheDsin G. The final latent HNC preparation was diluted with HBSS sufficient to yield a collagenase activity of approximateIy 20 ng/min when 1.0 mM mersalyl was added to an aliquot of the preparation. Aliquots (l.O2.0 ml) of the latent HNC preparation were then incubated for one hour at 37°C with either HOCl, cathepsin G or HOC1 in combination with cathepsin G followed by addition of 1.0 mM DFP and determination of collagenase activity. Aliquots of the latent HNC preparation lone unit of collagenase cleaves 1.0 pg of collagen/minute. 561
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were also treated sequentially with cathepsin G for one or two hours (terminated by addition of DFP), followed by addition of varied concentrations of HOC1 or mersalyl. Lmethionine (10 mM) was added to some of the treated aliquots prior to determination of collagenase activity. Collagenase assays. Triplicate 200 ul aliquots of treated or untreated HNC preparations were incubated for 18 hours (37°C) with reconstituted [3H]-labeled type I collagen fibrils in the presence of 1.0 mM DFP (10). Collagenase activity in each aliquot was calculated from radioactivity released into the aliquot and radioactivity released following incubation of comparable fibrils with 250 ug/ml clostridial collagenase (2).
RESULTS Activation
of latent HNC bv HOCl. To determine
optimal HOC1 concentrations
required for activation of latent HNC, aliquots of each specific granule derived preparation containing predominantly
latent HNC were treated for one hour with O-1 mM HOC1 (Figure
1). Significant increases in collagenase activity were noted in aliquots treated with lo-100 PM (final concentration)
HOCl.
Comparable results were obtained when HOC1 was added
in the presence of DFP (data not shown). Radioactivity release by collagen fibrils incubated with HBSS containing only lo-100 PM HOC1 or by HOC1 treated HNC preparations
to
which 2 mM EDTA was added did not exceed that noted for fibrils incubated with HBSS alone (data not shown). Activation of latent HNC bv catheDsin G. Depending on the amount of cathepsin G used and incubation time, incubation of latent HNC preparation with cathepsin G alone resulted in enhancement
of collagenase activity comparable to that noted for preparations incubated
CONCENTRATION
HOCL
(FM)
Figure 1. Effects of HOC1 on collagenase activity in specific granule derived latent HNC preparations. One ml aliquots of three different latent HNC preparations were treated for one hour (37°C) with concentrations of HOC1 shown on the abscissa. Collagenase activity was then determined in HOC1 treated and untreated preparations as described in the methods. Values depicted on graph represent mean (+ SEM) percent increase in collagenase activity of HNC preparations treated with indicated doses of HOC1 relative to activity in control (untreated) preparations. 562
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Table 1. Activation of latent HNC by Cathepsin G Treatment
Collagenase Activity (ng/rnin)
none 1.0 pg CG
10.5 + 1.36
100 2 13.6
13.3 5 .83
127 zt 8.4
2.5 ,ug CG
15.5 5 1.40
148 + 13.2
5.0 pg CG 10.0 pg CG
17.1 t .25
163 k 26.0
21.0 + 1.70
200 + 19.4
3.1 2 .28
29 !I 2.9
10.0 pg CG + EDTA*
Percent of Control
One ml aliquots of a specific granule derived latent HNC preparation were treated one hour (37°C) with O-10 pg puified cathepsin G, followed by addition of 1 mM DFP. Collagenase activities in the treated and untreated preparations were then determined as described in the methods section. Values in table denote mean (n =3) + SD collagenase activity in treated or untreated aliquots. “EDTA (1.0 mM) added prior to determination of coIlagenase activity.
with HOC1 alone (Table 1). Incubation
of HNC with cathepsin G beyond two hours did not
result in further increases in collagenase activity and incubation beyond eight hours resulted in loss of activity (data not shown). Effects of HOC1 scavenger on activated HNC. latent HNC by exogenous hypochlorite collagenase activity was determined
To determine whether activation
of
persisted in the presence of HOC1 scavenger,
in aliquots of HNC preparation
to which 10 mM L
methionine was added following one hour treatment of the preparation with 100 uM HOCl. Addition
of L-methionine
resulted in significant, but not complete attenuation
mediated HNC activation (Figure 2). L-methionine HNC preparations
of HOCI-
had no effect on collagenase activity in
treated with cathepsin G (Figure 2) or mersalyl (data not shown).
Combined affects of HOC1 and cathepsin G on HNC activation.
Treatment
of the
latent HNC preparation with HOC1 and cathepsin G simultaneously resulted in significantly greater collagenase activity relative to preparations treated with HOC1 or cathepsin G alone (Figure 3). Comparable
but slightly higher incremental
increases in collagenase activity
were observed when latent HNC preparations were treated sequentially with cathepsin G for one hour (terminated
with DFP) then 50 I.LM HOCl. Addition
of L-methionine
following
treatment of HNC preparations with HOC1 and cathepsin G resulted in collagenase activity comparable to preparations in HNC preparations
treated with cathepsin G alone (Figure 3). Collagenase activity
treated concurrently
with HOC1 and cathepsin G in the presence of 563
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BIOCHEMICAL
AND BIOPHYSICAL
ncontrol
RESEARCH COMMUNICATIONS 0 Control E;gt,epsin
fS8 Cathepsin
G *
G
ET!3Cothepmn G + HOC1 ~Cothepsm G -DFP+HOCI CHI Cathepsin C +HOCL-Methi
Methionine
Figure 2. Effects of an HOC1 scavenger on HNC activity. Aliquots (2.0 ml) of latent HNC preparation were incubated for one hour (37°C) in the presence or absence of either 100 PM HOC1 or 10 bg/ml cathepsin G. Following incubation, 1 mM DFP was added to each aliquot, followed by addition of Lmethionine (10 mM final concentration) to half of each aliquot. Bar heights denote mean (n=3)+SD collagenase activities determined for each treated and untreated sample, determined as described in the methods section. Comparable results were obtained in identical experiments using two other latent HNC preparations. Figure 3. Combined Effects of HOC1 and Cathepsin G on HNC Activity. Aliquots (2.0 ml) of each latent HNC preparation were incubated one hour (37°C) in the presence or absence of either 100 yM HOCI, 10 pg/‘ml cathepsin G, or 100 PM HOC1 and 10 pg/ml cathepsin G simultaneously. An additional aliquot of each preparation was incubated one hour with 10 fig/ml of cathepsin G followed by addition of 1 mM DFP and 100 PM HOCl. Prior to determination of collagenase activity (described in methods), 10 mM L-methionine was added to half of the aliquot treated with HOC1 and cathepsin G concurrently. Bar heigths denote mean (n=3)tSEM percent increases in collagenase activity for treated aliquots relative to activity in corresponding control (untreated) preparations.
1.0 mM DFP was comparable to that noted for preparations
treated with HOC1 alone (data
not shown). Regardless of the dose of oxidant or organomercurial or mersalyl to aliquots of latent HNC pretreated
used, addition of HOC1
two hours with cathepsin G resulted in
collagenase activity exceeding that in the cathepsin G treated preparation latent HNC aliquots treated only with the corresponding concentration
as well as that in
of HOC1 or mersalyl
(Figure 4).
DISCUSSION Since imunoglobulin
latent
HNC
secreted
into
phagolysosomes
surrounding
tissue bound
may interact with both HOC1 and cathepsin G, the relative effects of these
PMN products on HNC activation confirm that HOC1 concentrations
are of significant interest. The results presented here comparable to those generated during PMN stimulation 564
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A-ACGtMedyl *- - -* hkrsoiyi olane
O-OCGWHOCL .-0 HOCL olone 404
6
0 A0
10
100
0
0
1
.l Mersolyl
HOC1 (/Ad)
2.5
(mM)
Figure 4. Enhancing effects of HOC1 (4A) or Organomercurial (4B) on HNC Preparations Treated or Untreated with Cathepsin G. Latent HNC preparation was incubated for two hours (37°C) in the presence or absence of 10 pg/ml cathepsin G. Following addition of 1 mM DFP, O-100 PM HOC1 or O-5 mM mersalyl (final concentrations) were added to 1.0 ml aliquots of both cathepsin G treated and untreated preparations prior to determination of collagenase activity. Values depicted on graphs represent mean (“SD) of triplicate determinations of collagenase activity for each sample. * p < .05 CG*SObM HOC1 vs 50 PM HOC1 alone.
with SAIgG (50pM)
(5) are capable of enhancing collagenase activity in preparations
of
latent HNC. These results also confirm that HOC1 can activate HNC in the absence of cathepsin G (4), as there was no detectable preparation,
cathepsin G activity in the final HNC
and addition of DFP to the latent HNC preparation did not alter the enhancing
affects of HOC1 on collagenase activity. Although initial cleavage of HNC by cathepsin G during cell lysis and preparation unlikely
as cell sonication
performed
of the specific granule extracts may have occurred, this is
and treatment
of the granules with Triton
were
in the presence of DFP.
The observed loss of collagenase activity following preparations
detergent
of HOCI-activated
addition of HOC1 scavenger to
HNC suggests a potential mechanism of regulating HNC
activity in vivo, in that loss of enzymatic activity may occur during diffusion activated HNC
away from the phagolysosome
scavenged. This observation organomercurial organomercurials,
mediated
into a milieu
where HOC1 is readily
is analagous to findings in a previous activation
HOC1 is thought
report
of HNC was shown to be reversible to mediate activation
cysteine sulfhydryl moeity contained within a PRCGVPD metal ion (12,13). The failure of methionine
of HOC1
in which (11). Like
of HNC by dissociating
the
sequence from the active site zinc
concentrations
one hundred-fold
in excess of
HOC1 to completely reverse HOC1 mediated enhancement of collagenase activity could be 565
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due to greater affinity organomercurial
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of the oxidant for the cysteine sulhydryl group.
activated zinc-metalloproteases
Alternatively,
have been shown to undergo autocatalytic
cleavage with retention of enzyme activity (14), and a similiar occurance for HOC1 activated HNC may account for a portion of the enzyme preparation
remaining active in the presence
of HOC1 scavenger. Although treatment of latent HNC with cathepsin G alone resulted in augmentation collagenase activity comparable
to that noted for HOC1 treated preparations,
of
the even
greater increase in collagenase activity noted following treatment with HOC1 and cathepsin G concurrently
suggests the effects of HOC1 and cathepsin G on HNC activity may be
additive. Augmented proteolytic activation of HNC by cathepsin G in the presence of HOC1 could account for these observations, but comparable were noted in preparations Furthermore,
if not higher collagenase activities
treated sequentially with cathepsin G, DFP, and then HOCl.
addition of HOC1 scavenger to preparations
HOC1 and cathepsin G simultaneously noted for preparations activity independent
which had been treated with
resulted in collagenase activity comparable to that
treated with cathepsin G alone, suggesting HOC1 effects on enzyme of those mediated by cathepsin G.
Higher collagenase activity observed in mixtures treated with HOC1 and cathepsin G in combination
could also be accounted for by activation
enzyme relative
to preparations
of a greater percentage of latent
treated with either agent alone.
However,
maximizing cathepsin G mediated HNC activation by two hour incubation with cathepsin demonstrated
G, the subsequent
addition
despite
of latent HNC
of HOC1 or mersalyl in concentrations
to optimize HNC activity resulted in collagenase activity which exceeded that
in the cathepsin G treated preparation the optimal concentrations
as well as latent HNC preparations
treated only with
of HOC1 or mersalyl. These results, suggesting independant
effects of cathepsin G and HOC1 on HNC activity that are additive, may prove to be an important consideration in developing interventions to abrogate tissue injury by neutrophils. Future studies correlating
molecular characteristics of HNC treated with HOC1 and/or
cathepsin G with characteristics of HNC generated in inflamed tissues may further clarify which of these potential mechanisms of regulating HNC activity are operative in vivo.
REFERENCES 1.
2. 3.
Murphy, G., Reynolds, J.J. Bretz, U., and Baggiolinz, M. (1977) Biochem J. 162,195 197. Chatham, W. W., Heck, L.W., and Blackburn, W.D., Jr. (1990) Arthritis and Rheumatism 33, 228-234. Weiss, S.J., Peppin, G., Ortiz, X., Ragsdale, C. and Test, S.T. (1985) Science 227, 747-749. 566
Vol.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
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2,
1992
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Saari, H., Suomalainen, K., Lindy, O., Kontinnen, Y., and Sorsa, T. (1990) Biochem. Biophys. Res. Comm. 171, 979-987. Chatham, W.W., Heck, L.W., and Blackburn, W.D., Jr. (1992) Matrix (supplement), In press. Capodici, C., Muthukumaran, G., Amorusu, M., and Berg, R.A. (1989) Inflammation 13, 245-258. Henson, P.M. and Johnston, R.B. (1987) J. Clin. Invest. 79, 669-674. Borregard, N., Heiple, J.M., Simons, E.R., and Clark, R.A. (1983) J. Cell Biol., 97, 52-61. Heck, L.W., Rostand, K.S., Hunter, F.A., and Bhown, A. (1986) Anal. Biochem., 158, 217-227. Johnson-Win& B. (1990) Anal. Biochem. 104, 175-181. Mookhtiar, K.A. and VanWart, H.E. (1990) Biochemistry 29, 10620-10627. Springman, E.B., Angleton, E.L., Birkedal-Hansen, H., and VanWart, H.E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 364-368. Hasty, K.A., Pourmotabbed, T.F., Goldberg, E.I., Thompson, J.P., Spinella, D.G., Stevens, R.M. and Mainardi, C.L. (1990) J. Biol. Chem. 265, 11421-11424. Grant, G., Eisen, A.Z., Marmar, B.L., Roswit, W.T. and Goldberg, G.I. (1987) J. Biol. Chem. 262, 5886-5889.
567
184,
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April
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2, 1992
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COMMUNICATIONS Pages
ADDITIVE
ENHANCEMENT OF NEUTROPHIL COLLAGENASE BY HOC1 AND CATHEPSIN G
560-567
ACTIVITY
W. Winn Chatham, Warren D. Blackburn, Jr., and Louis W. Heck Division of Clinical Immunology and Rheumatology Department of Medicine, The University of Alabama at Birmingham and Department of Veteran’s Affairs, Birmingham, Alabama Received
March
9,
1992
Summary: Using preparations of latent collagenase derived from neutrophil specific granule extracts, the relative effects of cathepsin G and HOC1 on activation of neutrophil collagenase were determined using a quantitative collagenase assay. Enhancement of collagenase activity by cathepsin G and physiologically relevant concentrations of HOC1 were comparable, but HOC1 mediated collagenase activation was reversible in the presence of HOC1 scavenger. Collagenase activity in preparations treated with cathepsin G and HOC1 simultaneously or sequentially was significantly greater than activity in preparations treated with HOC1 or cathepsin G alone. The results indicated additive, yet independant enhancing effects of HOC1 and cathepsin G on activity of neutrophil collagenase. c 1992Academic Press,1°C.
Human
neutrophil
collagenase is a metalloprotease
specific granules of neutrophils released extracellularly
(1). Depending
may be predominantly
activated (2). Ligands that induce significant
stored in latent form within
upon the PMN
activating ligand, HNC
latent or a significant generation
portion
may be
of activated HNC also induce
significant generation of HOC1 and several studies indicate that latent HNC can be activated by this compound including
(3,4,5).
Serine proteases with trypsin or chymotrypsin-like
the PMN azurophilic
granule constituent
activity,
cathepsin G, are also capable of
activating HNC (4). Results from at least one study suggest that cathepsin G contributes to HNC activation during PMN stimulation with phorbol esters (6); however, the respective roles of HOC1 and cathepsin G in facilitating
HNC activation during PMN stimulation by
physiologic ligands in vivo remains uncertain. Abbreviations collagenase; Sue-AAPPpNa,
used: PMN, polymorphonuclear leukocyte; HNC, human neutrophil SAIgG, surface associated IgG; DFP, diisopropyl fluorophosphate; succinyl-(alanyl)2-prolyl-phenylalanine-p-nitroanilide.
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of neutrophils with SAIgG , a model for PMN interactions with immune
complexes, results in release of activated HNC in association with significant generation of HOC1 and extracellular release of cathepsin G (1,5,7). Although these observations suggest that both HOC1 and cathepsin G potentially
contribute to activation of HNC in vivo, the
relative effects of these PMN products on HNC activation have not been determined using quantitative
measures of collagenase activity. Using a quantitative
preparations
of latent HNC that are free of cathepsin G activity, we determined
of HOC1 and cathepsin G, alone or in combination,
MATERIALS
collagenase assay and the effects
on HNC activation.
AND METHODS
Reapents and Materials. CM cellulose and DE cellulose were obtained from Pharmacia (Piscataway, N.J.). Percoll, DFP , L-methionine, mersalyl, and other chemical reagents were obtained from Sigma (St. Louis, MO). Sue-AAPPpNa was obtained from Cal Biochem (La Jolla, Ca.). Clostridial collagenase was obtained from Worthington Biochemical (Freehold, N.J.). Preparation of latent HNC from neutroDhi1 soecific granules. Neutrophils were prepared from blood of normal donors using established methods (1). For each preparation, 3 x lo8 PMN were suspended in buffer containing 15 mM CaCl,, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl, and 10 mM Pipes, pH 7.4, and sonicated in the presence of 1.0 mM NaN,, 5 mM Lmethionine, and 1.0 mM DFP. Specific granules in the cell lysate were then isolated on Percoll gradients as previously described (8). Following separation from the Percoll by ultracentrifugation (35,000 g x 2 hr), the granules were suspended in 3.0 ml of HBSS with 1.0 mM DFP, to which 0.1% Triton X-100 was added. The granule contents (average protein content = 600 pg/ml) were dialyzed against 50 mM sodium acetate, pH 6.0 with 15 mM CaCl,, then applied over a 1.5 x 3 cm column of CM cellulose equilibrated with the same buffer. The exclusion fraction from the column was collected and dialyzed against 10 mM Tris with 1 mM CaCl,, pH 8.0, then applied over a 1.5 by 3 cm DE cellulose column equilibrated in the same buffer. Following three column washes, collagenase adherant to the column was eluted with 50 mM NaCl in 10 mM Tris/l mM CaCl, and the eluted mixture (mean protein concentration = 12 pg/ml) was dialyzed into HBSS. Specific collagenase activity in mersalyl (1 mM) treated aliquots of the final HNC preparation averaged 0.024 units’/pg (a 140-fold enhancement relative to the specific granule extract) and was consistently three to four times higher than that in aliquots not treated with mersalyl. No cathepsin G activity could be detected in the final HNC preparation during a 24 hour incubation with Sue-AAPPpNa . Preuaration of Catheosin G. Neutrophil cathepsin G was purified from human neutrophils as previously prescribed (9). The cathepsin G preparation had no evidence of collagenase activity and 1.0 ug released 83.4 pmole/min of p-nitrophenol from Sue-AAPPpNa (37°C). Activation of latent HNC by HOC1 and/or CatheDsin G. The final latent HNC preparation was diluted with HBSS sufficient to yield a collagenase activity of approximateIy 20 ng/min when 1.0 mM mersalyl was added to an aliquot of the preparation. Aliquots (l.O2.0 ml) of the latent HNC preparation were then incubated for one hour at 37°C with either HOCl, cathepsin G or HOC1 in combination with cathepsin G followed by addition of 1.0 mM DFP and determination of collagenase activity. Aliquots of the latent HNC preparation lone unit of collagenase cleaves 1.0 pg of collagen/minute. 561
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were also treated sequentially with cathepsin G for one or two hours (terminated by addition of DFP), followed by addition of varied concentrations of HOC1 or mersalyl. Lmethionine (10 mM) was added to some of the treated aliquots prior to determination of collagenase activity. Collagenase assays. Triplicate 200 ul aliquots of treated or untreated HNC preparations were incubated for 18 hours (37°C) with reconstituted [3H]-labeled type I collagen fibrils in the presence of 1.0 mM DFP (10). Collagenase activity in each aliquot was calculated from radioactivity released into the aliquot and radioactivity released following incubation of comparable fibrils with 250 ug/ml clostridial collagenase (2).
RESULTS Activation
of latent HNC bv HOCl. To determine
optimal HOC1 concentrations
required for activation of latent HNC, aliquots of each specific granule derived preparation containing predominantly
latent HNC were treated for one hour with O-1 mM HOC1 (Figure
1). Significant increases in collagenase activity were noted in aliquots treated with lo-100 PM (final concentration)
HOCl.
Comparable results were obtained when HOC1 was added
in the presence of DFP (data not shown). Radioactivity release by collagen fibrils incubated with HBSS containing only lo-100 PM HOC1 or by HOC1 treated HNC preparations
to
which 2 mM EDTA was added did not exceed that noted for fibrils incubated with HBSS alone (data not shown). Activation of latent HNC bv catheDsin G. Depending on the amount of cathepsin G used and incubation time, incubation of latent HNC preparation with cathepsin G alone resulted in enhancement
of collagenase activity comparable to that noted for preparations incubated
CONCENTRATION
HOCL
(FM)
Figure 1. Effects of HOC1 on collagenase activity in specific granule derived latent HNC preparations. One ml aliquots of three different latent HNC preparations were treated for one hour (37°C) with concentrations of HOC1 shown on the abscissa. Collagenase activity was then determined in HOC1 treated and untreated preparations as described in the methods. Values depicted on graph represent mean (+ SEM) percent increase in collagenase activity of HNC preparations treated with indicated doses of HOC1 relative to activity in control (untreated) preparations. 562
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Table 1. Activation of latent HNC by Cathepsin G Treatment
Collagenase Activity (ng/rnin)
none 1.0 pg CG
10.5 + 1.36
100 2 13.6
13.3 5 .83
127 zt 8.4
2.5 ,ug CG
15.5 5 1.40
148 + 13.2
5.0 pg CG 10.0 pg CG
17.1 t .25
163 k 26.0
21.0 + 1.70
200 + 19.4
3.1 2 .28
29 !I 2.9
10.0 pg CG + EDTA*
Percent of Control
One ml aliquots of a specific granule derived latent HNC preparation were treated one hour (37°C) with O-10 pg puified cathepsin G, followed by addition of 1 mM DFP. Collagenase activities in the treated and untreated preparations were then determined as described in the methods section. Values in table denote mean (n =3) + SD collagenase activity in treated or untreated aliquots. “EDTA (1.0 mM) added prior to determination of coIlagenase activity.
with HOC1 alone (Table 1). Incubation
of HNC with cathepsin G beyond two hours did not
result in further increases in collagenase activity and incubation beyond eight hours resulted in loss of activity (data not shown). Effects of HOC1 scavenger on activated HNC. latent HNC by exogenous hypochlorite collagenase activity was determined
To determine whether activation
of
persisted in the presence of HOC1 scavenger,
in aliquots of HNC preparation
to which 10 mM L
methionine was added following one hour treatment of the preparation with 100 uM HOCl. Addition
of L-methionine
resulted in significant, but not complete attenuation
mediated HNC activation (Figure 2). L-methionine HNC preparations
of HOCI-
had no effect on collagenase activity in
treated with cathepsin G (Figure 2) or mersalyl (data not shown).
Combined affects of HOC1 and cathepsin G on HNC activation.
Treatment
of the
latent HNC preparation with HOC1 and cathepsin G simultaneously resulted in significantly greater collagenase activity relative to preparations treated with HOC1 or cathepsin G alone (Figure 3). Comparable
but slightly higher incremental
increases in collagenase activity
were observed when latent HNC preparations were treated sequentially with cathepsin G for one hour (terminated
with DFP) then 50 I.LM HOCl. Addition
of L-methionine
following
treatment of HNC preparations with HOC1 and cathepsin G resulted in collagenase activity comparable to preparations in HNC preparations
treated with cathepsin G alone (Figure 3). Collagenase activity
treated concurrently
with HOC1 and cathepsin G in the presence of 563
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ncontrol
RESEARCH COMMUNICATIONS 0 Control E;gt,epsin
fS8 Cathepsin
G *
G
ET!3Cothepmn G + HOC1 ~Cothepsm G -DFP+HOCI CHI Cathepsin C +HOCL-Methi
Methionine
Figure 2. Effects of an HOC1 scavenger on HNC activity. Aliquots (2.0 ml) of latent HNC preparation were incubated for one hour (37°C) in the presence or absence of either 100 PM HOC1 or 10 bg/ml cathepsin G. Following incubation, 1 mM DFP was added to each aliquot, followed by addition of Lmethionine (10 mM final concentration) to half of each aliquot. Bar heights denote mean (n=3)+SD collagenase activities determined for each treated and untreated sample, determined as described in the methods section. Comparable results were obtained in identical experiments using two other latent HNC preparations. Figure 3. Combined Effects of HOC1 and Cathepsin G on HNC Activity. Aliquots (2.0 ml) of each latent HNC preparation were incubated one hour (37°C) in the presence or absence of either 100 yM HOCI, 10 pg/‘ml cathepsin G, or 100 PM HOC1 and 10 pg/ml cathepsin G simultaneously. An additional aliquot of each preparation was incubated one hour with 10 fig/ml of cathepsin G followed by addition of 1 mM DFP and 100 PM HOCl. Prior to determination of collagenase activity (described in methods), 10 mM L-methionine was added to half of the aliquot treated with HOC1 and cathepsin G concurrently. Bar heigths denote mean (n=3)tSEM percent increases in collagenase activity for treated aliquots relative to activity in corresponding control (untreated) preparations.
1.0 mM DFP was comparable to that noted for preparations
treated with HOC1 alone (data
not shown). Regardless of the dose of oxidant or organomercurial or mersalyl to aliquots of latent HNC pretreated
used, addition of HOC1
two hours with cathepsin G resulted in
collagenase activity exceeding that in the cathepsin G treated preparation latent HNC aliquots treated only with the corresponding concentration
as well as that in
of HOC1 or mersalyl
(Figure 4).
DISCUSSION Since imunoglobulin
latent
HNC
secreted
into
phagolysosomes
surrounding
tissue bound
may interact with both HOC1 and cathepsin G, the relative effects of these
PMN products on HNC activation confirm that HOC1 concentrations
are of significant interest. The results presented here comparable to those generated during PMN stimulation 564
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A-ACGtMedyl *- - -* hkrsoiyi olane
O-OCGWHOCL .-0 HOCL olone 404
6
0 A0
10
100
0
0
1
.l Mersolyl
HOC1 (/Ad)
2.5
(mM)
Figure 4. Enhancing effects of HOC1 (4A) or Organomercurial (4B) on HNC Preparations Treated or Untreated with Cathepsin G. Latent HNC preparation was incubated for two hours (37°C) in the presence or absence of 10 pg/ml cathepsin G. Following addition of 1 mM DFP, O-100 PM HOC1 or O-5 mM mersalyl (final concentrations) were added to 1.0 ml aliquots of both cathepsin G treated and untreated preparations prior to determination of collagenase activity. Values depicted on graphs represent mean (“SD) of triplicate determinations of collagenase activity for each sample. * p < .05 CG*SObM HOC1 vs 50 PM HOC1 alone.
with SAIgG (50pM)
(5) are capable of enhancing collagenase activity in preparations
of
latent HNC. These results also confirm that HOC1 can activate HNC in the absence of cathepsin G (4), as there was no detectable preparation,
cathepsin G activity in the final HNC
and addition of DFP to the latent HNC preparation did not alter the enhancing
affects of HOC1 on collagenase activity. Although initial cleavage of HNC by cathepsin G during cell lysis and preparation unlikely
as cell sonication
performed
of the specific granule extracts may have occurred, this is
and treatment
of the granules with Triton
were
in the presence of DFP.
The observed loss of collagenase activity following preparations
detergent
of HOCI-activated
addition of HOC1 scavenger to
HNC suggests a potential mechanism of regulating HNC
activity in vivo, in that loss of enzymatic activity may occur during diffusion activated HNC
away from the phagolysosome
scavenged. This observation organomercurial organomercurials,
mediated
into a milieu
where HOC1 is readily
is analagous to findings in a previous activation
HOC1 is thought
report
of HNC was shown to be reversible to mediate activation
cysteine sulfhydryl moeity contained within a PRCGVPD metal ion (12,13). The failure of methionine
of HOC1
in which (11). Like
of HNC by dissociating
the
sequence from the active site zinc
concentrations
one hundred-fold
in excess of
HOC1 to completely reverse HOC1 mediated enhancement of collagenase activity could be 565
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due to greater affinity organomercurial
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of the oxidant for the cysteine sulhydryl group.
activated zinc-metalloproteases
Alternatively,
have been shown to undergo autocatalytic
cleavage with retention of enzyme activity (14), and a similiar occurance for HOC1 activated HNC may account for a portion of the enzyme preparation
remaining active in the presence
of HOC1 scavenger. Although treatment of latent HNC with cathepsin G alone resulted in augmentation collagenase activity comparable
to that noted for HOC1 treated preparations,
of
the even
greater increase in collagenase activity noted following treatment with HOC1 and cathepsin G concurrently
suggests the effects of HOC1 and cathepsin G on HNC activity may be
additive. Augmented proteolytic activation of HNC by cathepsin G in the presence of HOC1 could account for these observations, but comparable were noted in preparations Furthermore,
if not higher collagenase activities
treated sequentially with cathepsin G, DFP, and then HOCl.
addition of HOC1 scavenger to preparations
HOC1 and cathepsin G simultaneously noted for preparations activity independent
which had been treated with
resulted in collagenase activity comparable to that
treated with cathepsin G alone, suggesting HOC1 effects on enzyme of those mediated by cathepsin G.
Higher collagenase activity observed in mixtures treated with HOC1 and cathepsin G in combination
could also be accounted for by activation
enzyme relative
to preparations
of a greater percentage of latent
treated with either agent alone.
However,
maximizing cathepsin G mediated HNC activation by two hour incubation with cathepsin demonstrated
G, the subsequent
addition
despite
of latent HNC
of HOC1 or mersalyl in concentrations
to optimize HNC activity resulted in collagenase activity which exceeded that
in the cathepsin G treated preparation the optimal concentrations
as well as latent HNC preparations
treated only with
of HOC1 or mersalyl. These results, suggesting independant
effects of cathepsin G and HOC1 on HNC activity that are additive, may prove to be an important consideration in developing interventions to abrogate tissue injury by neutrophils. Future studies correlating
molecular characteristics of HNC treated with HOC1 and/or
cathepsin G with characteristics of HNC generated in inflamed tissues may further clarify which of these potential mechanisms of regulating HNC activity are operative in vivo.
REFERENCES 1.
2. 3.
Murphy, G., Reynolds, J.J. Bretz, U., and Baggiolinz, M. (1977) Biochem J. 162,195 197. Chatham, W. W., Heck, L.W., and Blackburn, W.D., Jr. (1990) Arthritis and Rheumatism 33, 228-234. Weiss, S.J., Peppin, G., Ortiz, X., Ragsdale, C. and Test, S.T. (1985) Science 227, 747-749. 566
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AND
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Saari, H., Suomalainen, K., Lindy, O., Kontinnen, Y., and Sorsa, T. (1990) Biochem. Biophys. Res. Comm. 171, 979-987. Chatham, W.W., Heck, L.W., and Blackburn, W.D., Jr. (1992) Matrix (supplement), In press. Capodici, C., Muthukumaran, G., Amorusu, M., and Berg, R.A. (1989) Inflammation 13, 245-258. Henson, P.M. and Johnston, R.B. (1987) J. Clin. Invest. 79, 669-674. Borregard, N., Heiple, J.M., Simons, E.R., and Clark, R.A. (1983) J. Cell Biol., 97, 52-61. Heck, L.W., Rostand, K.S., Hunter, F.A., and Bhown, A. (1986) Anal. Biochem., 158, 217-227. Johnson-Win& B. (1990) Anal. Biochem. 104, 175-181. Mookhtiar, K.A. and VanWart, H.E. (1990) Biochemistry 29, 10620-10627. Springman, E.B., Angleton, E.L., Birkedal-Hansen, H., and VanWart, H.E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 364-368. Hasty, K.A., Pourmotabbed, T.F., Goldberg, E.I., Thompson, J.P., Spinella, D.G., Stevens, R.M. and Mainardi, C.L. (1990) J. Biol. Chem. 265, 11421-11424. Grant, G., Eisen, A.Z., Marmar, B.L., Roswit, W.T. and Goldberg, G.I. (1987) J. Biol. Chem. 262, 5886-5889.
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