Inhibition of Human Leukocyte Elastase Bound to Elastin: Relative Ineffectiveness and Two Mechanisms of Inhibitory Activity Heather M. Morrison, Howard G. Welgus, Robert A. Stockley, David Burnett, and Edward J. Campbell Respiratory and Critical Care and Dermatology Divisions, Department of Internal Medicine, Jewish Hospital at Washington University Medical Center, St. Louis, Missouri and Lung Immunobiochemical Research Laboratory, General Hospital, Birmingham, United Kingdom
Human leukocyte elastase (HLE) has been demonstrated on lung elastic fibers in areas of pulmonary emphysema. In vitro studies in our laboratory have shown that HLE-elastin complexes may be remarkably stable. We tested the possibility that elastin-bound HLE may retain catalytic activity in the presence of inhibitors that are effective against free HLE and found: (1) alpha-l-proteinase inhibitor (aIPI), antileukoprotease (ALP), and eglin C inhibited free HLE on an approximately 1:1 molar basis, measured with either 3H-elastin or a synthetic peptide substrate; (2) the ability of each inhibitor to control catalytic activity of HLE when complexed with elastin was impaired (e.g., ina 24-h assay, a 70-fold molar excess of alPI gave only 93 % inhibition of HLE); and (3) a chloromethyl' ketone inhibitor of HLE gave qualitatively similar results, although at the low enzyme concentrations used it was a less effective inhibitor of free and elastin-bound enzyme than were the polypeptide inhibitors. Further, we found evidence for two distinct mechanisms of inhibition of elastin-bound HLE. alPI and eglin C prevented elastin solubilization largely by enhancing net dissociation of HLE from the complexes; enzyme remaining bound to the substrate retained essentially full activity. In contrast, ALP and the chloromethyl ketone prevented elastin solubilization by binding to the complexes and inhibiting the enzyme in situ. These results may have implications regarding progressive elastin solubilization in vivo and should stimulate further investigation of enzyme activity in heterogeneous systems in which one or more reactants are insoluble.
Human leukocyte elastase (HLE) is a serine neutral proteinase found in peroxidase-containing cytoplasmic granules of polymorphonuclear neutrophils (1) and monocytes (2-5). It may also be found in alveolar macrophages (2-4,6). Both neutrophils and monocytes promptly release substantial amounts of the enzyme in response to appropriate stimuli (1, 5). Considerable evidence now implicates HLE in the pathogenesis
Key Kbrds: human leukocyte elastase, proteinase inhibitor, elastin (Received in original form June 12, 1989 and in revised form September 8, 1989)
Addresscorrespondence to: Edward J. Campbell, M.D., Division of Respiratory, Critical Care, and Occupational Pulmonary Medicine, Department of Medicine, University of Utah Health Sciences Center, 50 N. Medical Drive, Salt Lake City, UT 84132. Dr. Morrison's present address is: Chest Medical Unit, Papworth Hospital, Cambridge, United Kingdom. Dr. Campbell's present address is: Division of Respiratory, Critical Care, and Occupational Pulmonary Medicine, Department of Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah.
Abbreviations: alpha-l-proteinase inhibitor, aIPI; antileukoprotease, ALP; human leukocyte (neutrophil) elastase, HLE; methoxysuccinyl-talajj-proval-chloromethyl ketone, MSAP-CMK; methoxysuccinyl-talaj-pro-valparanitroanilide, MSAPN. Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 263-269, 1990
of injury to elastin fibers in the lung parenchyma, with subsequent development of pulmonary emphysema (7-9). Damiano and colleagues have demonstrated accumulation of HLE on interstitial elastin in areas of the human lung affected by emphysema (10); the local severity of emphysema was strongly correlated with the amount of HLE present. The rare neutrophils found in this study suggested that the enzyme, once associated with elastin, may be quite persistent. In vitro studies in our laboratory (11) substantiate this view, because HLE bound with high affinity to sites on elastin and showed little tendency to dissociate spontaneously. HLE bound to high-affinity sites on elastin is catalytically active (11) and therefore has the potential to perpetuate elastolytic injury. The ability of antielastases to inactivate HLE bound to elastin is thus of critical importance. Inhibitors of HLE, such as alpha-l-proteinase inhibitor (aIPI) and alpha-2-macroglobulin, may be of limited effectiveness in the pericellular microenvironment (12). In vitro studies have shown that neutrophils and monocytes in close apposition to extracellular matrix components can express HLE-mediated proteolytic activity in the presence of proteinase inhibitors (5, 13-21). Consequently, HLE released from neutrophils or monocytes in close proximity to interstitial elastin may be able to bind with high affinity to elastin while retaining catalytic activity.
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Previous studies have shown variability in the capacity of various inhibitors to inhibit HLE bound to elastin (22-24), but these studies have not addressed mechanisms underlying such inhibition. In this report, we compare the efficacy of a variety of inhibitors against catalytic activity of HLE bound to elastin. We show that all are relatively ineffectual against elastin-bound HLE, and we demonstrate two distinct mechanisms of inhibitory activity.
Materials and Methods Materials Bovine albumin, glucose oxidase, B-D( +)-glucose, sodium m~!abisulfite, potassium iodide, fluorescein isothiocyanate, Brij 35, and SephadexG-25 were obtained from Sigma Chemical Co. (St. Louis, MO). Trasylol-sepharose was prepared according to the procedure of Baugh and Travis (25). Elastase and Elastase Substrates T~e el~s~ase substrate methoxysuccinyl-(ala)2-pro-val-paramtroamhde (MSAPN) was purchased from Sigma. Tritiated bovine ligamentum nuchae elastin (Elastin Products Co., Pacific, MO) was prepared as described by Banda and colleagues (26) and had a specific activity of 907 dpm/ iig. Part of the same batch of elastin was fluoresceinated by dialyzing fluorescein isothiocyanate against elastin (25 mg fluorescein/g elastin, in 0.1 M carbonate/bicarbonate buffer; pH 9.5) at room temperature. Release of soluble tritium and of fluorescein from elastin were each linear with respect to both HLE added and time. Human leukocyte elastase (HLE) was prepared from purulent sputum (27) and was 100% active when titrated against the active site titrant Z-ala-ala-pro-aala-ONP (28). Na 125I (100 mCi/ml; Amersham, Arlington Heights, IL) was used to radio-iodinate a batch of the HLE using a modification of the Marchalonis method described earlier (6, 29). Proteinase Inhibitors Alpha-I-proteinase inhibitor (aIPI) was a gift from Dr. 1. Pierce (Washington University Medical Center, St. Louis, MO); Dr. 1. Kramps (University of Leiden, Netherlands) provided the antileukoprotease (ALP). Eglin C was a gift from Dr. H.-P. Schnebli (Ciba-Geigy Limited, Basel, Switzerland). Methoxysucciny1-(ala)2-pro-val-chloromethy1 ketone (MSAP-CMK) was from Enzyme System Products (Dublin, CA). The activities of porcine pancreatic elastase and trypsin were determined with the active site titrants Z-ala-ala-pro~ala-ONP and p-nitrophenyl p'-guanidinobenzoate, respectively (28, 30). Using these enzymes, the preparation of oPl was shown to be 63 % active. The activity of ALP and eglin C were measured using active site titrated HLE (31). All concentrations of HLE and inhibitors indicated refer to the amount of active protein present. Inhibitory Activity against Free HLE Various amounts of each inhibitor were preincubated with HLE for 15 min at 37° C. Residual catalytic activity was then
determined, using either 3H-elastin (320 p.g)1 or the peptide substrate MSAPN (final concentration, 0.8 mM). Assays using 3H-elastin were incubated for 24 h at 37° C, after ~hich supernatant fluids were counted in a liquid scintillanon counter (Rackbeta; LKB Instruments, Inc., Gaithersburg, MD). Assays employing MSAPN were incubated for 5 min at 37° C, after which the optical absorbance at 410 nm was ~easured. These and all subsequent assays were performed in phosphate-buffered normal saline (0.05 M phosphate, 0.15 M NaCI; pH 7.4), containing 0.067% Brij 35. Inhibitory Activity against HLE Bound to Elastin HLE-elastin complexes were prepared by incubating HLE (28.6 nM) with 3H-elastin (13.8 mg) at 0° C for 15 min and were then rapidly washed in ice-cold buffer. This incubation has previously been shown to allow equilibration of HLE binding to elastin, while not allowing significant catalysis of the substrate (11). The proportion of HLE remaining free, and hence the amount of HLE bound to 3H-elastin, was determined by measurement of the catalytic activity of the supernatant fluid against MSAPN after the incubation, as previously described (11). The HLE-3H -elastin complexes (equivalent to 320 p.g 3H-elastin) were then added to various amounts of each inhibitor (0 to 83.3 nM aIPI, eglin C, and ALP; 0 to 1.7 p.M MSAP-CMK) and were incubated at 37° C for 24 h. After incubation, the amount of 3H-elastin solubilized was determined. Effect of Inhibitors upon Dissociation of HLE from Elastin To gain insight into the mechanisms of inhibition of elastinbo~nd HLE, enzyme-substrate complexes were prepared usmg 125I-Iabeled HLE and elastin-fluorescein. This experimental design allowed assessment of the effect of inhibitors on: (1) the catalytic activity of HLE bound to elastin and (2) dissociation of HLE from elastin. HLE-elastin complexes were prepared by mixing 1251_ HLE (11.7 nM; sp act, 4,300 cpm/ng) with elastin-fluorescein (3 mg) at 0° C for 15 min. The enzyme-substrate complexes were washed rapidly 3 times. Complexes (equivalent to 100 p.g elastin-fluorescein) were added to each inhibitor or (as a control) to albumin. The final concentrations of aIPI, eglin C, ALP, and albumin were 31 nM; the final concentration of MSAP-CMK was 3.1 p.M. After incubation at 37° C, the pelleted elastin was washed twice with ice-cold buffer. The effect of the inhibitors on dissociation of HLE from elastin was determined by counting the pellets and supernatant fluids from the above assays in a gamma counter (Clinigamma; LKB Instruments). Elastolytic activity was determined by measuring solubilized fluorescein (A4 System, Farrand Optical, Aminco-Bowman, Inc., Silver Spring, MD; excitation filter = Corning 7-59 [wavelength approximately 380 nm]; emission filter = Corning 3-70 [wavelength lBec~use substrate "concentration" has no physical meaning for insoluble elastin, mass of substrate rather than concentration will be given.
Morrison, Welgus, Stockley et al.: Mechanisms of Inhibition of Elastin-bound Elastase
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Results Inhibitory Activity against Free HLE Data regarding the ability of aIPI, eglin C, and ALP to inhibit the degradation of 3H-elastin and MSAPN are shown in Figures 1 and 2. In all cases, preincubation of HLE with inhibitor prior to addition of substrate resulted in essentially linear inhibition profiles, Linear regression analysis of these results enabled the inhibitor:HLE molar ratio at functional equivalence
Figure 2. Activity of free HLE or elastin-bound HLE versus substrate in the presence of low molecular weight elastase inhibitors. The experimental procedure was as described for Figure 1; triangles = elastin substrate, circles = MSAPN substrate, and squares = HLE-elastin complexes. (a) Eglin C; (b) ALP; (c) MSAP-CMK. Note that the effectiveness of each inhibitor against elastin-bound HLE was relatively impaired.
(i.e., zero enzyme activity) to be determined (32), The results of these analyses are presented in Table 1. Figures 1 and 2 and Table 1 demonstrate that aIPI, eglin C, and ALP all inhibited free HLE at approximately a 1:1 molar basis when enzyme activity was assessed with the pep-
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TABLE 1
Inhibitor-Hl.E molar ratios at functional equivalence Molar Ratios* Inhibitor
alPI ALP Eglin C MSAP-CMK
MSAPNt
3H-elastint
1.08 0.94 1.22 30.01
1.47 1.59 1.12 25.70
* Inhibition profiles were drawn as in Figures 1 and 2. The inhibitor:HLE molar ratio at the point of functional equivalence (zero enzyme activity) was calculated by linear regression analysis. t Residual enzyme activity was determined by the addition of MSAPN or 3H-elastin.
tide substrate MSAPN. When enzyme activity was assessed with 3H-elastin, similar results were obtained. However, with alPI and ALP, greater amounts of inhibitor were required to abolish enzymatic activity when 3H-elastin was used as substrate (molar ratios at functional equivalence 36% and 69% greater, respectively). This discrepancy has been noted previously (31) and may reflect some degree of dissociation of inhibitor from enzyme in the more prolonged assay (24 h versus 5 min), or to other mechanisms. Nevertheless, the similar effectiveness of inhibition of enzymatic activity against the two substrates indicates that the HLE-inhibitor complexes are very stable over at least 24 h. For MSAP-CMK, the molar ratio that was required for complete inhibition of the enzyme concentration tested (20 nM) was more than an order of magnitude higher than for other inhibitors (Figure 2c and Table 1). The concentration of HLE used in all experiments was low to permit use of the sensitive substrate MSAPN, and the relatively low K of MSAP-CMK (as reported by Hornebeck and Schnebli [23]) could explain the high molar amounts of the inhibitor necessary to obtain functional equivalence in the present study. To confirm this possibility, we measured the residual HLE activity against MSAPN after incubation with a 10-fold molar excess of MSAP-CMK at progressive dilutions of the inhibitor and enzyme (HLE, 1.8 J.tM to 18 nM). At the highest concentrations tested (MSAP-CMK, 18 J.tM; HLE, 1.8 J.tM), HLE retained 3 % activity. When the concentrations of both enzyme and inhibitor were reduced 100-fold, the enzyme retained 41% activity, despite the MSAPCMK:HLE molar ratio remaining constant. Similar inhibition of free HLE at any particular MSAP-CMK concentration was noted, whether assessed by MSAPN or by 3H-elastin. These results confirm that high MSAP-CMK:HLE molar ratios are required for inhibition at the low (20 nM) HLE concentrations employed here, due to the relatively low K, of MSAP-CMK. Inhibitory Activity against HLE Bound to Elastin Figures 1 and 2 also show data regarding inhibition of HLE that had previously been allowed to bind to elastin. Each of the inhibitors was markedly less effectiveagainst HLE when complexed with elastin in comparison with its effectiveness against free HLE. Inhibition of HLE-elastin complexes by up to an approximately 5-fold molar excess of o.Pl (Figure 1a) was mark-
edly decreased in comparison with that of free HLE. Further increases, up to a 70-fold molar excess of aIPI, gradually increased inhibition but to only 93 % (Figure 1b). Similarly reduced effectiveness of eglin C (Figure 2a) as an inhibitor of elastin-bound HLE was observed. When the inhibitory activity of ALP was tested in an identical manner (figure 2b), similar results were obtained. For relatively high molar ratios of inhibitor to elastin-bound HLE, however, somewhat more effective inhibition was achieved than that observed with either alPI or eglin C. At a 9:1 molar ratio of ALP to HLE, for example, 95% inhibition of elastin-bound HLE was observed. Figure 2c shows similarly reduced effectiveness of MSAPCMK as an inhibitor of HLE after binding to elastin. Up to nearly 200-fold excess of inhibitor produced only 82 % inhibition of elastin-bound HLE. Taken together, the results in Figures 1 and 2 indicate that a proportion of elastin-bound HLE is relatively resistant to inhibition by a diverse group of inhibitors, even when the latter are present in marked excess. Mechanism of Inhibition of Elastin-bound HLE To gain insight into the relative resistance of elastin-bound HLE to inhibition, and to begin to explore mechanisms by which inhibition of elastin-bound HLE occurs, we examined the ability of various inhibitors to dissociate HLE from elastin. When 125I-HLE was bound to elastin-fluorescein and then incubated at 37° C, the amount of '25I-HLE persistently bound to elastin could be determined by counting the elastin pellets. The catalytic activity of the enzyme could be assessed in parallel by measuring fluorescent elastin peptides released from the substrate. Inhibition of elastin-bound HLE by all tested inhibitors was slow. Preliminary experiments with incubation times of 1, 3, and 6 h revealed that both dissociation of 125I-HLE from elastin, and also inhibition of catalytic activity, were minimal at 1 h and increased with prolongation of incubation time. By 6 h (Figure 3), substantial inhibition of catalytic activity was observed with all inhibitors tested. In the experiment shown, aIPI, ALP, eglin C, and albumin were present in a 7-fold molar excess relative to bound HLE, whereas MSAP-CMK was present in approximately 700-fold molar excess in the assay. In the control assays (no inhibitor present), the amount of catalytic activity was appropriate for the amount of enzyme bound at the end of the assay. As a further control, addition of albumin did not change either the amount of persistently bound HLE or the amount of observed catalytic activity. When a IPI was present (Figure 3), the amount of persistently bound HLE after 6 h was significantly reduced. However, the catalytic activity measured by release of fluorescent elastin peptides again was appropriate for the amount of persistently bound enzyme. Figure 3 also shows that similar results were obtained for eglin C. The results obtained when ALP and MSAP-CMK were used as inhibitors are also shown in Figure 3. In marked contrast to o.Pl and eglin C, these inhibitors did not enhance dissociation of HLE from elastin. However, the catalytic activity observed was markedly reduced in relation to the amount of HLE persistently bound to substrate (30.5% and 31.3% for ALP and MSAP-CMK, respectively).
Morrison, Welgus, Stockley et al.: Mechanisms of Inhibition of Elastin-bound Elastase
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Figure 3. Binding of HLE related to its catalytic activity in the
presence of inhibitors. 125I-HLE was bound to elastin-fluorescein at 0 0 C; the complexes were washed, and the amount of HLE bound was determined using a gamma counter. The complexes were incubated with the various inhibitors for over 6 h at 3r C, after which the HLE-elastin complexes were separated from the supernatants and washed. Elastin solubilization over the 6-h incubation was measured by fluorimetry of the supernatants. The amount of HLE remaining bound to elastin at 6 h was measured using a gamma counter. The amount of bound HLE that was active during the incubation was calculated by comparison with an HLE-elastin standard curve. Shaded bars = HLE bound; closed bars = HLE activity. Note alPI and eglin C dissociated HLE from the complexes, but enzyme remaining in complex was nearly fully active. In contrast, ALP and MSAP-CMK substantially inhibited the enzyme in situ without dissociating HLE from the complexes.
These results, taken together, indicate that alPI and eglin C enhance dissociation of HLE bound to elastin. However, even when measured over a several-hour period, these inhibitors have minimal capability to inhibit the enzyme while it is bound to substrate. In contrast, ALP and MSAPCMK do not appreciably enhance enzyme-substrate dissociation but are able to inhibit HLE while in complex with substrate.
Discussion The present results indicate that a heterogeneous group of proteinase inhibitors exhibits strikingly reduced effectiveness in inhibiting catalytic activity of HLE after the enzyme has been allowed to bind to elastin. Solubilization of elastin by HLE in the absence of proteinase inhibitors is linear with respect to time in this 24-h assay (11). Because even brief preincubation of HLE with these inhibitors resulted in functionally irreversible inhibition, it appears that catalytically important binding of HLE to elastin is highly stable over at least 24 h; thus, the various inhibitors were unable to arrest progressive elastin solubilization. Further, it appears that (in a functional sense) HLE does not leave the elastic fibers during catalytic action on this insoluble substrate, because the inhibitory activities of alPI and eglin C were impaired for the entire duration of the assay. We have previously estimated the turnover number of HLE against insoluble elastin to be 31.9 mol of tropoelastin equivalents/mol HLE/h (11). This approximated the minimal number of peptide bonds cleaved per enzyme molecule per
267
hour. Thus, despite moving among various sites of peptide bond scission on the elastic fibers, the enzyme does not freely interact with soluble proteinase inhibitors even during prolonged incubation. The physical meaning of this observation is unclear, because the biochemical nature of the highaffinity,catalytically active binding between HLE and elastin (11) is still poorly understood. The relative resistance of elastin-bound HLE to inhibition may relate to the very high local "concentration" of substrate on the surface of elastic fibers, or to other biochemical phenomena that are not yet understood. Further insights into these local processes await biochemical characterization of the interactions between HLE and elastin, as well as greater theoretical understanding of the surprising behavior of enzymes in heterogeneous systems (33, 34). The present results follow from recent work suggesting that HLE activity against insoluble elastin occurs at the surface of the elastic fiber and proceeds independently of substrate "concentration" (11). Only a small fraction of the available binding sites for HLE on elastin results in catalytic activity against the substrate; these binding interactions are very stable, and at such sites HLE displays a high affinity for substrate ([11]; equilibrium dissociation constant = 9 x 10-9 M). Our results are also in substantial agreement with other recent reports. Reilly and Travis (22) reported that o.Pl was less effective as an inhibitor of elastin-bound elastase than when HLE and o.Pl were incubated together prior to addition of elastin. Hornebeck and Schnebli (23) confirmed this observation and also demonstrated that eglin C (an inhibitor from the medicinal leech) and the synthetic inhibitor MSAP-CMK were both equally effective at inhibiting free or elastin-bound HLE. Our data differ somewhat from those in the latter report in that each inhibitor we tested was more effectiveagainst free than against elastin-bound enzyme; however, our results agree that a lesser discrepancy was noted for eglin C and MSAP-CMK than for alPI when inhibition of free and elastin-bound enzyme was compared. More recently, Bruch and Bieth (24) have shown that the bronchial inhibitor (or ALP) is a relatively efficient inhibitor of bound elastase and that it is capable of inactivating elastinbound HLE not inhibited by alPI. These observations are especially noteworthy in light of the findings of Damiano and colleagues (10) that immunoreactive HLE can be demonstrated on interstitial elastin in emphysematous human lungs, and that local presence of enzyme correlated with .local severity of pulmonary emphysema. The absence of neutrophils at such sites has suggested the possibility that HLE binding to lung elastin in vivo might be very persistent. Although no evidence for catalytic activity of the observed HLE was provided in the study by Damiano and colleagues, the present results and the studies cited above support the possibility that HLE persistently bound to elastin in vivo may be catalytically active for an undetermined duration (possibly many hours or longer), even in the presence of proteinase inhibitors. Substantial evidence suggests that HLE-mediated injury to lung elastin fibers may be important in the pathogenesis of pulmonary emphysema (7-9); moreover, the present results suggest one potential mechanism by which pulmonary emphysema might progress with time in laboratory animals after the intratracheal instillation of elastase (35, 36).
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For our results to have relevance to lung injury in vivo, HLE must reach (and bind to) elastic fibers prior to interacting with proteinase inhibitors. A substantial body of evidence in model systems in vitro indicates that at sites of close contact of neutrophils (13, 15-18, 20) and/or monocytes (5) with susceptible insoluble substrates, HLE can reach such substrates in a catalytically active state even when inhibitors are present. Potential mechanisms by which catalytic activity of HLE might exist in the immediate pericellular milieu have recently been reviewed (12, 15, 37). In addition to demonstrating relative ineffectiveness of inhibitors against elastin-bound HLE, the present work also begins to provide insights into the mechanisms by which such inhibition can be achieved. These results were somewhat surprising and were of considerable interest. Inhibition of elastin-bound HLE appeared to be achieved via one of two distinct mechanisms: (1) enhancement of net dissociation of HLE from elastin, with minimal inhibition' of HLE remaining bound to substrate or (2) inhibition of elastin-bound HLE in situ, without enhancing dissociation of the enzyme from substrate. o.Pl and eglin C were the two inhibitors tested that enhanced net dissociation ofHLE from elastin, yet were unable to inhibit HLE persistently bound to substrate. Presumably, the increased HLE dissociation was explained by competition between inhibitor and elastin for binding of enzyme, with catalytic inactivation of the enzyme occurring upon binding to inhibitor. Both o.Pl and eglin C have low equilibrium dissociation constants (K; = 3.3 X 10-14 [38] and 7.5 X 10-11 M [39], respectively). At inhibitor concentrations ofless than 3 nM, eglin C acts as a reversible, competitive inhibitor (40), but as a pseudo-irreversible inhibitor at higher concentrations (> 3 nM [39]). The concentration of eglin C used in all these experiments was greater than 3 nM. o.Pl forms covalent complexes with HLE (38). It is thus possible that these inhibitors could compete favorably with elastin for HLE binding, even near the surface of the elastic fiber. Explanation of the results obtained with ALP and MSAPCMK was more problematic. ALP is a cationic, competitive inhibitor (K = 1.87 X 10-10 M [41]) of low molecular weight. MSAP-CMK forms irreversible complexes with HLE, but much less rapidly than the polypeptide proteinase inhibitors. These inhibitors did not enhance dissociation of HLE from elastin. Instead, and in marked contrast to the effects of o.Pl and eglin C, these inhibitors arrested the catalytic activity of HLE remaining complexed to substrate. This was clearly demonstrated in experiments in which both bound enzyme and its catalytic activity could be measured. The mechanism underlying such in situ inhibition of HLE is of considerable interest and was not determined in our studies. MSAP-CMK and ALP are both of relatively low molecular weight (590 and 14,000 D, respectively). Although size may be an important factor in allowing an inhibitor to inactivate elastin-bound HLE in situ, it is not the sole prerequisite because eglin C (M, 8,100) fails to inhibit in this manner. It is possible that these inhibitors possess the correct combination of size, charge, and active site conformation to allow in situ inhibition, whereas o.Pl and eglin C rapidly form sta-
ble complexes with a small fraction of the enzyme molecules that are in transit between sites of peptide bond scission. The ability of ALP to inhibit HLE bound to elastin may be of importance in vivo, since Willems and associates have shown ALP associated with elastin fibers in alveolar septae of human lungs removed surgically (42). It is possible that one physiologic role of ALP is to inhibit HLE in situ on elastin, whereas that of o.Pl is to restrict elastolytic activity in solution (24). We believe that the present work should encourage further investigation of the biochemical mechanisms of elastase-elastin interactions as well as mechanisms of inhibition of the catalytic activity of elastases bound to insoluble substrates. Such work may have substantial biologic relevance to the pathogenesis of alveolar septal destruction; moreover, it may improve our level of understanding of enzymatic activity in biologically common heterogeneous systems in general (34), such as that which occurs during solubilization of other extracellular matrix macromolecules. Acknowledgments: This work was supported by a grant from the Medical Research Council of Great Britain; by Grants HL-29594 and AM-35805 from the U,S. Public Health Service, by Grants 1252 and 1742 from the Council for Tobacco Research, USA, Inc., and by the British Lung Foundation. Dr. Welgus is a recipient of U. S. Public Health Service Research Career Development Award AM-1525. Dr. Stockley is a Wolfson Research Fellow of the Royal College of Physicians of London. The writers also wish to acknowledge the excellent technical assistance of S. K. Endicott and M. A. Campbell.
References I. Havemann, K., and A. Janoff. 1978. Neutral Proteases of Human Polymorphonuclear Leukocytes. Baltimore: Urban and Schwartzenberg. 2. White, R. R., A. Janoff, R. Gordon, and E. J. Campbell. 1982. Evidence for in vivo internalization of human leukocyte elastase by human alveolar macrophages. Am. Rev. Respir. Dis. 125:779-781. 3. Hinman, L. M., C. A. Stevens, R. A. Matthay, and J. B. L. Gee. 1980. Elastase and lysozyme activities in human alveolar macrophages. Effects of cigarette smoking. Am. Rev. Respir. Dis. 121:263-271. 4. Campbell, E. J., R. R. White, R. M. Senior, R. J. Rodriguez, and C. Kuhn III. 1979. Receptor-mediated binding and internalization of leukocyte elastase by alveolar macrophages in vitro. J. Clin. Invest. 64:824-833. 5. Campbell, E. J., E. K. Silverman, and M. A. Campbell. 1989. Elastase and cathepsin G of human monocytes. Quantification of cellular content, release in response to stimuli, and heterogeneity in elastase-mediated proteolytic activity. J. Immuno/. (In press) 6. Campbell, E. J. 1982. Human leukocyte elastase, cathepsin G, and lactoferrin: a family of neutrophil granule glycoproteins which bind to an alveolar macrophage receptor. Proc. Nail. Acad. Sci. USA 79:6941-6945. 7. Janoff, A. 1985. State of the art. Elastases and emphysema. Am. Rev. Respir. Dis. 132:417-433. 8. Niewoehner, D. E. 1988. Cigarette smoking, lung inflammation, and the development of emphysema. J. Lab. Clin. Med.lll: 15-27. 9. Wewers, M. 1989. Pathogenesis of emphysema: assessment of basic science concepts through clinical investigation. Chest 95: 190-195. 10. Damiano, V. V., A. Tsang, U. Kucich et al. 1986. Immunolocalization of elastase in human emphysematous lungs. J. Clin. Invest. 78:482-493. II. Morrison, H. M., H. G. Welgus, R. A. Stockley, D. Burnett, and E. J. Campbell. 1989. Studies on the interaction between human leukocyte elastase and bovine elastin. Evidence for two classes of enzyme binding sites. (Submitted) 12. Campbell, E. J., R. M. Senior, and H. G. Welgus. 1987. Extracellular matrix injury during lung inflammation. Chest 92: 161-167. 13. Campbell, E. J., R. M. Senior, J. A. McDonald, and D. L. Cox. 1982. Proteolysis by neutrophils. Relative importance of cell-substrate contact and oxidative inactivation of proteinase inhibitors in vitro. J. Clin Invest. 70:845-852. 14. Campbell, E. J., and M. A. Campbell. 1987. Proteolysis by neutrophils while in contact with substrate: incomplete protection of substrate by proteinase inhibitors. In Pulmonary Emphysema and Proteolysis. Vol. II. C. Mittman andJ. C. Taylor, editors. Academic Press, New York. 235-244. 15. Campbell, E. J., and M. A. Campbell. 1988. Pericellular proteolysis by neutrophils in the presence of proteinase inhibitors: effects of substrate opsonization. J. Cell Bioi. 106:667-676.
Morrison, Welgus, Stockley et al.: Mechanisms of Inhibition of Elastin-bound Elastase
16. Sibille, Y., J. S. Lwebuga-Mukasa, L. Palomski, W.W. Merrill, D. H. Ingbar, and J. B. L. Gee. 1986. An in vitro model for polymorphonuclearleukocyte-induced injury to an extracellular matrix: relative contribution of oxidants and elastase to fibronectin release from amniotic membranes. Am. Rev. Respir. Dis. 134:134-140. 17. Weiss, S. J., and S. Regiani. 1984. Neutrophils degrade subendothelial matrices in the presence of alpha-I-proteinase inhibitor: cooperative use of lysosomal proteinases and oxygen metabolites. J. Clin. Invest. 73: 1297-1303. 18. Weiss, S. J., J. T. Curnutte, and S. Regiani. 1986. Neutrophil-mediated solubilization of the subendothelial matrix: oxidative and nonoxidative mechanisms of proteolysis used by normal and chronic granulomatous disease phagocytes. J. Immunol. 136:636-641. 19. Schalkwijk, J., W. B. Van den Berg, L. B. A. Van de Putte, and L. A. B. Joosten. 1987. Elastase secreted by activated polymorphonuclear leucocytes causes chondrocyte damage and matrix degradation in intact articular cartilage: escape from inactivation by alpha-I-proteinase inhibitor. Br. J. Exp. Pathol. 68:81-88. 20. Weitz, J. I., A. J. Huang, S. L. Landman, S. C. Nicholson, and S. C. Silverstein. 1988. Elastase-mediated fibrinogenolysis by chemoattractantstimulated neutrophils occurs in the presence of physiologic concentrations of antiproteinases. 1. Exp. Med. 166:1838-1850. 21. Johnson, K. J., and J. Varani. 1981. Substrate hydrolysis by immune complex-activated neutrophils: effect of physical presentation of complexes and protease inhibitors. J. Immunol. 127:1875-1879. 22. Reilly, C. F., and J. Travis. 1980. The degradation of human lung elastin by neutrophil proteinases. Biochim. Biophys. Acta 621: 147-157. 23. Hornebeck, W., and H. P. Schnebli. 1982. Effect of different elastase inhibitors on leukocyte elastase pre-adsorbed to elastin. Hoppe-Seyler's Z. Physiol. Chem. 363:455-458. 24. Bruch, M., and J. G. Bieth. 1986. Influence of elastin on the inhibition of leucocyte elastase by alpha I-proteinase inhibitor and bronchial inhibitor. Potent inhibition of elastin-bound elastase by bronchial inhibitor. Biochem. J. 238:269-273. 25. Baugh, R. J., and J. Travis. 1976. Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry 15:836-841. 26. Banda, M. J., H. F. Dovey, and Z. Werb. 1981. Elastinolytic enzymes. In Methods for Studying Mononuclear Phagocytes. D. O. Adams, P. J. Edelson, and H. Koren, editors. Academic Press, New York. 603-618. 27. Martodam, R. R., R. J. Baugh, D. Y. Twumasi, and I. E. Liener. 1979. A rapid procedure for the large scale purification of elastase and cathepsin G from human sputum. Prep. Biochem. 9: 15-31. 28. Powers, J. C., and B. F. Gupton. 1977. Reaction of serine proteases with
29. 30. 31.
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33. 34. 35. 36. 37. 38.
39. 40. 41. 42.
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aza-amino acid and aza-peptide derivatives. Methods Enzymol. 46: 208-216. Marchalonis, J. J. 1969. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 113:299-305. Chase, T., and E. Shaw. 1967. p-Nitrophenyl-p'-guanidinobenzoate HCI: a new active site titrant for trypsin. Biochem. Biophys. Res. Commun. 29:508-514. Kramps,1. A., H. M. Morrison, D. Burnett, 1. H. Dijkman, and R. A. Stockley. 1987. Determination of elastase inhibitory activity of alpha,proteinase inhibitor and antileukoprotease: different results using insoluble elastin or synthetic low molecular weight substrates. Scand. J. Clin. Lab. Invest. 47:405-410. Boudier, c.. A. Pelletier, G. Pauli, and 1. G. Bieth. 1983. The functional activity of alpha I-proteinase inhibitor in bronchoalveolar lavage fluids from healthy human smokers and non-smokers. Clin. Chim. Acta. 132: 309-315. Welgus, H. G., 1. 1. Jeffrey, and A. Z. Eisen. 1981. Human skin fibroblast collagenase: assessment of activation energy and deuterium isotope effect with collagenous substrates. J. BioI. Chem. 256:9516-9521. Kopelman, R. 1988. Fractal reaction kinetics. Science 241:1620-1626. Snider, G. L., and C. B. Sherter. 1977. A one-year study of the evolution of elastase-induced emphysema in hamsters. J. Appl. Physiol. 43:721-729. Kucich, U., P. Christner, G. Weinbaum, and J. Rosenbloom. 1980. Immunologic identification of elastin-derived peptides in the serum of dogs with experimental emphysema. Am. Rev. Respir. Dis. 122:461-466. Weiss, S. J. 1989. Tissue destruction by neutrophils. N. Engl. J. Med. 320:365-376. Beatty, K., N. Matheson, and J. Travis. 1984. Kinetic and chemical evidence for the inability of oxidized alpha -proteinase inhibitor to protect lung elastin from elastolytic degradation. Hoppe-Seyler's Z. Physiol. Chem. 365:731-736. Braun, N. J., J. L. Bodmer, G. D. Virca et al. 1987. Kinetic studies on the interaction of eglin C with human leukocyte elastase and cathepsin G. Bioi. Chem. Hoppe-Seyler. 368:299-308. Baici, A., and U. Seemuller. 1984. Kinetics of inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis. Biochem. J. 218:829-833. Smith, C. E., and D. A. Johnson. 1985. Human bronchial leukocyte proteinase inhibitor. Rapid isolation and kinetic analysis with human leukocyte proteinases. Biochem. J. 225:463-472. Willems, L. N. A., C. J. M. Otto-Verberne, J. A. Kramps, A. A. W. ten Have-Opbroek, and J. H. Dijkman. 1986. Detection ofanti-Ieukoprotease in connective tissue of lung. Histochemistry 86: 165-168.
Human leukocyte elastase (HLE) has been demonstrated on lung elastic fibers in areas of pulmonary emphysema. In vitro studies in our laboratory have shown that HLE-elastin complexes may be remarkably stable. We tested the possibility that elastin-bound HLE may retain catalytic activity in the presence of inhibitors that are effective against free HLE and found: (1) alpha-l-proteinase inhibitor (aIPI), antileukoprotease (ALP), and eglin C inhibited free HLE on an approximately 1:1 molar basis, measured with either 3H-elastin or a synthetic peptide substrate; (2) the ability of each inhibitor to control catalytic activity of HLE when complexed with elastin was impaired (e.g., ina 24-h assay, a 70-fold molar excess of alPI gave only 93 % inhibition of HLE); and (3) a chloromethyl' ketone inhibitor of HLE gave qualitatively similar results, although at the low enzyme concentrations used it was a less effective inhibitor of free and elastin-bound enzyme than were the polypeptide inhibitors. Further, we found evidence for two distinct mechanisms of inhibition of elastin-bound HLE. alPI and eglin C prevented elastin solubilization largely by enhancing net dissociation of HLE from the complexes; enzyme remaining bound to the substrate retained essentially full activity. In contrast, ALP and the chloromethyl ketone prevented elastin solubilization by binding to the complexes and inhibiting the enzyme in situ. These results may have implications regarding progressive elastin solubilization in vivo and should stimulate further investigation of enzyme activity in heterogeneous systems in which one or more reactants are insoluble.
Human leukocyte elastase (HLE) is a serine neutral proteinase found in peroxidase-containing cytoplasmic granules of polymorphonuclear neutrophils (1) and monocytes (2-5). It may also be found in alveolar macrophages (2-4,6). Both neutrophils and monocytes promptly release substantial amounts of the enzyme in response to appropriate stimuli (1, 5). Considerable evidence now implicates HLE in the pathogenesis
Key Kbrds: human leukocyte elastase, proteinase inhibitor, elastin (Received in original form June 12, 1989 and in revised form September 8, 1989)
Addresscorrespondence to: Edward J. Campbell, M.D., Division of Respiratory, Critical Care, and Occupational Pulmonary Medicine, Department of Medicine, University of Utah Health Sciences Center, 50 N. Medical Drive, Salt Lake City, UT 84132. Dr. Morrison's present address is: Chest Medical Unit, Papworth Hospital, Cambridge, United Kingdom. Dr. Campbell's present address is: Division of Respiratory, Critical Care, and Occupational Pulmonary Medicine, Department of Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah.
Abbreviations: alpha-l-proteinase inhibitor, aIPI; antileukoprotease, ALP; human leukocyte (neutrophil) elastase, HLE; methoxysuccinyl-talajj-proval-chloromethyl ketone, MSAP-CMK; methoxysuccinyl-talaj-pro-valparanitroanilide, MSAPN. Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 263-269, 1990
of injury to elastin fibers in the lung parenchyma, with subsequent development of pulmonary emphysema (7-9). Damiano and colleagues have demonstrated accumulation of HLE on interstitial elastin in areas of the human lung affected by emphysema (10); the local severity of emphysema was strongly correlated with the amount of HLE present. The rare neutrophils found in this study suggested that the enzyme, once associated with elastin, may be quite persistent. In vitro studies in our laboratory (11) substantiate this view, because HLE bound with high affinity to sites on elastin and showed little tendency to dissociate spontaneously. HLE bound to high-affinity sites on elastin is catalytically active (11) and therefore has the potential to perpetuate elastolytic injury. The ability of antielastases to inactivate HLE bound to elastin is thus of critical importance. Inhibitors of HLE, such as alpha-l-proteinase inhibitor (aIPI) and alpha-2-macroglobulin, may be of limited effectiveness in the pericellular microenvironment (12). In vitro studies have shown that neutrophils and monocytes in close apposition to extracellular matrix components can express HLE-mediated proteolytic activity in the presence of proteinase inhibitors (5, 13-21). Consequently, HLE released from neutrophils or monocytes in close proximity to interstitial elastin may be able to bind with high affinity to elastin while retaining catalytic activity.
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Previous studies have shown variability in the capacity of various inhibitors to inhibit HLE bound to elastin (22-24), but these studies have not addressed mechanisms underlying such inhibition. In this report, we compare the efficacy of a variety of inhibitors against catalytic activity of HLE bound to elastin. We show that all are relatively ineffectual against elastin-bound HLE, and we demonstrate two distinct mechanisms of inhibitory activity.
Materials and Methods Materials Bovine albumin, glucose oxidase, B-D( +)-glucose, sodium m~!abisulfite, potassium iodide, fluorescein isothiocyanate, Brij 35, and SephadexG-25 were obtained from Sigma Chemical Co. (St. Louis, MO). Trasylol-sepharose was prepared according to the procedure of Baugh and Travis (25). Elastase and Elastase Substrates T~e el~s~ase substrate methoxysuccinyl-(ala)2-pro-val-paramtroamhde (MSAPN) was purchased from Sigma. Tritiated bovine ligamentum nuchae elastin (Elastin Products Co., Pacific, MO) was prepared as described by Banda and colleagues (26) and had a specific activity of 907 dpm/ iig. Part of the same batch of elastin was fluoresceinated by dialyzing fluorescein isothiocyanate against elastin (25 mg fluorescein/g elastin, in 0.1 M carbonate/bicarbonate buffer; pH 9.5) at room temperature. Release of soluble tritium and of fluorescein from elastin were each linear with respect to both HLE added and time. Human leukocyte elastase (HLE) was prepared from purulent sputum (27) and was 100% active when titrated against the active site titrant Z-ala-ala-pro-aala-ONP (28). Na 125I (100 mCi/ml; Amersham, Arlington Heights, IL) was used to radio-iodinate a batch of the HLE using a modification of the Marchalonis method described earlier (6, 29). Proteinase Inhibitors Alpha-I-proteinase inhibitor (aIPI) was a gift from Dr. 1. Pierce (Washington University Medical Center, St. Louis, MO); Dr. 1. Kramps (University of Leiden, Netherlands) provided the antileukoprotease (ALP). Eglin C was a gift from Dr. H.-P. Schnebli (Ciba-Geigy Limited, Basel, Switzerland). Methoxysucciny1-(ala)2-pro-val-chloromethy1 ketone (MSAP-CMK) was from Enzyme System Products (Dublin, CA). The activities of porcine pancreatic elastase and trypsin were determined with the active site titrants Z-ala-ala-pro~ala-ONP and p-nitrophenyl p'-guanidinobenzoate, respectively (28, 30). Using these enzymes, the preparation of oPl was shown to be 63 % active. The activity of ALP and eglin C were measured using active site titrated HLE (31). All concentrations of HLE and inhibitors indicated refer to the amount of active protein present. Inhibitory Activity against Free HLE Various amounts of each inhibitor were preincubated with HLE for 15 min at 37° C. Residual catalytic activity was then
determined, using either 3H-elastin (320 p.g)1 or the peptide substrate MSAPN (final concentration, 0.8 mM). Assays using 3H-elastin were incubated for 24 h at 37° C, after ~hich supernatant fluids were counted in a liquid scintillanon counter (Rackbeta; LKB Instruments, Inc., Gaithersburg, MD). Assays employing MSAPN were incubated for 5 min at 37° C, after which the optical absorbance at 410 nm was ~easured. These and all subsequent assays were performed in phosphate-buffered normal saline (0.05 M phosphate, 0.15 M NaCI; pH 7.4), containing 0.067% Brij 35. Inhibitory Activity against HLE Bound to Elastin HLE-elastin complexes were prepared by incubating HLE (28.6 nM) with 3H-elastin (13.8 mg) at 0° C for 15 min and were then rapidly washed in ice-cold buffer. This incubation has previously been shown to allow equilibration of HLE binding to elastin, while not allowing significant catalysis of the substrate (11). The proportion of HLE remaining free, and hence the amount of HLE bound to 3H-elastin, was determined by measurement of the catalytic activity of the supernatant fluid against MSAPN after the incubation, as previously described (11). The HLE-3H -elastin complexes (equivalent to 320 p.g 3H-elastin) were then added to various amounts of each inhibitor (0 to 83.3 nM aIPI, eglin C, and ALP; 0 to 1.7 p.M MSAP-CMK) and were incubated at 37° C for 24 h. After incubation, the amount of 3H-elastin solubilized was determined. Effect of Inhibitors upon Dissociation of HLE from Elastin To gain insight into the mechanisms of inhibition of elastinbo~nd HLE, enzyme-substrate complexes were prepared usmg 125I-Iabeled HLE and elastin-fluorescein. This experimental design allowed assessment of the effect of inhibitors on: (1) the catalytic activity of HLE bound to elastin and (2) dissociation of HLE from elastin. HLE-elastin complexes were prepared by mixing 1251_ HLE (11.7 nM; sp act, 4,300 cpm/ng) with elastin-fluorescein (3 mg) at 0° C for 15 min. The enzyme-substrate complexes were washed rapidly 3 times. Complexes (equivalent to 100 p.g elastin-fluorescein) were added to each inhibitor or (as a control) to albumin. The final concentrations of aIPI, eglin C, ALP, and albumin were 31 nM; the final concentration of MSAP-CMK was 3.1 p.M. After incubation at 37° C, the pelleted elastin was washed twice with ice-cold buffer. The effect of the inhibitors on dissociation of HLE from elastin was determined by counting the pellets and supernatant fluids from the above assays in a gamma counter (Clinigamma; LKB Instruments). Elastolytic activity was determined by measuring solubilized fluorescein (A4 System, Farrand Optical, Aminco-Bowman, Inc., Silver Spring, MD; excitation filter = Corning 7-59 [wavelength approximately 380 nm]; emission filter = Corning 3-70 [wavelength lBec~use substrate "concentration" has no physical meaning for insoluble elastin, mass of substrate rather than concentration will be given.
Morrison, Welgus, Stockley et al.: Mechanisms of Inhibition of Elastin-bound Elastase
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Inhibitor: Enzyme Ratio Figure i. Activity of free HLE or elastin-bound HLE versus substrate in the presence of alPI. (a) HLE was preincubated with alPI and added to 3H-elastin (triangles) or MSAPN (circles). HLE-elastin complexes were prepared at 0° C as described in MATERIALS AND METHODS, then added to alPI (squares). The residual enzyme activity was determined after incubation at 37° C for 24 h eH-elastin) or 5 min (MSAPN). In each case, residual HLE activity was expressed relative to the activity of enzyme in the absence of inhibitor. Note that complete enzyme inhibition resulted at an approximately 1:1 molar ratio when HLE was pre incubated with the inhibitor; in contrast, nonlinear and incomplete inhibition was observed for inhibition of HLE-elastin complexes. (b) Methods as for (a). Note the inability of a 70-fold molar excess of o.Pl to completely inhibit elastin-bound HLE.
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approximately 520 nml). Standard curves were prepared to relate fluorescence released from elastin to the amount of active HLE present.
Results Inhibitory Activity against Free HLE Data regarding the ability of aIPI, eglin C, and ALP to inhibit the degradation of 3H-elastin and MSAPN are shown in Figures 1 and 2. In all cases, preincubation of HLE with inhibitor prior to addition of substrate resulted in essentially linear inhibition profiles, Linear regression analysis of these results enabled the inhibitor:HLE molar ratio at functional equivalence
Figure 2. Activity of free HLE or elastin-bound HLE versus substrate in the presence of low molecular weight elastase inhibitors. The experimental procedure was as described for Figure 1; triangles = elastin substrate, circles = MSAPN substrate, and squares = HLE-elastin complexes. (a) Eglin C; (b) ALP; (c) MSAP-CMK. Note that the effectiveness of each inhibitor against elastin-bound HLE was relatively impaired.
(i.e., zero enzyme activity) to be determined (32), The results of these analyses are presented in Table 1. Figures 1 and 2 and Table 1 demonstrate that aIPI, eglin C, and ALP all inhibited free HLE at approximately a 1:1 molar basis when enzyme activity was assessed with the pep-
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990
TABLE 1
Inhibitor-Hl.E molar ratios at functional equivalence Molar Ratios* Inhibitor
alPI ALP Eglin C MSAP-CMK
MSAPNt
3H-elastint
1.08 0.94 1.22 30.01
1.47 1.59 1.12 25.70
* Inhibition profiles were drawn as in Figures 1 and 2. The inhibitor:HLE molar ratio at the point of functional equivalence (zero enzyme activity) was calculated by linear regression analysis. t Residual enzyme activity was determined by the addition of MSAPN or 3H-elastin.
tide substrate MSAPN. When enzyme activity was assessed with 3H-elastin, similar results were obtained. However, with alPI and ALP, greater amounts of inhibitor were required to abolish enzymatic activity when 3H-elastin was used as substrate (molar ratios at functional equivalence 36% and 69% greater, respectively). This discrepancy has been noted previously (31) and may reflect some degree of dissociation of inhibitor from enzyme in the more prolonged assay (24 h versus 5 min), or to other mechanisms. Nevertheless, the similar effectiveness of inhibition of enzymatic activity against the two substrates indicates that the HLE-inhibitor complexes are very stable over at least 24 h. For MSAP-CMK, the molar ratio that was required for complete inhibition of the enzyme concentration tested (20 nM) was more than an order of magnitude higher than for other inhibitors (Figure 2c and Table 1). The concentration of HLE used in all experiments was low to permit use of the sensitive substrate MSAPN, and the relatively low K of MSAP-CMK (as reported by Hornebeck and Schnebli [23]) could explain the high molar amounts of the inhibitor necessary to obtain functional equivalence in the present study. To confirm this possibility, we measured the residual HLE activity against MSAPN after incubation with a 10-fold molar excess of MSAP-CMK at progressive dilutions of the inhibitor and enzyme (HLE, 1.8 J.tM to 18 nM). At the highest concentrations tested (MSAP-CMK, 18 J.tM; HLE, 1.8 J.tM), HLE retained 3 % activity. When the concentrations of both enzyme and inhibitor were reduced 100-fold, the enzyme retained 41% activity, despite the MSAPCMK:HLE molar ratio remaining constant. Similar inhibition of free HLE at any particular MSAP-CMK concentration was noted, whether assessed by MSAPN or by 3H-elastin. These results confirm that high MSAP-CMK:HLE molar ratios are required for inhibition at the low (20 nM) HLE concentrations employed here, due to the relatively low K, of MSAP-CMK. Inhibitory Activity against HLE Bound to Elastin Figures 1 and 2 also show data regarding inhibition of HLE that had previously been allowed to bind to elastin. Each of the inhibitors was markedly less effectiveagainst HLE when complexed with elastin in comparison with its effectiveness against free HLE. Inhibition of HLE-elastin complexes by up to an approximately 5-fold molar excess of o.Pl (Figure 1a) was mark-
edly decreased in comparison with that of free HLE. Further increases, up to a 70-fold molar excess of aIPI, gradually increased inhibition but to only 93 % (Figure 1b). Similarly reduced effectiveness of eglin C (Figure 2a) as an inhibitor of elastin-bound HLE was observed. When the inhibitory activity of ALP was tested in an identical manner (figure 2b), similar results were obtained. For relatively high molar ratios of inhibitor to elastin-bound HLE, however, somewhat more effective inhibition was achieved than that observed with either alPI or eglin C. At a 9:1 molar ratio of ALP to HLE, for example, 95% inhibition of elastin-bound HLE was observed. Figure 2c shows similarly reduced effectiveness of MSAPCMK as an inhibitor of HLE after binding to elastin. Up to nearly 200-fold excess of inhibitor produced only 82 % inhibition of elastin-bound HLE. Taken together, the results in Figures 1 and 2 indicate that a proportion of elastin-bound HLE is relatively resistant to inhibition by a diverse group of inhibitors, even when the latter are present in marked excess. Mechanism of Inhibition of Elastin-bound HLE To gain insight into the relative resistance of elastin-bound HLE to inhibition, and to begin to explore mechanisms by which inhibition of elastin-bound HLE occurs, we examined the ability of various inhibitors to dissociate HLE from elastin. When 125I-HLE was bound to elastin-fluorescein and then incubated at 37° C, the amount of '25I-HLE persistently bound to elastin could be determined by counting the elastin pellets. The catalytic activity of the enzyme could be assessed in parallel by measuring fluorescent elastin peptides released from the substrate. Inhibition of elastin-bound HLE by all tested inhibitors was slow. Preliminary experiments with incubation times of 1, 3, and 6 h revealed that both dissociation of 125I-HLE from elastin, and also inhibition of catalytic activity, were minimal at 1 h and increased with prolongation of incubation time. By 6 h (Figure 3), substantial inhibition of catalytic activity was observed with all inhibitors tested. In the experiment shown, aIPI, ALP, eglin C, and albumin were present in a 7-fold molar excess relative to bound HLE, whereas MSAP-CMK was present in approximately 700-fold molar excess in the assay. In the control assays (no inhibitor present), the amount of catalytic activity was appropriate for the amount of enzyme bound at the end of the assay. As a further control, addition of albumin did not change either the amount of persistently bound HLE or the amount of observed catalytic activity. When a IPI was present (Figure 3), the amount of persistently bound HLE after 6 h was significantly reduced. However, the catalytic activity measured by release of fluorescent elastin peptides again was appropriate for the amount of persistently bound enzyme. Figure 3 also shows that similar results were obtained for eglin C. The results obtained when ALP and MSAP-CMK were used as inhibitors are also shown in Figure 3. In marked contrast to o.Pl and eglin C, these inhibitors did not enhance dissociation of HLE from elastin. However, the catalytic activity observed was markedly reduced in relation to the amount of HLE persistently bound to substrate (30.5% and 31.3% for ALP and MSAP-CMK, respectively).
Morrison, Welgus, Stockley et al.: Mechanisms of Inhibition of Elastin-bound Elastase
24 , . . . . - - - - - - - - - - - - - - - - - - - - . 20 16
Control
BSA
(X1-PI
EgC
ALP
CMK
Figure 3. Binding of HLE related to its catalytic activity in the
presence of inhibitors. 125I-HLE was bound to elastin-fluorescein at 0 0 C; the complexes were washed, and the amount of HLE bound was determined using a gamma counter. The complexes were incubated with the various inhibitors for over 6 h at 3r C, after which the HLE-elastin complexes were separated from the supernatants and washed. Elastin solubilization over the 6-h incubation was measured by fluorimetry of the supernatants. The amount of HLE remaining bound to elastin at 6 h was measured using a gamma counter. The amount of bound HLE that was active during the incubation was calculated by comparison with an HLE-elastin standard curve. Shaded bars = HLE bound; closed bars = HLE activity. Note alPI and eglin C dissociated HLE from the complexes, but enzyme remaining in complex was nearly fully active. In contrast, ALP and MSAP-CMK substantially inhibited the enzyme in situ without dissociating HLE from the complexes.
These results, taken together, indicate that alPI and eglin C enhance dissociation of HLE bound to elastin. However, even when measured over a several-hour period, these inhibitors have minimal capability to inhibit the enzyme while it is bound to substrate. In contrast, ALP and MSAPCMK do not appreciably enhance enzyme-substrate dissociation but are able to inhibit HLE while in complex with substrate.
Discussion The present results indicate that a heterogeneous group of proteinase inhibitors exhibits strikingly reduced effectiveness in inhibiting catalytic activity of HLE after the enzyme has been allowed to bind to elastin. Solubilization of elastin by HLE in the absence of proteinase inhibitors is linear with respect to time in this 24-h assay (11). Because even brief preincubation of HLE with these inhibitors resulted in functionally irreversible inhibition, it appears that catalytically important binding of HLE to elastin is highly stable over at least 24 h; thus, the various inhibitors were unable to arrest progressive elastin solubilization. Further, it appears that (in a functional sense) HLE does not leave the elastic fibers during catalytic action on this insoluble substrate, because the inhibitory activities of alPI and eglin C were impaired for the entire duration of the assay. We have previously estimated the turnover number of HLE against insoluble elastin to be 31.9 mol of tropoelastin equivalents/mol HLE/h (11). This approximated the minimal number of peptide bonds cleaved per enzyme molecule per
267
hour. Thus, despite moving among various sites of peptide bond scission on the elastic fibers, the enzyme does not freely interact with soluble proteinase inhibitors even during prolonged incubation. The physical meaning of this observation is unclear, because the biochemical nature of the highaffinity,catalytically active binding between HLE and elastin (11) is still poorly understood. The relative resistance of elastin-bound HLE to inhibition may relate to the very high local "concentration" of substrate on the surface of elastic fibers, or to other biochemical phenomena that are not yet understood. Further insights into these local processes await biochemical characterization of the interactions between HLE and elastin, as well as greater theoretical understanding of the surprising behavior of enzymes in heterogeneous systems (33, 34). The present results follow from recent work suggesting that HLE activity against insoluble elastin occurs at the surface of the elastic fiber and proceeds independently of substrate "concentration" (11). Only a small fraction of the available binding sites for HLE on elastin results in catalytic activity against the substrate; these binding interactions are very stable, and at such sites HLE displays a high affinity for substrate ([11]; equilibrium dissociation constant = 9 x 10-9 M). Our results are also in substantial agreement with other recent reports. Reilly and Travis (22) reported that o.Pl was less effective as an inhibitor of elastin-bound elastase than when HLE and o.Pl were incubated together prior to addition of elastin. Hornebeck and Schnebli (23) confirmed this observation and also demonstrated that eglin C (an inhibitor from the medicinal leech) and the synthetic inhibitor MSAP-CMK were both equally effective at inhibiting free or elastin-bound HLE. Our data differ somewhat from those in the latter report in that each inhibitor we tested was more effectiveagainst free than against elastin-bound enzyme; however, our results agree that a lesser discrepancy was noted for eglin C and MSAP-CMK than for alPI when inhibition of free and elastin-bound enzyme was compared. More recently, Bruch and Bieth (24) have shown that the bronchial inhibitor (or ALP) is a relatively efficient inhibitor of bound elastase and that it is capable of inactivating elastinbound HLE not inhibited by alPI. These observations are especially noteworthy in light of the findings of Damiano and colleagues (10) that immunoreactive HLE can be demonstrated on interstitial elastin in emphysematous human lungs, and that local presence of enzyme correlated with .local severity of pulmonary emphysema. The absence of neutrophils at such sites has suggested the possibility that HLE binding to lung elastin in vivo might be very persistent. Although no evidence for catalytic activity of the observed HLE was provided in the study by Damiano and colleagues, the present results and the studies cited above support the possibility that HLE persistently bound to elastin in vivo may be catalytically active for an undetermined duration (possibly many hours or longer), even in the presence of proteinase inhibitors. Substantial evidence suggests that HLE-mediated injury to lung elastin fibers may be important in the pathogenesis of pulmonary emphysema (7-9); moreover, the present results suggest one potential mechanism by which pulmonary emphysema might progress with time in laboratory animals after the intratracheal instillation of elastase (35, 36).
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990
For our results to have relevance to lung injury in vivo, HLE must reach (and bind to) elastic fibers prior to interacting with proteinase inhibitors. A substantial body of evidence in model systems in vitro indicates that at sites of close contact of neutrophils (13, 15-18, 20) and/or monocytes (5) with susceptible insoluble substrates, HLE can reach such substrates in a catalytically active state even when inhibitors are present. Potential mechanisms by which catalytic activity of HLE might exist in the immediate pericellular milieu have recently been reviewed (12, 15, 37). In addition to demonstrating relative ineffectiveness of inhibitors against elastin-bound HLE, the present work also begins to provide insights into the mechanisms by which such inhibition can be achieved. These results were somewhat surprising and were of considerable interest. Inhibition of elastin-bound HLE appeared to be achieved via one of two distinct mechanisms: (1) enhancement of net dissociation of HLE from elastin, with minimal inhibition' of HLE remaining bound to substrate or (2) inhibition of elastin-bound HLE in situ, without enhancing dissociation of the enzyme from substrate. o.Pl and eglin C were the two inhibitors tested that enhanced net dissociation ofHLE from elastin, yet were unable to inhibit HLE persistently bound to substrate. Presumably, the increased HLE dissociation was explained by competition between inhibitor and elastin for binding of enzyme, with catalytic inactivation of the enzyme occurring upon binding to inhibitor. Both o.Pl and eglin C have low equilibrium dissociation constants (K; = 3.3 X 10-14 [38] and 7.5 X 10-11 M [39], respectively). At inhibitor concentrations ofless than 3 nM, eglin C acts as a reversible, competitive inhibitor (40), but as a pseudo-irreversible inhibitor at higher concentrations (> 3 nM [39]). The concentration of eglin C used in all these experiments was greater than 3 nM. o.Pl forms covalent complexes with HLE (38). It is thus possible that these inhibitors could compete favorably with elastin for HLE binding, even near the surface of the elastic fiber. Explanation of the results obtained with ALP and MSAPCMK was more problematic. ALP is a cationic, competitive inhibitor (K = 1.87 X 10-10 M [41]) of low molecular weight. MSAP-CMK forms irreversible complexes with HLE, but much less rapidly than the polypeptide proteinase inhibitors. These inhibitors did not enhance dissociation of HLE from elastin. Instead, and in marked contrast to the effects of o.Pl and eglin C, these inhibitors arrested the catalytic activity of HLE remaining complexed to substrate. This was clearly demonstrated in experiments in which both bound enzyme and its catalytic activity could be measured. The mechanism underlying such in situ inhibition of HLE is of considerable interest and was not determined in our studies. MSAP-CMK and ALP are both of relatively low molecular weight (590 and 14,000 D, respectively). Although size may be an important factor in allowing an inhibitor to inactivate elastin-bound HLE in situ, it is not the sole prerequisite because eglin C (M, 8,100) fails to inhibit in this manner. It is possible that these inhibitors possess the correct combination of size, charge, and active site conformation to allow in situ inhibition, whereas o.Pl and eglin C rapidly form sta-
ble complexes with a small fraction of the enzyme molecules that are in transit between sites of peptide bond scission. The ability of ALP to inhibit HLE bound to elastin may be of importance in vivo, since Willems and associates have shown ALP associated with elastin fibers in alveolar septae of human lungs removed surgically (42). It is possible that one physiologic role of ALP is to inhibit HLE in situ on elastin, whereas that of o.Pl is to restrict elastolytic activity in solution (24). We believe that the present work should encourage further investigation of the biochemical mechanisms of elastase-elastin interactions as well as mechanisms of inhibition of the catalytic activity of elastases bound to insoluble substrates. Such work may have substantial biologic relevance to the pathogenesis of alveolar septal destruction; moreover, it may improve our level of understanding of enzymatic activity in biologically common heterogeneous systems in general (34), such as that which occurs during solubilization of other extracellular matrix macromolecules. Acknowledgments: This work was supported by a grant from the Medical Research Council of Great Britain; by Grants HL-29594 and AM-35805 from the U,S. Public Health Service, by Grants 1252 and 1742 from the Council for Tobacco Research, USA, Inc., and by the British Lung Foundation. Dr. Welgus is a recipient of U. S. Public Health Service Research Career Development Award AM-1525. Dr. Stockley is a Wolfson Research Fellow of the Royal College of Physicians of London. The writers also wish to acknowledge the excellent technical assistance of S. K. Endicott and M. A. Campbell.
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