Induction of Emphysema in Hamsters by Intratracheal Instillation of Cathepsin 8 1- 3

MARVIN LESSER, MARIA L. PADILLA, and CHRISTOPHER CARDOZO

Introduction SUMMARY Current theories of pathogenesis suggest that pUlmonary emphysema develops In huPulmonary emphysema is a disabling mans because of progressive loss or derangement of lung elastin through a process mediated by disorder characterized anatomically by elastolytic enzymes released by inflammatory cells. Neutrophils are considered primary etiologic destruction of lung parenchyma distal to factors because these cells produce and release two potent serine proteinases that cause emphyseterminal bronchioles in the absence of ma when instilled Into the lungs of animals. It has been suggested that alveolar macrophages also fibrosis (1). Theories of pathogenesis sugcontribute to the development of emphysema through production of several enzymes with elastolytIe activity, including the lysosomal cysteine proteinases cathepsin B and cathepsin L, but this has gest that the destructive process involves not been verified experimentally. In the current study, we instilled 115l1gof active cathepsin B Into primarily lung elastin, and that elastin the lungs of hamsters three times at 48-h intervals. After 6 wk microscopic evaluation revealed that is lost or structurally altered through the lung sections of five of seven animals given cathepsin B contained focal areas of enlarged and activities of enzymes released within the distorted alveoli, In the absence of fibrosis, which were similar to changes seen in the lungs of lung by inflammatory cells recruited by animals given papain Intratracheally. Morphometrically, mean linear intercept (l1m) values were signifcomponents of cigarette smoke (2). Neuicantly higher (p < 0.025) in animals given cathepsin B (204.4 ± 20.8) as compared with control trophils are considered primary factors animals (173.2 ± 7.8), and Internal surface area (sqcm) values were significantly lower (935 ± 120 mediating connective tissue damage beversus 1,083 ± 56 In control animals), thereby confirming that airspace enlargement had developed cause these cells produce two potent serafter instillation of the enzyme. Lung volumes (ml) and compliance (mllcm H 0 ) were not significantine proteinases with elastolytic activity ly higher in animals given cathepsin B. The current findings, along with additional observations that cause emphysema when instilled inthat the number of alveolar macrophages are Increased in the lungs of cigarette smokers and that the cells contain higher levels of cathepsin B, suggest that alveolar macrophages may participate to the lungs of animals (2-5). Support in the development of emphysema and that cysteine protelnases contained within these cells could comes from observations that individuexert elastolytlc activity, thereby contributing to the process. als who lack alpha-l-proteinase inhibiAM REV RESPIR DIS 1992; 145:661-668 tor (A-I-PI), a potent inhibitor of neutrophil elastase in the lung, develop emphysema at an early age (6), particularly if they smoke cigarettes. However, the precise role of neutrophils and their physema. Studies designed to investigate and that activity of cathepsin Band elastolytic enzymes in the development the elastolytic properties of AM have cathepsin L, a cysteine proteinase simiof emphysema in humans remains un- shown that human AM homogenates, lar to cathepsin B, increases in AM of clear because the lungs of most patients culture media from cells maintained in rats following exposure to cigarette with emphysema contain normal levels vitro, and lysosomal extracts digest smoke (24, 25). Thus, cumulative findof A-I-PI, the major antiproteinase in the elastin (13-17). Although in these studies ings suggest that enzymes with elastolytic lungs (7). Also, although components of elastolytic activity was attributed to a activity produced by AM may be responcigarette smoke oxidize A-I-PI in vitro metalloproteinase and/or neutrophil sible for the development of emphyse(8), it is doubtful that unrestricted neu- elastase phagocytosed by AM, studies ma, and that cathepsin B may be one of trophil elastase activity occurs in vivo (9, using liveAM maintained in culture have the mediating factors. However, this 10). Based largely upon these observa- shown that the cells degrade elastin postulation has not been verified extions, it was recently suggested that neu- through the activities of one or more cys- perimentally. Therefore, in the current trophils may be the principal agents caus- teine proteinases and/or a metalloproing emphysema in individuals lacking teinase (10, 18). In one of these studies, A-I-PI, whereas alveolar macrophages degradation was attributed to cathepsin (AM) may be more important cells in in- B because significant inhibition of ac- (Received in original form March 1, 1991 and in dividuals with normal levelsof A-I-PI (2), tivity was observed with the diazometh- revised form September 17, 1991) which is the case for most patients with ylketone, Z-Phe-Ala-CHN2 (10).CathepFrom the Veterans Affairs Medical Center, sin B is a lysosomal enzyme that degrades emphysema. Bronx, New York, and the Department of MediAM accumulate in the lungs in re- several connective tissue components in- cine, The Mount Sinai Medical Center, New York, sponse to cigarette smoke in much greater cluding collagen, proteoglycans, and New York. numbers than do neutrophils (2). In elastin (19-21). Previous studies from this Supported by the VeteransAdministration and healthy smokers and smokers with foci laboratory have shown that levels of the New York Lung Association. 3 Correspondence and requests for reprints of early emphysema, these cells are found cathepsin B and cathepsin D, an asparshould be addressed to Marvin Lesser, M.D., Pulin large numbers in the walls of respira- tyl proteinase, are significantly elevated monary Section, Bronx Veterans Affairs Medical tory bronchioles (11, 12), the principal in AM and in bronchoalveolar lavage flu- Center, 130 West Kingsbridge Road, Bronx, NY sites of development of centrilobular em- id of healthy human smokers (22, 23), 10468. 2

1

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661

662

LESSER, PADILLA, AND CARDOZO

study we evaluated the potential importance of cathepsin B in the development of emphysema by determining if cathepsin B causes emphysema when instilled into the lungs of hamsters. We compared physiologic and pathologic changes caused by the agent with those induced with two different concentrations of papain, a cathepsin B-like cysteine proteinase of plant origin (26) previously shown to cause emphysema in this animal species (27). Methods

Materials Substrates, inhibitors, and other reagents. Brij 35, L-trans-epoxysuccinyl-L-leucylamido-(4guanidino) butane (E-64),7-amino-d-methylcoumarin, dimethyl sulfoxide (DMSO), pepstatin A, isopropanol, diisopropylfluorophosphate (DFP), 1,1O-phenanthroline, and dithiothreitol (DfT) were purchased from Sigma Chemical Co. (St. Louis, MO). Z-Phe-ArgNMec and Z-Arg-Arg-NMec were obtained from Cambridge Research Biochemicals (Valley Stream, NY). Z-Phe-Phe-CHN 2 was purchased from Enzyme Systems Products (Livermore, CA). Elastin-rhodamine was obtained from Elastin Products Co., Inc. (Pacific, MO). Enzymes. Cathepsin B, cathepsin D, and papain were purchased from Sigma. Lyophilized cathepsin B purified from bovine spleen (28) contained approximately 40070 protein, with the balance of material consisting primarily of sodium phosphate, sodium chloride, and approximately 6070 EDfA. Lyophilized cathepsin D purified from bovine spleen contained approximately 30% protein, with the balance consisting primarily of citrate buffer salts. Lyophilized, 2 x crystallized papain purified from Papaya latex contained approximately 80% protein, with the balance consisting primarily of sodium chloride and sodium acetate. Cathepsin L was purified from sheep liver by methods previously described (29, 30), with steps including wholetissue homogenization, ammonium sulfate fractionation, and chromatography on CMSephadex C-50, phenyl-Sepharose, and Sephadex 0-75. The enzyme was stored in 50% ethylene glycol. The absolute concentrations of cathepsin B, cathepsin L, and papain, expressed as micrograms of active enzyme, were determined by active-site titration with E-64 using Z-Phe-Arg-NMec as substrate (31). Enzyme assays. Enzyme assays were performed with synthetic substrates to evaluate cathepsin B activity and to determine if the preparation was contaminated with cathepsin L. The determinations were based upon observations that cathepsin B is active toward the peptide methy!coumarylamide substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec, whereas cathepsin L has little activity toward Z-Arg-Arg-NMec (32) and is selectively in-

hibited with low concentrations of Z-PhePhe-CHN 2 (33). In the assay, microgram amounts of cathepsin B or cathepsin L in acetate buffer (340 mlvl-scdium acetate and 60 mM-acetic acid, pH 5.5) containing 3 mM EDfA were preincubated at 37° C in 430 III water and 250 III acetate buffer containing freshly added DfT (final concentration: 3 mM). After 5 minutes, 50 III of 0.1% Brij 35, Z-Phe-Phe-CHN 2 in 0.1070 Brij 35 (final concentrations: 12,4, 1.33, or 0.44llM), or E-64, a specific inhibitor of cysteine proteinases, in 0.1% Brij 35 (final concentration: 0.5 llM) were added. After an additional 5 minutes, 250 III of Z-Phe-Arg-NMec or Z-Arg-ArgNMec (final concentrations of 5, 10, or 15 llM) were added and incubation was continued at 37° C for 5, 10, or 15 minutes. The reactions were stopped with 1 m1 of a solution containing 100 mM monochloroacetate, 30 mM sodium acetate, and 70 mM acetic acid (pH 4.5). Fluorescence of the free methylcoumarylamide was determined with a PerkinElmer LS-5 fluorescence spectrophotometer with an excitation wavelength of 370 nm and an emission wavelength of 460 nm. Zero on the machine was set with a mixture of buffer, substrate, and stopping reagent, and 1,000was arbitrarily set with buffer and stopping reagent containing 1 nmol methylcoumarylamide. Specific activity was expressed as nanomoles of substrate cleavedper hour per microgram of active enzyme. Determination of elastolytic activity. Elastin purified by neutral extraction from bovine neck ligament and covalently labeled with rhodamine-B-isothiocyanate (particle size 37 to 75 urn) was used to evaluate elastolytic activity of cathepsin B, cathepsin L, and papain. Elastin was washed once with 0.1% Triton X-100 and twice with acetate buffer. Then, 250 ug of elastin was suspended in 5 ml of acetate buffer (pH 5.5) containing DfT (final concentration: 3 mM) and either papain (0.036 to O.72llg), cathepsin B (40 or 80 ug), cathepsin L (3.5 ug), or cathepsin D (300 ug), In some tubes E-64 (10, 20, or 50 llM), DFP (1 mM) in isopropanol, 1,1O-phenanthroline (l mM) in methanol, pepstatin A (1 llM) in methanol, isopropanol, or methanol were preincubated for 20 minutes at room temperature with cathepsin B (40 ug; total volume: 1 ml) before addition of the substrate (total volume: 4 ml). The samples containing substrate were incubated at 37° C in a shaking water bath for 4 h. Some experiments with cathepsin B (40 ug) were carried out in 0.1 M Tris-HCl buffer at pH 7.0 after the substrate had been washed with the same buffer. Following incubation the samples were centrifuged at 4° C at 450 x g for 10 min. The supernatant was gently removed and filtered (Whatman #1). Soluble rhodamine-conjugated products were quantitated with a Perkin-Elmer LS-5 fluorescence spectrophotometer with an excitation wavelength of 550 nm and an emission wavelength of 580 nm. Zero was set with samples carried through the procedure without enzymes. A reading of

1,000 was arbitrarily set with a sample in which all of the elastin had been degraded by 1 mg papain.

Experimental Protocol Injection protocol. Male Syrian golden hamsters (Charles River Breeding Laboratories, Inc., Wilmington, MA) weighing 80 to 110g were housed in a unit providing filteredrecycled air and fed food and water ad libitum. Cathepsin B was dissolved in acetate buffer containing 3 mM EDTA and 3 mM DfT, pH 5.5, and placed on ice for 2 h to maximize enzyme activity by ensuring reduction of the active-site cysteine. Animals were randomly assigned to receive cathepsin B or buffer. After induction of anesthesia by intraperitoneal injection of pentobarbital, the animals were intubated with a polyethylene catheter under direct visualization and given 0.2 ml of acetate buffer or buffer containing cathepsin B (115 ug active enzyme) intratracheally. The agents were instilled slowly through the catheter while the animals were positioned on their backs on an animalrestraining board inclined at 45 degrees. An increase in respiratory rate after instillation was used to confirm that the test substances had entered the lungs. Distribution of the agents was optimized by gentle rotation of the animals from side to side. The procedure was performed three separate times at 48-h intervals. Eight animals were included in the control group and eight animals received cathepsin B. One of the animals receiving cathepsin B died during the second injection. The lungs of the animal demonstrated widespread hemorrhage and consolidation. In a separate experiment, male Syrian golden hamsters weighing 110to 120g were selected at random and given 0.2 ml of acetate buffer or buffer containing 36 or 72 ug papain by the same methods used to instill cathepsin B. Animals given papain received only one instillation. Four animals served as controls, four animals received 36 ug papain, and five animals received 72 ug papain. None of the animals died before they were killed. At 6 wk after instillation of the agents, the animals were anesthetized by intraperitoneal injection of pentobarbital and exsanguinated by transection of the abdominal aorta. Determination ofquasistatic lung volumes and compliance. To evaluate changes in pulmonary elasticity, quasistatic pressure-volume studies were performed. The chest was opened and the anterior chest wall was removed. The trachea was exposed and cut just below the larynx, and a small polyethylene catheter was secured in the trachea with a suture. Pressures were recorded using a water manometer. The lungs were not degassed before the maneuver. Air was slowly instilled with a lO-mlglass syringe until a steady reading of 25 em H 20 was obtained. Air was then slowly removed, and the volume removed was recorded for every 5-cm H 20 decrease in pressure until a pressure of - 20 em H 20 was reached. Two deflation pressure-volume curves were recorded for

663

CATHEPSIN B AND EMPHYSEMA

each animal and mean values were used for subsequent calculations. The recorded volumes agreed within 5010. Total lung volume represents the quantity of air removed between 25 and - 20 em H 2 0 . Quasistatic compliance was calculated over the steepest part of the deflation curve, which occurred between + 5 and 0 em H 20. Thus, compliance, expressed as milliliters per centimeter H 2 0 , represents the volume removed between + 5 and 0 em H 2 0 divided by 5. Anatomic studies and morphometry. The heart was removed, and the lungs, trachea, and esophagus were excised en bloc and weighed. A cannula was secured in the trachea with suture material and the lungs were fixed in inflation overnight at 30 em H 2 0 with 0.1 M phosphate buffer (pH 7.2) containing 4% formaldehyde and 1% glutaraldehyde. The cannula was removed and lung volume was determined by displacement using a 100-ml graduated cylinder filled with phosphate-buffered saline. Variation of volume measurements was generally less than 0.3 mI. A midcoronal section of the right lung was dehydrated and embedded in paraffin. Sections 5 11m in thickness were stained with hematoxylin and eosin or Masson's trichrome. The slides were coded and morphometric measurement of mean linear intercept (MLI) was performed with an eyepiece fitted with a cross hair disk at 100x magnification and corrected for shrinkage using a shrinkage factor of 1.35 (34). A total of 50 fields from all available lobes were evaluated. Fields were selected to exclude major airways or vessels. The internal surface area (ISA) was calculated by standard methods using lung volumes obtained by volume displacement (35). Analysis of bronchoalveolar lavage cell differentials. Using additional animals not evaluated for the presence of emphysema, bronchoalveolar lavage was performed to assess the inflammatory response following instillation of the test agents. For this study, animals were anesthetized with pentobarbital and exsanguinated by aortic transection 2 days or 4 wk after a single intratracheal injection of 0.2 ml of acetate buffer containing DTT (3 mM), or the same buffer containing cathepsin B (115 ug). Bronchoalveolar lavage was performed using a plastic catheter secured in the trachea. A total of 36 ml of phosphatebuffered saline containing no calcium or magnesium was instilled in six aliquots of 6 ml each. The samples were centrifuged at 450 x g, and cellpellets weresuspended in a small amount of phosphate-buffered saline. Airdried smears of the cell suspensions were stained with Wright-Giemsa and 100consecutive cells wereevaluated under oil-immersion magnification (x 1,000)to determine the percentage of AM and polymorphonuclear leukocytes (PMN) in the samples. Data analysis. All data are expressed as the mean ± SD. One-way analysis of variance was used for comparison of variables among three groups. For data from two groups of animals the Student's t test was used to es-

tablish statistical significance. Probability values of p';;;0.05wereconsidered significant. Results

Determination of Enzyme Activity Activities of cathepsin B and cathepsin L toward the substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec and the effects of the inhibitors Z-Phe-Phe-CHNz and E-64 are shown in table 1. In the absence of inhibitors, cathepsin B degraded Z-PheArg-NMec approximately 3.2 times more rapidly than Z-Arg-Arg-NMec. Under the same conditions, cathepsin L degraded Z-Phe-Arg-NMec approximately 80 times faster than Z-Arg-Arg-NMec. In the presence of different concentrations of Z-Phe-Phe-CHNz , inhibition of cathepsin B activity toward Z-Phe-Arg-NMec was comparable to that found with Z-ArgArg-NMec, with only 8OJo inhibition at a concentration of 0.44 JlM. In contrast, these concentrations of Z-Phe-Phe-CHNz inhibited essentially all cathepsin L activity. E-64 (0.5 JlM) inhibited all activity of cathepsin B and cathepsin L toward both substrates. Thus, the findings of equal inhibition of cathepsin B activity toward Z-Phe-Arg-NMec and Z-Arg-ArgNMec with all concentrations of Z-PhePhe-CHNz indicate that the cathepsin B preparation did not contain significant amounts of cathepsin L. Inhibition of all activity toward the substrates with E-64 indicates that degradation was due to cysteine proteinase activity. Determination of Elastolytic Activity Papain rapidly degraded rhodamine-

labeled elastin (table 2). Release of soluble rhodamine-labeled products increased linearly with amounts of enzyme ranging from 0.036 to 0.72 ug. Cathepsin B also degraded elastin, but the rate of degradation calculated per microgram of active enzyme was approximately 550 times slower than that of papain. Activity of cathepsin B was reduced 67, 72, and 96% with 10,20, and 50 JlME-64, respectively. Degradation was not reduced in the presence of DFP or pepstatin A, but was reduced by approximately 20% in the presence of 1,IO-phenanthroline (1 mM). Cathepsin B-mediated degradation of elastin was only reduced by 20% when incubation was carried out in 0.1 M TrisHCI buffer at pH 7.0. Cathepsin L degraded the substrate approximately 30 times more actively than did cathepsin B at pH 5.5. Cathepsin D did not degrade elastin.

Morphometric and Physiologic Studies Initial body weights of animals given buffer or cathepsin B were comparable (table 3). During the subsequent 6 wk, animals given cathepsin B gained less weight than did control animals, but the differences were not statistically significant. Initial body weights of all animals in the papain experiment were higher than those in the cathepsin B experiment, but initial weights did not differ significantly in animals given buffer alone or buffer containing 36 or 72 ug papain. Only animals given 72 Jlgpapain gained no weight during the course of the experiment. Total lung volumes (milliliters) mea-

TABLE 1 EFFECT OF INHIBITORS ON ACTIVITIES OF CATHEPSIN B AND CATHEPSIN L*

Enzyme Cathepsin B

Inhibitor

Concentration (PM)

Z-Phe-Phe-CHN2

o

E-64 Cathepsin L

Z-Phe-Phe-CHN,

E-64

0.44 1.33 4 12 0.5

o 0.44 1.33 4 12 0.5

Activity (nmoll/lglh) Z-Phe-Arg-NMec

Z-Arg·Arg-NMec

2,578 (0) 2,367 (8) 2,055 (20) 1,690 (34) 1,313 (49) 0(100)

803 (0) 745 (7) 662 (18) 500 (38) 410 (49) 0(100)

2,960 (0) 91 (97) 54 (98) 36 (99) 36 (99) 0(100)

36 (0) o (100) 0(100) o (100) 0(100) 0(100)

* Data are mean values from three experiments. Numbers in parentheses represent percent inhibition. The assays were performed in acetate buffer (pH 5.5) containing EDTA (3 mM), on (3 mM), and substrate (10 uM). Active enzyme (micrograms) was determined by active-site titration with E-64.

664

LESSER, PADILLA, AND CARDOZO

TABLE 2

tions coded to prevent viewer bias revealed that MLI values were significantly higher and ISA values were significantly lower in animals given cathepsin B (p < 0.025) as compared with animals given buffer (table 3). Similarly, MLI values were significantly higher and ISA values were significantly lower in animals given 36 and 72 J.1g papain. Also, MLI and ISA values were significantly different in animals given 36 versus 72 J.1g papain. Microscopically, the lungs of animals given buffer alone revealed minimal foci of inflammation consisting primarily of mononuclear cells. The airways showed small numbers of mucous cells and no obvious mucus. The lungs of animals given cathepsin B demonstrated mild interstitial inflammatory changes consisting primarily of mononuclear cells. Some fields contained foamy macrophages, hemosiderin-laden macrophages, and focally thickened pleura. Airways contained increased numbers of goblet cells and focal collections of mucus. Localized areas of airspace enlargement and distortion were seen in five of seven animals (figure 1), which tended to be peripheral and occasionally extended to the pleural surface (figure 2). Microscopically, the lungs of animals given papain showed thickening of alveolar walls due to increased numbers of mononuclear cells and fibrin. Hemosiderin-laden macrophages and foamy macrophages were present in some sections. Emphysematous changes were seen in all samples. Increased numbers of goblet cells were present along with increased amounts of luminal mucus. By 2 days after instillation of cathepsin B, the percentage of AM in bronchoalveolar lavage samples was decreased and the percentage of PMN was in-

DIGESTION OF ELASTIN-RHODAMINE' Amount (pg)

Enzyme

Elastin Degraded (pg)

Inhibitor

1.65 3.16 6.24 13.50 25.70

0.036 0.072 0.18 0.36 0.72

Papain

Cathepsin B

40 80 40 40 40 40 40 40

2.68 5.10 0.88 0.75 0.11 2.60 2.80 2.14

E-64 (10 ~M) E-64 (20 ~M) E-64 (50 ~M) DFP (1 mM) Pepstatin A (1 ~M) 1,10-Phenanthroline (1 mM)

3.5

Cathepsin L Cathepsin D

(67) (72) (96) (3) (0) (20)

6.84 0.21

300

• Oata represent mean values from two determinations performed in duplicate. The assays were performed in acetate buffer (pH 5.5) containing EOTA (3 mM) and (3 mM). Nn was included in the assay performed with cathepsin O. Incubation was performed at 37° C for 4 h in a shaking water bath. The amount (micrograms) of active papain. cathepsin B. and cathepsin L was determined by active-site titration with E-64.

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sured from pressure-volume deflation curves (25 to -20 em H 20) did not differ significantly in animals given cathepsin B compared with control animals (table 3). Total lung volumes determined from pressure-volume curves were higher in animals given papain, but the differences compared with control animals were not statistically significant. Quasistatic compliance expressed as milliliters per centimeter H 20, and calculated from the steepest part of the deflation curve ( + 5 to 0 em H 20), did not differ in animals given buffer or cathepsin B (table 3). Although compliance was higher in animals given 36 and 72 J.1g papain as compared with controls, the differences were not of statistical significance. Lung weights and lung weight/body

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weight ratios (070) of animals given cathepsin B did not differ significantly compared with control animals (table 3). In contrast, lung weights of animals given 36 J.1g papain were significantly higher than those of control and 72 J.1gpapain animals. Lung weight/body weight ratios (%) were significantly higher in animals given 36 and 72 J.1g papain as compared with controls. Lung volumes determined by water displacement after inflation-fixation were lower than those obtained from pressurevolume curves. Total lung volumes did not differ significantly in animals given cathepsin B or buffer (table 3). In the papain experiment, the total lung volume was significantly higher in 72 ug-papain animals (p < 0.025). Microscopic evaluation of lung sec-

TABLE 3 MORPHOMETRIC AND PHYSIOLOGIC MEASUREMENTS' Papain Experiment

Cathepsin B Experiment Parameter Animals, n Initial body weight, g Weight gain, g Lung volume from pressure-volume curve, ml Compliance, mllcm H2O Lung weight, g Lung/body weight ratio, % Lung volume from displacement, ml MlI, ~ ISA, sq cm

Control

Cathepsin B

Control

Papain (36 /lg)

Papain (72 /lg)

8 98.6 ± 4.5 31 ± 11.7

7 97.4 ± 3.6 20.7 ± 10.5

4 131.5 ± 8.4 9.3 ± 13.5

4 127.8 ± 11.2 14 ± 9.8

5 119 ± 6.8 0.5 ± 1.1

6.84 0.84 1.027 0.729 4.90 162 1,187

7.42 1.00 1.199 0.850 4.90 239.8 888

6.23 0.76 0.944 0.727 4.55 173.2 1,083

± ± ± ± ± ± ±

0.36 0.08 0.121 0.032 0.26 7.8 56

6.59 0.77 0.913 0.776 4.74 204.4 935

± ± ± ± ± ± ±

• Data are mean ± SO.

t p < 0.05 compared with control animals and animals given 72 I1g papain. ~p

< 0.05

compared with control animals.

0.70 0.10 0.076 0.064 0.45 20.8:1: 120:1:

± ± ± ± ± ± ±

0.64 0.10 0.168 0.053 0.81 8.8 99

± ± ± ± ± ± ±

0.88 0.16 0.008t 0.071:1: 0.88 36.1t 139t

7.69 1.05 1.023 0.858 6.10 308.5 646

± ± ± ± ± ± ±

0.66 0.14 0.051 0.045:1: 0.50:1: 19.9 32

665

CATHEPSIN B AND EMPHYSEMA

creased (table 4). Microscopically, 2 days after instillation of cathepsin B or buffer alone, lungsections demonstrated thickening of alveolar walls due to infiltration of neutrophils and mononuclear cellsand edema fluid. Alveolar spaces also contained increased numbers of the inflammatory cells. Several sections from animals given cathepsin B showed focal peribronchial inflammation. At 4 wk after instillation of the agents, lavage samples from animals givenbuffer or cathepsin B contained comparable percentages of AM and PMN. Discussion

Fig. 1. Photomicrographs of lung sections from animals given buffer (panel A) or cathepsin B (panel B). Enlarged, distorted airspaces intermixed with normal appearing alveoli are present in the sample from an animal given cathepsin B. (Hematoxylin-eosin stain, x100.)

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In this study we found that the intratracheal instillation of 115 ug of active cathepsin B three times at 48-hr intervals caused emphysematous changes in the lungs of hamsters. Microscopically, focal areas of airspace enlargement and distortion were apparent in five of seven animals and, although patchy and intermixed with areas of normal appearing lung, the changes were associated with a statistically significant increase in MLI and a significant decrease in ISA. The changes were less extensive than those induced with 36 or 72 ug papain. Minimal interstitial inflammation was present in the lungs of animals given cathepsin B, whereas in animals given papain alveolar walls werethickened because of infiltration of mononuclear cells, Previously it was shown that papain, a cysteine proteinase from the papaya plant very similar in structure to cathepsin B (26), causes emphysema in hamsters when given intratracheally (27), Studies evaluating mechanisms by which papain causes emphysema indicate that the major intrapulmonary connective tissue attacked by the agent is elastin (36). In the current study, the changes seen after instillation of cathepsin B were presumably also due to degradation of

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TABLE 4

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CELL DIFFERENTIALS IN BRONCHOALVEOLAR LAVAGE SAMPLES'

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n

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PMN

3 3

55 ± 12 97 ± 2

45 ± 12 3 ± 2

3 3

30 ± 1t 97 ± 2

70 ± 1t 3 ± 2

, Data are mean ± SO. with animals given buffer alone at the same time point.

t p < 0.025 compared

666

LESSER, PADILLA, AND CARDOZO

Fig. 2. Photomicrographs of lung sections nearthe pleural surface of animals given buffer (panel A) or cathepsin B (panel B). The section from an animal given cathepsin B shows extensive airspace enlargement. (Hematoxylin-eosin stain, x200.)

elastin, although the enzyme degrades other connective tissue elements including native collagen (19) and proteoglycans (20). By use of peptide methycoumarylamide substrates and the inhibitor Z-PhePhe-CHN 2 • we determined that the cathepsin B preparation used in the study was not contaminated with cathepsin L, the only other mammalian cysteine proteinase known to degrade elastin (21). Our findings agree with those obtained using cathepsin B and cathepsin L purified from human kidney, which demonstrated that 0.56 IlM Z-Phe-Phe-CHN 2 completely inhibited cathepsin L activity toward Z-Phe-Arg-NMec, but inhibited only 8070 of cathepsin B activity (37). By use of rhodamine-labeled elastin we also found that the cathepsin B preparation degraded elastin. Activity, based upon micrograms of active enzyme added to the assay mixture, was approximately 550 times lower than that of papain and approximately 30 times lower than that of cathepsin L. Nearly complete inhibition of activity toward elastin with 50 IlM E-64, coupled with findings of insignificant inhibition with DFP, pepstatin A, and 1,1O-phenanthroline, indicate that degradation of elastin and the development of emphysema was due to cathepsin B and not to contamination of the preparation with serine, metallo-, or aspartyl proteinases. Also, the presence of EDTA in the buffer in which cathepsin B was suspended would negate the effect of contaminating metalloproteinases. It seems doubtful that the changes caused by cathepsin B weredue to recruitment of PMN with secondary release of elastolytic enzymes by these cellsbecause, even though the percentage of PMN was increased in bronchoalveolar samples 2 days after instillation of cathepsin B, significant numbers of PMN were also found in lavage samples from animals given buffer alone and very few PMN were present in lavage.samples 4 wk after instillation of buffer or cathepsin B. In addition, large numbers of PMN appeared in lavage samples after instillation of cathepsin D and yet no emphysematous changes developed with this agent (unpublished observations). It is of interest that PMN also contain cathepsin B, although specificactivity is approximately four times lower than that found in AM (38). The significance of this finding is unclear because factors leading to alterations in intracellular content of cathepsin B in PMN or conditions mediating extracellular release have not been

667

CATHEPSIN B AND EMPHYSEMA

established. In the current study, we did not determine if the intratracheal instillation of cathepsin B affected levels of the enzyme in PMN or AM. Previously, wefound that cathepsin B levelsincreased in AM of hamsters with pancreatic elastase-induced emphysema, and that levels remained elevated for at least 105 days, whereas a decrease in enzyme activity occurred during this time in animals with bleomycin-induced pulmonary fibrosis (38). The findings of the current study further implicate AM as contributory factors in the development of emphysema in humans with smoke-induced disease. Macrophages synthesize and express cathepsin B (39), and levels of the enzyme are approximately three times higher in the cells of healthy cigarette smokers as compared with nonsmokers, and approximately 10 times higher in bronchoalveolar lavage fluid of smokers (22). In a study evaluating elastolytic activity of live AM, it was observed that approximately 800/0 of activity was due to cystein proteinase(s) (10). Although activity was attributed to cathepsin B because elastin degradation was significantly inhibited with the diazomethylketone, ZPhe-Ala-CHN 2 , it is possible that some or all of the activity was due to cathepsin L, because this enzyme is more subject to inhibition with this diazomethylketone (33). Cysteine proteinases produced by AM would presumably be primarily active in the microenvironment between cell and substrate. In vitro, elastolytic activity of live AM requires close contact with substrate (40), where activity occurs both intracellulaily and at the cell surface or in the immediate microenvironment (10). Although the pH in the microenvironment between nonstimulated and stimulated AM and connective tissue substrate is unknown, it was found that rat peritoneal macrophages adherent to collagen gels acidified the microenvironment between cell and connective tissue to pH values of less than 5 when the cells were stimulated in vivo with glycogen or in vitro with endotoxin (41). If cigarette smoke-stimulated AM behave similarly, an environment would be created that would favor cysteine proteinase activity, particularly because stimulated macrophages form a protein-tight seal at the periphery of their contact with surfaces and thereby create a closed compartment between the cell and target (42) that would exclude cystatins, the major inhibitors of cysteineproteinases, and other in-

hibitors. Also, osteoclasts, which are macrophage-like cells,activelyacidify the extracellular compartment during the process of bone resorption (43). In addition, cathepsin B (44) and cathepsin L (45) demonstrate short-lived activity at neutral pH. In the current study, we found that degradation of elastin by cathepsin B was reduced by only 20% when incubation was carried out in 0.1 M Tris-HCI buffer at pH 7.0 as compared with incubation in acetate buffer at pH 5.5. Although OTT was required in the current study to maximize cathepsin B activity, the presence of high levels of reduced glutathione, an antioxidant, in vivo suggests that the enzyme would be protected from oxidation in the lower respiratory tract (46). Undoubtedly, the precise role of cysteine proteinases and AM in the development of emphysema, if any, is complex and may involve a number of parameters including concentrations and activities of the enzymes at the sites of enzyme-substrate interaction and possible degradation of A-I-PI (47), which would augment neutrophil elastase activity. Other modifying factors could include amplifying and synergistic effects of other enzymes including plasminogen activator (48), interaction with endogenous inhibitors including cystatin C (49), changes in levels of enzymes and cystatins in association with cigarette smoke, and the effects of the enzymes on other connective tissue elements including collagen and proteoglycans. References 1. Snider GL, Kleinerman J, Thurlbeck WM, Bengali ZH. The definition of emphysema: report of a National Heart, Lung, and Blood Institute, Division of Lung Diseases Workshop. Am Rev Respir Dis 1985; 132:182-5. 2. Janoff A. Elastases and emphysema: current assessment of the protease-antiprotease hypothesis. Am Rev Respir Dis 1985; 132:417-33. 3. Janoff A, Sloan B, Weinbaum G, et al. Experimental emphysema induced with purified human neutrophil elastase: tissue localization of the instilled protease. Am Rev Respir Dis 1977; 115: 461-78. 4. Senior RM, Tegner H, Kuhn C, Ohlsson K, Starcher BC, Pierce JA. The induction of pulmonary emphysema with leukocyte elastase. Am Rev Respir Dis 1977; 116:469-75. 5. Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal JR. Porteinase 3: a distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988; 82: 1963-73. 6. Laurell CB, Eriksson S. The electrophoretic alpha l-globulin pattern of serum in alpha I-antitrypsin deficiency. Scand J Clin Invest 1963; 15:132-40. 7. Gadek JE, FellsGA, Zimmerman RL, Rennard

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