Goblet Cell Hyperplasia in Large Intrapulmonary Airways after Intratracheal Injection of Cathepsin B into Hamsters'?
CHRISTOPHER CARDOZO, MARIA L. PADILLA, HO-SOON H. CHOI, and MARVIN LESSER
Introduction
Goblet cellhyperplasia (GCH) is a notable histologic feature in the airways of smokers (1-4). Findings that the severity of GCR in small airways correlates with pack-years of smoking, decreased FEV 1/ FVC ratios (1), and shortness of breath (1, 3) suggest that the process contributes to airway obstruction in chronic bronchitis. Although the pathogenesis of GCR in humans remains unclear, in animals the process can be induced by inhalation of cigarette smoke, sulfur dioxide, or nebulized papain (5-7), or by the intratracheal instillation 0 f mild acids (8) or proteinases, including neutrophil elastase, cathepsin G, pancreatic elastase, and trypsin (9-11). In humans, neutrophils, which are present in increased numbers in the lungs of smokers (12) may contribute to GCR through release of several proteinases, including neutrophil elastase and cathepsin G. Alveolar macrophages (AM) represent another source of proteolytic activity in the lung because the cells produce severallysosomal proteinases with broad activity, including the cysteine proteinase cathepsin B (12, 13). The number of AM in bronchoalveolar lavage fluid (BALF) is increased fivefold in healthy smokers, and the total number of AM in the lungs of healthy smokers far exceeds the number ofneutrophils (12). Observations that levels of cathepsin B are elevated in AM and BALF from smokers (12), and that exposure of rats to cigarette smoke leads to increased levels of the enzyme in AM (13), introduce the possibility that the enzyme contributes to smoke-induced pulmonary disorders. Support comes from the recent observation that the intratracheal instillation of cathepsin B into hamsters caused morphometric changes consistent with emphysema (14). To investigate the possible role of cathepsin B in the development of GCR, we examined the effects of intratracheal instillation of cathepsin B on histologic findings and secretory cell numbers in the
SUMMARY Goblet cell hyperplasia (GCH) Is a frequent histologic finding In the airways of smokers. Experimental observations suggest that the process may be caused by Increased proteinase activity In the airways. To investigate the possible role of cathepsin B In the development of GCH, male Syrian golden hamsters were given three Intratracheal InJections of bovine spleen cathepsin B or buffer (pH 5.5) at 2·day Intervals. Six weeks later, we found by review of PAS-hematoxylln·stalned t-um sections of plastlc·embedded lung tissue that large IntrapUlmonary airways of animals given cathepsin B contained a significantly greater number of secretory cells per millimeter of airway (64.8 ± 7.3 versus 47.5 ± 10.3 for control animals, p < 0.005) In association with a significant Increase in the number of total cells per millimeter of airway, from 149 ± 14 for control animals to 164 ± 11 for cathepsln-B-treated animals (p < 0.025). No change was observed In the number of ciliated cells (93.9 ± 8.1/mm for control animals versus 94.8 ± 10.3/mm for cathepsln-B·treated animals) or other cells (3.0 ± 2.2/mm for control versus 4.3 ± 4.1/mm for cathepsin B), Indicating that selective expansion of the secretory cell population occurred. In contrast, in the main bronchi of animals given cathepsin B, no significant alterations were found In the number or percentage of secretory cells. The findings reveal that cathepsin B Induces secretory cell hyperplasia in hamsters and suggest the possibility that cysteine protelnases may contribute to GCH In smokers. AM REV RESPIR DIS 1992; 145:675-679
main bronchi and intrapulmonary airways of hamsters. Methods Cathepsin B Commercially available cathepsin B (EC 3.4.22.1) purified from bovine spleen was used for all experiments (Sigma Chemical Co., Saint Louis, MO). The enzyme was obtained as a lyophilized powder containing approximately 40070 protein, with the remainder made up primarily of sodium chloride, sodium phosphate, and EDTA. The purity ofthe preparation has been described elsewhere (14). The preparation contained no cathepsin L as determined by use of synthetic substrates and the inhibitor Z-Phe-Phe-CHN 2 (14, 15). Amounts of catalytically active cathepsin B were determined by active site titration with E-64 (16). For the injections, cathepsin B from a single lot was dissolved in acetate buffer at pH 5.5 (containing 400 mM sodium acetate, 3 mM EDTA, and 3mM DTT) and placed on ice for 1 to 2 h to fully activate the enzyme.
Animals Male Syrian golden hamsters (Charles River Breeding Laboratories Inc., Wilmington, MA) were housed in a facility providing filtered, recycled air and fed food and water ad libitum. For intratracheal instillations, animals were lightly anesthetized intraperitoneally with pentobarbital, and a plastic catheter was
passed transorally into the proximal treachea under direct visualization. The treatment solution was instilled slowly, and the animals were turned from side to side 10 times to ensure even distribution of the agents. The respiratory rate was determined before and after each intratracheal injection. An increase in respiratory rate was assumed to indicate that the test substance had entered the lungs.
Experimental Protocol Animals weighing approximately 100 g were selected at random and given cathepsin B (115 ug in 0.2 ml acetate buffer; eight animals) or acetate buffer (0.2 ml; eight animals) intratracheally on three occasions at 2-day intervals. One animal died during the second injection of cathepsin B. At autopsy, the lungs
(Received in original form March 1, 1991 and in revised form September 17, 1991) 1 From the Departments of Medicine and Pathology, The Mount Sinai School of Medicine, New York, and the Laboratory Service and Pulmonary Section of the Medical Service, the Veterans Affairs Medical Center, Bronx, New York. 2 Supported by the Department of Veterans Affairs and by the New York Lung Association. 3 Correspondence and requests for reprints should be addressed to Marvin Lesser,M.D., Chief, Pulmonary Section, VeteransAffairs Medical Center, 130West Kingsbridge Road, Bronx, NY 10468.
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showed diffuse hemorrhage and consolidation. The acetate buffer (pH 5.5) contained 400 mM sodium acetate, 3 mM EDTA, and 3 mM DTT. Six weeks later the animals were anesthetized with pentobarbital and exsanguinated by transection of the abdominal aorta. The lungs were removed and fixed in inflation overnight with a solution containing 4070 formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 (17), at a pressure of 30 cm H 2 O. Two blocks were cut from the left lung as described previously (18). Briefly, the first block, containing peripheral airways, was prepared by cutting the lung sagittally 3 mm medial to the lateral pleura. The second specimen, containing large intrapulmonary airways, was prepared by cutting the lung sagittaly 1 mm lateral to the hilus. Additional transverse sections were cut from the midleft main bronchus. Tissues were dehydrated in graded ethanol and embedded in JB-4 embedding medium (Polysciences Inc., Warrington, PA). Sections 1 urn thick were stained with toluidine blue or PAS-hematoxylin. Sections 5 urn thick of paraffin-embedded coronal sections of the right lung were stained with hematoxylin-eosin or Perl's iron stain. All histologic and morphometric assessments were performed blinded. The number of cells per millimeter of basement membrane was determined by viewing toluidine-bluestained sections under oil immersion at x 630 through an eyepiece fitted with a microbiology counting grid, which was calibrated with a stage micrometer. The number of epithelial cells within the grid was determined for each of five consecutive fields free of sharp curves or projections. For sections containing large intrapulmonary airways, only airways with a diameter greater than 0.5 mm were evaluated. Sections containing peripheral airways were not evaluated for epithelial cell density. Data are expressed as the number of cells per millimeter. Differential counts of epithelial cell subtypes were performed by examining PAShematoxylin-stained sections under oil immersion at X630. Only cells with a luminal border were evaluated. Cells were classified as ciliated by the presence of cilia or basal bodies, as secretory by the presence of three or more PAS-positive secretory granules, or as other in the absence of these features. At least 500 cells were examined for each level of the airway; multiple sections were examined when fewer than 500 cells were present on a single slide. The number of secretory cells per millimeter was determined by multiplying the total number of epithelial cells per millimeter by the proportion of secretory cells. The number of ciliated and "other" cells per millimeter was determined in a similar manner. Peripheral airways were not evaluated for cell differential counts or numbers of cells per millimeter of airway because of the curvature of these airways. To determine the number of iron-staining macrophages, sections were examined at X400 through an eyepiece fitted with a microbiolo-
CARDOZO, PADILLA, CHOI. AND LESSER
gy counting grid. Randomly selected fields free of airways or vessels were assessed for the number of blue-stained macrophages lying within the grid. A minimum of 15 fields were evaluated for each section. Data are expressed as the mean number of positively stained macrophages per field.
Statistics All data are expressed as mean ± SD. Comparisons were performed using Student's t test. Probability values of p < 0.05 were considered significant.
Results
Weight gain, pulmonary physiology, and histologic and morphometric examination of the lung parenchyma of the animals have been described elsewhere (14).
Histologic Examination Histologic reviewof PAS-stained plasticembedded sections of main stem bronchi revealed no significant changes in the bronchi of cathepsin B treated animals. Specifically,there wasno evidence of loss of epithelium, epithelial thickening or atypia, inflammation, or change in the thickness of adventitial or muscle layers or in numbers of mast cells. In contast, large intrapulmonary airwaysof cathepsin B-treated animals contained numerous foci of increased numbers of goblet cells (figure 1). In these areas, the epithelium appeared thickened, and the goblet cells appeared wider and more deeply stained than did those of control animals. The cells were often engorged with secretory granules (figure 1). Ciliated cells were often narrowed as if compressed by the adjacent goblet cells. There was no change in the number of inflammatory cells or mast cells, or in the thickness of the subepithelial layer. No epithelial sloughing was noted. Sections of peripheral airways from cathepsin B-treated animals contained increased numbers of cells with granular cytoplasm containing PAS-negative material, and increased numbers of ballooned cells with indistinct cell borders. No goblet cells were seen at this level of the airway. Cell Differentials The effects of intratracheal instillation of cathepsin B on cell numbers and cell differentials are shown in table 1. There was a significant increase in the total number of cells per millimeter and in the number and percentage of secretory cells (cells that contained three or more PASpositive secretory granules) in the large
intrapulmonary airways of animals given cathepsin B. No significant change was seen in the number of ciliated or other cells, indicating that the increase in the total number of epithelial cells was solely due to expansion of the secretory cell population. Further evidencethat a selective increase in the number of secretory cells occurred is provided by the finding that the increase in the total number of cells per millimeter was nearly the same as the increase in the number of secretory cells per millimeter. No significant change in the number or percentage of cells classified as "other," which included presecretory and preciliated cells (19), was observed. The significant decrease in the percentage of ciliated cells in the large intrapulmonary bronchi of cathepsin B-animals is presumably due to the increase in the proportion of secretory cells. Fewsignificant alterations in cell numbers and cell differentials were noted in the main bronchi of cathepsin B-animals (table 1). The tissue from one cathepsin B-treated animal was inadequate for evaluation; tissue from one control animal was inadequate for determination of cell differentials. No significant alteration in the number or percentage of secretory cells was noted in the main bronchi of these animals. The marginally significant increase in the total number of epithelial and ciliated cells in cathepsin B-treated animals probably represents a type II statistical error because the data are skewed by findings from one animal with an unusually high cell density, and because the differences disappear when data from this animal are not included in the analysis.
Iron-containing Macrophages In preliminary studies we noted increased numbers of pigment-laden macrophages in the lungs of hamsters given cathepsin B. Because the lungs of the animal that died during the second injection of cathepsin B contained diffuse hemorrhage at autopsy, we speculated that the pigment seen at 6 wk was hemosiderin. Accordingly, we reviewed paraffin sections stained with Perl's iron stain. Histologically, iron-positive macrophages appeared to be more numerous and more deeply stained in the lungs of cathepsin B-treated animals. However, quantitation of blue-stained macrophages revealed that the lungs of cathepsin B-treated animals contained slightly though not significantly more positivelystained macrophages than did the lungs of buffer-
GOBLET CELL HYPERPLASIA IN INTRAPULMONARY AIRWAYS AFTER CATHEPSIN B
Fig. 1. Top panel. Epithelium of the intrapulmonary airway of an animal given acetate buffer (pH, 5.5).The epithelium is made up of ciliated cells and secretory cells containing PAS-stained secretory granules. Bottompanel. Intrapulmonary airway epithelium of an animal given cathepsin B. The epithelium appears thickened and contains increased numbers of goblet cells, which are larger and contain increased numbers of granules. (PAS-hematoxylin· stained t-urn sections of plastic-embedded tissues, original magnification, 1,040.)
treated animals (5.3 ± 1.5, n 1.7, n = 8, respectively).
=
7; 4.4 ±
Discussion
We found that three intratracheal injections of cathepsin B caused significant increases in the number and percentage of PAS-positive secretory cells in large intrapulmonary airways of hamsters. We believethat this is the first report of secre-
tory cellhyperplasia caused by an enzyme contained within AM, and the first evidence that mammalian cysteine proteinases are capable of inducing this abnormality. The findings indicate that cathepsin B should be added to the growing list of proteinases capable of causing this abnormality in hamsters, which already includes human neutrophil elastase, porcine pancreatic elastase, trypsin,
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and cathepsin G (9-11). The findings provide support for the theory advanced by Christensen and coworkers (9) that increased levels of airway proteinases contribute to the development of GCH in smokers. Although previous studies have focused on the neutrophil proteinases, neutrophil elastase, and cathepsin G (10, 11, 18,20,21), findings that the levels of cathepsin B in homogenates of AM from smokers are increased two to three times, and that levels in BALF from smokers are increased 10-fold (12), suggest that AM also contribute to the proteinase burden in the airways. Alveolar macrophages are the predominant cells in BALF of healthy smokers and nonsmokers, and the number of AM in BALF from healthy smokers is approximately fivetimes higher than that found in nonsmokers, indicating that AM may be an important source of proteinases in the lung (12). Cathepsin B-induced secretory cell hyperplasia of large intrapulmonary airways occurred without significant alterations in the numbers of ciliated or other epithelial cells. The findings suggest that in this model, secretory cell hyperplasia occurs through selectiveexpansion of the secretory cell pool. Expansion presumably occurs through proliferation of a precursor cell. Support comes from findings that proliferation of nonciliated, nonsecretory cells occurs after intratracheal instillation of neturophil elastase (22). In contrast to our findings, studies evaluating the effects of human neutrophil elastase on hamster intrapulmonary airways found significant decreases in the percentage of nonciliated, nonsecretory cellswithout a change in the percentage of ciliated cells or the number of cells per millimeter of airway (18). We found that the epithelium of peripheral airways of cathepsin B-treated animals contained increased numbers of ballooned cells and cells with granular cytoplasm. In contrast, studies of the effects of human neutrophil elastase found no abnormalities at this level of the airway, whereas studies of the effects of porcine pancreatic elastase reported the presence of PAS-positive cells (9, 20). In our study, the number and percentage of PAS-positive secretory cells in the airways of buffer-treated animals decreased as the airways became smaller, and there were essentially no PASpositive secretory cells in the peripheral airways. These 0 bservations are in agreement with those obtained by Kennedy and coworkers (23) from light and electron microscopic examination of normal hamster airways. We found that epi-
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CARDOZO, PADILLA, CHOI, AND LESSER
TABLE 1 CELL DENSITY AND CELL DIFFERENTIAL COUNTS' Cell Numbers per Millimeter Epitheliumt Group Main bronchi Control Cathepsin B Intrapulmonary bronchi Control Cathepsin B
Cell Differential (%)
n
Total
Ciliated
Secretory
Other
7 6
153 ± 15 174 ± 2411
92.7 ± 10.0 107.3 ± 15.911
56.6 ± 10.5 62.4 ± 10.5
3.0 ± 2.2 3.6 ± 1.8
61.0 ± 4.1 61.9 ± 1.7
37.0 ± 4.2 35.9 ± 2.1
2.0 ± 1.6 2.2 ± 1.2
8 7
149 ± 14 164 ± 11§
93.9 ± 8.1 94.8 ± 10.3
47.5 ± 10.3 64.8 ± 7.3:1:
3.0 ± 2.2 4.3 ± 4.1
63.2 ± 5.0 57.8 ± 4.211
31.8 ± 4.9 39.6 ± 4.4:1:
5.0 ± 3.2 2.6 ± 2.5
Ciliated
Secretory
Other
• Data are mean values ± SO; n refers to the number of animals for which specimens were reviewed. Control animals were given acetate buffer (pH, 5.5); cathepsin-B-treated animals were given cathepsin B dissolved in acetate buffer (pH, 5.5). Methods for determination of numbers of epithelial cells and for determination of cell differentials are given in the text. t Number was eight for determination of total number of cells per millimeter for main bronchi from control animals. p < 0.005 compared with control animals. § p < 0.025 compared with control animals. IIp < 0.05 compared with control animals.
*
thelium of intrapulmonary bronchi of animals treated with buffer contained 31.8070 secretory cells, a finding in agreement with studies of normal hamster bronchi using similar methods, which found that 32.9% of cells with a luminal border were secretory cells (23). Of interest, we found no significant increase in the number or percentage of secretory cells in the main bronchi of cathepsin B-treated animals. Our findings are in agreement with 0 bservations that no change occurred in the number of tracheal goblet cells after the intratracheal instillation of human neutrophil elastase (24). Thus, the biology of epithelium at this level of the airway may be fundamentally different from that of epithelium more distal in the respiratory system. Support for this notion comes from electron microscopic studies using iron-conjugated lectins, which showed that significant differences exist in epithelial surface glycoconjugates expressed in hamster trachea and bronchi (25). Although gross pulmonary hemorrhage was seen in the lungs of the animal that died during the instillation of cathepsin B, no significant increase in the number of iron-stained macrophages was observed in the lungs of cathepsin B-treated animals 6 wk after instillation of the enzyme. However, macrophages in the lungs of cathepsin B-treated animals were more deeply stained, suggesting that some increase in iron pigments was present. Our observations differ from those of Lucey and coworkers (21) who found that the number of iron-laden macrophages was markedly increased 1 month after injection of human neutrophil elastase, and remained increased for at least 18 months. The reason(s) for the difference are unknown, but they might include variations in the amount of blood
entering the lung after injection or in the trophil alveolitis and intra-alveolar rates of clearance of pigment-laden hemorrhage, presumably associated with exudation of serum proteins, including macrophages. The mechanisms by which proteinases uz-macroglobulin. Evidence that procause OCH remain unknown. Findings tease-ai-macroglobulin complexes are that enzymes must be proteolytically ac- formed after instillation of proteases tive to cause OCH (10, 11) imply that comes from studies showing that in hyperplasia results from cleavage of one BALF samples obtained from hamsters or more proteins. Of interest in this re- after the intratracheal injection of 1251_ gard, the specificities of the proteinases human neutrophil elastase, a large porthat cause OCH vary significantly. Ca- tion of the enzyme is present as a 780thepsin B is a cysteine proteinase with kD complex (31). In contrast to the other enzymes shown broad specificity that cleaves peptide bonds between amino acids with vary- to cause OCH, cathepsin B is optimally ing properties (26). In contrast, neutro- active at pH 5.5 to 6.0. Thus, activity is phil elastase cleaves peptide bonds on the presumably limited to microenvironcarboxyl side of residues with aliphatic ments with acidic pH. Several observaside chains (27), whereas trypsin, anoth- tions suggest that such conditions exist er proteinase capable of causing OCH extracellularly, Microelectrode studies (10), cleaves on the carboxyl side of ba- have demonstrated that stimulated perisic amino acid residues. Thus, it seems toneal macrophages adherent to collagen unlikely that goblet cell hyperplasia gels acidify the microenvironment beresults from cleavage of a specific pep- tween the cell membrane and the gel (32). Thus, it is possible that inhalation of tide bond in a single protein. It is also possible that the stimulus is smoke stimulates AM adherent to respidue to proteinases complexed with U2- ratory epithelium to create an acidic macroglobulin or other airway proteinase microenvironment. Also, lacunar osinhibitors. Alphaz-macroglobulin inacti- teoclasts, macrophage-like cells, degrade vates a number of proteinases, including bone collagens through the action of cyscathepsin B, neutrophil elastase, trypsin, teine proteinases (33), and, under certain and cathepsin 0 (28-30). Alphas-mac- conditions, human AM degrade proteins roglobulin acts by "trapping" proteinases in extracellular matrices predominantly within the four subunits of the molecule, through the action of cysteine proteinases a process that is triggered by proteolytic acting largely at the cell surface or in the cleavage of a bait region (28). Thus, the pericellular microenvironment (34, 35). inhibitor only complexes with enzymes Findings that the enzyme retains signifithat are proteolytically active (28). It is cant activity at pH 7.0 (36) indicate that possible that the u2-macroglobulin-en- even slight reductions in the pH ofairzyme complex binds to constituents of way fluids may permit cathepsin B prothe epithelial cell membrane, thereby teolytic activity. Thus, if the pH of airproviding a stimulus for secretion or way lining fluid in humans is reduced, proliferation of goblet cells. Although as it is in cats and fetal lambs (37, 38), uz-macrcglobulin is not normally pres- it is possible that cathepsin B participates ent in significant amounts in the airway, in the degradation of extracellular proinstillation of proteinases induces neu- teins in vivo.
GOBLET CELL HYPERPLASIA IN INTRAPULMONARY AIRWAYS AFTER CATHEPSIN B
References 1. Nagai A, West WW, Paul JL, Thurlbeck WM. The National Institutes of Health intermittent positive-pressurebreathing trial: pathology studies. Am Rev Respir Dis 1985; 132:946-53. 2. Wright JL, Lawson LM, Pare PD, Wiggs BJ, Kennedy S, Hogg I'C, Morphology of peripheral airways in current smokers and ex-smokers. Am Rev Respir Dis 1983; 127:474-7. 3. Thurlbeck WM, Malaka D, Murphy K. Goblet cells in the peripheral airways in chronic bronchitis. Am Rev Respir Dis 1975; 112:65-9. 4. Cosio M, Ghezzo H, Hogg JC, et al. The relations between structural changes in small airways and pulmonary-function tests. N Engl J Med 1977; 298:1277-81. 5. Rogers DF, Jeffery PK. Inhibition of cigarette smoke-induced airway secretorycell hyperplasia by indomethacin, dexamethasone, prednisolone, or hydrocortisone in the rat. Exp Lung Res 1986; 10:285-98. 6. Spicer SS, Chakrin LW,Wardell JR. Effect of chronic sulfur dioxide inhalation on the carbohydrate histochemistry and histology of the canine respiratory tract. Am Rev Respir Dis 1974; 110: 13-24. 7. Goldring IP, Greenburg L, Park S-S, Ratner 1M. Pulmonary effects of sulfur dioxide exposure in the Syrian hamster. II. Combined with emphysema. Arch Environ Health 1970; 21:32-7. 8. Christensen TG, Lucey EC, Breuer R, Snider GL. Acid-inducedsecretory cellmetaplasia in hamster bronchi. Environ Res 1988; 45:78-90. 9. Christensen TO, Korthy AL, Snider GL, Hayes JA. Irreversible bronchial goblet cell metaplasia in hamsters with elastase-induced panacinar emphysema. J Clin Invest 1977; 59:397-404. 10. Breuer R, Lucey EC, Stone PJ, Christensen TO, Snider GL. Proteolytic activity of human neutrophil elastase and porcine pancreative trypsin causes bronchial secretory cell metaplasia in hamsters. Exp Lung Res 1985; 9:167-75. 11. Lucey EC, Stone PJ, Breuer R, et al. Effect of combined human neutrophil cathepsin G and elastase on inducti on of secretory cell metaplasia and emphysema in hamsters, with in vitro observations on elastolysis by these enzymes. Am Rev Respir Dis 1985; 132:362-6. 12. Chang JC, Lesser M, Yoo OH, Orlowski M. Increased cathepsin B-likeactivity in alveolar macrophages and bronchoalveolar lavage fluid from
smokers. Am Rev Respir Dis 1986; 134:538-41. 13. Gairola CG, Galicki NI, Cardozo C, L2:i Y-L, Lesser M. Cigarette smoke stimulates cathepsin B activity in alveolar macrophages of rats. J Lab Clin Med 1989; 114:419-25. 14. Lesser M, Padilla M, Cardozo C. Induction of emphysema in hamsters by intratracheal instillation of cathepsin B. Am Rev Respir Dis 1992(In Press). 15. Kirschke H, Wood L, Roisen FJ, Bird JWc. Activityof lysosomal cysteine proteinase during differentiation ofrat skeletal muscle. Biochem J 1983; 214:871-7. 16. Barrett AJ. Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol 1981; 80:535-60. 17. McDowellEM, Trump BE Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 1976; 100:405-14. 18. Breuer R, Christensen TO, Lucey EC, Stone PJ, Snider GL. Quantitative study of secretory cell metaplasia induced by human neutrophil elastase in the large bronchi of hamsters. J Lab Clin Med 1985; 105:635-40. 19. McDowell EM, Combs JW, Newkirk C. A quantitative light and electron microscopic study of hamster tracheal epithelium with special attention to so-called intermediate cells. Exp Lung Res 1983; 4:205-26. 20. Snider GL, Lucey EC, Christensen TO, et al. Emphysema and bronchial secretory cell metaplasia induced in hamsters by human neutrophil products. Am Rev Respir Dis 1984; 129:155-60. 21. Lucey ED, Stone PJ, Christensen TO, Breuer R, Snider GL. An 18-month study of the effects on hamster lungs 0 f intratracheally administered human neutrophil elastase. Exp Lung Res 1988; 14:671-86. 22. WeinbergKS, Hayes JA. Elastase-induced emphysema: asynchronous bronchial, alveolar and endothelial cell proli feration during acute response to injury. J Pathol 1982; 136:253-64. 23. Kennedy AR, Derosiers A, Terzaghi M, Little J8. Morphometric and histological analysis of the lungs of Syrian golden hamsters. J Anat 1978; 125:527-53. 24. Christensen TG, Breuer R, Lucey EC, Stone PJ, Snider GL. Regional difference in airway epithelial response to neutrophil elastase. Exp Lung Res 1989; 15:943-59. 25. Christensen TG, Breuer R, Lucey EC, Hornstra LJ, Stone PJ, Snider GL. Lectin cytochemis-
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try revealsdifferences between hamster trachea and bronchus in the composition of epithelial surface glycoconjugates and in the response of secretory cells to neutrophil elastase. Am J Respir Cell Mol BioI 1990; 3:61-9. 26. McKayMJ, Offermann MK, Barrett AJ, Bond JS. Action of human liver cathepsin B on the oxidized insulin B chain. Biochem J 1983;213:467-71. 27. Powers JC, Gupton BF, Harley AD, Nishino N, Whitely RJ. Specificity of porcine pancreatic elastase, human leukocyte elastase and cathepsin G. Inhibition with peptide chlorornethyl ketones. Biochim Biophys Acta 1977; 485:156-66. 28. Mason RW.Interaction oflysosomal cysteine proteinases with uz-macroglobulin: conclusive evidence for the endopeptidase activities of cathepsin Band H. Arch Biochem Biophys 1989; 273:367-74. 29. Barrett AJ, McDonald JK. Mammalian proteases Vol. 1. London: Academic Press, 1980; 165. 30. Barret AJ, McDonald JK. Mammalian proteases. Vol. 1. London: Academic Press, 1980; 182. 31. Stone PJ, Lucey EC, Calore JD, McMahon MP, Snider GL, Franzblau C. Defensesof the hamster lung against human neutrophil and porcine elastase. Respiration 1988; 54:1-15. 32. SilverA, Murrills RJ, Etherington OJ. Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp Cell Res 1988; 175:266-76. 33. Delaisse JM, Eeckhout Y, VaesG. In vivo and in vitro evidence for the involvement of cysteine proteinase in bone resorbtion. Biochem Biophys Res Commun 1984; 125:441-7. 34. Chapman HA, Reilly JJ, Kobzik L. Role of plasminogen activator in degradation of extracellular matrix protein by livehuman alveolar macrophages. Am Rev Respir Dis 1988; 137:412-9. 35. Chapman HA, Stone OL. Comparison oflive human neutrophil and alveolar macrophage elastolytic activity in vitro. J Clin Invest 1984;74:1693700. 36. Mort JS, Recklies AD, Poole AR. Extracellular presence of the lysosomal proteinase cathepsin B in rheumatoid synovium and its activity at neutral pH. Arthritis Rheum 1984; 27:509-15. 37. Adamson TM, Boyd RDH, Platt HS, Strang LB. Composition of alveolar liquid in the foetal lamb. J Physiol (Lond) 1977; 204:159-68. 38. Scarpelli EM. The surfactant system of the lung. Int Anesthesiol Clin 1977; 15:19-60.
CHRISTOPHER CARDOZO, MARIA L. PADILLA, HO-SOON H. CHOI, and MARVIN LESSER
Introduction
Goblet cellhyperplasia (GCH) is a notable histologic feature in the airways of smokers (1-4). Findings that the severity of GCR in small airways correlates with pack-years of smoking, decreased FEV 1/ FVC ratios (1), and shortness of breath (1, 3) suggest that the process contributes to airway obstruction in chronic bronchitis. Although the pathogenesis of GCR in humans remains unclear, in animals the process can be induced by inhalation of cigarette smoke, sulfur dioxide, or nebulized papain (5-7), or by the intratracheal instillation 0 f mild acids (8) or proteinases, including neutrophil elastase, cathepsin G, pancreatic elastase, and trypsin (9-11). In humans, neutrophils, which are present in increased numbers in the lungs of smokers (12) may contribute to GCR through release of several proteinases, including neutrophil elastase and cathepsin G. Alveolar macrophages (AM) represent another source of proteolytic activity in the lung because the cells produce severallysosomal proteinases with broad activity, including the cysteine proteinase cathepsin B (12, 13). The number of AM in bronchoalveolar lavage fluid (BALF) is increased fivefold in healthy smokers, and the total number of AM in the lungs of healthy smokers far exceeds the number ofneutrophils (12). Observations that levels of cathepsin B are elevated in AM and BALF from smokers (12), and that exposure of rats to cigarette smoke leads to increased levels of the enzyme in AM (13), introduce the possibility that the enzyme contributes to smoke-induced pulmonary disorders. Support comes from the recent observation that the intratracheal instillation of cathepsin B into hamsters caused morphometric changes consistent with emphysema (14). To investigate the possible role of cathepsin B in the development of GCR, we examined the effects of intratracheal instillation of cathepsin B on histologic findings and secretory cell numbers in the
SUMMARY Goblet cell hyperplasia (GCH) Is a frequent histologic finding In the airways of smokers. Experimental observations suggest that the process may be caused by Increased proteinase activity In the airways. To investigate the possible role of cathepsin B In the development of GCH, male Syrian golden hamsters were given three Intratracheal InJections of bovine spleen cathepsin B or buffer (pH 5.5) at 2·day Intervals. Six weeks later, we found by review of PAS-hematoxylln·stalned t-um sections of plastlc·embedded lung tissue that large IntrapUlmonary airways of animals given cathepsin B contained a significantly greater number of secretory cells per millimeter of airway (64.8 ± 7.3 versus 47.5 ± 10.3 for control animals, p < 0.005) In association with a significant Increase in the number of total cells per millimeter of airway, from 149 ± 14 for control animals to 164 ± 11 for cathepsln-B-treated animals (p < 0.025). No change was observed In the number of ciliated cells (93.9 ± 8.1/mm for control animals versus 94.8 ± 10.3/mm for cathepsln-B·treated animals) or other cells (3.0 ± 2.2/mm for control versus 4.3 ± 4.1/mm for cathepsin B), Indicating that selective expansion of the secretory cell population occurred. In contrast, in the main bronchi of animals given cathepsin B, no significant alterations were found In the number or percentage of secretory cells. The findings reveal that cathepsin B Induces secretory cell hyperplasia in hamsters and suggest the possibility that cysteine protelnases may contribute to GCH In smokers. AM REV RESPIR DIS 1992; 145:675-679
main bronchi and intrapulmonary airways of hamsters. Methods Cathepsin B Commercially available cathepsin B (EC 3.4.22.1) purified from bovine spleen was used for all experiments (Sigma Chemical Co., Saint Louis, MO). The enzyme was obtained as a lyophilized powder containing approximately 40070 protein, with the remainder made up primarily of sodium chloride, sodium phosphate, and EDTA. The purity ofthe preparation has been described elsewhere (14). The preparation contained no cathepsin L as determined by use of synthetic substrates and the inhibitor Z-Phe-Phe-CHN 2 (14, 15). Amounts of catalytically active cathepsin B were determined by active site titration with E-64 (16). For the injections, cathepsin B from a single lot was dissolved in acetate buffer at pH 5.5 (containing 400 mM sodium acetate, 3 mM EDTA, and 3mM DTT) and placed on ice for 1 to 2 h to fully activate the enzyme.
Animals Male Syrian golden hamsters (Charles River Breeding Laboratories Inc., Wilmington, MA) were housed in a facility providing filtered, recycled air and fed food and water ad libitum. For intratracheal instillations, animals were lightly anesthetized intraperitoneally with pentobarbital, and a plastic catheter was
passed transorally into the proximal treachea under direct visualization. The treatment solution was instilled slowly, and the animals were turned from side to side 10 times to ensure even distribution of the agents. The respiratory rate was determined before and after each intratracheal injection. An increase in respiratory rate was assumed to indicate that the test substance had entered the lungs.
Experimental Protocol Animals weighing approximately 100 g were selected at random and given cathepsin B (115 ug in 0.2 ml acetate buffer; eight animals) or acetate buffer (0.2 ml; eight animals) intratracheally on three occasions at 2-day intervals. One animal died during the second injection of cathepsin B. At autopsy, the lungs
(Received in original form March 1, 1991 and in revised form September 17, 1991) 1 From the Departments of Medicine and Pathology, The Mount Sinai School of Medicine, New York, and the Laboratory Service and Pulmonary Section of the Medical Service, the Veterans Affairs Medical Center, Bronx, New York. 2 Supported by the Department of Veterans Affairs and by the New York Lung Association. 3 Correspondence and requests for reprints should be addressed to Marvin Lesser,M.D., Chief, Pulmonary Section, VeteransAffairs Medical Center, 130West Kingsbridge Road, Bronx, NY 10468.
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showed diffuse hemorrhage and consolidation. The acetate buffer (pH 5.5) contained 400 mM sodium acetate, 3 mM EDTA, and 3 mM DTT. Six weeks later the animals were anesthetized with pentobarbital and exsanguinated by transection of the abdominal aorta. The lungs were removed and fixed in inflation overnight with a solution containing 4070 formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 (17), at a pressure of 30 cm H 2 O. Two blocks were cut from the left lung as described previously (18). Briefly, the first block, containing peripheral airways, was prepared by cutting the lung sagittally 3 mm medial to the lateral pleura. The second specimen, containing large intrapulmonary airways, was prepared by cutting the lung sagittaly 1 mm lateral to the hilus. Additional transverse sections were cut from the midleft main bronchus. Tissues were dehydrated in graded ethanol and embedded in JB-4 embedding medium (Polysciences Inc., Warrington, PA). Sections 1 urn thick were stained with toluidine blue or PAS-hematoxylin. Sections 5 urn thick of paraffin-embedded coronal sections of the right lung were stained with hematoxylin-eosin or Perl's iron stain. All histologic and morphometric assessments were performed blinded. The number of cells per millimeter of basement membrane was determined by viewing toluidine-bluestained sections under oil immersion at x 630 through an eyepiece fitted with a microbiology counting grid, which was calibrated with a stage micrometer. The number of epithelial cells within the grid was determined for each of five consecutive fields free of sharp curves or projections. For sections containing large intrapulmonary airways, only airways with a diameter greater than 0.5 mm were evaluated. Sections containing peripheral airways were not evaluated for epithelial cell density. Data are expressed as the number of cells per millimeter. Differential counts of epithelial cell subtypes were performed by examining PAShematoxylin-stained sections under oil immersion at X630. Only cells with a luminal border were evaluated. Cells were classified as ciliated by the presence of cilia or basal bodies, as secretory by the presence of three or more PAS-positive secretory granules, or as other in the absence of these features. At least 500 cells were examined for each level of the airway; multiple sections were examined when fewer than 500 cells were present on a single slide. The number of secretory cells per millimeter was determined by multiplying the total number of epithelial cells per millimeter by the proportion of secretory cells. The number of ciliated and "other" cells per millimeter was determined in a similar manner. Peripheral airways were not evaluated for cell differential counts or numbers of cells per millimeter of airway because of the curvature of these airways. To determine the number of iron-staining macrophages, sections were examined at X400 through an eyepiece fitted with a microbiolo-
CARDOZO, PADILLA, CHOI. AND LESSER
gy counting grid. Randomly selected fields free of airways or vessels were assessed for the number of blue-stained macrophages lying within the grid. A minimum of 15 fields were evaluated for each section. Data are expressed as the mean number of positively stained macrophages per field.
Statistics All data are expressed as mean ± SD. Comparisons were performed using Student's t test. Probability values of p < 0.05 were considered significant.
Results
Weight gain, pulmonary physiology, and histologic and morphometric examination of the lung parenchyma of the animals have been described elsewhere (14).
Histologic Examination Histologic reviewof PAS-stained plasticembedded sections of main stem bronchi revealed no significant changes in the bronchi of cathepsin B treated animals. Specifically,there wasno evidence of loss of epithelium, epithelial thickening or atypia, inflammation, or change in the thickness of adventitial or muscle layers or in numbers of mast cells. In contast, large intrapulmonary airwaysof cathepsin B-treated animals contained numerous foci of increased numbers of goblet cells (figure 1). In these areas, the epithelium appeared thickened, and the goblet cells appeared wider and more deeply stained than did those of control animals. The cells were often engorged with secretory granules (figure 1). Ciliated cells were often narrowed as if compressed by the adjacent goblet cells. There was no change in the number of inflammatory cells or mast cells, or in the thickness of the subepithelial layer. No epithelial sloughing was noted. Sections of peripheral airways from cathepsin B-treated animals contained increased numbers of cells with granular cytoplasm containing PAS-negative material, and increased numbers of ballooned cells with indistinct cell borders. No goblet cells were seen at this level of the airway. Cell Differentials The effects of intratracheal instillation of cathepsin B on cell numbers and cell differentials are shown in table 1. There was a significant increase in the total number of cells per millimeter and in the number and percentage of secretory cells (cells that contained three or more PASpositive secretory granules) in the large
intrapulmonary airways of animals given cathepsin B. No significant change was seen in the number of ciliated or other cells, indicating that the increase in the total number of epithelial cells was solely due to expansion of the secretory cell population. Further evidencethat a selective increase in the number of secretory cells occurred is provided by the finding that the increase in the total number of cells per millimeter was nearly the same as the increase in the number of secretory cells per millimeter. No significant change in the number or percentage of cells classified as "other," which included presecretory and preciliated cells (19), was observed. The significant decrease in the percentage of ciliated cells in the large intrapulmonary bronchi of cathepsin B-animals is presumably due to the increase in the proportion of secretory cells. Fewsignificant alterations in cell numbers and cell differentials were noted in the main bronchi of cathepsin B-animals (table 1). The tissue from one cathepsin B-treated animal was inadequate for evaluation; tissue from one control animal was inadequate for determination of cell differentials. No significant alteration in the number or percentage of secretory cells was noted in the main bronchi of these animals. The marginally significant increase in the total number of epithelial and ciliated cells in cathepsin B-treated animals probably represents a type II statistical error because the data are skewed by findings from one animal with an unusually high cell density, and because the differences disappear when data from this animal are not included in the analysis.
Iron-containing Macrophages In preliminary studies we noted increased numbers of pigment-laden macrophages in the lungs of hamsters given cathepsin B. Because the lungs of the animal that died during the second injection of cathepsin B contained diffuse hemorrhage at autopsy, we speculated that the pigment seen at 6 wk was hemosiderin. Accordingly, we reviewed paraffin sections stained with Perl's iron stain. Histologically, iron-positive macrophages appeared to be more numerous and more deeply stained in the lungs of cathepsin B-treated animals. However, quantitation of blue-stained macrophages revealed that the lungs of cathepsin B-treated animals contained slightly though not significantly more positivelystained macrophages than did the lungs of buffer-
GOBLET CELL HYPERPLASIA IN INTRAPULMONARY AIRWAYS AFTER CATHEPSIN B
Fig. 1. Top panel. Epithelium of the intrapulmonary airway of an animal given acetate buffer (pH, 5.5).The epithelium is made up of ciliated cells and secretory cells containing PAS-stained secretory granules. Bottompanel. Intrapulmonary airway epithelium of an animal given cathepsin B. The epithelium appears thickened and contains increased numbers of goblet cells, which are larger and contain increased numbers of granules. (PAS-hematoxylin· stained t-urn sections of plastic-embedded tissues, original magnification, 1,040.)
treated animals (5.3 ± 1.5, n 1.7, n = 8, respectively).
=
7; 4.4 ±
Discussion
We found that three intratracheal injections of cathepsin B caused significant increases in the number and percentage of PAS-positive secretory cells in large intrapulmonary airways of hamsters. We believethat this is the first report of secre-
tory cellhyperplasia caused by an enzyme contained within AM, and the first evidence that mammalian cysteine proteinases are capable of inducing this abnormality. The findings indicate that cathepsin B should be added to the growing list of proteinases capable of causing this abnormality in hamsters, which already includes human neutrophil elastase, porcine pancreatic elastase, trypsin,
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and cathepsin G (9-11). The findings provide support for the theory advanced by Christensen and coworkers (9) that increased levels of airway proteinases contribute to the development of GCH in smokers. Although previous studies have focused on the neutrophil proteinases, neutrophil elastase, and cathepsin G (10, 11, 18,20,21), findings that the levels of cathepsin B in homogenates of AM from smokers are increased two to three times, and that levels in BALF from smokers are increased 10-fold (12), suggest that AM also contribute to the proteinase burden in the airways. Alveolar macrophages are the predominant cells in BALF of healthy smokers and nonsmokers, and the number of AM in BALF from healthy smokers is approximately fivetimes higher than that found in nonsmokers, indicating that AM may be an important source of proteinases in the lung (12). Cathepsin B-induced secretory cell hyperplasia of large intrapulmonary airways occurred without significant alterations in the numbers of ciliated or other epithelial cells. The findings suggest that in this model, secretory cell hyperplasia occurs through selectiveexpansion of the secretory cell pool. Expansion presumably occurs through proliferation of a precursor cell. Support comes from findings that proliferation of nonciliated, nonsecretory cells occurs after intratracheal instillation of neturophil elastase (22). In contrast to our findings, studies evaluating the effects of human neutrophil elastase on hamster intrapulmonary airways found significant decreases in the percentage of nonciliated, nonsecretory cellswithout a change in the percentage of ciliated cells or the number of cells per millimeter of airway (18). We found that the epithelium of peripheral airways of cathepsin B-treated animals contained increased numbers of ballooned cells and cells with granular cytoplasm. In contrast, studies of the effects of human neutrophil elastase found no abnormalities at this level of the airway, whereas studies of the effects of porcine pancreatic elastase reported the presence of PAS-positive cells (9, 20). In our study, the number and percentage of PAS-positive secretory cells in the airways of buffer-treated animals decreased as the airways became smaller, and there were essentially no PASpositive secretory cells in the peripheral airways. These 0 bservations are in agreement with those obtained by Kennedy and coworkers (23) from light and electron microscopic examination of normal hamster airways. We found that epi-
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CARDOZO, PADILLA, CHOI, AND LESSER
TABLE 1 CELL DENSITY AND CELL DIFFERENTIAL COUNTS' Cell Numbers per Millimeter Epitheliumt Group Main bronchi Control Cathepsin B Intrapulmonary bronchi Control Cathepsin B
Cell Differential (%)
n
Total
Ciliated
Secretory
Other
7 6
153 ± 15 174 ± 2411
92.7 ± 10.0 107.3 ± 15.911
56.6 ± 10.5 62.4 ± 10.5
3.0 ± 2.2 3.6 ± 1.8
61.0 ± 4.1 61.9 ± 1.7
37.0 ± 4.2 35.9 ± 2.1
2.0 ± 1.6 2.2 ± 1.2
8 7
149 ± 14 164 ± 11§
93.9 ± 8.1 94.8 ± 10.3
47.5 ± 10.3 64.8 ± 7.3:1:
3.0 ± 2.2 4.3 ± 4.1
63.2 ± 5.0 57.8 ± 4.211
31.8 ± 4.9 39.6 ± 4.4:1:
5.0 ± 3.2 2.6 ± 2.5
Ciliated
Secretory
Other
• Data are mean values ± SO; n refers to the number of animals for which specimens were reviewed. Control animals were given acetate buffer (pH, 5.5); cathepsin-B-treated animals were given cathepsin B dissolved in acetate buffer (pH, 5.5). Methods for determination of numbers of epithelial cells and for determination of cell differentials are given in the text. t Number was eight for determination of total number of cells per millimeter for main bronchi from control animals. p < 0.005 compared with control animals. § p < 0.025 compared with control animals. IIp < 0.05 compared with control animals.
*
thelium of intrapulmonary bronchi of animals treated with buffer contained 31.8070 secretory cells, a finding in agreement with studies of normal hamster bronchi using similar methods, which found that 32.9% of cells with a luminal border were secretory cells (23). Of interest, we found no significant increase in the number or percentage of secretory cells in the main bronchi of cathepsin B-treated animals. Our findings are in agreement with 0 bservations that no change occurred in the number of tracheal goblet cells after the intratracheal instillation of human neutrophil elastase (24). Thus, the biology of epithelium at this level of the airway may be fundamentally different from that of epithelium more distal in the respiratory system. Support for this notion comes from electron microscopic studies using iron-conjugated lectins, which showed that significant differences exist in epithelial surface glycoconjugates expressed in hamster trachea and bronchi (25). Although gross pulmonary hemorrhage was seen in the lungs of the animal that died during the instillation of cathepsin B, no significant increase in the number of iron-stained macrophages was observed in the lungs of cathepsin B-treated animals 6 wk after instillation of the enzyme. However, macrophages in the lungs of cathepsin B-treated animals were more deeply stained, suggesting that some increase in iron pigments was present. Our observations differ from those of Lucey and coworkers (21) who found that the number of iron-laden macrophages was markedly increased 1 month after injection of human neutrophil elastase, and remained increased for at least 18 months. The reason(s) for the difference are unknown, but they might include variations in the amount of blood
entering the lung after injection or in the trophil alveolitis and intra-alveolar rates of clearance of pigment-laden hemorrhage, presumably associated with exudation of serum proteins, including macrophages. The mechanisms by which proteinases uz-macroglobulin. Evidence that procause OCH remain unknown. Findings tease-ai-macroglobulin complexes are that enzymes must be proteolytically ac- formed after instillation of proteases tive to cause OCH (10, 11) imply that comes from studies showing that in hyperplasia results from cleavage of one BALF samples obtained from hamsters or more proteins. Of interest in this re- after the intratracheal injection of 1251_ gard, the specificities of the proteinases human neutrophil elastase, a large porthat cause OCH vary significantly. Ca- tion of the enzyme is present as a 780thepsin B is a cysteine proteinase with kD complex (31). In contrast to the other enzymes shown broad specificity that cleaves peptide bonds between amino acids with vary- to cause OCH, cathepsin B is optimally ing properties (26). In contrast, neutro- active at pH 5.5 to 6.0. Thus, activity is phil elastase cleaves peptide bonds on the presumably limited to microenvironcarboxyl side of residues with aliphatic ments with acidic pH. Several observaside chains (27), whereas trypsin, anoth- tions suggest that such conditions exist er proteinase capable of causing OCH extracellularly, Microelectrode studies (10), cleaves on the carboxyl side of ba- have demonstrated that stimulated perisic amino acid residues. Thus, it seems toneal macrophages adherent to collagen unlikely that goblet cell hyperplasia gels acidify the microenvironment beresults from cleavage of a specific pep- tween the cell membrane and the gel (32). Thus, it is possible that inhalation of tide bond in a single protein. It is also possible that the stimulus is smoke stimulates AM adherent to respidue to proteinases complexed with U2- ratory epithelium to create an acidic macroglobulin or other airway proteinase microenvironment. Also, lacunar osinhibitors. Alphaz-macroglobulin inacti- teoclasts, macrophage-like cells, degrade vates a number of proteinases, including bone collagens through the action of cyscathepsin B, neutrophil elastase, trypsin, teine proteinases (33), and, under certain and cathepsin 0 (28-30). Alphas-mac- conditions, human AM degrade proteins roglobulin acts by "trapping" proteinases in extracellular matrices predominantly within the four subunits of the molecule, through the action of cysteine proteinases a process that is triggered by proteolytic acting largely at the cell surface or in the cleavage of a bait region (28). Thus, the pericellular microenvironment (34, 35). inhibitor only complexes with enzymes Findings that the enzyme retains signifithat are proteolytically active (28). It is cant activity at pH 7.0 (36) indicate that possible that the u2-macroglobulin-en- even slight reductions in the pH ofairzyme complex binds to constituents of way fluids may permit cathepsin B prothe epithelial cell membrane, thereby teolytic activity. Thus, if the pH of airproviding a stimulus for secretion or way lining fluid in humans is reduced, proliferation of goblet cells. Although as it is in cats and fetal lambs (37, 38), uz-macrcglobulin is not normally pres- it is possible that cathepsin B participates ent in significant amounts in the airway, in the degradation of extracellular proinstillation of proteinases induces neu- teins in vivo.
GOBLET CELL HYPERPLASIA IN INTRAPULMONARY AIRWAYS AFTER CATHEPSIN B
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