Feb 20, 1980 - macroglobulin-pancreatic elastase complexes were re- covered from the .... lavage fluid on a Sepharose 6B column (presented in Table II A).
Role of a-Macroglobulin-Elastase Complexes in the Pathogenesis of Elastase-induced Emphysema in Hamsters PHILLIP J. STONE, JAMES D. CALORE, GORDON L. SNIDER, and CARL FRANZBLAU, Departments of Biochemistry and Medicine, Boston University School of Medicine, Boston Veterans Administration Hospital, Boston, Massachusetts 02118
A B S T R A C T Radiolabeled, enzymatically active or chloromethyl ketone-inactivated porcine pancreatic elastase was endotracheally instilled into hamsters. Gel filtration of the bronchopulmonary lavage fluid revealed two major radioactive fractions: one, eluting at 780,000 daltons, corresponding to an a-macroglobulinpancreatic elastase complex, and another, at 68,000 daltons, corresponding to an a-1-protease inhibitorpancreatic elastase complex. Elastolytic activity was recovered in the bronchopulmonary lavage fluid up to 4 d after elastase instillation and was associated with the a-macroglobulin-pancreatic elastase complex. Small amounts of this complex were recovered 15 d after instillation. When 0 .10 8
16
-ME/ /f-2-M-Elastase / / y-=0.04+0.43x .O,p
0.10
o
20 0 2 jAg
_
/
y=0.1 1+0.023x
8
16
202
8
-P I-Elastase y=0.12+0.16x 0.01 ~~~~~~~~~~~~~~r=1.0,p< 16
20
Elastase/ml Plasma
FIGURE 2 Comparison of the functional antielastase profile of hamster plasma with normal human plasma (PiMM) and deficient human plasma (PiZZ). Plasma samples were incubated with 3H-Me-PE and chromatographed on Sepharose 6B columns as described in Fig. 3. The quantity of elastase complexed with a-M and a-l-PI was calculated using the specific radioactivity of the 3H-Me-PE as described in the footnotes to Table I. The equations of the corresponding linear regression lines and their r values are indicated. Two sets of data points for the hamster plot have been presented in greater detail in Table III.
added to the fraction of chromatographed hamster plasma, which contained 53,000-dalton material. After chromatography of this mixture, no enzyme activity was detected in any of the resulting fractions. 55% of the recovered radioactivity was found in 68,000-dalton material and the remainder was found in low molecular weight fractions. In other studies, 70±4% (mean±SE; n = 3) of the hamster a-l-PI-elastase recovered from lavage and 86±5% (mean±SE; n = 4) of the complex from plasma was precipitated by rabbit antiserum to porcine pancreatic elastase. Little of the ca-M-elastase recovered from hamster plasma or lavage was precipitated by the antiserum, presumably because the elastase is surrounded by the large macroglobulin molecule (11, 12).
DISCUSSION
What is the fate and mode of transport of elastase instilled into hamster's lungs? 1 h after instillation of an emphysema-inducing dose of 3H-Me-PE, 40% remained intact and was not complexed with antiprotease molecules. Subsequently, much of the radioactivity in lung lavage was associated with a-M-3H-MePE, while levels of this complex in the blood were quite low as compared with a-l-PI-elastase or low molecular weight material. Levels of lavageable a-M'H-Me-PE decreased rapidly, although traces could still be detected in lavage fluid 15 d after instillation. Possible explanations for the relatively low level in the blood include a short half-life for the a-M-elastase complex in the blood, which has been noted for human
TABLE IV Functional Antielastase Titer of Plasma from Hamsters and Normal and a-l-PI-deficient (PiZZ) Humans° 'sH-Me-PE recovered
as
complex oft Source of plasma
No. of samples
a-M
Normal humans (PiMM) Deficient humans (PiZZ) Hamsters
4 5 5
84±18 42±6 226±33
Percent a-l-PI
recovery§
269±41
76±3 81±6 95±11
a-M-elastase/ a-l-PI-elastase
pg
Antielastase titer/ml"
pg
36±7 84±12
0.3±0.1 1.2±0.4 2.7±0.8
353±59 78±13 310±45
5H-Me-PE were incubated and then loaded on molecular sieve columns as described in the text. 'H-Me-PE was recovered complexed with a-M and a-i-PI, quantified by scintillation spectrometry, and assessed for enzyme activity. Free enzyme (25,000 daltons) with full enzymatic activity was recovered, indicating overtitration of the antielastase present. a Plasma and saturating amounts of
t Mean±SE. § The recovery of tritium radioactivity in the eluted volume was compared with the level of radioactivity loaded on the column. "The sum of the amounts of elastase complexed with a-i-PI and a-M per milliliter of plasma.
928
P. J. Stone, J. D. Calore, G. L. Snider, and C. Franzblau
670.000
z 1.5r
DALTONS
0
(TH-YROGLOBULIN) I I I 68,000 DALTONS
* 780,000 DALTONS: *
Po
*
* *
_LLI
Ec
1.0 0.9 0.8 0.7 0.6
0.5 0.4
0.3 0.2 0.1
0
z 1.01-
7000
(ALBUMIN)
6000
z
0
/~ **
5000
:
4000
U-
-w4 -4U _UJ n
z
117,800 DALTONS
05.5
_.
_w
2
2000
C.)
(MYOGLOBIN)
en a
-tU)
3000
*
~
.
.
11000 0
"15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72
FRACTION NUMBER
FIGURE 3 The molecular weight distribution of complexes between 3H-Me-PE and hamster plasma. 1 ml of hamster plasma was incubated with 0.2 ml of water containing 18.6 ,ug of 3HMe-PE and placed on a Sepharose 6B column. The fractions (3 ml) were assessed for absorbance at 280 nm (-- -), radioactivity (-), and elastolytic activity (... ) (see the footnotes to Table III). Aliquots of those fractions containing material with a molecular weight of 25,000 daltons or less were rechromatographed on Sephadex G-100 columns to resolve 25,000-dalton material from low molecular weight material. Similar results were obtained whether the plasma was collected in heparin or acid citrate dextrose or whether serum was used. The elution positions of molecular weight standards are indicated.
a-2-macroglobulin-l31I-leukocyte elastase (13), and the sequestering of the large c-M-3H-Me-PE complex by the lung matrix with little clearance into the blood (3). Ohlsson and Laurell (13) found that substantial amounts of radioactivity were quickly localized in the liver after administration of '31I-leukocyte elastase. We found substantial amounts of radioactivity in the livers of hamsters after instillation of 3H-Me-PE and much lower levels after instillation of 3H-Me-CMK-PE (3). The a-M-3H-Me-PE complexes, whether obtained from elastase-hamster plasma mixtures or the lung lavage fluid, exhibited elastolytic activity against bovine ligament, lung, and aorta elastin. Blood was apparently the source of the a-M found in the lung lavage material, because incubation of 3H-Me-PE in vitro with concentrated nonhemorrhagic lung lavage fluid from hamsters that had not been treated with elastase produced relatively small amounts of a-M-3H-Me-PE as compared with a-l-PI-3H-Me-PE. A low dose of elastase instilled into the hamster's lungs produces no apparent hemorrhage or emphysema and, we believe, is inactivated by the endogenous lung antiprotease system, which contains little or no a-M-like inhibitor. Insignificant amounts of a-M-3H-Me-PE and free 3HMe-PE were found in their lung lavage fluid, and no
elastolytic activity could be detected in the fluid. The only complex found resembled a-l-PI-3H-Me-PE. The oversaturation of this endogenous antiprotease system by a large dose of elastase facilitated proteolytic degradation of capillary walls and hemorrhage (3). The formation of a-M-elastase complexes in the lung follows. Inactivation of 3H-Me-PE with N-acetyl-(L-alanyl)3L-alanine chloromethyl ketone resulted in a dramatic alteration of the rate and manner of clearance (3). Much of the 3H-Me-CMK-PE was excreted as an intact molecule of 25,000 daltons. As expected, little 3H-MeCMK-PE (1-2%) complexed with antiprotease molecules. Sandhaus and Janoff (14) found that 1251-elastase was bound to a-l-PI, while chloromethyl ketone-inhibited '25I-elastase was uncomplexed. Inasmuch as antiprotease molecules played little or no role in the clearance of 3H-Me-CMK-PE and significant amounts of free 3H-Me-PE were not found after 1 h in lavage fluid or plasma, the slower rate of clearance of 3H-MePE from the lungs as compared with 3H-Me-CMK-PE (3) suggests that the formation of a-l-PI-3H-Me-PE and a-M-3H-Me-PE retarded the clearance of elastase from the lung matrix. 15 d after instillation of 3H-MePE we were still able to recover small amounts of aa-Macroglobulin-Elastase Complexes 929
M-3H-Me-PE from hamsters by bronchopulmo- secreted by an alveolar macrophage or released by a nary lavage. neutrophil, will complex with a-l-PI and be rendered The finding of a low but significant amount of ra- inactive. If the amount of elastase elaborated exceeds dioactivity (1,200 cpm) in hamster lungs 144 d after the inhibitory capacity of a-l-PI, the type I cell may instillation of either dose of 3H-Me-PE but virtually be injured, and the elastase will gain access to the none (100 cpm) after 3H-Me-CMK-PE suggested the interstitium. During the course of entry of the elastase presence of 3H-Me-PE in some form (3). If some of into the interstitium, capillary endothelium may also the radioactivity in the lung matrix represented an be injured and hemorrhage may occur. As a result of a-M-elastase complex, the potential elastolytic activity the hemorrhage, circulating a-2-M and a-l-PI enter may be protected from a-l-PI inhibition, and the sub- the air spaces and combine with any free elastase. sequent release of elastase from the complexes may Damage to elastin fibers may continue as a result of lead to degradation of newly formed or older lung the elastolytic potential of the a-2-M-elastase comelastin (3, 15, 16). We (17) have shown that as little plexes, which are very large and may remain sequesas 0.006 ,ug of elastase binds to elastin and under op- tered in the lungs. It seems reasonable to think that timal conditions in vitro continues to degrade signif- this complex might serve to preserve the elastolytic icant amounts of the elastin for at least 4 wk. We did potential of the enzyme molecule for a long period of not find morphological evidence for pulmonary re- time. Deficiency of a-l-PI will result in the formation action to inactivated porcine elastase, suggesting that of higher levels of a-2-M-elastase complex. One imthe small amount of foreign protein was not injurious. plication of the role of the a-2-M-elastase complex in Since the discovery of a-l-PI deficiency and its as- the pathogenesis of emphysema is the prediction that sociation with pulmonary emphysema, workers have compounds that can inhibit elastase bound to a-2-M focused on the importance of absolute levels of anti- will be of greater benefit in inhibiting or preventing protease activity. For example, on the basis of the work the progression of emphysema than treatment with of Ihrig and co-workers (18), Lieberman (19) has sug- a-l-PI, which cannot inactivate elastase that has been gested that the ease with which emphysema is induced bound to a-2-M. in hamsters may be related to their low antiprotease Galdston et al. (15) have suggested that complexes level. It would appear from our work that the parti- of a-2-M and human neutrophil elastase may play a tioning of elastase between the antiproteases a-M and role in the pathogenesis of emphysema. Morse (22) has a-l-PI may play a key role in the pathogenesis of this hypothesized that a-2-M-elastase complex in the alenzyme-induced emphysema model. We have used veoli of humans might be ingested by macrophages radiolabeled elastase to determine the functional an- and concentrated centrilobularly. Alveolar macrotielastase profile and the titer of plasma and serum phages have been shown to concentrate elastase insamples. Because we find no free inactivated elastase stilled into dogs (23) and hamsters (3). Human alveolar molecules in these mixtures using gel filtration, we macrophages concentrate human neutrophil elastase believe that the sum of the elastase complexed with (24), much of which retains its enzymatic activity for a-l-PI and a-M accurately measures the functional at least 2 d (25). Alveolar macrophages may secrete inhibitory capacity of the sample. Although the ham- relatively large amounts of an elastase when stimulated ster appears to have an adequate antielastase titer, by such insults as cigarette smoke (26). Recent reports which greatly exceeds that of an a-l-PI-deficient (27, 28) indicate that cigarette smoke inactivates a-l(PiZZ) human, the major elastase inhibitor in the ham- PI molecules in the lungs of rats and humans. Johnson ster is a-M. a-l-PI apparently plays a significant role and Travis (29) have shown that myeloperoxidase from only after most of the a-M is saturated with elastase polymorphonuclear leukocytes readily inactivates a-l(Table IV). The a-2-M plays a more important role in PI; and we (30) have demonstrated that this a-l-PI binding small amounts of elastase added to plasma will not bind to and inactivate elastase. Such a comfrom a-l-PI-deficient (PiZZ) humans than normal bination of events might lead to the overwhelming of (PiMM) humans. Human leukocytic elastase has been endogenous pulmonary antiprotease defenses in smokfound to produce substantial pulmonary hemorrhage ers and facilitate the formation of a-2-M-elastase combut little emphysema in hamsters (20). Inasmuch as plexes, which themselves retain proteolytic activity Ohlsson and Olsson (21) have reported that the a-l-PI and may also degrade local a-l-PI. The enzymatic lifein human serum binds 92% of the added human leu- time of the elastase molecules appears to be greatly kocyte elastase and that a-2-M binds only 8% in vitro, extended by formation of a-2-M-elastase complexes, most of the leukocyte elastase introduced into the ham- which stabilizes the elastase against autolysis. Using ster's lungs may similarly be inactivated by a-1-PI. an immunohistochemical technique, Cassiman and coFinally, we wish to speculate on how imbalances of workers (31) have reported that human a-2-M is found the antielastase system might give rise to emphysema. associated with connective tissues of organs such as the Elastase, gaining access to the alveolar space, perhaps kidney and lungs. We suggest that the susceptibility P. J. Stone, J. D. Calore, G. L. Snider, and C. Franzblau 930
of some patients to emphysema may relate not only to the absolute concentrations of a-l-PI and a-2-M in the blood and lungs but also to their relative concentrations and their individual affinities for elastase. ACKNOWLEDGMENTS This work was supported by National Heart, Lung, and Blood Institute grants NIH-HL-19717 and NIH-HL-25229 and the Veterans Administration Research Service.
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Twumasi. 1979. Degradation of tropoelastin and elastin substrates by human neutrophil elastase, free and bound to alpha2-macroglobulin in serum of the M and Z (Pi) phenotypes for alpha1-antitrypsin. Am. Rev. Respir. Dis. 119: 435-441. 16. Christner, P., G. Weinbaum, B. Sloan, and J. Rosenbloom. 1978. Degradation of tropoelastin by proteases. Anal. Biochem. 88: 682-688. 17. Stone. P. J., G. Crombie, and C. Franzblau. 1977. The use of tritiated elastin for the determination of subnanogram amounts of elastase. Anal. Biochem. 80: 572577. 18. Ihrig, J., J. Kleinerman, and D. J. Rynbrandt. 1971. Serum antitrypsins in animals. Studies of species variations, components, and the influence of certain irritants. Am. Rev. Respir. Dis. 103: 377-389. 19. Lieberman, J. 1976. Elastase, collagenase, emphysema, and alpha1-antitrypsin deficiency. Chest. 70: 62-67. 20. Senior, R. M., H. Tegner, C. Kuhn, K. Ohlsson, B. C. Starcher, and J. A. Pierce. 1977. The induction of pulmonary emphysema with human leukocyte elastase. Am. Rev. Respir. Dis. 116: 469-475. 21. Ohlsson, K., and I. Olsson. 1974. Neutral proteases of human granulocytes. III. Interaction between human granulocyte elastase and plasma protease inhibitors. Scand. J. Clin. Lab. Invest. 34: 349-355. 22. Morse, J. 0. 1978. Alpha1-antitrypsin deficiency. N. Engi. J. Med. 299: 1099-1105. 23. Janoff, A., B. Sloan, G. Weinbaum, V. Damiano, R. A. Sandhaus, J. Elias, and P. Kimbel. 1977. Experimental emphysema induced with purified human neutrophil elastase: tissue localization of the instilled protease. Am. Rev. Respir. Dis. 115: 461-478. 24. Campbell, E. J., R. R. White, R. M. Senior, R. J. Rodriquez, and C. Kuhn. 1979. Receptor-mediated binding and internalization of leukocyte elastase by alveolar macrophages in vitro. J. Clin. Invest. 64: 824-833. 25. McGowan, S. E., P. J. Stone, and G. L. Snider. 1981. The fate of human leukocyte elastase incorporated into alveolar macrophages. Am. Rev. Respir. Dis. 123(Pt. 2): 226. (Abstr.) 26. Rodriquez, R. J., R. R. White, R. M. Senior, and E. A. Levine. 1977. Elastase release from human alveolar macrophages: Comparison between smokers and non-smokers. Science (Wash. D. C.). 198: 313-314. 27. Janoff, A., H. Carp, D. K. Lee, and R. T. Drew. 1979. Cigarette smoke inhalation decreases a1-antitrypsin activity in rat lung. Science (Wash. D. C.). 206:1313-1314. 28. Gadek, J. E., G. A. Fells, and R. G. Crystal. 1979. Cigarette smoking induces functional antiprotease deficiency in the lower respiratory tract of humans. Science (Wash. D. C.). 206: 1315-1316. 29. Johnson, D., and J. Travis. 1979. The oxidative inactivation of human a-l-proteinase inhibitor. Further evidence for methionine at the reactive center. J. Biol. Chem. 254: 4022-4026. 30. Clark, R. A., P. J. Stone, A. El Hag, J. D. Calore, and C. Franzblau. 1981. Myeloperoxidase-catalyzed inactivation of a-1-protease inhibitor by human neutrophils. J. Biol. Chem. 256: 3348-3353. 31. Cassiman, J. J., F. van Leuven, B. van der Schueren, and H. van den Berghe. 1980. Immunohistochemical localization of human a2-macroglobulin in connective tissue. Cell Tissue Res. 213:301-310.
a-Macroglobulin-Elastase Complexes
931
