A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e , Vol. 3 , No. 5 Copyright © 1973, Institute for Clinical Science

The Role of Proteolytic Enzymes in

the Pathogenesis o f Emphysema

G. WILLIAM ATKINSON, M.D.*

Thomas Jefferson University, Philadelphia, PA 19107

ABSTRACT

Alphai-antitrypsin (aiAT) deficiency states, which predispose one to the development of pulmonary emphysema, have been described with increasing frequency. While the precise physiologic role of this protein is not known, it appears certain that it protects the lung from proteolytic digestion by a number of enzymes. Data are now available which mimic this situation in the experi­mental animal and demonstrate the protective effect of aiAT. Mechanisms of this genetic model for autodigestion and a number of pathological and bio­chemical considerations are offered in this paper.

Laurell originally observed that the ab­sence of the alpha- 1 band on routine pro­tein electrophoresis strips was associated with pulmonary disease. 29 Subsequent studies by Eriksson, 5 elucidating the clin­ical nature of pulmonary emphysema asso­ciated with deficiency of alpha-l-antitryp- sin have led to increasing interest in the role of this acute phase protein, or lack of it, in the pathogenesis of chronic obstruc­tive pulmonary disease. Furthermore, what appears to be a genetic model for “auto­digestion of the lung” has led to studies focusing on this disease from various view­points.

The alpha-l-antitrypsin deficiency state represents less than five percent of the total emphysema population. 2 1 It differs from classical emphysema only in that it occurs

* Recipient of the Pulmonary Academic Award NHLIHT-116-72.

at a younger age and is evenly distributed between male and female. 6 Afflicted indi­viduals appear to have less of a bronchitic component than the emphysema popula­tion at large but, by all parameters of phys­iological testing no distinct abnormality of lung function separates aiAT deficient pa­tients from other emphysema patients. Alpha-l-antitrypsin deficient patients char­acteristically have radiological evidence of lower zone emphysema, 1 loss of elastic re­coil, and abnormal ventilation/perfusion ratios in the lower lobes in addition to evidence of obstructive lung disease by spirometry. 7 Studies of frequency depen­dent compliance and other sophisticated procedures have been reported and have only confirmed the similarity between aiAT deficient patients and the general population of emphysema patients. 8

Emphysematous destruction of the lower lobes with dilatation of the air spaces and

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destruction of lung parenchyma, not unlike the general emphysematous lung of pan- acinar emphysema, is the most common finding. 43 The loss of elastic tissue appears to be the common denominator for all the physiologic and pathologic abnormalities but, to date, the mechanism of such de­struction is poorly understood.

B iochem ical and G enetic C onsiderations

Alpha-l-antitrypsin (axAT) is one of a number of protease inhibitors found in se­rum. Other protease inhibitors are chymo- trypsin inhibitor (C l) , inter-alpha-trypsin inhibitor (ia-AT) and alpha-2-macroglob- ulin (A2 M ). Alpha-l-antitrypsin is a low molecular weight glycoprotein (M.W. 54,000) that migrates with the alpha-1- globulin fraction on routine protein electro­phoresis and is responsible for ninety per­cent of the anti-trypsin activity of serum. 6

It is not specific for trypsin since it has activity against elastase, 45 plasmin, 1 4 leuko­cyte protease, 31 kallikrein4 6 and thrombin. 14

It has little activity against proteases of plant origin.

Alpha-l-antitrypsin is an acute reaction protein that is produced in liver paren­chymal cells and can be identified in se­rum, lymph, semen, liver and lung tissue. 12

Blood levels increase in infection, with es­trogen therapy, during stress and in preg­nancy. 23 This rise is attributed to increased production.

Eight aiAT zones have been described by acid starch gel electrophoresis. Various combinations of these zones identify 17 variants in the so-called protease inhibitor (Pi) system. 9 These aiAT variants are in­herited as codominant alleles and are des­ignated by the two zones in which the major protein concentration exists. Anti- gen-antibody crossed electrophoresis has given similar results. 28

To date, the aiAT type that is unequiv­ocally associated with pulmonary emphy­

sema is type Pizz, whereas PiMM is the most common type in normals as well as the general emphysema population. 18 There is still debate over whether some of the het­erozygous variants, PiMZ, Pisz and PiFZ, predispose one to the development of pul­monary emphysema. Current evidence sup­ports the thesis that it does, but suggests that evolution of the disease takes longer and that it is generally less severe. 27

Alpha-l-antitrypsin protease activity is commonly measured in the laboratory by assay of its inhibitory effect on trypsin, hence the name. Benzoyl-D, L-arginine-p- nitroanalide ( BAPA ) is a suitable substrate which gives the yellow color of p-nitro- analine when reacted with trypsin. 22 Pro­tein concentration of aiAT is determined immunologically by either the radial im­munodiffusion technique of Mancini38 or by the “rocket” technique of Laurell. There is general correlation between the amount of immunologically identifiable protein and trypsin inhibitory capacity (T.I.C.) of se­rum.

Affected families and population surveys show a trimodal distribution of values in which very low values represent homozy­gous Pizz states and intermediate values correlate with heterozygosity, Pisz, PiFZ and PiMZ. Misidentification of the patient with intermediate deficiency as normal is an occasional problem because these pa­tients respond to infection and the other factors by increased production of aiAT and thus may have normal values under such conditions. To be sure of heterozygous deficiency either genetic studies of the family or identification of the Pi variant must be performed. Homozygously defi­cient patients of the Pizz type cannot sim­ilarly respond to reach levels which would be confused with intermediate values.

In addition to differential migration in an electrophoretic field, there is evidence to suggest that abnormal variants of aiAT are structurally different from normal.

R O L E O F P R O T E O L Y T IC E N Z Y M E S IN PA TH O G EN ESIS O F E M P H Y SE M A 3 4 7

Lieberman34 has observed large membrane bound globules crowding the rough endo­plasmic reticulum of hepatic cells in pa­tients with axAT deficiency. Similar changes are seen in juvenile cirrhosis asso­ciated with aiAT deficiency. These glob­ules are PAS positive, diastase resistant and contain a ^ T by immunofluorescent stain­ing. Changes have not been observed in normal livers. The reason for these changes is not known. Lieberman postulated that the aiAT molecule could not be released into the circulation owing to either the absence of a releasing factor or structural alteration which would not permit the mol­ecule to enter the extracellular space. Our data on one patient confirm Lieberman’s observation. Although it was possible to identify the globules as aiAT by immuno­fluorescent staining, little protease inhibitor activity was detected in sucrose, buffered saline, tween-80 or in 0.001 M HC1 extracts. Residual a, AT immunofluorescent activity remained in the unsoluabilized pellet. It is suggested that axAT in the liver of such patients is membrane bound and structur­ally different from that circulating in the blood.

Patients with aiAT deficiency of the Pizz type do not necessarily develop hepatic scarring nor have patients with juvenile cirrhosis been observed to have emphy­sema. Furthermore, there are a number of instances of aiAT deficient patients of the Pizz type who have neither pulmonary em­physema nor hepatic cirrhosis. This evi­dence suggests that there may be variants with the Pizz type itself and that the Pi variety may simply be a marker for a yet unidentified genetic system.

Lieberman suggested that abnormal pro­tein variants of aiAT deteriorate in vivo. He observed a “double ring” around all immunodiffusion wells of heterozygously deficient patients’ serum. 3 3 Lieberman pos­tulated that a portion of what appears to be a homogenous protein variant forms

dimers which migrate differently in im­munochemical gel. Confirmation of this thesis awaits turnover studies of specific Pi varieties and verification that the plates used contained monospecific antibody.

Present evidence indicates that aiAT binds reversibly to the lysine residue of trypsin. However, there is no free trypsin in the extracellular or vascular compart­ments of the body. More consideration must be given to the known actions of aiAT against other proteases, especially elastase. To date, the mechanism of action of axAT in vivo and its role in homeostasis is mere speculation with evidence to sup­port various theories.

PathophysiologySince Gross17 first described the destruc­

tive properties of papain in the lungs of experimental animals and Goldring15 ob­served elastic fiber destruction in the lungs of these animals, research has centered on the search for an endogenous enzyme with elastoproteinase activity. Janoff25 recently characterized an esteroprotease in granules of polymorphonuclear leukocytes and mac­rophages. He observed that this enzyme was active at physiologic pH and was more destructive to the lung of experimental an­imals than collagenase. This enzyme, leuko­cyte elastase, when purified was found to be one-tenth as active as pancreatic elas­tase and more active than papain, trypsin and chymotrypsin against synthetic sub­strates in vitro.

Alpha-l-antitrypsin has been shown to inhibit the fibrinolytic activity of leuko­cytes. However, Gans13 was unable to demonstrate lack of leukocyte fibrinolytic inhibition in aiAT deficiency states. Lieb­erman3 5 has recently clarified this area by characterizing four neutral proteases in ex­tracts of purulent sputum, only one of which is fibrinolysin. One is similar to leukoclastase, as described by Janoff, and the others are referred to as stable protease

F i g u r e 1. Photomicrographs of rat lung after instillation of 1 mg. of papain transtracheally showing patchy alveolar destruction similar to centrilobular emphysema, a. Control at 24 h. b. Control at 48 h. c. Control at 6 days. d. Test animal at 24 h. e. Test animal at 48 h. f. Test animal at 6 days.

B O L E O F PK O T E O L Y T IC E N Z Y M E S IN PA TH O G EN ESIS O F E M P H Y SE M A 3 4 9

and liable protease. These proteases were not present in non-purulent mucoid sputum and were unrelated to the infecting or­ganism.

Using hemoglobin substrates, it can be demonstrated in vitro that the proteolytic activity of the stable and liable protease is inhibited by normal serum, less so by het­erozygous a5 AT serum and only minimally by homozygous aiAT serum when equiv­alent dilutions are used. 32 These leukopro- teases in vivo cause disintegration of alve­olar structure in hamster lung.

Normal serum can be shown to inhibit the rate of spontaneous proteolysis of ho­mogenized human lungs. 30 Furthermore, spontaneous autolysis of atAT deficient lungs is higher than normal lungs and can be accentuated by the addition of leuko­cyte protease, an effect which is readily preventable by the addition of normal serum.

Intense macrophage activity has been observed when the lungs are exposed to cigarette smoke. McLaughlin and Tueller42

have observed a pigmented macrophage, thought to be a component of cigarette smoke, in lungs of patients with early em­physema. This pigmented macrophage is shed in the sputum of smokers and is ab­sent in non-smokers. It can be recovered continually from the lungs of patients as long as five years after cessation of smok­ing. Harris2 0 postulated release of lyso­somal enzymes from these macrophages as a factor which leads to alveolar damage in smokers.

Loss of elasticity is characteristic of all forms of pulmonary emphysema. Debate exists as to whether or not there is less elastic tissue per se, but it is clear that the elastic tissue of emphysematous lung is ab­normal. Keller26 has shown that, in contra­distinction to normal lung elastic tissue, neutral amino acid residues are decreased and polar amino acid residues are in­creased in emphysematous lung. This re­sults in decreased cross linking and loss of

overall elasticity. Similar changes have been observed in experimental emphysema.

Bacteria have been shown to produce in­activation of alpha-l-antitrypsin. Masho- witz has shown that species of Proteus and Pseudomonas produce large amounts of this so-called antitrypsin inactivator (A I) and suggests that lung destruction could result from AI overwhelming the protective protease inhibitor proteins of the aiAT de­ficient lung. 41

There appears to be no free elastolytic activity in the blood, as previously re­ported. With careful preparations, all elas- tase properties of blood are found in poly­morphonuclear leukocytes and monocytes. Loeven has observed higher pancreatic levels of elastoproteinase in senile emphy­sema and a number of other degenerative conditions. 3 6 This elastoproteinase attacks lung elastic tissue vigorously and increases destruction of lung in the experimental an­imal when superimposed pneumonia is present.

Werle has suggested that kallikrein, a potent substance active in the lung, may be responsible for some of the changes in pulmonary emphysema. 11 It is only slowly inhibited by aiAT, even when aiAT is in excess, but may be active in the pulmonary capillaries when aiAT is overwhelmed or in axAT deficiency states. Mustafa has suggested that the primary induction of protease release from macrophages and leukocytes may be caused by membrane associated biochemical reactions of lipase and has experimental evidence to confirm this reaction. 3 Dukor has demonstrated that lysosomal proteases are under the control of cyclic AMP and the release of these enzymes is inhibited by colchicine. 4

Experim ental Em physem aOne of the main thrusts of emphysema

research has been to develop an experi­mental animal model in which to study the pathogenesis of the disease. Gross instilled papain into the tracheas of rats and dem­

3 5 0 ATB3NSON

onstrated anatomical changes not unlike centrilobular emphysema. 1 7 Previous expo­sure to silica dust had a protective effect on these lungs. Goldring produced similar changes by aerosol and further demon­strated disruption of the elastic fibers. 15

Papain and other plant enzymes cause direct injury to the alveolus followed by inflammation, frank pneumonia and finally fibrosis and alveolar destruction (figure 1 ). When papain is directly instilled, centri­lobular emphysema is produced; when it is aerosolized, panacinar emphysema is the usual lesion. Hamsters, which do not have antitrypsin in their serum, show an intense reaction to proteases which are instilled into the lung. Persistance of alveolar mac­rophages is observed after the inflamma­tory reaction. The detrimental effect on lung of other plant proteases in enzyme detergents has been confirmed in the ex­perimental animal. 89 Alcalase and maxa- tase, proteases produced by B. Subtilis, produce focal emphysema in hamster lung but the lesions are less profound than those of papain.

Kimbel40 produced emphysema in dogs by aerosolization of leukocyte protease har­vested from human polymorphonuclear leukocytes, dog polymorphonuclear leuko­cytes and dog alveolar macrophages. Rab­bit PMN extracts had little activity and did not produce convincing lesions. Janoff25 has subsequently shown that rabbit leukocytes do not have demonstrable elastolytic pro­tease. Progesterone has been shown to pro­tect experimental animals from papain em­physema without a permanent rise in the T.I.C. or increase in serum aiAT . 2 4 Al­though the mechanism is not known, Lieb- erman surmised it is due to the anti-inflam­matory properties of progesterone rather than its ability to increase circulating aiAT . 30

Turpentine by injection and nitrogen di­oxide by inhalation, agents which are known to increase serum tryptic activity,

have been given in conjunction with pa­pain to experimental animals. 28 Although there are marked increases in serum tryptic activity which are seen transiently, these substances did not prevent the eventual development of emphysema.

Mandl has produced emphysema in rats by injecting thermolycin and pancreatic elastase directly into the systemic circula­tion. 8 9 No damage was seen in other or­gans. The loss of elasticity in the lungs of such animals was attributed to enzymatic attack of the valine and isolucine in the outer shell of elastin. In addition, a de­creased ratio of polar to nonpolar amino acids was observed in these lungs. When papain is injected systematically rather than administered locally to the lungs, the supporting capillary network of elastic tis­sue in the lung is destroyed. Harley19 sug­gested that this is the primary event in papain emphysema and that the alveolar damage and loss of capillary bed is a secondary phenomenon.

Nitrogen dioxide (N 0 2), a known pollu­tant found in cigarette smoke, increases proteolytic activity in lungs of exposed rats. Lunan37 demonstrated increased protein synthesis and increased protein soluble fil­trate in such lungs when they were inflated with BAPA substrate. Since N 0 2 oxidizes a-tocopherol and other fatty acid compo­nents of cell membranes, Lunan suggested that protease release in such animals is secondary to initial destruction of lipid membranes of cells which are undergoing rapid turnover.

The mechanics of papain lung in experi­mental animals have been studied by Park4 4 and Caldwell. 2 Increased lung com­pliance and decreased elastic recoil have been demonstrated by plethysmography without change in the airway resistance. Park maintains that emphysema in such animals can develop without predisposing airway disease. Caldwell concluded that the abnormal mechanical properties of

R O L E O F P R O T E O L Y T IC E N Z Y M E S IN PA TH O G EN ESIS O F E M P H Y S E M A 3 5 1

such lungs is due to the loss of tethering properties of the airway rather than to changes in the viscoelastic properties of the airway wall.

Sum m aryThe etiology and pathogenesis of pulmo­

nary emphysema is still unknown. How­ever, there is good evidence to suggest that autodigestion of lung parenchymea by pro­teolytic enzymes with elastase properties occurs in response to noxious inhalants and pulmonary infection. The source of these enzymes is most likely from endogenous polymorphonuclear leukocytes and macro­phages which migrate from the capillary bed to alveolar space and subsequently lose their cellular integrity. Alpha-1-antitrypsin, a potent protease inhibitor with elastase inhibitory properties, protects the lung from proteolytic damage, in most instances. However, it is postulated that emphysema can occur when this enzyme inhibitor is abnormally low as in the cases of aiAT deficiency.

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