Neurochemical Research (2) 233-246 (1977)
Overview
THE ROLE OF P R O T E O L Y T I C E N Z Y M E S IN D E M Y E L I N A T I O N IN E X P E R I M E N T A L ALLERGIC ENCEPHALOMYELITIS MARION EDMONDS SMITH Department of Neurology Veterans Administration Hospital Palo Alto, California Standord University School of Medicine Stanford, California 94305
Accepted November 23, 1976
For about 30 years the autoimmune disease experimental allergic encephalomyelitis (EAE) has been regularly produced in the laboratory in various species of animals to serve as a model of a cell-mediated demyelinating disease. It was recognized early that EAE and rabies postvaccinal encephalomyelitis were probably caused by identical immunologic mechanisms and agents, and were undoubtedly the same disease. A similar autoimmune process has been invoked as an explanation for the cause of other demyelinating diseases including postinfectious encephalomyelitis or acute disseminated encephalomyelitis, and perhaps multiple sclerosis (MS). Most of the early work on EAE is represented in two symposium collections (1,2).
EAE: P A T H O L O G I C A L D E S C R I P T I O N
Experimental allergic encephalomyelitis is produced by injection of susceptible animals with whole CNS tissue, white matter, purified myelin, or myelin basic protein in combination with Freund's complete 233 q~) 1977 Plenum Publishing Col~p., 227 West 17th Street, New York, N.Y. 100li. To promote freer access to published material in the spirit of the 1976 Copyright Law, Plenum sells reprint articles from all its journals. This availability underlines the fact that no part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher. Shipment is prompt; rate per article is $7.50.
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adjuvant. The animals most commonly used are rabbits, guinea pigs, certain susceptible strains of rats such as the Lewis rat, and monkeys. Only minimal clinical effects are usually observed for the first 10 days after injection; then the animal begins to lose weight and rapidly develops paralysis, which is often fatal in the rabbit, guinea pig, and monkey. Light-microscopic examination of CNS tissues from smaller animals with acute EAE reveals perivascular cuffing; the infiltrating cells have been identified as lymphocytes, small monocytes, and plasma cells (3). Demyelination occurs only in tissues in which infiltrating mononuclear cells are present. In the monkey the lesion is more severe, with large foci of hemorrhagic necroses often macroscopically visible, and the infiltrating cells contain many polymorphonuclear cells and eosinophils (4) in addition to mononuclear cells. The hyperacute lesion can also be produced in small animals by the use of pertussis vaccine
(5). Examination of the demyelinating lesion by electron microscopy has provided some understanding of the mechanisms by which the myelin sheath is destroyed. The infiltrating mononuclear cell surrounds the myelin sheath, which then becomes hydrated and splits at the intraperiod line. The major dense line may split also, and the separated lamellae cuff to form vesicular structures. The mononuclear cell appears to invade the sheath at the mesaxon and peels off the outer lamellae, which are then engulfed by phagocytosis. The myelin fragments are transformed into globoid lipid bodies within intracytoplasmic compartments (6). Hydration and vesiculation of the myelin sheath appear to result from the extracellular action of the mononuclear cells; then, after active stripping of the lamellae, the myelin debris becomes incorporated within phagocytic cells, where further digestion takes place.
E N Z Y M A T I C A C T I O N ON M Y E L I N . H I S T O R I C A L Although the morphological studies show that a complex series of events attend the complete dissolution of the myelin sheath, it has been suggested that an early perturbation, perhaps enzymatic, may initiate the degradative course. Since lipid is the major constituent of the myelin sheath, the possible role of lipases in myelin disintegration was first considered. In 1950, Morrison and Zamecnik (7) showed that blocks of spinal cord tissue incubated with Clostridium welchii filtrate or with various snake venoms containing lipases produced several kinds of lipid degeneration, including production of neutral lipids. More recently, significant increases have been measured in the levels of several lipases
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in the nervous system of EAE animals, such as might be expected to hydrolyze the lipids contained in myelin. These include aryl sulfatase (8) and phospholipase (9,10). Of late, however, more attention has been centered around the possibility that the first point of attack on the myelin sheath is protein that represents only 25-30% of the dry weight of myelin, but which is more vulnerable to attack in view of the properties of certain constituents.
N A T U R E OF M Y E L I N PROTEINS The proteins of the myelin sheath are relatively few in number compared to those in more metabolically active plasma membranes of most cells. Over 50% of the total myelin protein is composed of proteolipid protein, first described by Folch and Lees (11). This protein is highly complexed with lipids, some of which are covalently bound, and contains a large proportion of hydrophobic amino acids. Proteolipid protein is notable for its resistance to proteolytic enzymes, but may be partially hydrolyzed by trypsin in the presence of certain detergents (12), and by elastase (13) and pronase (14). The myelin basic protein, which comprises about 30% of the total myelin protein, is relatively small, with a molecular weight of about 18,000. In aqueous solution it has little if any tertiary structure, although the myelin membrane may impose some degree of order in situ. The amino acid sequence and other properties have been fully described (15). This protein, unlike the proteolipid protein, is hydrolyzed by a number of proteolytic enzymes including trypsin and pepsin (16,17), chymotrypsin, thermolysin, subtilysin, aminopeptidase, carboxypeptidase A and B (17), acid proteinase (18), pronase and elastase (14), and probably others. The remaining 20% of the myelin protein is made up of a group of higher-molecular-weight proteins, some of which may be contaminants as a result of the isolation process. Two of the higher-molecularweight proteins are believed to be associated with the myelin membrane, the Wolfgram protein, an acidic proteolipid (19), and a glycoprotein (20). Several enzymes also appear to be myelin specific. These proteins and the DM-20, which is separable by polyacrylamide gel electrophoresis under special conditions (21), have not been studied in regard to proteolytic enzyme susceptibility. If proteolysis is the first step in myelin breakdown, the myelin basic protein appears to be most vulnerable, not only in its isolated aqueous form, but also in situ in the myelin membrane. It is the only protein showing a decrease after incubation of whole myelin with trypsin.
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Although London and Vossenberg found that various lipids, especially acidic lipids, protect myelin basic protein from the proteolytic action of trypsin (22), others have found that low amounts of trypsin effectively remove much of the basic protein from purified myelin, while proteolipid and high-molecular-weight proteins remain unchanged (23,24).
P R O T E O L Y T I C E N Z Y M E S IN T H E CNS Consistent with the rapid turnover of most of its protein constituents, the CNS is notable for the high content of proteolytic enzymes. The identification of various endopeptidases and exopeptidases in the CNS has been reviewed (25,26), but relatively few of these have been purified and characterized. Acid proteinase in whole brain tissue was first reported by Kies and Schwimmer (27), and this enzyme has been further purified by several investigators (25). Acid proteinase or cathepsin D in the CNS was found predominantly in neurons, and resembles other lysosomal hydrolases in distribution (28). A neutral proteinase in brain tissue was described in 1954 by Ansell and Richter (29), and several investigators have reported the purification of this enzyme from animal brain (e.g., 30,31). Cathepsin A (carboxypeptidase A) (32), aminopeptidases, and arylamidases have also been identified (33). Aminopeptidase (34,35) and neutral proteinases (36), but not acid proteinase, are associated with highly purified myelin preparations. Proteolytic enzymes in the brain that might be involved in the formation and in/~ctivation of peptide hormones of the neuroendocrine system arid the vasoactive polypeptides are an important area of investigation; their characterization is essential to the further understanding of the action of these hormones and their regulation.
P R O T E O L Y T I C E N Z Y M E S IN D E M Y E L I N A T I N G DISEASES The possibility that proteolytic enzymes might be active agents in the demyelination process was suggested early by Kerekes et al. (37), who found a 62% increase in catheptic activity in hares injected with Freund's adjuvant and heterologous brain tissue. A marked intensification of proteolytic activity in periventricular white matter was observed histochemically in guinea pigs before EAE lesions developed and during the period of evolution of the lesion (38). That proteolytic enzymes are much increased in secondary demyelination, such as is seen in Wallerian degeneration, has been well documented (39,40). In 1969, a loss of basic
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protein was reported in MS plaques, as observed by histochemical methods (40), and this was consistent with the finding of increased proteolytic activity at the edges of the demyelinated area, along with an increased cell population and N A D H dehydrogenase activity (41). Hallpike et al. (40) tentatively suggested that the oligodendroglia or myelin itself may be the source of the enzymatic activity. In early studies on the purification of the encephalitogenic protein in the CNS, it was noted that, during acid extraction of acetone-defatted CNS, several smaller polypeptide fragments were obtained, whereas tissue defatted with chloroform-methanol or extracted below pH 2.0 yielded a larger encephalitogenic protein. The breakdown of the encephalitogen was attributed to the presence of acid proteinase, which was preserved by acetone extraction of the CNS (42). It was proposed at that time that such action of acid proteinase might be a key step in the breakdown of myelin in the disease state. This suggestion was given support by the finding of a 4-fold increase in acid proteinase at the edge of MS plaques (43), with a corresponding reduction of basic protein in the plaque (44).
PROTEOLYT1C E N Z Y M E S IN EAE The investigations of acid proteinase in demyelinative lesions were continued in animals with acute EAE. In the characteristic hyperacute lesions of the monkey, levels of acid proteinase were increased from two to 10 times that of apparently unaffected tissue of the same animal (4). A study of four lesions from some of the same monkeys in this laboratory by a different method revealed increases in the range of 152-I76% of the activity of analogous control areas (45). These increases were compared to increments in activity of two proteolytic enzymes with pH optima in a more neutral range, neutral proteinase and cathepsin A. The latter enzyme was shown to be increased in MS plaques, and was suggested as a marker for macrophages since the specific activity of cathepsin A in these cells was 30x that of white matter (46). In five EAE monkey lesions, neutral proteinase was 220-258% of control areas, and in the one lesion assayed for cathepsin A an increase of 8-fold was measured. Govindarajan et al. (47) also found markedly elevated cathepsin D, neutral proteinase, and cathepsin B1 levels in lesions of six monkeys with acute EAE. In brain stems of rats with acute EAE, acid proteinase was increased only 28%, neutral proteinase 12% (not significant), and cathepsin A was present at levels 258% of the level in brain stems of Freund's adjuvant-
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injected control rats. The differences in levels of increases between the monkey and rat tissue probably reflect the nature of the lesion in each animal. Whereas it was possible to dissect out the large areas of focal lesion tissue in the monkey, this was not possible in the rat, where the lesions are microscopic and diffuse; therefore, it was necessary to assay whole tissue. Increases in acid proteinase and cathepsin A were not seen up to 7 days after injection of the animals with the encephalitogenic material, but at 12-14 days, at which time acute clinical symptoms were obvious, the cathepsin A levels rose dramatically. In contrast to the Lewis rat, a strain susceptible to EAE, such increases were not seen in the brain stems of the injected Wistar rat, which is relatively nonsusceptible, although histological examination of the CNS tissue showed a very mild perivascular cuffing. The acid proteinase-specific activity was fairly constant in lymph nodes draining the site of injection from 4 days after injection until the acute stage, and did not differ greatly in Lewis or Wistar rats injected with Freund's adjuvant alone, or in uninjected controls. The size of the lymph node increased greatly after immunological stimulation; therefore, the total acid proteinase increased per lymph node. Lymph node cathepsin A, on the other hand, increased in specific activity and total activity over that of the uninjected control with the development of EAE, although there was no real difference in specific activity of lymph nodes of the Lewis and Wistar rat with acute EAE or the Freund's adjuvant-injected control (45). These results were not confirmed by Boehme et al. (48), who also observed a 55% increase in acid proteinase in spinal cord extracts of Lewis rats with acute EAE, but did not observe increas~es in cathepsin A. Bowen and Davison have pointed out that cathepsin A is not extracted from tissue in the alkaline range (32); in the experiments of Boehme et al., some extractions were done at pH 7.6. That a number of kinds of proteolytic enzymes are probably increased in EAE was demonstrated by Buletza and Smith (49), who found increased proteolytic activity throughout the pH range 3.5-8 in homogenates of spinal cords from rats with acute EAE. This increase was especially marked at pH 5-7 when casein was used as a substrate, and was also increased throughout the pH range using myelin basic protein as a substrate. The identification of the enzymes active at the various pH's was not made. The pH optimum of a particular enzyme may change with the substrate used. Although the pH optimum for acid proteinase is 3.8 with hemoglobin as a substrate, maximal activity for this enzyme may be seen at higher values using other substrates (50). The optimum pH for the acid proteinase hydrolysis of myelin basic protein, however, appears to coincide with that of hemoglobin (18).
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The source of the enzyme increases may be the CNS or the inflammatory cells. On the basis of the following indirect evidence, Smith et al. (45) suggested that the infiltrating cells of lymphoid origin might contain the observed enzyme increments: ~. The enzyme increases were much greater in the hyperacute concentrated EAE lesion of the monkey than in the diffuse microscopic lesion of the rat. 2. The enzyme increases were not seen until the onset of paralytic symptoms which coincided with the time of cellular infiltration. 3. The increments of enzyme activity in the EAE rat were greater with respect to cathepsin A than to acid proteinase, corresponding to the difference in levels of these enzymes between lymph nodes and brain stem. 4. In a demyelinative condition resulting from triethyl tin feeding, where infiltration of cells does not occur, levels of acid proteinase and cathepsin A were normal. Hirsch and Parks (51) confirmed these suppositions with elegant quantitative microhistochemical techniques. In the Lewis rat with acute EAE, only t h o s e areas that contained lesions showed increased acid proteinase, cathepsin A,/3-glucuronidase, and acid lipase-esterase, and these enzymes were precisely pinpointed to the loci of cellular infiltration. In one monkey lesion, levels in/3-glucuronidase, acid proteinase, dipeptidyl arylamidase iI, cathepsin A, acid phosphatase, neutral lipase, and D N A were sharply increased.
LYMPH NODE PROTEASES In view of the lymphoid origin of mononuclear cells migrating into the CNS in acute EAE (52), Buletza and Smith (49) assayed proteinase activity in homogenates of lymph nodes draining the site of injection in rats with acute EAE, and found very high proteolytic activity at p H 4-8 when basic protein was used as a substrate. These levels were not different in lymph nodes activated with Freund's adjuvant alone, suggesting that the nature of the challenging material injected does not confer specificity to the nature of the catabolic enzymes. When purified myelin was incubated at p H 7 with homogenates of inguinal and popliteal lymph nodes from rats with acute EAE, the myelin basic protein was selectively removed, while a continued increase in ninhydrin-positive material was released into the medium over a 4-h period of incubation. A fraction active in protease activity at p H 7 was prepared from these lymph nodes by ammonium sulfate precipita-
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tion. The enzyme activity was found to act selectively only on basic protein substrates including protamine sulfate, myelin basic protein, polylysine, and various histories. Much less activity was measured with cytochrome c and polyarginine. No activity was observed when casein, hemoglobin, egg albumin, bovine serum albumin, and ribonuclease were used as substrates (53). The p H optimum was 7 for rat myelin, beef basic protein, and histone. This enzyme has been purified about 80 times by gel filtration and ion exchange chromatography, appears to require a thiol group for activity, and is inhibited by phenylmethylsulfonyl fluoride and Trasylol (54). The neutral proteinase activity is present in lymphocytes prepared from chopped, sieved lymph nodes of rats with acute EAE. Such preparations are virtually free of macrophages. When these lymphocytes are incubated unbroken in isotonic solutions with purified myelin, myelin basic protein, or polylysine, a high level of proteolytic activity is obtained, while more activity is obtained from the lymphocytes upon homogenization (14). It is not clear whether the enzyme activity of the whole lymphocytes is a result of extracellular excretion or whether the enzyme is present on the plasma membrane exterior. Protease activity has been found on the rat lymph node lymphocyte cell surface (55), and it has been suggested that membrane-associated proteinases may participate in the killing of tumor cells by lymphocytes (56).
O T H E R P R O T E A S E S OF CNS A N D L E U K O C Y T E S Lysosomal enzymes from the brain alone are fully capable of destroying myelin basic protein. Purified brain cathepsin D cleaves purified myelin basic protein into three peptide fragments by hydrolysis of the two phenylalanine-phenylalanine linkages (57). Cathepsin A, also purified from brain, while not able to hydrolyze myelin basic protein by itself, can act on one of the three polypeptide fragments formed by acid proteinase (58). Thus the activity in cathepsin D is rate limiting in the stepwise cleavage by these two lysosomal enzymes. Such a concerted enzyme action is a feasible process by which normal turnover of myelin basic protein might occur, as well as in breakdown in pathological conditions. Immunologically active infiltrating cells, however, may exert their action by additional mechanisms. Human peripheral blood lymphocytes contain a number of proteases including cathepsin D, a neutral serine protease, and probably a thiol protease. The neutral protease activity was found bound to the surface of lymphocytes (59). Two unique types
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of cathepsin D-like proteases, apparently present only in rat thoracic duct lymphocytes and rat lymphoid tissues, have been described. Both enzymes differ from the rat liver cathepsin D by a greater sensitivity to pepstatin and their interconvertibility by mercaptoethanol (60). In the near future, proteolytic enzymes in normal and activated lymphocytes should become an important area of exploration. Other types of leukocytes should also be investigated for a possible role in demyelinative diseases. The cellular infiltrate in the hyperacute lesion of the monkey contained polymorphonuclear cells that are known to be a rich source of elastases (50,61). This enzyme is one of the few proteases found thus far that can cleave the proteolipid protein as well as the myelin basic protein.
P R O T E O L Y T I C E N Z Y M E S A N D T H E I R A C T I V I T Y IN MS LESIONS The original impetus for the investigation of proteolytic enzymes in demyelinative lesions in EAE was given by and has been paralleled by similar studies of the MS plaque. Following the original suggestions by Hallpike and Adams that proteases may have importance in the pathogenesis of the MS plaque (40), and the finding that increased levels of acid proteinase could be measured around the MS lesion (43), other workers were able to confirm the presence of higher acid proteinase levels in plaque border zones of active cellular destruction of myelin (62), as well as in normal-appearing white matter of brains of patients with MS (63). Using sensitive histochemical methods, Hirsch et al. (64) found elevated acid proteinase in and around plaques, although the increase in acid phosphatase was more dramatic. Increases in cathepsin A of 37 and 155% in chronic and acute MS plaques have also been reported (46). Cathepsins B1 and D were measured in lymphocytes obtained from MS patients and controls, and there was no difference in enzyme levels between these two groups (65). Cuzner et al., however, have reported that an increase in leukocyte neutral proteinase activity is associated with an attack of MS (66). Of special relevance is the nature of the infiltrating cells in the early MS plaque. The inflammatory cuff is composed chiefly of small lymphocytes, with plasma cells, histiocytes, and fatty macrophages also present (67), but the lymphocytes do not appear to penetrate much beyond the area of the blood vessels. Prineas (67) has suggested that any extracellular expression of the antigen must be restricted largely to the Virchow-Robin spaces. Extracellular factors, however, may penetrate
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beyond the area of the cell, as noted by Hirsch and Parks (51) in measuring/3-glucuronidase in EAE lesions of the monkey. Prineas (67) has noted that there are several features of the demyelinating MS plaque that histologically are quite different from those of the EAE lesion. Hirsch (68) has concluded that a special role for acid proteinase in MS plaques is unlikely because this enzyme is increased less markedly than is acid phosphatase, and has suggested that astrocytes with high lysosomal enzyme activity may b e active in myelin breakdown and removal of debris. Most MS plaques studied have been of long duration, while the early plaques may resemble more closely those of EAE, as suggested by Hirsch (68). Studies of early MS plaques will be necessary to ascertain whether proteases are specifically involved in the etiology of the early lesion. In view of the suspected immunological factors accompanying the recurrence of the demyelinative episode in MS, further investigation of the lymphoid cells and their enzyme activities in MS patients, especially those with different stages of the disease, is a promising field for further study.
CONCLUSIONS The evidence for a role for proteases, either as an initial or secondary feature in the demyelinative process, is only circumstantial at present. Increased levels of certain proteases at the site of the EAE lesion or MS plaque may be a consequence of the presence of infiltrating cells not of CNS origin, which contain proteases as nonfunctional passengers. In order to ascertain whether proteases are indeed a primary cause of demyelination, more information is needed on the kinds of enzymes operative, their properties, the nature of the physiological substrate or first point of attack, and the mechanism by which the substrate becomes available to the enzyme. The latter factor may be especially important in certain kinds of pathological myelin loss where no infiltration of cells occurs, as in conditions of white matter edema. An increase of fluid within the myelin sheath may facilitate entry of cerebral proteases that are normally excluded from the hydrophobic milieu of the myelin membrane. Such a mechanism may also be operative in cell-mediated demyelinating diseases including experimental allergic encephalomyelitis, in which edema may be an accompanying feature (69) and in which hydration of the myelin sheath occurs (6). If proteolytic enzymes are a causative feature of demyelination in EAE, the most likely morphological correlation of their initial action is
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the early splitting of the intraperiod and main period lines described by Lampert (6). This event could conceivably be caused by the extracellular expression of lysosomal enzymes or by a surface-bound protease in the invading mononuclear cell. Other enzymes, both of CNS origin and from the mononuclear cell, may then participate in the further disposition of the debris in the phagocytic process. The participation of glia, especially of oligodendroglia, in the demyelinative process appears to be much greater in MS plaques than in EAE lesions, as noted by Prineas (67). Further studies on proteinases and their role in disease will be of importance in devising a rationale for treatment. Many proteinase inhibitors have been identified, and the action of these .on cerebral proteases has been reviewed by Marks et al. (70). It is possible that such inhibitors may be useful to intervene in the course of degenerative CNS diseases of myelin.
REFERENCES I. KIES, M. W., and ALVORD, E. C., JR. (eds.). 1959. Allergic Encephalomyelitis, Charles C Thomas, Springfield, Illinois. 2. Research in Demyelinating Diseases. 1965. (SCHEINBERG,L. C., KIES, M. W., and ALVORD, E. C., JR., Conference Cochairmen.) Ann. N. Y. Acad. Sci. 122:1-570. 3. WAKSMAN, B. H., and ADAMS, R. D. 1962. A histologic study of the early lesion in experimental allergic encephalomyelitis in the guinea pig and rabbit. Am. J. Pathol. 41:135-162. 4. RAUCH, H. C., EINSTEIN, E. R., and CSEJTEY, J. 1973. Enzymatic degradation of myelin basic protein in central nervous system lesions of monkeys with experimental allergic encephalomyelitis. Neurobiology 3:195-205. 5. LEVINE, S., and SOWlNSKI, R. 1973. Hyperacute allergic encephalomyelitis. A localized form produced by passive transfer and pertussis vaccine. Am. J. Pathol. 73:247-258. 6. LAMPERT, P. 1967. Electron microscopic studies on ordinary and hyperacute experimental allergic encephalomyelitis. Acta Neuropathol. 9:99-126. 7. MORRISON, L. R., and ZAMECNII~,P. C. 1950. Experimental demyelination by means of enzymes, especially the alpha toxin of Clostridiurn welchii. Arch. Neurol. Psychiatry 63:367-381, 8. MAGGIO, B., MACCIONI, H. J., and CUMAR, F. A. 1973. Arylsulfatase A (EC. 3.1.6.1) activity in rat central nervous system during EAE. J. Neurochem. 20:503-510. 9. WOELK, H., and KANIC, K. 1974. Phospholipid metabolism in experimental allergic encephalomyelitis: Activity of brain phospholipase As towards specifically labelled glycero phospholipids. J. Neurochern. 23:739-743. 10. WOELK, H., KANIG, K., and PEILER-ICHIKAWA,K. 1974. Phospholipid metabolism in experimental allergic encephalomyelitis: Activity of mitochondrial phospholipase A 2 of rat brain towards specifically labelled 1,2-diacyl- 1-alk-l'-enyl-2 acyl- and 1-alkyl-2acyl-SN-glycero-3 phosphorylcholine. J. Neurochem. 23:745-750.
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11. FOLCH, J., and LEES, M. 1951. Proteolipides, a new type of tissue lipoproteins. Their isolation from brain. J. Biol. Chem. 191:807-817. 12. LEES, M. B., MESSINGER, B. F., and BURNHAM, J. D. 1967. Tryptic hydrolysis of brain proteolipid. Biochem. Biophys. Res. Commun. 28:185-190. 13. LEES, M. B., and CHAN, D. S. 1975. Proteolytic digestion of bovine brain white matter proteolipid. J. Neuroehem. 25:595-600. 14. SMITH, M. E. Unpublished observations. 15. CARNEGIE, P. R., and DUNKLEY, P. R. 1975. Basic proteins of central and peripheral nervous system myelin. Pages 95-135, in AGRANOFF, B. W., and APRISON, M. H. (eds.), Advances in Neurochemistry, Vol. I, Plenum Press, New York. 16. EYLAR, E. H., BROSTOFF, S., HASHIM, G., CACCAM, J., and BURNETT, P. 1971. Basic A1 protein of the myelin membrane. The complete amino acid sequence. J. Biol, Chem. 246:5770-5784. 17. CARNEGIE, P. R. 1971. Amino acid sequence of the encephalitogenic basic protein from human myelin. Biochem. J. 123:57-67. 18. EINSTEIN, E. R., CSEJTEY,J., and MARKS, N. 1968. Degradation of encephalitogen by purified brain acid proteinase. FEBS Lett. 1:191-195. 19. WOLFGRAM, F., and KOTORII, K. 1968. The composition of the myelin proteins of the central nervous system. J. Neurochem. 15:1281-1290. 20. QUARLES, R. H., EVERLV, J. L., and BRAINY, R. O. 1972. Demonstration of a glycoprotein which is associated with a purified myelin fraction from rat brain. Biochem. Biophys. Res. Commun. 47:491-497. 21. AGRAWAL, H. C., BURTON, R. M., FISHMAN, M. A., MITCHELL, R. F., and.PRENSKY, A. L. 1972. Partial characterization of a new myelin protein component. J. Neurochem. 19:2083-2089. 22. LONDON, Y., and VOSSENBERG, F. G. A. 1973. Specific interaction of central newous system myelin basic protein with lipids. Specific regions of the protein sequence protected from the proteolytic action of trypsin. Biochem. Biophys. Acta 307:478-490. 23. WOOD, J. G., DAWSON, R. M. C., and HAUSER, H. 1974. Effect of proteolytic attack on the structure of CNS myelin membrane. J. Neurochem. 22:637-643. 24. BANIK, N. L., and DAVISON, A. N. 1974. Lipid and basic protein interaction in myelin. Biochem. J. 143:39-45. 25. MARKS, N., and LAJTHA, A. 1971. Protein and polypeptide breakdown. Pages 49-139, in Lajtha, A. (ed.), Handbook of Neurochemistry, Vol. 5, Pt. A, Plenum Press, New York. 26. MARKS, N. 1968. Expopeptidases of the nervous system. Int. Rev. Neurobiol. 11:5797, Academic Press, New York. 2 7 . KIES, M. W., and SCHWIMMER, S. 1942. Observations on proteinase in brain. J. Biol. Chem. 145:685-691. 28. HIRSCH, H. E., and PARKS, M. E. 1973. The quantitative histochemistry of acid proteinase in the nervous system: Localization in neurons. J. Neurochem. 21:453-458. 29. ANSELL, G. B., and RICHTER, D. 1954. Evidence for a "neutral proteinase" in brain tissue. Biochim. Biophys. Acta 13:92-97. 30. RIEKKINEN, P. J., and RINNE, U. K. 1968. A new neutral proteinase from rat brain. Brain Res. 9:126-135. 31. BOSMANN, H. B. 1973. Protein catabolism. III. Proteolytic enzymes of guinea pig cerebral cortex and synaptosomal localization. Int. J. Pept. Protein Res. 5:135-147. 32. BOWEN, D. M., and DAVISON, A. N. 1973. Cathepsin A in human brain and spleen. Biochem. J. 131:417-419.
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33. MARKS, N., DATTA, R. K., and LAJTHA, A. 1968. Partial resolution of brain arylamidases and aminopeptidases. J. Biol. Chem. 243:2882-2889. 34. ADAMS, C. W. M., DAVISON, A. N., and GREGSON, N. A. 1963. Enzyme inactivity of myelin: Histochemical and biochemical evidence. J. Neurochem. 10:383-395. 35. BECK, C. S., HASINOFF, C. W., and SMITH, M. E. 1968. E-alanyl-fl-naphthylamidase in rat spinal cord myelin. J. Neurochem. 15:1297-1301. 36. REIKKINEN, P. J., and RUMSBY, M. G. 1972. Aminopeptidase and neutral proteinase activity associated with central nerve myelin preparation during purification. Brain Res. 41:512-517. 37. KEREKES, M. F., FESZT, T., and KOVACS, A. 1965. Catheptic activity in the cerebral tissue of the rabbit during allergic encephalomyelitis. Experientia 21:42-43. 38. BENETATO, G., GABRIELESCU, E., and BOROS, I. 1965. Histochemistry of cerebral proteases in experimental allergic encephalitis. Rev. Roum. Physiol. 2:379-384. (C. A. 64, 20401, 1966.) 39. PORCELLATI,G. 1972. Amino acid and protein metabolism in Wallerian degeneration. Pages 191-219, in LAJTHA, A. (ed.), Handbook of Neurochemistry, Vol. 7, Plenum Press, New York. 40. HALLPIKE, J. F., and ADAMS, C. W. M. 1969. Proteolysis and myelin breakdown: A review of recent histochemical and biochemical studies. Histochem. J. 1:559-578. 41. HALLPIKE, J. F., ADAMS, C. W. M., and BAYLISS, O. B. 1970. Histochemistry of myelin. VIII. Proteolytic activity around multiple sclerosis plaques. Histochem. J. 2:199-208. 42. ROBoz-EINSTEIN, E., CSEJTEV, J., DAVIS, W. J., LAJTHA, A., and MARKS, N. 1969. Enzymatic degradation of the encephalitogenic protein. Add. ad Int. Arch. Allergy 36:363-375. 43. EINSTEIN, E. R., DALAL, K. B., and CSFJTEY, J. 1970. Increased protease activity and changes in basic proteins and lipids in multiple sclerosis plaques. J. Neurol. Sci. 11:109-121. 44. EINSTEIN, E. R., CSEJTEY, J., DALAL, K. B., ADAMS, C. W. M., BAYLISS,O, B,, and HALLPIKE, J. F. 1972, Proteolytic activity and basic protein loss in and around multiple sclerosis plaques: Combined biochemical and histochemical observations. J. Neurochem. 19:653-662. 45. SMITH, M. E., SEDGEWICK, L. M., and TAGG, J. S. 1974. Proteolytic enzymes and experimental demyelination in the rat and monkey. J. Neurochem. 23:965-971. 46. BOWEN, D. M., and DAVISON, A. N. 1974. Macrophages and cathepsin A activity in multiple sclerosis brain. J. Neurol. Sci. 21:227-231. 47. GOVINDARAJAN, K. R., RAUCH, H. C., CLAUSEN, J., and EINSTEIN, E. R. 1974. Changes in cathepsins B-1 and D, neutral proteinase, and 2',3'-cyclic nucleotide-3'phosphohydrolase activities in monkey brain with experimental allergic encephalomyelitis. J. Neurol. Sci. 23:295-306. 48. BOEHME, D. H., FORDICE, M. W., and MARKS, N. 1974. Proteolytic activity in brain and spinal cord in sensitive and resistant strains of rat and mouse subjected to experimental allergic encephalomyelitis. Brain Res. 75:153-162. 49. BULETZA, G, F., JR., and SMITH, M. E. 1976. Enzymic hydrolysis of myelin basic protein and other proteins in central nervous system and lymphoid tissues from normal and demyelinating rats. Biochem. J. 156:627-633. 50. BARRETT, A. J. 1975. Lysosomal and related proteinases. Pages 467-801, in REICH, E., RIFKIN, D. B., and SHAW, E. (eds.), Proteases and Biologic'al Control, Cold Spring Harbor Conferences on Cell Proliferation, Vol. 2, Cold Spring Harbor.
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51. H1RSCH,H. E., and PARKS, M. E. 1975. Acid proteinases and other acid hydrolases in experimental allergic encephalomyelitis: Pinpointing the source. J. Neurochem. 24:853-858. 52. RAUCI-I,H. C., and EINSTEIN, E. R. 1974. Specific brain proteins: A biochemical and immunological review. Pages 283-343, in EHRENPREIS, S., and KOPIN, I. J. (eds.), Review ofNeuroscience, Vol. I, Raven Press, New York. 53. SMITH,M. E. 1976. A lymph node neutral proteinase acting on myelin basic protein. J. Neurochem. 27:1077-1082. 54. SMITH, M. E., and CHOW, S. H. 1976. Neutral proteinase in lymph nodes specific for basic protein, Trans. Am. Soc. Neurochem. 7:178. 55. TOKES, Z. A., and KIEFER, H. 1976. Estimation of cell surface associated protease activity and its application to lymphocytes. J. Supramol. Struct. 4:507-513. 56, ALLISON,A. C., and FERLUGA,J. 1976. How lymphocytes kill tumor cells. N. Engl. J. Med. 295:165-167. 57. BENUCK, M., MARKS, N., and HASHIM, G. A. 1975. Metabolic instability of myelin proteins. Breakdown of basic protein induced by brain cathepsin D. Eur. J. Biochem. 52:615-621. 58. MARKS, N., GRYNBAUM,A., and BENUCK, M. 1976. On the sequential cleavage of myelin basic protein by catepsins A and D. J. Neurochem. 27:765-768. 59. GRAYZEL, A. I., HATCHER, V. B., and LAZARUS,G. S. 1975. Protease activity of normal and PHA stimulated human lymphocytes, Cell Immunol. 18:210-219. 60. YAGO, N., and BOWERS, W. E. 1975. Unique cathepsin D-type proteases, in rat thoracic duct lymphocytes and in rat lymphoid tissues. J. Biol. Chem. 250:4749-4754. 61. JANOFF,A. 1972. Neutrophil proteases in inflammation. Ann. Rev. Med. 23:177-190. 62. RIEKKINEN,P. J., CLAUSEN,J., FREY, H. J., FOG, T., and RINNE, U. K. 1970. Acid proteinase activity of white matter and plaques in multiple sclerosis. Acta Neurol. Scand. 46:349-353. 63. CUZNER, M. L., and DAVISON,A. N. 1973. Changes in cerebral lysosomal enzyme activity and lipids in multiple sclerosis. J. Neurol. Sci. 19:29-36. 64. HIRSCH, H. E., DUQUETTE,P., and PARKS,M. E. 1976. The quantitative histochemistry of multiple sclerosis plaques: Acid proteinase and other acid hydrolases. J. Neurochem. 26:505-512. 65. GOVINDARAJAN,K. R., OEENER, H., CLAUSEN, J., FOG, T., and HYLLESTED, K. 1974. The lymphocytic cathepsins B-1 and D activities in multiple sclerosis. J. Neurol. Sci. 23:81-87. 66. CUZNER, M. L., McDONALD, W. I., RtJDGE, P., SMITH, M., BORSHELL, N., and DAVISON, A. N. 1975. Leucocyte proteinase activity and acute multiple sclerosis. J. Neurol. Sci. 26:107-111. 67. PRINEAS, J. 1975. Pathology of the early lesion in multiple sclerosis. Hum. Pathol. 6:531-554. 68. HIRSCH, H. E. 1976. The role of acid hydrolases in demyelination. Neurology 26(6), Pt. 2:39-41. 69. LEVINE, S., SIMON, J., and WENK, E. J. 1966. Edema of the spinal cord in experimental allergic encephalomyelitis. Proc. Soc. Exp. Biol. Med. 123:539-541. 70. MARKS, N., D'MONTE0 B., and LAJTHA, A. 1973. Inhibition of cerebral proteolytic enzymes. Tex. Rep. Biol. Med. 31:345-365.
Overview
THE ROLE OF P R O T E O L Y T I C E N Z Y M E S IN D E M Y E L I N A T I O N IN E X P E R I M E N T A L ALLERGIC ENCEPHALOMYELITIS MARION EDMONDS SMITH Department of Neurology Veterans Administration Hospital Palo Alto, California Standord University School of Medicine Stanford, California 94305
Accepted November 23, 1976
For about 30 years the autoimmune disease experimental allergic encephalomyelitis (EAE) has been regularly produced in the laboratory in various species of animals to serve as a model of a cell-mediated demyelinating disease. It was recognized early that EAE and rabies postvaccinal encephalomyelitis were probably caused by identical immunologic mechanisms and agents, and were undoubtedly the same disease. A similar autoimmune process has been invoked as an explanation for the cause of other demyelinating diseases including postinfectious encephalomyelitis or acute disseminated encephalomyelitis, and perhaps multiple sclerosis (MS). Most of the early work on EAE is represented in two symposium collections (1,2).
EAE: P A T H O L O G I C A L D E S C R I P T I O N
Experimental allergic encephalomyelitis is produced by injection of susceptible animals with whole CNS tissue, white matter, purified myelin, or myelin basic protein in combination with Freund's complete 233 q~) 1977 Plenum Publishing Col~p., 227 West 17th Street, New York, N.Y. 100li. To promote freer access to published material in the spirit of the 1976 Copyright Law, Plenum sells reprint articles from all its journals. This availability underlines the fact that no part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher. Shipment is prompt; rate per article is $7.50.
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adjuvant. The animals most commonly used are rabbits, guinea pigs, certain susceptible strains of rats such as the Lewis rat, and monkeys. Only minimal clinical effects are usually observed for the first 10 days after injection; then the animal begins to lose weight and rapidly develops paralysis, which is often fatal in the rabbit, guinea pig, and monkey. Light-microscopic examination of CNS tissues from smaller animals with acute EAE reveals perivascular cuffing; the infiltrating cells have been identified as lymphocytes, small monocytes, and plasma cells (3). Demyelination occurs only in tissues in which infiltrating mononuclear cells are present. In the monkey the lesion is more severe, with large foci of hemorrhagic necroses often macroscopically visible, and the infiltrating cells contain many polymorphonuclear cells and eosinophils (4) in addition to mononuclear cells. The hyperacute lesion can also be produced in small animals by the use of pertussis vaccine
(5). Examination of the demyelinating lesion by electron microscopy has provided some understanding of the mechanisms by which the myelin sheath is destroyed. The infiltrating mononuclear cell surrounds the myelin sheath, which then becomes hydrated and splits at the intraperiod line. The major dense line may split also, and the separated lamellae cuff to form vesicular structures. The mononuclear cell appears to invade the sheath at the mesaxon and peels off the outer lamellae, which are then engulfed by phagocytosis. The myelin fragments are transformed into globoid lipid bodies within intracytoplasmic compartments (6). Hydration and vesiculation of the myelin sheath appear to result from the extracellular action of the mononuclear cells; then, after active stripping of the lamellae, the myelin debris becomes incorporated within phagocytic cells, where further digestion takes place.
E N Z Y M A T I C A C T I O N ON M Y E L I N . H I S T O R I C A L Although the morphological studies show that a complex series of events attend the complete dissolution of the myelin sheath, it has been suggested that an early perturbation, perhaps enzymatic, may initiate the degradative course. Since lipid is the major constituent of the myelin sheath, the possible role of lipases in myelin disintegration was first considered. In 1950, Morrison and Zamecnik (7) showed that blocks of spinal cord tissue incubated with Clostridium welchii filtrate or with various snake venoms containing lipases produced several kinds of lipid degeneration, including production of neutral lipids. More recently, significant increases have been measured in the levels of several lipases
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in the nervous system of EAE animals, such as might be expected to hydrolyze the lipids contained in myelin. These include aryl sulfatase (8) and phospholipase (9,10). Of late, however, more attention has been centered around the possibility that the first point of attack on the myelin sheath is protein that represents only 25-30% of the dry weight of myelin, but which is more vulnerable to attack in view of the properties of certain constituents.
N A T U R E OF M Y E L I N PROTEINS The proteins of the myelin sheath are relatively few in number compared to those in more metabolically active plasma membranes of most cells. Over 50% of the total myelin protein is composed of proteolipid protein, first described by Folch and Lees (11). This protein is highly complexed with lipids, some of which are covalently bound, and contains a large proportion of hydrophobic amino acids. Proteolipid protein is notable for its resistance to proteolytic enzymes, but may be partially hydrolyzed by trypsin in the presence of certain detergents (12), and by elastase (13) and pronase (14). The myelin basic protein, which comprises about 30% of the total myelin protein, is relatively small, with a molecular weight of about 18,000. In aqueous solution it has little if any tertiary structure, although the myelin membrane may impose some degree of order in situ. The amino acid sequence and other properties have been fully described (15). This protein, unlike the proteolipid protein, is hydrolyzed by a number of proteolytic enzymes including trypsin and pepsin (16,17), chymotrypsin, thermolysin, subtilysin, aminopeptidase, carboxypeptidase A and B (17), acid proteinase (18), pronase and elastase (14), and probably others. The remaining 20% of the myelin protein is made up of a group of higher-molecular-weight proteins, some of which may be contaminants as a result of the isolation process. Two of the higher-molecularweight proteins are believed to be associated with the myelin membrane, the Wolfgram protein, an acidic proteolipid (19), and a glycoprotein (20). Several enzymes also appear to be myelin specific. These proteins and the DM-20, which is separable by polyacrylamide gel electrophoresis under special conditions (21), have not been studied in regard to proteolytic enzyme susceptibility. If proteolysis is the first step in myelin breakdown, the myelin basic protein appears to be most vulnerable, not only in its isolated aqueous form, but also in situ in the myelin membrane. It is the only protein showing a decrease after incubation of whole myelin with trypsin.
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Although London and Vossenberg found that various lipids, especially acidic lipids, protect myelin basic protein from the proteolytic action of trypsin (22), others have found that low amounts of trypsin effectively remove much of the basic protein from purified myelin, while proteolipid and high-molecular-weight proteins remain unchanged (23,24).
P R O T E O L Y T I C E N Z Y M E S IN T H E CNS Consistent with the rapid turnover of most of its protein constituents, the CNS is notable for the high content of proteolytic enzymes. The identification of various endopeptidases and exopeptidases in the CNS has been reviewed (25,26), but relatively few of these have been purified and characterized. Acid proteinase in whole brain tissue was first reported by Kies and Schwimmer (27), and this enzyme has been further purified by several investigators (25). Acid proteinase or cathepsin D in the CNS was found predominantly in neurons, and resembles other lysosomal hydrolases in distribution (28). A neutral proteinase in brain tissue was described in 1954 by Ansell and Richter (29), and several investigators have reported the purification of this enzyme from animal brain (e.g., 30,31). Cathepsin A (carboxypeptidase A) (32), aminopeptidases, and arylamidases have also been identified (33). Aminopeptidase (34,35) and neutral proteinases (36), but not acid proteinase, are associated with highly purified myelin preparations. Proteolytic enzymes in the brain that might be involved in the formation and in/~ctivation of peptide hormones of the neuroendocrine system arid the vasoactive polypeptides are an important area of investigation; their characterization is essential to the further understanding of the action of these hormones and their regulation.
P R O T E O L Y T I C E N Z Y M E S IN D E M Y E L I N A T I N G DISEASES The possibility that proteolytic enzymes might be active agents in the demyelination process was suggested early by Kerekes et al. (37), who found a 62% increase in catheptic activity in hares injected with Freund's adjuvant and heterologous brain tissue. A marked intensification of proteolytic activity in periventricular white matter was observed histochemically in guinea pigs before EAE lesions developed and during the period of evolution of the lesion (38). That proteolytic enzymes are much increased in secondary demyelination, such as is seen in Wallerian degeneration, has been well documented (39,40). In 1969, a loss of basic
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protein was reported in MS plaques, as observed by histochemical methods (40), and this was consistent with the finding of increased proteolytic activity at the edges of the demyelinated area, along with an increased cell population and N A D H dehydrogenase activity (41). Hallpike et al. (40) tentatively suggested that the oligodendroglia or myelin itself may be the source of the enzymatic activity. In early studies on the purification of the encephalitogenic protein in the CNS, it was noted that, during acid extraction of acetone-defatted CNS, several smaller polypeptide fragments were obtained, whereas tissue defatted with chloroform-methanol or extracted below pH 2.0 yielded a larger encephalitogenic protein. The breakdown of the encephalitogen was attributed to the presence of acid proteinase, which was preserved by acetone extraction of the CNS (42). It was proposed at that time that such action of acid proteinase might be a key step in the breakdown of myelin in the disease state. This suggestion was given support by the finding of a 4-fold increase in acid proteinase at the edge of MS plaques (43), with a corresponding reduction of basic protein in the plaque (44).
PROTEOLYT1C E N Z Y M E S IN EAE The investigations of acid proteinase in demyelinative lesions were continued in animals with acute EAE. In the characteristic hyperacute lesions of the monkey, levels of acid proteinase were increased from two to 10 times that of apparently unaffected tissue of the same animal (4). A study of four lesions from some of the same monkeys in this laboratory by a different method revealed increases in the range of 152-I76% of the activity of analogous control areas (45). These increases were compared to increments in activity of two proteolytic enzymes with pH optima in a more neutral range, neutral proteinase and cathepsin A. The latter enzyme was shown to be increased in MS plaques, and was suggested as a marker for macrophages since the specific activity of cathepsin A in these cells was 30x that of white matter (46). In five EAE monkey lesions, neutral proteinase was 220-258% of control areas, and in the one lesion assayed for cathepsin A an increase of 8-fold was measured. Govindarajan et al. (47) also found markedly elevated cathepsin D, neutral proteinase, and cathepsin B1 levels in lesions of six monkeys with acute EAE. In brain stems of rats with acute EAE, acid proteinase was increased only 28%, neutral proteinase 12% (not significant), and cathepsin A was present at levels 258% of the level in brain stems of Freund's adjuvant-
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injected control rats. The differences in levels of increases between the monkey and rat tissue probably reflect the nature of the lesion in each animal. Whereas it was possible to dissect out the large areas of focal lesion tissue in the monkey, this was not possible in the rat, where the lesions are microscopic and diffuse; therefore, it was necessary to assay whole tissue. Increases in acid proteinase and cathepsin A were not seen up to 7 days after injection of the animals with the encephalitogenic material, but at 12-14 days, at which time acute clinical symptoms were obvious, the cathepsin A levels rose dramatically. In contrast to the Lewis rat, a strain susceptible to EAE, such increases were not seen in the brain stems of the injected Wistar rat, which is relatively nonsusceptible, although histological examination of the CNS tissue showed a very mild perivascular cuffing. The acid proteinase-specific activity was fairly constant in lymph nodes draining the site of injection from 4 days after injection until the acute stage, and did not differ greatly in Lewis or Wistar rats injected with Freund's adjuvant alone, or in uninjected controls. The size of the lymph node increased greatly after immunological stimulation; therefore, the total acid proteinase increased per lymph node. Lymph node cathepsin A, on the other hand, increased in specific activity and total activity over that of the uninjected control with the development of EAE, although there was no real difference in specific activity of lymph nodes of the Lewis and Wistar rat with acute EAE or the Freund's adjuvant-injected control (45). These results were not confirmed by Boehme et al. (48), who also observed a 55% increase in acid proteinase in spinal cord extracts of Lewis rats with acute EAE, but did not observe increas~es in cathepsin A. Bowen and Davison have pointed out that cathepsin A is not extracted from tissue in the alkaline range (32); in the experiments of Boehme et al., some extractions were done at pH 7.6. That a number of kinds of proteolytic enzymes are probably increased in EAE was demonstrated by Buletza and Smith (49), who found increased proteolytic activity throughout the pH range 3.5-8 in homogenates of spinal cords from rats with acute EAE. This increase was especially marked at pH 5-7 when casein was used as a substrate, and was also increased throughout the pH range using myelin basic protein as a substrate. The identification of the enzymes active at the various pH's was not made. The pH optimum of a particular enzyme may change with the substrate used. Although the pH optimum for acid proteinase is 3.8 with hemoglobin as a substrate, maximal activity for this enzyme may be seen at higher values using other substrates (50). The optimum pH for the acid proteinase hydrolysis of myelin basic protein, however, appears to coincide with that of hemoglobin (18).
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The source of the enzyme increases may be the CNS or the inflammatory cells. On the basis of the following indirect evidence, Smith et al. (45) suggested that the infiltrating cells of lymphoid origin might contain the observed enzyme increments: ~. The enzyme increases were much greater in the hyperacute concentrated EAE lesion of the monkey than in the diffuse microscopic lesion of the rat. 2. The enzyme increases were not seen until the onset of paralytic symptoms which coincided with the time of cellular infiltration. 3. The increments of enzyme activity in the EAE rat were greater with respect to cathepsin A than to acid proteinase, corresponding to the difference in levels of these enzymes between lymph nodes and brain stem. 4. In a demyelinative condition resulting from triethyl tin feeding, where infiltration of cells does not occur, levels of acid proteinase and cathepsin A were normal. Hirsch and Parks (51) confirmed these suppositions with elegant quantitative microhistochemical techniques. In the Lewis rat with acute EAE, only t h o s e areas that contained lesions showed increased acid proteinase, cathepsin A,/3-glucuronidase, and acid lipase-esterase, and these enzymes were precisely pinpointed to the loci of cellular infiltration. In one monkey lesion, levels in/3-glucuronidase, acid proteinase, dipeptidyl arylamidase iI, cathepsin A, acid phosphatase, neutral lipase, and D N A were sharply increased.
LYMPH NODE PROTEASES In view of the lymphoid origin of mononuclear cells migrating into the CNS in acute EAE (52), Buletza and Smith (49) assayed proteinase activity in homogenates of lymph nodes draining the site of injection in rats with acute EAE, and found very high proteolytic activity at p H 4-8 when basic protein was used as a substrate. These levels were not different in lymph nodes activated with Freund's adjuvant alone, suggesting that the nature of the challenging material injected does not confer specificity to the nature of the catabolic enzymes. When purified myelin was incubated at p H 7 with homogenates of inguinal and popliteal lymph nodes from rats with acute EAE, the myelin basic protein was selectively removed, while a continued increase in ninhydrin-positive material was released into the medium over a 4-h period of incubation. A fraction active in protease activity at p H 7 was prepared from these lymph nodes by ammonium sulfate precipita-
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tion. The enzyme activity was found to act selectively only on basic protein substrates including protamine sulfate, myelin basic protein, polylysine, and various histories. Much less activity was measured with cytochrome c and polyarginine. No activity was observed when casein, hemoglobin, egg albumin, bovine serum albumin, and ribonuclease were used as substrates (53). The p H optimum was 7 for rat myelin, beef basic protein, and histone. This enzyme has been purified about 80 times by gel filtration and ion exchange chromatography, appears to require a thiol group for activity, and is inhibited by phenylmethylsulfonyl fluoride and Trasylol (54). The neutral proteinase activity is present in lymphocytes prepared from chopped, sieved lymph nodes of rats with acute EAE. Such preparations are virtually free of macrophages. When these lymphocytes are incubated unbroken in isotonic solutions with purified myelin, myelin basic protein, or polylysine, a high level of proteolytic activity is obtained, while more activity is obtained from the lymphocytes upon homogenization (14). It is not clear whether the enzyme activity of the whole lymphocytes is a result of extracellular excretion or whether the enzyme is present on the plasma membrane exterior. Protease activity has been found on the rat lymph node lymphocyte cell surface (55), and it has been suggested that membrane-associated proteinases may participate in the killing of tumor cells by lymphocytes (56).
O T H E R P R O T E A S E S OF CNS A N D L E U K O C Y T E S Lysosomal enzymes from the brain alone are fully capable of destroying myelin basic protein. Purified brain cathepsin D cleaves purified myelin basic protein into three peptide fragments by hydrolysis of the two phenylalanine-phenylalanine linkages (57). Cathepsin A, also purified from brain, while not able to hydrolyze myelin basic protein by itself, can act on one of the three polypeptide fragments formed by acid proteinase (58). Thus the activity in cathepsin D is rate limiting in the stepwise cleavage by these two lysosomal enzymes. Such a concerted enzyme action is a feasible process by which normal turnover of myelin basic protein might occur, as well as in breakdown in pathological conditions. Immunologically active infiltrating cells, however, may exert their action by additional mechanisms. Human peripheral blood lymphocytes contain a number of proteases including cathepsin D, a neutral serine protease, and probably a thiol protease. The neutral protease activity was found bound to the surface of lymphocytes (59). Two unique types
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of cathepsin D-like proteases, apparently present only in rat thoracic duct lymphocytes and rat lymphoid tissues, have been described. Both enzymes differ from the rat liver cathepsin D by a greater sensitivity to pepstatin and their interconvertibility by mercaptoethanol (60). In the near future, proteolytic enzymes in normal and activated lymphocytes should become an important area of exploration. Other types of leukocytes should also be investigated for a possible role in demyelinative diseases. The cellular infiltrate in the hyperacute lesion of the monkey contained polymorphonuclear cells that are known to be a rich source of elastases (50,61). This enzyme is one of the few proteases found thus far that can cleave the proteolipid protein as well as the myelin basic protein.
P R O T E O L Y T I C E N Z Y M E S A N D T H E I R A C T I V I T Y IN MS LESIONS The original impetus for the investigation of proteolytic enzymes in demyelinative lesions in EAE was given by and has been paralleled by similar studies of the MS plaque. Following the original suggestions by Hallpike and Adams that proteases may have importance in the pathogenesis of the MS plaque (40), and the finding that increased levels of acid proteinase could be measured around the MS lesion (43), other workers were able to confirm the presence of higher acid proteinase levels in plaque border zones of active cellular destruction of myelin (62), as well as in normal-appearing white matter of brains of patients with MS (63). Using sensitive histochemical methods, Hirsch et al. (64) found elevated acid proteinase in and around plaques, although the increase in acid phosphatase was more dramatic. Increases in cathepsin A of 37 and 155% in chronic and acute MS plaques have also been reported (46). Cathepsins B1 and D were measured in lymphocytes obtained from MS patients and controls, and there was no difference in enzyme levels between these two groups (65). Cuzner et al., however, have reported that an increase in leukocyte neutral proteinase activity is associated with an attack of MS (66). Of special relevance is the nature of the infiltrating cells in the early MS plaque. The inflammatory cuff is composed chiefly of small lymphocytes, with plasma cells, histiocytes, and fatty macrophages also present (67), but the lymphocytes do not appear to penetrate much beyond the area of the blood vessels. Prineas (67) has suggested that any extracellular expression of the antigen must be restricted largely to the Virchow-Robin spaces. Extracellular factors, however, may penetrate
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beyond the area of the cell, as noted by Hirsch and Parks (51) in measuring/3-glucuronidase in EAE lesions of the monkey. Prineas (67) has noted that there are several features of the demyelinating MS plaque that histologically are quite different from those of the EAE lesion. Hirsch (68) has concluded that a special role for acid proteinase in MS plaques is unlikely because this enzyme is increased less markedly than is acid phosphatase, and has suggested that astrocytes with high lysosomal enzyme activity may b e active in myelin breakdown and removal of debris. Most MS plaques studied have been of long duration, while the early plaques may resemble more closely those of EAE, as suggested by Hirsch (68). Studies of early MS plaques will be necessary to ascertain whether proteases are specifically involved in the etiology of the early lesion. In view of the suspected immunological factors accompanying the recurrence of the demyelinative episode in MS, further investigation of the lymphoid cells and their enzyme activities in MS patients, especially those with different stages of the disease, is a promising field for further study.
CONCLUSIONS The evidence for a role for proteases, either as an initial or secondary feature in the demyelinative process, is only circumstantial at present. Increased levels of certain proteases at the site of the EAE lesion or MS plaque may be a consequence of the presence of infiltrating cells not of CNS origin, which contain proteases as nonfunctional passengers. In order to ascertain whether proteases are indeed a primary cause of demyelination, more information is needed on the kinds of enzymes operative, their properties, the nature of the physiological substrate or first point of attack, and the mechanism by which the substrate becomes available to the enzyme. The latter factor may be especially important in certain kinds of pathological myelin loss where no infiltration of cells occurs, as in conditions of white matter edema. An increase of fluid within the myelin sheath may facilitate entry of cerebral proteases that are normally excluded from the hydrophobic milieu of the myelin membrane. Such a mechanism may also be operative in cell-mediated demyelinating diseases including experimental allergic encephalomyelitis, in which edema may be an accompanying feature (69) and in which hydration of the myelin sheath occurs (6). If proteolytic enzymes are a causative feature of demyelination in EAE, the most likely morphological correlation of their initial action is
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the early splitting of the intraperiod and main period lines described by Lampert (6). This event could conceivably be caused by the extracellular expression of lysosomal enzymes or by a surface-bound protease in the invading mononuclear cell. Other enzymes, both of CNS origin and from the mononuclear cell, may then participate in the further disposition of the debris in the phagocytic process. The participation of glia, especially of oligodendroglia, in the demyelinative process appears to be much greater in MS plaques than in EAE lesions, as noted by Prineas (67). Further studies on proteinases and their role in disease will be of importance in devising a rationale for treatment. Many proteinase inhibitors have been identified, and the action of these .on cerebral proteases has been reviewed by Marks et al. (70). It is possible that such inhibitors may be useful to intervene in the course of degenerative CNS diseases of myelin.
REFERENCES I. KIES, M. W., and ALVORD, E. C., JR. (eds.). 1959. Allergic Encephalomyelitis, Charles C Thomas, Springfield, Illinois. 2. Research in Demyelinating Diseases. 1965. (SCHEINBERG,L. C., KIES, M. W., and ALVORD, E. C., JR., Conference Cochairmen.) Ann. N. Y. Acad. Sci. 122:1-570. 3. WAKSMAN, B. H., and ADAMS, R. D. 1962. A histologic study of the early lesion in experimental allergic encephalomyelitis in the guinea pig and rabbit. Am. J. Pathol. 41:135-162. 4. RAUCH, H. C., EINSTEIN, E. R., and CSEJTEY, J. 1973. Enzymatic degradation of myelin basic protein in central nervous system lesions of monkeys with experimental allergic encephalomyelitis. Neurobiology 3:195-205. 5. LEVINE, S., and SOWlNSKI, R. 1973. Hyperacute allergic encephalomyelitis. A localized form produced by passive transfer and pertussis vaccine. Am. J. Pathol. 73:247-258. 6. LAMPERT, P. 1967. Electron microscopic studies on ordinary and hyperacute experimental allergic encephalomyelitis. Acta Neuropathol. 9:99-126. 7. MORRISON, L. R., and ZAMECNII~,P. C. 1950. Experimental demyelination by means of enzymes, especially the alpha toxin of Clostridiurn welchii. Arch. Neurol. Psychiatry 63:367-381, 8. MAGGIO, B., MACCIONI, H. J., and CUMAR, F. A. 1973. Arylsulfatase A (EC. 3.1.6.1) activity in rat central nervous system during EAE. J. Neurochem. 20:503-510. 9. WOELK, H., and KANIC, K. 1974. Phospholipid metabolism in experimental allergic encephalomyelitis: Activity of brain phospholipase As towards specifically labelled glycero phospholipids. J. Neurochern. 23:739-743. 10. WOELK, H., KANIG, K., and PEILER-ICHIKAWA,K. 1974. Phospholipid metabolism in experimental allergic encephalomyelitis: Activity of mitochondrial phospholipase A 2 of rat brain towards specifically labelled 1,2-diacyl- 1-alk-l'-enyl-2 acyl- and 1-alkyl-2acyl-SN-glycero-3 phosphorylcholine. J. Neurochem. 23:745-750.
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33. MARKS, N., DATTA, R. K., and LAJTHA, A. 1968. Partial resolution of brain arylamidases and aminopeptidases. J. Biol. Chem. 243:2882-2889. 34. ADAMS, C. W. M., DAVISON, A. N., and GREGSON, N. A. 1963. Enzyme inactivity of myelin: Histochemical and biochemical evidence. J. Neurochem. 10:383-395. 35. BECK, C. S., HASINOFF, C. W., and SMITH, M. E. 1968. E-alanyl-fl-naphthylamidase in rat spinal cord myelin. J. Neurochem. 15:1297-1301. 36. REIKKINEN, P. J., and RUMSBY, M. G. 1972. Aminopeptidase and neutral proteinase activity associated with central nerve myelin preparation during purification. Brain Res. 41:512-517. 37. KEREKES, M. F., FESZT, T., and KOVACS, A. 1965. Catheptic activity in the cerebral tissue of the rabbit during allergic encephalomyelitis. Experientia 21:42-43. 38. BENETATO, G., GABRIELESCU, E., and BOROS, I. 1965. Histochemistry of cerebral proteases in experimental allergic encephalitis. Rev. Roum. Physiol. 2:379-384. (C. A. 64, 20401, 1966.) 39. PORCELLATI,G. 1972. Amino acid and protein metabolism in Wallerian degeneration. Pages 191-219, in LAJTHA, A. (ed.), Handbook of Neurochemistry, Vol. 7, Plenum Press, New York. 40. HALLPIKE, J. F., and ADAMS, C. W. M. 1969. Proteolysis and myelin breakdown: A review of recent histochemical and biochemical studies. Histochem. J. 1:559-578. 41. HALLPIKE, J. F., ADAMS, C. W. M., and BAYLISS, O. B. 1970. Histochemistry of myelin. VIII. Proteolytic activity around multiple sclerosis plaques. Histochem. J. 2:199-208. 42. ROBoz-EINSTEIN, E., CSEJTEV, J., DAVIS, W. J., LAJTHA, A., and MARKS, N. 1969. Enzymatic degradation of the encephalitogenic protein. Add. ad Int. Arch. Allergy 36:363-375. 43. EINSTEIN, E. R., DALAL, K. B., and CSFJTEY, J. 1970. Increased protease activity and changes in basic proteins and lipids in multiple sclerosis plaques. J. Neurol. Sci. 11:109-121. 44. EINSTEIN, E. R., CSEJTEY, J., DALAL, K. B., ADAMS, C. W. M., BAYLISS,O, B,, and HALLPIKE, J. F. 1972, Proteolytic activity and basic protein loss in and around multiple sclerosis plaques: Combined biochemical and histochemical observations. J. Neurochem. 19:653-662. 45. SMITH, M. E., SEDGEWICK, L. M., and TAGG, J. S. 1974. Proteolytic enzymes and experimental demyelination in the rat and monkey. J. Neurochem. 23:965-971. 46. BOWEN, D. M., and DAVISON, A. N. 1974. Macrophages and cathepsin A activity in multiple sclerosis brain. J. Neurol. Sci. 21:227-231. 47. GOVINDARAJAN, K. R., RAUCH, H. C., CLAUSEN, J., and EINSTEIN, E. R. 1974. Changes in cathepsins B-1 and D, neutral proteinase, and 2',3'-cyclic nucleotide-3'phosphohydrolase activities in monkey brain with experimental allergic encephalomyelitis. J. Neurol. Sci. 23:295-306. 48. BOEHME, D. H., FORDICE, M. W., and MARKS, N. 1974. Proteolytic activity in brain and spinal cord in sensitive and resistant strains of rat and mouse subjected to experimental allergic encephalomyelitis. Brain Res. 75:153-162. 49. BULETZA, G, F., JR., and SMITH, M. E. 1976. Enzymic hydrolysis of myelin basic protein and other proteins in central nervous system and lymphoid tissues from normal and demyelinating rats. Biochem. J. 156:627-633. 50. BARRETT, A. J. 1975. Lysosomal and related proteinases. Pages 467-801, in REICH, E., RIFKIN, D. B., and SHAW, E. (eds.), Proteases and Biologic'al Control, Cold Spring Harbor Conferences on Cell Proliferation, Vol. 2, Cold Spring Harbor.
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