57
Biochimica et Biophysica Acta, 584 (1979) 57--65 © Elsevier/North-Holland Biomedical Press
BBA 28846
LYSOSOMAL ENZYME SECRETION BYMACROPHAGES DURING INTRACELLULAR STORAGE OF PARTICLES
R.T. DEAN, W. HYLTON and A.C. ALLISON
Division of Cell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ (U.K.) (Received September 4th, 1978)
Key words: Lysosomal secretion; ~-N-Acetylglucosaminidase; Lactate dehydrogenase; Macrophage
Summary Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase ~-N-acetylglucosaminidase. Thus macrophage lysosomal enzyme secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both N-acetyl-/~-D-glucosaminidase and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.
Introduction It is now well known that macrophages in culture can selectively release lysosomal acid hydrolases in response to various stimuli, such as phagocytosable particles and the complement component C3b [ 1 ]. The relevance of such secretion to the degradative events in, and to the maintenance of, chronic inflammatory lesions, has been discussed extensively [2]. Previous work has concerned secretion occurring while the stimuli are present in the culture medium. Thus it has not been clear whether secretion can take place while the stimulating agents are present within the cells (for instance, after endocytosis) but no longer outside the cells. This situation obtains in many chronic inflammatory lesions (such as those characterised by the Abbreviation:
Hexosaminidase, fl-N-acetylglucosaminidase(EC 3.2.1.30).
58 long-term persistence within macrophages of the non-degradable Goup A strep. tococcal cell well materials [3] ). The present work demonstrates that lysosomal enzyme secretion does occur while some, but not all, endocytosed particles are stored within macrophages in culture, in the absence of extracellular particles. Materials and Methods Materials for tissue culture. Petri dishes were from Sterilin, Teddington, Middlesex. Medium 199 and sera were from BioCult Laboratories, Glasgow, U.K. Biochemical reagents. Penicillin, streptomycin, zymosan from Saccharomyces cerevisiae yeast, were obtained from Sigma Chemical Co., Surbiton, Surrey, U.K.; dextran sulphate (DS 500) was from Serva; polystyrene latex particles (0.81 um diameter: 5%, v/v) were from Difco, U.S.A.; p-nitrophenyl-2acetamido-2-deoxy-fl-D-glucopyranoside from Koch-Light Labs., Colnbrook, Bucks, U.K.; heparin, preservative free, from Paines and Byrne, Greenford, Middlesex, U.K.; pyruvate and reduced nicotinamide adenine dinucleotide from Boehringer Mannheim Gmbh, F.R.G. and Triton X-100, from BDH Ltd., Poole, Dorset, U.K. Chrysotile asbestos (UICC Standard reference sample of Rhodesian Chrysotile) was used as starting material. A sample of short-fibre asbestos prepared from this by milling was also used. Macrophage collection and culture. Macrophages were obtained by peritoneal lavage of normal Swiss mice (T.O. strain) with 5 ml cold Medium 199 containing 100 I.U./ml penicillin and streptomycin and 10 I.U./ml of heparin. 5-ml samples of the peritoneal exudate cell suspension (containing 0.75 • 106--1.3 • 106 cells/ml) were placed in 50-mm Petri dishes and incubated at 37°C in a humidified atmosphere of 5% carbon dioxide and air for 1--2 h. Nonadherent cells were then removed by washing four times with phosphate-buffered saline. After washing, the cells were cultured in 5 ml Medium 199 containing 10% (v/v) heat-inactivated swine serum. The heat inactivation was performed at 56°C for 30 min. Cultures gave a sheet of spread cells within a few hours, and experimental treatments began after incubation overnight (at which point the cells sheets were considered to be established). At this point, the medium was changed, and stimuli applied as described below. The commencement of stimulation is referred to as 0 h in Table I. Triplicate cultures were used in all experiments, and the biochemical data are expressed as mean +S.D. At the end of the incubation period, medium was collected, and the cells lysed with saline containing 0.1% (v/v) Triton X-100. Each time point on the figures refers to a separate set of cultures. The cells were then scraped off with sterile silicone rubber bungs. Enzyme assays. All assays were conducted under conditions giving linear release of product in relation to the amount of sample used and the time of incubation. Lactate dehydrogenase (EC 1.1.1.27) was assayed by determining the rate of oxidation of reduced nicotinamide adenine dinucleotide at 340 nm according to the Biochemica LDH test combination./3-N-Acetylglucosaminidase (hexosaminidase, EC 3.2.1.30) was assayed using p-nitrophenyl-2-acetamido-2~ deoxy-f~-D-glucopyranoside as substrate in 0.1 M citrate/phosphate buffer, pH
59 4.5 [4]. Results are given as means +S.D., both on the figures and in the tables. Macrophage stimuli. These were suspended directly in Medium 199 containing 10% heat-inactivated swine serum at the following concentrations known from previous work to be effective [1,2,5]: zymosan, 50 gg/ml; latex, 2 ul of the 5% (v/v) stock/ml of culture medium; chrysotile asbestos, 50 pg/ml; dextran sulphate, 100 ug/ml. To prepare serum containing activated complement components, zymosan (10 mg/ml) was incubated in unheated foetal calf serum in the presence of 0.25 M e~aminocaproic acid (to inhibit the carboxypeptidase which degrades split C3) for 30 min at 37°C [6]. The zymosan was removed by centrifugation, and the serum collected. It was used at 1% (v/v) in the cultures, in the presence of 10% heat-inactivated swine serum. Sucrose was presented in some experiments at 20 mM. Presentation of stimuli to macrophages. Stimuli were given to the cultures for various times, up to 24 h, depending on the experiment. After the chosen period, the medium was removed, and preserved for assay. One set of cultures/ stimulant was harvested at this point. The remaining cultures were vigorously washed four times with phosphate-buffered saline, to remove non-internalised particles and stimuli. Light microscopy indicated that very few or no zymosan and latex particles remained outside the cells after such washing. In contrast significant amounts of the long fibres in the normal chrysotile asbestos remained attached to the cell surface, consistent with earlier suggestions that such particles are not well phagocytosed [7]. However, with the milled shortfibre asbestos, very few particles remained attached to the cell surface after washing, while the cells became vacuolated, consistent with the material having been endocytosed. After washing, cultures received fresh medium, normally without stimuli, to allow secretion to proceed in the absence of exogenous stimuli. Occasionally, the fresh medium given after washing contained the same concentration of the stimuli present in the first period. Cultures were harvested after chosen periods of further incubation.
Results Table I shows the results of an experiment in which cells were exposed for 24 h to various stimuli, allowing substantial phagocytosis. After 24 h one set of cultures was harvested, and columns 1A and B show that during this period, zymosan, zymosan-activated serum, and to a lesser extent, dextran sulphate, had induced secretion of lysosomal hexosaminidase (not accompanied by equivalent release of lactate dehydrogenase). In this experiment some of the control hydrolase release seems to be selective as it exceeds the lactate dehydrogenase release. Such 'basal' release is often found, and to a variable extent. It probably depends on the degree of stimulation (by infection etc.) to which the macrophages have been exposed in vivo. In general, the lactate dehydrogenase release was quite low and thus hexosaminidase release is not attributable to cell death. The activated serum showed some degree of lytic effect on the cells, as indicated by the quite high percentage lactate dehydrogenase release (column 1B). Data presented later show that the cultures stimulated with
60 TABLE I SECRETION OF HEXOSAMINIDASE DURING EXPOSURE OF MACROPHAGES STIMULI, AND IN RESPONSE TO INTRACELLULAR STORAGE OF ZYMOSAN
TO VARIOUS
Cultures ( t r i p l i c a t e ) w e r e e i t h e r h a r v e s t e d after 24 h o f s t i m u l a t i o n , w h i c h c o m m e n c e d after f o r m a t i o n o f t h e cell s h e e t (data in c o l u m n s 1 A and B) or w a s h e d f o u r t i m e s and h a r v e s t e d after a further 24 h i n c u b a t i o n in e i t h e r s t i m u l a n t - c o n t a i n i n g ( c o l u m n s 3 A and B) or n o r m a l m e d i u m ( c o l u m n s 2 A and B). M e d i u m e n z y m e activities are e x p r e s s e d as p e r c e n t o f the t o t a l in the h a r v e s t e d cultures. T o t a l h e x o s a m i n i d a s e activities in c o n t r o l c u l t u r e s in t h e p r e s e n t w o r k ranged f r o m 3 5 0 to 6 0 0 n m o l / h per plate and w e r e i n c r e a s e d b y up t o 6 0 % b y c o n t i n u o u s e x p o s u r e t o z y m o s a n for 48 h (see Fig. 3). T o t a l l a c t a t e d e h y d r o genase ( L D H ) a c t i v i t i e s in c o n t r o l s ranged f r o m 2 5 0 t o 4 5 0 m u n i t s / p l a t e and w e r e i n c r e a s e d b y up to 50% b y c o n t i n u o u s e x p o s u r e t o z y m o s a n for 48 h. Stimulus
Control (medium only) Dextran sulphate (100 #g/ml) Activated serum (1%, v / v ) Zymosan (50 Dg/ml)
C o l u m n s 1 A and B (release during c u l t u r e from 0--24 h with stimuli)
C o l u m n s 2 A and B (release b e t w e e n 24--48 h without stimuli)
C o l u m n s 3 A and B (release b e t w e e n 24---48 h w i t h s t i m u l i )
Hexosaminidase
LDH
Hexosaminidase
LDH
Hexosaminldase
LDH
3 4 . 5 -+ 6.7
11.1 + 1.2
2 9 . 0 ± 10.9
1 6 . 7 -+ 4 . 2
2 9 . 0 + 10.9
16.7 ± 4.2
4 2 . 4 +_ 2.5
2 5 . 0 -+ 3.2
29.6 ± 5.96
16.7 + 5.1
43.1 + 1 2 . 2
2 0 . 0 ± 3.2
6 5 . 4 ± 0.8
5 0 . 0 ± 4.7
26.9 ± 4 . 4 7
9.1 ± 3.2
82.6 ± 2.64
4 2 . 4 ± 3.8
79.1 -+ 0.3
3 7 . 5 ± 4.3
53.2 ± 1 . 1 0
9.1 ± 2.1
6 2 . 6 + 1.3
16.1 ± 2.2
zymosan and asbestos not only remain viable, but actually produce increased amounts of hexosaminidase and lactate dehydrogenase. The remaining cells from this experiment were washed extensively to remove external stimuli, and then given fresh medium either with (columns 3A and B, Table I) or without (columns 2A and B) the original stimulant. In the latter case, endocytosis of, and stimulation by, the agent in question continued, and thus the behaviour of the cells during this second phase (columns 3A and B) and quite similar to that during the first 24 h phase (results o f columns 1A and B). However, the most important observations are those in columns 2A and B, describing the behaviour of cells cultivated without external stimuli during the second phase (24--48 h). Cells which had been exposed to zymosan for 24 h, continued to secrete large amounts o f hexosaminidase selectively during the second phase (24---48 h) in the absence of zymosan (Table I, columns 2A and B). This release was almost as substantial as that when zymosan was reintroduced after washing (columns 3A and B). In contrast the secretion during this same period (from 24 to 48 h after establishmen¢ o f the cell sheet) by cells initially exposed to dextran sulphate or activated serum, and then cultured in the absence of stimuli, was no greater than that by control cells (columns 2A and B). Dextran sulphate and activated serum in the medium were capable of inducing secretion in this same period (columns 3A and B). Thus the presence within macrophages of zymosan, but not of dextran sulphate or activated serum, allows continued secretion of lysosomal enzymes in the absence of further exogenous stimulus.
61
Table II shows that even a short (2 h) pulse of endocytosis of zymosan, or of short-fibre asbestos, allowed secretion during a subsequent 24 h period in the absence of the stimulus. However, normal chrysotile asbestos given for up to 24 h, was relatively ineffective in inducing release over a subsequent 24 h period in the virtual absence of asbestos, even though it induced release during the first 24 h (data not shown). This suggests that the large particles act mainly at the plasma membrane, from which they are largely removed by washing. In contrast, the short-fibre form is substantially endocytosed, and presumably canact in lysosomes in the absence of exogenous asbestos. Several repetitions of such experiments confirmed that macrophages storing zymosan or short-fibre asbestos remain viable, and secrete lysosomal enzymes selectively, while those pretreated with dextran sulphate or activated serum do not continue to do so in the absence of stimuli. Sucrose (which induces vacuolation and increased lysosomal enzyme activity within cells, but not secretion [8]) and latex (which is phagocytosed, reaches the lysosomes, but does not induce secretion [9] ), given for 24 h, were also incapable of inducing secretion while stored in the cells, over a succeeding 24 h period in the absence of exogenous sucrose or latex (release of hexosaminidase between 2 and 4% only). Next the dependence of secretion during storage on the quantity of endocytosed material was assessed. To achieve variations in the endocytosed quantity standard amounts of zymosan and short-fibre asbestos were given for varied lengths of time before a 24 h period in the absence of particles. Controls were incubated and washed in parallel, but without particles at any stage. Fig. 1 shows that in such experiments selective release of hexosaminidase but not lactate dehydrogenase was observed. A phagocytic pulse of 2--4 h resulted in maximal release. Release of lactate dehydrogenase and hexosaminidase in control cultures treated in parallel were indistinguishable from each other, or from the lactate dehydrogenase release in the stimulated cultures (Fig. 1). Similarly, Fig. 2 compares the release of hexosaminidase in cells in particle-free medium following various lengths of exposure to zymosan with the release in controls. Hexosaminidase release is maximal in response to a phagocytic pulse of about 1--2 h, but is relatively constant in controls. Several other experiments over different time periods confirmed the selectivity of the hydrolase release, and the length of endocytosis required for maximal secretion in a subsequent 24 h period. T A B L E II E F F E C T S OF S T O R A G E OF ZYMOSAN A N D S H O R T - F I B R E ASBESTOS, ON S E C R E T I O N OF H E X O S A M I N I D A S E BY M O U S E M A C R O P H A G E
Macrophages e n d o c y t o s e d t h e p a r t i c l e s f o r 2 h, a n d w e r e t h e n w a s h e d a n d i n c u b a t e d f o r 24 h in fresh m e d i u m w i t h o u t particles b e f o r e h a r v e s t i n g . L D H , l a c t a t e d e h y d r o g e n a s e , n . d . , n o t detectable. Stimulus
None (medium only) Zymosan (50 ~g/ml) Short-fibre c h r y s o t i l e asbestos ( 1 0 0 p g / m l )
Percentage release during 24 h f o l l o w i n g a 2 h period of e n d o c y t o s i s Hexosaminidase
LDH
3.86 _+ 3 . 5 7 5 4 . 4 _+ 2 . 7 5 4 7 . 6 + 5.49
n.d. 2 3 . 5 -+ 6 . 7 3 8.01 -+ 6.98
62 7060-
5040-
30-
20-
10--
O--
//
I
I
l
i
i
i
I
0
2
4
6
8
10
12
,,
24
Fig. 1. R e l e a s e o f e n z y m e s b y m a c r o p h a g e s during 2 4 h c u l t i v a t i o n a f t e r e x p o s u r e t o short-fibre a s b e s t o s . Abscissa: d u r a t i o n o f e x p o s u r e t o p a r t i c l e s ( h o u r s ) . Ordinate: P e r c e n t o f e n z y m e a c t i v i t y in c u l t u r e m e d i u m ; o l a c t a t e d e h y d z o g e n a s e ; o, h e x o s a m i n i d a s e .
Finally, the dependence o f secretion and cellular enzyme levels on time in culture after a phagocytic pulse was investigated. After a 2 h pulse o f zymosan (Fig. 3) hydrolase secretion from the washed cells continued over long periods (at least to 72 h). The initial release was accounted for by loss from the cells (see Fig. 3) but after a lag of about 24 h, the cellular activity of hexosaminidase, and thus the total activity in the cultures, began to rise. Thus the percentage release o f hexosaminidase rose progressively to 71% at 24 h, but although secretion continued to 72 h, because of increased intracellular activity, the percent release fell after 24 h. In control cultures studied in parallel, the total hexosaminidase activity declined slightly between 24 and 72 h, while its release slowly rose to about 30% by 72 h. Such cultures commence with higher activity than those which have received the phagocytic pulse, since during the latter there is considerable secretion (as in Table I). The stimulated release was a selective secretion, since lactate dehydrogenase release was no greater than in 50--
40--
]:
I
30--
20--
10--
O--
I
0
I
15
I
30
I
60
I
90
I
120
Fig. 2. R e l e a s e o f h e x o s a m i n i d a s e b y m a c r o p h a g e s during 2 4 h c u l t i v a t i o n after e x p o s u r e t o z y m o s a n or c o n t r o l m e d i u m for various l e n g t h s o f t i m e . Abscissa: d u r a t i o n o f e x p o s u r e t o z y m o s a n ( e ) or c o n t r o l m e d i u m ( o ) , in m i n u t e s . Ordinate: p e r c e n t release o f h e x o s a m i n i d a s e into c u l t u r e m e d i u m .
63 480--
400--
320--
l
240--
160-
80.
O-
I
' 4
8
12 16
24
48
72
Fig. 3. T i m e course o f cellular and m e d i u m activity o f h e x o s a m i n i d a s e after a 2 h p u l s e o f z y m o s a n . Abscissa: time in culture after t h e end o f p h a g o c y t i c pulse ( h o u r s ) . o m e d i u m a c t i v i t y ; o, cellular activity. Ordinate: n m o l p r o d u c t / h per plate. Separate groups of cultures w e r e harvested at each t i m e specified.
the control cells. Indeed, lactate dehydrogenase activity increased progressively in the cultures exposed to zymosan, confirming their viability. The response of macrophages to a 2 h pulse of short-fibre asbestos was quite similar (Fig. 4). A prolonged selective secretion occurred, and although cellular hydrolase levels fell throughout, there was again a large increase in the total hexosaminidase activity in the system between 24 and 96 h. The lactate dehydrogenase activity also rose considerably in the stimulated cultures, and a relatively low degree of release of lactate dehydrogenase occurred, equivalent to the release of hexosaminidase in control cultures (which reached a maximum of 40% at 96 h). Thus again hydrolase release was selective, and associated with increased culture levels of the enzyme, and also of lactate dehydrogenase.
480, 400. o 320-
240-
160-
80-
ol
/ 0
24
I
I
I
48
72
96
Fig. 4. T h e t i m e c o u r s e o f cellular and m e d i u m activity o f h e x o s a m i n i d a s e after a 2 h p u l s e o f short-fibre asbestos. Abscissa: t i m e in culture after t h e end o f t h e p h a g o c y t i c pulse ( h o u r s ) . o, m e d i u m a c t i v i t y ; 0, cellular activity. Ordinate: n m o l p r o d u e t / h Per plate. Separate groups o f cultures w e r e harvested at each t i m e specified.
64 Discussion This work shows that cultured mouse macrophages storing zymosan or shortfibre asbestos (but not latex), secrete lysosomal hydrolase activity in the absence of extracellular particles. Under these conditions secretion is not dependent on simultaneous phagocytosis. It probably results directly from the presence of the particles in the lysosomes, though other interpretations remain possible. An effect on the plasma membrane which lasts after the agents have been removed from the extracellular phase seems unlikely for two reasons. Firstly, dextran sulphate and activated complement do not induce release after they have been removed from the extracellular phase. They presumably neither leave a lasting stimulus on the plasma membrane, nor accumulate sufficiently within the cells. Secondly, UICC Chrysotile asbestos though interacting with the plasma membrane, was not phagocytosed much and induced little secretion after the cells were washed, unlike short fibre Chrysotile which was effective from within the cells. Since normal Chrysotile asbestos is a potent inducer of secretion only when presented continuously [1,211] it may exert its effects largely on the plasma membrane, while the short-fibre asbestos may act both on the plasma membrane, and when stored, on the lysosomal membrane. It has been shown previously that storage of certain particles within macrophage and fibroblast lysosomes results also in secretion of several neutral proteinases, which are probably not lysosomal [10,11]. The elevation of total culture activities of hexosaminidase and lactate dehydrogenase in response to storage, extends earlier related observations [ 1,2,8,9 ]. Several of the stimuli causing increased lysosomal enzyme levels, though accumulating in lysosomes do not induce secretion. The mechanism of elevation of the enzymes is still unknown [1,3,9,12], but one possibility stems from the recent observation of one of us [14] that storage of zymosan and sucrose inhibits degradation of endogenous proteins in cultivated macrophages. These agents probably act by reducing lysosomal activity in protein degradation, and in so doing may reduce the degradation rate of both lysosomal and cytosol enzymes. In contrast to the mechanism of induction of secretion from within cells, it is thought that some secretion of lysosomal enzymes (by both macrophages and neutrophils) accompanies the process of phagocytosis, as primary lysosomes fuse with the membrane of the phagosome while it is still open to the exterior [12]. In addition secretion by phagocytes may be induced by some agents which act at the cell surface, particularly when the phagocytic capacity of the cells has been abolished with the agent active on microfilaments, cytochalasin B [12,13]. The effectiveness in inducing secretion of the various particles used in the present work parallels exactly earlier observations on secretion in response to externally presented agents [1,2,15]. Thus similar inducing mechanisms may be involved, acting either on plasma membrane or lysosomal membrane, or both. Indeed a mechanism capable of perturbing the plasma membrane from the exterior so as to favour fusion would be expected to exert a similar effect from within lysosomal membranes. For the interior of the lysosomal membrane, just like the external face of the plasma membrane, is external to the
65 cytoplasm, and the two membranes are in dynamic continuity. Further the lysosomal membrane is quite similar to the plasma membrane in composition, and quite unlike most other intracellular membranes [12]. The effects of the particles on lysosomal enzyme levels and secretion were long lasting, and this is to be expected as both contain non-digestible components. The endocytosed materials remained associated with the cells throughout the experiments and were apparently not exocytosed, in agreement with other work on fibroblasts and macrophages [10,16]. The hydrolase secretion is thus likely to be relevant to the maintenance of chronic inflammatory reactions [2] induced by particulate non-digestible materials such as zymosan, asbestos [17] and bacterial components [3]. In agreement with this, cell walls from group A streptococci which are non-degradable and can induce chronic inflammation [3] can activate macrophages so that they can lyse certain cells, while group D cell walls (which are digestible) exert no such effects [18]. In addition it is known that group A streptococcal cell walls can induce prolonged lysosomal enzyme release [9]. References 1 Davies, P. and Allison, A.C. (1976) in Lysosomes in Biology and Pathology (Dingle, J.T. and Dean, R.T., eds.), Vol. 5, PP. 61--98, North-Holland, Amsterdam 2 Davies, P. and Allison, A.C. (1976) in Immunology of the Macrophage (Nelson, D.S., ed.), pp. 427-461, A c a d e m i c Press, New York 3 Smialowicz, R.J. and Schwab, J.H. (1977) Infect Immun. 17,591--598 4 Woollen, J.W., Heyworth, R. and Walker, P.G. (1961) Biochem. J. 78, 111--116 5 Schorlemmer, H.U., Burger, R., Hylton, W. and Allison, A.C. (1977) Clin. Immunol. Immunopathol. 7, 88---96 6 Vallota, E.H. and Mdller-Eberhsxd, H.J. (1973) J. Exp. Med. 137, 1109--1123 7 AlLison, A.C. (1973) in Biological Effects of Asbestos, pp. 89--93, International Agency for Research on Cancer, Lyon 8 Cohn, Z.A. and Ehrenreich, Z.A. (1969) J. Exp. Med. 129, 201--225 9 Davies, P., Page, R.C. and Allison, A.C. (1974) J. Exp. Med. 139, 1262--1282 10 Werb, Z. and Dingle, J.T. (1976) Lysosomes in Biology and Pathology (Dingle, J.T. and Dean, R.T., eds.), Vol. 5, pp. 127--156, North-Holland, Amsterdam 11 Hamilton, J., VassalU, J.-D. and Reich, E. (1976) J. Exp. Med. 114, 1689--1691 12 Dean, R.T. and Bazrett, A.J. (1976) Essays Biochem. 12, 1--40 1.3 Temple, A., Loewi, G., Davies, P. and Howard, A. (1973) Immunology 2 4 , 6 5 5 - - 6 7 0 14 Dean, R.T. in Protein Turnover and Lysosomal Function (Segal, H.L. and Doyle, D., eds.), A c a d e m i c Press, N e w York, in Press 15 Schorlemmer, H.U., Edwards, J.H., Davies, P. and Allison, A.C. (1977) Clin. Exp. Immunol. 2 7 , 1 9 8 - 207 16 Riley, P.A. and Dean, R.T. (1978) Exp. Cell. Biol. 46, 367--373 17 Schorlemmer, H.U., Davies, P., Hylton, W., Guglg, M. and Allison, A.C. (1977) Br. J. Exp. Pathol. 58, 315--326 18 Smialowicz, R.J. and Schwab, J.H. (1977) Infect. Immun. 17,599--606
Biochimica et Biophysica Acta, 584 (1979) 57--65 © Elsevier/North-Holland Biomedical Press
BBA 28846
LYSOSOMAL ENZYME SECRETION BYMACROPHAGES DURING INTRACELLULAR STORAGE OF PARTICLES
R.T. DEAN, W. HYLTON and A.C. ALLISON
Division of Cell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ (U.K.) (Received September 4th, 1978)
Key words: Lysosomal secretion; ~-N-Acetylglucosaminidase; Lactate dehydrogenase; Macrophage
Summary Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase ~-N-acetylglucosaminidase. Thus macrophage lysosomal enzyme secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both N-acetyl-/~-D-glucosaminidase and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.
Introduction It is now well known that macrophages in culture can selectively release lysosomal acid hydrolases in response to various stimuli, such as phagocytosable particles and the complement component C3b [ 1 ]. The relevance of such secretion to the degradative events in, and to the maintenance of, chronic inflammatory lesions, has been discussed extensively [2]. Previous work has concerned secretion occurring while the stimuli are present in the culture medium. Thus it has not been clear whether secretion can take place while the stimulating agents are present within the cells (for instance, after endocytosis) but no longer outside the cells. This situation obtains in many chronic inflammatory lesions (such as those characterised by the Abbreviation:
Hexosaminidase, fl-N-acetylglucosaminidase(EC 3.2.1.30).
58 long-term persistence within macrophages of the non-degradable Goup A strep. tococcal cell well materials [3] ). The present work demonstrates that lysosomal enzyme secretion does occur while some, but not all, endocytosed particles are stored within macrophages in culture, in the absence of extracellular particles. Materials and Methods Materials for tissue culture. Petri dishes were from Sterilin, Teddington, Middlesex. Medium 199 and sera were from BioCult Laboratories, Glasgow, U.K. Biochemical reagents. Penicillin, streptomycin, zymosan from Saccharomyces cerevisiae yeast, were obtained from Sigma Chemical Co., Surbiton, Surrey, U.K.; dextran sulphate (DS 500) was from Serva; polystyrene latex particles (0.81 um diameter: 5%, v/v) were from Difco, U.S.A.; p-nitrophenyl-2acetamido-2-deoxy-fl-D-glucopyranoside from Koch-Light Labs., Colnbrook, Bucks, U.K.; heparin, preservative free, from Paines and Byrne, Greenford, Middlesex, U.K.; pyruvate and reduced nicotinamide adenine dinucleotide from Boehringer Mannheim Gmbh, F.R.G. and Triton X-100, from BDH Ltd., Poole, Dorset, U.K. Chrysotile asbestos (UICC Standard reference sample of Rhodesian Chrysotile) was used as starting material. A sample of short-fibre asbestos prepared from this by milling was also used. Macrophage collection and culture. Macrophages were obtained by peritoneal lavage of normal Swiss mice (T.O. strain) with 5 ml cold Medium 199 containing 100 I.U./ml penicillin and streptomycin and 10 I.U./ml of heparin. 5-ml samples of the peritoneal exudate cell suspension (containing 0.75 • 106--1.3 • 106 cells/ml) were placed in 50-mm Petri dishes and incubated at 37°C in a humidified atmosphere of 5% carbon dioxide and air for 1--2 h. Nonadherent cells were then removed by washing four times with phosphate-buffered saline. After washing, the cells were cultured in 5 ml Medium 199 containing 10% (v/v) heat-inactivated swine serum. The heat inactivation was performed at 56°C for 30 min. Cultures gave a sheet of spread cells within a few hours, and experimental treatments began after incubation overnight (at which point the cells sheets were considered to be established). At this point, the medium was changed, and stimuli applied as described below. The commencement of stimulation is referred to as 0 h in Table I. Triplicate cultures were used in all experiments, and the biochemical data are expressed as mean +S.D. At the end of the incubation period, medium was collected, and the cells lysed with saline containing 0.1% (v/v) Triton X-100. Each time point on the figures refers to a separate set of cultures. The cells were then scraped off with sterile silicone rubber bungs. Enzyme assays. All assays were conducted under conditions giving linear release of product in relation to the amount of sample used and the time of incubation. Lactate dehydrogenase (EC 1.1.1.27) was assayed by determining the rate of oxidation of reduced nicotinamide adenine dinucleotide at 340 nm according to the Biochemica LDH test combination./3-N-Acetylglucosaminidase (hexosaminidase, EC 3.2.1.30) was assayed using p-nitrophenyl-2-acetamido-2~ deoxy-f~-D-glucopyranoside as substrate in 0.1 M citrate/phosphate buffer, pH
59 4.5 [4]. Results are given as means +S.D., both on the figures and in the tables. Macrophage stimuli. These were suspended directly in Medium 199 containing 10% heat-inactivated swine serum at the following concentrations known from previous work to be effective [1,2,5]: zymosan, 50 gg/ml; latex, 2 ul of the 5% (v/v) stock/ml of culture medium; chrysotile asbestos, 50 pg/ml; dextran sulphate, 100 ug/ml. To prepare serum containing activated complement components, zymosan (10 mg/ml) was incubated in unheated foetal calf serum in the presence of 0.25 M e~aminocaproic acid (to inhibit the carboxypeptidase which degrades split C3) for 30 min at 37°C [6]. The zymosan was removed by centrifugation, and the serum collected. It was used at 1% (v/v) in the cultures, in the presence of 10% heat-inactivated swine serum. Sucrose was presented in some experiments at 20 mM. Presentation of stimuli to macrophages. Stimuli were given to the cultures for various times, up to 24 h, depending on the experiment. After the chosen period, the medium was removed, and preserved for assay. One set of cultures/ stimulant was harvested at this point. The remaining cultures were vigorously washed four times with phosphate-buffered saline, to remove non-internalised particles and stimuli. Light microscopy indicated that very few or no zymosan and latex particles remained outside the cells after such washing. In contrast significant amounts of the long fibres in the normal chrysotile asbestos remained attached to the cell surface, consistent with earlier suggestions that such particles are not well phagocytosed [7]. However, with the milled shortfibre asbestos, very few particles remained attached to the cell surface after washing, while the cells became vacuolated, consistent with the material having been endocytosed. After washing, cultures received fresh medium, normally without stimuli, to allow secretion to proceed in the absence of exogenous stimuli. Occasionally, the fresh medium given after washing contained the same concentration of the stimuli present in the first period. Cultures were harvested after chosen periods of further incubation.
Results Table I shows the results of an experiment in which cells were exposed for 24 h to various stimuli, allowing substantial phagocytosis. After 24 h one set of cultures was harvested, and columns 1A and B show that during this period, zymosan, zymosan-activated serum, and to a lesser extent, dextran sulphate, had induced secretion of lysosomal hexosaminidase (not accompanied by equivalent release of lactate dehydrogenase). In this experiment some of the control hydrolase release seems to be selective as it exceeds the lactate dehydrogenase release. Such 'basal' release is often found, and to a variable extent. It probably depends on the degree of stimulation (by infection etc.) to which the macrophages have been exposed in vivo. In general, the lactate dehydrogenase release was quite low and thus hexosaminidase release is not attributable to cell death. The activated serum showed some degree of lytic effect on the cells, as indicated by the quite high percentage lactate dehydrogenase release (column 1B). Data presented later show that the cultures stimulated with
60 TABLE I SECRETION OF HEXOSAMINIDASE DURING EXPOSURE OF MACROPHAGES STIMULI, AND IN RESPONSE TO INTRACELLULAR STORAGE OF ZYMOSAN
TO VARIOUS
Cultures ( t r i p l i c a t e ) w e r e e i t h e r h a r v e s t e d after 24 h o f s t i m u l a t i o n , w h i c h c o m m e n c e d after f o r m a t i o n o f t h e cell s h e e t (data in c o l u m n s 1 A and B) or w a s h e d f o u r t i m e s and h a r v e s t e d after a further 24 h i n c u b a t i o n in e i t h e r s t i m u l a n t - c o n t a i n i n g ( c o l u m n s 3 A and B) or n o r m a l m e d i u m ( c o l u m n s 2 A and B). M e d i u m e n z y m e activities are e x p r e s s e d as p e r c e n t o f the t o t a l in the h a r v e s t e d cultures. T o t a l h e x o s a m i n i d a s e activities in c o n t r o l c u l t u r e s in t h e p r e s e n t w o r k ranged f r o m 3 5 0 to 6 0 0 n m o l / h per plate and w e r e i n c r e a s e d b y up t o 6 0 % b y c o n t i n u o u s e x p o s u r e t o z y m o s a n for 48 h (see Fig. 3). T o t a l l a c t a t e d e h y d r o genase ( L D H ) a c t i v i t i e s in c o n t r o l s ranged f r o m 2 5 0 t o 4 5 0 m u n i t s / p l a t e and w e r e i n c r e a s e d b y up to 50% b y c o n t i n u o u s e x p o s u r e t o z y m o s a n for 48 h. Stimulus
Control (medium only) Dextran sulphate (100 #g/ml) Activated serum (1%, v / v ) Zymosan (50 Dg/ml)
C o l u m n s 1 A and B (release during c u l t u r e from 0--24 h with stimuli)
C o l u m n s 2 A and B (release b e t w e e n 24--48 h without stimuli)
C o l u m n s 3 A and B (release b e t w e e n 24---48 h w i t h s t i m u l i )
Hexosaminidase
LDH
Hexosaminidase
LDH
Hexosaminldase
LDH
3 4 . 5 -+ 6.7
11.1 + 1.2
2 9 . 0 ± 10.9
1 6 . 7 -+ 4 . 2
2 9 . 0 + 10.9
16.7 ± 4.2
4 2 . 4 +_ 2.5
2 5 . 0 -+ 3.2
29.6 ± 5.96
16.7 + 5.1
43.1 + 1 2 . 2
2 0 . 0 ± 3.2
6 5 . 4 ± 0.8
5 0 . 0 ± 4.7
26.9 ± 4 . 4 7
9.1 ± 3.2
82.6 ± 2.64
4 2 . 4 ± 3.8
79.1 -+ 0.3
3 7 . 5 ± 4.3
53.2 ± 1 . 1 0
9.1 ± 2.1
6 2 . 6 + 1.3
16.1 ± 2.2
zymosan and asbestos not only remain viable, but actually produce increased amounts of hexosaminidase and lactate dehydrogenase. The remaining cells from this experiment were washed extensively to remove external stimuli, and then given fresh medium either with (columns 3A and B, Table I) or without (columns 2A and B) the original stimulant. In the latter case, endocytosis of, and stimulation by, the agent in question continued, and thus the behaviour of the cells during this second phase (columns 3A and B) and quite similar to that during the first 24 h phase (results o f columns 1A and B). However, the most important observations are those in columns 2A and B, describing the behaviour of cells cultivated without external stimuli during the second phase (24--48 h). Cells which had been exposed to zymosan for 24 h, continued to secrete large amounts o f hexosaminidase selectively during the second phase (24---48 h) in the absence of zymosan (Table I, columns 2A and B). This release was almost as substantial as that when zymosan was reintroduced after washing (columns 3A and B). In contrast the secretion during this same period (from 24 to 48 h after establishmen¢ o f the cell sheet) by cells initially exposed to dextran sulphate or activated serum, and then cultured in the absence of stimuli, was no greater than that by control cells (columns 2A and B). Dextran sulphate and activated serum in the medium were capable of inducing secretion in this same period (columns 3A and B). Thus the presence within macrophages of zymosan, but not of dextran sulphate or activated serum, allows continued secretion of lysosomal enzymes in the absence of further exogenous stimulus.
61
Table II shows that even a short (2 h) pulse of endocytosis of zymosan, or of short-fibre asbestos, allowed secretion during a subsequent 24 h period in the absence of the stimulus. However, normal chrysotile asbestos given for up to 24 h, was relatively ineffective in inducing release over a subsequent 24 h period in the virtual absence of asbestos, even though it induced release during the first 24 h (data not shown). This suggests that the large particles act mainly at the plasma membrane, from which they are largely removed by washing. In contrast, the short-fibre form is substantially endocytosed, and presumably canact in lysosomes in the absence of exogenous asbestos. Several repetitions of such experiments confirmed that macrophages storing zymosan or short-fibre asbestos remain viable, and secrete lysosomal enzymes selectively, while those pretreated with dextran sulphate or activated serum do not continue to do so in the absence of stimuli. Sucrose (which induces vacuolation and increased lysosomal enzyme activity within cells, but not secretion [8]) and latex (which is phagocytosed, reaches the lysosomes, but does not induce secretion [9] ), given for 24 h, were also incapable of inducing secretion while stored in the cells, over a succeeding 24 h period in the absence of exogenous sucrose or latex (release of hexosaminidase between 2 and 4% only). Next the dependence of secretion during storage on the quantity of endocytosed material was assessed. To achieve variations in the endocytosed quantity standard amounts of zymosan and short-fibre asbestos were given for varied lengths of time before a 24 h period in the absence of particles. Controls were incubated and washed in parallel, but without particles at any stage. Fig. 1 shows that in such experiments selective release of hexosaminidase but not lactate dehydrogenase was observed. A phagocytic pulse of 2--4 h resulted in maximal release. Release of lactate dehydrogenase and hexosaminidase in control cultures treated in parallel were indistinguishable from each other, or from the lactate dehydrogenase release in the stimulated cultures (Fig. 1). Similarly, Fig. 2 compares the release of hexosaminidase in cells in particle-free medium following various lengths of exposure to zymosan with the release in controls. Hexosaminidase release is maximal in response to a phagocytic pulse of about 1--2 h, but is relatively constant in controls. Several other experiments over different time periods confirmed the selectivity of the hydrolase release, and the length of endocytosis required for maximal secretion in a subsequent 24 h period. T A B L E II E F F E C T S OF S T O R A G E OF ZYMOSAN A N D S H O R T - F I B R E ASBESTOS, ON S E C R E T I O N OF H E X O S A M I N I D A S E BY M O U S E M A C R O P H A G E
Macrophages e n d o c y t o s e d t h e p a r t i c l e s f o r 2 h, a n d w e r e t h e n w a s h e d a n d i n c u b a t e d f o r 24 h in fresh m e d i u m w i t h o u t particles b e f o r e h a r v e s t i n g . L D H , l a c t a t e d e h y d r o g e n a s e , n . d . , n o t detectable. Stimulus
None (medium only) Zymosan (50 ~g/ml) Short-fibre c h r y s o t i l e asbestos ( 1 0 0 p g / m l )
Percentage release during 24 h f o l l o w i n g a 2 h period of e n d o c y t o s i s Hexosaminidase
LDH
3.86 _+ 3 . 5 7 5 4 . 4 _+ 2 . 7 5 4 7 . 6 + 5.49
n.d. 2 3 . 5 -+ 6 . 7 3 8.01 -+ 6.98
62 7060-
5040-
30-
20-
10--
O--
//
I
I
l
i
i
i
I
0
2
4
6
8
10
12
,,
24
Fig. 1. R e l e a s e o f e n z y m e s b y m a c r o p h a g e s during 2 4 h c u l t i v a t i o n a f t e r e x p o s u r e t o short-fibre a s b e s t o s . Abscissa: d u r a t i o n o f e x p o s u r e t o p a r t i c l e s ( h o u r s ) . Ordinate: P e r c e n t o f e n z y m e a c t i v i t y in c u l t u r e m e d i u m ; o l a c t a t e d e h y d z o g e n a s e ; o, h e x o s a m i n i d a s e .
Finally, the dependence o f secretion and cellular enzyme levels on time in culture after a phagocytic pulse was investigated. After a 2 h pulse o f zymosan (Fig. 3) hydrolase secretion from the washed cells continued over long periods (at least to 72 h). The initial release was accounted for by loss from the cells (see Fig. 3) but after a lag of about 24 h, the cellular activity of hexosaminidase, and thus the total activity in the cultures, began to rise. Thus the percentage release o f hexosaminidase rose progressively to 71% at 24 h, but although secretion continued to 72 h, because of increased intracellular activity, the percent release fell after 24 h. In control cultures studied in parallel, the total hexosaminidase activity declined slightly between 24 and 72 h, while its release slowly rose to about 30% by 72 h. Such cultures commence with higher activity than those which have received the phagocytic pulse, since during the latter there is considerable secretion (as in Table I). The stimulated release was a selective secretion, since lactate dehydrogenase release was no greater than in 50--
40--
]:
I
30--
20--
10--
O--
I
0
I
15
I
30
I
60
I
90
I
120
Fig. 2. R e l e a s e o f h e x o s a m i n i d a s e b y m a c r o p h a g e s during 2 4 h c u l t i v a t i o n after e x p o s u r e t o z y m o s a n or c o n t r o l m e d i u m for various l e n g t h s o f t i m e . Abscissa: d u r a t i o n o f e x p o s u r e t o z y m o s a n ( e ) or c o n t r o l m e d i u m ( o ) , in m i n u t e s . Ordinate: p e r c e n t release o f h e x o s a m i n i d a s e into c u l t u r e m e d i u m .
63 480--
400--
320--
l
240--
160-
80.
O-
I
' 4
8
12 16
24
48
72
Fig. 3. T i m e course o f cellular and m e d i u m activity o f h e x o s a m i n i d a s e after a 2 h p u l s e o f z y m o s a n . Abscissa: time in culture after t h e end o f p h a g o c y t i c pulse ( h o u r s ) . o m e d i u m a c t i v i t y ; o, cellular activity. Ordinate: n m o l p r o d u c t / h per plate. Separate groups of cultures w e r e harvested at each t i m e specified.
the control cells. Indeed, lactate dehydrogenase activity increased progressively in the cultures exposed to zymosan, confirming their viability. The response of macrophages to a 2 h pulse of short-fibre asbestos was quite similar (Fig. 4). A prolonged selective secretion occurred, and although cellular hydrolase levels fell throughout, there was again a large increase in the total hexosaminidase activity in the system between 24 and 96 h. The lactate dehydrogenase activity also rose considerably in the stimulated cultures, and a relatively low degree of release of lactate dehydrogenase occurred, equivalent to the release of hexosaminidase in control cultures (which reached a maximum of 40% at 96 h). Thus again hydrolase release was selective, and associated with increased culture levels of the enzyme, and also of lactate dehydrogenase.
480, 400. o 320-
240-
160-
80-
ol
/ 0
24
I
I
I
48
72
96
Fig. 4. T h e t i m e c o u r s e o f cellular and m e d i u m activity o f h e x o s a m i n i d a s e after a 2 h p u l s e o f short-fibre asbestos. Abscissa: t i m e in culture after t h e end o f t h e p h a g o c y t i c pulse ( h o u r s ) . o, m e d i u m a c t i v i t y ; 0, cellular activity. Ordinate: n m o l p r o d u e t / h Per plate. Separate groups o f cultures w e r e harvested at each t i m e specified.
64 Discussion This work shows that cultured mouse macrophages storing zymosan or shortfibre asbestos (but not latex), secrete lysosomal hydrolase activity in the absence of extracellular particles. Under these conditions secretion is not dependent on simultaneous phagocytosis. It probably results directly from the presence of the particles in the lysosomes, though other interpretations remain possible. An effect on the plasma membrane which lasts after the agents have been removed from the extracellular phase seems unlikely for two reasons. Firstly, dextran sulphate and activated complement do not induce release after they have been removed from the extracellular phase. They presumably neither leave a lasting stimulus on the plasma membrane, nor accumulate sufficiently within the cells. Secondly, UICC Chrysotile asbestos though interacting with the plasma membrane, was not phagocytosed much and induced little secretion after the cells were washed, unlike short fibre Chrysotile which was effective from within the cells. Since normal Chrysotile asbestos is a potent inducer of secretion only when presented continuously [1,211] it may exert its effects largely on the plasma membrane, while the short-fibre asbestos may act both on the plasma membrane, and when stored, on the lysosomal membrane. It has been shown previously that storage of certain particles within macrophage and fibroblast lysosomes results also in secretion of several neutral proteinases, which are probably not lysosomal [10,11]. The elevation of total culture activities of hexosaminidase and lactate dehydrogenase in response to storage, extends earlier related observations [ 1,2,8,9 ]. Several of the stimuli causing increased lysosomal enzyme levels, though accumulating in lysosomes do not induce secretion. The mechanism of elevation of the enzymes is still unknown [1,3,9,12], but one possibility stems from the recent observation of one of us [14] that storage of zymosan and sucrose inhibits degradation of endogenous proteins in cultivated macrophages. These agents probably act by reducing lysosomal activity in protein degradation, and in so doing may reduce the degradation rate of both lysosomal and cytosol enzymes. In contrast to the mechanism of induction of secretion from within cells, it is thought that some secretion of lysosomal enzymes (by both macrophages and neutrophils) accompanies the process of phagocytosis, as primary lysosomes fuse with the membrane of the phagosome while it is still open to the exterior [12]. In addition secretion by phagocytes may be induced by some agents which act at the cell surface, particularly when the phagocytic capacity of the cells has been abolished with the agent active on microfilaments, cytochalasin B [12,13]. The effectiveness in inducing secretion of the various particles used in the present work parallels exactly earlier observations on secretion in response to externally presented agents [1,2,15]. Thus similar inducing mechanisms may be involved, acting either on plasma membrane or lysosomal membrane, or both. Indeed a mechanism capable of perturbing the plasma membrane from the exterior so as to favour fusion would be expected to exert a similar effect from within lysosomal membranes. For the interior of the lysosomal membrane, just like the external face of the plasma membrane, is external to the
65 cytoplasm, and the two membranes are in dynamic continuity. Further the lysosomal membrane is quite similar to the plasma membrane in composition, and quite unlike most other intracellular membranes [12]. The effects of the particles on lysosomal enzyme levels and secretion were long lasting, and this is to be expected as both contain non-digestible components. The endocytosed materials remained associated with the cells throughout the experiments and were apparently not exocytosed, in agreement with other work on fibroblasts and macrophages [10,16]. The hydrolase secretion is thus likely to be relevant to the maintenance of chronic inflammatory reactions [2] induced by particulate non-digestible materials such as zymosan, asbestos [17] and bacterial components [3]. In agreement with this, cell walls from group A streptococci which are non-degradable and can induce chronic inflammation [3] can activate macrophages so that they can lyse certain cells, while group D cell walls (which are digestible) exert no such effects [18]. In addition it is known that group A streptococcal cell walls can induce prolonged lysosomal enzyme release [9]. References 1 Davies, P. and Allison, A.C. (1976) in Lysosomes in Biology and Pathology (Dingle, J.T. and Dean, R.T., eds.), Vol. 5, PP. 61--98, North-Holland, Amsterdam 2 Davies, P. and Allison, A.C. (1976) in Immunology of the Macrophage (Nelson, D.S., ed.), pp. 427-461, A c a d e m i c Press, New York 3 Smialowicz, R.J. and Schwab, J.H. (1977) Infect Immun. 17,591--598 4 Woollen, J.W., Heyworth, R. and Walker, P.G. (1961) Biochem. J. 78, 111--116 5 Schorlemmer, H.U., Burger, R., Hylton, W. and Allison, A.C. (1977) Clin. Immunol. Immunopathol. 7, 88---96 6 Vallota, E.H. and Mdller-Eberhsxd, H.J. (1973) J. Exp. Med. 137, 1109--1123 7 AlLison, A.C. (1973) in Biological Effects of Asbestos, pp. 89--93, International Agency for Research on Cancer, Lyon 8 Cohn, Z.A. and Ehrenreich, Z.A. (1969) J. Exp. Med. 129, 201--225 9 Davies, P., Page, R.C. and Allison, A.C. (1974) J. Exp. Med. 139, 1262--1282 10 Werb, Z. and Dingle, J.T. (1976) Lysosomes in Biology and Pathology (Dingle, J.T. and Dean, R.T., eds.), Vol. 5, pp. 127--156, North-Holland, Amsterdam 11 Hamilton, J., VassalU, J.-D. and Reich, E. (1976) J. Exp. Med. 114, 1689--1691 12 Dean, R.T. and Bazrett, A.J. (1976) Essays Biochem. 12, 1--40 1.3 Temple, A., Loewi, G., Davies, P. and Howard, A. (1973) Immunology 2 4 , 6 5 5 - - 6 7 0 14 Dean, R.T. in Protein Turnover and Lysosomal Function (Segal, H.L. and Doyle, D., eds.), A c a d e m i c Press, N e w York, in Press 15 Schorlemmer, H.U., Edwards, J.H., Davies, P. and Allison, A.C. (1977) Clin. Exp. Immunol. 2 7 , 1 9 8 - 207 16 Riley, P.A. and Dean, R.T. (1978) Exp. Cell. Biol. 46, 367--373 17 Schorlemmer, H.U., Davies, P., Hylton, W., Guglg, M. and Allison, A.C. (1977) Br. J. Exp. Pathol. 58, 315--326 18 Smialowicz, R.J. and Schwab, J.H. (1977) Infect. Immun. 17,599--606
