Histochemistry59, 225-232 (1979)
Histochemistry 9 by Springer-Verlag 1979
Cytoehemieal Analysis of Organelle Degradation in Phagosomes and Apoptotic Cells of the Mucoid Epithelium of Mice N. Pipan and M. Sterle Institute of Human Biology,MedicalFaculty,YU-61000 Ljubljana,Lipi~eva2, Yugoslavia
Summary. The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2-6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes. Introduction The very common phenomenon of sequestration and digestion by cells of parts of their own cytoplasm which is known as autophagy has as yet no satisfactory explanation. The question whether in the autophagic processes the degradation of organelles occurs at random or whether there is a selective mechanism has remained unanswered (Arstila et al., 1971; Ericsson, 1969a; Ericsson, 1969b; Ericsson, 1969c; Farquhar, 1971; Helminen and Ericsson, 1971; Pipan and Marn, 1972). It is well established that the frequency of autophagic vacuoles increases markedly under a variety of conditions, and that it is the usual response to injuries (Arstila et al., 1971; Ericsson, 1969a; Holtzman, 1976), but whether it contributes directly to cell death has not yet been proven. Mucoid cells of the stomach epithelium of mice contain different phagoand lysosomes (Pipan, 1972; Pipan and Marn, 1972; Pipan, 1974) and a special type of cellular death known as apoptosis, found in normal and pathological
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c o n d i t i o n s ( K e r r , 1972; K e r r a n d Searle, 1972; K e r r et al., 1972; K e r r , 1973; K e r r et al., 1974; P i p a n , 1976), o c c u r s also in t h e s e cells. F o r this r e a s o n t h e y r e p r e s e n t a c o n v e n i e n t m o d e l for the s t u d y o f d e g r a d a t i o n p r o c e s s e s a n d cell d e a t h in p h y s i o l o g i c a l c o n d i t i o n s . W e a n a l y s e d the s e q u e s t r a t i o n r a t e o f m i t o c h o n d r i a a n d p e r o x i s o m e s in t h e p h a g o l y s o s o m e s o f m o c o i d e p i t h e l i a l cells u s i n g c y t o c h e m i c a l m e t h o d s . T h e initial m o r p h o l o g i c a l a n d p h y s i o l o g i c a l e v e n t s o f a p o p t o s i s a r e n o t v e r y well u n d e r s t o o d ( W y l l i e et al., 1977). I n m u c o i d cells e x h i b i t i n g a p o p t o t i c d e a t h we a n a l y s e d c y t o c h e m i c a l l y t h e a c t i v i t y o f c y t o c h r o m e o x i d a s e a n d in this w a y t h e f u n c t i o n a l state o f m i t o c h o n d r i a w a s tested. W e w e r e also i n t e r e s t e d to see if a p o p t o t i c cells c o n t a i n p h a g o - a n d l y s o s o m e s a n d w h a t h a p p e n s t o t h e d e a d cells a f t e r t h e y are e x t r u d e d f r o m t h e m u c o i d e p i t h e l i u m .
Material and Methods The tissue from the stomach of 2-6 day old mice was taken for this investigation. For the analysis of peroxisomal catalase and lysosomal acid phosphatase, slices of tissue were fixed for 2 h in cold 2.5% glutaraldehyde with 4% paraformaldehyde in 0.2 M cacodylate buffer at pH 7.2-7.4 followed by overnight rinsing in 0.1 M cacodylate buffer with 0.3 M sucrose. For catalase, the tissue was incubated in the alkaline 3.Ydiaminobenzidine (DAB) medium, containing KCN described by Novikoff et al. (1972). The medium is prepared by dissolving 20 mg DABtetrahydrochloride in 9.3 ml 0.05 M propandiol buffer, pH 9.0; after adjusting the pH to 9.7 with 1 N NaOH, 0.5 ml 0.1 N KCN and 0.2 ml 2.4% H202 are added. The incubation time was 60 min at 37~ C. Following incubation, the tissue was rinsed several times in 7.5% sucrose. For control, the incubation medium without H202 was used. After rinsing, the tissue was treated with buffered 1% OsO4, dehydrated in ethanol and embedded in Epon. For cytochrome oxidase, the method of Seligman et al. (1968) was used. Small tissue slices were fixed for 60 min in 4% paraformaldehyde in 0.05 M sodiumphosphate buffer at pH 7.4. After fixation the tissue was washed for 15 min in 0.22 M sucrose 0.05 M phosphate buffer, at pH 7.4. The tissue was incubated for 60 rain at 37~ C in a medium containing 5 mg diaminobenzidine tetrahydrochloride, 9 ml 0.05 M Na-phosphate buffer, 1 ml catalase (20 mg/ml), 10 mg cytochrome C, 750 mg sucrose. The control material was incubated in 0.01 M KCN; following incubation the tissue was rinsed in 0.05 M phosphate buffer, pH 7.4 for 15 rain and postfixed in 1% buffered osmium tetroxide, dehydrated and embedded in Epon. For simultaneous analysis of cytochrome oxidase and catalase the tissue was fixed in the same way as for cytochrome oxidase, only using 0.05 M propandiol buffer instead of sodium phosphate buffer. The tissue was incubated for 90 min at 37~ C in the same medium as for catalase at pH 9.7 but KCN was omitted and 0.2 ml 2.5% H202 added. Following incubation, the tissue was rinsed in 0.05 M propandiol buffer at pH 9, posffixed in I% OsO4, dehydrated and embedded in Epon. For acid phosphatase the tissue was incubated in a medium (Barka and Anderson, 1962) containing 5 mI 1.2 Na-c~-glycerophosphate, pH 5, 5 ml 0.1 M tris-maleate buffer, pH 5, 5 ml water, 10 ml 0.2% Pb(NO3)2, and 7.5% sucrose. The incubation time was 35 rain at 37~ C. The control material was incubated without Na-c~-glycerophosphate. Following incubation the tissue was washed in twice distilled water, postfixed with 1% KMnO4, washed, dehydrated and embedded in Epon. Thin sections were prepared with an LKB Ultratome. The sections were examined after staining with lead citrate and uranyl acetate in a Siemens Elmiskop I at 80 kV or in a Jeol T8 microscope at 80 kV.
Results I n t h e s m a l l a n d large a u t o p h a g o s o m e s o f m u c o i d cells m a i n l y m i t o c h o n d r i a a n d E R a r e s e q u e s t e r e d . A l t h o u g h m u c o i d cells c o n t a i n n u m e r o u s p e r o x i s o m e s
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Fig. 1. Cytochrome oxidase reaction product in cristae of the mitochondrium (M1) in an autophagosome and in unsequestered mitochondria (M:) of a mocoid cell from a 6 day old mouse, x 35,000 Fig. 2. a Detail from Figure 2b showing mitochondria with reaction product in the intramembraneous space of the cristae (arrows). x 50,000. h A spherical apoptotic mucoid cell in which most mitochondria (M) contain reaction product of cytochrome oxidase. Nucleus (N) is pycnotic, plasmalema is well-preserved (arrow). Six day old mouse, x 11,000
Fig. 3. a Detail from Figure 3 b showing mitochondria with visible cristal membranes without reaction product of cytochrome oxidase (arrows). x 50000. b Spherical apoptotic mucoid cell of a 6 day old mouse in which most mitochondria are swollen and their cristae contain no reaction product of cytochrome oxidase, x 11,000 Fig. 4. Endocytosed apoptotic cell in the heterophagosome of a sister mucoid cell from a 6 day old mouse contains swollen mitochondria (M) without reaction product of cytochrome oxidase. Reaction product is in mitochondria (M1) of the surrounding celt. An ingested apoptotic body has well-preserved plasmalema, x 11,000
Fig. 5. A big phagosome in a mucoid cell of a 6 day old mouse in which catalase and cytochrome oxidase were analysed simultaneously. Peroxisomes (P) contain catalase, swollen mitochondria (M) are without reaction product of cytochrome oxidase, rest of a nucleus (N) ; normal peroxisomes (P1) and mitochondria (M1) with typical reaction product, x 15,000 Fig. 6. Apoptotic bodies in heterophagosomes of mucoid cells of a 6 day old mouse with reaction product of acid phosphatase in incorporated lysosome (L), Golgi vacuoles (GA) and secondary lysosomes (L~) which surround apoptotic bodies. Mitochondria (M) in heterophagosomes are partly swollen, x 14,000
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these organelles were found only exceptionally inside phagosomes. Autophagocytosed mitochondria with well-preserved morphology show normal activity of cytochrome oxidase and the reaction product fills the intramembraneous space between the outer and inner mitochondrial membrane (Fig. 1). Apoptotic cells which are extruded from the epithelium contain more or less well-preserved organelles but they show also the signs of lytic processes. Inspite of these observations phagolysosomes do not seem to contribute essentially to their death (Fig. 2a, b and 3a, b). The reaction product of cytochrome oxidase fills the intramenbraneous space of cristae (Fig. 2a) until the mitochondria become swollen (Fig. 3 b). Some or all apoptotic cells are later ingested by sister cells and the final degradation of organelles occurs inside lysosomes. In the apoptotic cells phagocytosed by sister cells their mitochondria exhibit different activity of cytochrome oxidase. In Figure 4a heterophagosome with one whole apoptotic cells can be seen but the reaction product can no longer be observed in lightly swollen mitochondria. Heterophagosomes contain different numbers of peroxisomes which can be identified by the presence of catalase. Catalase can be demonstrated in lysosomes in which the mitochondria have already been destroyed (Fig. 5). The degradation of phagocytosed material can probably be initiated in several ways. Figure 6 suggests two different possibilities; either the lysosomes are sequestered in the phagosomes, like any other cell organelle, or by the secondary lysosomes which encircle the phagosomes. A direct fusion of the phagosome and the secondary lysosome was not observed, but it can be presumed. In phagosomes containing either normal or swollen mitochondria, acid phosphatase could not be identified (Fig. 6).
Discussion
Although mucoid cells contain numerous peroxisomes (Pipan and P~eni~nik, 1976) in autophagosomes these organelles were found only exceptionally. We followed especially the sequestration rate of peroxisomes because Locke and Mc Mahon (1971), who studied selectivity in autophagic processes, found that autophagosomes of fatty body cells engulf first of all the peroxisomes and later the mitochondria and other cell components. On the other hand Deter's study of autophagocytosis in hepatocytes induced by glucagon showed that the frequency of peroxisomes was much lower than would be expected on the basis of their quantity in these cells (Deter, 1971). The selectivity rules obviously change from cell to cell and probably depend, even in the same type of cell, upon their functional state. The quantity of organelles cannot, in itself, be essential for the selective mechanism. Dysfunction of the organelle seems also not to be the primary signal for the sequestration in the autophagosome. Although in our investigation only one of the numerous mitochondrial enzymes was taken into consideration, well-preserved activity of cytochrome oxidase gives indirect information about the functional state of sequestered organelles. The reaction product of cytochrome oxidase in the mitochondria of spherical
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apoptotic cells ejected from the mucoid epithelium provides interesting additional information to the results of Wyllie et al. (1977), in which it was found that intracellular ATP is maintained until late in the process of apoptotic death. Both results indicate that apoptotic death does not initiate energy deficiency in the cell. Phago- and lysosomes also do not seem to be directly involved in apoptotic death although they are present in such cells. Contrary to our results, Hurle and Hinchliffe (1977) found that apoptotic death of mesenchymal cells follows the state in which large lysosomes appear. Apoptotic bodies can be endocytosed by sister cells (Kerr et al., 1972; Pipan, 1976; Wyllie et al., 1977). During our observations we came to the conclusion that sister cells endocytose only membrane-bounded cells or their membranebounded fragments but never free cell debris which is also found in the stomach lumina. For the explanation of this, the results of Pricer and Ashwell (1971), obtained on hepatocytes, seem to be relevant. They have reported that isolated hepatic plasmalema binds neuraminidase-treated glycoproteins more avidly that the intact ones. It is possible to conclude from these data that hepatocyte surfaces carry receptors with greater affinity for asialoglycoproteins. Changes in the membrane glycoproteins which play an important role in cell to cell contact must probably occur in apoptotic death because such cells lose their connection to neighbour cells and leave the epithelium as shperical bodies. The nature of membrane changes in apoptotic bodies is still unknown (Wyllie et al., 1977) but the presumption that apoptotic cells become asialated and so in some way induce endocytosis through the receptors of living cells seems very attractive, although it was not proven in our analysis. The deterioration of the engulfed material is possible because lysosomal enzymes reach heterophagosomes in different ways. The initial degradation of mitochondria probably goes on without lysosomal enzymes and is induced only be sequestration into phagosomes and by loss of normal metabolic conditions.
References Arstila, A., Jauregni, J., Chang, J., Trump, B.: Studies on cellular autophagocytosis. Lab. Invest. 24, 162-174 (i971) Barka, T., Anderson, P.J. : Histochemical methods for acid phosphatase using hexazonium pararosanillin as coupler. J. Histochem. Cytochem. 10, 741 (1962) Deter, R.L.: Quantitative characterization of dense bodies, autophagic vacuoles and acid bearing particles population during the early phases of glucagon induced autophagy in rat liver. J. Cell Biol. 48, 473~489 (1971) Ericsson, J.L.E. : Mechanism of cellular autophagy. In: Lysosomes in biology and pathology. Dingle, J.T., Fell, H.B. (eds.), Vol. 2, pp. 345-394. Amsterdam: North Holland Publishing Co. 1969a Ericsson, J.L.E.: Studies on induced cellular autophagy. I. Electron microscopy of ceils with in vivo labelled lysosomes. Exp. Cell Res. 55, 95 106 (1969b) Ericsson, J.L.E.: Studies on induced cellular autophagy. II. Characterization of the membrane bordering autophagosomes in parenchymal liver cells. Exp. Cell Res. 56, 393-405 (1969c) Farquhar, M.G.: Processing of secretory products by cells of the anterior pituitary gland. Mem. Soc. Endocrinol. 19, 79 124 (1971) Helminen, H.J., Ericsson, J.L.E. : Ultrastructural studies on prostatic involution in the rat. Mechanism of autophagy in epithelial cells with special reference to the rough surfaced endoplasmic reticulum. J. Ultrastruct. Res. 36, 708-724 (1971)
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Holtzman, E.: Lysosomes: A survey. Cell biology monographs V, pp. 64-67. Wien, New York: Springer 1976 Hurle, J., Hinchliffe, J.R.: Cytochemical and stereoscan studies of cell death in the posterior necrotic zone (PNZ) of the embryonic chick wing-bud. Proc. R. Micr. Soc. 12/5, 214 (197~/) Kerr, J.F.R.: Shrinkage necrosis. A distinct mode of cellular death. J. Pathol. 10fi, 13-20 (1971) Kerr, J.F.R., Searle, J. : Digestion of cellular fragments with phagolysosomes in carcinomous cells. J. Pathol. 108, 55 58 (1972) Kerr, J.F.R., Wyllie, A.H., Currie, A.R. : Apoptosis: A basic biological phenomenon with wideranging implications in tissue kinetics. Br. J. Cancer 26, 239-257 (1972) Kerr, J.F.R., Harmon, B., Searle, J.: An electron-microscope study of cell deletion in the anur+n tadpole tail during spontaneous metamorphosis with special reference to apoptosis of striated muscle fibres. J. Cell Sci. 14, 571-585 (1974) Kerr, J.F.R.: Some iysosome functions in liver cells reacting to sublethal injury. In: Lysosomes in biology and pathology. Dingle, J.T. (ed.), Vol. 3, pp. 365-394. Amsterdam: North Holland PuNishing Co. 1973 Locke, M., Mc Mahon, J.T.: The origin and fate of microbodies in the fat body of an insect. J. Cell Biol. 48, 61-78 (I971) Novikoff, A.B., Novikoff, P.M., Davis, C., Qnintana, N.: Studies on microperoxisomes. II. A cytochemical method for light and electron microscopy. J. Histochem. Cytochem. 20, 1006-1023 (1972) Pipan, N. : Autophagie in den Driisenzellen des Magens bei jungen M/iusen. Z. Zellforsch. 127, 258-269 (1972) Pipan, N., Marn, M.: Autophagie und Glykogenverteilung im Magenepithel der Maus wfihrend der Differenzierung. Z. Mikroskop. Anat. Forsch. 85, 421-437 (1972) Pipan, N. : Lizosomski sistem celic ~revesnega in ~elod~nega epitela ter proksimalnih tubulov pri mini med ontogenezo. Biol. Vest. 22, 143-154 (1974) Pipan, N. : Death and Phagocytosis of epithelial cells in developing mouse kidney. Cytobiologie 13, 435-441 (1976) Pipan, N., P~eni~nik, M. : Dynamics of microperoxisomes in some developing mammalian tissues. Abstracts, The first international congress on cell biology, Boston. J. Cell Biol. 70, 420a (1976) Pricer, W.E., Ashwell, G. : The binding of disalylated glycoproteins by plasma membranes of rat liver. J. Biol. Chem. 246, 4825-4833 (i971) Seligman, A.M., Karnovsky, M.J., Wasserkrug, H.L., Hanker, J.S.: Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). J. Cell Biol. 38, 1 (1968) Wyllie, A.H., Robertson, M.G., Wadell, A.W., Bird, C.,Curries, A.R. : Cell death in living tissue: Morphology and mechanism of apoptosis. Proc. R. Micr. Soc. 12/5, 210 (1977)
Received February 24, 1978
Histochemistry 9 by Springer-Verlag 1979
Cytoehemieal Analysis of Organelle Degradation in Phagosomes and Apoptotic Cells of the Mucoid Epithelium of Mice N. Pipan and M. Sterle Institute of Human Biology,MedicalFaculty,YU-61000 Ljubljana,Lipi~eva2, Yugoslavia
Summary. The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2-6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes. Introduction The very common phenomenon of sequestration and digestion by cells of parts of their own cytoplasm which is known as autophagy has as yet no satisfactory explanation. The question whether in the autophagic processes the degradation of organelles occurs at random or whether there is a selective mechanism has remained unanswered (Arstila et al., 1971; Ericsson, 1969a; Ericsson, 1969b; Ericsson, 1969c; Farquhar, 1971; Helminen and Ericsson, 1971; Pipan and Marn, 1972). It is well established that the frequency of autophagic vacuoles increases markedly under a variety of conditions, and that it is the usual response to injuries (Arstila et al., 1971; Ericsson, 1969a; Holtzman, 1976), but whether it contributes directly to cell death has not yet been proven. Mucoid cells of the stomach epithelium of mice contain different phagoand lysosomes (Pipan, 1972; Pipan and Marn, 1972; Pipan, 1974) and a special type of cellular death known as apoptosis, found in normal and pathological
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c o n d i t i o n s ( K e r r , 1972; K e r r a n d Searle, 1972; K e r r et al., 1972; K e r r , 1973; K e r r et al., 1974; P i p a n , 1976), o c c u r s also in t h e s e cells. F o r this r e a s o n t h e y r e p r e s e n t a c o n v e n i e n t m o d e l for the s t u d y o f d e g r a d a t i o n p r o c e s s e s a n d cell d e a t h in p h y s i o l o g i c a l c o n d i t i o n s . W e a n a l y s e d the s e q u e s t r a t i o n r a t e o f m i t o c h o n d r i a a n d p e r o x i s o m e s in t h e p h a g o l y s o s o m e s o f m o c o i d e p i t h e l i a l cells u s i n g c y t o c h e m i c a l m e t h o d s . T h e initial m o r p h o l o g i c a l a n d p h y s i o l o g i c a l e v e n t s o f a p o p t o s i s a r e n o t v e r y well u n d e r s t o o d ( W y l l i e et al., 1977). I n m u c o i d cells e x h i b i t i n g a p o p t o t i c d e a t h we a n a l y s e d c y t o c h e m i c a l l y t h e a c t i v i t y o f c y t o c h r o m e o x i d a s e a n d in this w a y t h e f u n c t i o n a l state o f m i t o c h o n d r i a w a s tested. W e w e r e also i n t e r e s t e d to see if a p o p t o t i c cells c o n t a i n p h a g o - a n d l y s o s o m e s a n d w h a t h a p p e n s t o t h e d e a d cells a f t e r t h e y are e x t r u d e d f r o m t h e m u c o i d e p i t h e l i u m .
Material and Methods The tissue from the stomach of 2-6 day old mice was taken for this investigation. For the analysis of peroxisomal catalase and lysosomal acid phosphatase, slices of tissue were fixed for 2 h in cold 2.5% glutaraldehyde with 4% paraformaldehyde in 0.2 M cacodylate buffer at pH 7.2-7.4 followed by overnight rinsing in 0.1 M cacodylate buffer with 0.3 M sucrose. For catalase, the tissue was incubated in the alkaline 3.Ydiaminobenzidine (DAB) medium, containing KCN described by Novikoff et al. (1972). The medium is prepared by dissolving 20 mg DABtetrahydrochloride in 9.3 ml 0.05 M propandiol buffer, pH 9.0; after adjusting the pH to 9.7 with 1 N NaOH, 0.5 ml 0.1 N KCN and 0.2 ml 2.4% H202 are added. The incubation time was 60 min at 37~ C. Following incubation, the tissue was rinsed several times in 7.5% sucrose. For control, the incubation medium without H202 was used. After rinsing, the tissue was treated with buffered 1% OsO4, dehydrated in ethanol and embedded in Epon. For cytochrome oxidase, the method of Seligman et al. (1968) was used. Small tissue slices were fixed for 60 min in 4% paraformaldehyde in 0.05 M sodiumphosphate buffer at pH 7.4. After fixation the tissue was washed for 15 min in 0.22 M sucrose 0.05 M phosphate buffer, at pH 7.4. The tissue was incubated for 60 rain at 37~ C in a medium containing 5 mg diaminobenzidine tetrahydrochloride, 9 ml 0.05 M Na-phosphate buffer, 1 ml catalase (20 mg/ml), 10 mg cytochrome C, 750 mg sucrose. The control material was incubated in 0.01 M KCN; following incubation the tissue was rinsed in 0.05 M phosphate buffer, pH 7.4 for 15 rain and postfixed in 1% buffered osmium tetroxide, dehydrated and embedded in Epon. For simultaneous analysis of cytochrome oxidase and catalase the tissue was fixed in the same way as for cytochrome oxidase, only using 0.05 M propandiol buffer instead of sodium phosphate buffer. The tissue was incubated for 90 min at 37~ C in the same medium as for catalase at pH 9.7 but KCN was omitted and 0.2 ml 2.5% H202 added. Following incubation, the tissue was rinsed in 0.05 M propandiol buffer at pH 9, posffixed in I% OsO4, dehydrated and embedded in Epon. For acid phosphatase the tissue was incubated in a medium (Barka and Anderson, 1962) containing 5 mI 1.2 Na-c~-glycerophosphate, pH 5, 5 ml 0.1 M tris-maleate buffer, pH 5, 5 ml water, 10 ml 0.2% Pb(NO3)2, and 7.5% sucrose. The incubation time was 35 rain at 37~ C. The control material was incubated without Na-c~-glycerophosphate. Following incubation the tissue was washed in twice distilled water, postfixed with 1% KMnO4, washed, dehydrated and embedded in Epon. Thin sections were prepared with an LKB Ultratome. The sections were examined after staining with lead citrate and uranyl acetate in a Siemens Elmiskop I at 80 kV or in a Jeol T8 microscope at 80 kV.
Results I n t h e s m a l l a n d large a u t o p h a g o s o m e s o f m u c o i d cells m a i n l y m i t o c h o n d r i a a n d E R a r e s e q u e s t e r e d . A l t h o u g h m u c o i d cells c o n t a i n n u m e r o u s p e r o x i s o m e s
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Fig. 1. Cytochrome oxidase reaction product in cristae of the mitochondrium (M1) in an autophagosome and in unsequestered mitochondria (M:) of a mocoid cell from a 6 day old mouse, x 35,000 Fig. 2. a Detail from Figure 2b showing mitochondria with reaction product in the intramembraneous space of the cristae (arrows). x 50,000. h A spherical apoptotic mucoid cell in which most mitochondria (M) contain reaction product of cytochrome oxidase. Nucleus (N) is pycnotic, plasmalema is well-preserved (arrow). Six day old mouse, x 11,000
Fig. 3. a Detail from Figure 3 b showing mitochondria with visible cristal membranes without reaction product of cytochrome oxidase (arrows). x 50000. b Spherical apoptotic mucoid cell of a 6 day old mouse in which most mitochondria are swollen and their cristae contain no reaction product of cytochrome oxidase, x 11,000 Fig. 4. Endocytosed apoptotic cell in the heterophagosome of a sister mucoid cell from a 6 day old mouse contains swollen mitochondria (M) without reaction product of cytochrome oxidase. Reaction product is in mitochondria (M1) of the surrounding celt. An ingested apoptotic body has well-preserved plasmalema, x 11,000
Fig. 5. A big phagosome in a mucoid cell of a 6 day old mouse in which catalase and cytochrome oxidase were analysed simultaneously. Peroxisomes (P) contain catalase, swollen mitochondria (M) are without reaction product of cytochrome oxidase, rest of a nucleus (N) ; normal peroxisomes (P1) and mitochondria (M1) with typical reaction product, x 15,000 Fig. 6. Apoptotic bodies in heterophagosomes of mucoid cells of a 6 day old mouse with reaction product of acid phosphatase in incorporated lysosome (L), Golgi vacuoles (GA) and secondary lysosomes (L~) which surround apoptotic bodies. Mitochondria (M) in heterophagosomes are partly swollen, x 14,000
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these organelles were found only exceptionally inside phagosomes. Autophagocytosed mitochondria with well-preserved morphology show normal activity of cytochrome oxidase and the reaction product fills the intramembraneous space between the outer and inner mitochondrial membrane (Fig. 1). Apoptotic cells which are extruded from the epithelium contain more or less well-preserved organelles but they show also the signs of lytic processes. Inspite of these observations phagolysosomes do not seem to contribute essentially to their death (Fig. 2a, b and 3a, b). The reaction product of cytochrome oxidase fills the intramenbraneous space of cristae (Fig. 2a) until the mitochondria become swollen (Fig. 3 b). Some or all apoptotic cells are later ingested by sister cells and the final degradation of organelles occurs inside lysosomes. In the apoptotic cells phagocytosed by sister cells their mitochondria exhibit different activity of cytochrome oxidase. In Figure 4a heterophagosome with one whole apoptotic cells can be seen but the reaction product can no longer be observed in lightly swollen mitochondria. Heterophagosomes contain different numbers of peroxisomes which can be identified by the presence of catalase. Catalase can be demonstrated in lysosomes in which the mitochondria have already been destroyed (Fig. 5). The degradation of phagocytosed material can probably be initiated in several ways. Figure 6 suggests two different possibilities; either the lysosomes are sequestered in the phagosomes, like any other cell organelle, or by the secondary lysosomes which encircle the phagosomes. A direct fusion of the phagosome and the secondary lysosome was not observed, but it can be presumed. In phagosomes containing either normal or swollen mitochondria, acid phosphatase could not be identified (Fig. 6).
Discussion
Although mucoid cells contain numerous peroxisomes (Pipan and P~eni~nik, 1976) in autophagosomes these organelles were found only exceptionally. We followed especially the sequestration rate of peroxisomes because Locke and Mc Mahon (1971), who studied selectivity in autophagic processes, found that autophagosomes of fatty body cells engulf first of all the peroxisomes and later the mitochondria and other cell components. On the other hand Deter's study of autophagocytosis in hepatocytes induced by glucagon showed that the frequency of peroxisomes was much lower than would be expected on the basis of their quantity in these cells (Deter, 1971). The selectivity rules obviously change from cell to cell and probably depend, even in the same type of cell, upon their functional state. The quantity of organelles cannot, in itself, be essential for the selective mechanism. Dysfunction of the organelle seems also not to be the primary signal for the sequestration in the autophagosome. Although in our investigation only one of the numerous mitochondrial enzymes was taken into consideration, well-preserved activity of cytochrome oxidase gives indirect information about the functional state of sequestered organelles. The reaction product of cytochrome oxidase in the mitochondria of spherical
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apoptotic cells ejected from the mucoid epithelium provides interesting additional information to the results of Wyllie et al. (1977), in which it was found that intracellular ATP is maintained until late in the process of apoptotic death. Both results indicate that apoptotic death does not initiate energy deficiency in the cell. Phago- and lysosomes also do not seem to be directly involved in apoptotic death although they are present in such cells. Contrary to our results, Hurle and Hinchliffe (1977) found that apoptotic death of mesenchymal cells follows the state in which large lysosomes appear. Apoptotic bodies can be endocytosed by sister cells (Kerr et al., 1972; Pipan, 1976; Wyllie et al., 1977). During our observations we came to the conclusion that sister cells endocytose only membrane-bounded cells or their membranebounded fragments but never free cell debris which is also found in the stomach lumina. For the explanation of this, the results of Pricer and Ashwell (1971), obtained on hepatocytes, seem to be relevant. They have reported that isolated hepatic plasmalema binds neuraminidase-treated glycoproteins more avidly that the intact ones. It is possible to conclude from these data that hepatocyte surfaces carry receptors with greater affinity for asialoglycoproteins. Changes in the membrane glycoproteins which play an important role in cell to cell contact must probably occur in apoptotic death because such cells lose their connection to neighbour cells and leave the epithelium as shperical bodies. The nature of membrane changes in apoptotic bodies is still unknown (Wyllie et al., 1977) but the presumption that apoptotic cells become asialated and so in some way induce endocytosis through the receptors of living cells seems very attractive, although it was not proven in our analysis. The deterioration of the engulfed material is possible because lysosomal enzymes reach heterophagosomes in different ways. The initial degradation of mitochondria probably goes on without lysosomal enzymes and is induced only be sequestration into phagosomes and by loss of normal metabolic conditions.
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Received February 24, 1978
