Cell Tissue Res. 196, 237-247 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
Identification of G6PDH-Active Sinusoidal Cells as Kupffer Cells in the Rat Liver* * * W. Hosemann, H.F. Teutsch and D. Sasse AnatomischesInstitut der UniversitfitFreiburg, LehrstuhlAnatomie III, Freiburg i.Br., Bundesrepublik Deutschland
Summary. The aim of this study was to identify the G6PDH-active sinusoidal cells in the rat liver described by Rieder et al. (1978). Because of their number and distribution in the liver parenchyma, endothelial cells and pit cells could be excluded. Fat-storing cells were specifically marked by vital staining with vitamin A and identified by fluorescence microscopy. Kupffer cells could be detected after vital staining with carmine. Both staining methods allowed a subsequent incubation for the demonstration of G 6 P D H activity in the same unfixed cryostat section. Whereas more than 80 % of the fluorescent particles were found outside the enzyme-positive cells, all G6PDH-active cells contained carmine particles. After counting the G6PDH-active cells, an estimation of 0.217 x 108 cells/g liver tissue was obtained. The results indicate that high G 6 P D H activity is c o m m o n to all Kupffer cells, and is therefore a highly specific marker enzyme for this class of sinusoidal liver cells. Key words: Liver - Kupffer cells - G 6 P D H activity - Histochemistry - Rat.
Recently Rieder et al. (1978) described liver sinusoidal cells in the rat which react intensely after incubation in an improved medium for the histochemical demonstration of glucose-6-phosphate-dehydrogenase (G6PDH) activity. The cells are found in both male and female rats but, because of the less pronounced and more evenly distributed parenchymal activity, are more readily identifiable in the male. An uneven pattern of distribution of these highly reactive cells was described; they are concentrated in the periportal region (zone 1) of the liver acinus, and their number decreases toward the region of the terminal hepatic venule (zone 3). Send offprint requests to: Prof. Dr. D. Sasse, Anatomisches Institut der Universit~it Freiburg/Br.,
Lehrstuhl Anatomie III, AlbertstraBe 17, D-7800 Freiburg/Br., Federal Republic of Germany * The essential parts of this study will be presented as an Inaugural-Dissertation to the Medical Faculty of the University of Freiburg by W. Hosemann ** Supported by a grant from the SFB 46 ("Molgrudent")
0302-766X/79/0196/0237/$02.20
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F o u r classes o f sinusoidal cells are possible candidates for the origin o f the high enzyme activity: 1. endothelial cells, 2. Kupffer cells, 3. fat-storing cells, and 4. pit cells. These different types o f sinusoidal lining cells can be distinguished by their functional characteristics and ultrastructure. As described by Wisse (1977), endothelial cells are evenly distributed in the liver p a r e n c h y m a ; they have a streamlined shape and bulge only slightly into the sinusoidal lumen. According to the m o r p h o m e t r i c determinations o f Blouin (1977), endothelial cells contribute 44.4 ~o to the volume o f non-hepatocytes. Kupffer cells are highly variable in shape, most o f their total surface area being exposed to the sinusoidal blood stream. Kupffer cells are characterized by their phagocytotic capacity for rather large particles (0.1 Ixm, Wisse, 1977; 0.8~tm, W i d m a n n et al., 1972; W i d m a n n and Fahimi, 1976). They represent 33.3 ~ o f the volume o f n o n p a r e n c h y m a l cells. Fatstoring cells (22.2 V o l ~ ) , which were first described by Ito and N e m o t o (1952), are situated in the space o f Disse. They are characterized by their intracytoplasmic fat droplets and are t h o u g h t to occupy fixed positions evenly distributed along the sinusoidal wall. The fourth type o f sinusoidal cell was described by Wisse et al. (1976) as the pit cell, the origin and function o f which remain hypothetical. They are considered to have no preferential location in the liver parenchyma, and contribute less than 5 ~o to n o n p a r e n c h y m a l cell suspensions (Sleyster et al., 1977). It was the aim o f the present study to determine the identitiy o f the highly G 6 P D H - a c t i v e sinusoidal cells. In preliminary assays it was found that other histochemical reactions c o m m o n l y used to characterize different types o f sinusoidal cells interfere with the demonstration o f G 6 P D H . A n attempt was therefore made to combine vital staining with the technique o f G 6 P D H demonstration. Furthermore, it was intended to find out whether the high G 6 P D H activity is a transient indication o f a particular function, or whether it should be regarded as a permanent characteristic o f one class o f sinusoidal cell in the rat liver.
Materials and Methods Twelve male and two female adult Wistar rats (200-300 g) were kept at constant room temperature (+22~ and a day/night rhythm (light period: 7 a.m. - 7 p.m.). All animals had free access to Altromin | and water. The animals were divided into four groups. (a) During the morning two animals were injected intravenously with 1 ml of a carmine solution (2.5g carmine and 1 g Na2CO 3 in 100ml water) according to Romeis (1968) w The animals were killed 90 min after the last injection. (b) Eight animals were each injected subcutaneously on four consecutive days with 0.5 ml vitamin A/oil solution (330000 IU/kg, Wake, 1974). 14 days after the last injection the animals were killed. (c) Three animals were injected subcutaneously with a vitamin A/oil solution (as in b.). After 14 days the carmine solution was injected twice (as in a.). (d) One control rat received no injection. All animals were killed by removing the liver under deep ether anesthesia. Small blocks of liver were frozen in isopentane cooled with liquid nitrogen. Blocks not immediately used were stored in airtight tubes at - 70~C. Cryostat sections, 8 Ixm thick, were thawed on coverslips and incubated either directly or after photography for fluorescence. The demonstration of G6PDH activity was carried out by the method described by Rieder et al. (1978). The sections were incubated for 15min at + 37~ in the following medium: 10 mM G6P; 0.8 mM NADP; 5 mM NBT; 5 mM MgC12;20 ~ PVA; 50 mM Tris-HCl-buffer, pH 7.4; 10 mM NAN3;0.325mM PMS. After incubation sections were washed in a 0.9 ~ NaCI solution (1 min; + 37~ fixed in a mixture of 4 ~ formaldehyde, 2 ~ CaC12 and 7.5 ~ PVP (20 min; + 0~ again washed in distilled water (2 • 5 min) and mounted in glycerine jelly.
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Fig. 1. Male rat. G6PDH-active nonparenchymal cells preferentially located in zone 1 of the liver acinus. • Photography and Identification of the Cells Vitamin A fluorescence was photographed in unstained cryostat sections with a Wild M20 microscope on a transmitted light base III equipped for automatic photomicrographic and polaroid photography (polaroid film type 107). After fluorescence photography and incubation for the demonstration of G6PDH activity, the same area was again photographed in bright field illumination (polaroid film type 665). Only those fluorescence photomicrographs were used in which the structures of the vessel walls allowed strict coordination with the second bright field photomicrograph. This coordination was carried out with a transparent foil on which the central points of the G6PDH-active cells were marked. Around these points idealized cells were described as circles with a radius equivalent to an absolute measurement of 4.6 ~tm.
Results A f t e r i n c u b a t i o n for the d e m o n s t r a t i o n o f G 6 P D H activity, s e x - d e p e n d e n t d i s t r i b u t i o n p a t t e r n s were f o u n d in the livers o f male a n d female rats. W h i l e in the livers o f females there is a high activity in the p e r i v e n o u s zone (zone 3) o f the liver acinus with a clear g r a d i e n t in the direction o f the p e r i p o r t a l area, in the male r a t liver a r a t h e r m o d e r a t e activity is seen, which seems to be fairly evenly d i s t r i b u t e d t h r o u g h o u t the entire p a r e n c h y m a . I n b o t h sexes highly reactive sinusoidal cells are d e m o n s t r a b l e , the n u m b e r o f which is clearly higher in the p e r i p o r t a l a r e a (c.f. R i e d e r et al., 1978). Since, owing to the less intensely staining p a r e n c h y m a l " b a c k g r o u n d " , the m a r k e d s i n u s o i d a l cells are m o r e easily o b s e r v e d in the liver o f male rats, further investigations were carried o u t o n livers o f male rats only (Fig. 1).
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Fig. 2a and b. Male rat, identical areas, a Vitamin A fluorescence,circles indicating the positions of G6PDH-active ceils, demonstrated in b. x 140
Calculation ofG6PDH-Active Sinusoidal Cells. For the calculation of the number of positively reacting sinusoidal cells, five microscope fields from sections of livers from the rats of group a were examined. With the use of a zoomlens an area of parenchyma was in each case chosen in such a way that opposite points o f the field included terminal afferent vessels. In this way, it was made certain that all three zones of the liver acinus had been taken into consideration. On the basis of an average number of 115 G6PDH-positive sinusoidal cells/area (800-980 lam in diameter), an average value of 0.217 • 108 cells/cm 3 liver volume was calculated. Carmine Storage and G6PDH Activity. In rats injected with carmine, Kupffer cells were marked by their bright red inclusions. After incubation for G6PDH activity the carmine-containing cells showed blue formazan particles. The majority of enzyme-positive cells exhibited large amounts of carmine, although some contained only a few particles. Sometimes extracellular carmine accumulations without formazan were demonstrable. Vitamin A Storage and G6PDH Activity. Under ultraviolet light unfixed cryostat sections of livers of animals treated with vitamin A revealed a rapidly fading yellowgreen fluorescence in the form of small circles. No zonal distribution pattern could be observed. Since peanut oil, used here as the solvent for vitamin A, does not autofluoresce, spurious fluorescence due to contaminants had to be strictly avoided. Because of the subsequent histochemical procedure, fluorescence microscopic examination had to be carried out directly on unfixed cryostat sections without any mounting medium and without coverslips. The photographs of specific fluorescence had to be taken within a few minutes because, in the course of drying, fluorescence of the parenchymal cell borders occurs which interferes with the fluorescence of the vitamin A droplets. The specific fluorescence was therefore immediately recorded on a highly sensitive film.
Kupffer Cells of the Rat Liver
241 100 %
N = 376
81,g %
Fig. 3. Column 1. Fields of G6PDH-active cells without fluorescent particles. Column 2. Fields of G6PDH-active cells in contact with fluorescent particles. Column 3. Fields of G6PDH-active cells containing fluorescent particles
13,8%
1
2
3
Calculation of the Number of Fluorescent Droplets. Five fields of view from different rats of group b were examined for calculation of the number of vitamin A fluorescent particles. F r o m these values an average number of 0.268 • 108/cm 3 vitamin A fluorescent particles was obtained. In 47 fluorescence photomicrographs of seven rats the central points of 376 G6PDH-active sinusoidal cells of the same area were inserted. Around these points circles with diameters equivalent to 9.2 Ixm were drawn, corresponding to the size of the enzyme-positive cells (Fig. 2a, b). 308 G6PDH-active cells marked in this manner showed no coincidence with fluorescent particles; 52 cells had contact with fluorescent particles, and, in 16 cells, fluorescent particles were found within the circle (Fig. 3). Discussion In the present study two different methods for vitally staining nonparenchymal liver cells were used: (i) carmine staining for the demonstration of Kupffer cells, and (ii) injection of vitamin A for the demonstration of fat-storing cells. The results indicate that in all G6PDH-active cells there was a marked accumulation of carmine, whereas in more than 80 ~o of the cells marked by vitamin A fluorescence no enzyme activity was demonstrable. It needs to be established whether these results, taken together with those already reported in the literature, allow a clear identification of the G6PDH-active sinusoidal cells. The problem of independence of the different sinusoidal cells has been the source of much discussion, it having been widely held that 'endothelial cells' and 'Kupffer cells' are not separate cell types but two terms for different
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functional states of the same cell (Riittner et al., 1956; Aterman, 1958, 1963; Popper and Schaffner, 1961; Nicolescou and Rouiller, 1967; Ito and Shibasaki, 1968; Fahimi, 1970). On the basis of recent results, especially those obtained with the electron microscope, the sinusoidal cells of the liver can be classified in the following way: 1) In the sinusoids of rat liver there are four different cell classes; they differ in morphology (Nakane, 1963; Wake, 1974; Fahimi et al., 1976; Widmann and Fahimi, 1976; Blouin, 1977; Blouin et al., 1977; Satodate et al., 1977; further references in Wisse, 1977), in enzyme content (Knook and Sleyster, 1976b), in their capacity for endocytosis (Bronfenmajer et al., 1966; Satodate et al., 1977; Wisse, 1972, 1974b, 1977), and in their different responses to experimental conditions (Satsuki in Ito and Nemoto, 1952; Wake, 1974; Wisse, 1974a, b, 1977; Emeis and Planqu~, 1976; Kent et al., 1977a, b; Widmann and Fahimi, 1976; Muto and Fujita, 1977; Sleyster et al., 1977). 2) Under normal and experimental conditions, transitional cell forms have never been detected, either between endothelial and Kupffer cells (Wisse, 1970, 1972, 1974a, b, 1977; Widmann et al., 1972; Ogawa et al., 1973; Widmann and Fahimi, 1976; Motta, 1977; Naito and Wisse, 1977), or between blood cells, monocytes and Kupffer cells (Wisse, 1974a, b; Widmann and Fahimi, 1975, 1976; Naito and Wisse, 1977). 3) Mitoses are found in all cell classes; there are therefore four independent, selfproliferating types of cell in the liver sinusoids (Widmann et al., 1972; Ogawa et al., 1973; Wisse, 1974a, b, 1977; Widmann and Fahimi, 1975, 1976; Kent et al., 1976, 1977a, b; Wisse et al., 1976; Naito and Wisse, 1977). Each of these cell types may possibly be the site of high G6PDH activity. Endothelial Cells. A specific marker for the demonstration of endothelial cells has
not yet been found (Emeis and Planqu6, 1976). Electron microscopically they are seen as cells of streamlined shape, with fiat processes which show typical fenestrations (Wisse, 1972). While, according to Widman et al. (1972), endothelial cells contribute 48 ~ to the volume of nonhepatocytes, determinations made in nonparenchymal cell suspensions have led to even higher values of 50-70 ~ (Emeis and Planqu6, 1976; Knook and Sleyster, 1976a; Knook et al., 1977; Sleyster et al., 1977). Endothelial cells are capable of endocytosis, but this ability shows a fundamental difference from the behavior of the Kupffer cell: the endothelial cells are only able to endocytose small particles up to a diameter of 1000A (Widmann and Fahimi, 1975, 1976; Wisse, 1977) and have, in comparison with Kupffer cells, a much lower capacity for endocytosis (Widmann et al., 1972; Wisse, 1972, 1974b; Ogawa et al., 1973; Widmann and Fahimi, 1976; Knook et al., 1977). Since carmine particles have a diameter of only 20.5A (Lison and Smulders, 1948), theoretically they can be taken up by endothelial cells. In the light microscope, however, only large quantities of intracellularly accumulated carmine are visible, a fact which follows from the high storage capacity of Kupffer cells. The following observations serve as arguments against the possibility that endothelial cells are the sites of high nonparenchymal G6PDH activity. Endothelial cells are evenly distributed in the liver acinus, whereas G6PDH-active cells are mainly located in the periportal area. Endothelial ceils are fiat, but G6PDH-active cells
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bulge into the sinusoidal lumen. Endothelial cells, as viewed with the light microscope, are not stained with carmine, while G6PDH-active cells always contain a marked carmine accumulation. Pit Cells comprise a new sinusoidal cell class, knowledge of the morphology and especially the function of which has until now remained fragmentary (Wisse et al., 1976; Wisse, 1977). The pit cells can be excluded as sites of high G6PDH activity because 1) they are considered to be evenly distributed in the liver parenchyma, 2) they do not endocytose and are therefore not stainable by carmine, as are the G6PDHactive cells, and 3) they are too few in number (less than 0.027 x 108 cells/cm a liver volume, as calculated from the data of Knook and Sleyster, 1976b, and of Sleyster et al., 1977). The number of G6PDH-active cells is ten times higher. Fat-Storing Cells are situated exclusively in the space of Disse. Often these cells are compressed into recesses between hepatocytes (Ito and Nemoto, 1952; Ito and Shibasaki, 1968; Wake, 1971; Muto, 1975). They send characteristic cytoplasmic processes into the space of Disse (Ito and Shibasaki, 1968; Wisse, 1970, 1974a, 1977; Muto, 1975; Muto et al., 1977; Naito and Wisse, 1977). In normal rats fat-storing cells contain an average of 25.3 Vol ~ of fat droplets (compared to 0.37 Vol ~o in Kupffer cells) (Blouin et al., 1977). Depending on their location in the liver acinus and on the amount of fat stored, the perikarya bulge into the sinusoidal lumen (Ito and Nemoto, 1952; Ikejiri and Tanikawa, 1977). Fatstoring cells in the perivenous zone (zone 3) of the liver acinus often show very few fat droplets, so that they have been called "leere Fettspeicherzellen" (Ito and Nemoto, 1952). According to Wisse (1977), fat-storing cells are evenly distributed in the liver parenchyma. They represent 10 to 25 ~o of the nonparenchymal cells of the liver (calculated from data of Ito and Nemoto, 1952, Bronfenmajer et al., 1966; Weibel et al., 1969; Greengard et al., 1972; Widmann et al., 1972; Ogawa et al., 1973 and Munthe-Kaas et al., 1976). Fat-storing cells probably have several functions. They are known to be sites of fat metabolism, since fat synthesis and occurrence of fat droplets have been observed (Ito and Nemoto, 1952; Bronfenmajer et al., 1966; Ito and Shibasaki, 1968). Moreover, fat-storing cells may have a mechanical function insofar as they support the sinusoidal wall (Ito and Shibasaki, 1968; Wisse, 1970, 1977; Muto et al., 1977). There is even some evidence of their ability to produce fibrous tissue, since, in areas of damaged liver parenchyma, local accumulations of fat-storing cells that synthesize collagen are to be seen. Because transitional forms between fat-storing cells and fibroblasts were observed, fat-storing cells were interpreted as resting fibroblasts, which might be responsible for "intralobular" fibrogenesis (Mac Gee and Patrick, 1972; Kent et al., 1976, 1977a, b; Wisse, 1977). 90 ~ of all the vitamin A in the body is stored in the liver (Popper and Schaffner, 1961). Detailed studies were undertaken on the function of fat-storing cells as storage sites of vitamin A. After application of vitamin A the number of fat-storing cells increases, as well as the number of fat droplets per cell (Wake, 1971 ; Hirosawa and Yamada, 1973; Kobayashi et al., 1973; Kent et al., 1976; Ikejiri and Tanikawa, 1977; Kusumoto and Fujita, 1977). Wake (1971) suggested that vitamin A enters the fat-storing cells via the Kupffer cells, because blockage of the reticuloendothelial system influences the amount of vitamin A stored in the fatstoring cells. Furthermore, the vitamin A fat-storing cells can be stained with Sudan
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III, as well as with gold chloride (Kupffer, 1876, modified by Wake, 1971; Kusumoto and Fujita, 1977). These latter techniques, however, are not compatible with the histochemical demonstration of G6PDH activity. At concentrations of more than 1.5 lag/g liver, vitamin A gives rise to a yellowgreen fluorescence with an absorption maximum at 328 nm, vanishing in 10 to 20 seconds (Popper, 1944). Therefore vitamin A fluorescence must be recorded on highly sensitive films. A special problem arose in connection with the localization of G6PDH-active ceils and fluorescent fat-storing cells in the same area. It was assumed that the volume of a G6PDH-active cell would correspond to that of a Kupffer cell which, according to Greengard et al. (1972), has a volume of 290 txm3. If this volume were spherical, it would have a radius of 4.1 Ixm. Owing to technical difficulties, it was only possible to describe circles with a radius corresponding to 4.6 ~tm, i.e. a cell with a volume of 407 I.tm3. But even when cells of this volume were taken as the standard, coincidence of G6PDH activity and fluorescent droplets was seen in only 4.3 ~o of the cases. This might be due to minor errors in the coordination of the photographs, or it could be accounted for either by the presence of small amounts of vitamin A in the Kupffer cells (Linder et al., 1971; Wake, 1971; Hirosawa and Yamada, 1973), or by the fact that Kupffer cells and fat-storing cells lie on occasion in such close contact (Wake, 1971; Wisse, 1974a; Ikejiri and Tanikawa, 1977) that they appear to be one and the same cell. On the basis of our own results and the data obtained from the literature, fatstoring cells can be excluded as sites of high G6PDH activity, since enzyme-positive cells are marked by carmine and fat-storing cells do not incorporate this stain. Moreover, in most cases a clear separation of vitamin A-positive and G6PDHactive cells was quite obvious. Finally, fat-storing cells are evenly distributed and G6PDH-positive cells occur predominantly in the periportal area. Kupffer Cells are characterized electron microscopically by a perikaryon rich in cytoplasm, which bulges into the sinusoidal lumen, and by specialized surfaces (Wallraff, 1969; Wisse and Knook, 1977). Because of their high phagocytotic capacity, Kupffer cells can be specifically marked under experimental conditions. Histochemically Kupffer cells are characterized by a high endogenous peroxidase activity (Wisse, 1974a; Widmann and Fahimi, 1976). 2.1 ~o of the liver volume consists of Kupffer cells (Blouin et al., 1977). Single cells have an average volume of 290 ~tm3 (Greengard et al., 1972); after isolation they appear spherical with a diameter of 11 Ixm (Knook and Sleyster, 1976b). The self-proliferating Kupffer cells (North, 1969; Ogawa et al., 1973; Wisse, 1974b, 1977; Widmann and Fahimi, 1975, 1976) represent 30 to 40 ~o of the total number of sinusoidal cells (Popper and Schaffner, 1961 ; Widmann et al., 1972). After isolation of the nonparenchymal cells, 20 to 30 ~ are found to be Kupffer cells (Emeis and Planqu6, 1976; Knook and Sleyster, 1976a; Knook et al., 1977; Sleyster et al., 1977). This corresponds to an absolute value of 0.16 x 108-0.28 x 108 Kupffer cells/cm 3 liver tissue (Loud, 1968; Weibel et al., 1969; Greengard et al., 1972; Knook and Sleyster, 1976b; Munthe-Kaas et al., 1976; Knook et al., 1977). Kupffer cells are not evenly distributed in the liver parenchyma, but are situated predominantly in the periportal zone 1 of the liver acinus (Carr, 1973; Wisse, 1974a, 1977). After exclusion of endothelial cells, pit cells and fat-storing cells as sites of high G6PDH activity, the following arguments support the suggestion that Kupffer cells
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are the morphological sites of this enzyme activity. 1) The histological correspondence of the same zonal accumulation of Kupffer and G6PDH-active cells in the periportal area of the liver acinus. 2) The cytological correspondence of intracellular accumulation of carmine and formazan deposits after application of the histochemical techniques. The comparison between the calculated number of 0.217 • 108 G6PDH-active cells/cm 3 liver in this study and the value of 0.16-0.28 x 108 Kupffer cells given in the literature indicates that the histochemical demonstration of G6PDH activity does not merely identify a certain fraction of the Kupffer cells, but reveals a histochemical characteristic common to all these cells. A high activity of G6PDH in Kupffer cells was also reported without further detail by Schmidt et al. (1977). This enzymatic equipment may well be responsible for the ability of the Kupffer cell to effect phagocytosis. Since in polymorphonuclear leucocytes the NADPH generating pentose phosphate shunt is stimulated by phagocytosis (Rossi and Zatti, 1966; Selvaraj and Sbarra, 1966; Patriarca et al., 1971; van Berkel and Kruijt, 1977), similar behavior can be expected in Kupffer cells. Possibly G6PDH activity yields the substrate for the membrane-bound NADPH-oxidase which is supposed to play a role in the synthesis of H20 2 within the phagocytotic vacuoles (van Berkel and Kruijt, 1977; van Berkel and Koster, 1977). Acknowledgement. The authors are grateful to Dr. F. Steel for revising the English manuscript. References Aterman, K. : Some observations on the sinusoidal cells of the liver. Acta Anat. (Basel) 32, 193-213 (1958) Aterman, K.: Liver sinusoids and sinusoidal cells. In: The liver (Ch. Rouiller, ed.), Vol. 1, pp. 61-136 New York-London: Academic Press 1963 Berkel, T.J.C. van, K oster, J.F.: Biochemical characteristics of non-parenchymal liver cells. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), pp. 299-306. Elsevier: NorthHolland Biomedical Press 1977 Berkel, T_I.C. van, Kruijt, J.K.: Distribution and some properties on NADPH and NADH oxidase in parenchymal and non-parenchymal liver cells. Arch. Biochem. Biophys. 179, 8-14 (1977) Blouin, A.: Morphometry of liver sinusoidal cells. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), 61-71. Elsevier: North-Holland Biomedical Press 1977 Blouin, A., Bolender, R., Weibel, E.: Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma - a stereological study. J. Cell Biol. 72, 441-445 (1977) Bronfenmajer, S., Schaffner, F., Popper, H.: Fat storing cells (lipocytes) in human liver. Arch. Path. 82, 447453 (1966) Carr, I.: The macrophage a review of ultrastructure and function. London-New York: Academic Press 1973 Emeis, J.J., Planqur, B.: Heterogeneity of cells isolated from rat liver by pronase digestion: ultrastructure, cytochemistry and cell structure. J. Reticuloendothel. Soc. 20, 11-29 (1976) Fahimi, H.D.: The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver. J. Cell Biol. 47, 247-262 (1970) Fahimi, H.D., Gray, B.A., Herzog, V.K.: Cytochemical localization of catalase and peroxidase in sinusoidal cells of rat liver. Lab. Invest. 34, 192-201 (1976) Greengard, O., Federman, M., Knox, W.E.: Cytomorphometry of developing rat liver and its application to enzymic differentiation. J. Cell Biol. 52, 261-272 (1972) Hirosawa, K., Yamada, E.: The localization of the vitamin A in the mouse liver as revealed by electron microscope radioautography. J. Electron Microsc. 22, 337-346 (1973) Ikejiri, N., Tanikawa, K.: Effects of vitamin A and oestrogen on the sinusoidal cells in rat liver. In:
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Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), pp. 83-92. Elsevier: North-Holland Biomedical Press 1977 Ito, T., Nemoto, M.: Ober die Kupfferschen Sternzellen und die "Fettspeicherungszellen" ("fat storing cells") in der Blutkapillarenwand der menschlichen Leber. Okajimas Folia Anat. Jap. 24, 243-258 (1952) Ito, T., Shibasaki, S.: Electron microscopic study on the hepatic sinusoidal wall and the fat storing cells in the normal human liver. Arch. Histol. Jap. 29, 137-192 (1968) Kent, G., Gay, S., Inouye, T., Bahu, R., Minick, O.T., Popper, H.: Vitamin A - containing lipocytes and formation of type III collagen in liver injury. Proc. Natl. Acad. Sci. USA 73, 3719-3722 (1976) Kent, G., Inouye, T., Minick, O.T., Bahu, R.M.: Role of lipocytes (perisinusoidal cells) in fibrogenesis. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), pp. 73-82. Elsevier: North-Holland Biomedical Press 1977b Kent, G., Minick, T., Inouye, T.: Nature and role of vitamin A - storing cells. J. Cell Biol. 75, HM 936 (1977a) Knook, D.L., Blansjaar, N., Sleyster, E.CH.: Isolation and characterization of Kupffer and endothelial cells from the rat liver. Exp. Cell Res. 109, 317-329 (1977) Knook, D.L., Sleyster, E.CH.: Lysosomal enzyme activities in parenchymal and nonparenchymal liver cells isolated from young, adult and old rats. Mech. Aging Development 5, 389-397 (1976a) Knook, D.L., Sleyster, E.CH.: Separating of Kupffer and endothelial cells of the rat liver by centrifugal elutriation. Exp. Cell Res. 99, 444 449 (1976b) Kobayashi, K., Takahashi, T., Shibasaki, S.: Cytological studies of fat-storing cells in the liver of rats given large doses of vitamin A. Nature New Biol. 243, 186-188 (1973) Kupffer, C. von: Ueber Sternzellen in der Leber - briefliche Mittheilung an Prof. Waldeyer. Arch. Mikr. Anat. 12, 353-358 (1876) Kusumoto, Y., Fujita, T.: Vitamin A uptake cells distributed in the liver and other organs. Arch. Histol. Jap. 40, 121-136 (1977) Linder, M.C., Anderson, G.H., Ascarelli, I.: Quantitative distribution of vitamin A in Kupffer cell and hepatic population of rat liver. J. Biol. Chem. 2.46, 5538-5540 (1971) Lison, L., Smulders, J.: Discriminating'athrocytes' in the reticuloendothelial system. Nature 162, 65q56 (1948) Loud, A.V.: A quantitative stereological description of the ultrastructure of normal rat liver parenchymal cells. J. Cell Biol. 37, 27-46 (1968) MacGee, J. O'D., Patrick, R.S.: The role of perisinusoidal cells in hepatic fibrogenesis, an electron microscopic study of acute carbon tetrachloride liver injury. Lab. Invest. 26, 429~40 (1972) Motta, P.: Kupffer cells as revealed by scanning electron microscopy. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), pp. 93-102. Elsevier: North-Holland Biomedical Press 1977 Munthe-Kass, A.C., Berg, T., Seljelid, R.: Distribution of lysosomal enzymes in different types of rat liver cells. Exp. Cell Res. 99, 146-154 (1976) Muto, M.: A scanning electron microscopic study on endothelial cells and Kupffer cells in rat liver sinusoids. Arch. Histol. Jap. 37, 369-386 (1975) Muto, M., Fujita, T.: Phagocytic activities of the Kupffer cell: a scanning electron microscope study. In: Kupffer cells and other liver sinusoidal cells (E.Wisse and D.L. Knook, eds.), pp. 109-119. Elsevier: North-Holland Biomedical Press 1977 Muto, M., Nishi, M., Fujita, T.: Scanning electron microscopy of human liver sinusoids. Arch. Histol. Jap. 40, 137-151 (1977) Naito, M., Wisse, E.: Observations on the fine structure and cytochemistry of sinusoidal cells in fetal and neonatal rat liver. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), pp. 497-505. Elsevier: North-Holland Biomedical Press 1977 Nakane, P.K.: Ito's "fat storing cell" of the mouse liver. Anat. Rec. 145, 265-266 (1963) Nicolescu, P., Rouiller, CH.: Beziehungen zwischen den Endothelzellen der Lebersinusoide und den von Kupffer'schen Sternzellen. Z. Zellforsch. 76, 313-338 (1967) North, R.J.: The mitotic potential of fixed phagocytes in the liver as revealed during the development of cellular immunity. J. Exp. Med. 130, 315-326 (1969) Ogawa, K., Minase, T., Yokoyama, S., Ono6, T.: An ultrastructural study of peroxidatic and phagocytic activities of two types of sinusoidal lining cells in rat liver. Tohoku J. Exp. Med. 111, 253-269 (1973) Patriarca, P., Cramer, R., Moncalvo, S., Rossi, F., Romeo, D.: Enzymatic basis of metabolic stimulation.
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in leukocytes during phagocytosis: the role of activated NADPH oxidase. Arch. Biochem. Biophys. 145, 255-262 (1971) Popper, H.: Distribution of vitamin A in tissue as visualized by fluorescence microscopy. Physiol. Rev. 24, 205-224 (1944) Popper, H., Schaffner, F.: Die Leber, Struktur und Funktion. Stuttgart: Georg Thieme 1961 Rieder, H., Teutsch, H.F., Sasse, D.: NADP-dependent dehydrogenases in rat liver parenchyma, methodological studies on the qualitative histochemistry of G6PDH, 6PGDH, malic enzyme and ICDH. Histochemistry 56, 283-298 (1978) Romeis, B.: Mikroskopische Technik. Mfinchen-Wien: Oldenbourg Verlag 1968 Rossi, F., Zatti, M.: Effect of phagocytosis on the carbohydrate metabolism of polymorphonuclear leukocytes. Biochim. Biophys. Acta 121, 110-119 (1966) Rfittner, J.R., Brunner, H.E., Vogel, A.P.: Untersuchungen fiber die Kupfferschen Zellen der Rattenleber. Schweiz. Z. Path. Bakt. 19, 738-747 (1956) Satodate, R., Sasou, S., Oikawa, K., Hatakeyama, N., Katsura, S.: Scanning electron microscopical studies on the Kupffer cell in phagocytic activity. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), pp. 121-129. Elsevier: North Holland Biomedical Press 1977 Schmidt, E., Schmidt, F.W., Vonnahme, FJ.: Aspekte von Struktur und Funktion der Leber. Muzed 1, 150-162 (1977) Selvaraj, R.J., Sbarra, A.J.: Relationship of glycolytic and oxidative metabolism to particle entry and destruction in phagocytosing cells. Nature 211, 1272-1276 (1966) Sleyster, E.CH., Westerhuis, F.G., Knook, D.L.: The purification of nonparenchymal liver cell classes by centrifugal elutriation. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), pp. 289-298. Elsevier: North-Holland Biomedical Press 1977 Wake, K.: "Sternzellen" in the liver: perisinusoidal cells with special reference to storage of vitamin A. Am. J. Anat. 132, 429462 (1971) Wake, K.: Development of vitamin A-rich lipid droplets in multivesicular bodies of rat liver stellate cells. J. Cell Biol. 63, 683-691 (1974) Wallraff, J.: Verdauungsapparat, Atmungsapparat: Disse'scher Raum, Lebersinusoide, von Kupffersche Sternzelle. In: Handbuch der mikroskopischen Anatomic des Menschen (J. Wallraff, ed.), Bd. V, 4. Teil, S. 178-207. Berlin-Heidelberg-New York: Springer 1969 Weibel, E.R., Str~iubli, W., Gn/igi, H.R., Hess, F.A. : Correlated morphometric and biochemical studies on the liver cell; morphometric model, stereologic methods and normal morphometric data for rat liver cells. J. Cell Biol. 42, 68-91 (1969) Widmann, J.J., Cotran, R.S., Fahimi, H.D.: Mononuclear phagocytes (Kupffer cells) and endothelial cells - identification of two functional cell types in rat liver sinusoids by endogenous peroxidase activity. J. Cell Biol. 52, 159-170 (1972) Widmann, J.J., Fahimi, H.D.: Proliferation of mononuclear phagocytes (Kupffer cells) and endothelial cells in regenerating rat liver. Am. J. Pathol. 811, 349-366 (1975) Widmann, J.J., Fahimi, H.D.: Proliferation of endothelial cells in estrogen-stimulated rat liver - a light and electron microscopic cytochemical study. Lab. Invest. 34, 141-149 (1976) Wisse, E.: An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids. J. Ultrastruct. Res. 31, 125-150 (1970) Wisse, E.: An ultrastructural characterization of the endothelial cell in the rat liver sinusoid under normal and various experimental conditions, as a contribution to the distinction between endothelial and Kupffer cells. J. Ultrastruct. Res. 38, 528-562 (1972) Wisse, E.: Observations on the fine structure and peroxidase cytochemistry of normal rat liver Kupffer cells. J. Ultrastruct. Res. 46, 393426 (1974a) Wisse, E.: Kupffer cell reactions in rat liver under various conditions as observed in the electron microscope. J. Ultrastruct. Res. 46, 499-520 (1974b) Wisse, E.: Ultrastructure and function of Kupffer cells and other sinusoidal cells in the liver. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), pp. 33-60. Elsevier: NorthHolland Biomedical Press 1977 Wisse, E., Knook, D.L. (eds.): Kupffer cells and other liver sinusoidal cells. Elsevier: North-Holland Biomedical Press 1977 Wisse, E., Noordende, J.M. van't, Meulen, J. van der, Daems, W.T.H.: The pit cell: description of a new type of cell occurring in rat liver sinusoids and peripheral blood. Cell Tissue Res. 173, 423435 (1976) Accepted November 5, 1978
Cell and Tissue Research 9 by Springer-Verlag 1979
Identification of G6PDH-Active Sinusoidal Cells as Kupffer Cells in the Rat Liver* * * W. Hosemann, H.F. Teutsch and D. Sasse AnatomischesInstitut der UniversitfitFreiburg, LehrstuhlAnatomie III, Freiburg i.Br., Bundesrepublik Deutschland
Summary. The aim of this study was to identify the G6PDH-active sinusoidal cells in the rat liver described by Rieder et al. (1978). Because of their number and distribution in the liver parenchyma, endothelial cells and pit cells could be excluded. Fat-storing cells were specifically marked by vital staining with vitamin A and identified by fluorescence microscopy. Kupffer cells could be detected after vital staining with carmine. Both staining methods allowed a subsequent incubation for the demonstration of G 6 P D H activity in the same unfixed cryostat section. Whereas more than 80 % of the fluorescent particles were found outside the enzyme-positive cells, all G6PDH-active cells contained carmine particles. After counting the G6PDH-active cells, an estimation of 0.217 x 108 cells/g liver tissue was obtained. The results indicate that high G 6 P D H activity is c o m m o n to all Kupffer cells, and is therefore a highly specific marker enzyme for this class of sinusoidal liver cells. Key words: Liver - Kupffer cells - G 6 P D H activity - Histochemistry - Rat.
Recently Rieder et al. (1978) described liver sinusoidal cells in the rat which react intensely after incubation in an improved medium for the histochemical demonstration of glucose-6-phosphate-dehydrogenase (G6PDH) activity. The cells are found in both male and female rats but, because of the less pronounced and more evenly distributed parenchymal activity, are more readily identifiable in the male. An uneven pattern of distribution of these highly reactive cells was described; they are concentrated in the periportal region (zone 1) of the liver acinus, and their number decreases toward the region of the terminal hepatic venule (zone 3). Send offprint requests to: Prof. Dr. D. Sasse, Anatomisches Institut der Universit~it Freiburg/Br.,
Lehrstuhl Anatomie III, AlbertstraBe 17, D-7800 Freiburg/Br., Federal Republic of Germany * The essential parts of this study will be presented as an Inaugural-Dissertation to the Medical Faculty of the University of Freiburg by W. Hosemann ** Supported by a grant from the SFB 46 ("Molgrudent")
0302-766X/79/0196/0237/$02.20
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F o u r classes o f sinusoidal cells are possible candidates for the origin o f the high enzyme activity: 1. endothelial cells, 2. Kupffer cells, 3. fat-storing cells, and 4. pit cells. These different types o f sinusoidal lining cells can be distinguished by their functional characteristics and ultrastructure. As described by Wisse (1977), endothelial cells are evenly distributed in the liver p a r e n c h y m a ; they have a streamlined shape and bulge only slightly into the sinusoidal lumen. According to the m o r p h o m e t r i c determinations o f Blouin (1977), endothelial cells contribute 44.4 ~o to the volume o f non-hepatocytes. Kupffer cells are highly variable in shape, most o f their total surface area being exposed to the sinusoidal blood stream. Kupffer cells are characterized by their phagocytotic capacity for rather large particles (0.1 Ixm, Wisse, 1977; 0.8~tm, W i d m a n n et al., 1972; W i d m a n n and Fahimi, 1976). They represent 33.3 ~ o f the volume o f n o n p a r e n c h y m a l cells. Fatstoring cells (22.2 V o l ~ ) , which were first described by Ito and N e m o t o (1952), are situated in the space o f Disse. They are characterized by their intracytoplasmic fat droplets and are t h o u g h t to occupy fixed positions evenly distributed along the sinusoidal wall. The fourth type o f sinusoidal cell was described by Wisse et al. (1976) as the pit cell, the origin and function o f which remain hypothetical. They are considered to have no preferential location in the liver parenchyma, and contribute less than 5 ~o to n o n p a r e n c h y m a l cell suspensions (Sleyster et al., 1977). It was the aim o f the present study to determine the identitiy o f the highly G 6 P D H - a c t i v e sinusoidal cells. In preliminary assays it was found that other histochemical reactions c o m m o n l y used to characterize different types o f sinusoidal cells interfere with the demonstration o f G 6 P D H . A n attempt was therefore made to combine vital staining with the technique o f G 6 P D H demonstration. Furthermore, it was intended to find out whether the high G 6 P D H activity is a transient indication o f a particular function, or whether it should be regarded as a permanent characteristic o f one class o f sinusoidal cell in the rat liver.
Materials and Methods Twelve male and two female adult Wistar rats (200-300 g) were kept at constant room temperature (+22~ and a day/night rhythm (light period: 7 a.m. - 7 p.m.). All animals had free access to Altromin | and water. The animals were divided into four groups. (a) During the morning two animals were injected intravenously with 1 ml of a carmine solution (2.5g carmine and 1 g Na2CO 3 in 100ml water) according to Romeis (1968) w The animals were killed 90 min after the last injection. (b) Eight animals were each injected subcutaneously on four consecutive days with 0.5 ml vitamin A/oil solution (330000 IU/kg, Wake, 1974). 14 days after the last injection the animals were killed. (c) Three animals were injected subcutaneously with a vitamin A/oil solution (as in b.). After 14 days the carmine solution was injected twice (as in a.). (d) One control rat received no injection. All animals were killed by removing the liver under deep ether anesthesia. Small blocks of liver were frozen in isopentane cooled with liquid nitrogen. Blocks not immediately used were stored in airtight tubes at - 70~C. Cryostat sections, 8 Ixm thick, were thawed on coverslips and incubated either directly or after photography for fluorescence. The demonstration of G6PDH activity was carried out by the method described by Rieder et al. (1978). The sections were incubated for 15min at + 37~ in the following medium: 10 mM G6P; 0.8 mM NADP; 5 mM NBT; 5 mM MgC12;20 ~ PVA; 50 mM Tris-HCl-buffer, pH 7.4; 10 mM NAN3;0.325mM PMS. After incubation sections were washed in a 0.9 ~ NaCI solution (1 min; + 37~ fixed in a mixture of 4 ~ formaldehyde, 2 ~ CaC12 and 7.5 ~ PVP (20 min; + 0~ again washed in distilled water (2 • 5 min) and mounted in glycerine jelly.
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Fig. 1. Male rat. G6PDH-active nonparenchymal cells preferentially located in zone 1 of the liver acinus. • Photography and Identification of the Cells Vitamin A fluorescence was photographed in unstained cryostat sections with a Wild M20 microscope on a transmitted light base III equipped for automatic photomicrographic and polaroid photography (polaroid film type 107). After fluorescence photography and incubation for the demonstration of G6PDH activity, the same area was again photographed in bright field illumination (polaroid film type 665). Only those fluorescence photomicrographs were used in which the structures of the vessel walls allowed strict coordination with the second bright field photomicrograph. This coordination was carried out with a transparent foil on which the central points of the G6PDH-active cells were marked. Around these points idealized cells were described as circles with a radius equivalent to an absolute measurement of 4.6 ~tm.
Results A f t e r i n c u b a t i o n for the d e m o n s t r a t i o n o f G 6 P D H activity, s e x - d e p e n d e n t d i s t r i b u t i o n p a t t e r n s were f o u n d in the livers o f male a n d female rats. W h i l e in the livers o f females there is a high activity in the p e r i v e n o u s zone (zone 3) o f the liver acinus with a clear g r a d i e n t in the direction o f the p e r i p o r t a l area, in the male r a t liver a r a t h e r m o d e r a t e activity is seen, which seems to be fairly evenly d i s t r i b u t e d t h r o u g h o u t the entire p a r e n c h y m a . I n b o t h sexes highly reactive sinusoidal cells are d e m o n s t r a b l e , the n u m b e r o f which is clearly higher in the p e r i p o r t a l a r e a (c.f. R i e d e r et al., 1978). Since, owing to the less intensely staining p a r e n c h y m a l " b a c k g r o u n d " , the m a r k e d s i n u s o i d a l cells are m o r e easily o b s e r v e d in the liver o f male rats, further investigations were carried o u t o n livers o f male rats only (Fig. 1).
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Fig. 2a and b. Male rat, identical areas, a Vitamin A fluorescence,circles indicating the positions of G6PDH-active ceils, demonstrated in b. x 140
Calculation ofG6PDH-Active Sinusoidal Cells. For the calculation of the number of positively reacting sinusoidal cells, five microscope fields from sections of livers from the rats of group a were examined. With the use of a zoomlens an area of parenchyma was in each case chosen in such a way that opposite points o f the field included terminal afferent vessels. In this way, it was made certain that all three zones of the liver acinus had been taken into consideration. On the basis of an average number of 115 G6PDH-positive sinusoidal cells/area (800-980 lam in diameter), an average value of 0.217 • 108 cells/cm 3 liver volume was calculated. Carmine Storage and G6PDH Activity. In rats injected with carmine, Kupffer cells were marked by their bright red inclusions. After incubation for G6PDH activity the carmine-containing cells showed blue formazan particles. The majority of enzyme-positive cells exhibited large amounts of carmine, although some contained only a few particles. Sometimes extracellular carmine accumulations without formazan were demonstrable. Vitamin A Storage and G6PDH Activity. Under ultraviolet light unfixed cryostat sections of livers of animals treated with vitamin A revealed a rapidly fading yellowgreen fluorescence in the form of small circles. No zonal distribution pattern could be observed. Since peanut oil, used here as the solvent for vitamin A, does not autofluoresce, spurious fluorescence due to contaminants had to be strictly avoided. Because of the subsequent histochemical procedure, fluorescence microscopic examination had to be carried out directly on unfixed cryostat sections without any mounting medium and without coverslips. The photographs of specific fluorescence had to be taken within a few minutes because, in the course of drying, fluorescence of the parenchymal cell borders occurs which interferes with the fluorescence of the vitamin A droplets. The specific fluorescence was therefore immediately recorded on a highly sensitive film.
Kupffer Cells of the Rat Liver
241 100 %
N = 376
81,g %
Fig. 3. Column 1. Fields of G6PDH-active cells without fluorescent particles. Column 2. Fields of G6PDH-active cells in contact with fluorescent particles. Column 3. Fields of G6PDH-active cells containing fluorescent particles
13,8%
1
2
3
Calculation of the Number of Fluorescent Droplets. Five fields of view from different rats of group b were examined for calculation of the number of vitamin A fluorescent particles. F r o m these values an average number of 0.268 • 108/cm 3 vitamin A fluorescent particles was obtained. In 47 fluorescence photomicrographs of seven rats the central points of 376 G6PDH-active sinusoidal cells of the same area were inserted. Around these points circles with diameters equivalent to 9.2 Ixm were drawn, corresponding to the size of the enzyme-positive cells (Fig. 2a, b). 308 G6PDH-active cells marked in this manner showed no coincidence with fluorescent particles; 52 cells had contact with fluorescent particles, and, in 16 cells, fluorescent particles were found within the circle (Fig. 3). Discussion In the present study two different methods for vitally staining nonparenchymal liver cells were used: (i) carmine staining for the demonstration of Kupffer cells, and (ii) injection of vitamin A for the demonstration of fat-storing cells. The results indicate that in all G6PDH-active cells there was a marked accumulation of carmine, whereas in more than 80 ~o of the cells marked by vitamin A fluorescence no enzyme activity was demonstrable. It needs to be established whether these results, taken together with those already reported in the literature, allow a clear identification of the G6PDH-active sinusoidal cells. The problem of independence of the different sinusoidal cells has been the source of much discussion, it having been widely held that 'endothelial cells' and 'Kupffer cells' are not separate cell types but two terms for different
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functional states of the same cell (Riittner et al., 1956; Aterman, 1958, 1963; Popper and Schaffner, 1961; Nicolescou and Rouiller, 1967; Ito and Shibasaki, 1968; Fahimi, 1970). On the basis of recent results, especially those obtained with the electron microscope, the sinusoidal cells of the liver can be classified in the following way: 1) In the sinusoids of rat liver there are four different cell classes; they differ in morphology (Nakane, 1963; Wake, 1974; Fahimi et al., 1976; Widmann and Fahimi, 1976; Blouin, 1977; Blouin et al., 1977; Satodate et al., 1977; further references in Wisse, 1977), in enzyme content (Knook and Sleyster, 1976b), in their capacity for endocytosis (Bronfenmajer et al., 1966; Satodate et al., 1977; Wisse, 1972, 1974b, 1977), and in their different responses to experimental conditions (Satsuki in Ito and Nemoto, 1952; Wake, 1974; Wisse, 1974a, b, 1977; Emeis and Planqu~, 1976; Kent et al., 1977a, b; Widmann and Fahimi, 1976; Muto and Fujita, 1977; Sleyster et al., 1977). 2) Under normal and experimental conditions, transitional cell forms have never been detected, either between endothelial and Kupffer cells (Wisse, 1970, 1972, 1974a, b, 1977; Widmann et al., 1972; Ogawa et al., 1973; Widmann and Fahimi, 1976; Motta, 1977; Naito and Wisse, 1977), or between blood cells, monocytes and Kupffer cells (Wisse, 1974a, b; Widmann and Fahimi, 1975, 1976; Naito and Wisse, 1977). 3) Mitoses are found in all cell classes; there are therefore four independent, selfproliferating types of cell in the liver sinusoids (Widmann et al., 1972; Ogawa et al., 1973; Wisse, 1974a, b, 1977; Widmann and Fahimi, 1975, 1976; Kent et al., 1976, 1977a, b; Wisse et al., 1976; Naito and Wisse, 1977). Each of these cell types may possibly be the site of high G6PDH activity. Endothelial Cells. A specific marker for the demonstration of endothelial cells has
not yet been found (Emeis and Planqu6, 1976). Electron microscopically they are seen as cells of streamlined shape, with fiat processes which show typical fenestrations (Wisse, 1972). While, according to Widman et al. (1972), endothelial cells contribute 48 ~ to the volume of nonhepatocytes, determinations made in nonparenchymal cell suspensions have led to even higher values of 50-70 ~ (Emeis and Planqu6, 1976; Knook and Sleyster, 1976a; Knook et al., 1977; Sleyster et al., 1977). Endothelial cells are capable of endocytosis, but this ability shows a fundamental difference from the behavior of the Kupffer cell: the endothelial cells are only able to endocytose small particles up to a diameter of 1000A (Widmann and Fahimi, 1975, 1976; Wisse, 1977) and have, in comparison with Kupffer cells, a much lower capacity for endocytosis (Widmann et al., 1972; Wisse, 1972, 1974b; Ogawa et al., 1973; Widmann and Fahimi, 1976; Knook et al., 1977). Since carmine particles have a diameter of only 20.5A (Lison and Smulders, 1948), theoretically they can be taken up by endothelial cells. In the light microscope, however, only large quantities of intracellularly accumulated carmine are visible, a fact which follows from the high storage capacity of Kupffer cells. The following observations serve as arguments against the possibility that endothelial cells are the sites of high nonparenchymal G6PDH activity. Endothelial cells are evenly distributed in the liver acinus, whereas G6PDH-active cells are mainly located in the periportal area. Endothelial ceils are fiat, but G6PDH-active cells
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bulge into the sinusoidal lumen. Endothelial cells, as viewed with the light microscope, are not stained with carmine, while G6PDH-active cells always contain a marked carmine accumulation. Pit Cells comprise a new sinusoidal cell class, knowledge of the morphology and especially the function of which has until now remained fragmentary (Wisse et al., 1976; Wisse, 1977). The pit cells can be excluded as sites of high G6PDH activity because 1) they are considered to be evenly distributed in the liver parenchyma, 2) they do not endocytose and are therefore not stainable by carmine, as are the G6PDHactive cells, and 3) they are too few in number (less than 0.027 x 108 cells/cm a liver volume, as calculated from the data of Knook and Sleyster, 1976b, and of Sleyster et al., 1977). The number of G6PDH-active cells is ten times higher. Fat-Storing Cells are situated exclusively in the space of Disse. Often these cells are compressed into recesses between hepatocytes (Ito and Nemoto, 1952; Ito and Shibasaki, 1968; Wake, 1971; Muto, 1975). They send characteristic cytoplasmic processes into the space of Disse (Ito and Shibasaki, 1968; Wisse, 1970, 1974a, 1977; Muto, 1975; Muto et al., 1977; Naito and Wisse, 1977). In normal rats fat-storing cells contain an average of 25.3 Vol ~ of fat droplets (compared to 0.37 Vol ~o in Kupffer cells) (Blouin et al., 1977). Depending on their location in the liver acinus and on the amount of fat stored, the perikarya bulge into the sinusoidal lumen (Ito and Nemoto, 1952; Ikejiri and Tanikawa, 1977). Fatstoring cells in the perivenous zone (zone 3) of the liver acinus often show very few fat droplets, so that they have been called "leere Fettspeicherzellen" (Ito and Nemoto, 1952). According to Wisse (1977), fat-storing cells are evenly distributed in the liver parenchyma. They represent 10 to 25 ~o of the nonparenchymal cells of the liver (calculated from data of Ito and Nemoto, 1952, Bronfenmajer et al., 1966; Weibel et al., 1969; Greengard et al., 1972; Widmann et al., 1972; Ogawa et al., 1973 and Munthe-Kaas et al., 1976). Fat-storing cells probably have several functions. They are known to be sites of fat metabolism, since fat synthesis and occurrence of fat droplets have been observed (Ito and Nemoto, 1952; Bronfenmajer et al., 1966; Ito and Shibasaki, 1968). Moreover, fat-storing cells may have a mechanical function insofar as they support the sinusoidal wall (Ito and Shibasaki, 1968; Wisse, 1970, 1977; Muto et al., 1977). There is even some evidence of their ability to produce fibrous tissue, since, in areas of damaged liver parenchyma, local accumulations of fat-storing cells that synthesize collagen are to be seen. Because transitional forms between fat-storing cells and fibroblasts were observed, fat-storing cells were interpreted as resting fibroblasts, which might be responsible for "intralobular" fibrogenesis (Mac Gee and Patrick, 1972; Kent et al., 1976, 1977a, b; Wisse, 1977). 90 ~ of all the vitamin A in the body is stored in the liver (Popper and Schaffner, 1961). Detailed studies were undertaken on the function of fat-storing cells as storage sites of vitamin A. After application of vitamin A the number of fat-storing cells increases, as well as the number of fat droplets per cell (Wake, 1971 ; Hirosawa and Yamada, 1973; Kobayashi et al., 1973; Kent et al., 1976; Ikejiri and Tanikawa, 1977; Kusumoto and Fujita, 1977). Wake (1971) suggested that vitamin A enters the fat-storing cells via the Kupffer cells, because blockage of the reticuloendothelial system influences the amount of vitamin A stored in the fatstoring cells. Furthermore, the vitamin A fat-storing cells can be stained with Sudan
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III, as well as with gold chloride (Kupffer, 1876, modified by Wake, 1971; Kusumoto and Fujita, 1977). These latter techniques, however, are not compatible with the histochemical demonstration of G6PDH activity. At concentrations of more than 1.5 lag/g liver, vitamin A gives rise to a yellowgreen fluorescence with an absorption maximum at 328 nm, vanishing in 10 to 20 seconds (Popper, 1944). Therefore vitamin A fluorescence must be recorded on highly sensitive films. A special problem arose in connection with the localization of G6PDH-active ceils and fluorescent fat-storing cells in the same area. It was assumed that the volume of a G6PDH-active cell would correspond to that of a Kupffer cell which, according to Greengard et al. (1972), has a volume of 290 txm3. If this volume were spherical, it would have a radius of 4.1 Ixm. Owing to technical difficulties, it was only possible to describe circles with a radius corresponding to 4.6 ~tm, i.e. a cell with a volume of 407 I.tm3. But even when cells of this volume were taken as the standard, coincidence of G6PDH activity and fluorescent droplets was seen in only 4.3 ~o of the cases. This might be due to minor errors in the coordination of the photographs, or it could be accounted for either by the presence of small amounts of vitamin A in the Kupffer cells (Linder et al., 1971; Wake, 1971; Hirosawa and Yamada, 1973), or by the fact that Kupffer cells and fat-storing cells lie on occasion in such close contact (Wake, 1971; Wisse, 1974a; Ikejiri and Tanikawa, 1977) that they appear to be one and the same cell. On the basis of our own results and the data obtained from the literature, fatstoring cells can be excluded as sites of high G6PDH activity, since enzyme-positive cells are marked by carmine and fat-storing cells do not incorporate this stain. Moreover, in most cases a clear separation of vitamin A-positive and G6PDHactive cells was quite obvious. Finally, fat-storing cells are evenly distributed and G6PDH-positive cells occur predominantly in the periportal area. Kupffer Cells are characterized electron microscopically by a perikaryon rich in cytoplasm, which bulges into the sinusoidal lumen, and by specialized surfaces (Wallraff, 1969; Wisse and Knook, 1977). Because of their high phagocytotic capacity, Kupffer cells can be specifically marked under experimental conditions. Histochemically Kupffer cells are characterized by a high endogenous peroxidase activity (Wisse, 1974a; Widmann and Fahimi, 1976). 2.1 ~o of the liver volume consists of Kupffer cells (Blouin et al., 1977). Single cells have an average volume of 290 ~tm3 (Greengard et al., 1972); after isolation they appear spherical with a diameter of 11 Ixm (Knook and Sleyster, 1976b). The self-proliferating Kupffer cells (North, 1969; Ogawa et al., 1973; Wisse, 1974b, 1977; Widmann and Fahimi, 1975, 1976) represent 30 to 40 ~o of the total number of sinusoidal cells (Popper and Schaffner, 1961 ; Widmann et al., 1972). After isolation of the nonparenchymal cells, 20 to 30 ~ are found to be Kupffer cells (Emeis and Planqu6, 1976; Knook and Sleyster, 1976a; Knook et al., 1977; Sleyster et al., 1977). This corresponds to an absolute value of 0.16 x 108-0.28 x 108 Kupffer cells/cm 3 liver tissue (Loud, 1968; Weibel et al., 1969; Greengard et al., 1972; Knook and Sleyster, 1976b; Munthe-Kaas et al., 1976; Knook et al., 1977). Kupffer cells are not evenly distributed in the liver parenchyma, but are situated predominantly in the periportal zone 1 of the liver acinus (Carr, 1973; Wisse, 1974a, 1977). After exclusion of endothelial cells, pit cells and fat-storing cells as sites of high G6PDH activity, the following arguments support the suggestion that Kupffer cells
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are the morphological sites of this enzyme activity. 1) The histological correspondence of the same zonal accumulation of Kupffer and G6PDH-active cells in the periportal area of the liver acinus. 2) The cytological correspondence of intracellular accumulation of carmine and formazan deposits after application of the histochemical techniques. The comparison between the calculated number of 0.217 • 108 G6PDH-active cells/cm 3 liver in this study and the value of 0.16-0.28 x 108 Kupffer cells given in the literature indicates that the histochemical demonstration of G6PDH activity does not merely identify a certain fraction of the Kupffer cells, but reveals a histochemical characteristic common to all these cells. A high activity of G6PDH in Kupffer cells was also reported without further detail by Schmidt et al. (1977). This enzymatic equipment may well be responsible for the ability of the Kupffer cell to effect phagocytosis. Since in polymorphonuclear leucocytes the NADPH generating pentose phosphate shunt is stimulated by phagocytosis (Rossi and Zatti, 1966; Selvaraj and Sbarra, 1966; Patriarca et al., 1971; van Berkel and Kruijt, 1977), similar behavior can be expected in Kupffer cells. Possibly G6PDH activity yields the substrate for the membrane-bound NADPH-oxidase which is supposed to play a role in the synthesis of H20 2 within the phagocytotic vacuoles (van Berkel and Kruijt, 1977; van Berkel and Koster, 1977). Acknowledgement. The authors are grateful to Dr. F. Steel for revising the English manuscript. References Aterman, K. : Some observations on the sinusoidal cells of the liver. Acta Anat. (Basel) 32, 193-213 (1958) Aterman, K.: Liver sinusoids and sinusoidal cells. In: The liver (Ch. Rouiller, ed.), Vol. 1, pp. 61-136 New York-London: Academic Press 1963 Berkel, T.J.C. van, K oster, J.F.: Biochemical characteristics of non-parenchymal liver cells. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), pp. 299-306. Elsevier: NorthHolland Biomedical Press 1977 Berkel, T_I.C. van, Kruijt, J.K.: Distribution and some properties on NADPH and NADH oxidase in parenchymal and non-parenchymal liver cells. Arch. Biochem. Biophys. 179, 8-14 (1977) Blouin, A.: Morphometry of liver sinusoidal cells. In: Kupffer cells and other liver sinusoidal cells (E. Wisse and D.L. Knook, eds.), 61-71. Elsevier: North-Holland Biomedical Press 1977 Blouin, A., Bolender, R., Weibel, E.: Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma - a stereological study. J. Cell Biol. 72, 441-445 (1977) Bronfenmajer, S., Schaffner, F., Popper, H.: Fat storing cells (lipocytes) in human liver. Arch. Path. 82, 447453 (1966) Carr, I.: The macrophage a review of ultrastructure and function. London-New York: Academic Press 1973 Emeis, J.J., Planqur, B.: Heterogeneity of cells isolated from rat liver by pronase digestion: ultrastructure, cytochemistry and cell structure. J. Reticuloendothel. Soc. 20, 11-29 (1976) Fahimi, H.D.: The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver. J. Cell Biol. 47, 247-262 (1970) Fahimi, H.D., Gray, B.A., Herzog, V.K.: Cytochemical localization of catalase and peroxidase in sinusoidal cells of rat liver. Lab. Invest. 34, 192-201 (1976) Greengard, O., Federman, M., Knox, W.E.: Cytomorphometry of developing rat liver and its application to enzymic differentiation. J. Cell Biol. 52, 261-272 (1972) Hirosawa, K., Yamada, E.: The localization of the vitamin A in the mouse liver as revealed by electron microscope radioautography. J. Electron Microsc. 22, 337-346 (1973) Ikejiri, N., Tanikawa, K.: Effects of vitamin A and oestrogen on the sinusoidal cells in rat liver. In:
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