Bioscience Reports, Vof. 12, No. 6, 1992
The Simultaneous Exposition of Galactose and Mannose-Specific Receptors on Rat Liver Macrophages is Developmentally Regulated Alessandro Lentini, Laura Falasea, Franeesco Autuori, and Luciana Dini m Received June 28, 1992 We studied the simultaneous binding of galactose and mannose-exposing ligands in sinusoidal rat liver cells during development and aging. The galactose-specific receptors were visualized using 17 nm diameter colloidal gold particles coupled with Lactosylated bovine serum albumine (LacBSA), while mannose-specific receptors were localized by means of 5 nm diameter particles adsorbed with mannan. We observed the presence of four different classes of Kupffer cells in relation to the ligands bound. The percentage of each group of Kupffer cells varied in relation to the age of the subject from which the sample was taken. There were few double-labelled cells in the livers from newborn rats, with numbers increasing with age to adulthood, and decreasing again in the older animals. Cells without labelling were in the majority after birth, but they decreased in number up to adulthood and increased again during subsequent aging. The numbers of single-labelled cells did not change significantly during liver maturation. We hypothesize that the exposition of galactose and mannose-specific receptorial systems is regulated by developmental conditions. KEY WORDS: Galactose-specific receptor; mannose-specific receptor; Kupffer ceils; development;
aging.
INTRODUCTION In liver the hepatocytes are by far the most prominent class of cells, in terms of both size and total volume and relative number. In fact, the non parenchymal liver cells (i.e. Kupffer cells, endothelial cells, fat storing cells, Ito cells and Pit cells) represent only a small percentage of the entire liver mass. Increasing knowledge of their functions has, however, revealed their importance to liver physiology. In recent years, it has been amply demonstrated that the functions of hepatocytes and sinusoidal cells differ according to the different zones of the hepatic acinus [1, 2]. Kupffer cells do not constitute a homogeneous population of 1 Department of Biology, University of Rome "Tor Vergata" Via E. Carnevale 00173 Rome. z To whom correspondence should be addressed. 453 0144-8463/92/1200-0453506.50/0~) 1992 Plenum Publishing Corporation
Lentini, Falasca, Autuori and Dini
454
cells [3]. Sleyster and Knook [3] observed a correlation between functional heterogeneity, i.e. endocytic and lysosomal enzyme activity, and the localization of the cells in the liver acinus. Periportal Kupffer cells were larger and showed greater lysosomal enzyme activities than midzonal and perivenous Kupffer cells [4,5,6]. The only differences so far found in the hepatic endothelial cell population have, on the other hand, been morphological. Although these differences may be related to functional activities, molecular data confirming this hypothesis are still scarce [7, 8, 9]. One of the major functions of sinusoidal liver cells is the removal of macromolecules, bacteria and particles from the circulation. Not all the phagocytic activity of Kupffer and endothelial cells is nonspecific, since a large variety of receptors are expressed o n their cell surface [10, 11]. Among them, the galactose and mannose-specific receptors are the greatest in number [12, 13]. The galactosespecific receptors on Kupffer and endothelial cells recognize the penultimate non-reducing galactose residues of the oligosaccharide chains which are made free after desialylation. It is also well known that receptors with a similar binding specificity (known as asialoglycoprotein receptor, ASGP-R) are present on the surface of hepatocytes [14]. Circulating ASGP are internalized by hepatocytes by means of ASGP-R which is structurally and immunologically different from the galactose-specific receptors of Kupffer and endothelial cells [15]. Also mannoseterminated glycoproteins are removed from the blood by liver specific receptors. Sinusoidal cells in particular show a great capacity for endocytosing these ligands [13, 16]. The cytochemical binding and internalization pattern of mannose and galactose-exposing ligands on Kupffer cells visualized by means of protein-gold complexes is similar [12, 17]. In order to investigate whether there is a heterogeneity regarding to the carbohydrate-recognizing systems, we studied the ability of Kupffer cells to bind the mannose and galactose-exposing ligands simultaneously. Since Kupffer cells modulate the number of receptors on their surface in an age-dependent manner [17, 18, 19] (i.e. the number of receptors gradually increases during post natal life while during aging there is a considerable loss of receptor activity), we also investigated any influence of liver maturation (postnatal development and aging) on their binding capacity. MATERIALS AND METHODS Materials
Newborn (24 hours after birth), 6, 10, 15-day-old, adult (2-month-old) and old (24-month-old) male Wistar rats were used. Mannan from yeast and bovine serum albumine (BSA) were purchased from Sigma (USA); Lactosylated bovine serum albumine (LacBSA) from Soeckebort (Sweden); Eagle's medium from Flow (USA); All other chemicals were from Merck (Germany). Methods
Colloidal gold solutions with particles in 5 nm diameter (Au5) or 17 nm (Au17) were prepared by reduction of chloroauric acid with white phosphorous or
Kupffercells heterogeneity
455
sodium citrate, respectively [20]. Mannan was coupled to gold particles 5 nm in diameter (Man-Au5) and LacBSA to gold particles 17 nm in diameter (LacBSAAu17) following the method described by Horisberger and Rosset [20]. Ligand saturation was found at 150/~g of mannan/ml Au5 and 14/tg of LacBSA/ml AU17.
Localization of Mannose- and Galactose-Specific Receptors on Rat Liver Cells in situ The binding capacity of livers was investigated by in situ experiments, in which livers were slightly prefixed before ligand administration, as detailed elsewhere [18]. 400/zl of Man-Au5 and 400/tl of LacBSA-Au17 were injected into the perfusion medium for adult and aged animals. Decreasing amounts of standard gold-complexes solution were used for younger animals in relation to the size of the liver. In this way we were sure that the gold complexes were evenly distributed in the organ, allowing Kupffer cells to be in contact with the ligands. The ligands were always injected in a saturating concentration, detected by the presence of gold granules in the perfusate after passage in the liver. The organs were further fixed with 0.5% glutaraldehyde in a s-Collidine buffer (pH 7.2) and then cut into small pieces (samples of the liver were chosen at random), postfixed with 2% OsO4, dehydrated and embedded in epoxy resin. Ultrathin sections were obtained with a Reichert Ultracut ultramicrotome. Samples, stained with uranyl acetate and lead citrate, were observed under a Philips EM 400 T electron microscope. For each age three different animals were used and non-consecutive ultrathin sections were cut from four different specimens to make sure that we were not counting the same cell twice or more. More than 100 Kupffer ceils binding one, both or no ligands were counted. The cell surface density of the two receptors is very high (about 70-80%), thus it was practically impossible for us to have cut Kupffer cells which had not come into contact with any gold particles at all. The percentage presence of the different classes of Kupffer cells was then evaluated. Inhibition of ligand binding was achieved by incubation in the presence of the competing monosaccharides N-acetyl-D-galactosamine (GalNAc) and mannose in a final concentration of 80mM. Further control binding experiments were performed on a cell line (U937 monocyte like) known not to express the receptors.
Quantitative Evaluation of Gold Particles Bound The number of gold particles bound per #m of Kupffer cells plasma membrane was calculated on at least 20 electron micrographs for each group. Micrographs were taken at random of the sinusoidal faces of the Kupffer cells sectioned through their nuclei. The length of the plasma membrane was calculated using a Tektronik 4051 computer, equipped with a graphic tablet.
456
Lentini, Falasca, Auluori and Dini RESULTS
The simultaneous localization of galactose- and mannose-specific receptors by means of different-sized protein-gold complexes, revealed a heterogeneity in the Kupffer cell population. As shown in Fig. 1, at all the ages studied we observed four different classes of macrophages in relation to the ligands bound: two classes of macrophages, binding either Man-Au5 or LacBSA-Au17 but not both particles, one class of macrophages binding both ligands, and the last class of Kupffer cells without labelling. The percentage of double labelled macrophages gradually increase from the early stages of development up to 15 days after birth, when the value is comparable with that of adult animals. The percentage of double labelled cells decreased remarkably as age increased, to become similar to that of newborn rats at 24-month-old. The number of cells without binding activity followed an inverse pattern during development and aging, i.e. the number of cells that were completely lacking binding sites decreased from birth up to 15-day-old rats, remaining stable during the adult life and increasing during the aging. Conversely, the percentage of cells with only one of the two receptors did not change significatively during development. Therefore the simultaneous exposition of the two receptor systems seems to be age-dependent. Table 1 shows the quantification of gold particles bound on the Kupffer cell surface in in situ simultaneous localization of both galactose and mannose specific receptors during the entire life span Of rat. The mean values are comparable to those previously measured with binding experiments of single labelling [17, 18, 19], thus indicating that the simultaneous use of different sized protein-gold complexes does not affect the binding capacity of Kupffer cells. We observed a low variability (less than 10%) from rat to rat in the binding experiments. In in situ binding experiments the values measured are not affected by the use of collagenase as it is the case for the experiments with isolated Kupffer cells. Therefore, our data reflects the real in vivo situation of the Kupffer cells receptor expression at the time of the sacrifice. %
I . Galactose/ma~zzese
68-
[] none
[ ] 6a]actose
38-
N
6
18
15
A
fir
days
Fig. 1. Percentage of four Kupffer cell populations detected by their receptorial properties during postnatal development and aging ( N = newborn; 6, 10, 15 days after birth; A = adult; AR = aged rat).
Kupffer cells heterogeneity
457
Table 1. Number of LacBSA-Aulv particles per equatbrial Kupffer cell section and number of mannan-Au5 particles per #m of plasma membrane of Kupffer cells in in situ simultaneous localization of galactose and mannose specificbinding sites Age
LacBSA-Au17
Mannan-Au5
Newborn 6 days 10 days 15 days 2-month-old 24-month-old
9 ~ 5 (26) 31 • 10 (30) 75 • 25 (28) 130 • 31 (29) 127 • 28 (30) 81 5:27 (28)
1.4 • 0.7 (22) 2.3 5:0.6 (21) 5.8 i 1.7 (19) 12.3 d: 2.1 (25) 14.8 i 3.6 (22) 6.3 i 2.1 (25)
Each value is the mean of two different experiments ~ SD (n) number of cells examined
Figure 2 and Fig. 3 show electron micrographs of Kupffer and endothelial cells respectively labelled simultaneously with LacBSA-Au17 and Man-Au5 particles. The gold particles at all ages examined are not colocalized on Kupffer cells and their distribution is not restricted to specialized regions of the plasma membrane, whereas on endothelial cells binding activities are mainly concentrated in the coated regions of the cell surface. The specifcity of the binding sites described is demonstrated in the inhibition control experiments, in which the gold ligands are not binding to the cell surface of Kupffer (Fig. 2e,f) or endothelial cells (Fig. 3e,f) in the presence of competing saccharides (GalNac and mannose 80 mM final concentration). No binding sites were detected when a cultured cell line (U937 monocyte like) was incubated with a mixture of both ligands for 30 min at ice temperature to avoid possible endocytic phenomena (data not shown). The number of binding sites is strictly related to the stage of liver maturation. In fact, as reported in Table 2, the morphometry on Kupffer cells in in situ binding experiments of single labelling has revealed that the cell surface expression of the galactose and mannose-specific receptors is modulated in relation to the stage of liver maturation. The number of gold granules bound to the cell surface of Kupffer cells (both for galactose- and mannose-exposing ligands) increases gradually from birth to 15th day of rat life, when the adult values are reached, to decrease in aged rats to values comparable to those measured during perinatal age. The different number of both gold particles observed on the cell surface of Kupffer cells from adult (Fig. 2a) and 15-day-old (Fig. 2c) rats with respect to cells from newborn (Fig. 2b) and 24-month-old (Fig. 2d) animals is related to this difference. It should be also kept in mind that the number of galactose-specific binding sites is always greater than that of mannose-specific binding sites. The cell surface modulation of binding sites described for Kupffer cells was not observed on endothelial cells. In fact, the binding activity shown in Fig. 3 for endothelial cells from adult (a) and 15-day-old (c) rats is similar to that observed on endothelial cells from newborn (b) and aged (d) rats.
458
Lentini, Falasca, Autuori and Dini
Fig. 2. In situ double labelling experiments for galactose- (LacBSA-Aul7, arrowheads) and mannose- (mann-Aus, arrows) specific receptors on Kupffer cell plasma membranes from adult (a), newborn (b), 15-day-old(c) and aged (d) rats. The two receptors are not colocalized in the same region of the plasma membrane. The inhibition achieved by preincubation with N-acetyl-D-galactosamine (80mM final concentration) is almost complete (e= adult; f= newborn). Bars = 1 #m. DISCUSSION Our results confirmed the existence of significant differences in the cells of hepatic sinusoids as far as the simultaneous binding of galactose and mannose-exposing molecules was concerned. W e can divide the liver m a c r o p h a g e population into four different classes with respect to m a n n o s e and galactose binding capacity. A t each age studied we found cells which bind both m a n n o s e and glactose-exposing particles, cells which bind only one ligand, and others which express no binding capacity. These results are in a g r e e m e n t with others [4], which d e m o n s t r a t e d heterogeneity in the binding capacity of galactose terminating particles for Kupffer cells and of m a n y lectins bound differently to the different vascular
Kupffer cells heterogeneity
459
Fig, 3. In situ double labelling experiments for galactose- (lacBSA-Au17, arrowheads) and mannose- (mann-Aus, arrows) specific receptors on endothelial cell plasma membranes from adult (a), newborn (b), 15-day-old (c), and aged (d) rats. The gold granules are mainly localized in the coated regions of the plasma membrane and are both found in the same coated pits. No gold particles are bound on endothelial cells (e ~ adult; f = newborn) after preincubation with N-acetyl-D-galactosamine(80 mM final concentration). Bars -~ 1 #m. segments [7]. It is well known that macrophages, in different tissues, are heterogeneous in morphology, enzyme activity and cell surface properties I21, 22]. In particular liver macrophages express different characteristics and different surface antigens [23] within the same population: the Kupffer cell population contains cells which are different in size and possibly in density (the average diameter varying from 10 # m (small cells) to 14/am (large cells) [4]). The presence of Kupffer cells showing different receptorial patterns may be due to the presence in the liver of different populations of macrophages, which are detectable not morphologically but by their receptorial expression. These different populations of Kupffer cells could be related to different physiological needs, even though the physiological role played by the receptorial system
Lentini, Falasca, Autuori and Dini
460
2. Percentage of binding of colloidal gold particles coupled with LacBSA (LacBSA-Aus) and with mannan (Mannan-Au5) per /tm of Kupffer cells plasma membrane, in in situ binding experiments of single labeling Table
Age Newborn 6 gg 10 gg 15 gg 2-month-old 24-month-old
LacBSA-Au5 Mannan-Au5 27-2 40-5 59.5 97.4 100.0 36-7
10-9 21-9 26-6 89-0 100-0 37-5
Each value is the mean of three different experiments. S.D. does not exceed 10%. The value of the 2-raonth-old rat is taken as 100% binding.
specific for galactose is not clear at the present time. Very recently, a role has been proposed for the asialoglycoprotein receptor of hepatocytes in the removal of apoptotic hepatocytes [24]. Conversely the activity of the mannose-specific receptors is thought to be linked to the antibacterial defence and the plasma clearance of lysosomal enzymes [25, 26]. Differences in the Kupffer cells' functional activity have already been demonstrated, i.e. Kupffer cells showed a greater phagocytic activity in pericentral regions of the liver lobule [27]. The use of phagocytosis markers, such as latex particles or polymer microspheres, resulted in at most 64% of macrophages labelled, even when high doses of particles were injected [28, 29]. The different receptorial properties, described in the present paper, could be the expression of differentiative maturation in the Kupffer cell population itself. In fact, we showed not only the existence of heterogeneity in the Kupffer cell population but also that this heterogeneity is modified throughout the entire life of rats. In fact, during the early stages of development (newborn until 6 days after birth) double-labelled Kupffer cells are present in low percentages. These cells become the majority of the population at 10-15 days after birth, when their number is comparable with that of the adult, their number then decreases drastically with senescence. The unlabelled cells, on the contrary, largely decrease from neonatal until adult age, and then become the most numerous type in old animals, as they are for the neonatal ones. The cells which are positive for one of the two ligands used, do not vary in number in a significant way. These data could be explained in terms of different stages of maturation in the same cell population. In particular, the different receptorial properties could be the expression of differentiating processes in the Kupffer cell population itself. This hypothesis could be seen as supporting our previous studies of single labelling in which the expression of galactose and mannose-specific receptors are strictly related to liver maturation [17, 18].
Kupffer cells heterogeneity
461
REFERENCES 1. Jungermann, K. (1988) Seminars in Liver Disease 8:329-341. 2. Wisse, E., Geerts, A., Bouwens, L., van Bossuyt, H., Vanderkerken, K. and van Goethern, F. (1989) In: Cells of the Hepatic Sinusoid, Vol. 2 (E. Wisse, D. L. Knook, K. Decker eds), Kupffer Cell Foundation, Rijswijk: 1-9. 3. Sleyster, E. C. and Knook, D. L. (1982) Lab. Invest. 47:484-490. 4. Hardonk, M. J., Van Goor, H., Sherphof, G. L. and Daemen, T. (1986) In: Cells of the Hepatic Sinusoid, Vol. 2 (E. Wisse, D. L. Knook and K. Decker eds) Kupffer Cell Foundation, Rijswijk: 434-438 (1989). 5. Bouwens, L., Baekeland, M., De Zanger, R. and Wisse, E. (1986). Hepatology 6:718-722. 6. Sherwood, E. R. and Williams, D. L. (1988) In: Sinusoids in human liver: health and disease: (P. Bioulac-Sage and C. Balabaud eds) 359-368. 7. Barber~i-Guillerm, E., Arrue, J. M., Ballesteros, J. and Vidal Vanaclocha, F. (1986) J. Ultrastruct. Molec. Struct. Res. 97:197-206. 8. De Zanger, R. B. and Wisse, E. (1982) In: Sinusoidal Liver Cells (D. L. Knook and E. Wisse eds) Elsevier, Amsterdam: 69-76. 9. Wisse, E., De Zanger, R. B., Charels, K., van der Smissen, P. and McCuskey, R. S. (1985) Hepatology 5: 683-692. 10. Naito, M., Kodama, T., Matsumoto, A. and Takahashi, K. (1991) In: Cells of the Hapatic Sinusoid Vol. 3 (E. Wisse, D. L. Knook and R. S. McCuskey eds) Kupffer Cell Foundation, Rijswijk, 497-501. 11. Wardle, E. M. (1987) Liver 7:63-70. 12. Kolb-Bachofen, V., Schlepper-Schafer, J. and Vogell, J. (1982) Cell 29:859-866. 13. Praaning-VanDalen, D. P., De Leeuw, A. M., Brouwer, A. and Knook, D. L. (1987) Hepatology 7: 672-679. 14. Schwartz, A. L. (1984) CRC Crit. Rev. Biochem. 16:207-233. 15.. Roos, P., Hartman, H. J., Schlepper-Schaefer, J., Kolb, H. and Kolb-Bachofen, V. (1985) Biochim. Biophys. Acta 847: 115-121. 16. Stahl, P. D., Wileman, T. E., Diment, S. and Shepherd, V. L. (1984) Biol. Cell 51:215-218. 17. Dini, L., Lentini, A. and Conti-Devirgiliis, L. (1990) Mech. Ageing Dev. 56: 117-128. 18. Dini, L. and Kolb-Bachofen, V. (1987) Eur. J. Cell Biol. 44:144-150. 19. Dini, L. and Conti-Devirgiliis. (1989) Mech. Ageing Dev. 50:57-69. 20. Horisberger, M. and Rosset, J. (1977) J. Histochem. Cytochem. 25:295-305. 21. Forster, O. and Landy, M. (1981) Heterogeneity of mononuclear phagocytes. London, Academic Press. 22. Marahan, P. S. (1980) Reticuloendothel. Soc. 27:223-228. 23. Takeya, M., Hsiao, L., Shimokawa, Y. and Takahashi, K. (1989) J. Histochem. Cytochem. 37: 635-641. 24. Dini, L., Autuori, F., Lentini, A., Oliverio, S. and Piacentini, M. (1992) FEBS 2:174-178. 25. Eshdat, Y. and Sharon, N. (1984) Biol. Cell 51:259-266. 26. Schlesinger, P. H., Rodman, J. S., Doebber, T. W., Stahl, P. D., Lee, Y. C., Stowell, C. P. and Kuhlenschmidt, T. B. (1980) Biochem. J. 192:597-606. 27. Te Koppele, J. M. and Thurman, R. G. (1990) Am. J. Physiol. 259:G814-G821. 28. Spitzer, J. J., Spolarics, Z., Bautista, A. P., Schuler, A. and Lang, C. H. (1991) In: Cells of the Hepatic Sinusoid, Vol. 3 (E. Wisse, D. L. Knook and R. S. McKuskey eds) Kupffer Cell Foundation, Rijswijk: 68-72. 29. Tabata, Y. and Ikada, Y. (1988) Biomaterials 9:356-362.
The Simultaneous Exposition of Galactose and Mannose-Specific Receptors on Rat Liver Macrophages is Developmentally Regulated Alessandro Lentini, Laura Falasea, Franeesco Autuori, and Luciana Dini m Received June 28, 1992 We studied the simultaneous binding of galactose and mannose-exposing ligands in sinusoidal rat liver cells during development and aging. The galactose-specific receptors were visualized using 17 nm diameter colloidal gold particles coupled with Lactosylated bovine serum albumine (LacBSA), while mannose-specific receptors were localized by means of 5 nm diameter particles adsorbed with mannan. We observed the presence of four different classes of Kupffer cells in relation to the ligands bound. The percentage of each group of Kupffer cells varied in relation to the age of the subject from which the sample was taken. There were few double-labelled cells in the livers from newborn rats, with numbers increasing with age to adulthood, and decreasing again in the older animals. Cells without labelling were in the majority after birth, but they decreased in number up to adulthood and increased again during subsequent aging. The numbers of single-labelled cells did not change significantly during liver maturation. We hypothesize that the exposition of galactose and mannose-specific receptorial systems is regulated by developmental conditions. KEY WORDS: Galactose-specific receptor; mannose-specific receptor; Kupffer ceils; development;
aging.
INTRODUCTION In liver the hepatocytes are by far the most prominent class of cells, in terms of both size and total volume and relative number. In fact, the non parenchymal liver cells (i.e. Kupffer cells, endothelial cells, fat storing cells, Ito cells and Pit cells) represent only a small percentage of the entire liver mass. Increasing knowledge of their functions has, however, revealed their importance to liver physiology. In recent years, it has been amply demonstrated that the functions of hepatocytes and sinusoidal cells differ according to the different zones of the hepatic acinus [1, 2]. Kupffer cells do not constitute a homogeneous population of 1 Department of Biology, University of Rome "Tor Vergata" Via E. Carnevale 00173 Rome. z To whom correspondence should be addressed. 453 0144-8463/92/1200-0453506.50/0~) 1992 Plenum Publishing Corporation
Lentini, Falasca, Autuori and Dini
454
cells [3]. Sleyster and Knook [3] observed a correlation between functional heterogeneity, i.e. endocytic and lysosomal enzyme activity, and the localization of the cells in the liver acinus. Periportal Kupffer cells were larger and showed greater lysosomal enzyme activities than midzonal and perivenous Kupffer cells [4,5,6]. The only differences so far found in the hepatic endothelial cell population have, on the other hand, been morphological. Although these differences may be related to functional activities, molecular data confirming this hypothesis are still scarce [7, 8, 9]. One of the major functions of sinusoidal liver cells is the removal of macromolecules, bacteria and particles from the circulation. Not all the phagocytic activity of Kupffer and endothelial cells is nonspecific, since a large variety of receptors are expressed o n their cell surface [10, 11]. Among them, the galactose and mannose-specific receptors are the greatest in number [12, 13]. The galactosespecific receptors on Kupffer and endothelial cells recognize the penultimate non-reducing galactose residues of the oligosaccharide chains which are made free after desialylation. It is also well known that receptors with a similar binding specificity (known as asialoglycoprotein receptor, ASGP-R) are present on the surface of hepatocytes [14]. Circulating ASGP are internalized by hepatocytes by means of ASGP-R which is structurally and immunologically different from the galactose-specific receptors of Kupffer and endothelial cells [15]. Also mannoseterminated glycoproteins are removed from the blood by liver specific receptors. Sinusoidal cells in particular show a great capacity for endocytosing these ligands [13, 16]. The cytochemical binding and internalization pattern of mannose and galactose-exposing ligands on Kupffer cells visualized by means of protein-gold complexes is similar [12, 17]. In order to investigate whether there is a heterogeneity regarding to the carbohydrate-recognizing systems, we studied the ability of Kupffer cells to bind the mannose and galactose-exposing ligands simultaneously. Since Kupffer cells modulate the number of receptors on their surface in an age-dependent manner [17, 18, 19] (i.e. the number of receptors gradually increases during post natal life while during aging there is a considerable loss of receptor activity), we also investigated any influence of liver maturation (postnatal development and aging) on their binding capacity. MATERIALS AND METHODS Materials
Newborn (24 hours after birth), 6, 10, 15-day-old, adult (2-month-old) and old (24-month-old) male Wistar rats were used. Mannan from yeast and bovine serum albumine (BSA) were purchased from Sigma (USA); Lactosylated bovine serum albumine (LacBSA) from Soeckebort (Sweden); Eagle's medium from Flow (USA); All other chemicals were from Merck (Germany). Methods
Colloidal gold solutions with particles in 5 nm diameter (Au5) or 17 nm (Au17) were prepared by reduction of chloroauric acid with white phosphorous or
Kupffercells heterogeneity
455
sodium citrate, respectively [20]. Mannan was coupled to gold particles 5 nm in diameter (Man-Au5) and LacBSA to gold particles 17 nm in diameter (LacBSAAu17) following the method described by Horisberger and Rosset [20]. Ligand saturation was found at 150/~g of mannan/ml Au5 and 14/tg of LacBSA/ml AU17.
Localization of Mannose- and Galactose-Specific Receptors on Rat Liver Cells in situ The binding capacity of livers was investigated by in situ experiments, in which livers were slightly prefixed before ligand administration, as detailed elsewhere [18]. 400/zl of Man-Au5 and 400/tl of LacBSA-Au17 were injected into the perfusion medium for adult and aged animals. Decreasing amounts of standard gold-complexes solution were used for younger animals in relation to the size of the liver. In this way we were sure that the gold complexes were evenly distributed in the organ, allowing Kupffer cells to be in contact with the ligands. The ligands were always injected in a saturating concentration, detected by the presence of gold granules in the perfusate after passage in the liver. The organs were further fixed with 0.5% glutaraldehyde in a s-Collidine buffer (pH 7.2) and then cut into small pieces (samples of the liver were chosen at random), postfixed with 2% OsO4, dehydrated and embedded in epoxy resin. Ultrathin sections were obtained with a Reichert Ultracut ultramicrotome. Samples, stained with uranyl acetate and lead citrate, were observed under a Philips EM 400 T electron microscope. For each age three different animals were used and non-consecutive ultrathin sections were cut from four different specimens to make sure that we were not counting the same cell twice or more. More than 100 Kupffer ceils binding one, both or no ligands were counted. The cell surface density of the two receptors is very high (about 70-80%), thus it was practically impossible for us to have cut Kupffer cells which had not come into contact with any gold particles at all. The percentage presence of the different classes of Kupffer cells was then evaluated. Inhibition of ligand binding was achieved by incubation in the presence of the competing monosaccharides N-acetyl-D-galactosamine (GalNAc) and mannose in a final concentration of 80mM. Further control binding experiments were performed on a cell line (U937 monocyte like) known not to express the receptors.
Quantitative Evaluation of Gold Particles Bound The number of gold particles bound per #m of Kupffer cells plasma membrane was calculated on at least 20 electron micrographs for each group. Micrographs were taken at random of the sinusoidal faces of the Kupffer cells sectioned through their nuclei. The length of the plasma membrane was calculated using a Tektronik 4051 computer, equipped with a graphic tablet.
456
Lentini, Falasca, Auluori and Dini RESULTS
The simultaneous localization of galactose- and mannose-specific receptors by means of different-sized protein-gold complexes, revealed a heterogeneity in the Kupffer cell population. As shown in Fig. 1, at all the ages studied we observed four different classes of macrophages in relation to the ligands bound: two classes of macrophages, binding either Man-Au5 or LacBSA-Au17 but not both particles, one class of macrophages binding both ligands, and the last class of Kupffer cells without labelling. The percentage of double labelled macrophages gradually increase from the early stages of development up to 15 days after birth, when the value is comparable with that of adult animals. The percentage of double labelled cells decreased remarkably as age increased, to become similar to that of newborn rats at 24-month-old. The number of cells without binding activity followed an inverse pattern during development and aging, i.e. the number of cells that were completely lacking binding sites decreased from birth up to 15-day-old rats, remaining stable during the adult life and increasing during the aging. Conversely, the percentage of cells with only one of the two receptors did not change significatively during development. Therefore the simultaneous exposition of the two receptor systems seems to be age-dependent. Table 1 shows the quantification of gold particles bound on the Kupffer cell surface in in situ simultaneous localization of both galactose and mannose specific receptors during the entire life span Of rat. The mean values are comparable to those previously measured with binding experiments of single labelling [17, 18, 19], thus indicating that the simultaneous use of different sized protein-gold complexes does not affect the binding capacity of Kupffer cells. We observed a low variability (less than 10%) from rat to rat in the binding experiments. In in situ binding experiments the values measured are not affected by the use of collagenase as it is the case for the experiments with isolated Kupffer cells. Therefore, our data reflects the real in vivo situation of the Kupffer cells receptor expression at the time of the sacrifice. %
I . Galactose/ma~zzese
68-
[] none
[ ] 6a]actose
38-
N
6
18
15
A
fir
days
Fig. 1. Percentage of four Kupffer cell populations detected by their receptorial properties during postnatal development and aging ( N = newborn; 6, 10, 15 days after birth; A = adult; AR = aged rat).
Kupffer cells heterogeneity
457
Table 1. Number of LacBSA-Aulv particles per equatbrial Kupffer cell section and number of mannan-Au5 particles per #m of plasma membrane of Kupffer cells in in situ simultaneous localization of galactose and mannose specificbinding sites Age
LacBSA-Au17
Mannan-Au5
Newborn 6 days 10 days 15 days 2-month-old 24-month-old
9 ~ 5 (26) 31 • 10 (30) 75 • 25 (28) 130 • 31 (29) 127 • 28 (30) 81 5:27 (28)
1.4 • 0.7 (22) 2.3 5:0.6 (21) 5.8 i 1.7 (19) 12.3 d: 2.1 (25) 14.8 i 3.6 (22) 6.3 i 2.1 (25)
Each value is the mean of two different experiments ~ SD (n) number of cells examined
Figure 2 and Fig. 3 show electron micrographs of Kupffer and endothelial cells respectively labelled simultaneously with LacBSA-Au17 and Man-Au5 particles. The gold particles at all ages examined are not colocalized on Kupffer cells and their distribution is not restricted to specialized regions of the plasma membrane, whereas on endothelial cells binding activities are mainly concentrated in the coated regions of the cell surface. The specifcity of the binding sites described is demonstrated in the inhibition control experiments, in which the gold ligands are not binding to the cell surface of Kupffer (Fig. 2e,f) or endothelial cells (Fig. 3e,f) in the presence of competing saccharides (GalNac and mannose 80 mM final concentration). No binding sites were detected when a cultured cell line (U937 monocyte like) was incubated with a mixture of both ligands for 30 min at ice temperature to avoid possible endocytic phenomena (data not shown). The number of binding sites is strictly related to the stage of liver maturation. In fact, as reported in Table 2, the morphometry on Kupffer cells in in situ binding experiments of single labelling has revealed that the cell surface expression of the galactose and mannose-specific receptors is modulated in relation to the stage of liver maturation. The number of gold granules bound to the cell surface of Kupffer cells (both for galactose- and mannose-exposing ligands) increases gradually from birth to 15th day of rat life, when the adult values are reached, to decrease in aged rats to values comparable to those measured during perinatal age. The different number of both gold particles observed on the cell surface of Kupffer cells from adult (Fig. 2a) and 15-day-old (Fig. 2c) rats with respect to cells from newborn (Fig. 2b) and 24-month-old (Fig. 2d) animals is related to this difference. It should be also kept in mind that the number of galactose-specific binding sites is always greater than that of mannose-specific binding sites. The cell surface modulation of binding sites described for Kupffer cells was not observed on endothelial cells. In fact, the binding activity shown in Fig. 3 for endothelial cells from adult (a) and 15-day-old (c) rats is similar to that observed on endothelial cells from newborn (b) and aged (d) rats.
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Fig. 2. In situ double labelling experiments for galactose- (LacBSA-Aul7, arrowheads) and mannose- (mann-Aus, arrows) specific receptors on Kupffer cell plasma membranes from adult (a), newborn (b), 15-day-old(c) and aged (d) rats. The two receptors are not colocalized in the same region of the plasma membrane. The inhibition achieved by preincubation with N-acetyl-D-galactosamine (80mM final concentration) is almost complete (e= adult; f= newborn). Bars = 1 #m. DISCUSSION Our results confirmed the existence of significant differences in the cells of hepatic sinusoids as far as the simultaneous binding of galactose and mannose-exposing molecules was concerned. W e can divide the liver m a c r o p h a g e population into four different classes with respect to m a n n o s e and galactose binding capacity. A t each age studied we found cells which bind both m a n n o s e and glactose-exposing particles, cells which bind only one ligand, and others which express no binding capacity. These results are in a g r e e m e n t with others [4], which d e m o n s t r a t e d heterogeneity in the binding capacity of galactose terminating particles for Kupffer cells and of m a n y lectins bound differently to the different vascular
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Fig, 3. In situ double labelling experiments for galactose- (lacBSA-Au17, arrowheads) and mannose- (mann-Aus, arrows) specific receptors on endothelial cell plasma membranes from adult (a), newborn (b), 15-day-old (c), and aged (d) rats. The gold granules are mainly localized in the coated regions of the plasma membrane and are both found in the same coated pits. No gold particles are bound on endothelial cells (e ~ adult; f = newborn) after preincubation with N-acetyl-D-galactosamine(80 mM final concentration). Bars -~ 1 #m. segments [7]. It is well known that macrophages, in different tissues, are heterogeneous in morphology, enzyme activity and cell surface properties I21, 22]. In particular liver macrophages express different characteristics and different surface antigens [23] within the same population: the Kupffer cell population contains cells which are different in size and possibly in density (the average diameter varying from 10 # m (small cells) to 14/am (large cells) [4]). The presence of Kupffer cells showing different receptorial patterns may be due to the presence in the liver of different populations of macrophages, which are detectable not morphologically but by their receptorial expression. These different populations of Kupffer cells could be related to different physiological needs, even though the physiological role played by the receptorial system
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2. Percentage of binding of colloidal gold particles coupled with LacBSA (LacBSA-Aus) and with mannan (Mannan-Au5) per /tm of Kupffer cells plasma membrane, in in situ binding experiments of single labeling Table
Age Newborn 6 gg 10 gg 15 gg 2-month-old 24-month-old
LacBSA-Au5 Mannan-Au5 27-2 40-5 59.5 97.4 100.0 36-7
10-9 21-9 26-6 89-0 100-0 37-5
Each value is the mean of three different experiments. S.D. does not exceed 10%. The value of the 2-raonth-old rat is taken as 100% binding.
specific for galactose is not clear at the present time. Very recently, a role has been proposed for the asialoglycoprotein receptor of hepatocytes in the removal of apoptotic hepatocytes [24]. Conversely the activity of the mannose-specific receptors is thought to be linked to the antibacterial defence and the plasma clearance of lysosomal enzymes [25, 26]. Differences in the Kupffer cells' functional activity have already been demonstrated, i.e. Kupffer cells showed a greater phagocytic activity in pericentral regions of the liver lobule [27]. The use of phagocytosis markers, such as latex particles or polymer microspheres, resulted in at most 64% of macrophages labelled, even when high doses of particles were injected [28, 29]. The different receptorial properties, described in the present paper, could be the expression of differentiative maturation in the Kupffer cell population itself. In fact, we showed not only the existence of heterogeneity in the Kupffer cell population but also that this heterogeneity is modified throughout the entire life of rats. In fact, during the early stages of development (newborn until 6 days after birth) double-labelled Kupffer cells are present in low percentages. These cells become the majority of the population at 10-15 days after birth, when their number is comparable with that of the adult, their number then decreases drastically with senescence. The unlabelled cells, on the contrary, largely decrease from neonatal until adult age, and then become the most numerous type in old animals, as they are for the neonatal ones. The cells which are positive for one of the two ligands used, do not vary in number in a significant way. These data could be explained in terms of different stages of maturation in the same cell population. In particular, the different receptorial properties could be the expression of differentiating processes in the Kupffer cell population itself. This hypothesis could be seen as supporting our previous studies of single labelling in which the expression of galactose and mannose-specific receptors are strictly related to liver maturation [17, 18].
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