Histochemistry

Histochemistry (1985) 83:165 169

9 Springer-Verlag 1985

Distribution of albumin in normal and regenerating livers of mice A light microscopic immunohistochemical and autoradiographic study* H.-V. Tuczek, P. Fritz, A. Grau, A Mischlinsky, T. Wagner, and G. Wegner Department of Clinical and Experimental Pathology Robert-Bosch-Hospital, Auerbachstral3e 110, D-7000 Stuttgart 50, Federal Republic of Germany Accepted May 11, 1985

Summary. The conditions affecting the immunohistochemical identification of albumin in livers of male NMRI-mice were investigated by light microscopy. In normal livers albumin is randomly distributed, revealing a pancytoplasmic nearly homogen reaction in groups of hepatocytes or single parenchymal cells. However, combined autoradiographic studies after pulse labelling with 3H-valin and perfusion experiments with human albumin indicate that this distribution is caused by albumin from blood plasma and does not reflect true protein synthesis. After perfusion of the livers followed by immunohistochemical amplification techniques which allowed to dilute the primary antibody up to 1:30,000, albumin could be detected nearly in all liver parenchymal cells as granular deposits decreasing in its density from periportal fields towards the terminal hepatic venules. In regenerating livers due to partial hepatectomy no remarkable differences in granular albumin deposits between G1- and S-phase of the cell cycle could be detected as was demonstrated by combined immunohistochemistry and 3H-dThd-autoradiography. However, during mitosis the content of albumin was often considerably reduced.

granular final reaction product (FRP) covering the cytoplasm of albumin producing hepatocytes. In general at the level of light microscope the immunohistochemical detection of albumin is described as a pancytoplasmic homogen FRP within the cytoplasm of randomly distributed groups of hepatocytes or single parenchymal cells (Hamashima et al. 1964; Chandrasekharan 1968; Lane 1969; Fetdmann et al. 1972; Horne et al. 1972; Brozman 1977; Guillouzo et al. 1978). However, albumin is demonstrable in a nonrandom manner in all hepatocytes if the liver is flushed free of plasma by a preceeding perfusion (Le Bouton and Masse 1980a, b; Geuze et al. 1981 ; Yokata and Fahimi 1981; Le Bouton 1982; Pignal etal. 1982; Clement et al. 1983) and under special experimental conditions, e.g. during fasting for example a granular FRP was described (Yokata and Fahimi 1981 ; Le Bouton 1982). The following light microscopic studies were performed in order to correlate the immunohistochemically identified albumin content of normal and regenerating livers with the molecular biologic conception of intracellular package and transport of secretory proteins.

Material and methods Introduction With the exception of the immunoglobulins the liver synthesizes nearly all plasma proteins. Among these, albumin represents the most abundant secretory protein (Peters 1962a, b; Rothschild et al. 1969) and albumin m - R N A is found to be about 10% of the total rat liver poly(A)-containing RNA population (Tse et al. 1978). Like other secretory proteins (for review see Palade 1975) albumin is assembled on the polysomes of the rough endoplasmic reticulum, segregates to its lumen, followed by passage through the smooth endoplasmic reticulum to the cisternae of the Golgi apparatus where it is concentrated and subsequently transported within membrane vesicles to the cell surface (Peters 1962a, b, 1977; Peters et al. 1962; Glaumann and Ericsson 1970; Jamison and Palade 1977; Strous et al. 1983; Sztul et al. 1983). It might be expected from this kind of intracellular arrangement and transport of secretory proteins that the immunohistochemical identification of albumin reveals a * Supported by a grant from the Robert-Bosch-Foundation, Stuttgart, Federal Republic of Germany

29 male NMRI-mice (body weight 32.9_+2.1 g (SE) and 4 months of age) with free access to water and food (Altromin) were used. Each group consisted of 4-5 animals and all operations were performed between 5.00 and 7.30 h p.m. First experimental series: Each animal of the group received a pulse

label of 185 kBq 3H-valin ((3,4(N)-3H)valin, specific activity 1~221 GBq/mM, Amersham Buchler, Braunschweig, FRG) per g body weight intraperitoneally and was killed after 30 rain. The livers were fixed by immersion in Bouin's solution for about 15 h and subsequently embedded in paraplast plus. Sections of 4 Ixm were cut by a semiautomatic microtome (LKB, HistoRange). Albumin was localized immunohistochemically according to the peroxidase-anti-peroxidase-(PAP)-method of Sternberger et al. (1970) using rabbit-anti-mouse-albumin (1:2,000, Capel, Malvern, USA) as primary antibody. In order to suppress background-staining the NaCl-content of Tris-saline buffer was raised up to 0.5 mM (Grube 1980). Peroxidase was assayed with ethylcarbazol (3-amino-9-ethylcarbazol, Sigma, Taufkirchen, FRG) dissolved in PBS-buffer, pH 5.1 containing 0.1% tween 80 (Porstmann et al. 1981). Non-specific immunohistochemical reactions were excluded by omitting the specific antiserum or using human-albumin, antiherpes, anti-candida, or anti-AFP instead of anti-mouse-albumin. In the autoradiographic series the slices were washed with inactive valin (25 mM) for 2 x 60 miu prior to immunohistochemistry.

166

Fig. 1. a Randomly distributed mouse albumin in untreated livers of mice (anti-mouse albumin 1:2,000) x 150. b Nearly complete disappearance of albumin after perfusion with 3 ml 0.15 M NaC1 (anti-mouse albumin 1:2,000) x 150. c Randomly distributed human albumin in livers of mice after perfusion with 3 ml 0.15 M NaC1 followed by 3 ml human albumin (anti-human albumin 1:4,000) x 150

Subsequently autoradiography was performed using the stripping film technique (Kodak A R 10, time of exposition l l d at 4 ~ C). Finally, if required, the samples were slightly counterstained with hematoxylin. The silver grain density covering albumin-positive or -negative areas of the cytoplasm of hepatocytes was determined by an ocular grid. Thereby 80 squares of 100 gm 2 were evaluated from each liver. In additional series without subsequent autoradiography the percentage of anti-albumin-positive parenchymal cells was determined by counting 5 x 1,000 hepatocytes/liver in order to compare the results with those after previous perfusion with salt solution or human albumin (see below).

Second experimental series: In these experiments the livers were perfused via a small catheter (outer diameter 0.61 ram) tightened into the vena porta. One group of animals recieved 3 ml 0.15 M NaCI followed by 3 ml Bouin's fixative. Another group was additionally perfused with 3 ml human albumin between the salt solution and the fixative fluid. Beyond this livers were fixed by immersion for about 15 h and subsequently washed in 70% ethanol containing one drop of a 25% N H 3 solution/100 ml until the yellow colour of the picrinic acid disappeared completely. This washing procedure was performed under gentle shaking generally lasting for 5-7 days. As could be shown in recently performed experinrents (not demonstrated here) similar results were obtained with depolymerisized 4% paraformaldehyde as fixative. The immunohistochemical procedure was quite the same as in the first experimental group using anti-mouse-albumin (1:2,000) or anti-human-albumin (1:4,000) respectively. The percentage of albumin-positive hepatocytes was determined for both groups as described above. After perfusion with salt solution no significant evidence of mouse albumin could be detected by immunohistochemistry used in this experimental arrangement. Therefore we employed an amplification technique involving two essential steps : 1. the primary antibody was incubated for 48 h at 4 ~ C (Vandesande 1979). 2. the double bridge technique of Vacca (1980) was applied. The combination of these variations of the PAP-method allowed to dilute the primary antibody against mouse albumin up to 1 : 20,000-1 : 30,000 resulting in a granular final reaction product.

In detail, the method finally used was the following : 1. Block endogenous peroxidase by treatment the sections with 0.3% (or 1%) H2Oz/methanol for 30 rain. 2. Wash in 0.5 M Tris-saline, pH 7.6 for 10 rain. 3. Demasque the antigens by incubating at 37~ for 30 min with 0.1% trypsin and 0.1% CaC12. 4. Repeat Step 2. 5. To reduce non-specific staining, preincubate for 10 rain with unspecific swine serum 1 : 20. Drain off but do not wash. 6. Apply the primary antibody (anti-mouse-albumin ] :20,000 or 1 : 30,000) for 48 h at 4 ~ C. 7. Wash slides in 0.5 M Tris-saline, pH 7.6 for 30 rain. 8. Repeat Step 5. 9. Incubate with swine-anti-rabbit-serum 1 : 100 for 30 min. 10. Repeat Step 7. 11. Repeat Step 5. 12. Treat sections with rabbit-PAP-complex 1:100 for 30 min (in the dark). 13. Repeat Step 7-12. 14. Repeat Step 7. 15. Incubate the sections for 10 min in a mixture containing 12 mt of a stock solution (10 mg etylcarbazol/6 ml DMSO) filled up ad 100 ml with 0.2 M PBS-buffer, pH 5.1 and supplemented with 0.1% tween 80. 16. Rinse in aqua dest. and drain excess of water. 17. If required counterstain gently with hematoxylin. 18. Mount the sections with glycerin-gelatine. If not indicated otherwise all procedures were carried out at room temperature.

Third experimental series: In this study two groups of mice were subjected to a 2/3-hepatectomy. About 48 h and 72 h after the operation the livers were perfused with 3 ml 0.15 M NaC1 followed by 1.5 ml NaCl-solution containing 111 kBq (3H)dThd (6-3H-thy midine, specific activity 185 MBq, Amersham Buchler, Braunschweig, FRG). Time allowed for incorporation was 5 rain. Thereafter the livers were perfused with 2 ml of 0.5 M inactive thymidine followed by 3 ml Bouin's fixative. The immunohistochemical procedures were the same as described above employing the amplifica-

167 Table 1. Per cent of albumin positive hepatocytes In untreated livers of mice (anti-mouse albumin 1:2,000) After perfusion with NaCI (anti-mouse albumin 1 : 2,000) After perfusion with NaC1 followed by human albumin (anti-human albumin 1 : 4,000)

m = 16.8+_11.6 (SE) m=0.98 +_1.29 (SE) m = 18.3+9.4 (SE)

Table 2. Silver grain density in untreated livers of mice 30 min after a pulse label with 3H-valin

Covering albumin positive hepatocytes

m = 12.7+_5.9 (SE)

or

surrounding liver parenchymal cells

m = 13.2_+ 6.2 (SE)

Fig. 3a, b. Granular deposits of albumin in regenerating livers of mice 48 h after partial hepatectomy (anti-mouse albumin 1:30,000). As can be shown in simultaneously performed 3H-dThd autoradiograms (a), no differences in albumin content is visible between S-phase (arrow) and Gl-phase of the cell cycle, whereas in mitosis (b) the granular albumin deposits are often reduced (arrow) x 555

Fig. 2. Granular deposits of albumin in hepatocytes decreasing in its density from portal fields towards terminal hepatic venules after perfusion of the livers followed by a modified intensifying PAPmethod (anti-mouse albumin 1:30,000, x 216

tion technique. Subsequently autoradiography was performed as already described using the stripping film technique (Kodak ARI 0, time of exposition 18d at 4~ C). Results

In non perfused immersion fixed livers of mice a pancytoplasmic, almost homogen reaction against anti-albumin occurs in randomly distributed groups of hepatocytes or single parenchymal cells if the indirect peroxidase-anti-peroxidase-(PAP)-method of Sternberger et al. (1970) is applied (Fig. I a). A b o u t 17% of the hepatocytes stained immunohistochemically positive for albumin (Table 1), whereas the surrounding parenchymal cells were negative or sometimes revealed a very few, scarcely perceptible granular deposits.

Likewise, in Kupffer cells a positive reaction was detectable. However, the combination of immunohistochemistry and autoradiography after a pulse label with 3H-valin yields no differences in protein synthesis between albumin-positive or -negative areas as might be deducted from the almost identical silver grain densities covering the cytoplasm of these two populations of liver cells (Table 2). In perfused livers which were flushed free from blood plasma, the positive reaction for albumin disappeared almost completely (Fig. 1 b, Table 1) and no albumin could be demonstrated in Kupffer cells. The randomly distributed groups of hepatocytes strongly reacting against anti-human-albumin reappeared if the livers were subsequently perfused with human albumin (Fig. I c, Table 1). However, employing immunohistochemical amplification techniques (see Material and methods) yield a positive final reaction product (FRP) even if anti-mouse-albumin as the primary antibody is diluted up to I : 30,000. Thereby a granular F R P is identified by light microscopy in the cytoplasm of nearly all hepatocytes decreasing in its density from the portal fields towards the terminal hepatic venules of the liver lobules (Fig. 2). The granules seem to traverse the cytoplasm and sometimes to join to the surface of the liver cells. In regenerating livers about 48 h and 72 h following a two-thirds hepatectomy the combination of immunohistochemistry and autoradiography after a short pulse label with 3H-thymidine reveals no decisive differences between hepatocytes in G~- or S-phase of the cell cycle (Fig. 3 a). During mitosis the granular F R P is often reduced

168 (Fig. 3b), indicating that during this phase (or short before in Gz) the content of albumin diminishes.

Discussion The results of our experiments clearly indicate that the immunohistochemical identified random distribution of albumin in clusters of hepatocytes or single liver parenchymal cells does not reflect true protein synthesis, since in simultaneously performed 3H-valin autoradiograms the silver grain density does not differ from the surrounding albumin-negative hepatocytes. In addition, after perfusion of the livers with saline, to remove interfering blood plasma, the irregularly distributed hepatocytes staining almost pancytoplasmic positive for albumin were highly reduced and reappeared after perfusion with h u m a n albumin as could be easily demonstrated with the corresponding antibody. Therefore these random distribution of albumin within the lobules of non-perfused livers may be due to a passive influx of serum albumin or to non species-specific receptors involved in catabolism of albumin or albumin-bound substances (Weisiger et al. 1981; Trevisan et al. 1982). However, after perfusion of the livers and modification of the PAP-method nearly all hepatocytes stained positive for albumin. Likewise transferrin, ferritin, and lactoferrin were also randomly distributed in non-perfused livers (Mason and Taylor 1978), whereas Morris et al. (1979) established a non random arrangement of angiotensinogen preferable near the terminal hepatic venules after perfusion. Additionally the influx of proteins from blood plasma into liver cells seems to depend on the plasma levels because c~-fetoprotein, known to occur at low levels during liver regeneration (Kuhlmann 1979) could be demonstrated in our experiments after perfusion of partially hepatectomized mice (results not shown) only in a few scattered hepatocytes as described in non perfused livers elsewhere (Tuczek et al. 1981, 1984). Our results confirm that the immunohistochemical identification of hepatic proteins depends widely of the methods employed. In the experiments presented here, combining perfusion of the liver with immunohistochemical amplification techniques, a granular final reaction product (FRP) could be demonstrated by light microscope in the cytoplasm of nearly all hepatocytes. Obviously these granular deposits represent intracytoplasmic membrane vesicles, where albumin is concentrated enough to be detected by highly diluted antibodies. Thereby the density of the F R P decreases from the portal fields towards the terminal hepatic venules, thus corresponding well with the intralobular distribution of protein synthesis after labelling with 3H-leucin (LeBouton 1968, 1969) and the concept of R a p p a p o r t (1976) concerning intralobular areas of different metabolic activities. However, after a fasting period of 16 h or 18 h, respectively, no differences of immunohistochemical demonstrable albumin could be established within the liver lobule of rats (Yokata and Fahimi 1981) or the albumin content was found to be higher especially around the terminal hepatic venules (LeBouton 1982). These differences may be due in part to a higher rate of protein turnover and secretory activity in the periportal zone I of Rappaport's liver lobule. In regenerating livers no differences in granular albumin deposits could be detected between G1- and S-phase of the cell cycle, indicating that albumin containing vesicles are not significantly diminished during DNA-synthesis. How-

ever during mitosis often a striking reduction of albumin containing granules was ascertainable, pointing at a lower albumin content of dividing hepatocytes. Thus our results agree with those of LeBouton and Masse (1980) indicating that perfusion of livers is a prerequisite for immunohistochemical identification of albumin. Moreover the granular reaction products of albumin obtained by the immunohistochemical methods presented here are in accord with the molecular biologic conception that secretory proteins are transported in a series of membrane vesicles towards the cell surface. Acknowledgements. The authors would like to thank particularly Prof. Dr. U. Klotz for constructive criticism and Mrs. B. Ahlborn for valuable assistance.

References Brozman M (1977) The localization of serum albumin in human liver cells. Acta Histochem 39:89-100 Chandrasekharan N (1968) Cytological studies of plasma albumin in the rat liver. Histochemie 13 : 11-15 Clement B, Rissel M, Latiner MF, Guillouzo A (1983) Fixation and membrane permeabilization of hepatocytes for immunolocalization of albumin. 2 i6me Symposium Internationale sur les Techniques Immunoenzymatiques. Cannes 16-18 March Feldmann G, Penand-Laurencin J, Crassons J, Benhamon JP (1972) Albumin synthesis by human liver cells: its morphological demonstration. Gastroenterology 63:1036--1048 Geuze JJ, Slot JW, Brands R (1981) The occurrence of albumin in the rat liver. A light and electron microscope immunocytochemical study. Cell Biol Int Rep 5 : 463 Glaumann H, Ericsson JLE (1970) Evidence for the participation of the Golgi apparatus in the intracellular transport of nascent albumin in the liver cell. J Cell Biol 47:555-567 Grube D (1980) Immunoreactivities of gastrin (G)-cells by ionic interactions. II. Non-specific binding of immunoglobulins to G-cells by ionic interactions. Histochemistry 66:149-167 Guillouzo A, Belanger L, Beaumont C, Valet JP, Briggs R, Chiu JF (1978) Cellular and subcellular immunolocalization of alpha-fetoprotein and albumin in rat liver. Reevaluation of various experimental conditions. J Histochem Cytochem 26:948 959 Hamashima Y, Harter JG, Coons AH (1964) The localization of albumin and fibrinogen in human liver cells. J Cell Biol 20: 271-279 Home CHW, Patrick RS, Macsween RNM, Henderson AR (1972) Albumin content of hepatocytes in experimental cirrhosis. Experentia 28 : 306-307 Jamieson JD, Palade GE (1977) Production of secretory proteins in animal cells. In: Brinkley BR, Porter KR (eds) International cell biology. The Rockefeller University Press, New York, pp 308-317 Kuhlmann WD (1979) Immunoperoxidase labelling of alphat-fetoprotein (AFP) in normal and regenerating livers of a low and a high AFP producing mouse strain. Histochemistry 64 : 67-75 Lane RS (1969) The cellular distribution of albumin in normal rat liver demonstrated by immuno-fluorescent staining. Clin Sci 36:157-159 LeBouton AV (1968) Heterogeneity of protein metabolism between liver cells as studied by radioautography. Curt Mod Biol 2:111-114 LeBouton AV (1969) Relations and extend of the zone of intensified protein metabolism in the liver acinus. Curr Mod Biol 3:4-8 LeBouton AV (1982) Routine overnight starvation and immunocytochemistry of hepatocyte albumin content. Cell Tissue Res 227 : 423-427

169 LeBouton AV, Masse MP (1980 a) A random arrangement of albumin-containinghepatocytes seen with histo-immunologicmethods. I. Verification of the artifact. Anat Rec 197:183 194 LeBouton AV, Masse JP (1980b) A random arrangement of albumin-containinghepatocytes seen with histo-immunologicmethods. II. Conditions that produce the artifact. Anat Rec 197:195 203 Mason DY, Taylor CR (1978) Distribution of transferrin, ferritin, and lactoferrin in human tissues. J Clin Pathol 31:316-327 Morris BJ, Iwamoto HS, Reid IA (1979) Localization of angiotensinogen in rat liver by immunohistochemistry. Endocrinology 105 : 796-800 Palade G (1975) Intracellular aspects of the process of protein synthesis. Science 189:347-358 Peters T Jr (1962a) The biosynthesis of rat serum albumin, l. Properties of rat albumin and its occurrence in liver cell fractions. JBiolChem237:1181 1185 Peters T Jr (1962b) The biosynthesis of rat serum albumin. I1. Intracellular phenomena in the secretion of newly formed albumin. J Biol Chem 237:1186-1189 Peters T Jr (1977) Intracellular albumin transport. In: Rosiner VM, Oratz M, Rothschild MA (eds) Albumin structures, function and uses. Pergamon Press, New York, pp 30~332 Peters T Jr, Fleischer B, Fleischer S (1962) The biosynthesis of rat serum albumin. IV Apparent passage of albumin through the Golgi apparatus during secretion. J Biol Chem 246: 240-244 Pignal F, Maurice M, Feldmann G (1982) Immunoperoxidase localization of albumin and fibrinogen in rat liver fxed by perfusion or immersion: Effect of saponin on the intracellular penetration of labeled antibodies. J Histochem Cytochem 30:1004-1014 Porstmann B, Porstmann T, Gaede D, Nagel E, Egger E (1981) Temperature dependent rises in activity of horse-radish peroxidase caused by non-ionic detergens and its use in enzyme-immunoassay. Clin Chim Acta 109 : 175-i 81 Rappaport AM (1976) The microcirculatory acinar concept of normal and pathological hepatic structure. Beitr Pathol 157:215 243 Rothschild MA, Oratz M, Schreiber SS (1969) Serum albumin. Am J Dig Dis 14:711-714 Sternberger LA, Hardy PH, Cuculin JJ, Meyer HG (1970) The unlabeled antibody enzyme method of immunohistochemistry.

Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-anti-horseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315-333 Strous G JAM, Willemsen R, Kerkhof van P, Slot JW, Geuze H J, Harvey FL (1983) Vesicular stomatitis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same Golgi vesicles. J Cell Biol 97:1815-1822 Sztul LS, Howell KE, Palade GE (1983) Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes. J Cell Biol 97:1582-1591 Trevisan A, Gudat F, Guggenheim R, Krey G, Dfirmiiller U, Liiond G, D/iggelin M, Landmann J, Tondelli P, Bianchi L (1982) Demonstration of albumin receptors on isolated human hepatocytes by light and scanning electron microscopy. Hepatology 2:832-835 Tse TPH, Morris HP, Taylor JM (1978) Molecular basis of reduced albumin synthesis in Morris hepatoma 7777. Biochemistry 17:3121-3128 Tuczek H-V, Fritz P, Wagner T, Braun U, Grau A, Wegner G (1981) Synthesis of alpha-fetoprotein (AFP) and cell proliferation in regenerating livers of NMRI mice after partial hepatectomy. An immunohistochemical and autoradiographic study with 3H-thymidine. Virchows Arch Cell Pathol 38:229237 Tuczek H-V, Fritz P, Wagner T, Grau A, Braun U, Wegner G (1984) Investigations concerning the correlation between liver cell proliferation, production of ~-fetoprotein and DNA-synthesis of lymphocytes in the spleen of NMRI-mice. An autoradiographic and immunohistochemical study. Pathol Res Pract 178:335-338 Vacca LL (1982) Double bridge technique of immuno cytochemistry. In: Bullock GR, Petrusz P (eds) Techniques in immuno cytochemistry, vol I. Academic Press, London, pp 155-182 Vandesande F (1979) A critical review of immunocytochemical methods for light microscopy. J Neurosci Methods 1 : 3-23 Weisinger R, Gollan J, Ockner R (1980) Receptor for albumin on the liver cell surface may mediate uptake of fatty acids and other albumin-bound substances. Science 211 : 1048-1050 Yokata S, Fahimi HD (1981) Immunocytochemical localization of albumin in the secretory apparatus of rat liver parenchymal cells. Proc Natt Acad Sci USA 78 : 4970~4974