Histopathological examinations of allogenic liver transplants concentrate primarily on alterations to the portal areas of the liver lobules. In those studies, the most characteristic histological features are a mixed inflammatory infiltrate, inflammatory damage of the small and medium-sized bile ducts, and inflammation of the venous endothelial cells, including functional cholestasis (1-4). Parenchymal lesions occurring together with these features of rejection are hepatocyte ballooning and centrilobular necrosis (4-6). A morphometric analysis of the infiltrating cells revealed that the number of lymphocytes, monocytes, and macrophages increased in liver allografts, and many of these cells migrate into the space of Disse during the first 7 days (7). Macrophages in the liver are the Kupffer cells (KCs*), which play a key role in the immunological response of allograft rejection. At the light and electron microscopic levels, KCs in rat liver can be distinguished from sinusoidal endothelial cells by the specific peroxidase staining using the 3,3′-diaminobenzidine method (8). It is well known that activated KCs secrete multifunctional cytokines such as interleukins (IL)-1 and 6 as well as tumor necrosis factor (TNF)-α (9-11). Recently, Green et al. (12) reported in a biochemical study using cultured hepatocytes that IL-6 inhibits hepatocyte taurocholate uptake together with Na+,K+-ATPase activity, but that it does not influence bile canalicular (BC) Mg++-ATPase activity in the apical membrane. TNF-α has no effect on both enzymes. Green et al. (12) concluded from these results that these effects of TNF-α in vivo are caused indirectly by IL-6.
The basolaterally located Na+,K+-ATPase plays an important role in homeostasis (13) and provides the driving force for the sodium-dependent uptake of taurocholate that occurs via the basolateral sodium-dependent taurocholate cotransport system (14-17). For hepatobiliary excretion, the apical membrane exhibits ATP-dependent transport systems that are important in the detoxification of endogenous and exogenous compounds (for review, see Vore  and, more recently, Mayer et al. ).
To prevent rejection, immunosuppressive drugs such as cyclosporine (CsA) are widely used. CsA influences the signal transduction system within the immunocompetent lymphocytes by inhibiting activator genes, which are known to affect the synthesis of cytokines (20, 21). However, CsA therapy is associated with numerous side effects in different organs (22, 23). The inhibition of ATP-dependent carriers in the canalicular membrane of hepatocytes by CsA has been described in various reports (24-29), as has the inhibition of biliary secretion of cholesterol and phospholipids (30). These processes were dose-dependent (30).
On the basis of the above-mentioned results described by Green et al. (12) concerning the incubation of cultured hepatocytes with IL-6, we expected similar effects in liver allografts. Therefore, in this study we have quantitatively determined the number and size of peroxidase-stained KCs. In liver allograft recipients, functional cholestasis is described during the course of rejection (1, 6). Na+,K+-ATPase is the cotransporter for hepatocyte taurocholate uptake; therefore, the activity of this enzyme may prevent cholestasis in allograft rejection. To assess the mechanism of cholestasis, we investigated whether Na+,K+-ATPase activity is changed during acute liver rejection and whether BC Mg++-ATPase activity is affected by this process.
MATERIALS AND METHODS
Animals. This study was conducted in compliance with the German Animal Protection Law under the permit issued by the state government, Darmstadt, FRG. Livers of male Lewis RT11 (LEW) rats and blood group D Agouti RT1a (DA) rats weighing 250-350 g were given transplants according to the following donor-recipient combination (n=4): the isogenic LEW to LEW combination controlled the success of the operation; the allogenic DA to LEW combination represented the rejection model. For immunosuppression of the allograft, we administered 1 mg/kg body weight of CsA intraperitoneally per day from the first to the tenth postoperative day. The operative technique was performed using rearterialization as described by Hanisch et al. (31). A kinetic of the body weight of each animal from the first to the last postoperative day was established.
Fixation. For light and electron microscopic studies, the livers were fixed via the portal vein with a fixative containing 0.25% glutaraldehyde and 2% sucrose in 100 mM PIPES buffer (piperazine-N,N′-bis(2-ethanesulfonic acid)) at a pH of 7.4 for 5 min. After fixation, 50-μm sections were cut with a microslicer (Dosaka EM Co., Kyoto, Japan) and collected in 100 mM PIPES buffer at a pH of 7.4.
KCs. For the demonstration of peroxidase activity, we used the specific 3,3′-diaminobenzidine procedure staining the nuclear envelope and the rough endoplasmic reticulum. The incubation was carried out in a medium containing 2.5 mM 3,3′-diaminobenzidine and 0.002% H2O2 in 100 mM PIPES buffer at a pH of 6.5 for 30 min (8). The microslicer sections were postfixed in 2% aqueous osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon 812. Semithin sections were observed without any counterstaining, and ultrathin sections were counterstained with lead citrate for 1 min.
An analysis of peroxidase-stained KCs within semithin sections was conducted using the texture analysis system (Leitz, Wetzlar, FRG) based on a video-image analyzer. The number of KCs per mm2 was calculated in a section area of 40,000 μm2. Forty randomly chosen areas per animal were investigated. For the determination of KC size, 50 cross-sections per animal were measured planimetrically on the texture analysis system screen using an interactive pen. In addition to the isogenic transplanted livers (LEW to LEW), livers of nontransplanted DA rats served as control for analysis of KC size and number.
Statistics. For statistical analysis of the data concerning the KCs, the Kruskal-Wallis one-way analysis of variance on ranks was used. To isolate the groups that differed from the others, Dunn's pairwise multiple comparison procedures were applied.
Hepatocytes. Na+,K+-ATPase activity was shown using the cerium technique as described recently by Angermüller et al. (32). Sections were preincubated for 30 min at 37°C in a medium without substrate. Incubation was carried out in a medium containing 5 mM Na-ATP (Sigma, Munich, Germany), 3 mM CeCl3, 10 mM MgCl2, and 20 mM KCl in 100 mM PIPES at a pH of 7.8 for 60 min at 37°C. For the demonstration of BC Mg++-ATPase activity, we applied a modified cerium technique: sections were preincubated for 30 min at 37°C in a medium containing 3 mM CeCl3 and 2 mM MgCl2 in 100 mM acetate buffer at a pH of 5.0. Incubation was performed in the same medium now containing 5 mM Na-ATP as substrate.
Postincubation processing for ATPases. All sections were postfixed with reduced osmium containing 1% aqueous osmium tetroxide and 1.5% potassium ferrocyanide, dehydrated in graded ethanol, and embedded in Epon 812. Ultrathin sections were examined without counterstaining in a Philips EM 301 electron microscope.
Cytochemical controls for ATPases. Cytochemical controls were carried out with the following media: (1) incubation medium without substrate, (2) preincubation medium and incubation medium together with 2.5 mM ouabain, the specific inhibitor of Na+,K+-ATPase, and (3) preincubation medium and incubation medium together with 10 mM tetramisole, an inhibitor of alkaline phosphatase.
The body weights showed statistically significant differences between the rejection group and the two other groups (Fig. 1). During the first 3 postoperative days, all animals lost body weight continuously. In the isogenic control group (LEW to LEW), a continuous increase of body weight was noticed after the fourth postoperative day. Likewise, a continuous increase of body weight was observed after the fourth postoperative day in the allogenic group with immunosuppression, but the increase was less prominent. However, in the rejection group, a continuous decrease of body weight until the last day was observed.
KCs. In the livers of all groups, KCs could be identified by their specific peroxidase reaction product in the nuclear envelope and in the rough endoplasmic reticulum (Fig. 2). In contrast, monocytes showed peroxidase activity only in cytoplasmic granules (Fig. 3), and sinusoidal endothelial cells were completely peroxidase negative. In the LEW to LEW group and in the CsA-treated group, KCs were located mainly in the sinusoids, and their number corresponded to that in the nontransplanted rat liver. In the rejection group, the number of KCs increased significantly (P<0.05) from an average of 12 KCs in control liver to 56 KCs in the allograft in an area of 1 mm2(Fig. 4). In the rejection group, many KCs had migrated through fenestrations of the sinusoidal endothelial cells into the enlarged space of Disse (Fig. 2). KCs in mitosis were not found. Rough endoplasmic reticulum and Golgi complexes were pronouncedly developed (Figs. 2 and 3), indicating the production of soluble molecules, whereas the few lysosomes represent only a low phagocytic activity. KC size of liver isograft and allograft was slightly enlarged compared with control and CsA-treated livers (Fig. 5).
Hepatocytes Na+,K+-ATPase: Liver isografts showed normal liver architecture on the 10th postoperative day. In electron micrographs, a strong Na+,K+-ATPase activity was demonstrated at the basolateral plasma membrane, while the apical domain did not show any reaction product (Fig. 6a). Ouabain, the specific inhibitor of Na+,K+-ATPase, reduced the reaction product substantially but did not abolish enzyme activity completely (Fig. 6b). In liver allografts, a strong cellular infiltration of lymphocytes was observed in portal and central regions. Only in the midzone did hepatic cells form the typical parenchymal branches. In these areas, the perisinusoidal space of Disse was enormously enlarged, containing mainly activated KCs. Some hepatocytes had lost contact with neighboring cells, ballooning and displaying swollen mitochondria and nuclei. Na+,K+-ATPase activity had been abolished in the basolateral plasma membrane (Fig. 7). In livers of allogenic transplanted rats treated with a very low CsA dose, only some immunocompetent cells were seen in portal areas and occasionally in the sinusoidal lumen. The space of Disse had normal size, and neither monocytes nor lymphocytes were now discovered there. Na+,K+-ATPase activity was preserved in the basolateral membrane (Fig. 8a), whereas the apical domain was free of reaction product (Fig. 8b).
BC Mg++-ATPase: In the isogenic transplanted liver, the ultrastructure of the bile canaliculi exhibited normal features, with microvilli protruding into the lumen of the small biliary space. Using an acid value of pH 5 together with ATP as substrate, we were able to selectively demonstrate the BC Mg++-ATPase activity in the apical plasma membrane of hepatocytes (Fig. 9a). Lysosomes and the rigid lamella of the Golgi complex containing acid phosphatases did not show any reaction product. In the allogenic transplanted livers, BC Mg++-ATPase activity had not been extinguished from the apical hepatic plasma membrane (Fig. 10, a-d). The morphology of the bile canaliculi had changed: dilated bile canaliculi without any microvilli (Fig. 10b) and obstructed bile canaliculi surrounded by homogenous cytoplasm without any cell organelles (Fig. 10c) were observed. In some areas, the number of canaliculi had increased (Fig. 10d). In allogenic transplanted livers of rats treated with CsA, BC Mg++-ATPase activity was as strong as in liver isografts (Fig. 11, a and b). In cytochemical control sections, alkaline phosphatase activity, also located in the bile canaliculi, was excluded using tetramisole in the preincubation medium and the incubation medium. Tetramisole has no influence on the reaction product of BC Mg++-ATPase activity.
In the present study, we examined a rat liver rejection model using a DA to LEW strain combination. We performed quantitative analyses of the number and size of peroxidasestained KCs in association with an electron microscopic study revealing the activity of Na+,K+-ATPase and of BC Mg++-ATPase. The majority of the KCs was found in the dilated space of Disse. There are two different possibilities of activation of these macrophages: first, an increased protein synthesis of bioactive substances together with a slight enhancement of the cell size, and second, an increased phagocytosis together with an enormous number of great lysosomes and an enormous enlargement of KC size, as shown recently in reoxygenated rat liver (33). As a morphological sign for the “switch on” of the synthesis of bioactive proteins, a particularly well-defined rough endoplasmic reticulum and many well-constructed Golgi complexes developed in such KCs as seen in Figures 2 and 3. In comparison with these activated macrophages, inactive KCs as seen in hypoxic livers were small and flattened, resembling endothelial cells (33).
Stimulated KCs express a wide variety of hepatotoxic substances, including cytokines such as IL-1, IL-6, and TNF-α (9). These mediators are not constitutively expressed, but a cytokine network causes their release. In fact, TNF-α induces IL-1 expression and vice versa. IL-1 and TNF-α both induce IL-6 expression (10). TNF-α and macrophage colony-stimulating factor are able to activate the transcription factor NF-κB in rat KCs, thereby suggesting an autostimulatory loop for TNF-α production (34).
In liver diseases, cytokines are involved from the onset of intrahepatic immune responses, exerting specific proinflammatory effects (35, 36) and up-regulation of both intercellular adhesion molecule-1 gene expression and protein secretion (37). Recently, Green et al. (12) investigated the effects of IL-6 on transport functions in cultured hepatocytes and detected a 55% decrease of Na+,K+-ATPase activity but not of BC Mg++-ATPase activity. The inhibition of Na+,K+-ATPase activity by IL-6 provides a putative mechanism for the observed inhibition of sodium-dependent taurocholate uptake. Hepatocyte bile acid uptake involves two transport systems: the first is an Na+-coupled cotransport system that uses the Na+ gradient across the sinusoidal membrane as the driving force. The second appears to be a Na+-independent system (38, 39). Na+,K+-ATPase activity is also inhibited in rat liver after hypoxia but recovered after reperfusion, as we have documented recently in an electron microscopic study (32). The abolition of enzyme activity causes a loss of the sodium-potassium pump that results in a disturbed homeostasis and in a reduced activity of symport and antiport membrane transport systems (40). The loss of Na+,K+-ATPase activity described in our experiments may be responsible for the decreased uptake of bile acids into hepatocytes and for cholestasis, and seems to be in accordance with clinicopathological characteristics of acute allograft rejection concerning the marked elevation of serum bile acid (41). In a different study, we investigated the effect of bile duct ligature on the activity of Na+,K+-ATPase. Cholestasis by bile duct ligature had no effect on the activity of Na+,K+-ATPase.6 Thus, we conclude that the loss of Na+,K+-ATPase activity in the hepatocellular plasma membrane is not only an indirect consequence of cholestasis, but is also directly associated with rejection.
In the apical membrane, several distinct ATP-dependent transport carriers for bile have been described: (1) P-glycoprotein (GP-170), which is responsible for the efflux of lipophilic, weakly basic, polycyclic compounds (42); (2) a transporter for non-bile acid organic anions (43); (3) a transporter for cysteinyl leukotrienes (44), and (4) the bile salt carrier for taurocholate (45-47). In hepatocytes of transport-deficient TR- mutant rats, multidrug resistance protein is not detectable in the canalicular membrane, but is detectable at the lateral plasma membrane. However, the protein is present in both membrane segments in normal rat liver (19).
Using a modified cerium method, we were able to demonstrate selectively BC Mg++-ATPase activity in the canalicular membrane. In accordance with the results of Green et al. (12), who demonstrated that IL-6 has no influence on BC Mg++-ATPase activity, we could also determine this enzyme activity in the rejection model. However, we did not find any activity of BC Mg++-ATPase in the lateral part of the hepatic plasma membrane, as shown by Mayer et al. (19). In our experiments, CsA did not affect the existence of BC Mg++-ATPase activity. It must be emphasized that the dose of CsA applied in our transplantation model is much less than the dose used in other studies (48). Pathomorphological changes of the bile canaliculi were dilatation as well as obstruction. For this process, an impairment of the actin filaments extending into the core of the microvilli forming the bile canaliculi must be responsible (49).
In conclusion, we demonstrated in a rejection model of rat liver an increasing number of activated KCs together with the loss of Na+,K+-ATPase activity in the basolateral domain of the hepatic plasma membrane. In contrast, the BC Mg++-ATPase activity in the apical domain of the hepatic plasma membrane was not influenced. The present results and those of Green and co-workers (12) concerning these two ATPases suggest a similar mechanism of interaction between activated macrophages and hepatocytes on the one hand and cultured hepatocytes treated with IL-6 on the other hand. Therefore, we assume that activated KCs release cytokines including IL-6, which inactivates Na+,K+-ATPase in vivo as well. As Na+,K+-ATPase is the cotransporter for hepatocyte taurocholate uptake, this process may be one of the key mechanisms for cholestasis during hepatic allograft rejection.
Acknowledgments. The excellent technical assistance of Barbara Lenschow is gratefully acknowledged.
This study was supported by a grant (An 192/1-2) from Deutsche Forschungsgemeinschaft, Bonn Bad-Godesberg.
Abbreviations: BC, bile canalicular; CsA, cyclosporine; DA, blood group D Agouti RT1; IL, interleukin; KC, Kupffer cell; LEW, Lewis; TNF, tumor necrosis factor.
Landmann L, Angermüller S, Rahner C, Stieger B. Expression, distribution, and activity of Na+,K+-ATPase in normal and cholestatic rat liver. Manuscript submitted for publication.
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