Observation and characterisation of macrophages in zebrafish liver.

Kupffer cells are liver-resident macrophages that play an important role in mediating immune-related functions in mammals and humans. They are well-known for their capacity to phagocytose large amounts of waste complexes, cell debris, microbial particles and even malignant cells. Location, appearance and functional aspects are important features used to identify these characteristic cells of the liver sinusoid. To-date, there is limited information on the occurrence of macrophages in zebrafish liver. Therefore, we aimed to characterise the ultrastructural and functional aspects of liver-associated macrophages in the zebrafish model by taking advantage of the latest advances in zebrafish genetics and multimodal correlative imaging. Herein, we report on the occurrence of macrophages within the zebrafish liver exhibiting conventional ultrastructural features (e.g. presence of pseudopodia, extensive lysosomal apparatus, a phagolysosome and making up ∼3% of the liver volume). Intriguingly, these cells were not located within the sinusoidal vascular bed of hepatic tissue but instead resided between hepatocytes and lacked phagocytic function. While our results demonstrated the presence and structural similarities with liver macrophages from other experimental models, their functional characteristics were distinctly different from Kupffer cells that have been described in rodents and humans. These findings illustrate that the innate immune system of the zebrafish liver has some distinctly different characteristics compared to other animal experimental models. This conclusion underpins our call for future studies in order to have a better understanding of the physiological role of macrophages residing between the parenchymal cells of the zebrafish liver.

Impact of renal ischemia/reperfusion injury on the rat Kupffer cell as a remote cell: A biochemical, histological, immunohistochemical, and electron microscopic study.

Almost all transplanted solid organs are exposed to some degree of ischemia-reperfusion (IR) damage. It is interesting to know that this IR damage affects various remote tissues including the liver and resulted in serious adverse effects. Liver injury triggers different responses of liver tissue especially Kupffer cells (KCs). The goal of this current study is to assess the biochemical and morphological changes of hepatic KCs after the induction of renal ischemia-reperfusion (RIR) and point out their role in remote liver injury after RIR. Sixteen male Sprague-Dawley rats were randomly divided into two equal groups: Group I; sham group. Group II; renal ischemia reperfusion (IR) group in which rats were exposed to renal ischemia for 45 min followed by renal reperfusion for 48 h. Three rats from each group were subjected to charcoal injection to evaluate KCs activity. Specimens of rat liver from each group were obtained and processed for biochemical, light microscopic and ultramicroscopic examination. The current results showed elevated serum levels of AST and ALT. The liver HGF-α protein expression increased in IR group compared to the sham group. In IR group, numerous charcoal labeled KCs were observed mainly localized around the central vein. Scanning electron micrographs showed complex primary and secondary foot process of the KCs. Ultrastructural study showed KCs with multiple cytoplasmic vacuoles, lysosomes and mitochondria, rough endoplasmic reticulum and ribosomes. Immuno-histochemical study showed more tumor necrosis factor-α (TNF-α) expression in KCs than the sham group. These results collectively demonstrated that renal IR produced biochemical and morphological changes in the liver KCs and theses cells might have a role in the remote liver injury after renal IR. This might be one of the mechanisms through which RIR affects the liver.

Cellular and stromal elements organization in the liver of grass carp, Ctenopharyngodon idella (Cypriniformes: Cyprinidae).

Fish liver is considered as a key organ that controls various life functions. The cellular and stromal elements of liver of eighteen specimens of adult grass carp were investigated by light- and electron- microscopy and enzyme histochemistry. The liver was formed of two lobes with a long process extended from the right lobe. Serial paraffin section of the liver identified different kinds of vascular- biliary structures as follows: 1) pancreatic-venous-biliary-arteriolar tracts (P-VBAT); 2) venous-biliary-arteriolar tracts, (VBAT); 3) pancreatic-venous-biliary tracts (P-VBT); 4) venous-biliary tracts (VBT); 5) venous-arteriolar tracts (VAT); 6) isolated veins named as venous tracts (VT); 7) isolated bile ducts, named as biliary tracts (BT); 8) biliary-arteriolar tracts (BAT); 9) pancreatic-biliary tracts (P-BT); 10) pancreatic- venous tracts (P-VT). Macrophages aggregates were associated with VBT and P-BT. The hepatic parenchyma was consisted of many populations of cells. Histochemically, the hepatocytes were strongly reacted with PAS, and Best's carmine. Moreover, strong staining patterns for acid phosphatase, ATPase, and alkaline phosphatase were demonstrated in hepatocytes. The hepatic satellate (Ito) cells were observed in the space of Disse and between hepatocytes. Rodlet cells and eosinophilic granular/ mast cells were encountered in the liver of grass carp. The sinusoids were lined by fenestrated endothelial cells and Kupffer cells. Moreover, dendritic-like cells were demonstrated in the sinusoids and perisinusoidal connective tissue. The biliary duct system was constituted of bile canaliculi, ductules, and bile ducts. Telocytes with their characteristics telopodes were located around bile ducts. The current findings are offering fundamental data on histology of grass carp liver.

[Inhibition of nuclear factor kappa B mediated by miRNA plasmid carried by novel polyethyleneimine nanogel particles in Kupffer cells of rats].

OBJECTIVE: To test a novel gene carrier, named polyethyleneimine nanogel particles (PEI-NP), for delivering the mircroRNA (miRNA) into Kupffer cells (KCs) for silencing the expression of nuclear transcription factor κB (NF-κB) P65. METHODS: The capacity of PEI-NPs to carry miRNA was tested by gel electrophoresis. RAW264.7 cell line was transfected with the cationic polymers which mixed together PEI-NP and miRNA. Then, the cytotoxicity of the RAW264.7 cells was detected by cell counting kit-8 (CCK-8); the cell apoptosis and transfection efficiency were analyzed by flow cytometry combined with annexin V-FITC/PI staining; the gene and protein levels of NF-κB P65 were determined respectively by real-time quantitative PCR (qRT-PCR) and Western blotting. The distribution of the cationic polymers in RAW264.7 cells and liver KCs was observed by transmission electron microscope (TEM). The protein level of NF-κB P65 in KCs was detected by immunohistochemistry. RESULTS: PEI-NPs were able to completely combine with miRNA at the least mass ratio of 20:1. The highest transfection efficiency of 48 hours was (33.63±1.94)% in vitro. The protein level of NF-κB P65 at 48 hours of PEI-NP/miRNA transfection into RAW264.7 cells was significantly inhibited. TEM showed the cationic polymers in RAW264.7 cytoplasm and only in KCs of liver tissue after transfection. Immunohistochemistry indicated that the protein expression of NF-κB P65 in KCs was significantly lower than that in control group. CONCLUSION: Macrophages and KCs were successfully transfected with the cationic polymers in vitro and in vivo, respectively. The expression of NF-κB P65 was knocked down significantly after transfection.

Liver gender dimorphism--insights from quantitative morphology.

It was shown recently that many genes are differentially expressed in the liver of males and females, thus strengthening the concept of liver gender dimorphism. This dimorphism exists in many pathological scenarios, from regeneration to fibrosis, which has led to the development of gender hepatology. Nevertheless, it is still unknown if gender dimorphism occurs in the structure of the normal liver. In recent years, it has been shown that, compared with male, the female rat liver bears less fibrotic tissue, more Kupffer cells (per volume unit) and has higher hepatocellularity, including binucleated hepatocytes (per volume unit). Our hypothesis is that the human liver also hides a gender dimorphic pattern. Baseline differences in fibrotic tissue would contribute to explain severe liver fibrosis in men. As to the disparity of Kupffer cells, this would clarify the stronger response to post-surgery infections in women, and it could be equated when appraising the higher susceptibility to alcohol. Regarding differences in hepatocytes, they not only justify existing differences in some liver parameters (e.g., transaminases and bilirubin), but they could also account for the higher regenerative potential of the female liver. The structural dimorphism in the human liver would sustain the concept of gender hepatology and, eventually, should be considered in the context of liver transplantation.

Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells.

Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1(gt/gt) mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1(gt/gt) liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1(gt/gt) Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1(gt/gt) mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage.

The mechanism of anti-CD20-mediated B cell depletion revealed by intravital imaging.

Anti-CD20 Ab therapy has proven successful for treating B cell malignancies and a number of autoimmune diseases. However, how anti-CD20 Abs operate in vivo to mediate B cell depletion is not fully understood. In particular, the anatomical location, the type of effector cells, and the mechanism underlying anti-CD20 therapy remain uncertain. Here, we found that the liver is a major site for B cell depletion and that recirculation accounts for the decrease in B cell numbers observed in secondary lymphoid organs. Using intravital imaging, we established that, upon anti-CD20 treatment, Kupffer cells (KCs) mediate the abrupt arrest and subsequent engulfment of B cells circulating in the liver sinusoids. KCs were also effective in depleting malignant B cells in a model of spontaneous lymphoma. Our results identify Ab-dependent cellular phagocytosis by KCs as a primary mechanism of anti-CD20 therapy and provide an experimental framework for optimizing the efficacy of therapeutic Abs.

Rapid and efficient clearance of blood-borne virus by liver sinusoidal endothelium.

The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity.

Effect of preischemic treatment with fenofibrate, a peroxisome proliferator-activated receptor-α ligand, on hepatic ischemia-reperfusion injury in rats.

Liver ischemia/reperfusion (I/R) injury is a serious clinical problem. The reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) are important mediators in liver I/R injury. This study was designed to investigate the effect of preischemic treatment with fenofibrate (Peroxisome proliferator-activated receptor- α agonist) on the oxidative stress and inflammatory response to hepatic I/R injury in rats. Hepatic I/R was induced by clamping the blood supply of the left lateral and median lobes of the liver for 60 min, followed by reperfusion for 4 h. Each animal group was pretreated with a single dose of fenofibrate (50 mg/kg body weight) intraperitoneally 1 h before ischemia. At the end of reperfusion, blood samples and liver tissues were obtained to assess serum alanine aminotransferase (ALT), TNF-α, hepatic malondialdehyde (MDA) and superoxide dismutase activity (SOD). Liver specimens were obtained and processed for light and electron microscopic study. Hepatic I/R induced a significant elevation of serum ALT and TNF-α with significant elevation of hepatic MDA and reduction of SOD activity. Histopathological examination revealed hepatic inflammation, necrosis and apoptosis. Preischemic treatment with fenofibrate at a dose of 50 mg/kg significantly attenuated the biochemical and structural alterations of I/R-induced liver injury.

The biological properties of iron oxide core high-density lipoprotein in experimental atherosclerosis.

Lipoproteins are a family of plasma nanoparticles responsible for the transportation of lipids throughout the body. High-density lipoprotein (HDL), the smallest of the lipoprotein family, measures 7-13 nm in diameter and consists of a cholesteryl ester and triglyceride core that is covered with a monolayer of phospholipids and apolipoproteins. We have developed an iron oxide core HDL nanoparticle (FeO-HDL), which has a lipid based fluorophore incorporated in the phospholipid layer. This nanoparticle provides contrast for optical imaging, magnetic resonance imaging (MRI) and transmission electron microscopy (TEM). Consequently, FeO-HDL can be visualized on the anatomical, cellular and sub-cellular level. In the current study we show that the biophysical features of FeO-HDL closely resemble those of native HDL and that FeO-HDL possess the ability to mimic HDL characteristics both in vitro as well as in vivo. We demonstrate that FeO-HDL can be applied to image HDL interactions and to investigate disease settings where HDL plays a key function. More generally, we have demonstrated a multimodal approach to study the behavior of biomaterials in vitro as well as in vivo. The approach allowed us to study nanoparticle dynamics in circulation, as well as nanoparticle targeting and uptake by tissues and cells of interest. Moreover, we were able to qualitatively assess nanoparticle excretion, critical for translating nanotechnologies to the clinic.

Responses to cadmium intoxication in the liver of the wall lizard Podarcis sicula.

This study examined the cytological and molecular effects of cadmium, a toxic heavy metal, in the liver of the Italian wall lizard Podarcis sicula. Cadmium was administered in single dose, by diet, to induce a concentration comparable with that measured in animals living in contaminated sites. For comparison, cadmium was also administered in multiple doses by food (chronic) or in a single dose intraperitoneally (i.p.); the effects were followed at regular time intervals up to 30 days post treatments. Atomic absorption spectrometry analysis demonstrated cadmium ion uptake and accumulation in the parenchyma with an estimated half-life of approximately 8 days. Cytological analyses revealed that the metal induced oedema, activated metallothionein expression in Kupffer cells and extracellular matrix production in fat storing cells. It also caused swelling and alteration in lipid and sugar metabolism in hepatocytes. In conclusion, in the wall lizard cadmium is toxic to the liver even at very low concentrations, the response is not strictly dose and time dependent and almost no recovery occurs in short (30 days) time periods.

Iron distribution in the liver and duodenum during seasonal iron overload in Svalbard reindeer.

Seasonal iron overload in Svalbard reindeer was studied by light and electron microscopy and by X-ray microanalysis. The hepatic iron overload was of two types. The first type was characterized by massive siderosis of both parenchymal and non-parenchymal cells caused by a diet very rich in iron but low in energy and protein. Hepatocytes contained a moderate amount of free ferritin particles in the cytosol together with numerous siderosomes. This pattern is similar to that seen in primary haemochromatosis and thalassaemia. Kupffer cells contained large quantities of cytosolic ferritin, siderosomes and lysosomes with disintegrating red blood cells as seen in thalassaemia. The second type was characterized by massive non-parenchymal siderosis caused by an energy- and protein-poor diet with normal iron concentration. Hepatocytes contained little cytosolic ferritin and few siderosomes, but there were abundant electron-dense bodies without iron (i.e., autophagosomes). Kupffer cells were as described above. Ferritin was also present within the duodenal mucosa of these animals, located within enterocytes and lamina propria macrophages, as well as in the extracellular space and capillary and lacteal lumina. Ferritin was also present in the acinar cells of submucosal Brunner's glands. Changes consistent with exchange of ferritin particles between different cell types were observed. The role of ferritin as a possible iron transporter in this condition is discussed.

Cellular organization of normal mouse liver: a histological, quantitative immunocytochemical, and fine structural analysis.

The cellular organization of normal mouse liver was studied using light and electron microscopy and quantitative immunocytochemical techniques. The general histological organization of the mouse liver is similar to livers of other mammalian species, with a lobular organization based on the distributions of portal areas and central venules. The parenchymal hepatocytes were detected with immunocytochemical techniques to recognize albumin or biotin containing cells. The macrophage Kupffer cells were identified with F4-80 immunocytochemistry, Ito stellate cells were identified with GFAP immunocytochemistry, and endothelial cells were labeled with the CD-34 antibody. Kupffer cells were labeled with intravascularly administered fluorescently labeled latex microspheres of both large (0.5 mum) and small (0.03 mum) diameters, while endothelial cells were labeled only with small diameter microspheres. Neither hepatocytes nor Ito stellate cells were labeled by intravascularly administered latex microspheres. The principal fine structural features of hepatocytes and non-parenchymal cells of mouse liver are similar to those reported for rat. Counts of immunocytochemically labeled cells with stained nuclei indicated that hepatocytes constituted approximately 52% of all labeled cells, Kupffer cells about 18%, Ito cells about 8%, and endothelial cells about 22% of all labeled cells. Approximately, 35% of the hepatocytes contained two nuclei; none of the Kupffer or Ito cells were double nucleated. The presence of canaliculi and a bile duct system appear similar to that reported for other species. The cellular organization of the mouse liver is quite similar to that of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver structure and function.

Ultrastructure of oval cells in children with chronic hepatitis B, with special emphasis on the stage of liver fibrosis: the first pediatric study.

AIM: To investigate the ultrastructure of oval cells in children with chronic hepatitis B, with special emphasis on their location in areas of collagen fibroplasia. METHODS: Morphological investigations were conducted on biopsy material obtained from 40 children, aged 3-16 years with chronic hepatitis B. The stage of fibrosis was assessed histologically using the arbitrary semiquantitative numerical scoring system proposed by Ishak et al. The material for ultrastructural investigation was fixed in glutaraldehyde and paraformaldehyde and processed for transmission-electron microscopic analysis. RESULTS: Ultrastructural examination of biopsy specimens obtained from children with chronic hepatitis B showed the presence of two types of oval cells, the hepatic progenitor cells and intermediate hepatic-like cells. These cells were present in the parenchyma and were seen most commonly in areas of intense periportal fibrosis (at least stage 2 according to Ishak et al) and in the vicinity of the limiting plate of the lobule. The activated nonparenchymal hepatic cells, i.e. transformed hepatic stellate cells and Kupffer cells were seen in close proximity to the intermediate hepatic-like cells. CONCLUSION: We found a distinct relationship between the prevalence of oval cells (hepatic progenitor cells and intermediate hepatocyte-like cells) and fibrosis stage in pediatric patients with chronic hepatitis B.

An adult case with Hunter's syndrome presenting prominent hepatic failure: light and electron microscopic features of the liver.

We describe a 40-year-old male patient with Hunter's syndrome. His main manifestations were ascites and esophageal varices due to cirrhotic liver. We obtained hepatic biopsy samples and examined them. Ultrastructurally, the features of the hepatocytes and Kupffer cells were the same as those reported in young patients. The passage of 40 years led to gradual progression to fibrosis, and ultimately liver cirrhosis. Namely, with a longer survival time, the complications of liver cirrhosis become more remarkable. Hepatic fibrosis in Hunter's syndrome is slowly progressive and patients who are expected to have a longer life span should be continuously monitored for hepatic complications.

Plasma chitotriosidase in male Fabry patients: a marker for monitoring lipid-laden macrophages and their correction by enzyme replacement therapy.

Increased plasma chitotriosidase is a well established surrogate marker for the occurrence of lipid-laden macrophages in the glycosphingolipidosis Gaucher disease. The complete lack of surrogate markers for Fabry disease, X-linked globotriaosylceramidosis stemming from deficiency in the lysosomal alpha-galactosidase A (AGA), prompted us to study chitotriosidase in this disorder. In male Fabry patients plasma chitotriosidase is significantly elevated, consistent with the presence of lipid-laden macrophages in several tissues. Increased levels are detectable at very young age and precede clinical manifestations. No strict correlation exists with severity of disease manifestations. Upon therapy with either of the two available recombinant AGA preparations, plasma chitotriosidase levels are nicely normalized in male Fabry patients. However, in patients developing neutralizing antibodies towards AGA, reduction in plasma chitotriosidase is hampered. In sharp contrast to the situation in male patients, females heterozygous for AGA deficiency show no significantly elevated plasma chitotriosidase. This suggests that circulating endogenous AGA in heterozygotes is sufficient to supplement enzyme-deficient macrophages. In conclusion, for the first time a biological marker for lipid-laden cells in Fabry patients is demonstrated; elevated plasma chitotriosidase levels reflecting lipid-laden macrophages. Corrections in this marker illustrate the efficacy of enzyme replacement therapy in clearing the lipid accumulation in this particular cell type.

Ultrastructure of Kupffer cells and hepatocytes in the Dubin-Johnson syndrome: a case report.

Ultrastructure of Kupffer cells and hepatocytes in liver bioptate was evaluated in a 17-year-old boy with Dubin-Johnson syndrome (DJS). The liver tissue obtained by needle biopsy was fixed in glutaraldehyde and paraformaldehyde and routinely processed for electron microscopic analysis. The ultrastructural examinations of liver bioptate revealed the accumulation of membrane-bound, electron-dense lysosomal granules within the cytoplasm of hepatocytes, characteristic of DJS. They were located mainly in the vicinity of the biliary pole, and preferentially in the centrilobular region that corresponded to the pigment deposits seen under light microscope. The presence of the granules was accompanied by dilated elements of the granular endoplasmic reticulum and paracrystalline mitochondrial inclusions as well as dilation of the bile canaliculi. The changes in hepatocytes co-existed with marked stimulation and enhanced phagocytic activity of Kupffer cells. This was manifested in the accumulation of pigment deposits within their cytoplasm that corresponded to those observed in hepatocytes. Hyperactive pericentral Kupffer cells which are involved in the response to pigmentary material originating from disintegrated hepatocytes may play an essential role in the development of DJS.

Rapid Kupffer cell death after intravenous injection of adenovirus vectors.

When adenovirus vectors are injected intravenously, they are quickly taken up by Kupffer cells in the liver. We report that this causes rapid necrosis of Kupffer cells in mice at doses of 10(11) particles/kg or higher. By 10 min after intravenous vector injection, Kupffer cells were permeable to propidium iodide and trypan blue. This coincided with a sharp rise in serum lactate dehydrogenase. Ultrastructural examination showed degeneration of Kupffer cells, including complete disappearance of chromatin by 1 h. After an initial intravenous injection of vector, dead Kupffer cells were unable to take up a second dose of vector, and hepatic transgene expression from the second dose was augmented. Death of Kupffer cells did not affect serum levels of IL-6 or IL-12. There was no immediate change in the number of Kupffer cells in the liver, but a significant decline was found by 4 h after injection of vector. Interestingly, substantial numbers of vector-containing Kupffer cells were found in pulmonary capillaries, indicating that they had been swept out of the liver. Together these results show that an intravenous injection of adenovirus vector causes synchronous and surprisingly rapid Kupffer cell death.

The greater susceptibility of North Ronaldsay sheep compared with Cambridge sheep to copper-induced oxidative stress, mitochondrial damage and hepatic stellate cell activation.

Sheep of the semi-feral North Ronaldsay (copper-sensitive) and domesticated Cambridge (copper-tolerant) breeds were compared in respect of pathological changes and protein expression in the liver as a result of excessive dietary copper. Acute mitochondrial damage and hepatic stellate cell (HSC) activation with collagen synthesis occurred in response to moderate copper overload in North Ronaldsay but not in Cambridge sheep. Mitochondrial degradative changes occurred either as ballooning degeneration and rupture with subsequent autophagic degradation or as mitochondrial matrical condensation (pyknosis). In North Ronaldsay sheep prolonged exposure to copper produced mitochondrial hyperplasia and hypertrophy, and nuclear damage with necrosis. Cytosolic isocitrate dehydrogenase (IDH), an enzyme responsive to oxidative stress, was induced in the liver of Cambridge sheep receiving a Cu-supplemented diet but was undetectable in the non-supplemented control sheep. Conversely, IDH was detected at similar levels in both control and copper-supplemented North Ronaldsay sheep, indicating a lower threshold response, and an enhanced susceptibility, to oxidative stress. "Upregulation" of mitochondrial thioredoxin-dependent peroxidase reductase (antioxidant protein-1) in the hepatic cytosol of the North Ronaldsay (but not Cambridge) sheep affirmed the increased susceptibility of the mitochondria to Cu-induced oxidative stress in this breed. Likewise the upregulation of cathepsin-D indicated increased lysosomal activity and HSC activation. The findings may be relevant to copper toxicosis in human infants.

Enhanced expression of the mannose receptor by endothelial cells of the liver and spleen microvascular beds in the macrophage-deficient PU.1 null mouse.

Mice null for the haematopoietic lineage-specific transcription factor PU.1 lack mature Mphi and are compromised in their ability to clear cellular debris from the blood circulation. We investigated the possibility that non-professional phagocytes may partially compensate for the lack of Mphi in clearance functions. In the absence of Kupffer cells (resident liver Mphi) in the PU.1 null mice, electron microscopy revealed ingested debris in sinusoidal endothelial cells and hepatocytes although debris was also seen free in blood vessels. To investigate whether an increased clearance function of non-professional phagocytes might be linked to expression of Mphi-associated phagocytic and pinocytic receptors by other cells in PU.1 null mouse, we examined expression of several candidate proteins by immunocytochemistry and Western blotting. We found mannose receptor (MR) comparably expressed in PU.1 null and PU.1+ mice liver and spleen whereas class A scavenger receptor was substantially reduced and complement receptor 3 was absent in PU.1 null animals. By morphometric analysis, liver and spleen sinusoidal endothelial cells were seen to express significantly more MR in the PU.1 null mouse. This study provides the first evidence of apparently compensatory alterations in the microvasculature of the Mphi-deficient PU.1 null mouse.