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Colorectal cancer (CRC) is a common malignant disease and the severe nature of cases in men and women who develop colorectal cancer makes this an important socio-economic health issue. Major challenges such as understanding and modeling colorectal cancer pathways rely on our understanding of simple models such as outlined in this paper. We discuss that the development of novel standardized approaches of multidimensional (correlative) biomolecular microscopy methods facilitates the collection of (sub) cellular tissue information in the early onset of colorectal liver metastasis and that this approach will be crucial in designing new effective strategies for CRC treatment. The application of X-ray micro-computed tomography and its potential in correlative imaging of the liver vasculature will be discussed.

Liver sinusoids harbor populations of 2 important types of immunocompetent cells, Kupffer cells (KCs) and natural killer (NK) cells, which are thought to play an important role in controlling hepatic metastasis in the first 24 hr upon arrival of the tumor cells in the liver. We studied the early interaction of KCs, NK and CC531s colon carcinoma cells in a syngeneic rat model by confocal laser scanning microscopy. Results showed a minority of KCs (19% periportal and 7% pericentral) involved in the interaction with 94% of tumor cells and effecting the phagocytosis of 92% of them. NK cell depletion decreased the phagocytosis of tumor cells by KCs by 33% over a period of 24 hr, leaving 35% of the cancer cells free, as compared to 6% in NK-positive rats. Surviving cancer cells were primarily located close to the Glisson capsule, suggesting that metastasis would initiate from this region.

Hepatic NK cells are more cytotoxic than blood NK cells against tumor cells. To understand the basis of this difference in cytotoxicity we analyzed RNA derived from freshly isolated rat blood and hepatic NK cells [high-density (HD) and low-density subpopulations] by high-density oligonucleotide arrays (Affymetrix), containing about 9,000 genes and expressed sequence tags. IL-2-treated blood NK (A-NK) cells and IL-2-treated hepatic HD cells were used as a reference of NK cell activation. About 150 genes and expressed sequence tags were differentially expressed between hepatic and blood NK cells. Surprisingly, more than half of the increased expressed genes in hepatic NK cells were not increased in A-NK cells. Differentially expressed genes like the stem cell factor receptor c-kit and the chemokine receptor CCR5 can contribute to the homing and differentiation of hepatic NK cells in the liver sinusoids. Several of the differentially expressed genes can possibly contribute to the enhanced cytotoxic activity of hepatic NK cells: cell membrane receptors like NKG2D, NKG2C, CD94, ecto-ATPase; signaling molecules like phosphatidylinositol 3-kinase; granule-associated effector molecules like granzymes and defensin NP3. Moreover, it appears that redirection of cytotoxic granules and increase in intracellular Ca2+ are convergence points of several of these genes.

It is now well established that angiogenesis in multiple myeloma (MM) is associated with poor prognosis. The exact function of the newly formed vessels in MM is, however, a matter of debate. It is believed that, in contrast to solid tumor growth, the bone marrow (BM) is a sufficiently vascularized organ to support expansion of the MM clone with no need for additional blood vessels. From this point of view, it could be that MM-associated angiogenesis is rather an epiphenomenon and that the newly formed microvessels form a chaotic network that does not contribute to the blood flow. We investigated whether these newly formed microvessels in MM are connected to the blood circulation. The intravenously injected ferritin 30 min prior to sacrifice was detected in 100% of the BM vessels of control mice. In MM-bearing mice, the ferritin tracer was found in 31% of the MM-associated vessels, indicating a connection with the peripheral blood circulation in these vessels. We conclude that, comparable to the situation in solid tumors, at least part of the tumor-associated microvessels in MM is functionally connected to the blood circulation and, therefore, can participate in the transport of nutrients and in the dissemination of MM cells.

Liver sinusoidal endothelial cells (LSECs) can optimally be imaged by whole mount transmission electron microscopy (TEM). However, TEM allows only investigation of vacuum-resistant specimens and this usually implies the study of chemically fixed and dried specimens. Cryo-electron microscopy (cryo-EM) can be used as a good alternative for imaging samples as whole mounts. Cryo-EM offers the opportunity to study intact, living cells while avoiding fixation, dehydration and drying, at the same time preserving all solubles and water as vitrified ice. Therefore, we compared the different results obtained when LSECs were vitrified using different vitrification conditions. We collected evidence that manual blotting at ambient conditions and vitrification by the guided drop method results in the production of artefacts in LSECs, such as the loss of fenestrae, formation of gaps and lack of structural details in the cytoplasm. We attribute these artefacts to temperature and osmotic effects during sample preparation just prior to vitrification. By contrast, by using an environmentally controlled glove box and a vitrification robot (37 degrees C and 100% relative humidity), these specific structural artefacts were nearly absent, illustrating the importance of controlled sample preparation. Moreover, data on glutaraldehyde-fixed cells and obtained by using different vitrification methods suggested that chemical prefixation is not essential when vitrification is performed under controlled conditions. Conditioned vitrification therefore equals chemical fixation in preserving and imaging cellular fine structure. Unfixed, vitrified LSECs show fenestrae and fenestrae-associated cytoskeleton rings, indicating that these structures are not artefacts resulting from chemical fixation.

Liver sinusoidal endothelial cells (LSECs) possess fenestrae arranged in sieve plates. Hepatic endothelial fenestrae are open pores approximately 100 to 200 nm in diameter. Alterations in their number or diameter by hormones, xenobiotics, and diseases have important implications for hepatic microcirculation and function. Numerous reports of hepatotoxin-induced defenestration suggest that the cytoskeleton and the energy status of hepatic endothelial cells play a key role in the regulation of fenestrae. Therefore, we investigated the effect of antimycin A, an inhibitor of mitochondrial energy production, on the number of fenestrae in cultured LSECs using high-resolution microscopy and immunocytochemistry. Prolonged incubation (greater than 30 min) with antimycin A resulted in defenestrated cells and coincided with the appearance of F-actin dots, whereas the distribution of G-actin remained unchanged. Adenosine triphosphate (ATP) was depleted dramatically to less than 5% within 30 minutes within the LSECs. After treatment with antimycin A, unusual elevated fenestrated complexes were apparent, organized as a meshwork of anastomosing fenestrae at the center of and above the sieve plates. The position and appearance of these novel structures and their association with defenestration suggest that they are implicated in the process of defenestration. In conclusion, the results of experiments with antimycin A suggest that ATP is needed to maintain fenestrae and the underlying fenestrae-associated cytoskeleton rings that maintain fenestrae patency. Antimycin A-induced defenestration of LSECs is associated with the development of a structure in the sieve plate that appears to be intrinsically involved in defenestration.

This review provides a detailed overview of the current state of knowledge about the ultrastructure and dynamics of liver sinusoidal endothelial fenestrae. Various aspects of liver sinusoidal endothelial fenestrae regarding their structure, origin, species specificity, dynamics and formation will be explored. In addition, the role of liver sinusoidal endothelial fenestrae in relation to lipoprotein metabolism, fibrosis and cancer will be approached.

AIM:Several triggering receptors have been described to be involved in natural killer (NK) cell-mediated target cytotoxicity. In these studies, NK cells deriv ed from blood or spleen were used. Pit cells are liver-specific NK cells that possess a higher level of natural cytotoxicity and a different morphology when compared to blood NK cells. The aim of this study was to characterize the role of the NK triggering molecules NKR P1A, ANK61 antigen, and CD45 in pit cell medi ated killing of target cells.METHODS:( 51) Crrelease and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively.RESULTS:Flow cytometric analysis showed that pit cells expressed CD45, NK R P1A, and ANK61 antigen. Treatment of pit cells with monoclonal antibody (mAb)to CD45 (ANK74) not only inhibited CC531s or YAC 1 target lysis but also apopt osis induced by pit cells. The mAbs to NKR P1A (3.2.3) and ANK61 antigen (ANK61 )had no effect on pit cell mediated CC531s or YAC 1 target cytolysis or apopto sis, while they did increase the Fcgamma receptor positive (FcgammaR(+)) P815 cytolysi s and apoptosis. This enhanced cytotoxicity could be inhibited by 3,4 dichloroi socoumarin, an inhibitor of granzymes.CONCLUSION:These results indicate that CD45 participates in pit cell med iated CC531s and YAC-1 target cytolysis and apoptosis. NKR-P1A and ANK61 antigen on pit cells function as activation structures against Fc gammaR( +) P 815 cells, which was mediated by the perforin/granzyme pathway.

The atomic force microscope (AFM) is a powerful tool to investigate surface and submembranous structures of living cells under physiological conditions at high resolution. These properties enabled us to study the interaction between live hepatic natural killer (NK) cells, also called pit cells, and colon carcinoma cells in vitro by AFM. In addition, the staining for filamentous actin and DNA was performed and served as a reference, because actin and nuclear observations at the light microscopic level during the cytotoxic interaction between these two cell types have been presented earlier. In this study, we collected evidence that conjugation of hepatic NK cells with CC531s colon carcinoma cells results in a decreased binding of CC531s cells to the substratum as probed with the AFM in contact mode as early as 10 min after cell contact (n = 11). To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was performed on hepatic NK/CC531s cell conjugates, resulting in identical observations (n = 3). In contrast, the first cytotoxic signs, as determined with the nuclear staining dye Hoechst 33342, could be observed 3 h after the start of the co-culture. This study illustrates that the AFM can be used to probe early cytotoxic effects of effector to target cell contact in nearby physiological conditions. Other routine cytotoxicity tests detect the first cytotoxic effects after 1.5-3 h co-incubation at the earliest.

Hepatic natural killer (NK) cells are located in the liver sinusoids adherent to the endothelium. Human and rat hepatic NK cells induce cytolysis in tumor cells that are resistant to splenic or blood NK cells. To investigate the mechanism of cell death, we examined the capacity of isolated, pure (90%) rat hepatic NK cells to kill the splenic/blood NK-resistant mastocytoma cell line P815. Cell death was observed and quantified by fluorescence and transmission electron microscopy, DNA fragmentation, and 51Cr release. RNA and protein expression were determined by real time reverse transcription-polymerase chain reaction and Western blotting. Compared with splenic NK cells, hepatic NK cells expressed higher levels of perforin and granzyme B and readily induced apoptosis in P815 cells. Although P815 cells succumbed to recombinant Fas ligand (FasL) or isolated perforin/granzyme B, hepatic NK cells used only the granule pathway to kill this target. In addition, hepatic NK cells and sinusoidal endothelial cells strongly expressed the granzyme B inhibitor, protease inhibitor 9 (PI-9)/serine PI-6 (SPI-6), and P815 cells and hepatocytes were negative. Transfection of target cells with this inhibitor resulted in complete resistance to hepatic NK cell-induced apoptosis. In conclusion, hepatic NK cells kill splenic/blood NK-resistant/FasL-sensitive tumor cells exclusively by the perforin/granzyme pathway. Serine protease inhibitor PI-9/SPI-6 expression in liver sinusoidal endothelial cells may protect the liver microenvironment from this highly active perforin/granzyme pathway used to kill metastasizing cancer cells.

21-Aminosteroids are potent anti-inflammatory and antioxidant drugs that provide remarkable endothelial protection in different models of tissue ischemia-reperfusion and inflammation. The effects of 21-aminosteroids in sepsis, a highly inflammatory condition leading to panendothelial activation and injury, are largely uninvestigated. We therefore explored the effects of the 21-aminosteroid U74386G on hepatic blood flow, endothelial cell function, and sinusoidal structure in a canine model of fluid-resuscitated, hyperdynamic endotoxic shock.Following invasive hemodynamic monitoring and placement of ultrasonic flow probes around the common hepatic artery and the portal vein, 12 anesthetized dogs received 2 mg/kg iv of Escherichia coli endotoxin, followed by generous saline infusion, before randomization into two groups. One group (N = 6) received U74389G as an iv bolus of 80 microg/kg, followed by a continuous infusion of 10 microg/kg. min. The other group (N = 6) received an equivalent volume of vehicle. Hyaluronic acid was measured in plasma for in vivo evaluation of endothelial cell function. Liver biopsies were taken after 4 h of endotoxic shock and prepared for light and electron microscopic examination.Compared with the vehicle-treated controls, U74389G maintained a higher blood flow in the hepatic artery and in the portal vein, without markedly influencing the systemic hemodynamic response. The endotoxin-induced increase in plasma hyaluronic acid levels was significantly attenuated following U74389G treatment (70 +/- 14 vs 188 +/- 24 ng/mL after 3 h of endotoxic shock; P0.05). Morphological studies showed that the U74389G-treated group had less sinusoidal endothelial cell damage together with a dramatic reduction of neutrophil infiltration into the liver tissue.U74389G can preserve the functional and structural integrity of endothelial cells in the hepatic sinusoid during hyperdynamic endotoxic shock. This endothelial-protective effect was associated with a better maintained hepatic blood flow and a significant attenuation of inflammatory liver injury.

Hepatic sinusoidal endothelial cells differ from other endothelial cells. They possess open fenestrae that are grouped in sieve plates and lack a basal lamina. Fenestrae measure about 150nm, occur at a frequency of 9–13/mm2, and occupy 6%– 8% of the endothelial surface (porosity). These filter characteristics determine the exchange between the blood and the parenchymal cells, and are influencing the transport of lipoproteins including cholesterol and vitamin A. Forced sieving and endothelial massage are thought to enhance the passage of lipoproteins and to refresh the fluids in the space of Disse. Alcohol, nicotine, and other agents affect fenestrae in different ways and contribute either to fatty liver or hyperlipidemia. Furthermore, sinusoidal endothelial cells have: 1) a large capacity for pinocytosis via coated pits and macropinocytotic vesicles, 2) a variety of adhesion molecules, and receptors (scavenger, hyaluronan, collagen, Fc), and 3) a large capacity to digest material by numerous lysosomes. Endothelial cells secrete a limited amount of cytokines, but are vulnerable to products released by Kupffer cells, as in the case of endotoxemia or liver preservation. Capillarization of sinusoids includes the loss of fenestrae and the formation of a basal lamina and occurs in pathological conditions.

Pit cells are liver-specific natural killer (NK) cells and belong to the group of sinusoidal cells, together with Kupffer, endothelial, and fat-storing cells. Pit cells are lymphoid cells containing specific granules, classifying them also as large granular lymphocytes (LGL). They probably originate from the bone marrow, circulate in the blood, and marginate in the liver, where they develop into pit cells by lowering their density and increasing the number of granules, which decrease in size. Pit cells remain in the liver about 2 weeks and are dependent on Kupffer cells. Pit cells also proliferate locally, when stimulated with interleukin-2, biological response modifiers, or other agents. Pit cells adhere to tumor target cells during killing. They possess a high level of natural cytotoxicity against a variety of tumor cell lines, which is comparable to the cytotoxicity level of lymphokine-activated killer (LAK) cells. Tumor cell killing is synergistically enhanced when pit cells attack tumor cells together with Kupffer cells. Further investigations are needed to clarify the mechanisms of pit cell cytotoxicity and the role of these cells in killing virus-infected cells, such as during viral hepatitis.

Liver sinusoidal endothelial cells (LEC) contain fenestrae, which control the exchange of fluids, solutes, and particles between the sinusoidal blood and the microvillous surface of the parenchymal cells. The surface of LEC can be imaged by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM and AFM images of LEC can be used to study dynamic changes in fenestrae by comparing specimens fixed after different experimental treatments. In this article, we report the different results obtained when contact (using a constant force) or noncontact (amplitude detection) imaging on the same cells was applied. Special attention was paid on the optimalization of the image acquisition of fenestrae, because quality SEM examinations of fenestrae have already extensively been described. The following advances and conclusions are presented here: 1) High-resolution imaging of slightly fixed LEC in fluid can be performed in noncontact AFM; 2) correct acquisition of images of fenestrae with regard to their size (O, ±200 nm) and shape (oval, without deformation) under liquid was possible with noncontact AFM, which was hitherto only feasible with fixed, dried, and coated LEC in contact AFM or SEM; 3) this mode of operation is more gentle to cells than contact mode; 4) images of LEC obtained in noncontact mode are of higher quality and are devoid of smearing artefacts, prominently present in contact-mode images; 5) it is of great importance to optimize feedback and scan parameters to obtain correct surface information; and 6) LEC isolated and cultured by our method are physiologically responsive and represent an ideal object for AFM studies, because the cells are thin, smooth, and well attached to the culture substrate and show dynamic fine structural details, such as fenestrae and coated pits, which cannot be seen by light microscopy. © 1997 John Wiley & Sons, Inc. Int J Imaging Syst Technol, 8, 162–167, 1997

Previous studies showed that blood large granular lymphocytes (LGL), which possess natural killer (NK) activity, develop within rat liver sinusoids into high-density (HD) and subsequently into low-density (LD) pit cells which show an increasing level and spectrum of tumor cytotoxicity. In this study, we investigated the role of adhesion molecules, such as CD2, CD11a, CD18, and CD54 in the recruitment of pit cells to the liver. Immunostaining for electron microscopy, and two color flow cytometry showed that most pit cells expressed CD2, CD11a, CD18, and CD54. After intravenous injections into rats with anti-CD2, anti-CD11a, and anti-CD18 antibodies, the number of pit cells per square millimeter in frozen sections of liver tissue decreased. Treatment of rats with zymosan increased the number of pit cells fivefold, whereas subsequent treatment with anti-adhesion-molecule antibodies resulted in approximately 60% lower number of pit cells. Anti-CD54, supposed to block CD54 expression on sinusoidal endothelial cells, also decreased the number of pit cells. The number of blood LGL was, however, not affected by these antibodies. These results indicate that blocking of CD2, CD11a, CD18, and CD54 antigens on blood LGL and/or liver endothelium decreased the number of pit cells in the liver. These adhesion molecules therefore play an important role in the recruitment of pit cells in the liver.

Transforming growth factor β (TGF-β), a potent fibrogenic cytokine, is secreted in latent form. We examined which cell type in both normal and carbon tetrachloride (CC1 4 )-induced fibrotic rat liver bears surface type II IGF/mannose 6-phosphate (IGF-II/M6P) receptor, known to facilitate activation of TGF-β. In addition, the role of the IGF-II/M6P receptor in activation of latent TGF-β was investigated in a coculture system with sinusoidal endothelial cells. Northern hybridization analysis for IGF-II/M6P receptor messenger RNA (mRNA) was performed on total RNA of different isolated and purified liver cell types. In normal liver, cells expressed little IGF-II/M6P receptor mRNA. In fibrotic liver, we found significant expression only in fat-storing cells. The presence of IGF-II/M6P receptors was established by [ 125 I]IGF-II binding assays on freshly isolated fat-storing cells from normal and CC1 4 -exposed rat livers. We found specific binding of [ 125 I]IGF-II only on CC1 4 exposed fat-storing cells. As determined by polyacrylamide gel electrophoresis after affinity labeling, the specific binding involved 220 kD type II IGF receptors. Scatchard analysis revealed the presence of 3,043 ± 1,378 IGF-II/M6P high-affinity receptors/fat-storing cell, with a Kd of 387 ± 165 pmol/L. With a mink lung epithelial cell (Mv 1 Lu) proliferation inhibition assay, inhibition of proliferation (a measure of active TGF-β function) was determined using conditioned media of activated fat-storing cells, cocultures of fat-storing cells, and endothelial cells and pure endothelial cell cultures. We found that conditioned media of cocultures of fat-storing cells and endothelial cells inhibits the growth of Mv 1 Lu cells more strongly than conditioned media of homotypic cultures. Addition of neutralizing anti-TGF-β antibodies neutralizes this effect. The contribution of IGF-II/M6P receptors on the cell membrane of activated fat-storing cells to the activation of latent TGF-β was demonstrated by incubating the cells with M6P, or antibodies directed to the IGFII/M6P receptor, both of which diminishes activation of TGF-β. We conclude that IGF-II/M6P receptors are present on activated fat-storing cells and that the presence of this receptor at the cell surface is necessary, but not sufficient, for activation of latent TGF-β. Other factors, derived from endothelial cells, are involved.

In the present study, we have investigated the correlation between hepatic fibrosis in rats subjected to bile duct ligation, the numbers of Kupffer and fat-storing cells, and the level of endotoxin in both the portal and systemic circulation. The extent of hepatic fibrosis was measured by morphometry. Kupffer cells were identified by indirect immunoperoxidase staining using ED-2 anti-macrophage antibody. Fat-storing cells were stained with DE-B-5 anti-desmin antibody. Endotoxin levels were determined by the Limulus Lysate test. Following bile duct ligation, connective tissue septa rapidly developed in periportal areas. After 1 week, the volume density of connective tissue had increased from 0.6 +/- 0.1% in control animals to 3.8 +/- 1.1%. After 2 weeks, this volume increased to 19.9 +/- 1.3%, and after 3 weeks to 34.3% +/- 2.7%. The number of periportal fat-storing cells increased 2.8-fold during the first 2 weeks, whereas pericentral fat-storing cells increased only 1.7-fold. After 2 weeks, no further increase was observed. During the first week of bile duct ligation, the number of Kupffer cells increased nearly two-fold. Thereafter, no further increase was detected. In control rats, only two of ten rats showed low amounts of endotoxin in the portal blood. Portal endotoxemia increased with time after bile duct ligation. After 3 weeks, all rats were positive. The measured endotoxin levels were approximately 7 times higher than in control rats. We conclude that the development of fibrosis secondary to experimental bile duct ligation is accompanied by protal endotoxemia, and increases in the numbers of Kupffer and periportal fat-storing cells. We found a significant correlation between portal endotoxemia, the number of Kupffer and fat-storing cells, and the extent of fibrous septa, supporting the view that high endotoxemia levels coincide with Kupffer cell activation and fibrogenesis.

Tanji et al1 have demonstrated in a rat model that interleukin-2 (IL-2) gene transfer to the liver prevents liver metastasis of colon carcinoma cells. IL-2 gene transfer was performed by intraportal injection of an adenovirus containing the human IL-2 gene (AdCMVhIL-2), before the administration of RCN-9 syngeneic colon carcinoma cells. Controls included injection of either adenovirus containing the -galactosidase gene or PBS. The authors suggested that hepatic natural killer (NK) cells, which are located in the liver sinusoids,2,3 are responsible for this effect. Indeed, they demonstrated that hepatic NK cells isolated from AdCMVhIL-2 rats showed an increased cytotoxic activity against the NK-sensitive YAC-1 target cell line, confirming the results of Shiratori et al.4 However, they were unable to show an increased cytotoxic effect of these isolated hepatic NK cells against the RCN-9 colon carcinoma cells.1 In other words, the in vivo effects of preventing liver metastasis of RCN-9 colon carcinoma cells in AdCMVIL-2 treated rats did not coincide with an increased activity of hepatic NK cells of these rats against the same target cell line.

The aim of the present study was to identify the cell types that express collagen alpha 1(I), alpha 1(III) and alpha 1(IV), fibronectin and laminin B1 genes in normal rat liver. Parenchymal, sinusoidal endothelial, Kupffer and fat-storing (Ito) cells were isolated and purified. Total RNA of the freshly isolated cells was subjected to Northern hybridization analysis. We also compared the steady state levels of specific mRNAs in freshly isolated fat-storing cells to the levels in myofibroblast-like cells obtained from purified fat-storing cells cultured for two passages. The average purity of each cell preparation, and the percentage of contaminating cells, were determined by transmission electron microscopy and by examining the presence of vitamin A-autofluorescent cells. Fibronectin and collagen alpha 1(III) mRNAs were detected in total RNA of purified parenchymal cells. In poly(A)+ enriched RNA, small amounts of collagen alpha 1(I) mRNA were also present. In total RNA of freshly isolated fat-storing cells, collagen alpha 1(III), alpha 1(IV), and laminin B1 transcripts were found, whereas collagen alpha 1(I) and fibronectin mRNAs were not detected. Cultured fat-storing cells, however, did contain high levels of collagen alpha 1(I) and fibronectin mRNAs. The molecular size of the latter transcript was larger than the fibronectin transcript found in parenchymal cells and the whole liver. Endothelial cells contained small amounts of alpha 1(IV) mRNA. Kupffer cells did not contain the investigated transcripts. We conclude that normal parenchymal, fat-storing and endothelial cells each express a typical pattern of connective tissue molecules. When fat-storing cells are allowed to differentiate into myofibroblast-like cells, they express high levels of collagen alpha 1(I) and fibronectin mRNAs, in addition to collagen alpha 1(III) and alpha 1(IV), and laminin B1 chain mRNAs.

The motility and deformability of lymphokine-activated killer cells, purified by their adherence to plastic (A-LAK cells), was investigated in vivo and in vitro. In vitro, A-LAK cells were observed as intrinsically motile cells. They continuously changed their shape while forming protopods or pseudopods and crawled over the culture surface. A-LAK cells were able to migrate across micropores of 3 μm diameter, which was three times smaller than the average diameter of an A-LAK cell. Even in the absence of serum factors and of interleukin-2 (IL-2), more than half of the inoculated cells migrated across such micropore membranes within 18 h. Electron microscopic examination of these micropore membranes showed that the A-LAK cells were highly deformable. A-LAK cells also migrated across a confluent monolayer of endothelium-like 10T1/2 cells. After 24 h incubation on the monolayers, about 20% of the A-LAK cells were found underneath the monolayer. There, they actively moved in the narrow space between the monolayer and the bottom of the culture dish. In vivo IL-2-activated cells showing the large granular lymphocyte morphology accumulated in the hepatic microvasculature. Electron microscopic observations indicate that these cells did not accumulate by mechanical entrapment due to rigidity and size but rather by adherence to the endothelial lining of the sinusoidal capillaries. This also appeared to be the case for adoptively transferred A-LAK cells, which were seen to be attached to the capillary wall. These observations stress the importance of adhesive entrapment to explain the accumulation of intravenously transferred LAK cells in the vascular beds.

There is now sufficient evidence to consider the pit cells as a hepatic, intrasinusoidal population of large granular lymphocytes. They should be considered as resident sinusoidal cells of the liver, even if they depend on a continuous influx of precursor cells from the bone marrow, since they differ in several phenotypic and functional respects from their circulating (precursor) counterparts. There is growing evidence that a comparable population of activated or further differentiated, liver-specific pit cells (or LGL) is not only present in the rat but also in the human liver. Pit cells exert high spontaneous cytotoxic activity against tumor cell lines and may act as a primary defense barrier to metastasizing tumor cells and to virus infections. The total number of pit cells in the liver can be greatly increased by the administration of interleukin-2 or by a variety of biological response modifiers, which may represent a mechanism of defense against disease. Their involvement in liver pathologies deserves more attention.

Background: We have previously shown that the size of sinusoidal fenestrae is a critical determinant of hepatocyte transduction after adenoviral transfer (Lievens et al. Gene Therapy 2004; 11: 1523-1531). Previous in vitro studies have demonstrated that ethanol increases the size of fenestrae. The diameter of fenestrae in New Zealand White (NZW) rabbits is approximately 1.7-fold smaller than in C57BL/6 mice. Therefore, the hypothesis of this study was that intravenous administration of ethanol before transfer may increase transgene expression in NZW rabbits but not in C57BL/6 mice.

The hepatotropism and intrahepatic distribution of adenoviral vectors may be species dependent. Hepatocyte transduction was evaluated in three rabbit strains after transfer with E1E3E4-deleted adenoviral vectors containing a hepatocyte specific α1 -antitrypsin promoter driven expression cassette (AdAT4). Intravenous administration of 4 × 1012 particles/kg of AdAT4 induced human apo A-I levels above 40 mg/dl in Dutch Belt, but below 1 mg/dl in New Zealand White and Fauve de Bourgogne rabbits. Human apo A-I levels did not correlate with transgene DNA levels, but with human apo A-I mRNA/DNA ratio in the liver. This human apo A-I mRNA/DNA ratio in the liver was 1.6 ± 0.37 at day 14 in Dutch Belt rabbits and was 77-fold (p

Colorectal cancer cells can induce apoptosis in cells of various tissues [1]. Apoptosis can be induced by a number of factors such as Fas inducing apoptosis through the Fas/FasL pathway; other factors involve the TRAIL pathway and TNF. It is known that metastasizing colon cancer cells express more FasL then primary carcinoma cells [2]. We investigated whether a rat colon carcinoma cell line CC531s could induce apoptosis in liver sinusoidal endothelial cells (LSECs). LSECs and CC531s were co-cultured for 18 hrs and cells were visualized by SEM and TEM. Apoptosis was visualized by markers such as Hoechst and Propidium iodide. Furthermore, cells were recorded by time lapse video microscopy with and without an antagonistic antibody for FasL.

Comparative Hepatology is an Open Access, peer-reviewed online journal for liver oriented research, publishing basic and applied studies, in biology, veterinary and human medicine. The journal is published by BioMed Central.

Hepatic natural killer (NK) cells, also known as pit cells, are located in the liver sinusoids, adhering to the endothelial cells (LSECs), and are thus in a strategic position to kill arriving metastasizing tumor cells [1-3]. NK cells of different tissue origin (blood, spleen, liver) appear to have different levels of cytotoxicity. Lower levels can be enhanced by lymphokines such as interleukin-2 (IL-2) or IL-12, providing lymphokine-activated killer (LAK) cells [1]. P815 mastocytoma cells were found to be resistant to the induction of cytolysis (quantified by 51Cr release) by NK cells from spleen or blood, but are sensitive to hepatic NK and LAK cells [[1,3] and references therein]. Hepatic NK cells therefore might be considered as naturally activated LAK cells. Cytotoxic lymphocytes (NK cells, LAK cells, cytotoxic T cells, NK-T cells) use the FasL and the perforin/granzyme pathway to kill target cells [3]. FasL on effector cells binds Fas present on the target cell membrane, which results in oligomerization of Fas and activation of caspase 8. Perforin and granzymes, of which granzyme B is the most potent, reside in granules of the cytotoxic lymphocytes and are released by exocytosis. Intracellular delivery of granzyme B results in the initiation of the caspase cascade by proteolytic activation of caspase 3, either directly [4] or through a mitochondrium-dependent pathway [5]. Caspases play a central role in the execution of apoptosis [4]. In this study, we investigated the mechanism hepatic NK cells use to kill P815 cells.

Liver sinusoidal endothelial cells (LSECs) possess fenestrae whose number can be increased both in vitro and in situ by depolymerizing the actin cytoskeleton [1]. Specially designed liposomes can be targeted with a high efficiency to LSECs. These liposomes, which were surface grafted with poly-anionized albumin (Aco-HSA) [2], can be filled with various substances, such as microfilament-disrupting drugs. This technique opens up attractive possibilities to modulate the liver sieve of LSECs with liposome-encapsulated microfilament-disrupting drugs in vivo. The goal of this study was to alter the sieve's porosity by using cytochalasin B-loaded Aco-HSA liposomes. The increase in the liver sieve porosity induced by cytochalasin B (CB) may be exploited therapeutically to improve the extraction of atherogenic lipoproteins from the circulation; or to enhance the efficiency of liposome-mediated gene or drug delivery to hepatocytes.

During hepatic metastasis, tumor cells are exposed to the sinusoidal environment, involving endothelial cells, Kupffer cells, pit cells (NK cells) and fat-storing cells [1]. It is known that Kupffer cells and pit cells play a direct role in killing metastasizing colon carcinoma cells [1,2]. Only few studies describe the interactions of Kupffer cells and tumor cells within the first 24 hrs of metastasis [3-6]. The importance of this early phase lies in the substantial but not complete killing of tumor cells. Also, at later stages, when different elements of host defense are involved, it is apparent that the local immune system is not capable of preventing metastasis. Investigating rare cellular events is facilitated by studying the full depth of thick sections with confocal laser scanning microscopy. Complicated preparation and histochemical procedures and a low sampling-volume [7] characterize classical microscopic methods. In contrast, confocal laser scanning microscopy (CLSM) has the ability to study thick sections (100 micrometers), gathering 3D information and allowing the use of different fluorescent probes for different variables [8]. Localization of cells can be done by immunohistochemistry, by labeling cells in vitro prior to injection or by labeling them in vivo. The anti-metastatic function of Kupffer cells was studied by labeling tumor cells with the lipophilic probe DiO in vitro and Kupffer cells with fluorescent latex particles in vivo.

BACKGROUND: Hepatic natural killer (NK) cells, the most cytotoxic cells of the natural occurring NK cells, are located in the liver sinusoids and are thus in a strategic position to kill arriving metastasising tumour cells, like colon carcinoma cells. It is known that major histocompatibility complex (MHC) class I on tumour cells negatively regulates NK cell-mediated cytolysis, but this is found using blood- or spleen-derived NK cells. Therefore, using isolated rat hepatic NK cells and the syngeneic colon carcinoma cell line CC531s, we investigated whether this protective role of MHC class I is also operative in hepatic NK cells, and addressed the mechanism of MHC class I protection. RESULTS: When MHC class I on CC531s cells was masked by preincubation with monoclonal antibody OX18, hepatic NK cell-mediated cytolysis (51Cr release) as well as apoptosis (DNA fragmentation, nucleus condensation and fragmentation) increased. When hepatic NK cells were preincubated with the granzyme inhibitor 3,4-dichloroisocoumarin, or when extracellular Ca2+ was chelated by ethylene glycol-bis(beta-aminoethyl ether)-N, N-tetraacetic acid, the enhanced cytolysis and apoptosis were completely inhibited. The involvement of the perforin/granzyme pathway was confirmed by showing that the enhanced cytolysis was caspase-independent. CONCLUSIONS: MHC class I expression protects CC531s colon carcinoma cells from hepatic NK cell-mediated apoptosis and cytolysis, by blocking the perforin/granzyme pathway.

This review provides a detailed overview of the current state of knowledge about the ultrastructure and dynamics of liver sinusoidal endothelial fenestrae. Various aspects of liver sinusoidal endothelial fenestrae regarding their structure, origin, species specificity, dynamics and formation will be explored. In addition, the role of liver sinusoidal endothelial fenestrae in relation to lipoprotein metabolism, fibrosis and cancer will be approached.

Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied. Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers. (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs.

There is s growing evidence that pit cells are highly active, liver-specific NK cells. Pit cells are located in the liver sinusoids and can be easily isolated and purified by liver sinusoidal lavage and a magnetic separation method. Furthermore, pit cells can be separated into a LD and HD fraction by 45% iso-osmotic Per coll gradient centrifugation. These two populations are shown to differ morphol ogically, phenotypically and functionally from each other and from blood NK cells. LD pit cells contain more rod-cored vesicles and more, but smaller granules than blood NK cells although both of them share LGL morphology. Phenotypically, LD cells have a higher expression of LFA-1 and a lower expression of asialo-GM 1 molecules compared to blood or spleen NK cells. Functionally, pit cells are more cytotoxic against several tumor cell lines as compared to blood NK cells, and are able to kill-NK-resistant but LAK-sensitive P815 cells. These data indicate that pit cells are a kind of naturally activated NK cells and their cytotoxic function is comparable to IL-2 in vitro activated NK cells. The characteristics of HD cells are intermediate between LD pit cells and blood NK cells. Pit cells most probably originate from blood NK cells, although they show mitosis in the liver after certain stimuli. The recruitment of pit cells in the liver is mediated by adhesion molecules. A major challenge is to achieve a better understanding of the mechanisms of pit cell cytotoxicity and the cooperation between pit cells and other cells in the liver, i.e. Kc, Ec and LAL. Moreover, since pit cells are located in a strategic position in the hepatic sinusoids, they represent a first line of cellular defense against metastasing colon cancer cells. The role of pit cells in a number of liver pathologies deserves more attention.

Interaction of colon carcinoma cells with rat hepatic sinusoidal cells during early stages of metastasis

At ovulation, the mouse oocyte is arrested at metaphase of the second meiotic division. Since microtubules are thermo-and chemosensitive structures, the effects of 1.5 M dimethylsulphoxide and 1.5 M 1,2-propanediol were studied at room temperature on the morphology of the meiotic spindle. Oocytes incubated at 37 degrees C or at room temperature served to estimate the effect of temperature in the experiment. The meiotic spindle was visualized by immunogold-silver staining of microtubules. In the control group at 37 degrees C, 88% of oocytes had normal spindles. After incubation at room temperature for the same time, 89% of oocytes showed abnormal spindles. In the oocytes exposed to dimethyl-sulphoxide or 1,2-propanediol at room temperature a protective effect on spindle morphology could be recognized. Subsequent incubation at 37 degrees C resulted in partial restoration of the observed abnormalities after cooling to room temperature and after exposure to dimethylsulphoxide. Incubation at 37 degrees C after exposure to 1,2-propanediol at room temperature induced spindle absence in the majority of oocytes. Although this latter condition allowed fertilization without increased incidence of ploidy abnormalities, a role for 1,2-propanediol as an activating agent is hypothesized.

In an attempt to elucidate the role of fat-storing cells (FSCs) in liver fibrosis, we investigated the collagen synthesis by FSCs freshly isolated from rats treated with CCl4, with vitamin A, and from untreated rats. FSCs from CCl4-treated rats contained a small number of lipid droplets and an abundant rough endoplasmic reticulum (RER), while those from vitamin A-treated rats showed numerous large lipid droplets and scanty RER. The population doubling times of FSCs isolated from normal, CCl4-treated, and vitamin A-treated rats were 38 +/- 4.3, 24 +/- 2.5, and 48 +/- 6.3 hr, respectively. The rate of collagen synthesis by FSCs from CCl4-treated rats was four- to sixfold enhanced, while collagen synthesis by FSCs from vitamin A-treated rats was suppressed. The ratio of collagen type I to type III produced by FSCs from CCl4 rats was enhanced as compared with control rats (94.7:5.3 vs 87.6:12.4). Therefore, FSCs can be considered to play an important role in the pathogenesis of liver fibrosis.

Fat-storing cells were isolated and purified from livers of normal adult rats and maintained in primary culture. By light and electron microscopy it was established that they underwent phenotypic changes into cells with the ultrastructural characteristics of myofibroblasts, between the third and sixth day in culture. These morphological changes were accompanied by a 2-fold increase of l-[ 3 H]proline incorporation into secretory proteins and an 11-fold increase into secreted collagenase-sensitive proteins. In contrast, incorporation into ceil layer-associated proteins and into cell layer-associated collagenase-sensitive proteins was not significantly elevated. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with fluorography, demonstrated that the main collagen type secreted by the myofibroblast-like cells was collagen type I. Collagen types III and IV, and fibronectin were present in lesser amounts. The similarity between the well known in vivo alterations of fat-storing cells under pathological conditions and the spontaneous in vitro differentiation described in this study, makes primary cultures of fat-storing cells a valuable tool for studying their role in chronic liver disease.

Osteocalcin was purified by gel chromatography from a crude extract obtained after decalcification of rat incisors. The apparent molecular weight, as determined by 5-15% SDS-polyacrylamide gel electrophoresis, was 18,000, and amino acid analysis revealed 60 γ-carboxyglutamic acid residues per 1000. Antisera against osteocalcin, raised in rabbits, reacted specifically with osteocalcin when investigated by immuno-electroblotting of dentin crude extract. 4-μm cryosections of formaldehyde-fixed tooth germs showed positive immunocytochemical staining for osteocalcin in dentin and odontoblasts. The staining of the mantle dentin at the coronal sides of the tooth germs was more intense than that of the adjacent circumpulpal dentin, while the odontoblasts involved in the formation of mantle dentin showed stronger immunoreactivity than did odontoblasts involved in circumpulpal dentin formation. This marked difference was not observed on the root sides of the tooth germs. In 1-μm cryosections, osteocalcin immunoreactivity was found evenly distributed throughout the entire cell body, with the exception of the Golgi region, which was less intensely stained, while the nucleus and the cell process were negative. The positive staining reaction with anti-osteocalcin antiserum was found in dentin from the very onset of its formation in the fetus. ln conclusion, our results demonstrate the presence of osteocalcin in odontoblasts and dentin. Its immunocytochemical localization may be compatible with a distinct role in early dentinogenesis.

The influence of bile salts on the binding and uptake of Salmonella abortus equi lipopolysaccharide by cultured Kupffer cells was studied. In control preparations, the percentage of cell-associated lipopolysaccharide increased with time and reached a plateau after about 2 h incubation at 37 degrees C. About 1.2 micrograms lipopolysaccharide was associated with 10(6) Kupffer cells at this time interval. In the presence of 0.3, 0.6 and 1 mumol bile salts/ml the cell-associated lipopolysaccharide was respectively, about 5%, 13% and 29% lower than in control cultures. In the presence of 1 mumol bile salts/ml, the association of lipopolysaccharide to cells at 0 degrees C was about 25% lower than in controls. Preincubation of Kupffer cells with 1 mumol bile salts/ml, with or without lipopolysaccharide, did not affect cell-associated lipopolysaccharide after removal of the bile salts. The rate of secretion of radioactivity by Kupffer cells was not influenced by the presence of bile salts during the uptake or the secretion periods. Bile acids proved to inactivate lipopolysaccharide. From these observations it was concluded that low concentrations of bile salts influence the binding and uptake of lipopolysaccharide by Kupffer cells. It was, therefore, considered likely that, in patients with obstructive jaundice, the high serum bile acid level accounts for spill-over of portal lipopolysaccharide into the systemic blood.

The distribution of adrenergic nerves in guinea pig and rat liver was studied by the immunolocalization of fibers containing tyrosine hydroxylase and dopamine beta-hydroxylase, enzymes involved in the synthesis of catecholamines. In both species, adrenergic fibers were identified within portal tracts, often in close proximity to hepatic artery branches. In guinea pig liver, but not rat liver, abundant intraacinar fibers were identified; fibers were also seen within the walls of terminal hepatic vein radicles and larger hepatic veins. The presence of peptidergic nerves containing the regulatory peptide neuropeptide tyrosine and the C-flanking peptide CPON was investigated by indirect immunofluorescence. The distribution of these nerves was similar to that of tyrosine hydroxylase- and dopamine beta-hydroxylase-positive nerves and showed the same species difference. The effector sympathetic nature of tyrosine hydroxylase- and neuropeptide tyrosine-positive fibers in rat liver was confirmed by chemical denervation studies using 6-hydroxydopamine.

The structural unit of the liver is classically named the liver lobule and is defined as a unit of parenchymal tissue characterized by peripheral branches of the portal vein and hepatic artery, and by a centrilobular branch of the hepatic vein, i.e. the central vein. The blood enters the lobule via the portal tracts through sinusoidal inlets and, after interaction with the parenchymal tissue during passage through the hepatic sinusoids, leaves the lobule through the central veins [Wisse and De Leeuw, 1984].

The influence on diaminobenzidine staining of four variables: prefixation in aldehyde, temperature and pH of incubation, and H2O2 concentration, was investigated in catalase-, as well as in peroxydase-containing material. Catalase from five different sources and five types of peroxidase were examined. It is concluded: (a) when cells are incubated without prior fixation, in a DAB medium at room temperature and pH 7.3 with 0.003% H2O2, peroxidases produce a visible cytochemical stain, while catalases do not; (b) the cytochemical reaction elicited by catalases is stimulated by prior aldehyde fixation in specified conditions, and incubation at 45 degrees C and pH 9.7 with 0.06% H2O2; (c) under the latter circumstances several peroxidases also stain. Ultrastructural preservation is satisfactory in tissues incubated prior to fixation.