Dianzhong Luo

Structure and Function of Sinusoidal Lining Cells in the Liver

The hepatic sinusoid harbors 4 different cells: endothelial cells (100, 101), Kupffer cells (96, 102, 103), fat-storing cells (34, 51, 93), and pit cells (14, 107. 108). Each cell type has its own specific morphology and functions, and no transitional stages exist between the cells. These cells have the potential to proliferate locally, either in normal or in special conditions, that is, experiments or disease. Sinusoidal cells form a functional unit together with the parenchymal cells. Isolation protocols exist for all sinusoidal cells. Endothelial cells filter the fluids, exchanged between the sinusoid and the space of Disse through fenestrae (100), which measure 175 nm in diameter and are grouped in sieve plates. Fenestrae occupy 6-8% of the surface (106). No intact basal lamina is present under these cells (100). Various factors change the number and diameter of fenestrae [pressure, alcohol, serotonin, and nicotin; for a review, see Fraser et al (32)]. These changes mainly affect the passage of lipoproteins, which contain cholesterol and vitamin A among other components. Fat-storing cells are pericytes, located in the space of Disse, with long, contractile processes, which probably influence liver (sinusoidal) blood flow. Fat-storing cells possess characteristic fat droplets, which contain a large part of the bodys depot of vitamin A (91, 93). These cells play a major role in the synthesis of extracellular matrix (ECM) (34, 39-41). Strongly reduced levels of vitamin A occur in alcoholic livers developing fibrosis (56). Vitamin A deficiency transforms fat-storing cells into myofibroblast-like cells with enhanced ECM production (38). Kupffer cells accumulate in periportal areas. They specifically endocytose endotoxin (70), which activates these macrophages. Lipopolysaccharide, together with interferon γ, belongs to the most potent activators of Kupffer cells (28). As a result of activation, these cells secrete oxygen radicals, tumor necrosis factor, interleukin 1, interleukin 6, and a series of eicosanoids (28) and become cytotoxic against tumor cells [e.g., colon carcinoma cells (19, 22, 48)]. Toxic secretory products can cause necrosis of the liver parenchyma, which constitutes a crucial factor in liver transplantation (55). Pit cells possess characteristic azurophylic granules and display a high level of spontaneous cytolytic activity against various tumor cells, identifying themselves as natural killer cells (10). The number and cytotoxicity of pit cells can be considerably enhanced with biological response modifiers, such as Zymosan or interleukin 2 (8). Pit cell proliferation occurs within the liver, but recent evidence indicates that blood large granular lymphocytes develop into pit cells in 2 steps involving high- and low-density pit cells (88). Kupffer cells control the motility, adherence, viability, and cytotoxicity of pit cells (89), whereas cytotoxicity against tumor cells is synergistically enhanced (80, 81).

Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway

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.

Involvement of LFA-1 in hepatic NK cell (pit cell)-mediated cytolysis and apoptosis of colon carcinoma cells

BACKGROUND/AIMS Previous studies have shown that hepatic natural killer (NK) cells, also called pit cells, have a higher cytotoxicity against certain tumor cells and have a higher expression of the cell adhesion molecule CD11a as compared with blood NK cells. We further investigated the involvement of the adhesion molecules, reported to be involved in target cell killing by blood NK cells, in pit cell-mediated colon carcinoma cell killing. METHODS 51Cr-release and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively. Adhesion of pit cells to CC531s monolayers was quantitated. RESULTS Flow cytometric analysis showed that pit cells expressed CD2, CD11a, CD18 and CD54. CC531s cells expressed only CD54. Treatment of freshly isolated pit cells with monoclonal antibodies (mAbs) to CD11a and CD18 inhibited not only the pit cell-mediated CC531s cytolysis but also the pit cell-induced apoptosis of CC531s cells. The combination of mAbs to CD11a, CD18 and CD54 further increased the inhibition of pit cell-mediated CC531s cytolysis and apoptosis. Anti-CD2 mAb did not affect these processes. The binding of pit cells to CC531s cells was also inhibited by anti-CD11a, and CD18 mAbs, but not by anti-CD2 mAb. Anti-CD54 mAb reduced the target cell killing and the binding only slightly. CONCLUSIONS These results indicate that CD11a/CD18 (LFA-1) present on pit cells plays an important role in pit cell-mediated target cell adhesion, lysis and apoptosis. This finding might explain why pit cells, which have a higher expression of LFA-1 as compared to blood NK cells, are more cytotoxic against tumor cells as compared to blood NK cells.

The number and distribution of hepatic natural killer cells (pit cells) in normal rat liver : an immunohistochemical study

Pit cells are a unique population of cells in sinusoids and peripheral blood, which can be considered natural killer (NK) cells with large granular lymphocyte (LGL) morphology. The aim of this study was to investigate the use of the monoclonal antibody (MAb) 3.2.3 as a specific marker of rat pit cells to detect their number and distribution in the liver. The number of 3.2.3‐positive cells was comparable to the number of LGL in liver low‐density (LD) and high‐density (HD) pit cell fractions and in blood lymphocytes (P > 0.05). Immunoelectron microscopy showed that nearly all LGL in hepatic LD and HD and in blood fractions were 3.2.3 positive. Using MAb 3.2.3 immunoperoxidase staining, the mean number of pit cells in liver frozen sections was determined to be 13.7 ± 1.1/mm2. The number of pit cells was similar in the different liver lobes (P > 0.05). Reduced nicotinamide‐adenine dinucleotide (NADH) oxidase histochemical staining, to visualize a portal to central vein gradient, combined with immunostaining was used to analyze the lobular distribution of pit cells. We found that 61.3% (17.1/mm2) of pit cells were in the periportal area and 38.7% (10.8/mm2) in the central area. We conclude that MAb 3.2.3 can be used as a specific marker of rat pit cells and therefore can be used to quantify rat pit cell number in various experimental models.

Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells.

Abstract Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase.

High‐density oligonucleotide array analysis reveals extensive differences between freshly isolated blood and hepatic natural killer cells

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.

Endothelial Cells of the Hepatic Sinusoids: A Review

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.

MHC class I expression protects rat colon carcinoma cells from hepatic natural killer cell-mediated apoptosis and cytolysis, by blocking the perforin/granzyme pathway

BackgroundHepatic 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.ResultsWhen 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(β-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.ConclusionsMHC class I expression protects CC531s colon carcinoma cells from hepatic NK cell-mediated apoptosis and cytolysis, by blocking the perforin/granzyme pathway.

Pit cells exclusively kill P815 tumor cells by the perforin/granzyme pathway

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.

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

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