HIV-1 infection induces functional alterations in human liver endothelial cells in primary culture.

OBJECTIVES: Since human liver endothelial cells allow HIV-1 multiplication in vitro, we investigated whether HIV induced functional alterations in these cells in primary culture. DESIGN: Direct evidence of the replication of HIV in endothelial cells is sparse, but clotting abnormalities and thrombi, which suggest the existence of an endothelial dysfunction, have been observed in HIV-infected patients. We therefore studied the storage and release of endothelial-specific factors in primary cultures of liver endothelial cells infected with HIV, as well as their cytoskeleton, pinocytic and phagocytic properties. METHODS: Intracellular storage of von Willebrand's factor (vWF) was determined by immunofluorescence and computer image analysis. Excretion of vWF, protein S and endothelin-1 was measured using an enzyme-linked immunosorbent assay and radioimmunoassay. Cytoskeletal constituents were studied by light microscopy. The pinocytosis of acetylated low-density lipoproteins and the phagocytosis of latex beads were analysed under light and electron microscopy. RESULTS: The synthesis of vWF is markedly decreased in HIV-infected liver endothelial cells, as is the excretion of endothelin-1. In contrast, the excretion of protein S remains unaffected and the cytoskeletal network appears to be unaltered. Pinocytosis and phagocytosis are preserved. CONCLUSIONS: HIV infection triggers non-lethal functional alterations in cultured human liver sinusoidal endothelial cells, with a selective impairment in the storage and/or the excretion of endothelial-specific factors such as vWF. This functional modulation could play a role in the pathophysiology of HIV-induced disease.

Kupffer and endothelial liver cell damage renders A/J mice susceptible to mouse hepatitis virus type 3.

Damage to the Kupffer and endothelial cells of the liver sinusoids induced by the administration of sublethal doses of frog virus 3 (FV 3) renders A/J mice which are genetically resistant to mouse hepatitis virus type 3 (MHV 3) highly susceptible to this virus. Liver histopathology of these animals revealed typical necrotic foci containing MHV 3-specific antigens. FV 3-pretreated mice, after MHV 3 infection, showed higher levels of serum transaminase (GPT) than controls, and MHV 3 replicated more rapidly and to higher titres. Our results bear out the important role of the liver sinusoidal lining in protecting against hepatocyte infection and its direct involvement in the resistance of A/J mice to MHV 3 infection.

Permissivity of primary cultures of human Kupffer cells for HIV-1.

Kupffer cells (liver macrophages) represent the largest reservoir of fixed macrophages in the body. Accordingly, we have undertaken a study to evaluate their susceptibility to human immunodeficiency virus type 1 (HIV-1). Five-day-old primary cultures of Kupffer cells (KC) were infected with HIV-1, and as the infection progressed, syncytia appeared. Within the cells, viral proteins were detected by immunofluorescence using monoclonal antibodies directed against gp120 and p24. Electron microscopic examinations revealed the presence of typical Lentivirinae particles. The particles released from KC in the extracellular medium showed reverse transcriptase activity and p24 antigen; they could infect lymphocytic cells and were neutralized by a HIV+ patient's serum or an anti-gp120 monoclonal antibody. Our results thus demonstrate that the interaction of HIV-1 with KC in vitro leads to a productive infection. They suggest that the KC may be involved in the pathogenesis of HIV-1 infection and may (i) participate in the transmission of the infection to the peripheral blood cells, (ii) play a role in the depletion of uninfected CD4+ cells.

Increased susceptibility of mice to MHV 3 infection induced by hypercholesterolemic diet: impairment of Kupffer cell function.

Nutritionally induced hypercholesterolemia in A/J mice causes susceptibility to Mouse Hepatitis type 3 (MHV 3), whereas normal A/J mice are fully resistant. A/J mice fed with a hypercholesterolemic diet for 15 to 60 days develop 5 to 7 days after MHV 3 infection an acute hepatitis which led to high levels of mortality. A direct relationship was found between the high levels of plasma and hepatic cholesterol and the mortality. In attempting to define the dietary-induced physiological changes which led to the loss of resistance, the Kupffer cells were shown to exhibit an impairment of functions in their ability to become activated by LPS in order to take up C3-coated IgM opsonized sheep red blood cells, C3(IgM)SRBC, or 3H-thymidine Escherichia coli, and the susceptibility to interferon (IFN) for the induction of an antiviral state. Peritoneal macrophages which were studied in comparison with the Kupffer cells showed no impaired functions. The findings presented here indicate an inhibition of host resistance, by nutritional hypercholesterolemia, of A/J mice to MHV 3 infection and that, at least one site of impairment occurs specifically at the stage of Kupffer cells function.

Inhibition of mouse hepatitis virus type 3 multiplication in activated Kupffer cells.

Mouse Hepatitis Virus type 3 (MHV3) multiplication in isolated murine Kupffer cells was partially inhibited by pretreatment of the cells with lipopolysaccharide (LPS). Supernatants of LPS-treated Kupffer cells contained large amounts of interferon. Inhibition of MHV3 multiplication was also observed when normal Kupffer cells were cultivated in a medium containing supernatants of LPS-treated Kupffer cells. In addition to the antiviral effect of the released interferon, there seems to be another effect of LPS, since Kupffer cells cultured in medium containing anti-interferon alpha beta antibodies were partially activated by LPS to inhibit MHV3 replication. The in vivo consequences of these effects for the local immunity of the liver against MHV3 infection are discussed.

Interaction of mistletoe lectin I with Kupffer cells and endothelial cells of mouse liver.

The in vitro-formation of blebs by endothelial and Kupffer cells of the mouse liver after treatment with the toxic lectin I from mistletoe are demonstrated by means of scanning electron microscopy. The interaction of toxic lectins with nonparenchymal liver cells can be of importance concerning the elimination of these lectins in vivo.

C3-mediated phagocytosis induced in murine Kupffer cells by "in vitro" activation with endotoxin.

Binding and phagocytosis of red blood cells by Kupffer cells, mediated by C3 receptors, have been studied by incubating sheep red blood cells opsonized with C3, and Kupffer cells isolated from rat and mouse livers. Sheep red blood cell-binding was followed by internalization and phagocytosis only after preincubation of Kupffer cells with bacterial endotoxin. In both species of cells there was a direct relationship between the value of the phagocytic index and the amount of endotoxin needed to stimulate the cells. Increased phagocytosis was related to an increase in the number of phagocytosing cells; it did not depend on an increase in the number of red blood cells internalized per Kupffer cell. C3-mediated phagocytosis, together with the intrinsic and extrinsic antiviral activities, and the synthesis of interferon which take place in activated cells may play an important role in non-specific immunity.

[Hepatic macrophages and virus]. / Macrophages hépatiques et virus.

Kupffer cells are located at strategic positions in the liver sinusoids. They are involved in the clearance of about 90% of foreign particles namely viruses and play an important role in the pathogenesis since their interaction with viruses represents a major determinant of viremia. On the one hand, they may protect the host from the infection by taking up and degrading the viral particles as well as by presenting antigen to lymphocytes, on the other hand, they may enhance the infection by producing and disseminating viruses when they are permissive. When they are activated or when they produce interferon alpha, Kupffer cells exert an antiviral effect on adjacent cells. Moreover they participate to the inflammatory response by synthesizing numerous mediators. Their destruction as well as the alteration of their functional properties may have serious physiopathological consequences.

Leukotrienes as mediators in frog virus 3-induced hepatitis in rats.

The role of leukotrienes was investigated in frog virus 3-induced hepatitis in rats. Frog virus 3 elicited an enhanced generation of cysteinyl leukotrienes in vivo as monitored by measurement of N-acetyl-leukotriene E4 as the major endogenous metabolite of cysteinyl leukotrienes secreted into rat bile. N-Acetyl-leukotriene E4 concentrations were elevated for more than 4 hr after frog virus 3 injection. In vitro experiments using cultured rat liver Kupffer cells of high purity indicated that these cells can produce and metabolize leukotrienes and are thus a possible source of leukotrienes elicited in vivo by frog virus 3. The selective 5-lipoxygenase inhibitor AA 861 and the dual inhibitor of arachidonate lipoxygenase and cyclooxygenase, BW 755C, reduced the hepatocellular injury after a high dose of frog virus 3 by about 50 and 80%, respectively, as judged from plasma activities of ALT and sorbitol dehydrogenase at 24 hr after frog virus 3 administration. Our in vivo and in vitro studies argue in favor of an important role of leukotrienes as mediators in frog virus 3 hepatitis in rats.

Increase in the number of fenestrae in mouse endothelial liver cells by altering the cytoskeleton with cytochalasin B.

Endothelial cells of the hepatic sinusoid isolated from mice livers and maintained in culture display typical fenestrae grouped in sieve plates. Treatment with cytochalasin B led to no significant change in the mean diameter of the fenestrae but to an increase in their number and in the porosity of the cells (percentage of the cellular surface opened by the fenestrae) which attained up to 300% of that of the controls. Scanning electron microscopic observations of Triton-extracted cells revealed that these modifications were related to an alteration of the cytoskeleton. The effect of cytochalasin B could be reversed; 3 hr after removal of the drug, the cells recovered their original aspect with sieve plates scattered over their surface. These observations demonstrate that endothelial fenestrae are inducible structures and that the cytoskeleton seems to be involved in their formation.

Interaction between mouse hepatitis viruses and primary cultures of Kupffer and endothelial liver cells from resistant and susceptible inbred mouse strains.

Infection with mouse hepatitis virus type 3 (MHV 3) of primary cultures of Kupffer and endothelial cells from the livers of resistant (A/J) and susceptible (BALB/c) mice was followed by the appearance of typical syncytia and comparable yields of virus. Using cells from A/J mice there was a delay of about 24 to 36 h in the appearance of the first particles detected by electron microscopy, the maximum viral titre and the number and size of syncytia. The partial resistance to MHV 3 multiplication expressed by cells from A/J mice was also observed when they infected with a strain of low virulence (JHM strain). The delay in MHV multiplication found in the sinusoidal liver cells, mainly in the endothelial cells, may be an important factor in their resistance, by allowing time for the local and systemic responses to clear the infective particles as well as in determining their degree of hepatotropism.

Comparative ultrastructural study of rat livers preserved in Euro-Collins or University of Wisconsin solution.

University of Wisconsin solution greatly lengthens the time liver storage is possible compared with all previous solutions used. To test whether this improvement is related to better preservation of the endothelial cell, which is thought to be the most vulnerable cell type in cold storage, we compared time-related ultrastructural changes in rat livers stored in this solution or in Euro-Collins solution. Rat livers were harvested after combined arterial and portal perfusion with the cold-storage solution. They were then preserved for different lengths of time in the same solution at 4 degrees C before being perfusion-fixed and processed for light and electron microscopy. The first preservation damage was noted in endothelial cells; the time course of the lesions was similar in both solutions. After 2 hr of storage, enlarged and ruptured fenestrae with many gaps were observed. Swollen at 4 hr, the endothelial cells became stringlike at 10 hr, leading to stripped sinusoidal walls. Hepatocytes appeared better preserved in University of Wisconsin solution. The amount of glycogen, maintained near the control level at 24 hr in the latter, decreased dramatically between 0 and 4 hr in Euro-Collins solution, as ultrastructurally observed and biochemically confirmed. Furthermore, sinusoidal obstruction by blebs originating from the hepatocytes and quantified by image analysis on electron micrographs was markedly delayed. It was significantly less pronounced in University of Wisconsin solution at 24 hr than in Euro-Collins solution at 2 hr (p less than or equal to 0.05). Our findings confirm that endothelial cells are highly susceptible to preservation damage and show that University of Wisconsin solution does not improve preservation during storage.(ABSTRACT TRUNCATED AT 250 WORDS)

[Rat Kupffer cells cultures and their applications to experimental hepatology (author's transl)]. / Culture de cellules de Kupffer du rat. Intérêt en hépatologie expérimentale.

Non-parenchymal cells were obtained by perfusion of rat liver with 0.05% collagenase followed by incubation at 37 degrees C of liver fragments in collagenase in a rotating water-bath. Kupffer cells were separated from endothelial cells by centrifugal elutriation and could be kept alive for several days. They had the same morphology as in vivo and retained the same properties of phagocytosis and pinocytosis. Rat Kupffer cells cultures could be used to investigate a number of unsolved problems of hepatic pathophysiology.

[Isolation and culture of human Kupffer cells]. / Isolement et culture de cellules de Kupffer humaines.

The dissociation of human adult liver parenchyma by collagenase followed by cell separation with centrifugal elutriation allows the Kupffer cells to be isolated. These cells, which may be maintained in culture, retain their main morphological and functional features.

[Effect of a water-soluble derivative of silymarin on morphological and functional alterations of mouse hepatocytes induced by Frog Virus 3 (author's transl)]. / Wirkung eines wasserlöslichen Derivates von Silymarin auf die durch Frog-Virus 3 an Mäusehepatozyten hervorgerufenen morphologischen und funktionellen Veränderungen.

Pretreatment of mice with silymarin dihemisuccinate sodium salt (SHS-Na) 300 mg/kg 24 and 16 h before infection completely protected against the lethal effect of Frog Virus 3 (FV3). After the inoculation of 1 LD100 of virus it could be shown that: a) the same amount of radioactively labelled virus was found in the liver of control and SHS-Na treated animals, b) in SHS-Na pretreated animals the shut-off of macromolecular syntheses in the liver was considerably less extensive, c) the histological changes in the hepatocyte nuclei were attenuated in SHS-Na pretreated animals and the serum titer of the transaminases did no increase, d) in SHS-Na pretreated mice there were only limited functional alterations of the sinusoidal cells as studied by carbon phagocytosis and the serum titer of the lysosomal enzymes was only slightly modified, e) inhibition of protein synthesis during the period of pretreatment with SHS-Na did not decrease the level of protection. These results allow us to conclude that SHS-Na exerts its protective effect on the liver and that sinusoidal cells are most likely involved in this protection.

Phagocytosis, an unrecognized property of murine endothelial liver cells.

Impairment of the phagocytic capacities of Kupffer cells, as is found in Frog Virus 3 hepatitis of mice, allows the endothelial liver cells to take up intravenously inoculated latex particles of 1.0 micron diameter. In vitro experiments with cultivated endothelial cells isolated by collagenase perfusion of the liver and purified by centrifugal elutriation demonstrate that uptake occurs via a typical mechanism of phagocytosis involving pseudopodia. Ingestion of latex is inhibited by incubation of the cells at 4 degrees C and by treatment with cytochalasin B, whereas colchicine has no effect. These results demonstrate that: the Kupffer cells are not the only cells of the hepatic sinusoid capable of phagocytosis; and under conditions where the phagocytosis in Kupffer cells is impaired, the endothelial cells may participate in the clearance of large particles from the blood.

Studies on fenestral contraction in rat liver endothelial cells in culture.

Liver endothelial cells possess fenestrae, which are pores supported by a cytoskeleton ring composed of actin and myosin. Fenestrae are dynamic structures that can contract or dilate, although the mechanism for this phenomenon remains to be elucidated. Staining of actin and/or of myosin permitted measurement of fenestral diameter and area in cultured rat liver endothelial cells using digitized video-intensified fluorescence microscopy with image analysis. Within 1 minute of incubation with 0.1 micromol/L serotonin, fenestral diameter and area decreased by 24 +/- 5% and 56 +/- 7%, respectively. Contraction of fenestrae by serotonin was inhibited by chelation of extracellular Ca2+ with EGTA and by addition of Ca2+ channel blockers, such as dilthiazem and verapamil. The response of fenestrae to serotonin was mimicked by addition of a Ca2+ ionophore, A23187. Serotonin inhibited cAMP production, had no effect on inositol phosphate production, and activated phospholipase A2, causing release of arachidonic acid. These results suggest that contraction of fenestrae is associated with Ca2+ influx. In response to 0.1 micromol/serotonin, intracellular Ca2+ levels increased within 3 to 5 seconds from 150 nmol/L to >400 nmol/l followed by rapid phosphorylation of the 20-kd subunit of myosin light chain; both events dependent on extracellular Ca2+.