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The development of the sinusoids of fetal rat liver: morphology of endothelial cells, Kupffer cells, and the transmural migration of blood cells into the sinusoids.

The fine structural development of rat fetal liver sinusoids from 10 to 22 days gestation was studied. Colloidal carbon (Pelikan ink) was injected into 14-22 day gestation fetuses via the umbilical vein to assess the continuity of the sinusoidal lining and the phagocytic ability of the developing lining cells. Endothelial cells, devoid of an underlying basal lamina, form the bulk of the vascular lining at all gestational ages. These cells possess typical intercellular junctions and fenestrae with diaphragms before 17 days gestation. Transendothelial open fenestrations, typical of the adult liver, appear around 17 days gestation, increasing in number for the remainder of gestation. Although fenestrae possessing diaphragms are permeable to carbon before 16 days gestation, open fenestrations, first seen at 17 days gestation, allowed large amounts of carbon to reach the extravascular space. Endocytosis of carbon by endothelial cells was accomplished exclusively by large bristle-coated vesicles. Endothelial cells were also seen to be involved in transmural diapedesis of newly formed erythrocytes and megakaryocyte processes from the extravascular space by forming a temporary migration pore allowing these cells and processes to enter the circulation. At the end of gestation, blood-forming activity had nearly ceased, and only the space of Dissé separated the lining cells from the parenchymal cells. Kupffer cells were easily identified as early as 13 days gestation by their content of phagosomes and engulfed erythrocytes. The Kupffer cells are much more avid in the phagocytosis of carbon than are endothelial cells. Toward the end of gestation, some Kupffer cells develop a homogeneous "sticky coat" to carbon.

Animals

[Trend of molecular biological studies on the liver sinusoidal cells--Kupffer cells].

The Kupffer cells are sessile macrophages residing in the hepatic sinusoids. The Kupffer cells are well known as the first line of defence against pathogenic agents derived from not-self substances, in some cases even from self proteins. Indeed, Kupffer cells play an important roles in such a defence system: they act as an antigen presenting cells, and also produce various kinds of cytokines and chemical mediators such as prostaglandins, leukotrienes and platelet activating factor. In this article, we introduce these functions of Kupffer cells and discuss the possible pathway of the signal transduction.

Animals

Electron microscopy of Kupffer cells in the orthotopic porcine liver homograft during the late stage after transplantation (phagocytosis of host cells by Kupffer cells).

Electron microscopical observations of macrophages (Kupffer cells) in the orthotopic porcine liver homograft in later stages after transplantation are described and demonstrated. The findings show close topographical relations between the Kupffer cells and hepatocyte debris, erythrocytes, thrombocytes, lymphocytes and granulocytes. Besides an uptake of hepatocyte debris, a phagocytosis and degradation of apparently unaltered erythrocytes, thrombocytes and probably also lymphocytes by the macrophages were observed. The possible significance of these findings is discussed with regard to the immunological interactions between macrophages and lymphocytes, to the course of the rejection in the liver homograft, to the effect on the blood cells and platelets of the host organism, and thus to the long-term prognosis of liver transplantation.

Animals

Relation between localization and function of rat liver Kupffer cells.

Kupffer cells were found to be distributed over zone 1 (periportal), zone 2 (midzonal), and zone 3 (perivenous) of the rat liver acinus in a ratio of 4:3:2. After pronase digestion of the liver, two purified tractions of Kupffer cells could be obtained by centrifugal elutriation. Liver zone marking with methylene blue prior to cell isolation revealed that one fraction contained Kupffer cells from the periportal area; the other consisted mainly of such cells from the midzonal and perivenous areas. Periportal Kupffer cells were larger and showed higher lysosomal enzyme activities on a per cell basis as compared with midzonal and perivenous Kupffer cells. The immediate uptake of 0.31-micrometer. latex particles during a 2-minute perfusion of the liver was mainly accomplished by periportal Kupffer cells. The higher phagocytic activity of these cells was independent of the direction which latex was flushed through the liver. Seven days after in vivo administration of latex particles, at least 80 per cent of all Kupffer cells contained latex, but again periportal Kupffer cells showed a higher phagocytic activity in that they accumulated a relatively larger number of particles per cell. Latex phagocytosis increased the activity of cathepsin D in all Kupffer cells. Titration with pepstatin revealed that periportal Kupffer cells contained many more cathepsin D molecules than pepstatin revealed that periportal Kupffer cells contained many more cathepsin D molecules than midzonal and perivenous Kupffer cells. Latex particle uptake stimulated an increase in the molecular activity of cathepsin D in periportal cells as well as in the number of cathepsin D molecules in perivenous and midzonal cells. Kupffer cells show a functional heterogeneity that is related to their position in the liver acinus. Kupffer cells with a high endocytic activity, large and heterogeneous lysosomes, and high lysosomal enzyme activities are found in the periportal zone. This zonal heterogeneity can be reduced after phagocytosis of a triggering dose of latex.

Animals

Comparative study of cytotoxicity, tumor necrosis factor, and prostaglandin release after stimulation of rat Kupffer cells, murine Kupffer cells, and murine inflammatory liver macrophages.

Macrophages (Mphi) and Mphi-depleted (nonadherent) nonparenchymal cells (NPC) of the liver were examined for their cytotoxic potential against tumor cells, production of tumor necrosis factor (TNF), and release of prostaglandins (PG) following stimulation by lipopolysaccharide (LPS), interferon-gamma (IFN gamma), and zymosan. Resident murine liver macrophages had no natural cytotoxicity for the TNF-resistant target cell line P815. Activation of these cells was only obtained by a combination of IFN gamma and LPS. Inflammatory murine macrophages were in a primed stage and could be activated by LPS alone in the absence of IFN gamma. Rat resident macrophages resembled functionally the inflammatory macrophages of the mouse liver rather than the resident macrophages. They displayed natural cytotoxicity against all targets tested and were further activated by LPS in the absence of IFN gamma. Similar results were obtained with respect to macrophage-depleted nonadherent NPC: Mouse NPC had a low level of NK activity against Yac-1 cells. Treatment with pyran copolymer resulted in a strong increase of cytotoxicity against Yac-1; furthermore, a TNF-dependent killing of Wehi 164 and TNF-independent cytotoxicity against P815 cells were now acquired. In the rat NPC prepared from unstimulated animals expressed high levels of natural cytotoxicity against all targets. No major differences could be observed between inflammatory Mphi and Kupffer cells of rat and mouse liver with regard to TNF production and TNF-dependent killing of Wehi 164 tumor cells. The same was true for the spectrum of secreted prostanoids. Upon activation of all cell populations a marked shift toward the production of PGE2 occurred. Experiments involving the cyclooxygenase inhibitor indomethacin showed enhanced TNF-dependent tumor cell killing by nonactivated Mphi in the absence of prostanoid production.

Animals

Identification and induction of cytochrome P450 2E1 in rat Kupffer cells.

Kupffer cells, the resident macrophages of the liver, have a well characterized role in the removal of blood-born foreign substances by phagocytosis. Because Kupffer cells may contribute to hepatic xenobiotic metabolism, the current studies evaluated the presence and inducibility of P450 2E1 in rat Kupffer cells. Hepatocytes and Kupffer cells were isolated from the livers of control and acetone-treated (1% v/v acetone in the drinking water for 7 days) rats. P450 2E1 was immunochemically detectable at low levels in Kupffer cell homogenates from untreated rats and was induced greater than 10-fold by acetone-treatment. The presence of P450 2E1 in Kupffer cells from untreated rats was confirmed by inhibition of benzene hydroxylation with anti-P450 2E1 immunoglobulin G. Benzene hydroxylase activity was induced 16.3-fold in Kupffer cells isolated from acetone-treated rats and remained 70% inhibitable by anti-P450 2E1 antibody. The benzene hydroxylase activity of hepatocytes from the same animals was induced 3.9-fold by acetone treatment. The specific activity for benzene hydroxylation of Kupffer cell homogenates from acetone-treated rats was nearly equal to that for the hepatocytes from the same animals. The presence and inducibility of P450 2E1 in Kupffer cells suggests that, under conditions where P450 2E1 is induced, Kupffer cell-generated metabolites may contribute to Kupffer cell toxicity, as well as general hepatic injury.

Acetone

In vitro tumoricidal activity of resting and glucan-activated Kupffer cells.

Kupffer cells compose 80-90% of fixed tissue macrophages and have been suggested to play an important role in hepatic antitumor resistance. In the present study, the ability of resting and activated Kupffer cells to lyse syngeneic mammary adenocarcinoma BW10232 cells was evaluated. Activated Kupffer cells were isolated from C57Bl/6J mice following single of multiple intravenous (IV) injections of glucan (0.45 mg/mouse), a potent macrophage-activating agent. Mice receiving 5% (w/v) dextrose served as control. Resting Kupffer cells induced significant (P less than .05) 4% and 12% specific lysis of adenocarcinoma cells at target:effector ratios of 1:10 and 1:50, respectively. Kupffer-cell-mediated tumoricidal activity was depressed on day 1 following a single IV injection of glucan. By day 3 postglucan, the antitumor activity of Kupffer cells returned to control levels and was enhanced on days 5 and 10. Following multiple IV injections of glucan on days -5, -3, and -1, Kupffer-cell-mediated cytotoxicity was elevated on days 1 and 4. These observations demonstrate that resting Kupffer cells are significantly cytotoxic to adenocarcinoma cells at T:E ratios of 1:10 and 1:50 and following a transient inhibition of Kupffer-cell-mediated tumoricidal activity, glucan was effective in significantly enhancing the antitumor activity of Kupffer cells.

Adenocarcinoma

Effect of short-term ethanol treatment on voltage-dependent calcium channels in Kupffer cells.

Kupffer cells, the resident hepatic macrophages, are activated by calcium, and several reports indicate that their function (e.g., phagocytosis and cytokine production) is altered by ethanol. We recently found that Kupffer cells contain L-type voltage-dependent Ca2+ channels. The purpose of this study, therefore, was to evaluate the effect of short-term ethanol treatment on voltage-dependent Ca2+ channels in Kupffer cells. Kupffer cells were isolated from rats 2 hr after intragastric administration of ethanol (5 gm/kg intragastrically). Cytosolic free calcium concentration of cultured Kupffer cells was measured with the fluorescent Ca2+ indicator fura-2. In Kupffer cells isolated from control rats, partial replacement of extracellular Na+ by K+ caused an increase in cytosolic free calcium concentration in a concentration-dependent manner (half-maximal effect was observed with 81 mmol/L K+), presumably because of membrane depolarization. Acute ethanol treatment in vivo shifted the concentration-response curve for K+ to the right (half-maximal effect was observed with 94 mmol/L K+) and reduced the maximal elevation of cytosolic free calcium concentration by means of K+. Significantly, the dihydropyridine-type calcium channel agonist BAY K 8644 (1 mumol/L) shifted the concentration-response curve for K+ to the left in control and ethanol-treated groups (half-maximal effect was observed with 61 and 77 mmol/L K+, respectively). Moreover, the dihydropyridine-type calcium channel blocker nitrendipine (10 mumol/L) prevented the increase in cytosolic free calcium concentration in both groups. When extracellular Ca2+ was omitted from the incubation medium, the increases in cytosolic free calcium concentration due to depolarization were prevented completely. However, direct addition of ethanol to the cell cultures was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy

Fine structure and function of Kupffer cells.

Kupffer cells are macrophages that are attached to the luminal surface or inserted in the endothelial lining of hepatic sinusoids. In this site, Kupffer cells play a key role in host defense by removing foreign, toxic and infective substances from the portal blood and by releasing beneficial mediators. Under some conditions, toxic and vasoactive substances also are released from Kupffer cells which are thought to play a role in a variety of liver diseases. Many of these activities may be modulated by the levels of gut derived endotoxin normally present in the portal blood. The ultrastructural aspects of Kupffer cell structure function in situ are best studied using perfused-fixed livers. In fixed livers, transmission and scanning electron microscopy reveal Kupffer cells during health to be irregular in shape with their exposed surfaces presenting numerous microvilli, filopodia, and lamellopodia. Long filopodia penetrate endothelial fenestrae to secure Kupffer cells to the sinusoid lining. Specific membrane invaginations known as worm-like bodies or vermiform processes are seen in the cytoplasm of Kupffer cells as are numerous endocytotic vesicles and lysosomes which vary in density, shape and size. Sometimes, annulate lamellae connected to the rough endoplasmic reticulum also are found. The principal endocytic mechanisms of Kupffer cells are phagocytosis of particulates and cells, and bristle-coated micropinocytosis for fluid-phase endocytosis of smaller substances. Many of these events are mediated by specific receptors. In some species, Kupffer cells can be distinguished from other sinusoidal lining cells and monocytes by specific cytoplasmic staining or monoclonal antibodies. Kupffer cells have been shown to be of monocytic origin as well as having the capacity for self-replication.

Humans

Interdependence of tumor necrosis factor, prostaglandin E2, and protein synthesis in lipopolysaccharide-exposed rat Kupffer cells.

Kupffer cells are the main producers of tumor necrosis factor-alpha (TNF; cachectin) and eicosanoids in the liver exposed to lipopolysaccharide (endotoxin; LPS). A very rapid but transient release of TNF is followed by a slow, steady synthesis of prostaglandin E2 (PGE2). TNF itself is able to provoke eicosanoid synthesis in Kupffer cells; the rate and pattern of prostaglandin production are similar to those observed after treatment with LPS. Anti-TNF antibodies completely neutralize TNF action on Kupffer cells, thus ruling out any participation of contaminating LPS. LPS stimulation of PGE2 production in Kupffer cells is reduced by the antiserum to 50%, indicating an involvement of TNF in the stimulatory action of LPS. On the other hand, PGE2, a potent inhibitor of LPS-elicited TNF release, is able to suppress LPS- but not TNF-stimulated eicosanoid synthesis in rat Kupffer cells. In addition to this autocrine circuit, extrahepatic factors participate in the regulation of Kupffer cell activation: glucocorticoids not only inhibit TNF or prostaglandin production, they also reverse the LPS-specific changes in the prostaglandin pattern of Kupffer cells. LPS, TNF or cycloheximide when given alone in the concentration range applied in this study do not affect the viability of rat Kupffer cells. However, the combinations of cycloheximide and either LPS or TNF cause rapid death of the cultured cells. The cytolytic potential of either combination cannot be alleviated by treatment with glucocorticoids.

Animals

Regulation of endotoxin-induced IL-6 production in liver sinusoidal endothelial cells and Kupffer cells by IL-10.

Sinusoidal endothelial cells and Kupffer cells are the first cell populations in the liver that come into contact with gut-derived endotoxin in portal blood. Although endotoxin concentrations as high as 1 ng/ml are physiologically present in portal blood, no local inflammation is seen. We show that the proinflammatory cytokine IL-6, which is central to the development of inflammatory reactions in the liver, is produced by sinusoidal endothelial cells and Kupffer cells in response to low concentrations of endotoxin (100 pg/ml to 1 ng/ml). The anti-inflammatory cytokine IL-10 down-regulated endotoxin-induced IL-6 release in endothelial and Kupffer cells. Importantly, Kupffer cells secreted IL-10 after endotoxin stimulation and may therefore participate in the local regulation of inflammation. We have found that IL-6 secretion in Kupffer cells is tightly regulated by endogenous IL-10, because increased IL-6 secretion resulted when neutralizing antibodies to IL- 10 were added to resting and endotoxin-challenged Kupffer cells. Furthermore, repeated exposure of endothelial cells to endotoxin induced a state of tolerance which resulted in decreased release of IL-6 in response to a second endotoxin challenge. Our results support the notion that inflammatory reactions in the liver in response to endotoxin are down-regulated by local release of the anti-inflammatory cytokine IL-10 that is produced by Kupffer cells.

Animals

Release of peptide leukotrienes from rat Kupffer cells.

Kupffer cells isolated from the normal rat liver were incubated with calcium ionophore A23187, and the levels of peptide leukotrienes (LTC4, LTD4, and LTE4) contained in the culture supernatant were determined by the combined technique of reverse-phase high-performance liquid chromatography and radioimmunoassay. In response to A23187, Kupffer cells released LTC4, LTD4, and LTE4. After 10 min-preincubation of Kupffer cells with AA861, a 5-lipoxygenase inhibitor, the generation of LTC4, LTD4, and LTE4 from A23187-stimulated Kupffer cells was significantly suppressed. Platelet activating factor (PAF), a phospholipid mediator, significantly enhanced the release of LTC4, LTD4, and LTE4 from Kupffer cells stimulated with A23187. These results suggested that Kupffer cells may participate in inflammatory and immunologic events in the liver tissue by the release of peptide leukotrienes.

Animals

Effect of chronic ethanol feeding on nitric oxide synthesis by rat Kupffer cells.

Kupffer cells contribute to the important role of the liver defense mechanism through nitric oxide (NO) production. In this study, the effect of chronic ethanol administration on the ability of Kupffer cells to synthesize and release NO was investigated after stimulation with lipopolysaccharide (LPS). Male Wistar rats were chronically fed ethanol for 8 weeks according to the method described by DeCarli and Lieber et al. (J Nutr.91:331-336, 1967). Kupffer cells were isolated and cultured with LPS (1 micrograms/ml) for 24 hr. The levels of nitrite and nitrate, metabolites of NO, were determined in the culture medium, NO synthase (NOS) activity in Kupffer cells was determined by the method that measures conversion of [14C]arginine into [14C]citrulline. In control rats, a significant increase of nitrite and nitrate levels in culture medium was observed after LPS treatment. The magnitude of this increase was significantly smaller in chronic ethanol-fed rats. When the activity of NOS was determined, inducible NOS (iNOS) activity was higher than that of constitutive NOS, and LPS administration produced a significant elevation of iNOS activity in both control and chronic ethanol-fed rats. However, the elevation of iNOS activity by LPS stimulation was diminished by chronic ethanol administration. Distribution of iNOS in Kupffer cells as determined by an immunofluorescence method using a laser scanning confocal image system showed a lower expression of iNOS in chronic ethanol-fed rats even in the presence of LPS. These results demonstrate that the excessive production of NO by increased iNOS activity in Kupffer cells is diminished by chronic ethanol administration.

Animals

Antigen-presenting function and B7 expression of murine sinusoidal endothelial cells and Kupffer cells.

BACKGROUND & AIMS: Inflammatory liver disease as well as rejection of liver allografts are thought to be mediated by resident antigen-presenting cells in the liver. At the same time, in vivo antigen presentation in the liver appears to be a more tolerogenic than systemic antigen challenge. The aim of this study was to show and characterize the antigen-presenting capability of sinusoidal endothelial cells and Kupffer cells. METHODS: Purified murine sinusoidal endothelial cells and Kupffer cells were studied for their ability to serve as accessory cells and antigen-presenting cells by proliferation assays. They were also studied for their expression of interleukin 1 and the B7 costimulatory molecules by Northern blotting, polymerase chain reaction, and flow cytometry. RESULTS: Both cell types expressed interleukin 1 messenger RNA and could serve equally well as accessory and antigen-presenting cells. B7-2 messenger RNA and surface expression on sinusoidal endothelial cells and on Kupffer cells was shown. Antibodies to the B7 molecules inhibited antigen presentation. Addition of interleukin 10 as a regulatory cytokine secreted by Kupffer cells was suppressive. CONCLUSIONS: Sinusoidal endothelial cells carry functional B7-2 molecules and can serve as effective antigen-presenting cells. However, antigen presentation by sinusoidal endothelial cells may be locally down-regulated by interleukin 10.

Animals

Beta-glucuronidase and chloroacetate-esterase staining discriminates rat liver sinusoidal endothelial cells from Kupffer cells in primary culture.

Beta-glucuronidase and N-AS-D-chloroacetate esterase cytochemistry have been applied to rat liver sinusoidal endothelial cells and Kupffer cells. Both staining procedures allowed a clear-cut differentiation of either cell type. Kupffer cells which had been stained with beta-glucuronidase showed a positive reaction, whereas sinusoidal endothelial cells were completely negative. If the chloroacetate reaction was used, the former stained diffusely while the latter showed a characteristic granular staining pattern. Identity and purity of sinusoidal endothelial cells and Kupffer cells was validated by transmission and scanning electron microscopy as well as by the pattern of released eicosanoids which is characteristic for either cell type. These two staining techniques are a valuable addition to the peroxidase reaction commonly applied for differentiation.

Animals

In vitro antiviral properties of endotoxin-activated rat Kupffer cells.

Kupffer cells isolated from the liver of normal rats were checked for their antiviral activities. The intrinsic antiviral effect against vaccinia virus was high whether the cells were activated in vitro with endotoxin or not. The expression of the extrinsic antiviral activity measured by mixing isolated Kupffer cells with vaccinia-virus-infected target cells was remarkably enhanced by prior treatment of the Kupffer cells with LPS. Interferon was shown to be not responsible for that inhibitory activity. Different experimental data suggest that the virus replication in the target cells is blocked at a late stage in the replication cycle.

Animals

Involvement of prostaglandin E and adenosine 3', 5'-monophosphate in lipopolysaccharide-stimulated collagenase release by rat Kupffer cells.

Kupffer cells exposed to bacterial lipopolysaccharide in vitro synthesized collagenase and released the major portion of it into the extracellular space while the intracellular level of enzyme was not altered significantly. Cycloheximide prevented the appearance of collagenase in the medium indicating de novo synthesis. Indomethacin, an inhibitor of cyclooxygenase, also blocked collagenase synthesis. In line with this observation. Kupffer cells were found to synthesize substantial amounts of prostaglandin E2 when exposed to lipopolysaccharide; concomitantly, cellular cAMP levels were increased. Indomethacin was shown to abolish the stimulated cAMP formation. Addition to the culture medium of cAMP or dibutyryladenosine 3', 5'-monophosphate as well as of prostaglandin E2 or, to a lesser extent, prostaglandin E1 allowed indomethacin-inhibited cells to resume the production of collagenase. It is proposed that in rat Kupffer cells lipopolysaccharide-elicited collagenase synthesis and excretion is mediated sequentially by stimulated production of prostaglandin E2, enhanced adenylate cyclase activity and increased intracellular cAMP levels.

Animals

In vitro cytostatic properties of unactivated rat Kupffer cells.

Kupffer cells from the liver of normal rats were checked for their natural cytostatic capabilities using an in vitro target cell growth inhibition assay. A strong cytostatic effect was observed on an human tumor cell line and was shown to be exerted on various transformed or normal target cells with only small differences in their susceptibility. The inhibition of target cell proliferation was shown to depend on the effector/target cell ratio. Different experimental data suggest that an intimate membranal contact between Kupffer cells and target cells is required.

Animals