Expression of co-stimulatory molecules by Kupffer cells in chronic hepatitis of hepatitis C virus etiology

In this paper we show that in viral hepatitis most Kupffer cells (KCs) are activated and express high levels of CD80, CD40, and class-II MHC molecules, thus acquiring the phenotype of professional antigen presenting cells (APCs). Activated KCs display a close contact with CD4+ T lymphocytes and form KCs-T lymphocyte clusters. Clusters are found within the sinusoids, across the sinusoid wall, and within the liver parenchyma as well, as a consequence of transendothelial migration (TEM). The positivity of activated KCs for hepatitis C virus (HCV) antigens, which likely reflects phagocytosis of infected hepatocytes, suggests that KCs-T cell clusters represent the morphological expression of the functional interaction between KCs acting as professional APCs and antigen-experienced CD4+ T lymphocytes within the liver. These phenotypic and morphological changes are distinct features of livers in chronic hepatitis patients compared with controls.     

A study about the mechanism of hepatocyte injury induced by endotoxin--interaction of Kupffer cells and polymorphonuclear neutrophils (PMNs)

Focusing our attention on the TNF (tumor necrosis factor) produced by Kupffer cells (KCs). The role of KCs and polymorphonuclear neutrophils (PMNs) in endotoxin (LPS)-induced hepatocellular injury was investigated. This study used the culture fluid supernatant of KCs which had been stimulated with LPS as the "LPS stimulation supernatant", and evaluated hepatocellular injury as ornithine carbamyl transferase percent leakage. There was no difference between groups I (HCs: hepatocytes) and II (HCs + PMNs), but there were differences between groups I and III (HCs + LPS stimulation supernatant, groups II and IV (HCs + LPS stimulation supernatant + PMNs), and III and IV. There was no significant difference between III and V (III + anti-TNF antibody), but a difference was found between IV and VI (IV + anti-TNF antibody). These findings suggest that PMNs activated by KCs-generated TNF as well as KCs-derived humoral factors other than TNF play a role in the development of LPS-induced hepatocellular injury.     

Operative strategy in patients with MRI-identified dual pathology and temporal lobe epilepsy

The liver is the most common site of hematogenous metastases from colorectal carcinoma. Kupffer cells (KC), which line the hepatic sinusoids, may form the first line of defense against circulating tumor cells. The purpose of this study was to determine the effect of hepatic metastases and intra-abdominal tumor growth on KC binding of human colorectal carcinoma (HCRC) cells. MIP-101, a poorly metastatic cell line, and CX-1, a highly metastatic cell line, were injected intrasplenically into nude mice and KC were isolated by collagenase perfusion at varying intervals after injection. Conditioned media were collected from MIP-101, CCL 188 and CX-1 to determine their in vitro effect on KC function. KC from MIP-101 injected mice (14% liver metastases, 100% splenic tumors) bound a significantly greater number of MIP-101 and clone A cells than CX-1 cells in vitro. KC isolated from mice 5 weeks after CX-1 injection (100% liver metastases) also showed increased binding of MIP-101 and clone A cells compared to CX-1 cells. Similar results were obtained when tumor cell binding to normal human liver KC was compared to binding to KC from human livers from patients with hepatic metastasis from colorectal cancer. In contrast KC obtained from mice 3 weeks after CX-1 injection (44% liver metastases) showed significantly decreased binding of MIP-101 and clone A cells. The conditioned medium from CX-1 cells significantly decreased the in vitro binding of both MIP-101 and CX-1 by KC. These results indicate that the ability of KC to bind HCRC cells (which precedes phagocytosis and tumor cell killing) is a dynamic function and affected by concomitant tumor growth. HCRC cells may alter KC function via the production of specific tumor-derived soluble factors. In order to devise new and more effective therapeutic options in the treatment of liver metastases the nature of this tumor cell-KC interaction must be better understood.     

Repeatability of the Petrifilm HEC test and agreement with a hydrophobic grid membrane filtration method for the enumeration of Escherichia coli O157:H7 on beef carcasses

The review highlights recent advances in our understanding of basic mechanisms of reperfusion injury after warm hepatic ischemia. Kupffer cells play a central role as the initial cytotoxic cell type and as a source of many proinflammatory mediators. Subsequently, neutrophils are activated and recruited into the liver. Factors and conditions are outlined that determine whether neutrophils undergo apoptosis without causing damage or migrate out of the sinusoids and attack parenchymal cells. In addition to the inevitable inflammatory response during reperfusion, microcirculatory perfusion failure, due to an imbalance between the actions of vasodilators and vasoconstrictors, also has a serious impact on reperfusion injury. A better understanding of the basic pathophysiology will reveal potential targets for therapeutic interventions and will show us how to avoid risk factors that may aggravate reperfusion injury.     

Collagen type III and type IV detection in and around human hepatocellular carcinoma

Extracellular matrix proteins participate in tumor cell growth and progression. Their role in the extratumoral liver tissue needs to be elucidated. Eight patients with hepatocellular carcinoma on noncirrhotic livers are investigated by means of light microscopical and ultrastructural immunohistochemistry for collagen type III and type IV. In the tumor collagen type III, staining is weaker, and collagen type IV is increased. It is topographically located near perisinusoidal stromal cells. In the extratumoral liver tissue, the immunostaining for the two antibodies is stronger perisinusoidally. The number of Ito cells increases significantly in the extratumoral liver tissue. A lot of transitional cells are found there. Sinusoids in the extratumoral tissue are dilated and filled with lymphoid cells and platelets. The presence of matrix proteins between tumor cells is necessary to regulate their growth and differentiation. The increase in extracellular matrix content perisinusoidally in the extratumoral tissue probably erects a protective barrier against metastasizing tumor cells. There, collagen type III and type IV accumulation is probably initiated by signals coming from tumor cells, or from inflammatory cells and platelets in sinusoids.     

Apoptosis of neutrophils and their elimination by Kupffer cells in rat liver

The fate of neutrophils in the peripheral circulation is poorly understood. In this study, the role of Kupffer cells in eliminating aged and apoptotic neutrophils was investigated. Liver, spleen, lung, and blood samples from Wistar rats were examined by light and electron microscopy, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method, and immunohistochemistry after the intravenous injection of OK-432, a streptococcal preparation. Neutrophils were trapped predominantly in the periportal and midzonal regions of hepatic lobules and were in contact with endothelial cells and Kupffer cells, or were surrounded by Kupffer cells. The trapping of neutrophils peaked after 6 hours. Apoptotic neutrophils, with or without buds, were found in the lumen of hepatic sinusoids as early as 6 hours, reached maximal levels after 12 hours, and represented greater than 60% of the total number of neutrophils in the liver. The presence of apoptotic neutrophils was correlated with the degree of neutrophil phagocytosis. Double-staining showed that TUNEL-positive neutrophils were phagocytosed or encircled by ED1- or ED2-positive Kupffer cells. In contrast, apoptosis and phagocytosis of neutrophils were rare in the spleen, lung, and peripheral blood. These results suggested that the appearance of apoptotic neutrophils in the hepatic sinusoids and their rapid clearance by Kupffer cells occurs after the invasion of bacteria (i.e., bacteremia or bacteriotoxemia) or the release of inflammatory mediators into the blood stream. These findings have important implications for the regulation of neutrophil homeostasis, the limitation of inflammation and tissue injury, and provide insight into the physiological removal of circulating, senescent neutrophils.     

IFN-gamma-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones

NK cells, non-T non-B immune effector lymphocytes, are localized in many organs, including liver, as well as in the circulation. To investigate the regulatory mechanism of killing apparatus in hepatic NK cells, we established IL-2-dependent NK cell clones from liver lymphocytes of BALB/c nude mice. To generate the NK cell clones, we incubated liver lymphocytes with a high dose of IL-2 in the presence of irradiated Kupffer cells, as feeder cells and as the source of IL-12, originally identified as NK cell stimulatory factor. Unless liver lymphocytes were incubated with both IL-2 and Kupffer cells, no cell growth was observed. Hepatic NK cell clones were established from this cell line by limiting dilution. The surface phenotypes of cloned NK cells were IL-2R beta-chain+ CD16+ CD3- IgM-. The clones did not express NK2.1, which is expressed by a half of NK-enriched spleen cells of BALB/c mice. Although the cells contained dense granules reactive to mAb against perforin, they exerted no conventional cytolytic activity against YAC-1. They constitutively expressed Fas ligand (FasL) and specifically killed Fas-positive target cells by fragmenting DNA. This Fas-FasL-mediated killing activity was enhanced by IFN-gamma-inducing factor, a recently identified novel cytokine produced by activated Kupffer cells, but was not affected by other Kupffer cell-produced cytokines, such as IL-12, IL-1beta, and TNF-alpha. Taken together, these findings suggest that hepatic NK cells participate in the immune response as effector cells through the Fas-FasL system in collaboration with cytokines from Kupffer cells.     

Electron microscope study on the hepatic sinusoidal wall and fat-storing cells in the bat

The three cell types known to form the hepatic sinusoidal wall were electron microscopically observed in three kinds of bats captured in winter and summer. 1. The cytoplasmic extensions of sinusoidal endothelial cells consisted of continuous thicker parts ("cytoplasmic processes") and discontinuous thinner parts ("sieve plates"). The alternate disposition of the two parts was rather irregular, and the sizes, shapes and spacings of the fenestrae were variable. In the thinner parts with numerous small fenestrae, larger gaps were also mingled. The endothelium was simple-layered and devoid of basal lamina. Interendothelial junctions were found mainly between closely apposed margins of the "cytoplasmic processes" and agreed in structure with the "junctional complex" of WISSE (1970). 2. Kupffer cells, morphologically distinct from the endothelial cells, bulged strongly into the sinusoidal lumen. Provided with many microvillous pseudopods, they were stellate in appearance. They were fixed to the endothelial lining by small junctional areas which occurred between the Kupffer cell body and the "cytoplasmic processes" of the endothelium. 3. Fat-storing cells were located in the Disse's space. They generally contained only smaller amounts of lipid in a few droplets. So-called empty fat-storing cells were numerous, especially in winter bats. The perikaryonal cytoplasm revealed a large Golgi complex and well-developed granular endoplasmic reticulum. The three mesenchymal cell types of the sinusoidal wall possessed the centriole in common within the Golgi complex, but only the fat-storing cell was provided with the single cilium. Fat-storing cells extended cytoplasmic processes ramifying beneath the endothelial lining occasionally surrounding the sinusoids almost completely, and which seemed to reinforce the endothelial lining and to bring about the constriction of the sinusoid. In hypervitaminotic bats that daily received 6,000 I.U. vitamin A for three days, remarkable increase in size and number of lipid droplets was observed in slightly hypertrophic fat-storing cells, and the empty cells disappeared simulating an increased number of fat-storing cells. Suggestion was made of an antidotal function of fat-storing cells against excess vitamin A which might be considered a toxic agent in the broad sense. 4. The Disse's space of bat liver contained plasma cells, lymphocytes and macrophages, the latter too often being under migration through the endothelial fenestrae.     

The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver

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.     

Role of the donor liver in the origin of platelet disorders and hyperfibrinolysis in liver transplantation

We investigated the role of the donor liver in the origin of platelet disorders and hemostatic defects in liver transplantation. Eighteen pigs received an orthotopic or a heterotopic, auxiliary liver graft. Liver biopsies were taken for electron microscopic studies 5-10 min after reperfusion in nine animals. Blood samples were taken from the first hepatic outflow and from the systemic circulation before and 5 min after graft recirculation. Electron microscopy did not show any evidence of microthrombi or platelet aggregation in the graft, either after orthotopic liver transplantation or after heterotopic liver transplantation. Most blood platelets, which were lying free in the sinusoids, showed cell processes and many seemed to have lost their granulae, suggesting a degree of platelet activation. There were also signs of phagocytosis of platelets by the Kupffer cells. In the hepatic outflow, platelet count was significantly lower (p < 0.05) and fibrinolytic activity significantly higher (p < 0.01), than systemic post-reperfusion values. There were no important changes in the coagulation parameters. No significant changes were found between the effects on hemostasis of orthotopic and auxiliary graft reperfusion. In the second part of the study evidence for platelet activation was found after graft reperfusion in human liver transplantation. Plasma levels of platelet factor-4 and beta-thromboglobulin increased significantly after graft reperfusion. These studies suggest that platelet disorders and increased fibrinolytic activity are the major components of the hemostatic defect after graft recirculation in liver transplantation. Sequestration of platelets in the graft is probably due to the accumulation of (activated and degranulated) platelets in the sinusoids and phagocytosis by Kupffer cells.     

Effect of tuftsin on human Kupffer cell

BACKGROUND/AIMS: Kupffer cells are the most important category of reticuloendothelial cells which are critical for host defense in the liver. We investigated the effects of tuftsin (Thr-Lys-Pro-Arg) on human Kupffer cells. METHODOLOGY: Human Kupffer cells were obtained from the livers of patients with colon cancer. Phagocytosis assay was done by microscopic counting of the number of Kupffer cells that engulfed fluorescent particle(s), and the number of the particles engulfed per Kupffer cell when Kupffer cells were incubated with and without tuftsin. Effect of tuftsin on the release of tumor necrosis factor from Kupffer cells was also studied. RESULTS: Phagocytosis was enhanced significantly by tuftsin. The greatest effect on percentage of phagocytic cells was observed at 1.0 microg/ml of tuftsin. The mean number of particles engulfed per Kupffer cell was also increased with tuftsin 1.0 microg/ml. Tumor necrosis factor release was also significantly increased; the greatest effect was observed at 1.0 microg/ml of tuftsin. CONCLUSIONS: Tuftsin enhances phagocytic activity and tumor necrosis factor release of human Kupffer cells, which are advantageous for host defense against invading microorganisms and tumor cells.     

Early after/depolarizations and triggered activity: mechanisms and autonomic regulation

Although the clearance and distribution of ligand molecules in circulation represent the function of hepatic sinusoidal cells, these mechanisms revealed a network that is more intricate than would at first seem, since several receptors are common to not only one type of cell, but also to two or three types of cells in the liver. In the case of latex particles in which their uptake by a particular cell type seems to be determined by their size, sinusoidal endothelial cells are able to internalize particles up to 0.23 microns under physiologic conditions, in vivo, and larger particles are taken up by Kupffer cells. However, when the phagocytic function of Kupffer cells is impaired by frog virus 3 or alcohol, endothelial cells have been found to take up particles larger than 1 micron in diameter after the injection of an excess amount of latex particles. Endothelial cells would thus constitute a second line of defense in the liver in that they remove foreign materials from the blood when Kupffer cell phagocytic function is totally disturbed. This potential role may not, however, be fully expressed under physiologic conditions when Kupffer cells are active in clearing foreign substance from the circulation. The functions of liver sinusoidal cells are varied and complex and these cells can be regarded as "a sinusoidal cell unit." This cellular interaction must be taken into account for any quantitative analysis.     

Isolation and primary culture of rat Kupffer cells

The aim of this study was to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells. Kupffer cells were isolated following sequential pronase/collagenase digestion of the liver and enrichment of a non-parenchymal cell fraction by a single-density gradient centrifugation step using 30% metrizamide. Kupffer cells were isolated and further purified from this cell fraction by centrifugal elutriation. Kupffer cells were isolated at 1017 g at 48-110 mL/min. All Kupffer cell fractions exhibited phagocytosis of 3 microm latex beads. Kupffer cell fractions isolated at 48 and 60 mL/min were predominantly ED2 negative while later fractions (80-110 mL/min) were ED2 positive. Kupffer cells were adherent in culture after 2 h. This method for Kupffer cell isolation resulted in a yield of 80-120 x 10(6) Kupffer cells per liver.     

Dogs infected with a human granulocytotropic Ehrlichia spp. (Rickettsiales: Ehrlichieae)

In contrast to the anticipation that in sepsis granulocyte colony-stimulating factor (G-CSF) would overactivate the nonspecific immune system by recruiting and priming leukocytes with consequent aggravation of inflammatory tissue lesions, recombinant (r) G-CSF pretreatment was protective in various experimental non-neutropenic models of inflammation. The mechanisms of protection, however, are not fully understood. Using intravital fluorescence microscopy, we show that rG-CSF enhances leukocyte endothelial cell interaction within the microvasculature of normal rat livers, whereas rG-CSF pretreatment of animals exposed to lipopolysaccharide (LPS) attenuates the LPS-induced leukocytic response, including stasis in sinusoids as well as rolling and adherence in postsinusoidal venules with subsequent tissue infiltration. Moreover, rG-CSF, which did not affect Kupffer cell activity in normal rat livers, reduced the immediate activation of Kupffer cells on LPS exposure, as indicated in vivo by the delayed adherence/phagocytosis of intra-arterially administered latex particles associated with attenuation of proinflammatory cytokine release (tumor necrosis factor alpha and interleukin-6). Finally, rG-CSF reduced LPS-induced nutritive perfusion failure and hepatocellular excretory dysfunction. This study provides evidence for a distinct, possibly tumor necrosis factor alpha-dependent modulation of LPS-induced cellular response within the liver by rG-CSF, thereby achieving protection against microcirculatory perfusion failure and hepatic dysfunction.     

Orthodontic wire: a continuing evolution

Previous studies have shown that large doses of vitamin A potentiate chemical-induced liver injury and that the Kupffer cell is directly involved in this potentiation. Therefore, these studies were designed to determine if Kupffer cells isolated from vitamin A treated male Sprague-Dawley rats (75 mg/kg/day for 3-7 days as all- trans-retinol) had altered activity and function. Respiratory activity of Kupffer cells isolated from rats treated with vitamin A for 3 to 7 days markedly increased. Similarly, phagocytic activity was significantly elevated (up to 9-fold) after exposure to vitamin A for 3 to 7 days. Production of reactive oxygen species, measured by luminol-enhanced chemiluminescence of Kupffer cells isolated after 7 days of vitamin A exposure, was significantly higher than that of control cells when stimulated with opsonized zymosan. Also, the release of superoxide anion by individual Kupffer cells isolated from vitamin A treated rats was nearly three times greater than that of control cells. Basal production of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2) production were significantly elevated in Kupffer cells isolated from rats treated with vitamin A. Lastly, peripheral blood monocytes (PBMC) isolated from rats treated with vitamin A for 7 days had a significantly greater respiratory activity, as well as TNF-alpha and PGE2 production, than PBMC isolated from control rats. Our data suggest that large doses of vitamin A enhance both Kupffer cell and PBMC function. Upregulation of the activity by these phagocytic cells may play a role in the vitamin A potentiation of chemical-induced liver injury.     

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 Chinese hamster cells

A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed "growth inhibiting factor" (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity of GIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.     

Thrombopoietin and its receptor, c-mpl, are constitutively expressed by mouse liver endothelial cells: evidence of thrombopoietin as a growth factor for liver endothelial cells

Present data suggest that the primary site of thrombopoietin (TPO) mRNA is the liver. Previously, we reported that specific murine liver endothelial cells (LEC-1) located in the hepatic sinusoids support in vitro megakaryocytopoiesis from murine hematopoietic stem cells suggesting that these cells may be a source of TPO. We report here that TPO and its receptor, c-mpl, are coexpressed on cloned LEC-1. Enzyme-linked immunosorbent assay (ELISA), biological assay, and flow cytometry studies confirmed the expression of both TPO and its receptor, respectively, at the protein level. TPO activity was enhanced in supernatants from LEC-1 treated with tumor necrosis factor (TNF)-alpha and gamma-interferon (INF). Our results show that TPO through its receptor stimulated the growth of LEC-1 in vitro. These observations establish LEC-1 as a novel source of TPO in the liver. To our knowledge, this is the first report that liver endothelial cells express both TPO and its receptor, c-mpl, and our findings indicate that this cytokine constitutes a growth factor for liver endothelial cells in vitro.     

Endothelial cells potentiate oxidant-mediated Kupffer cell phagocytic killing

Phagocytosis and killing of circulating organisms by Kupffer cells (KCs) are discrete, important components of host defense. However, the killing mechanism(s) are not fully understood, and the potential role of adjacent nonparenchymal cells such as hepatic endothelial cells has not been defined. Rat KCs -/+ an hepatic endothelial cell enriched cellular fraction (HECEF) were incubated with Candida parapsilosis and assayed for phagocytosis and phagocytic killing by validated fluorochromatic vital staining. The role of reactive oxygen metabolites in KC phagocytic functions was examined by inhibition with superoxide dismutase and/or catalase. Diphenyleneiodonium and allopurinol were used to examine the potential roles of NADPH oxidase and xanthine oxidase, respectively, in generating these toxic oxidants. Coculture with HECEF increased KC phagocytic activity (from 75% to 88%) and candidacidal activity (from 20% to 31%). Superoxide dismutase, catalase, diphenyleneiodonium, or allopurinol caused inhibition of candidacidal activity, but did not affect phagocytosis, and did not block the potentiation of phagocytosis or of killing caused by coculture with HECEF. Reactive oxygen intermediates generated by both NADPH oxidase and xanthine oxidase-dependent pathways are important in KC killing of Candida parapsilosis. In vitro, KC phagocytosis and killing are potentiated (via a non-oxidant-mediated mechanism) by coculture with a preparation of hepatic non-parenchymal cells composed primarily of endothelial cells.