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L Bouwens

Publications and source records attributed to L Bouwens.

At least 55 records · Page 3Linked to original sources
PubMed1994

Cytochemical localization of NADPH-diaphorase in the four types of pancreatic islet cell.

NADPH-diaphorase activity, which has been previously reported to be associated with the enzyme nitric oxide synthase (NOS), was localized cytochemically in the pancreatic islets of normal rats. All islet cells types, i.e. insulin-, glucagon-, somatostatin- and pancreatic polypeptide-immunoreactive cells, expressed NAD-PH-diaphorase histochemical activity, whereas the exocrine tissue was almost negative. In streptozotocin-treated rats, only the surviving non-beta cells in the islet periphery were stained. Isolated beta and non-beta cells also expressed intense NADPH-diaphorase activity. By electron microscopy, the enzyme was localized primarily on membranes of the endoplasmic reticulum and nuclear envelope, as previously reported for neurons. In addition the enzyme activity was found in the cis-region of the Golgi complex. These results suggest that the four types of endocrine cells of the islets of Langerhans may contain the NOS-enzyme and thus constitutively produce nitric oxide.

Amino Acid Oxidoreductases↗
PubMed1994

Beta-cell proliferation in normal and streptozotocin-treated newborn rats: site, dynamics and capacity.

Regeneration of neonatal beta cells after streptozotocin (STZ)-induced destruction may be due to either replication from pre-existing intra-islet beta cells or extra-islet precursor cells. To further investigate this issue, beta-cell growth was analysed in normal and streptozotocin-treated newborn rats (100 micrograms/g body weight) at several time points during the first 20 days of life. Beta cells were identified by insulin immunostaining, non-isotopic in situ hybridization for rat preproinsulin mRNA, and electron microscopy. Their proliferative activity was recorded by bromodeoxyuridine-pulse labelling. Beta-cell size and total volume were determined by computerized morphometry. In normal rats, there was a threefold increase in total beta-cell volume during the first 5 days of life, with no further expansion till day 20. The bromodeoxyuridine labelling index of the intra-islet beta cells was smaller than that of the extra-islet beta cells (2-3% vs 15-20%). Comparison of the cell birth rate, calculated from the beta-cell labelling index, with the observed increase in beta-cell volume suggested that in normal neonatal rats proliferation of the intra-islet beta-cell population could account for only 10% of the observed expansion. Administration of streptozotocin at birth resulted in more than 90% reduction of the total beta-cell volume at day 2 which then increased to 39% of the normal value by day 20. During this period of partial regeneration, which restored normoglycaemia, the labelling index of intra-islet beta cells was higher than in normal rats (9% vs 2%, p < 0.001), whereas no change was seen in the extra-islet beta-cell labelling index.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas.

Cytokeratins (CKs) serve as immunocytochemical markers of epithelial cells. We found that duct cells of the neonatal and adult rat pancreas express CKs 7, 19, and 20. Because pancreatic endocrine cells are thought to derive from duct cells, we examined their expression of CKs 7, 19, and 20 during the neonatal period and the proliferative activity of the different cell types in and around islets. Immunocytochemical analysis revealed that the islets of the neonatal pancreas, in contrast to those of adults, strongly expressed CKs 7, 19, and 20, particularly within a peripheral mantle zone that was continuous with the epithelium of adjacent ductules. This pattern was found during the first 2 weeks of life when significant islet growth occurred as determined by morphometry and bromodeoxyuridine (BrdU) labeling. Moreover, BrdU labeling kinetics indicated that the growth of neonatal islets occurred in the peripheral mantle region characterized by intense CK20 and CK19 immunoreactivity and not in the region composed of differentiated endocrine cells. These observations strongly suggest that proliferating ductal cells associated with islets represent a pool of islet beta- and non-beta-precursor cells.

Animals↗
PubMed1994

Effect of irradiation on hepatic natural killer cells.

The rat liver contains a population of natural killer cells consisting of two morphologically and functionally different subsets, a low-density and a high-density fraction. In this work we describe the influence of low-dose radiation on hepatic natural killer activity. The effect on the cytotoxicity against YAC-1 lymphoma and CC531 colon adenocarcinoma tumor cells was measured in chromium-51 assays, and morphological changes were analyzed by means of electron microscopy. The low-density natural killer fraction showed increased cytotoxicity against YAC-1 which was associated with an increased binding of natural killer cells to the YAC-1 tumor cells shortly after irradiation. These phenomena were paralleled by an increased number of multivesicular bodies and cytoplasmic granules with an electron-lucent halo. In contrast, the other hepatic natural killer cell fraction, the high-density natural killer cells, did not show increased cytotoxicity, binding or morphological alterations. The radiation-stimulated lysis of YAC-1 cells was also observed when in vivo irradiated cells were isolated and tested immediately for in vitro lysis of YAC-1 cells. Sixteen hours after in vitro or in vivo irradiation, the cytotoxicity of hepatic natural killer cells against YAC-1 was no longer enhanced. The cytolysis of and binding of hepatic natural killer cells to CC531 colon adenocarcinoma cells was not stimulated by in vitro irradiation. From these experiments, we conclude that low-dose radiation stimulates the cytotoxicity of hepatic low-density natural killer cells against YAC-1 lymphoma cells immediately after irradiation as the result of enhanced binding of the cells to the tumor cells, in addition to the synthesis of new cytotoxic granules.

Adenocarcinoma↗
PubMed1993

Origin and differentiation of hepatic natural killer cells (pit cells).

Liver sinusoids contain a population of large granular lymphocytes or natural killer cells, originally termed pit cells. After isolation and purification, these cells were separated into a low-density and a high-density fraction. The liver low-density fraction differs significantly in morphology and function from cells of the blood, whereas the liver high-density fraction shows intermediate properties. In this study we demonstrate that this morphological and functional heterogeneity is based on subsequent steps of differentiation of the large granular lymphocytes within the liver. When cell proliferation was suppressed by sublethal total body irradiation, the life span of the hepatic large granular lymphocytes could be determined: high-density and low-density populations were totally depleted within 1 and 2 wk after irradiation, respectively. By using intravenous asialo-GM1 anti-serum to deplete animals of asialo-GM1-positive cells, we found that the depletion of the asialo-GM1-positive cells preceded the depletion of asialo-GM1-negative hepatic low-density large granular lymphocytes by approximately 1 wk. Direct evidence that the asialo-GM1-positive high-density large granular lymphocytes are precursors of the low-density large granular lymphocytes was given by adoptive transfer experiments with fluorescent-labeled high-density cells. Three days after their injection, labeled large granular lymphocytes were found in the hepatic low-density fraction of the recipient rat, and these cells had developed morphological characteristics of low-density large granular lymphocytes. It is concluded therefore that marginating blood large granular lymphocytes differentiate through high-density large granular lymphocytes into the typical liver specific low-density large granular lymphocytes or pit cells.

Animals↗
PubMed1992

High deformability and motility of lymphokine-activated killer cells in vitro and in vivo.

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 microns 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.

Animals↗
PubMed1992

Pit cells in the liver.

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.

Animals↗
PubMed1992

Liver cell heterogeneity: functions of non-parenchymal cells.

The normal hepatic sinusoid is formed or lined by four different cell types, each with its specific phenotypic characteristics, functions and topography. Endothelial cells constitute the closed lining or wall of the capillary. They contain small fenestrations to allow the free diffusion of substances, but not of particles like chylomicrons, between the blood and the hepatocyte surface. This filtering effect regulates the fat uptake by the liver. Sinusoidal endothelial cells also have a pronounced endocytotic capacity which makes them an important part of the reticuloendothelial system. They are also active in the secretion of bioactive factors and extracellular matrix components of the liver. Recently, a zonal heterogeneity of the endothelial lining has been reported with regard to its filtering capacity (fenestration) and binding capacity for lectins and cells. Kupffer cells are intrasinusoidally located tissue macrophages with a pronounced endocytotic capacity. They are potent mediators of the inflammatory response by the secretion of a variety of bioactive factors and play an important part in the immune defense. A zonal heterogeneity has been established with regard to the endocytotic capacity and cytotoxic function. Pit cells are now known to represent a liver-associated population of large granular lymphocytes. They have the capacity to kill tumor cells and probably also play a role in the antiviral defense of the liver. In addition, pit cells may have a growth-regulatory function of the liver. They are known to be numerically more prominent in the periportal region, as is also the case for Kupffer cells. Fat-storing or Ito cells are present in the perisinusoidal space of Disse and are thought to represent the main hepatic source of extracellular matrix components. They are also the main site of vitamin-A storage. Fat-storing cells are more numerous in the periportal region than in the central region of the hepatic acinus. The periportal cells also store higher amounts of vitamin A. Sinusoidal cells may be considered to represent a functional unit at the border line between the hepatocytes or parenchymal cells and the blood. They participate in various liver functions and liver pathologies and our knowledge about this is growing. The heterogeneity of these cell types and possible cooperations between them and the hepatocytes may add to our understanding of liver functions.

Animals↗
PubMed1990

Characterization of a phenotypically and functionally distinct subset of large granular lymphocytes (pit cells) in rat liver sinusoids.

Pit cells, or large granular lymphocytes, with natural tumoricidal activity are found in the sinusoids of normal rat liver. Hepatic large granular lymphocytes are heterogeneous and can be subdivided into two subsets. These subsets were compared with peripheral-blood large granular lymphocytes and were found to differ phenotypically and functionally. Phenotypical differences included lower expression of the asialo-GM1 marker of natural killer cells, lower cellular density and many more small cytoplasmic granules in hepatic large, granular lymphocytes. Low-density hepatic large, granular lymphocytes were five to eight times more cytotoxic than blood large, granular lymphocytes on a per-cell basis as measured against YAC-1 cells and colon carcinoma cells. In addition, hepatic large, granular lymphocytes were able to lyse P-815 target cells, which are resistant to blood natural killer cells. Large, granular lymphocytes isolated from the liver also contained a subset with intermediate phenotypical and functional characteristics, possibly representing a transitional form between blood and "liver-specific" large, granular lymphocytes. The liver thus contains a specific population of highly activated or further differentiated large granular lymphocytes.

Animals↗
PubMed1990

Electron microscopic observations on the accumulation of large granular lymphocytes (pit cells) and Kupffer cells in the liver of rats treated with continuous infusion of interleukin-2.

Treatment schedules were investigated for in vivo induction of lymphokine-activated killer cells in the rat liver. Treatment of rats with continuous systemic or regional infusion of recombinant human interleukin-2 with a dose of 4 to 8 x 10(4) U/day during 7 days, resulted in an increase in number of large granular lymphocytes or pit cells in the liver up to 43 times normal. Kupffer cells, nongranular lymphocytes, monocytes and neutrophils also increased in number, but with a maximal fivefold increase this was much less pronounced than for large granular lymphocytes. Kupffer cells showed morphological signs of activation and were frequently seen in mitosis. Frequent mitoses were also observed for large granular lymphocytes, but not for other leukocytes. This indicates that the effect of interleukin-2 treatment on hepatic (sinusoidal) cells was primarily directed to large granular lymphocytes and Kupffer cells. The large granular lymphocyte accumulation occurred mainly intrasinusoidally, but they were also frequently observed in the space of Disse where they are not found in control rats. This may be explained partly by the observed damage or gaps in the endothelial lining. The intrasinusoidal large granular lymphocytes adhered to the endothelium and to Kupffer cells. Higher responses, for all cell types, were found when interleukin-2 was administered regionally, that is, through the hepatic artery rather than through the systemic route (jugular vein), although the differences were not statistically significant. Doses below 4 x 10(4) U/day did not result in significant increases of large granular lymphocytes in the liver.

Animals↗
PubMed1990

Ultrastructure and function of hepatic fat-storing and pit cells.

The present paper reviews the literature on the ultrastructure and function of sinusoidal fat-storing cells and pit cells in the mammalian liver. Ultrastructurally, fat-storing cells are characterized by the presence of cytoplasmic fat droplets, well developed rough endoplasmic reticulum; a Golgi complex; multivesicular bodies; one or two centrioles; and few, rather small, lysosomes. These lysosomes are sometimes associated with fat droplets. Fat-storing cells may bear a cilium and project characteristic cytoplasmic processes into the space of Disse. These processes contain microtubules and filaments. Fat-storing cells are the main storage site of retinol esters in the mammalian body. Moreover, these cells have the potential of synthesizing several connective tissue components including the collagens type I, III, and IV; fibronectin; laminin; heparan sulfate; chondroitin sulfate; and dermatan sulfate. Pit cells are polarized cells, with most organelles localized at one site of the nucleus near the cytocentre. They are characterized electron microscopically by the presence of dense cytoplasmic granules with a specific ultrastructure, by rod-cored vesicles, and by multivesicular bodies. It has recently been shown that pit cells have natural killer activity to certain tumor cells and have many features in common with large granular lymphocytes. They therefore may act in the liver as a first line of defense against neoplasia, metastasis, and viral infections.

Animals↗
PubMed1988

Natural cytotoxicity of rat hepatic natural killer cells and macrophages against a syngeneic colon adenocarcinoma.

The in vitro cytotoxic activity of two types of hepatic sinusoidal cells, i.e., natural killer (NK) cells and macrophages (Kupffer cells), was tested against a syngeneic rat colon adenocarcinoma cell line (DHD-K12). Purified hepatic NK cells (85% cells with large granular lymphocyte morphology) were spontaneously cytolytic, whereas Kupffer cells (90% pure) were not able to kill the DHD-K12 cells. This carcinoma cell line was found to be resistant to the action of mouse recombinant tumor necrosis factor which is considered as the major cytolytic molecule secreted by macrophages. However, colon carcinoma cells were readily lysed by soluble factors present in the culture supernatant of NK cells. It is postulated that hepatic NK cells, which are strategically located within the lumen of the sinusoids, may form a first line of defense to metastasizing colon carcinoma cells.

Adenocarcinoma↗
PubMed1988

Cell population kinetics of Kupffer cells during the onset of fibrosis in rat liver by chronic carbon tetrachloride administration.

The cell population kinetics of Kupffer cells (KCs) were investigated during the induction of fibrosis in rat liver by carbon tetrachloride. KCs, characterized microscopically by their specific peroxidase pattern, increased in number during the first 24 h after CCl4 injection. After repeated injections of the toxin the number of KCs increased logarithmically. After 9 weeks of CCl4 treatment, the KC population reached a new steady state with a 2.6-times-higher cell number than in control animals. Local KC proliferation was demonstrated by the metaphase arrest technique using vinblastine. It was calculated that at 24 h and at 72 h after one injection of CCl4, local proliferation did not account for the entire growth of the population. This means that, in addition to local proliferation of KCs, an influx of mononuclear phagocytes into the liver sinusoids occurred.

Animals↗
PubMed1988

Proliferation and phenotypic expression of non-parenchymal liver cells.

The four types of non-parenchymal sinusoid lining cells can be identified in situ and can be isolated for in vitro studies: the fenestrated endothelial cells, the phagocytic Kupffer cells, the fibroblast-like fat-storing cells, and the 'natural killer' pit cells. In situ the last three sinusoidal cell types can be seen to proliferate and increase in number in experimental models of liver pathology, such as inflammations and fibrosis. The understanding of the population dynamics of these cells--their mitotic activity, turnover time, and migration--may therefore have clinical applications. In the present article our data concerning the proliferation kinetics of Kupffer and pit cells in rat liver are reviewed.

Animals↗
PubMed1988

Structural and functional aspects of Kupffer cells.

Methods are now available to characterize Kupffer cells both in vivo and in vitro. They can be isolated and purified from the liver and their functional activities studied in maintenance culture. Kupffer cells are not merely phagocytic cells or scavengers but they can also produce and secrete a variety of substances that can regulate the functions of other cells. They can also regulate the immune system and be directly cytotoxic to tumor cells and parasites. In the light of these reported functions, the clinical importance of Kupffer cells in a variety of liver diseases deserves to be better explored. Although the origin and cytokinetics of Kupffer cells, and specially their relation to the Mononuclear Phagocyte System (MPS), are still not completely understood, it has now become clear that Kupffer cells are not end-cells incapable of self-proliferation. Under various experimental conditions Kupffer cells have been shown to respond to activation by strong mitotic proliferation. On the other hand, bone marrow-derived cells can migrate to the liver under certain conditions and differentiate into liver macrophages, thus becoming Kupffer cells.

Animals↗
PubMed1987

Large granular lymphocytes or "pit cells" from rat liver: isolation, ultrastructural characterization and natural killer activity.

Considerable numbers of large granular lymphocytes (LGL) were isolated from rat liver by a simple method consisting of sinusoidal lavage at elevated (50 cm water column) perfusion pressure. This method gave a yield comparable with the enzymatic dissociation method commonly used for the isolation of nonparenchymal liver cells, but was shorter in time and had the advantage of avoiding the potentially harmful effects of the dissociating enzymes. The isolated LGL were highly cytotoxic against YAC-1 lymphoma cells and this cytolytic activity was blocked by treatment of the effector cells with an antibody against natural killer cells (anti-asialo GM1). We characterized the hepatic LGL as nonphagocytic, nonadherent, peroxidase-negative and acid phosphatase-positive cells which could be enriched in the low-density fraction of a Percoll gradient. At the light microscopic level, they showed characteristic azurophilic granules which corresponded to strongly osmiophilic granules with a specific morphology in electron microscopy. It is concluded that these LGL are identical to the "pit cells" which were formerly described by electron microscopy in situ as normal components of the liver sinusoids and which are easily recognized by their fine structure. It is also proposed that the liver may represent one of the major natural killer organs.

Animals↗
PubMed1987

Immuno-electron microscopic characterization of large granular lymphocytes (natural killer cells) from rat liver.

The immunocytochemical characteristics of large granular lymphocytes (LGL), isolated from the liver sinusoids of euthymic and athymic (nude) rats, were investigated in electron microscopy by the immunoperoxidase technique. The LGL were found positive for MRC OX-8 (natural killer cells and cytotoxic/suppressor T cells) and negative for MRC OX-19 (pan-T marker) in both rat strains. The LGL were heterogeneous in the expression of the natural killer cell marker asialo-GM1 which was found on 56% of the LGL from euthymic and on 71% of the LGL from athymic rats. LGL were easily distinguished from the other cells in the preparations, "conventional" lymphocytes and monocytes, thanks to their highly characteristic ultrastructural features, in particular by the presence of specific electron-dense cytoplasmic granules and rod-cored vesicles. These features have been described formerly for the so-called "pit cells" and are more reliable than the classical LGL characteristics at the light microscopic level, i.e. the presence of azurophilic granules. Our results give further support for the existence of an important population of natural killer cells in the liver sinusoids.

Animals↗