Rod-cored Vesicles Research Articles

Pit cells as liver-associated natural killer cells: morphology and function.

Pit cells are one type of hepatic sinusoidal cells, defined morphologically as large granular lymphocytes (LGLs) and functionally as liver-associated natural killer (NK) cells. They are situated inside the sinusoidal lumen, adhering to the endothelial cells and Kupffer cells. They contain multivesicular body-related dense granules and rod-cored vesicles. The number and size of granules and vesicles differ between hepatic NK cells and peripheral blood cells, suggesting possible differentiation of the latter into the former in the microenvironment of the liver. In addition to NK cells, natural killer T (NKT) cells are also abundant in the liver. They share several morphological properties with NK cells, and at least some are probably observed as pit cells under an electron microscope. NK cells recognize target cells with their surface receptors such as inhibitory and activating receptors. They exert antitumor functions by exocytosis of perforin/granzyme-containing granules, induction of death receptor-mediated apoptosis in target cells, and production of various cytokines that augment the activities of other immune cells. NKT cells play important roles in initiating and assisting the function of NK cells by producing interferon-gamma.

On the cell biology of pit cells, the liver-specific NK cells.

There is s growing evidence that pit cells are highly active, liver-specific NK cells. Pit cells are located in the liver sinusoids and can be easily isolated and purified by liver sinusoidal lavage and a magnetic separation method. Furthermore, pit cells can be separated into a LD and HD fraction by 45% iso-osmotic Per coll gradient centrifugation. These two populations are shown to differ morphol ogically, phenotypically and functionally from each other and from blood NK cells. LD pit cells contain more rod-cored vesicles and more, but smaller granules than blood NK cells although both of them share LGL morphology. Phenotypically, LD cells have a higher expression of LFA-1 and a lower expression of asialo-GM 1 molecules compared to blood or spleen NK cells. Functionally, pit cells are more cytotoxic against several tumor cell lines as compared to blood NK cells, and are able to kill-NK-resistant but LAK-sensitive P815 cells. These data indicate that pit cells are a kind of naturally activated NK cells and their cytotoxic function is comparable to IL-2 in vitro activated NK cells. The characteristics of HD cells are intermediate between LD pit cells and blood NK cells. Pit cells most probably originate from blood NK cells, although they show mitosis in the liver after certain stimuli. The recruitment of pit cells in the liver is mediated by adhesion molecules. A major challenge is to achieve a better understanding of the mechanisms of pit cell cytotoxicity and the cooperation between pit cells and other cells in the liver, i.e. Kc, Ec and LAL. Moreover, since pit cells are located in a strategic position in the hepatic sinusoids, they represent a first line of cellular defense against metastasing colon cancer cells. The role of pit cells in a number of liver pathologies deserves more attention.

Thy1(+) Lymphocytes with LAK-like Activity Are Present in Rat Liver Sinusoids.

Lymphocytes with cytotoxic activity have been identified in sinusoids of rat liver. We investigated the effects of aging on the cytotoxicity of liver sinusoidal lymphocytes in the rat and attempted to identify the cells responsible for cytotoxicity using a Thy1 monoclonal antibody. Natural Killer (NK) activity against YAC1 peaked at the age of 8 weeks, while cytotoxicity against NK-resistant Lymphokine Activated Killer (LAK)-sensitive P815 increased gradually with age. A cold target inhibition assay showed that lymphocytes cytotoxic for P815 and AH109 were also present in the liver sinusoids of aged rats. When these cells were fractionated by the Percoll discontinuous density gradient method, cytotoxic cells were found to comprise large granular lymphocytes (LGLs) rich in low- and middle-density fractions. Cytotoxicity against various targets increased when CD5(+) cells were removed using CD5-conjugated beads. Cytotoxicity against P815, AH109 and AH130 was decreased as compared with control lymphocytes when CD5(-) Thy1(+) lymphocytes were removed using CD5, Thy1-conjugated beads. Immunohistologic examination revealed Thy1(+) LGLs with rod-cored vesicles and electron-dense granules in the liver sinusoids. We consider that LAK-like cells with a Thy1 phenotype are present in the liver sinusoids and suggest that they have a broad range of cytotoxicity.

Quantitative analysis of rod-cored vesicles and dense granules of large granular lymphocytes in the liver, spleen, and peripheral blood of rats

Large granular lymphocytes (LGL) comprise a natural defense system in the liver and exert an inhibitory effect on tumor cell metastasis. In order to demonstrate the maturation of LGL in the liver from the morphological aspect, we evaluated electron-microscopically the frequency of 0.2 micron vesicles (rod-cored and "empty" vesicles) and dense granules in LGL from the liver, spleen, and peripheral blood of the rat. Both of these cell organelles are characteristic to LGL and may relate to natural killer-mediated cytolysis. On the average, there were 12.7 of the 0.2 micron vesicles and 4.3 rod-cored vesicles (RCV) per cell section in the liver, 6.6 0.2 micron vesicles and 1.6 RCV in the spleen, and 8.6 0.2 micron vesicles and 0.9 RCV in the peripheral blood. The number of 0.2 micron vesicles per cell section ranged from 0 to 19 with the exception of a few higher instances. Therefore, LGL were divided into vesicle-rich (> 9 0.2 micron vesicles per cell section) and vesicle-poor (< 8 per cell section) populations. Hepatic LGL consisted mainly of a vesicle-rich population while splenic LGL consisted mainly of a vesicle-poor population, and peripheral blood contained equal proportions of both populations. In addition to diversity with regard to the number of 0.2 micron vesicles, LGL obtained from various organs also displayed heterogeneity in the number and size of dense granules.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis of rod-cored vesicles and dense granules of large granular lymphocytes in the liver, spleen, and peripheral blood of rats

Quantitative analysis of rod-cored vesicles and dense granules of large granular lymphocytes in the liver, spleen, and peripheral blood of rats

網内系学説と現在の肝類洞壁細胞像

The wall of hepatic sinusoids is a morphological and functional unit made up of four different cell populations, i.e., endothelial cells, Kupffer cells, perisinusoidal stellate cells (fat-storing cells), and lymphocytes including pit cells (liver-associated LGLs).The core element of this unit is the endothelial lining which provides a bed for other sinusoidal cells. Endothelial cells are highly fenestrated and sieve the fluids exchanged between the sinusoidal lumen and the space of Disse. Presence of numerous receptors on endothelial cells supports the receptor-mediated endocytosis of various substances. Adhesion molecules are expressed on the surface of these cells. Kupffer cells are resident macrophages, and clear endotoxin from the blood stream. When activated, Kupffer cells secrete metabolites of arachidonic acid, TNF, IL-1, IFN, and reactive oxygen products. Stellate cells are located perisinusoidally surrounding the sinusoidal tubes and, on the other hand, send thorn-like microprojections to parenchymal cells. Stellate cells store vitamin A and produce Type I, III and IV collagens and other extracellular matrix. Pit cells are characterized by the presence of dense granules and rod-cored vesicles. After administration of BRMs, number of pit cells increases adhering to the endothelial lining and liver-associated NK activity is augmented. Paracrine and autocrine secretions of mediators and cytokines from sinusoidal cells modulate functions of the sinusoidal wall and parenchymal cells.Currently the RES is restricted only to the system of macrophages. From the modern concept of sinusoidal cells in the liver, however, phagocytosis is only the trigger for starting the cascade of broad RES functions beyond the single cell line. The liver RES is not only Kupffer cells but the sinusoidal wall itself which provides a microenvironment between the blood stream and parenchymal cells. The RES is specifically organized tissue in the body, as suggested by early investigators.

The intracellular distribution of cell organelles in natural killer cells during the cytolysis of bound tumor cells, with special reference to the rod-cored vesicles.

The secretory function of the natural killer (NK) cells has been considered to be essential for cytolytic activity against their target cells. In this study, the distribution and spatial relationship of various cell organelles which participate in the secretory process, i.e., Golgi apparatus, vesicles, granules and microtubules, were examined ultrastructurally in non-treated and OK-432-activated rat NK cells bound to the tumor cells, with special reference to the rod-cored vesicles which are the most characteristic structure of the NK cells. Rod-cored vesicles and their closely related structures, "empty" vesicles, were derived from the end portion of the Golgi trans cisternae and became accumulated in the central area which was surrounded by the Golgi apparatus, nucleus and contact surface. Some of the vesicles appeared to be further transported to the contact area along the microtubules extending from the centrioles toward the bound targets. The access of the vesicles to the contact surface occurred at that portion where subplasmalemmal actin lattice was thin. The distribution of the dense granules and multivesicular bodies was similar to that of the vesicles, but the area of their occurrence was a little wider. At the outer aspect of the Golgi apparatus was situated the endoplasmic reticulum from which transitional vesicles came to the inner or cis cisternae of the apparatus. The present observations indicate that the cell organelles of the conjugated NK cells are purposefully arranged in the cytoplasm in such order that the generated rod-cored vesicles and "empty" ones are efficiently directed toward the bound tumor cells.

Ultrastructural characteristics of lymphokine-activated killer cells of the rat in comparison with natural killer cells.

Lymphokine-activated killer (LAK) cells with a broad spectrum of tumor cell killing have been reported to be related to natural killer (NK) cells morphologically and phenotypically. We here examine the ultrastructural characteristics of LAK cells of the rat, comparing them to those of normal and OK-432-activated NK cells. Results show that, five days after the culturing of spleen lymphocytes with human recombinant interleukin-2, there were induced LAK cells, which were large granular lymphocytes and had a cytotoxic capacity against NK-resistant P-815 tumor cells. They were larger in size than NK cells and richer in cell organelles such as ribosomes, rough endoplasmic reticulum, a Golgi apparatus, granules and vesicles. The granules of LAK cells were shown to be related to multivesicular bodies as those of NK cells; they included multivesicular bodies, fully dense granules and intermediate forms between them. The average numbers and sizes of the granules and the proportion of multivesicular bodies and intermediate forms among the total granules were greater in LAK cells than in NK cells. The density of the small vesicles packed in multivesicular bodies and intermediate forms was much higher in LAK cells. At the contacting surface of the LAK cells bound to the target cells, exocytosis of multivesicular bodies was shown to occur. We recognized here two populations of LAK cells with different types of vesicles, one containing rod-cored vesicles and the other a new type of vesicles termed "demilune-cored vesicles". The latter vesicles were the same in size as the rod-cored ones and contained a dense core located eccentrically. Between these two populations of LAK cells, there was no difference concerning the profile of the dense granules. The present study indicates that, although LAK and NK cells share several ultrastructural features, the former show markedly enriched cell organelles, which indicate an accelerated metabolism of the cell for continuous proliferation.

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.

Liver-associated large granular lymphocytes: morphological and functional aspects.

Large granular lymphocytes (LGLs) differ from other lymphocytes in their recirculation pattern and are distributed preferentially in nonlymphoid organs such as the liver and lung. The liver-associated LGLs adhere strongly to the sinusoidal endothelium and show a natural killer (NK) cytotoxicity against incoming metastatic tumor cells; this reaction occurs very rapidly because, in contrast to the immune response, it does not require complex processes in the lymphoid tissue. They have been extensively studied morphologically in terms of pit cells. LGLs have two characteristic cell organelles which participate in the NK cytolysis, i. e., dense granules and rod-cored vesicles. The former are lysosomes derived from multivesicular bodies and contain pore-forming proteins. The latter are the secretory vesicles exclusively seen in LGLs and are markedly increased in number when the NK function is augmented by biological response modifiers. These two structures are believed to be exocytosed in the space between LGL and the conjugated tumor cell. The microenvironment of the liver sinusoids, which includes Kupffer cells, endothelial cells and other lymphocytes, is considered to regulate the function of the liver-associated LGLs. Liver-associated LGLs, as well as Kupffer cells, are intrinsically involved in the defense system of the liver under various physiological and pathological conditions.

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.

Pit cells in extrahepatic organs of the rat.

Electron microscopic examination of the extrahepatic distribution of pit cells, a cell type found in the liver, revealed their existence in several other organs of the rat. They were relatively frequent in lungs, spleen (red pulp), small intestine, epididymis, trachea, and peripheral blood; much fewer in bone marrow and thymus (medulla); and nonexistent in lymph nodes, spleen (white pulp), and thymus (cortex). The pit cells in these organs, as well as in the liver, contained characteristic dense granules and rod-cored vesicles in the cytoplasm. Our observations suggest that pit cells circulating in the peripheral blood adhere to the endothelium of capillaries in the various organs and migrate into the tissue, where they have some special immunological function.

&lt;b&gt;A striking increase in rod-cored vesicles in pit cells (natural killer cells) and augmentation of the liver-associated natural killer activity by a streptococcal preparation &lt;/b&gt;&lt;b&gt;(OK-432) &lt;/b&gt;

&lt;b&gt;A striking increase in rod-cored vesicles in pit cells (natural killer cells) and augmentation of the liver-associated natural killer activity by a streptococcal preparation &lt;/b&gt;&lt;b&gt;(OK-432) &lt;/b&gt;

Distribution and morphological characteristics of the pit cells in the liver of the rat.

Pit cells, on which almost no further contributions have been presented since the first report by Wisse et al. (1976), are described in detail in the rat liver. These cells show several characteristic features: 1) "rod-cored vesicles", a new type of vesicular inclusion observed first in our study; 2) electron-dense granules, which we consider to arise from multivesicular bodies by the accumulation of dense material; and 3) well-developed pseudopodia. Although these features clearly differentiate pit cells from conventional lymphocytes, these two cell types display similarities (i) in a number of ultrastructural features, (ii) in the pattern of their intralobular distribution, and (iii) in their presence in the spleen and peripheral blood.