Correlative Fine Structural,Behavioral, and Histochemical Analysis of Ascidian Blood Cells

Abstract The blood cells of a solitary ascidian, Halocynthia roretzi, were examined by electron microscopy (EM) with reference to their appearance by light microscopy (LM). In addition, their movement and stainability by vital dyes was observed by phase-contrast microscopy, and their stainability by Giemsa was also examined. Nine cell types were recognized: vacuolated cells, hyaline amoebocytes, small amoebocytes, granular amoebocytes, macrogranular cells, globular cells, lymphocyte-like cells, large basophilic cells and large granular cells. Vacuolated cells were found to possess various numbers of vacuoles containing strongly electron-dense materials and could be divided into at least three subgroups. Granular amoebocytes contained microfilaments and many granules of uniform size. Hyaline amoebocytes and small amoebocytes seemed to be specialized as phagocytes. Macrogranular cells and globular cells were not well characterized. In the blood of adult individuals, hemoblasts were rarely found, although lymphocyte-like cells were present. Each of two large cells, large basophilic cells and large granular cells, possessed novel granules or vacuoles, whose functions remain to be elucidated. The possible functions and relationships of these cells among various ascidian species are discussed.     

Immunocytochemical demonstration of a humoral defence factor in blood cells (amoebocytes) of the pond snail,Lymnaea stagnalis

Summary Monolayers of blood cells (amoebocytes) and sections of connective tissue and of amoebocyte pellets of the freshwater pulmonate gastropod Lymnaea stagnalis were stained immunocytochemically with antisera to snail agglutinin/opsonin. The presence of this substance was demonstrated light microscopically both in the cytoplasm and on the cell membrane of amoebocytes. This suggests that amoebocytes synthesize agglutinin/opsonin, and bear it at their surface as receptors for foreign materials.     

The blood cells of the sea squirt, Ciona intestinalis: Morphology,differential counts,and in vitro phagocytic activity

The sea squirt, Ciona intestinalis, contains several types of blood cells: stem cells, hyaline, granular, and refractile amoebocytes, signet ring cells, morula cells, small and large compartment cells, and orange cells. Of these cell types, only the hyaline and granular amoebocytes are capable of phagocytosing formalized sheep erythrocytes in vitro. After the addition of erythrocytes to blood cell monolayers, the attachment and ingestion of these particles occurs rapidly. The interrelationships of the various blood cell types are discussed.     

Recognition of foreignness by blood cells of the freshwater snail Lymnaea stagnalis, with special reference to the role and structure of the cell coat

The structure and function of the cell coat of the blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were studied with ultrahistochemical tests, including concanavalin A (Con A) labeling, and with in vitro phagocytosis experiments. The cell coat is intensely stained by ruthenium red and tannic acid. The cells possess binding sites for Con A. Proteolytic enzymes destroy the receptors for Con A and totally inhibit the phagocytic activity of amoebocytes. Incubation experiments with proteases, carbohydrases, and inhibition sugars revealed that (1) the Con A binding sites are anchored in the plasma membrane by proteins, and (2) glucose, fructose, mannose, and to a lesser extent N-acetylglucosamine and N-acetylgalactosamine, inhibit the binding of Con A to amoebocytes, suggesting that these carbohydrates might form part of these binding sites.     

Amoebocytes in mesoglea of Polypodium hydriforme

Mesogleal amoebocytes of free-living Polypodium hydriforme have been studied with transmission electron microscope. The amoebocytes have numerous processes and contain cytoplasmic vacuoles with fibrous material of different density. The phenomenon of cell death (apoptosis) of mesogleal amoebocytes is described. Chromatin of dying cells becomes condensed forming picnotic "caps" in the nucleus. No mitotic cells were encountered among mesogleal amoebocytes. The origin and functions of mesogleal amoebocytes of P. hydriforme are discussed.     

The characterization of granular amoebocytes and their possible roles in the asexual reproduction of the polystyelid ascidian,Polyzoa vesiculiphora

The blood cells in the bud and the zooid of the polystyelid ascidian, Polyzoa vesiculiphora, were examined by means of light and electron microscopy to identify the cells that have been named trophocytes. The large blood cells were abundant in the mesenchymal space of the bud, but not in that of the functional zooid. They contained glycogen particles, lipid droplets, large protein granules and autophagosomes in their cytoplasm and were identified as granular amoebocytes. The majority of these cells were specifically phagocytized by phagocytes during bud development and disappeared. These results indicate that the granular amoebocytes virtually represent trophocytes in Polyzoa and may participate in bud development via nutrient supply to the developing tissues.     

The origin of the endothelial cells: an evo-devo approach for the invertebrate/vertebrate transition of the circulatory system

Circulatory systems of vertebrate and invertebrate metazoans are very different. Large vessels of invertebrates are constituted of spaces and lacunae located between the basement membranes of endodermal and mesodermal epithelia, and they lack an endothelial lining. Myoepithelial differentation of the coelomic cells covering hemal spaces is a frequent event, and myoepithelial cells often form microvessels in some large invertebrates. There is no phylogenetic theory about the origin of the endothelial cells in vertebrates. We herein propose that endothelial cells originated from a type of specialized blood cells, called amoebocytes, that adhere to the vascular basement membrane. The transition between amoebocytes and endothelium involved the acquisition of an epithelial phenotype. We suggest that immunological cooperation was the earliest function of these protoendothelial cells. Furthermore, their ability to transiently recover the migratory, invasive phenotype of amoebocytes (i.e., the angiogenic phenotype) allowed for vascular growth from the original visceral areas to the well-developed somatic areas of vertebrates (especially the tail, head, and neural tube). We also hypothesize that pericytes and smooth muscle cells derived from myoepithelial cells detached from the coelomic lining. As the origin of blood cells in invertebrates is probably coelomic, our hypothesis relates the origin of all the elements of the circulatory system with the coelomic wall. We have collected from the literature a number of comparative and developmental data supporting our hypothesis, for example the localization of the vascular endothelial growth factor receptor-2 ortholog in hemocytes of Drosophila or the fact that circulating progenitors can differentiate into endothelial cells even in adult vertebrates.     

Antioxidant Enzymatic Activity of Coelomocytes of the Far East Sea Cucumber Eupentacta fraudatrix

The basal activity of superoxide dismutase (SOD), glutathione reductase (GR), and catalase in a pool of coelomocytes as well as in the fraction of amoebocytes and the mixed fraction of amoebocytes and morula-like cells of the sea cucumber Eupentacta fraudatrix is studied. For SOD and catalase, pH optima are in the range of values of pH optimum for tissues of mammals, the pH optimum for GR is shifted to a more acidic region in comparison with the latter enzymes. Temperature optima for all studied enzymes are higher than the usual temperature values of the sea cucumber habitation. A pronounced temperature dependence of all three enzymes is revealed. In coelomocytes, the activities of SOD and catalase, but not of GR, are lower than in the fraction of amoebocytes, but higher than in the mixed fraction of amoebocytes and morula-like cells. The rate of production of active forms of oxygen (AFO) is three times higher in amoebocytes than in the fraction relatively enriched in morula-like cells. Apparently, the main part of the SOD and catalase activities, as well as AFO production in coelomocytes is located in amoebocytes, which confirms the existence of cytophagic function in the latter cells as well as argues in favor of functional differentiation between individual types of coelomocytes.     

Regulation of histogenesis in the axial organ of echinoderms (Echinodermata) according to findings from diffusion chamber cultivation]

Fragments of axial organs of different echinoderms were cultivated together with the gonad epithelium in the diffusion chambers made of millipore filters impermeable for cells (VUFS). Under these conditions, the histogenesis of amoebocytes suffered definite changes: the number of middle amoebocytes increased 2-3 times and that of large amoebocytes decreased correspondingly. Similar results were obtained not only under the direct contact of two tissues, but also under the contact realized through the millipore filter impermeable for cells. Corticosteroid agents of vertebrates did not influence the histogenesis of amoebocytes in Echinodermata.     

Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.     

Analysis of the role of leukocyte function-associated antigen-1 in activation of human influenza virus-specific T cell clones

The role of leukocyte function-associated Ag-1 (LFA-1) in intercellular adhesion is well documented. Previously, we demonstrated that the LFA-1 molecule (CD11a/CD18) can also regulate the induction of proliferation of peripheral blood T cells. In these studies, we observed opposite effects of antibodies against CD11a (LFA-1-alpha-chain) or CD18 (LFA-1-beta-chain). Here, we determined the effects of anti-CD11a and anti-CD18 mAb on proliferation of cloned influenza virus-specific T cells. Anti-CD18 mAb had similar inhibiting effects on the proliferative response of T cell clones induced by immobilized anti-CD3 mAb as it had on the response of peripheral blood T cells. In contrast to its costimulatory effect on resting peripheral blood T cells, anti-CD11a mAb did not increase the proliferation of cloned T cells. Similar differences in effects of anti-CD11a and anti-CD18 mAb were observed when proliferation of the T cell clones was induced by immobilized anti-TCR mAb. When proliferation was induced by influenza virus presented by monocytes as APC, both anti-CD11a and anti-CD18 mAb inhibited T cell proliferation. However, when EBV-transformed B cells were used as APC, neither anti-CD11a nor anti-CD18 mAb inhibited proliferation. These results demonstrate that the effects of antibodies against CD11a (LFA-1-alpha) or CD18 (LFA-1-beta) on T cell proliferation depend on 1) the stage of activation of the T cells, 2) the activation stimulus and its requirement for intercellular adhesion involving LFA-1, and 3) the type of cell used to present Ag.     

Scanning x-ray microscopy of living and freeze-dried blood cells in two vanadium-rich ascidian species,Phallusia mammillata and Ascidia sydneiensis samea

Some ascidians (sea squirts) accumulate the transitional metal vanadium in their blood cells at concentrations of up to 350 mM, about 10(7) times its concentration found in seawater. There are approximately 10 different types of blood cell in ascidians. The identity of the true vanadium-containing blood cell (vanadocyte) is controversial and little is known about the subcellular distribution of vanadium. A scanning x-ray microscope installed at the ID21 beamline of the European Synchroton Radiation Facility to visualize vanadium in ascidian blood cells. Without fixation, freezing or staining realized the visualization of vanadium localized in living signet ring cells and vacuolated amoebocytes of two vanadium-rich ascidian species, Phallusia mammillata and Ascidia sydneiensis samea. A combination of transmission and fluorescence images of signet ring cells suggested that in both species the vacuoles contain vanadium.     

Inactivated bovine herpesvirus 1 induces apoptotic cell death of mitogen-stimulated bovine peripheral blood mononuclear cells.

Bovine herpesvirus 1 (BHV-1) is able to inhibit the proliferation of bovine peripheral blood mononuclear cells. Here, we have demonstrated that live BHV-1 and, interestingly, inactivated BHV-1 can induce apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.     

Cytochemical properties of Botryllus schlosseri haemocytes: indications for morpho-functional characterisation

In the present study, we carried out a detailed light microscopy investigation of the cytochemical properties of the haemocytes of the colonial ascidian Botryllus schlosseri, using new cytochemical stains and enzymatic markers, a panel of antibodies and lectins as probes to characterise Botryllus blood cells further. Results indicate that lymphocyte-like cells are circulating undifferentiated cells recognised by anti-CD34 antibody and there are at least two defined haemocyte differentiation pathways: i) phagocytes, represented by hyaline amoebocytes and macrophage-like cells, which share similar staining properties, the same hydrolytic enzyme content as well as the presence of detectable cytochrome-c-oxidase activity, recognition by anti-CD39 and Narcissus pseudonarcissus agglutinin; ii) cytotoxic cell line, represented by granular amoebocytes and morula cells which have vacuoles stained by Ehrlich's stain and Neutral Red; DOPA-containing protein are present inside morula cell vacuoles. Pigment cells and nephrocytes are involved in catabolite storage but their relationships with other cell types are less clear.     

中国鲎变形细胞超微结构的进一步观察

中国鲎的变形细胞可在血液中发育成熟,根据其超微结构,可分为原始变形细胞、幼变形细胞和变形细胞三个阶段。 变形细胞受到外环境或内毒素的刺激后,第一型颗粒出现一系列变化,最终把内容物释放到细胞外。与此同时,细胞表面形成若干胞质性突起,边缘带分散断裂,质膜下和胞质性突起中出现微丝。第二型颗粒无变化,提示它们和血液凝胶化过程无直接关系。     

Differences between blood cells of juvenile and adult specimens of the pond snail Lymnaea stagnalis

Summary Blood cells (amoebocytes) of juvenile and adult specimens of the pond snail Lymnaea stagnalis were compared. Juvenile snails contain fewer circulating amoebocytes per l haemolymph. However, a higher percentage of these cells shows mitotic activity, as determined by incorporation of ³H-thymidine in vitro. Relatively more amoebocytes of juvenile snails have the characteristics of less differentiated cells: they are small and round with few inclusions, a high nucleus-to-cytoplasm ratio, and a high pyronin stainability. Enzyme cytochemical studies showed that acid phosphatase (AcP), non-specific esterase (NSE), and alkaline phosphatase (AlP) are present in all amoebocytes of juvenile and adult snails. AcP activity is relatively weak. NSE activity is dispersed throughout the cytoplasm and occasionally found in granules, whereas AlP is clearly localized in granules. Differences between the two age groups were found only for the enzyme peroxidase (PO). In juvenile snails a lower percentage of the cells is positive and the granules that contain the activity are less abundant than in amoebocytes of adults. It is suggested that, due to the above-mentioned characteristics of the amoebocytes, the activity of the internal defence system in juvenile L. stagnalis is on a lower level than that in adult snails. This might be an explanation for the fact that juvenile L. stagnalis are highly susceptible to infection by the schistosome Trichobilharzia ocellata, whereas adult snails are less susceptible.     

A Quantitative,Comparative Study of Element Variations Found within the Range of Blood Cells from the Tropical Ascidian Phallusia philippinensis,using the Nuclear Microscope

The present study examines the concentrations of vanadium, bromine and sulphur contained within cryofixed/freeze-dried blood cells of the ascidian Phallusia philippinensis. Elemental profiles of seven cell types were obtained using the National University of Singapore nuclear microscope, in order to ascertain the cell types predominantly involved in accumulation. Morula cells were found to contain the following mean values (in ppm dry weight); 7878 vanadium, 34484 bromine and 61078 sulphur. Signet ring cells contained 5191 vanadium, 23945 bromine and 15281 sulphur. Compartment cells had 606 vanadium, 20700 bromine and 24309 sulphur. Other less abundant cell types such as lymphocytes, macrogranular amoebocytes, carotenoid pigment cells and granular amoebocytes were also analysed and found to contain (in ppm) 4384, 6652, 2366 and 10246 vanadium, 19652, 15630, 5964 and 11735 bromine and 13289, 15309, 3106 and 42968 sulphur, respectively. Sulphur occurred in high levels in all cell types, which could indicate its involvement in the vanadium concentration process, while bromine, incorporated into complexes, may be utilised for anti-fouling rather than as a deterrent to predators.     

Influence of aging on intracellular free calcium and proliferation of mouse T-cell subsets from various lymphoid organs.

The influence of aging on T-cell activation and proliferation was examined in lymphocytes derived from peripheral blood, spleen, and lymph nodes of WBB6F1 C57B1/6J x WB/Re) mice. Following activation with anti-CD3 monoclonal antibodies, the greatest age-related changes were seen in CD4+ cells derived from spleens of 27- to 30-month-old mice. These CD4+ lymphocytes showed reduced [Ca2+]i signaling and decreased proliferation in the presence of exogenous interleukin 2. CD8+ cells from spleens of old animals showed reduced [Ca2+]i but not altered proliferation. Both CD4+ and CD8+ cells derived from peripheral blood of old mice showed decreased peak [Ca2+]i, but no defect in cell proliferation. In contrast, age-related deficits in either [Ca2+]i or proliferation were not observed in CD4+ and CD8+ cells from lymph nodes. Additionally, the percentage of CD4+ cells was decreased in all lymphoid organs from old mice, while the percentage of CD8+ cells was similar in lymphoid organs of old and young mice. Old mice had a significant increase in expression of Pgp-1 in CD4+ cells from spleen and peripheral blood and CD8+ cells derived from lymph node. Our studies indicate that there are differential effects of aging in T lymphocytes derived from different lymphoid organs in mice. Among the cell sources and subsets examined, the age-related changes noted in CD4+ cells from mouse peripheral blood were the most similar to those previously observed in the corresponding peripheral blood lymphocyte subset in humans.     

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller)

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.     

Effects of infection of EGFP-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage 1 h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians.