Hyaline Amoebocytes Research Articles

Searching for the Origin and the Differentiation of Haemocytes before and after Larval Settlement of the Colonial Ascidian Botryllus schlosseri: An Ultrastructural Viewpoint

The colonial ascidian Botryllus schlosseri possesses an innate immunity, which plays fundamental roles in its survival, adaptability, worldwide spread and ecological success. Three lines of differentiation pathways of circulating haemocytes are known to be present in the haemolymph, starting from undifferentiated haemoblasts: (i) the phagocytic line (hyaline amoebocytes and macrophage-like cells), (ii) the cytotoxic line (granular amoebocytes and morula cells) and (iii) the storage cell line (pigment cells and nephrocytes). Many questions remain about their origin, and thus, observations during various stages of development were undertaken in this study. Haemocytes were detected beginning from the early tailbud embryo stage. Haemoblasts were always present and morula cells were the first differentiated haemocytes detected. In both the next stage, just before hatching, and the swimming tadpole larva stage, hyaline amoebocytes and pigment cells were also recognisable. Some morula cells containing active phenoloxidase migrated from the haemolymph into the tunic after having crossed the epidermis, and this behaviour could be related to the preparation of a defensive function for spatial competition. During larval metamorphosis, macrophage-like cells appeared with their phagosomes positive to acid phosphatase activity and containing apoptotic cells from tail tissue degeneration. After metamorphosis, in the filter-feeding oozoid stage, nephrocytes involved in nitrogen catabolism finally appeared. In both the subendostylar sinus and the peripheral blind-sac vessels (ampullae), clusters of haemoblasts were recognisable, some of which showed incipient specialisations, considering the hypothesis of the presence of putative niches of haemolymph stem cells.

Ciona robusta hemocyte populational dynamics and PO-dependent cytotoxic activity

Hemocyte populations from the ascidian Ciona robusta, separated through a Percoll discontinuous density gradient, are further characterized by May-Grünwald-Giemsa staining and a cytochemical reaction for phenoloxidase. Variability in cell density, acidophilic property and phenoloxidase activity suggest multiple hemocyte type populations, cell lineages and morphotypes that may be involved in distinct cellular responses. Therefore, unilocular refractile granulocytes, typical of this ascidian species, enriched in a fraction separated from the hemolymph show in vitro phenoloxidase-dependent cytotoxic activity against mammalian erythrocytes and a tumor cell lineage, in addition the properties listed above indicate relationships with vacuolated signet ring cells. Finally, bromo-deoxyuridine with, diamino-phenylindole fluorescent reaction and May-Grünwald-Giemsa staining show that in the hemolymph there are hyaline amoebocytes and granulocytes with potential proliferating activity. Present findings and reviewed images of previously reported inflammatory hemocytes in the tunic and pharynx allow us to speculate on theoretical outlines of hemocyte differentiation pathways.

The haemocytes of the colonial aplousobranch ascidian Diplosoma listerianum: Structural, cytochemical and functional analyses

Diplosoma listerianum is a colonial aplousobranch ascidian of the family Didemnidae that is native to the northeast Atlantic and exhibits a cosmopolitan distribution in temperate waters. It lacks a shared colonial circulation crossing the tunic, and the zooids are connected only by the common tunic. In the present study, the haemocytes of this ascidian were analysed via light and electron microscopy. Their phagocytic and enzymatic activities, staining and immunostaining properties, and lectin affinity were examined with various classical methods reconsidered and modified for small marine invertebrates. Eight morphotypes were identified in reference to corresponding cell types described in other ascidians: undifferentiated cells (haemoblasts), storage cells for nitrogenous catabolites (nephrocytes) and immunocytes. The immunocytes are involved in immune responses, acting as (1) phagocytes, rich in hydrolases and involved in the clearance of both foreign particles and effete cells (hyaline amoebocytes and macrophage-like cells); (2) cytotoxic cells, able to degranulate and induce cytotoxicity through the release of the enzyme phenoloxidase after an immune stimulus (granular amoebocytes and morula cells); and (3) basophilic cells with an affinity for ConA and NPA that contain heparin and histamine and that show sensitivity to the compound 48/80, promoting their degranulation (mast cell-like granulocytes). In addition, a particular cell type showing exceptional development of the Golgi apparatus and large vacuoles containing a filamentous material has been recognised (spherule cell), for which a role in tunic repair and fibrogenesis has been hypothesised.

Exposure of coelomocytes from the earthworm Eisenia hortensis to Cu, Cd, and dimethylbenz[a]anthracene: An in vitro study examining reactive oxygen species production and immune response inhibition

The principal aims of this study were to investigate the in vitro effects of copper (Cu), cadmium (Cd) and the polycyclic aromatic hydrocarbon (PAH) dimethylbenz[a]anthracene (DMBA) on reactive oxygen species (ROS) production and the effect of DMBA on immune function (phagocytosis) in coelomocyte samples isolated from the earthworm Eisenia hortensis . Using a fluorogenic indicator of ROS, dihydrorhodamine 123, we show that earthworm coelomocytes are induced to produce significant increases in ROS when exposed to Cu or Cd at 32–250 μM, an effect diminished by the chelator ethylenediaminetetraacetic acid (EDTA). DMBA also induced significant ROS production in the range of 20–400 μM compared to controls. Testing the effect of DMBA on cellular immune mechanisms, we used a phagocytosis assay to reveal an immunosuppressive effect of DMBA (50–200 μM) on the capacity of hyaline amoebocytes to ingest fluorescent Escherichia coli . These studies illustrate the relative ease and efficiency of these in vitro procedures for testing the toxicological effects of environmental pollutants, including heavy metals and PAHs, using earthworm coelomocytes as indicators of oxidative stress and immune response impairment.

Vertebrate cytokines interleukin 12 and gamma interferon, but not interleukin 10, enhance phagocytosis in the annelid Eisenia hortensis

Phagocytosis assays employing class I [interleukin 12 (IL-12)], and class II [gamma interferon (gIFN) and IL-10] human recombinant cytokines were carried out to determine the biological effects of these molecules on innate immune responses in the earthworm Eisenia hortensis. Coelomocytes from E. hortensis were pre-incubated with the cytokines for 16–20 h in vitro followed by introduction of Escherichia coli expressing green fluorescent protein ( E. coli/GFP). The pro-inflammatory cytokines IL-12 and gIFN stimulated statistically significant ( p ⩽ 0.05) enhanced phagocytosis of E. coli/GFP by hyaline amoebocytes as determined by flow cytometry; 10 out of 21 earthworms (48%) responded to IL-12, while eight out of 21 (38%) responded to gIFN. In contrast, the anti-inflammatory cytokine IL-10 neither stimulated nor inhibited phagocytosis in nine earthworms tested. These results demonstrate that vertebrate pro-inflammatory cytokines influence invertebrate cellular responses of immune cells causing enhanced phagocytic activity in earthworm coelomocytes.

Characteristics of coelomocytes of the stubby earthworm, Allolobophora chlorotica (Sav.)

Coelomic fluid and cells (coelomocytes) were extruded by the electric shock (1 min, 4.5 V) from field-collected individuals of Allolobophora chlorotica (Sav.). Coelomocytes were subjected to bright field and fluorescence light as well as electron microscopy (transmission and scanning). Results of all methods combined, revealed two distinct populations of circulating cells in the coelomic fluid: (1) autofluorescent eleocytes/chloragocytes filled with numerous large granules (chloragosomes) containing fluorophores, mainly riboflavin; and (2) amoebocytes, which can be differentiated into: (a) hyaline amoebocytes, firmly spreading in form of crinoline-like membranes; (b) granular amoebocytes with numerous dark granules spreading in form of fine pseudopodia. Flow cytometry confirmed the presence of highly complex autofluorescent cells (eleocytes/chloragocytes) and two cohorts of not-autofluorescence much less complex amoebocytes.

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.

RGD-containing molecules induce macropinocytosis in ascidian hyaline amoebocytes

Phagocytes of the compound ascidian Botryllus schlosseri are capable of constitutive macropinocytosis (MP) at sites of membrane ruffling along the leading edge. This gives rise to the formation of initially irregular vesicles which then move to the inside of the cells and acquire a more regular morphology. Both phagocyte spreading and MP are enhanced by the recognition of molecules containing the sequence Arg-Gly-Asp (RGD): this suggests that, as in mammals, integrin activation is involved in the induction of both cell spreading and endocytosis. The occurrence of MP is associated with increased oxygen consumption and a rise in the production of superoxide anion, as indicated by nitroblue tetrazolium reduction, and ATP, as indicated by increased cytochrome oxidase activity. On the whole, our results indicate the conservation of common mechanisms of MP induction throughout the Chordate phylum.

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.

Annual changes in coelomocytes of four earthworm speciesThe 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Summary Coelomocytes (extruded via dorsal pores of earthworms subjected to 5V electric shock) of adult Allolobophora chlorotica, Lumbricus terrestris, Dendrobaena veneta and Eisenia fetida consisted of species-specific proportions of hyaline amoebocytes, granular amoebocytes, and eleocytes. In all species a significant increase of granular amoebocytes with a parallel decrease of hyaline amoebocytes was recorded by the end of Spring and beginning of Summer, both in animals from the natural environment and in those kept in the laboratory at the room temperature. It indicates that coelomocyte proliferation/maturation is dependent mainly on the annual endogenous rhythm, which is relatively stable even at atypical ambient temperatures. During four successive seasons viability and functions (plastic adherence or neutral red pinocytosis) were recorded in coelomocytes of A. chlorotica assayed at various in vitro temperatures. In coelomocytes retrieved from animals freshly collected from their natural environment, coelomocyte activity was better at low temperatures (0 °C and 10 °C) during the Winter, while during other seasons (especially in the Summer) it was best at 22 °C. Such seasonal effects were abolished in coelomocytes from animals tested after one-month acclimation to the room temperature, as in all seasons cell functions were best at 22 °C. These observations indicate that earthworm coelomocytes can thermally adapt. This phenomenon was also recorded in L. terrestris, D. veneta and E. fetida . In conclusion, earthworm coelomocyte proliferation/maturation seems to be endogenously controlled while coelomocyte activity seems to be more plastic and subjected to thermal adaptation.

Annual changes in coelomocytes of four earthworm species: The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Coelomocytes (extruded via dorsal pores of earthworms subjected to 5V electric shock) of adult Allolobophora chlorotica, Lumbricus terrestris, Dendrobaena veneta and Eisenia fetida consisted of species-specific proportions of hyaline amoebocytes, granular amoebocytes, and eleocytes. In all species a significant increase of granular amoebocytes with a parallel decrease of hyaline amoebocytes was recorded by the end of Spring and beginning of Summer, both in animals from the natural environment and in those kept in the laboratory at the room temperature. It indicates that coelomocyte proliferation/maturation is dependent mainly on the annual endogenous rhythm, which is relatively stable even at atypical ambient temperatures. During four successive seasons viability and functions (plastic adherence or neutral red pinocytosis) were recorded in coelomocytes of A. chlorotica assayed at various in vitro temperatures. In coelomocytes retrieved from animals freshly collected from their natural environment, coelomocyte activity was better at low temperatures (0 °C and 10 °C) during the Winter, while during other seasons (especially in the Summer) it was best at 22 °C. Such seasonal effects were abolished in coelomocytes from animals tested after one-month acclimation to the room temperature, as in all seasons cell functions were best at 22 °C. These observations indicate that earthworm coelomocytes can thermally adapt. This phenomenon was also recorded in L. terrestris, D. veneta and E. fetida. In conclusion, earthworm coelomocyte proliferation/maturation seems to be endogenously controlled while coelomocyte activity seems to be more plastic and subjected to thermal adaptation.

Cell Types Involved in Allogeneic Contact Reactions of the Solitary Ascidian, Halocynthia roretzi

The blood cells of a solitary ascidian, Halocynthia roretzi exhibit the allogeneic cellular reaction in vitro denoted as the “contact reaction”(Fuke, 1980). Nine cell types have been recognized in the blood and body fluid of H. roretzi (Fuke and Fukumoto, 1993). In the present study, the precise role of each cell type in allogeneic reactions is investigated in vitro. The vacuolated cells show devacuolation after contact with almost all other cell types from different reactive animals. These cells include hyaline amoebocytes, granular amoebocytes, macrogranular cells, small amoebocytes and giant cells (large basophilic cells), as well as vacuolated cells. The hyaline amoebocytes and small amoebocytes, which belong to the phagocyte series exhibit contact reactions with the cells of phagocyte series from other specimens of H. roretzi. They also show a contact reaction when in contact with granular amoebocytes. Therefore, almost all cell types have the materials responsible for individuality on their cell surface and can directly show the allogeneic cellular reactivity when they contact each other. To determine whether the contact reaction is involved in cell death, the loss of plasma membrane integrity was examined using fluorescent dyes. The number of cells showing uptake of ethidium bromide increased immediately after mixing of allogeneic cells. Almost the same cell types as those showing allogeneic behavior by light microscopy, as described above, exhibited loss of cell membrane integrity.These results are discussed in the context of immune systems in invertebrates and vertebrates.

Antibacterial Properties of Isolated Amoebocytes From the Sea Anemone Actinia equina.

The antimicrobial defenses of anthozoans were investigated in vitro by extracting amoebocytes from the mesenteric filaments of the beadlet anemone, Actinia equina, and testing for their ability to phagocytose and kill the gram-negative bacterium Psychrobacter immobilis. Only the hyaline amoebocytes exhibited phagocytosis in vitro, with about 40% seen to ingest one or more bacteria over 45 min. Mixed cultures of viable amoebocytes were further found to produce O2- ions and other reactive oxygen species (ROS) after stimulation with phorbol myristate acetate or lipopolysaccharide. Co-incubation of viable amoebocytes with P. immobilis for 3 h in vitro resulted in reduced growth of the bacterium compared to saline-incubated bacteria, but because the growth of P. immobilis was also impaired by lysed control amoebocytes, the contribution made to bacterial killing by ROS could not be evaluated. Instead, as confirmed by additional experiments using lysate supernatants of the amoebocytes, it appears that the cells contain soluble bactericidal factors. The nature of these agents is at present unknown, although preliminary tests indicate that killing is not mediated by lysozyme.

New data on phagocytes and phagocytosis in the compound ascidianBotryllus schlosseri(Tunicata, Ascidiacea)

Abstract In the compound ascidian Botryllus schlosseri, phagocytes change their morphology and frequency during the colonial blastogenic cycle. In the regression phase, characterized by intense phagocytosis, zooids of the old generation are progressively resorbed, the frequency of hyaline amoebocytes falls abruptly, and the frequency of macrophage‐like cells increases significantly. Moreover, the hyaline amoebocytes engulf foreign materials, withdraw their cytoplasmic projections, and change their shape from flat and fusiform to spherical or ovoidal: all these processes suggest that hyaline amoebocytes represent the precursors of macrophage‐like cells. During regression, a significant increase in blood levels of hydrogen peroxide and acid phosphatase is observed, in agreement with data obtained in in vitro experiments showing an increase in reactive oxygen metabolite production, nitrite ion release, and acid phosphatase secretion associated with phagocytosis.

Antibacterial activity in the blood cells of the solitary ascidian, Ciona intestinalis, in vitro

AbstractThe antibacterial properties of the blood cells of the solitary ascidian, Ciona intestinalis, were investigated in vitro. The results show that blood cell lysate supernatants have potent antibacterial activity against a wide range of bacteria. Activity was strongest against Gram positive bacteria, where typically there was a 95‐99% reduction in bacterial viability within 1 h. By contrast, Gram negative bacteria were less susceptible to the antibacterial effects, with the viability of these bacteria generally reduced by approximately 20‐40% after 3 h. Activity against both types of bacteria was further found to be heat‐stable (even after heating to 100°C for 10 min or freezing to −20°C for up to 1 month), Ca2+‐but not Mg2+‐dependent, and active at very low titres. Comparison of the antibacterial vigour of lysate supernatants made from separated blood cell populations revealed that activity resides mainly in the morula cells with some, albeit weaker, activity also detectable in the hyaline amoebocytes. No activity was present in the plasma. Preliminary investigations into the mode of action of the active factors indicate that the response is not due to lysozyme, is not directly lytic in character, and is not attributable to agglutination. Instead, as preliminary characterization by gel filtration on Sephadex G‐75 revealed 4 main protein peaks, with antibacterial activity confined to peak 3 (molecular weight range of 8‐15 kDa), the phenomenon seems to be due to the action of one or more low molecular weight proteins. The location, action, and release of these factors appear to differ from those of the cecropins and defensins described in other animal groups. © 1995 Wiley‐Liss, Inc.

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.

Histoenzymatic staining and characterization of the colonial ascidianBotryllus schlösserihemocytes

Abstract The distribution pattern of some hydrolytic and oxidizing enzymes among Botryllus schlosseri hemocytes has been investigated histochemically. Signet‐ring cells, macrophages, and some hyaline amoebocytes were found to stain positively for phosphatases, glycosidases, and esterases, and morula cells and some granular amoebocytes for arylsulfatase, peroxidase, and phenoloxidase. Our results suggest two differentiation pathways for these blood cell types: one involves hyaline amoebocytes, signet‐ring cells, and macrophages and leads to phagocytic cells; the other involves granular amoebocytes and morula cells and leads to cells responsible for inflammatory responses.

Preliminary investigations on the possible antimicrobial properties of tunicate blood cell vanadium

AbstractTunicates have the unique ability to accumulate and store metals such as vanadium. In the sea squirt, Ciona intestinalis, this element is stored in phagocytic blood cells termed granular amoebocytes and vacuolar hyaline amoebocytes. Incubation of bacteria and amoebocytes together results in the phagocytosis of these particles with subsequent fusion of the vanadium‐containing vaeuoles with the bacteria‐laden phagosomes. This process may be important in the intracellular digestion of such microorganisms.

Inflammatory Responses in Annelids

The cellular inflammatory responses of annelids, which include chemotaxis, phagocytosis, encapsulation, and various phases of wound healing, are effected by coelomocytes, particularly the hyaline amoebocytes. Coelomic epithelial cells and specialized nephridial cells also participate in phagocytosis. A number of factors related to inflammation in vertebrates have been identified in annelids, i.e., histamine, kallikrein, lysosomal enzymes, agglutinins, lysins and antibacterial substances. However, their precise function in annelid inflammatory responses has yet to be determined.

Ultrastructural and cytochemical studies on the blood cells of the sea squirt, Ciona intestinalis. I. Stem cells and amoebocytes.

The ultrastructure and cytochemistry of the stem cell and amoebocytic blood cells of the sea squirt, Ciona intestinalis, are described. The stem cells are characterised by a high nuclear: cytoplasmic ratio and on undifferentiated cytoplasm. In thin sections, four types of amoebocytes can be demonstrated: non-vacuolar and vacuolar hyaline amoebocytes, granular amoebocytes, and refractile amoebocytes. The cytoplasm of non-vacuolar amoebocytes contains many vesicles, which although structurally similar to primary lysosomes are in most cases acid phosphatase-negative. The granular amoebocytes enclose several microtubule-containing granules and vacuoles with electron-dense vanadium deposits, while the refractive amoebocytes are characterised by their pleomorphic inclusions and spike-like pseudopodia. Both the granules and inclusions of these amoebocyte types are peroxidase- and acid phosphatase-negative indicating that they are probably not lysosomes. The possible interrelationships of the four amoebocyte types are discussed, and it is suggested that the vacuolar hyaline amoebocytes may differentiate into granular amoebocytes.