Involvement of tyrosine kinase and phosphatidylinositol 3-kinase in phagocytosis by ascidian hemocytes

It has been proposed that protein tyrosine phosphorylation plays important roles in signal transduction in mammalian T- and B-cells and monocytes. During our investigations on the ascidian host defense system, we have shown that the monoclonal antibody A74 strongly inhibits both phagocytosis of sheep red blood cells (SRBCs) by hemocytes and hemocyte aggregation, and that the A74 antigen protein has two immunoreceptor tyrosine-based activation motifs and several other motifs that are thought to function in signal transduction in mammals. In this study, we found that the A74 antibody strongly inhibited phagocytosis by ascidian hemocytes of yeast cells, as strongly as that of SRBCs, but not that of latex beads. We also found that herbimycin A and an erbstatin analog, tyrosine kinase inhibitors, and wortmannin, a specific inhibitor for phosphatidylinositol 3-kinase (PI3-kinase), inhibited the phagocytosis of yeast cells. We investigated which hemocyte proteins were specifically tyrosine-phosphorylated during phagocytosis by ascidian hemocytes and found that a protein with a molecular mass of 100 kDa was specifically tyrosine-phosphorylated upon phagocytosis; its tyrosine phosphorylation was inhibited by the A74 antibody. These results strongly suggest that both tyrosine kinase and PI3-kinase play important roles in phagocytosis by ascidian hemocytes.     

Cloning and characterization of integrin alpha subunits from the solitary ascidian, Halocynthia roretzi

Recent molecular and biochemical analysis has revealed the presence of an opsonic complement system in the solitary ascidian, Halocynthia roretzi, composed of at least C3, two mannan binding protein-associated serine proteases, and factor B. To elucidate further the structure and function of this apparently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are members of the leukocyte integrin family in mammals. Using degenerate primers, we isolated two integrin alpha subunits (alpha(Hr1) and alpha(Hr2)) from the hemocyte mRNA of H. roretzi, by RT-PCR, and the entire coding sequence of alpha(Hr1) was determined from cDNA clones. alpha(Hr1) contains an I domain, the inserted domain characteristic of a subset of mammalian alpha subunits, including the leukocyte integrin family. A phylogenetic tree constructed for the alpha subunits also supports the ancestral position of alpha(Hr1) in the monophyletic cluster of I domain-containing alpha integrins. The alpha(Hr1) gene shows hemocyte-specific expression on Northern blot analysis. Western blot analysis and immunocytochemical staining of the hemocytes of H. roretzi using anti-alpha(Hr1) Ab showed that alpha(Hr1) subunits exist on the surface of a subpopulation of phagocytic hemocytes. Furthermore, anti-alpha(Hr1) Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, of yeast cells by ascidian hemocytes. These observations strongly suggest that alpha(Hr1) constitutes an integrin molecule on the hemocytes of H. roretzi that functions as an ancestral form of CR3 and CR4 and mediates phagocytosis in the primitive complement system of the ascidian.     

Common cell-surface antigens functioning in self-recognition reactions by both somatic cells and gametes in the solitary ascidian Halocynthia roretzi

The "contact reaction" is an extremely rapid allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the solitary ascidian Halocynthia roretzi. It has been proposed that regulation of the alloreactivity of hemocytes may be involved in preference for fertilization or self-sterility in this species. To identify the receptors and target ligands involved both in self-recognition by somatic cells and self-discrimination by gametes, we produced monoclonal antibodies (mAbs) that inhibit the ACR mediated by hemocytes and tested their effects on fertilization. Six different mAbs that inhibit the ACR were prepared and categorized into three groups. Although all three mAbs seemed to have the same ability to inhibit the ACR, almost constant and statistically significant inhibition (CRB1.1) and infrequent but significant inhibition (CRB2.1, and CRB3.1) of the ACR were observed in the same pairs of animals. Pretreatment of the unfertilized eggs with CRB1.1, CRB2.1, and CRB3.1, resulted in the constant and statistically significant inhibition, infrequent but significant inhibition, and no inhibition, respectively, of fertilization. Antigens recognized by CRB1.1 (CRB1.1 antigens) were detected on the cell surface of all types of hemocytes and on the vitelline coat and follicle cells of unfertilized eggs. CRB2.1 and CRB3.1 antigens were detected on the surface of certain types of hemocytes and follicle cells, but not on the vitelline coat. CRB mAbs were directed against different epitopes in the N-linked glycan on glycoproteins. These common carbohydrate antigens on somatic cells and gametes may function in some recognition processes in ACR and fertilization in H. roretzi.     

Mutual and directional allogeneic cytotoxic reaction of hemocytes in the solitary ascidian Halocynthia roretzi revealed by one-step quantitative fluorimetric assay

A novel one-step microplate cytotoxicity assay using the cytoplasmic fluorescent viability dye calcein AM was established for simple, rapid, sensitive, and quantitative measurements of the allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the ascidian Halocynthia roretzi. The mutual and directional ACR was distinguishable by the assay using the hemocytes from pairs of animals with different alloreactivities. The ACR assay may allow more precise genetic analysis of the gene that controls alloreactivity of hemocytes, since the mutual and directional ACR may be related to levels of expression or numbers of the gene product or products on the target cells. The directional ACR will be useful in elucidating the cellular and molecular mechanisms of self-recognition in H. roretzi, since it allowed us to equate hemocytes from one animal with "effector cells" and those from the other animal of the pair with "target cells". In addition, the quantitative ACR assay in a large number of samples is possible and it will allow production of monoclonal antibodies that may recognize receptors or ligands functioning in self-recognition processes by the H. roretzi hemocytes.     

Zymosan induces production of superoxide anions by hemocytes of the solitary ascidian Halocynthia roretzi

Reactive oxygen intermediates (ROIs), including superoxide anions and hydrogen peroxide, are generated by phagocytes in invertebrates, as well as in vertebrates. To understand the molecular mechanisms underlying the generation of ROIs by hemocytes of the solitary ascidian Halocynthia roretzi, we established a method of measuring ROIs using luminol-dependent chemiluminescence (LDCL). LDCL analyses revealed that both zymosan and phorbol myristate acetate (PMA), but not lipopolysaccharide, beta1,3-glucan, or formylpeptide, induced the generation of ROIs by H. roretzi hemocytes. The zymosan-induced LDCL was markedly inhibited by the addition of superoxide dismutase (SOD) or H. roretzi plasma. A calcium-chelating reagent, BAPTA-AM, completely inhibited the zymosan-induced LDCL. On the other hand, the PMA-induced LDCL was only slightly inhibited by the addition of SOD or BAPTA-AM. Spectroscopic analysis at a low temperature revealed that H. roretzi hemocytes had absorption spectra specific for type b cytochrome, a component of the NADPH oxidase complex in mammalian phagocytes. These results strongly suggest that H. roretzi hemocytes generate superoxide anions upon phagocytosis and that intracellular calcium ions and possibly an NADPH oxidase complex are involved in their generation by H. roretzi hemocytes.     

Role of divalent cations in plasma opsonin-dependent and-independent erythrophagocytosis by hemocytes of the Asian clam,Corbicula fluminea

The in vitro phagocytosis-promoting properties of hemolymph from the freshwater clam, Corbicula fluminea, are described. Hemocytes were capable of phagocytosing aldehyde-fixed erythrocytes (RBCs) of seven vertebrate species with equal facility, but only in the presence of homologous clam plasma. The plasma factors mediating erythrophagocytosis were heat sensitive. Pretreatment (opsonizing) of target RBCs with plasma also resulted in enhancement of hemocyte particle uptake in the absence of plasma. Opsonin-dependent phagocytosis required the presence of divalent cations, especially calcium, although not in free ionic form. Evidence suggests that the plasma opsonin may normally exist as a divalent cation-macromolecular complex since opsonizing activity was retained after dialysis against Tris-buffered saline (TBS), but was lost following TBS/EDTA or TBS/EGTA dialysis. We also have identified an opsonin-independent phagocytosis mechanism in which Corbicula hemocytes are able to ingest nonopsonized RBCs in the absence of homologous plasma. Extracellular calcium or magnesium in the incubation medium is needed for particle uptake, although the direct binding of free ions to the target RBC surface does not appear to be mediating enhanced phagocytosis. From the present data, it is concluded that hemocyte recognition of aldehyde-fixed RBCs can be accomplished by either of two mechanisms: (1) by the coating of cells with plasma factors capable of triggering the phagocytic process (opsonization) or (2) by a plasma opsonin-independent mechanism in which extracellular divalent cations (e.g., Ca²⁺ or Mg²⁺) in the incubation buffer stimulate uptake of nonopsonized RBCs. The factors regulating in vitro erythrophagocytosis by clam hemocytes are considered to be analogous to those involved in nonimmune opsonin-dependent and -independent phagocytosis in mammalian macrophages.     

A unique primary structure, cDNA cloning and function of a galactose-specific lectin from ascidian plasma.

The complete amino acid sequence of a galactose-specific lectin from the plasma of the ascidian Halocynthia roretzi has been determined by sequential Edman degradation analysis of peptide fragments derived by proteolytic fragmentation and chemical cleavage of the reductive S-pyridylethylated lectin. Peptide fragments were separated by reverse-phase HPLC. The N-terminal and C-terminal amino acid sequences were determined by Edman degradation and enzymatic digestion. The H. roretzi plasma lectin is a single-chain protein consisting of 327 amino acids and four disulfide bonds, one of which was found to be cross-linked intramolecularly. A comparison of the amino acid sequence of the H. roretzi plasma lectin with the sequences of other proteins reveals that the H. roretzi lectin has a structure consisting of a twice-repeated sequence, a fibrinogen-related sequence and a C-type lectin-homologous sequence. The above amino acid sequence was verified by cDNA cloning of this lectin. Three cDNA clones that have single ORFs encoding the lectin precursor were isolated from an H. roretzi hepatopancreas cDNA library. The deduced amino acid sequences in the three cDNA clones contain the same sequence of the mature lectin molecule and the same putative signal sequence. In addition, it was demonstrated that this lectin can enhance phagocytosis by H. roretzi hemocytes. Thus, the plasma lectin is constructed into an oligomer structure via intermolecular disulfide bonds and plays a role in the biological defense of H. roretzi as a defense molecule.     

Schistosoma mansoni modulation of phagocytosis in Biomphalaria glabrata

Both short-term (3 hr) exposure of Biomphalaria glabrata snails (M-line and 13-16-R1) to Schistosoma mansoni (PR1) miracidia and in vitro incubation of parasite sporocysts with host hemolymph components altered host phagocytic ability. Hemocytes obtained from susceptible (M-line) snails that had been exposed to parasite miracidia for 3 hr showed reduced levels of phagocytosis of yeast cells in vitro compared to hemocytes from unexposed individuals. Incubation of whole hemolymph with sporocysts in vitro also reduced yeast phagocytosis in this susceptible strain. In contrast, resistant (13-16-R1) hemocytes showed increased levels of yeast phagocytosis after in vitro incubation with the parasite, and the opsonic properties of 13-16-R1 plasma were greater after exposure of snails to miracidia. These strain-specific effects of S. mansoni on host hemocyte phagocytosis and plasma opsonization were seen only when both plasma and hemocytes were present at the time of exposure to the parasite.     

Biomphalaria glabrata: influence of calcium, lectins, and plasma factors on in vitro phagocytic behavior of hemocytes of noninfected or Schistosoma mansoni-infected snails.

In vitro phagocytosis of erythrocytes by hemocytes of B. glabrata, intermediate host of S. mansoni, is strongly influenced by calcium, several lectins, and plasma factors. Our results indicate that two different mechanisms of non-self-recognition in B. glabrata may occur: (1) In the presence of calcium, phagocytosis occurs in noninfected and in infected snails without involvement of any other substances, and hemocytes of schistosome resistant as well as those of susceptible snails are able to recognize and phagocytose the target cells. (2) In the absence of calcium, phagocytosis occurs if bridging molecules (heterologous lectins in our assays) were present for which effector and target cells possess binding sites or if target cells were plasma coated prior to the assays. In suspensions in homologous plasma, hemocytes of both snail strains, infected or noninfected, subsequently showed phagocytic activities of about 70-80%. Preincubation of target cells in homologous plasma resulted in similar high phagocytic activities of hemocytes even in the absence of plasma during the standard assay. In these assays, a significantly higher proportion of hemocytes of resistant snails phagocytosed plasma-opsonized erythrocytes, whereas hemocytes of susceptible snails internalized less erythrocytes per cell and needed 60 min to phagocytose at percentages equivalent to that of resistant hemocytes within 10 min. Preincubation of erythrocytes in resistant plasma significantly increased the subsequent phagocytic activity of susceptible hemocytes, whereas preincubation of erythrocytes in susceptible plasma decreased the phagocytosis level of resistant hemocytes.     

Impaired defense mechanisms in bay mussels, Mytilus edulis, with hemic neoplasia

Immunocompetence of bay mussels, Mytilus edulis, with hemic neoplasia was investigated with an in vitro yeast phagocytosis assay and by in vivo clearance from the blood of injected Cytophaga sp. bacteria. The yeast phagocytosis assay was conducted with hemocytes maintained in 90% plasma. Neoplastic hemocytes, characterized by enlarged nuclei and scant cytoplasm, failed to phagocytose yeast cells. In contrast, greater than 90% of hemocytes from unaffected animals and morphologically normal hemocytes from mussels with the disease phagocytosed yeast. Substitution of normal plasma with that from a mussel with advanced disease (essentially 100% neoplastic hemocytes) did not affect the phagocytic capability of normal hemocytes. Conversely, normal plasma did not enhance the phagocytic capabilities of neoplastic cells. Mussels with advanced disease showed reduced bacterial clearance; control or lightly affected mussels (less than 11% neoplastic hemocytes) cleared greater than 90% of injected bacteria in 4 hr, while mussels with advanced disease cleared 44-83%. These experiments indicate that mussels with advanced hemic neoplasia have compromised defense systems. This may account for the reported mortality in mussels and other bivalve molluscs with hemic neoplasia.     

First report on histology and ultrastructure of an intrahemocytic paramyxean parasite (IPP) from tunicate Halocynthia roretzi in Korea

In 2004, epizootiological studies were conducted on mass mortalities of tunicates Halocynthia roretzi in Goje, Korea. The clinical characteristics of infected H. roretzi were weakness of the tunic, loss of elasticity, and finally death involving a rupture of the tunic. Histological studies revealed severe hemocyte infiltration in the connective tissue surrounding the intestine and mantle of infected H. roretzi. Hypertrophied eosinophilic hemocytes containing several cytoplasmic vacuoles were observed in the connective tissue surrounding the intestine, gill and mantle. Ultrastructural examination revealed the presence of a parasite in the cytoplasm of hemocytes. Secondary cells were observed in the primary cell of the parasite. Spore formation within primary cells suggests that the parasite may be an intrahemocytic paramyxean parasite (IPP) and may cause mass mortality of H. roretzi.     

Integrin βν-mediated phagocytosis of apoptotic cells in Drosophila embryos

To identify molecules that play roles in the clearance of apoptotic cells by Drosophila phagocytes, we examined a series of monoclonal antibodies raised against larval hemocytes for effects on phagocytosis in vitro. One antibody that inhibited phagocytosis recognized terribly reduced optic lobes (Trol), a core protein of the perlecan-type proteoglycan, and the level of phagocytosis in embryos of a Trol-lacking fly line was lower than in a control line. The treatment of a hemocyte cell line with a recombinant Trol protein containing the amino acid sequence RGD augmented the phosphorylation of focal adhesion kinase, a hallmark of integrin activation. A loss of integrin βν, one of the two β subunits of Drosophila integrin, brought about a reduction in the level of apoptotic cell clearance in embryos. The presence of integrin βν at the surface of embryonic hemocytes was confirmed, and forced expression of integrin βν in hemocytes of an integrin βν-lacking fly line recovered the defective phenotype of phagocytosis. Finally, the level of phagocytosis in a fly line that lacks both integrin βν and Draper, another receptor required for the phagocytosis of apoptotic cells, was lower than that in a fly line lacking either protein. We suggest that integrin βν serves as a phagocytosis receptor responsible for the clearance of apoptotic cells in Drosophila, independent of Draper.     

Blockades of phospholipase A(2) and platelet-activating factor receptors reduce the hemocyte phagocytosis in Rhodnius prolixus: in vitro experiments

The hemocytes phagocytosis in response to microorganisms may play an important role in the cellular immune responses of insects. Here, we have evaluated the effects of the platelet-activating factor (PAF) and eicosanoids in the phagocytosis of hemocyte monolayers of Rhodnius prolixus to the yeast Saccharomyces cerevisiae. Experiments showed that the phagocytosis of yeast cells by Rhodnius hemocytes is very efficient in both controls and cells treated with PAF and arachidonic acid. Phagocytosis of yeast particles is significantly blocked when the specific phopholipase A(2) inhibitor, dexamethasone, is applied on the hemocytes. By contrast, dexamethasone-pretreated hemocyte monolayers exhibit a drastic increase in the quantity of yeast cell-hemocyte internalization when the cells are treated by arachidonic acid. In addition, phagocytosis presents significant reduction in hemocyte monolayers treated with a specific PAF receptor antagonist, WEB 2086. Nevertheless, inhibition of phagocytosis with WEB 2086 is counteracted by the treatment of the hemocyte monolayers with PAF. In conclusion, phagocytosis of yeast cells by hemocytes is related to the activation of PAF receptors and eicosanoid pathways in the bloodsucking bug, R. prolixus.     

Complement receptor type three-dependent degradation of opsonized erythrocytes by mouse macrophages

The role of the complement receptor type 3 (CR3) on thioglycollate-elicited peritoneal macrophages (TG-PM) in the destruction of opsonized particles was studied. We found that sheep red blood cells (E) that were opsonized with an IgM monoclonal anti-Forssman antibody and complement (E-IgM-C) were lysed by TG-PM, whereas there was little lysis of E pretreated with either the antibody or the complement source alone. Furthermore, this lysis could be inhibited by anti-CR3 monoclonal antibodies that had previously been shown to inhibit binding of E-IgM-C to the CR3. Kinetic studies of phagocytosis and lysis indicated that lysis of E-IgM-C occurs after phagocytosis, suggesting that lysis is an intracellular event. Further findings suggested that intra-cellular lysis was promoted by CR3 bound to the phagocytosed target, because a monoclonal anti-CR3 antibody decreased the rate of phagocytosis of E-IgM-C but not its magnitude, whereas the rate and extent of lysis were strikingly inhibited. Furthermore, TG-PM that had already internalized unopsonized E selectively lysed E-IgM-C that were added later. These data confirm that the interaction of the CR3 with its ligand on E-IgM-C promotes rapid phagocytosis, and further suggest that the CR3 facilitates degradation of the target particle once internalization has occurred.     

Receptors for unopsonized particles: the role of alveolar macrophage scavenger receptors

The lung is constantly exposed to potentially pathogenic particles and microorganisms. Alveolar macrophage (AM) binding of inhaled environmental particles is a critical first step in phagocytosis and clearance, and must be accomplished without the benefit of opsonization by specific antibodies. Opsonin-independent phagocytosis is initiated by direct recognition of phagocytic target. The identities of receptors on AMs that mediate unopsonized particle binding were, until recently, not known. Using flow cytometry, monoclonal antibody and expression cloning techniques we have found a major role for the scavenger receptor, MARCO in AM binding of particles and bacteria. In this review we will discuss the role of scavenger receptors in AM binding of unopsonized particles and the use of flow cytomety in analyzing AM-particle interaction. We will also discuss other non-scavenger receptors involved in opsonin-independent phagocytosis.     

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A(2)

Phagocytosis is a hemocytic behavior against bacterial infection. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits immune responses of target insects and causes hemolymph septicemia. This study analyzed how X. nematophila could inhibit phagocytosis to increase its pathogenicity. Granular cells and plasmatocytes were the main phagocytic hemocytes of Spodoptera exigua determined by observing fluorescence-labeled bacteria in the cytosol. X. nematophila significantly inhibited phagocytosis of both hemocytes, while heat-killed X. nematophila lost its inhibitory potency. However, co-injection of X. nematophila with arachidonic acid did not show any significant inhibition of hemocyte phagocytosis. In fact, hemocytes of S. exigua infected with X. nematophila showed significant reduction in phospholipase A(2) (PLA(2)) activity. Dexamethasone, a specific PLA(2) inhibitor, significantly inhibited phagocytosis of both cell types. However, the inhibitory effect of dexamethasone was recovered by addition of arachidonic acid. Incubation of hemocytes with benzylideneacetone, a metabolite of X. nematophila, inhibited phagocytosis in a dose-dependent manner. These results suggest that X. nematophila produces and secretes PLA(2) inhibitor(s), which in turn inhibit the phagocytic response of hemocytes.     

Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins

Phagocytosis by monocytes or neutrophils can be enhanced by interaction with several proteins or synthetic peptides containing the Arg-Gly-Asp sequence. Recently we showed that an mAb, B6H12, specifically inhibited this enhancement of neutrophil phagocytosis by inhibiting Arg-Gly-Asp binding to the leukocyte response integrin (Gresham, H. D., J. L. Goodwin, P. M. Allen, D. C. Anderson, and E. J. Brown. 1989. J. Cell Biol. 108:1935-1943). Now, we have purified the antigen recognized by B6H12 to homogeneity. Surprisingly, it is a 50-kD molecule that is expressed on the plasma membranes of all hematopoietic cells, including erythrocytes, which express no known integrins. On platelets and placenta, but not on erythrocytes, this protein is associated with an integrin that can be recognized by an anti-beta 3 antibody. In addition, both the anti-beta 3 and several mAbs recognizing the 50-kD protein inhibit Arg-Gly-Asp stimulation of phagocytosis. These data demonstrate an association between integrins and the 50-kD protein on several cell types. For this reason, we call it Integrin-associated Protein (IAP). We hypothesize that IAP may play a role in signal transduction for enhanced phagocytosis by Arg-Gly-Asp ligands.     

Cellular immune response in Rhodnius prolixus: role of ecdysone in hemocyte phagocytosis

In this paper we investigate in vivo and in vitro effects of orally administered azadirachtin and ecdysone on the phagocytic responses of Rhodnius prolixus 5th-instar larval hemocytes to the yeast Saccharomyces cerevisiae. Groups of insects fed non-treated blood (control) and insects that received azadirachtin plus ecdysone in the blood meal were inoculated with yeast cells in the hemocele. The injected yeast cells disappeared rapidly from the hemolymph, being removed completely by 90min after inoculation. In the insects treated only with azadirachtin the clearance of free yeast circulating particles was significantly delayed compared to the two previously mentioned groups. It was demonstrated that the binding of yeast cells to hemocytes was reduced in the insects treated only with azadirachtin in comparison to both non-treated control and azadirachtin plus ecdysone-treated groups. Phagocytosis occurred when yeast cells were added to hemocyte monolayers prepared with hemolymph from blood fed insects, treated or not with azadirachtin plus ecdysone, so that yeast cells were rapidly bound to hemocytes and internalized in high numbers. By contrast, insects treated with azadirachtin exhibited a drastic reduction in the quantity of yeast cell-hemocyte binding and subsequent internalization. In all groups, the hemocytes attached to the glass slides were predominantly plasmatocytes. The magnitude and speed of the cellular response suggests that hemocyte phagocytosis is one of the main driving forces for the clearance of free circulating yeast cells from the hemolymph. We propose that ecdysone modulates phagocytosis in R. prolixus larvae, and that this effect is antagonized by azadirachtin.     

Functional analysis of plasma prophenoloxidase system in the marine mussel Perna viridis

The role of prophenoloxidase (proPO) system in recognition and phagocytosis of yeast cells by hemocytes was examined in vitro using whole plasma and proPO system isolated from the plasma of the marine mussel, Perna viridis. The proPO was isolated from the plasma by ammonium sulphate precipitation and gel filtration. The final proPO preparation was homogeneous in native PAGE, and could be activated by trypsin, α-chymotrypsin and pronase-E. Laminarin (a polymer of β-1, 3-glucan) and lipopolysaccharides (LPS) from diverse bacterial species effectively activated the isolated proPO, demonstrating the ability of this proenzyme to interact directly with microbial surface components. The susceptibility of proPO activation to inhibition by serine protease inhibitors such as soybean trypsin inhibitor (STI) or p-nitrophenyl-p′-guanidinobenzoate (p-NPGB), indicates that the isolated fraction may contain an integral serine protease domain in an inactive state. The presence of laminarin- or LPS-activated whole plasma of P. viridis facilitated adherence of yeast cells to hemocyte surface as well as eventually stimulated phagocytic uptake of the target cells by hemocytes, and no such hemocytic response was recorded with STI controls. This and other results strongly suggest that the intermediary factors generated during activation of plasma proPO system by non-self molecules play a key role in recognition and opsono-phagocytosis by hemocytes. However, the proPO system isolated from P. viridis plasma, after activation with microbial surface components, failed to show an opsonic effect.     

cDNA microarray analyses reveal candidate marker genes for the detection of ascidian disease in Korea

A serious disease of the ascidian Halocynthia roretzi has been spread extensively among Korean aquaculture sites. To reveal the cause of the disease and establish a monitoring system for it, we constructed a cDNA microarray spotted with 2,688 cDNAs derived from H. roretzi hemocyte cDNA libraries to detect genes differentially expressed in hemocytes between diseased and non-diseased ascidians. We detected 21 genes showing increased expression and 16 genes showing decreased expression in hemocytes from diseased ascidians compared with those from non-diseased ascidians. RT-PCR analyses confirmed that the expression levels of genes encoding astacin, lysozyme, ribosomal protein PO, and ubiquitin-ribosomal protein L40e fusion protein were increased in hemocytes from diseased ascidians, while those of genes encoding HSP40, HSP70, fibronectin, carboxypeptidase and lactate dehydrogenase were decreased. These genes were expressed not only in hemocytes but also in various other tissues in ascidians. Furthermore, the expression of glutathione-S transferase omega, which is known to be up-regulated in H. roretzi hemocytes during inflammatory responses, was strongly increased in hemocytes from diseased ascidians. These gene expression profiles suggest that immune and inflammatory reactions occur in the hemocytes of diseased ascidians. These genes will be good markers for detecting and monitoring this disease of ascidians in Korean aquaculture sites.