Opsonin-independent and -dependent Phagocytosis in the Ascidian Halocynthia roretzi: Galactose-specific Lectin and Complement C3 Function as Target-dependent Opsonins

To clarify the molecular mechanisms of phagocytosis, we have been preparing monoclonal antibodies that inhibit phagocytosis by the hemocytes of the ascidian Halocynthia roretzi. A monoclonal antibody, RA5, inhibited the phagocytosis of non-treated sheep red blood cells (SRBCs) and yeast cells. It was demonstrated that the phagocytosis by the hemocytes was enhanced by pretreatment of target cells, SRBCs or yeast cells, with H. roretzi plasma. However, the RA5 antibody was unable to inhibit the phagocytosis of plasma-treated target cells. These results strongly suggest that the molecule recognized with the RA5 antibody is involved in the opsonin-independent phagocytosis. Western blot analysis showed that this antibody recognized a 200 kDa protein in H. roretzi hemocytes. On the other hand, flow cytometry analyses showed that a galactose-specific lectin (Gal-lectin) and complement C3 (AsC3), present in H. roretzi plasma, can bind to SRBCs and yeast cells, respectively, to enhance the phagocytosis of the respective target cells. Thus, H. roretzi hemocytes undergo opsonin-independent and -dependent phagocytosis, and Gal-lectin and AsC3 both function in the opsonin-dependent phagocytosis.     

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.     

Agglutinins in the hemolymph of the hard clam, Mercenaria mercenaria.

Lectins in the serum of the clam Mercenaria mercenaria agglutinate some red blood cells, bacteria, and yeast. The interaction of these substances with particles is affected by sugars, ions, temperature, and alteration of particle surfaces. Lectins are not needed for phagocytosis of foreign particles in vitro. In M. mercenaria these recognition molecules do not enhance defense mechanisms.     

Host defense mechanisms of cephalopods

Humoral and cellular mechanisms of defense have been described for cephalopods, a relatively advanced group of mollusks. Typical of other mollusks, cephalopod agglutinins are the most documented component of humoral immunity. Lectins, which have agglutinating properties, have been described and characterized from octopuses. Agglutinins from cephalopod hemolymph have also been shown to agglutinate a variety of vertebrate red blood cells, as well as potential bacterial pathogens. Hemocytes are the primary component of cellular immunity. Although the hemocyte role in phagocytosis has been extensively studied in other mollusks, the mechanisms of phagocytosis have not been described extensively for cephalopods. Cephalopod hemocytes have phagocytic capabilities and may function in encapsulation and neutralization of foreign substances; however, the effects of environmental factors and the full extent of phagocytic capabilities of cephalopod hemocytes have not been reported. Hemocytes from cephalopods have a role in wound healing and inflammation which have been reported in detail by several investigators.     

Quantitative assessment of phagocytic activity of hemocytes in the prawn, Penaeus merguiensis, by flow cytometric analysis

BACKGROUND: The blood cells of crustaceans are involved in phagocytosis of invading microorganisms, contributing to their defense mechanisms. In this study, phagocytic activity of hemocytes of the prawn, Penaeus merguiensis, was quantitated by means of flow cytometric analysis. METHOD: This study was done in vitro. Hemolymph, which was extracted from prawns, was mixed with an equal volume of anticoagulant. Heat-killed Escherichia coli prestained with propidium iodide (PI) was then added. Hemocytes were fixed at various time intervals for flow cytometric analysis. This study was supplemented with electron micrographs using transmission electron microscopy (TEM), which showed three populations of hemocytes. RESULTS: It was observed that those hemocytes that were more active engulfed and digested bacteria readily, thus having higher red fluorescence intensity. The phagocytic activity was expressed as fluorescence unit or engulfed E. coli number per hemocyte. CONCLUSIONS: With this approach, the phagocytic and cellular activity of individual hemocyte populations could be studied quantitatively.     

Production of reactive oxygen species by hemocytes from the cattle tick Boophilus microplus.

We have investigated the phagocytic activity and the production of reactive oxygen species (ROS) by hemocytes from the cattle tick Boophilus microplus. Two main types of hemocytes were detected in tick hemolymph: plasmatocytes and granulocytes. The plasmocytes were the most abundant cells, being responsible for the in vivo phagocytosis of yeast. ROS production was evaluated by luminol-amplified luminescence and phenol red oxidation. The luminescence increased when hemocytes were incubated with bacteria, zymosan, or phorbol 12-miristate 13-acetate (PMA). The luminescence was inhibited by superoxide dismutase and catalase, which are antioxidant enzymes that remove superoxide and hydrogen peroxide, respectively. The phenol red oxidation assay also showed an increase in the level of hydrogen peroxide produced by hemocytes stimulated with bacteria and PMA. Taken all together, our data indicate that tick hemocytes are able to produce ROS during the phagocytic process similarly to vertebrate phagocytes.     

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.     

Acidification of the phagosome in Crassostrea virginica hemocytes following engulfment of zymosan

Phagocytic hemocytes are responsible for engulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, Crassostrea virginica. Since rapid acidification of the phagosome lumen is typically essential for activation of hydrolytic and reactive oxygen intermediate (ROI) producing enzymes in vertebrate cells, we measured phagosomal pH in oyster hemocytes by using the emission fluorescence of two fluorescent probes, rhodamine and Oregon Green 488 (OG 488), conjugated to zymosan to determine whether oyster hemocyte phagosomes become acidified after phagocytosis of zymosan. The average pH of 1079 phagosomes within 277 hemocytes 1 h after phagocytosis of zymosan was 3.9 +/- 0.03. Observations of 141 hemocytes with internalized zymosan by light microscopy revealed that, over a 60-min time period, 51% of highly granular hemocytes became partially granular, and 29% became agranular. In addition, 83% of partially granular hemocytes containing zymosan at time = 0 became agranular within 60 min. A comparison revealed that the phagosomes of agranular hemocytes were much more acidic (pH 3.1 +/- 0.02) than those of highly granular hemocytes (4.9 +/- 0.02; P < 0.05). These values are significantly lower than most reported in the literature for blood cells from metazoan organisms.     

Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca, Cephalopoda)

Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods.     

Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.     

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.     

Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes

The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.     

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.     

Microscopical studies on the hemocytes of bivalves and their phagocytic interaction with selected bacteria

Hemocytes represent one of the most important defense mechanisms against foreign material in Mollusca. The morphology, hematological parameters and behaviour of hemolymph cells were studied in the southern quahogMercenaria campechiensis, the eastern oysterCrassostrea virginica, and the blood arkAnadara ovalis challenged with the bacteriaVibrio vulnificus andV. anguillarum. Two general classes of hemocytes (granular and agranular) exist inC. virginica andM. campechiensis. In contrast,A. ovalis possesses 3 general classes (granular, agranular and erythrocytes). Three types of granules were identified by light microscopy. When hemolymph cells were studied by transmission electron microscopy, the cytoplasm of hemolymph cells was noted to contain many organelles, including electron dense granules. Both agranular and granular hemolymph cells were capable of colchicine-sensitive pseudopodial movement and spreading. The results indicate that marine bivalves possess hemolymph blood cells which may play a role in the internal defense paralleling mammalian phagocytes. The morphology of these cells, as determined by light, scanning and transmission electron microscopy, showed some similarity to mammalian-mononuclear phagocytes. The sub-cellular events of molluscan hemocyte phagocytosis ofV. vulnificus andV. anguillarum were studied by both scanning and transmission electron microscopy. The role of these cells and the factors which govern their behavior are of economic and public health importance.     

Components of the cellular defense and detoxification system of the common cuttlefish Sepia officinalis (Mollusca,Cephalopoda)

Endocytotic-active cells in the branchial heart complex of Sepia officinalis were studied by in situ injection of different types of xenobiotics and by in vitro perfusion of the organ complex with a bacterial suspension. The rhogocytes (ovoid cells) ingest particles of all tested sizes by endocytosis and phagocytosis. The hemocytes of the circulating blood and the adhesive hemocytes in the wall of the branchial heart incorporate all tested kinds of foreign materials, including bacterial cells due to phagocytosis achieved by the triangular mesenchymatic cells. The ultrastructural findings also give strong evidence that the triangular mesenchymatic cells are fixed hemocytes that have migrated into the branchial heart tissue. The ingestion and digestion of allogeneic substances and bacteria or their debris by rhogocytes and/or all (forms of) hemocytes suggests the involvement of these either fixed or mobile endocytotic-active cells in the defense and detoxification system of cephalopods.     

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.     

Interactions between the cellular and humoral immune responses in Drosophila

Drosophila has highly efficient defenses against infection. These include both cellular immune responses, such as the phagocytosis of invading microorganisms, and humoral immune responses, such as the secretion of antimicrobial peptides into the hemolymph [1] [2]. These defense systems are thought to interact, but the nature and extent of these interactions is not known. Here we describe a method for inhibiting phagocytosis in Drosophila blood cells (hemocytes) by injecting polystyrene beads into the body cavity. This treatment does not in itself make a fly susceptible to Escherichia coli infection. However, when performed on flies carrying the mutation immune deficiency (imd), which affects the humoral immune response [3], the treatment results in a striking decrease in resistance to infection. We therefore carried out a sensitized genetic screen to identify immunocompromised mutants by co-injecting beads and E. coli. From this screen, we identified a new gene we have named red shirt and identified the caspase Dredd as a regulator of the Drosophila immune response. The observation that mutants with defects in the humoral immune response are further immunocompromised by blocking phagocytosis, and thus inhibiting the cellular immune response, shows that the Drosophila cellular and humoral immune responses act in concert to fight infection.     

A thraustochytrid protist isolated from Mercenaria mercenaria: molecular characterization and host defense responses

A previously undescribed thraustochytrid protist, designated C9G, was isolated from the gills of a clam, Mercenaria mercenaria, collected from the Bay of Fundy, Canada. Sequence data analysis showed C9G to be related to the clam pathogen QPX, quahog parasite unknown; however, it is not enveloped by secreted mucoid material as is the case for QPX. Clam hemocytes recognized and phagocytized C9G in vitro in the absence of plasma recognition factors. Hemocytes were also capable of killing ingested C9G, as shown by the use of a tetrazolium reduction viability assay. The mechanisms underlying intracellular antimicrobial activity are not yet established, but no detectable cytotoxic reactive oxygen species were generated during phagocytosis of C9G. Clam plasma proteins were shown to inhibit C9G growth at concentrations similar to those in unfractionated hemolymph.     

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.