Evidence for perforin-like activity associated with earthworm leukocytes

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.     

Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida)

In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes - four times, in comparison to the control. In three cases: of the coelomic fluid, the homogenates from cocoons and the extract from coelomocytes, the antibody against HEWL (hen egg white lysozyme) recognized only one protein with a molecular mass of about 14.4 kDa. In the coelomic fluid, apart from the protein with molecular mass of 14.4 kDa the antibody directed against human lysozyme recognized an additional protein of 22 kDa. Using the bioautography technique after electrophoretic resolution of native proteins in acidic polyacrylamide gels, two lytic zones of M. luteus were observed in the case of the coelomic fluid and three after the analysis of the extract of coelomocytes and the egg homogenates. The results indicated the existence of several forms of lysozyme with a different electric charge in the analyzed D. veneta samples. The highest lysozyme activity in the intestine of D. veneta was observed in the midgut. The antibody directed against human lysozyme indicated a strong positive signal in epidermal and midgut cells of earthworm.     

Amassin,an olfactomedin protein,mediates the massive intercellular adhesion of sea urchin coelomocytes

Sea urchins have a fluid-filled body cavity, the coelom, containing four types of immunocytes called coelomocytes. Within minutes after coelomic fluid is removed from the body cavity, a massive cell-cell adhesion of coelomocytes occurs. This event is referred to as clotting. Clotting is thought to be a defense mechanism against loss of coelomic fluid if the body wall is punctured, and it may also function in the cellular encapsulation of foreign material and microbes. Here we show that this intercoelomocyte adhesion is mediated by amassin, a coelomic plasma protein with a relative molecular mass (Mr) of 75 kD. Amassin forms large disulfide-bonded aggregates that adhere coelomocytes to each other. One half of the amassin protein comprises an olfactomedin (OLF) domain. Structural predictions show that amassin and other OLF domain-containing vertebrate proteins share a common architecture. This suggests that other proteins of the OLF family may function in intercellular adhesion. These findings are the first to demonstrate a function for a protein of the OLF family.     

Identité immunologique et métabolique entre le liquide coelomique,les coelomocytes et les ovocytes de Perinereis cultrifera (Annélide Polychète)

Immunological study of Perinereis cultrifera reveals the existence of an identical antigen in the coelomic fluid of females (oocyte diameter > 140 (μm), in oocytes, and in coelomocytes. This factor is not found in the body fluid of males or young females.

The elution patterns obtained after Sephadex chromatography shows a similar glycoprotein fraction (fraction I) in the coelomic fluid, in coelomocytes, and in soluble oocyte extracts. This fraction includes the main part of the antigenic components of the coelomic fluid.

Gas chromatography reveals that identical monosaccharides are present, albeit in varying proportions, in samples of fraction I obtained from the different sources.

The metabolic interrelationships of coelomocytes, coelomic fluid and oocytes is discussed. Glycoprotein synthesized by coelomocytes may be discharged into the coelomic fluid and contribute to the development of the cortical alveoli of the oocytes. No evidence of an involvement of this material in yolk synthesis has been obtained.     

Expression of SpC3, the sea urchin complement component,in response to lipopolysaccharide

The homologue of the vertebrate complement component C3 that is expressed in the coelomocytes of the purple sea urchin, Strongylocentrotus purpuratus, designated SpC3, was investigated for changes in response to immune challenge or injury. Immunoquiescent animals were used in this study because they have reduced or no detectable SpC3 in their coelomocytes or coelomic fluid (CF). Animals were injected with lipopolysaccharide (LPS) or sterile sea water (SSW, injury control). Changes in the amounts of SpC3 in coelomic fluid and in coelomocytes were then followed over time by Western blots and ELISA. Changes in mRNA from the SpC3 gene (Sp064) were also followed by RT-PCR. Although all animals responded to injury with increased levels of SpC3 in the coelomic fluid, those challenged with LPS had greater amounts of SpC3 in both CF and coelomocytes than those receiving SSW. In most of the animals receiving LPS, initial increases in SpC3 were observed within 1 h post-injection, while the earliest response in the animals receiving SSW was 6 h. The appearance of SpC3 in the coelomocytes was delayed compared to its appearance in CF, and was first detected several days after challenge. Changes in mRNA from the Sp064 gene paralleled the appearance of SpC3 in the coelomic fluid. Increases in the number of coelomocytes per milliliter of CF and in the percentage of coelomocytes that were SpC3+ also occurred after challenge with LPS or in response to injury, with a slightly greater increase in response to LPS. Although the changes in SpC3 were not as great as those identified previously for human C3 expressed in macrophages, the kinetics of the response are similar to that of acute-phase reactants in mammals.     

Earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge

Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.E.L. Cooper has been supported by The Alexander von Humboldt Foundation, a GAAC grant from the Federal Republic of Germany and two grants from NATO, a Cooperative Research grant (971128) and an Advanced Research Workshop grant (976680)     

Coelomocytes and post-traumatic response in the common sea star Asterias rubens

Coelomocytes are recognized as the main cellular component of the echinoderm immune system. They are the first line of defense and their number and type can vary dramatically during infections or following injury. Sea stars have been used as a model system to study the regeneration process after autotomy or predation. In the present study we examined the cellular and biochemical responses of coelomocytes from the European sea star Asterias rubens to traumatic stress using immunochemical and biochemical approaches. In terms of trauma and post-traumatic stress period, here we consider the experimental arm amputation and the repair phase involved in the first 24 hours post-amputation, which mimicked a natural predation event. Four cell morphotypes were distinguishable in the coelomic fluid of both control and post-traumatic-stressed animals (phagocytes, amoebocytes, vibratile cells, hemocytes), but phagocytes were the major components, accounting for about 95% of the total population. Thus, the effects measured relate to the overall population of coelomocytes. A modest increase in the total number of freely circulating coelomocytes was observed 6 hours post-amputation. Interestingly, a monoclonal antibody (McAb) to a sea urchin embryo adhesion protein (toposome) cross-reacted with isolated sea star coelomocytes and stained the coelomic epithelium of control animals with an increase in trauma-stressed arms. In addition, coelomocytes from trauma-stressed animals showed a time-dependent increase in Hsp70 levels, as detected by both immunocytochemistry and immunoblotting within 24 hours after arm tip amputation, with a peak at 6 hours after amputation. Our findings indicate a clear role for coelomocytes and classic stress molecules in the post-traumatic stress associated with the early repair phase of regeneration.     

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L)

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens′, was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.     

The analysis of cellular elements in coelomic fluid during early regeneration of of the starfish Asterias rubens L

Three main cell types were found in the coelomic fluid (CF) of intact starfishes: agranulocytes (55-80%) varying in size and form (spherical and ovoid) and with occasional pseudopodia, granulocytes (15-45%), and small cells (up to 2 %) with a high nuclear-cytoplasmic ratio. The starfish response to injury depends on the degree of coelomic fluid loss. After a slight wounding, when only insignificant portion of CF is lost, the cellular composition of circulating fluid changed only slightly. Unlike, a significant injury resulted in rising the share of small cells, regarded presumably as young cells. Besides, after injury the functional characteristics of SF also changed: the proportion of cells with decondensed chromatin and stained nucleoli increased, and coelomocytes acquired ability to form nets at adhesion. Moreover, some new cell types can be found (fusiform cells), with granulocyte proportion in nets increasing. We suppose that after slight wounding circulating coelomocytes may restore from the existing store of differentiated cells beyond the circulation, whereas after significant injury young undifferentiated coelomocytes are involved in the process of restoration.     

Sites of expression of mRNA for lysenin, a protein isolated from the coelomic fluid of the earthworm Eisenia foetida

Lysenin is a 33-kDa protein of 297 amino acids that was originally purified from the coelomic fluid of the earthworm Eisenia foetida. It binds specifically to sphingomyelin. In this study, we attempted to identify the site of synthesis of lysenin in the earthworm. We detected the expression of mRNA for lysenin and the presence of immunoreactive lysenin in the large coelomocytes and in the free large chloragocytes present in the lumen of the typhlosole, a depression in the dorsal wall of the intestine. These coelomocytes and chloragocytes seemed to be mature and separate from the chloragogen tissue that lined the typhlosole. The free large chloragocytes in the typhlosole contained numerous vacuoles. The nuclei were small and irregular in shape, and glycogen granules and mitochondria were occasionally found between vacuoles. The chloragocytes of the chloragogen tissue that surrounded the coelomic side of the intestine and the dorsal blood vessel did not react with the lysenin antiserum and no expression of lysenin mRNA was detected in these cells. Furthermore, no evidence of the protein or of the mRNA was found in the cells of the pharyngeal gland. Our findings suggest that lysenin is produced in the free large chloragocytes in the lumen of the typhlosole.     

The characteristic of the coelomic fluid and coelomic epithelium cell populations of starfish Asterias rubens L., capable to attach and spread on various substrates

Cultivation is one of the methods modeling processes occurring in vivo. The success of cultivation, in particular, is defined by a substratum choice. We studied the ability of coelomocytes and coelomic epithelial cells to attach and spread to fibronectin, laminin, polylysine, and glass. Qualitative composition of heterogeneous populations of coelomocytes and epithelial cells was determined after staining the cells with rhodamine-phalloidin and DAPI, and changes in the composition of populations evaluated in response to injury. Seven relative classes of coelomocytes has been identified, three of which has been shown to participate in the formation of clot during primary repair of wounds. There was a change in the proportion of these cells, attached to specific ligands in response to the injury. In coelomic epithelium 8 relative classes of cells has been identified, two of which are likely to be candidates for the role of progenitor cells for coelomocytes--coelomocyte-like and small epithelial cells with high nuclear-cytoplasmic ratio. The enrichment with the small cells in population of attached coelomic epithelium cells has been revealed when seeding on laminin. Continued viability of epithelial cells has been shown when cultured on laminin during 2 months.     

Functional morphology of vibratile urnae in the synaptid holothuroid Leptosynapta inhaerens (Echinodermata)

Summary Vibratile urnae of Leptosynapta inhaerens are organized in three longitudinal bands along the mesenteries. An individual of 10 cm length usually houses about 4500 urnae. These are minute (300 Gmm high), S-shaped and hollow peritoneal organs consisting of an intracoelomic projection of the body wall mesothelium supported by a thin connective tissue layer. The urnal cavity is strongly ciliated. Each urna harbours a clump of coelomocytes at the lower part of its aperture. The clump is attached to the urna through spot-like desmosomes occurring between its inner-most coelomocytes and apical urnal cells. Clumps and urnae form functional units. Urnal cilia produce steady water currents through urnal cavities and whirls along urnal bands. The particulate material conveyed by the coelomic fluid enters the urnal cavity and is either trapped by coelomocyte pseudopodia or agglutinated by a mucoid substance that covers the clump's outer surface. Depending on individuals, clearance of coelomic fluid occurs from 2 to 3 h after experimental injection of particulate material. The effectiveness of coelomic fluid clearance appears to be due to the particular organization and location of urnae, viz. in longitudinal bands along the mesenteries.     

Characteristics of populations of the coelomic fluid and coelomic epithelium cells from the starfish <Emphasis Type="Italic">Asterias rubens</Emphasis> L. able attach to and spread on various substrates

Cultivation is one of the methods of modeling processes occurring in vivo. The success of cultivation, in particular, depends on the choice of substrate. We studied the ability of coelomocytes and coelomic epithelial cells to be attached and spread on fibronectin, laminin, polylysine, and glass. The qualitative composition of heterogeneous populations of coelomocytes and epithelial cells was determined after staining the cells with rhodamine-phalloidin and DAPI, and changes in the composition of populations were evaluated in response to trauma. Seven arbitrary classes of coelomocytes have been identified, three of them being shown to participate in clot formation during primary wound reparation. There was a change in the percentage of these cells attached to specific ligands in response to trauma. In coelomic epithelium, eight arbitrary classes of cells have been identified, two of them being probable candidates for the role of progenitor cells for coelomocytes—coelomocyte-like and small epithelial cells with a high nuclear-cytoplasmic ratio. Enrichment with the small cells in a population of attached coelomic epithelium cells has been revealed when seeding on laminin. Preservation of viability of epithelial cells has been shown when cultured on laminin for 2 months.     

Proteome characterization of sea star coelomocytes--the innate immune effector cells of echinoderms

Sea star coelomic fluid is in contact with all internal organs, carrying signaling molecules and a large population of circulating cells, the coelomocytes. These cells, also known as echinoderm blood cells, are responsible for the innate immune responses and are also known to have an important role in the first stage of regeneration, i.e. wound closure, necessary to prevent disruption of the body fluid balance and to limit the invasion of pathogens. This study focuses on the proteome characterization of these multifunctional cells. The identification of 358 proteins was achieved using a combination of two techniques for protein separation (1-D SDS-PAGE followed by nanoLC and 2-D SDS-PAGE) and MALDI-TOF/TOF MS for protein identification. To our knowledge, the present report represents the first comprehensive list of sea star coelomocyte proteins, constituting an important database to validate many echinoderm-predicted proteins. Evidence for new pathways in these particular echinoderm cells are also described, and thus representing a valuable resource to stimulate future studies aiming to unravel the homology with vertebrate immune cells and particularly the origins of the immune system itself.     

Cell cooperation in coelomocyte cytotoxic activity of Paracentrotus lividus coelomocytes

The coelomic fluid from the sea urchin Paracentrotus lividus contains several coelomocyte types including amoebocytes and uncoloured spherulocytes involved in immune defences. In the present paper, we show a Ca(2+)-dependent cytotoxic activity for the unfractionated coelomocytes assayed in vitro, with rabbit erythrocytes and the K562 tumour cell line. In a plaque-forming assay, whole coelomocyte preparations as well as density gradient separated coelomocyte populations revealed that cell populations enriched in uncoloured spherulocytes, exerted high cytotoxic activity by releasing lysins in the presence of amoebocytes. This cooperative effect could be dependent on soluble factors released by amoebocytes. With regard to this, we show that an enhanced cytotoxic activity was found by adding the supernatant from sonicated amoebocytes or hemocyte culture medium into spherulocyte preparations.     

Comparative immunological analysis of echinoderm cellular and humoral defense factors

Coelomocyte are found in the fluid filling coelomic cavity of echinoderms and depending on species can be a mixture of several morphologically different types. There are among them: granular and agranular amoebocytes, morula cells, vibratile and lymphocyte-like cells. All these cells take part in cellular response to immune challenges through phagocytosis, clotting, encapsulation of foreign particles, cytotoxicity, and the production of antimicrobial agents, such as reactive oxygen and nitric oxide. The data are given on a variety of humoral factors found in the coelomic fluid, including different types of lectines, agglutinins, hemolysins, acute phase proteins and antimicrobial factors. The discussion on cooperation between cellular and humoral arms of defense reactions during inflammation reveals the crucial role of coelomocytes in immune response. It is suggested that the sea urchin complement system (that is homologous to the alternative pathway in vertebrates) is appeared initially in echinoderms as a protein cascade that points to opsonization of foreign cells and particles, augmenting their phagocytosis and subsequent destruction by coelomocytes. So the identification of a simple complement system as a part of the echinoderm immune response shows that these animals as well as all invertebrate deuterostomes share innate immune system homologies with vertebrates. Studying the simpler immune response demonstrated by echinoderms is important for understanding the ancestral deuterostome defense system and reconstructing the evolution of immune system in higher vertebrates.     

Isolation and identification of diadenosine 5',5'-P1,P4-tetraphosphate binding proteins using magnetic bio-panning

We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap(4)A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap(4)A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap(4)A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.     

Coelomocyte Morphology in the Holothurians Apostichopus japonicus (Aspidochirota: Stichopodidae) and Cucumaria japonica (Dendrochirota: Cucumariidae)

The cellular composition of the coelomic fluid of the Far Eastern holothurinans Apostichopus japonicus and Cucumaria japonica was studied using light and transmission electron microscopy and histochemistry. In the coelomic fluid of A. japonicus, the following types of coelomocytes were distinguished: progenitor cells; amoebocytes; vacuolated cells; small (or young) morula cells; morula cells of type I, type II, and type III; crystal cells; and vibratile cells. In the coelomic fluid of C. japonicawere found progenitor cells, amoebocytes, vacuolated cells, morula cells of type I and type II, crystal cells, and hemocytes containing a respiratory pigment. The issue of stem cell type, which gives rise to coelomocytes, is discussed.     

Comparative analysis of behavior and proliferative activity in culture of cells of coelomic fluid and of cells of various tissues of the sea star <Emphasis Type="Italic">Asterias rubens</Emphasis> L. Isolated from normal and injured animals

The sources of coelomocytes in Asteroidea are suggested to be the coelomic epithelium, the axial organ, or Tiedemann’s bodies. To study the problem of whether the cells are replenished at the expense of divisions, we analyzed the incorporation of bromodeoxyuridine (BrdU) in vivo into cells from different tissues of the sea star Asterias rubens L. To study differentiation in vitro, methods of isolating and cultivating cells from various tissues were elaborated and an analysis of the behavior and incorporation of BrdU in culture was performed. The reproduced BrdU incorporation was detected in coelomic epithelial cells. The behavior of coelomocytes and the coelomic epithelial cells in culture depended on the time after the injury of the animals in which the cells were isolated, whereas, for the axial organ and Tiedemann’s bodies, no differences were revealed. After 2 months of cultivation, the formation of BrdU-incorporating, colony-like cells with high nuclear-cytoplasmic ratios was characteristic of coelomic epithelial cells. Thus, the most prospective object for studying the processes of A. rubens cell differentiation in vitro seems to be the coelomic epithelium.     

Gene expression profiling of coelomic cells and discovery of immune-related genes in the earthworm,Eisenia andrei,using expressed sequence tags

The coelomic cells of the earthworm consist of leukocytes, chlorogocytes, and coelomocytes, which play an important role in innate immunity reactions. To gain insight into the expression profiles of coelomic cells of the earthworm, Eisenia andrei, we analyzed 1151 expressed sequence tags (ESTs) derived from the cDNA library of the coelomic cells. Among the 1151 ESTs analyzed, 493 ESTs (42.8%) showed a significant similarity to known genes and represented 164 unique genes, of which 93 ESTs were singletons and 71 ESTs manifested as two or more ESTs. From the 164 unique genes sequenced, we found 24 immune-related and cell defense genes. Furthermore, real-time PCR analysis showed that levels of lysenin-related proteins mRNA in coelomic cells of E. andrei were upregulated after the injection of Bacillus subtilis bacteria. This EST data-set would provide a valuable resource for future researches of earthworm immune system.