Fundamental aspects of arm repair phase in two echinoderm models.

Regeneration is a post-embryonic developmental process that ensures complete morphological and functional restoration of lost body parts. The repair phase is a key step for the effectiveness of the subsequent regenerative process: in vertebrates, efficient re-epithelialisation, rapid inflammatory/immune response and post-injury tissue remodelling are fundamental aspects for the success of this phase, their impairment leading to an inhibition or total prevention of regeneration. Among deuterostomes, echinoderms display a unique combination of striking regenerative abilities and diversity of useful experimental models, although still largely unexplored. Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here used to comparatively investigate the main repair phase events after injury as well as the presence and expression of immune system and extracellular matrix (i.e. collagen) molecules using both microscopy and molecular tools. Our results showed that emergency reaction and re-epithelialisation are similar in both echinoderm models, being faster and more effective than in mammals. Moreover, in comparison to the latter, both echinoderms showed delayed and less abundant collagen deposition at the wound site (absence of fibrosis). The gene expression patterns of molecules related to the immune response, such as Ese-fib-like (starfishes) and Afi-ficolin (brittle stars), were described for the first time during echinoderm regeneration providing promising starting points to investigate the immune system role in these regeneration models. Overall, the similarities in repair events and timing within the echinoderms and the differences with what has been reported in mammals suggest that effective repair processes in echinoderms play an important role for their subsequent ability to regenerate. Targeted molecular and functional analyses will shed light on the evolution of these abilities in the deuterostomian lineage.

Two macrophage migration inhibitory factors regulate starfish larval immune cell chemotaxis.

Immune cell recruitment is critical step in the inflammatory response and associated diseases. However, the underlying regulatory mechanisms are poorly understood in invertebrates. Mesenchyme cells of the starfish larvae, which allowed Metchnikoff to complete his landmark experiments, are important model for analysis of immune cell migration. The present study investigated the role of macrophage migration inhibitory factor (MIF)--an evolutionarily conserved cytokine that is functionally similar to chemokines--in the larvae of the starfish Patiria (Asterina) pectinifera, which were found to possess two orthologs, ApMIF1 and ApMIF2. ApMIF1 and ApMIF2 clustered with mammalian MIF and its homolog D-dopachrome tautomerase (DDT), respectively, in the phylogenetic analysis. In contrast to the functional similarity between mammalian MIF and DDT, ApMIF1 knockdown resulted in the excessive recruitment of mesenchyme cells in vivo, whereas ApMIF2 deficiency inhibited the recruitment of these cells to foreign bodies. Mesenchyme cells migrated along a gradient of recombinant ApMIF2 in vitro, whereas recombinant ApMIF1 completely blocked ApMIF2-induced directed migration. Moreover, the expression patterns of ApMIF1 and ApMIF2 messenger RNA in bacteria-challenged mesenchyme cells were consistent with in vivo observations of cell behaviors. These results indicate that ApMIF1 and ApMIF2 act as chemotactic inhibitory and stimulatory factors, respectively, and coordinately regulate mesenchyme cell recruitment during the immune response in starfish larvae. This is the first report describing opposing functions for MIF- and DDT-like molecules. Our findings provide novel insight into the mechanisms underlying immune regulation in invertebrates.

Starfish ApDOCK protein essentially functions in larval defense system operated by mesenchyme cells.

In larvae of the starfish, Asterina pectinifera, mesenchyme cells operate in the defense system through various behaviors. We have investigated mesenchyme cell dynamics during the immune response by identifying ApDOCK, a new member of the DOCK180 superfamily protein. In 4-day-old bipinnaria larvae processed for morpholino oligonucleotide-mediated knockdown of ApDOCK, injection of inorganic foreign substances revealed that (1) mesenchyme cells fail to undergo either directed migration toward a large oil-droplet or persistent spreading on the oil-droplet after contact; (2) neither uptake of micro-beads nor cell-to-cell fusion on the large oil-droplet differed from that of mesenchyme cells from control larvae. Similar behaviors were also recorded in experiments where bacteria were injected. Under culture conditions, the expression level of ApDOCK mRNA was significantly associated with the immunological behavior of mesenchyme cells. Apparently, the mesenchyme cells from ApDOCK loss-of-function larvae exhibited insufficient lamellipodium formation via lack of fibrous form of actin organization at the leading edge. These results suggest that the migratory congregation and persistence of encapsulation of larval mesenchyme cells are intracellularly regulated by ApDOCK protein, and this regulation is associated with organization of cytoskeletal actin.

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.

Starfish, Asterias amurensis and Asterina pectinifera, as potential sources of Th1 immunity-stimulating adjuvants.

Saponin is the generic name of steroid or triterpene glycosides, and the capacities of some saponins to stimulate both Th1 immune response and production of cytotoxic T cells are useful as vaccine components against intracellular pathogens. Because saponins have been found commonly in starfish, we assessed the potential of starfish, Asterias amurensis and Asterina pectinifera, as adjuvant sources. Crude starfish saponins had hemolytic activities (EC(50)=10 to 100 µg/ml) and thin layer chromatography indicated heterogeneity of their constituents. When starfish saponis were subcutaneously injected into mice with ovalbumin (OVA), OVA-specific IgG, especially IgG2a instead of IgG1 was produced in mouse blood, suggesting starfish saponins stimulated Th1 type immunity and they were potential sources of new adjuvants.

Characterization of Coelomic Fluid Cell Types in the Starfish Marthasterias glacialis Using a Flow Cytometry/Imaging Combined Approach.

Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.

New insights into the evolutionary origins of the recombination-activating gene proteins and V(D)J recombination.

The adaptive immune system of jawed vertebrates relies on V(D)J recombination as one of the main processes to generate the diverse array of receptors necessary for the recognition of a wide range of pathogens. The DNA cleavage reaction necessary for the assembly of the antigen receptor genes from an array of potential gene segments is mediated by the recombination-activating gene proteins RAG1 and RAG2. The RAG proteins have been proposed to originate from a transposable element (TE) as they share mechanistic and structural similarities with several families of transposases and are themselves capable of mediating transposition. A number of RAG-like proteins and TEs with sequence similarity to RAG1 and RAG2 have been identified, but only recently has their function begun to be characterized, revealing mechanistic links to the vertebrate RAGs. Of particular significance is the discovery of ProtoRAG, a transposon superfamily found in the genome of the basal chordate amphioxus. ProtoRAG has many of the sequence and mechanistic features predicted for the ancestral RAG transposon and is likely to be an evolutionary relative of RAG1 and RAG2. In addition, early observations suggesting that RAG1 is able to mediate V(D)J recombination in the absence of RAG2 have been confirmed, implying independent evolutionary origins for the two RAG genes. Here, recent progress in identifying and characterizing RAG-like proteins and the TEs that encode them is summarized and a refined model for the evolution of V(D)J recombination and the RAG proteins is presented.

Alteration of cellular immune responses in the seastar Asterias rubens following dietary exposure to cadmium.

Several parameters of cellular immunity in seastars fed Cd-contaminated mussels were analyzed. The accumulation of cadmium in the seastars did not alter the concentration of amoebocytes in the coelomic fluid. On the contrary, the immune cells showed a reduced phagocytic activity and an increased production of reactive oxygen species. These effects may lead to an inability of the seastars to cope with bacterial infections and to oxidative damages to self tissue that could threaten the survival of the animals.

Increased phagocytic activity in contaminated seastars (Asterias rubens) collected in the Southern Bight of the North Sea.

In invertebrates, the phagocytosis exerted by immune cells constitutes the main line of internal defence against offending microorganisms. This study assessed during two consecutive years the phagocytic activity in seastars (Asterias rubens) collected in stations along the Belgian and Dutch coasts. The contamination of these seastars by metals and PCBs were measured in parallel. Increased phagocytic activities were found in seastars collected in the plume of the Scheldt river. This correlated with the contamination of seastars by metals, mainly Pb, but not with the contamination by the analysed PCB congeners. Furthermore, the relationship between phagocytosis and metal contamination was reproducible from one year to another. The possible mechanisms explaining this effect are discussed in light of a direct or indirect link between phagocytic activity and metal contamination of seastars.

Purification and biochemical characterization of an invertebrate interleukin 1.

Interleukin 1 (IL-1) is a major immunoregulatory protein released by macrophages with many host defense related properties. That IL-1 has been found in the invertebrates attests to its importance in homeostasis. The first step in comparing the vertebrate protein to its invertebrate correlate is to purify the protein to study. We have purified to homogeneity IL-1 isolated from the coelomic fluid of the starfish Asterias forbesi. The IL-1 had isoelectric points of 7.4, 5.4 and 4.8. The pI 4.8 species had a molecular weight of 22,000 and the pI 7.4 and 5.4 species both had Mr of 17,000. Higher Mr forms were also found. These molecules were biologically active in the human melanoma A375 cytotoxicity assay for IL-1, and were also able to stimulate murine dermal fibroblast proliferation, protein synthesis, and PGE2 production. The pI 4.8 and 5.4 forms were purified to homogeneity and the amino acid composition was determined. The pI 4.8 and 5.4 species were purified more than 200-fold to specific activities of 3 x 10(6) and 1 x 10(6) units mg-1, respectively. The pI 7.4 form was isolated and partial N-terminal sequence analysis was performed. The similarities of molecular weight, isoelectric points and biological properties between vertebrate and invertebrate IL-1 show that it is an important, evolutionarily stable host defense molecule.

Differentiation of immune cells challenged by bacteria in the common European starfish, Asterias rubens (Echinodermata).

Amoebocytes are the main effector cells of the echinoderm immune system. In starfishes, a taxon in which bacterial diseases have been rarely reported, amoebocytes are considered to be the only circulating and immune cell type. The present paper addresses the question of amoebocyte differentiation in the starfish Asterias rubens when challenged by bacteria. Starfishes were injected with FITC-coupled bacteria (Micrococcus luteus). Amoebocytes were collected at regular time intervals for 24 h. The cytometric characteristics and the phagocytic activity were studied by flow cytometry. Three amoebocyte groups of different size were identified. The cell concentrations of the two largest and more numerous of these groups (G2 and G3) were modulated by immune stimulation while the group of smallest, less numerous, cells (G1) was unaffected. All of these cell groups were phagocytic but their kinetics of cell activation and bacteria ingestion differed. G1 cells showed the lowest phagocytic activity while G3 cells had the highest and fastest phagocytic activity. Starfish amoebocytes appear to be segregated in three groups, two of them (G2 and G3) being immunomodulated and one of them presenting a very fast reaction to bacteria. It is suggested that the high efficiency of the immune system in starfishes is related to this fast reaction.

Effect of in vivo inoculation of bacteria on the spontaneous cytotoxicity of axial organ cells from Asterias rubens.

In vivo inoculation of heat-killed bacteria in the coelomic cavity of starfish induces an increase of spontaneous cytotoxicity developed by axial organ (an ancestral lymphoid organ) cells, phenomenon demonstrated by an in vitro [51Cr]-release assay. This observation corroborates the existence of a sophisticated immune system in the echinoderms, a first-rate model for phylogenic study of the immune response.

Spontaneous and induced cytotoxicity of axial organ cells from Asterias rubens (Asterid--echinoderm).

Spontaneously occurring cytotoxicity of starfish axial organ cells on target cells has been studied. It appears that in an allogeneic system no cytotoxic phenomenon occurs after 4 or 6 h of culture, whereas spontaneous cytotoxicity toward malignant vertebrate target cells is shown and reaches its maximum at 37 degrees C. In a certain way, the axial organ cell population includes cells, which may be compared with vertebrate natural killer cells. On the other hand, it must also be noted that axial organ cells are able to exert in a few cases, an induced cytotoxicity on MBL2 cells. This phenomenon appears at the end of the fourth day of culture.

MLR-like reaction between axial organ cells from asterids.

The uptake of [3H]thymidine by axial organ (AO) cells arising from two starfishes and mixed together, in a ratio of 50/50 was studied. Mixed leucocyte reaction (MLR) combinations gave a positive response at the fifth day of culture in about 20% of the tested combination (index: 1.8-6.7). No positive reaction was observed in an allogeneic system. More significant results seem to be obtained with the nonadherent nylon-wool subpopulation cells from the two species, rather than with the whole axial organ cells.

Kinetics of tumor cell cytostasis by sea star factor-activated macrophages.

Macrophages obtained from Wistar/Furth (W/Fu) and Fischer (F344) rats receiving intraperitoneal injection of 0.2 mg of sea star factor (SSF) exert a profound suppressive effect on proliferation of tumor cells in vitro. The cytostatic effect of these activated macrophages is not immunologically specific, and is demonstrably effective against syngeneic, allogeneic, or xenogeneic targets (the latter of murine origin, P815-X2 of DBA/2 strain). Evidence suggests that cytostasis can be resolved into three discrete temporally progressive steps: cytoadhesion, macrophage-dependent, and macrophage-independent states. Interference with the first stage by the use of high molecular weight dextran blocks development of macrophage-dependent cytostasis. Suppression of this second stage by selective macrophage toxicity using silica blocks the final event of irreversible macrophage-independent cytostasis. Taken together, these data suggest that suppression of tumor cell division by SSF-activated macrophages requires direct contact for a relatively prolonged period between viable effector macrophages and tumor target cells to achieve maximal cytostasis.

Reactions induced in vertebrates by invertebrate cell suspensions. II/Non adherent axial organ cells as effector cells.

In contrast to starfish axial organ cells, coelomocytes of Asterias rubens do not induce a significant reaction of angiogenesis after subcutaneous injection into irradiated mice. Axial organ cells are separated into adhering and nonadhering cells by attachment ot plastic surface. Nonadhering cells consisting mainly of small lymphocyte-like cells appear to be effector cells in the reaction. Phytogenetic implications of these observations are discussed.

Immunoglobulin-bearing blood leucocytes in the Pacific hagfish.

The occurrence of immunoglobulin (Ig) bearing leucocytes in the blood of the Pacific hagfish, Eptatretus stoutii , was examined using a murine monoclonal antibody (45.3) and a rabbit antiserum specific for hagfish serum Ig. Binding of antibody 45.3 to hagfish leucocytes assessed by radioimmunoassay was inhibited by preincubation of antibody with purified serum Ig thus verifying the presence of cell surface Ig cross reactive with serum Ig. The monoclonal antibody identified approximately 65% of blood leucocytes as Ig+ve while the rabbit antiserum indicated 81% Ig+ve cells. Both antibody preparations failed to react specifically with cells from mouse, horned shark, tunicate or sea star; this indicates the distinctive nature of hagfish Ig. The high percentage of blood cells bearing surface Ig in the hagfish raises the possibility that lymphocyte divergence to separate B and T pathways may not have occurred in this most primitive vertebrate. Alternatively, an Ig-like specificity characteristic of both "T" and "B" lymphocytes may have been detected. In any event, a subset of Ig negative leucocytes is evident in hagfish.

Complement-like activity in the sea star, Asterias forbesi.

The coelomic fluid (CF) of Asterias forbesi was examined for the presence of complement-like activity. Cell-free CF contains hemolytic activity for unsensitized rabbit erythrocytes (E) but not unsensitized or antibody-coated sheep E. Target E sensitivity is not dependent upon phylogenetic proximity of erythrocyte species to Asterias. The activity is optimally assayed at 25 degrees C, has a sigmoidal dose-response curve, and is heat-sensitive (56 degrees C). The activity is divalent cation dependent, although Ca++ and Mg++ are not required. Nonlytic CF does not contain an inhibitor; rather, combining nonlytic and lytic CFs results in an increase in hemolysis. Activity can be consumed by treatment with zymosan, pronase, trypsin, and PMSF. Enzymatic desialidation converts sheep E into activators of the hemolytic pathway. No phospholipase activity is present in CF. Biological function is demonstrated by bactericidal activity against Vibrio tubiashii by lytic CF. These results suggest that a primitive complement system is operating in Asterias.

Reactions induced in vertebrates by invertebrate cell suspensions. I/Specific effects of sea star axial organ cells injection.

Starfish axial organ cell suspensions injected subcutaneously into irradiated mice induce a characteristic angiogenesis reaction. The size of the reaction appears to be dependent upon the number of injected starfish cells. Axial organ cells also provoke a splenomegaly positive test in 16 day old chick embryos. No significant angiogenesis nor splenomegaly were induced by heat killed axial organ cells, ovocytes or branchial digestive organ cells. These results support our hypothesis that echinoid axial organ represents an ancestral primary lymphoid organ.

Contrasting effects of coplanar versus non-coplanar PCB congeners on immunomodulation and CYP1A levels (determined using an adapted ELISA method) in the common sea star Asterias rubens L.

Biological effects of two structurally contrasting PCB congeners (coplanar 77 and non coplanar 153) were investigated by measuring the induction of CYP1A immunopositive protein (CYP1A IPP) in the pyloric caeca and the production of reactive oxygen species (ROS) by amoebocytes in the common sea star Asterias rubens. CYP1A IPP was quantified using a specially designed ELISA which uses competitive binding between sea stars and trout CYP1A IPPs. Only the coplanar congener had a significant effect on the two considered biological responses. Intensity of the effects was dose-dependent. However, the highest dose of PCB 77 induced a dramatic decrease of ROS production. It is concluded that coplanar PCBs straightforwardly affect key biological processes such as the immune system and mixed-function oxidase (MFO) system.