Search

Many aspects of the cellular immune system in the marine sponge Callyspongia diffusa, have been defined by using artificially transplanted allogeneic tissues. Rejections show specificity of 'non-self' recognition, cytotoxic effector responses and short-term immunological memory. Histological investigations reveal a generalized mesohyl migration to the graft zone where archaeocytes line up at the allogeneic interface. Monoclonal antibodies (mAbs) raised to sponge cells have shown that little or no allogeneic cell mixing occurs at the graft interface and that certain mesohyl cell types do not appear to be directly involved in graft rejections. However, all mesohyl cell types are present in autograft fusion zones and in inflammatory responses to injury. The involvement of only some of the mesohyl cell types in graft rejections suggests specific interactions of an effector 'immunocyte'.

Experiments were carried out to test the hypothesis that fibronectin is involved in reaggregation of dissociated sponge cells. Cells from the siliceous sponge Geodia cydonium were extracted with urea to solubilize fibronectin from cells of higher multicellular organisms. The crude extract was further fractionated by DNA, heparin, and collagen affinity chromatography; they were termed Geodia fibronectin like fractions. The fibronectin like fractions contained a series of proteins with molecular weights different from that of the genuine fibronectin. The Geodia fibronectin like fractions did not react with antiserum, produced against human fibronectin, under formation of a precipitin line. Using this antiserum the sponge cells could not be specifically labeled with FITC-anti-IgG antiserum. Radioimmunoprecipitation experiments revealed that the Geodia fractions contain--if at all--0.1% fibronectin or fibronectin like protein at the most. In the crucial experiments it was shown that the Geodia fibronectinlike fractions, human fibronectin, and antifibronectin antiserum exerted no influence on adhesion of Geodia cells either in the absence or in the presence of the soluble aggregation factor. Based on these findings, we conclude that fibronectin is apparently not present on Geodia cells and does not play a role in aggregation of this biological system.

A D-galactose-specific lectin, purified from the marine sponge Geodia cydonium, is present on the cell surface of mucoid cells, free choanocytes and choanocyte clusters, as revealed first, by the adhesion assay which is based on the formation of "rosettes" with erythrocytes, and second, by immunofluorescence studies. Using the same techniques no lectin could be identified on the surface of archaeocytes. Rosette formation was inhibited in the presence of 20 mM D-lactose as well as after preincubation of erythrocytes with purified lectin. Titration experiments in a hemagglutination assay showed that the highest level of extractable lectin (5% of the total protein) is found in mucoid cells, lower concentrations are determined in choanocyte clusters (0.07%), free choanocytes (0.05%) and archaeocytes (0.01%). Only the mucoid cells were found to synthesize lectin which is secreted and subsequently transferred to the cell surface of other cell types. As one consequence of the binding of the lectin to the cell surface of aggregation-deficient choanocytes or archaeocytes, the conversion of these cells to aggregation-susceptible ones is observed. These results support previous evidence that the lectin is involved in the reaggregation process of single cells in the homologous biological system.

Allograft rejection processes of two taxonomically related sponge species, Polymastia robusta and Polymastia mamillaris, have been studied at the cellular level. Grafts in parabiosis have been made by pairing papillae. They were investigated using histology and scanning and transmission electron microscopy. In Polymatia robusta, allografts undergo progressive rejection with adhesion. A few days after grafting, archaeocytes and collencytes migrate toward the zone of contact and fall into line on either side of this zone. The narrow space which separates the grafted papillae is progressively filled up with collagen fibers thanks to which the parabionts are firmly held together. Allografts of Polymastia mamillaris never fuse to each other; the specimens are completely and directly rejected. In this species, the cell migration to the zone of contact is not followed by their alignment nor by deposition of a collagen layer. Autografts made in similar conditions succeed in all cases in both species. These experiments demonstrate for the first time that sponges, even taxonomically closely related, may develop different but defined processes of rejection.

Molecules binding sulfated polysaccharides were detected as lectins in cholate lysates of cells from twelve sponge species. Each species exhibited a unique binding profile. The pattern of binding indicated that the specificity was most probably determined by the orientation of the sulfate groups on the polysaccharide chains. Cells from each of the three species examined in more detail were found to express sulfated polysaccharide-binding molecules at their surface and at least one of the polysaccharides recognized was found to inhibit the reaggregation of cells from each species. Moreover, in all but one instance, lectins for the inhibitory polysaccharide were both detected in cell lysates and shown to be expressed at the cell surface. Sulfated polysaccharides, therefore, appeared to be involved in cell interaction events in the Porifera. This conclusion was confirmed by the isolation via ion exchange chromatography of an endogenous polysaccharide from an O. tenuis cell extract. This molecule contained uronic acid and hexose units in a ratio of 2:1, 11.9% sulfur and less than 0.5% protein. It inhibited the aggregation of O. tenuis cells and the agglutination of dextran-sulfate- and polyvinyl-sulfate-coupled erythrocytes by O. tenuis cell lysates. O. tenuis cell aggregation was also inhibited by polyvinyl sulfate and dextran sulfate and molecules binding these compounds were expressed on the surface of O. tenuis cells. Thus, is was probable that the cell surface receptor for polyvinyl sulfate and dextran sulfate and isolated sponge sulfated polysaccharide are one and the same. Finally, using a dextran sulfate affinity procedure, a 35 kD dextran-sulfate-binding protein was isolated from the surface of O. tenuis cells. The possibility that the polysaccharide isolated from O. tenuis cell extracts in the absence of calcium is the monomeric form of a cell aggregation-enhancing factor is discussed.

In contrast to cells from other multicellular organisms, cells from the marine sponge Geodia cydonium are provided with Ca++-dependent adhesion mechanisms only (W.E.G. Müller; Intern. Rev. Cytol. 77; 1982, 129). Two different mechanisms have been discovered by us, which were termed primary aggregation and secondary aggregation. In previous reports, we described that two macromolecules (aggregation factor [sAF] and aggregation receptor [AR]) are involved in the secondary aggregation of sponge cells. The sAF was bound to a high-molecular-weight particle and was termed aggregation complex. The aggregation complex was shown to consist of two further functional subunits: UDP-glucuronosyltransferase and UDP-beta-D-galactosyltransferase. The AR with a molecular weight of approximately 17,000 was found to be a glycoprotein with D-glucuronic acid as the terminal sugar moiety. Data are presented from in vitro and in vivo experiments with the Geodia system, indicating that cell aggregation and cell separation are controlled first by alteration of the binding capacity of the aggregation receptor and secondly by an additional molecule (anti-aggregation receptor), which can decrease the interaction between the aggregation factor and the aggregation receptor. Recently we succeeded in the identification and isolation of the primary aggregation factor (pAF) from the same sponge species. This pAF is a glycoprotein that is firmly associated with the cell membrane. The Mr of the native pAF was 36,000; under denatured conditions three protein species were identified in the pAF preparation. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF. A new, very exciting era began with the discovery that sponges are already provided with transplantation immunopotentialities.

The localization of two carohydrate binding proteins, so-called lectins, was studied in the sponge tissue of Axinella polypoides by light and immunofluorescence microscopy. They do not occur at the cellular surface of any cell type, but they are stored in vesicles of the "spherulous cells". After short formaldehyde fixation spherulous cells can be isolated and they release the active lectins upon lysis in distilled water. Electron microscopical studies of spherulous cells show that they contain almost nothing else but a small nucleus and vesicles of different size and number. Small vesicles are full of an electron dense material, whereas the content of large vesicles has a fluffy and fibrillar structure. Spherulous cells are large and tightly packed in the outer layer of the ectosome and in the meshwork of the spongin fibres of the central axis. The are small and scattered in the inner layer of the ectosome, and they are found throughout the choanosome. The function of the lectins is not clearly defined, and different alternatives such as participation in glycoprotein synthesis, immunological defense, or carbohydrate transport are possible.

The aggregation factor (AF) of the marine sponge Geodia cydonium recognizes the aggregation receptor (AR) which is inserted in the plasma membrane, under formation of species-specific aggregates. The specific cell-binding fragment of the AF was used to investigate for the first time the phosphoinositide metabolism in a lower avertebrate system. We found that after binding of the cell-binding fragment to the aggregation receptor a strong and rapid stimulation of the phosphate incorporation into phosphatidylinositol occurs followed by an increased turnover of phosphoinositides in the Geodia cells. The consequences of an increased degradation of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol-1,4,5-trisphosphate and diacylglycerol are 2-fold. First, after the addition of the extracellular stimulus the cytosolic Ca2+ concentration rises, resulting in a rapid increased Ca2+ efflux rate. The functional consequence of the increase of the extracellular Ca2+ level is an initiation of the aggregate formation that is mediated by the collagen assembly factor (= primary aggregation factor). Second, some experimental evidences are presented, showing that the other second messenger formed, diacylglycerol, causes a translocation of protein kinase C within the cell. Incubation of Geodia cells with the cell-binding fragment of the AF, or with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, resulted within 5 min after treatment in a 70% decrease in protein kinase C activity in the cytosolic fraction and in a 700% increase in enzyme activity in the membrane fraction. It is proposed that by membrane association protein kinase C becomes activated. As a result of this event a series of cellular proteins are phosphorylated, a process which ultimately leads to an unusually strong induction of DNA polymerase alpha activity. From these data we conclude that inositol trisphosphate and protein kinase C also play a fundamental role in cellular signal transduction in lower eukaryotes.

Freshwater sponges, Corvomeyenia carolinensis Harrison, were placed into tap water to induce degenerative reduction body formation. Reduction bodies were examined using light and electron microscopy in order to define their histochemical and ultrastructural characteristics. The reduction body of freshwater sponges is an extremely simple developmental system consisting primarily of an archeocyte reserve delimited by a simple squamous pinacoderm. The freshwater sponge reduction body displays many similarities to overwintering phases of marine sponges. The system presents an unusually straightforward vehicle for investigations of degeneration and regeneration as processes in developmental biology and may represent a reasonable vehicle in which to examine the process of the genesis of lysosomes.

In the sponge Lubomirskia baicalensis, a representative of the endemic fauna in the Baikal Lake, male specimens were first discovered and successive stages of their spermatogenesis were followed at light and electron microscopic levels. Male sex cells develop in cysts surrounded with a membrane consisting of cells which resemble pinacocytes by their ultrastructure. One sponge, during the period a spermatogenesis, contains in its tissue testicular follicles at various stages of the male sex cells maturation, within every follicle the development occurring more or less synchronously. As a result of transformations taking place in the ultrastructural organization of the spermatid, a primitive type of spermatozoon is forming, without acrosome, with large amount of cytoplasm containing small mitochondria in it.

Spongilla lacustris exhibits a type of alternative hermaphroditism, new to the phylum, in which a sponge may be exclusively male or female during the period of sexual reproduction one year and the opposite sex the next year. This form of sexuality may facilitate larva production and thus dispersal following colonization of a new habitat. Gametogenesis occurs shortly after gemmule hatching in both males and females but slightly later in males.

We have recently shown that the aggregation factor (AF) from the sponge Geodia cydonium stimulates DNA synthesis in quiescent, dissociated cells from the same organism; this event was correlated with the release of the two second messengers: inositol trisphosphate and diacylglycerol. Here we describe that after binding of the AF to the plasma membrane-bound aggregation receptor, a rapid and drastic increase in the incorporation of 32Pi into a series of proteins in the pore complex-lamina fraction occurs. Addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to quiescent cells resulted in a similar stimulation of phosphorylation of nuclear proteins. Among them we have selected one protein with a polypeptide Mr of 170,000 (pp170) for detailed studies. By immunoblotting pp170 was identified as DNA topoisomerase II. In vitro studies with nuclei and purified, homogeneous protein kinase C together with the required activators of this enzyme also showed a phosphorylation of pp170. After phosphorylation, DNA topoisomerase II activity was found to be 2.5-fold that of the non-phosphorylated enzyme. From these data we conclude that protein kinase C is involved in AF induced transmembrane signalling, ultimately leading to an initiation of DNA synthesis.

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA-initiation phase the cyclic AMP- and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10(6) cells to 0.3 pmol/10(6) cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10(6) cells. The activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. The soluble as well as the particulate enzyme activities were checked in vitro. The cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.

We have studied Microciona prolifera cells as a model for inflammation and secretion. Dissociated in Ca-, Mg-free seawater with 2.5 mM EDTA, the cells aggregate when exposed to Ca (greater than 5 mM) and Ca ionophores. Extracellular Ca is not required over the course of aggregation; brief pulses of Ca suffice. Aggregation was induced by A23187 in excess EDTA after cells were prepared by pulse Ca. It appeared that Ca ionophore stimulated the secretion of Microciona aggregation factor (MAF) to a locus or in a form inaccessible to external EDTA. Pulse-induced aggregation depended on MAF because it was inhibited by MAF fragments, which are ligands for MAF-binding sites. Sponge cells were preloaded with three fluorescent dyes that monitor aspects of stimulus-secretion coupling: 1) 3,3'-dipropylthiadicarbocyanine iodide (dis-C3-(5)), a carbocyanine dye presumed to report changes in membrane potential; 2) 9-aminoacridine (9AA), which presumably reports secretion from acid vesicles; and 3) chlortetracycline (CTC), presumed to report mobilization of membrane-associated Ca. Exposure of cells either to constant Ca or to pulse Ca stimuli caused prompt decreases in the fluorescence of cells with diS-C3-(5) and increases in fluorescence of cells with 9AA. In contrast, although constant Ca provoked decreases in fluorescence of cells with CTC, a pulse Ca was without effect. Moreover, inhibitors of stimulus-response coupling (e.g., aspirin, sodium salicylate, 5 mM; diclofenac, 100 microM) inhibited sponge aggregation induced by either constant or pulse stimuli. In contrast, like the endogenous mediator of inflammation, leukotriene B4, trienoic alkyl catechols (urushiol) from poison ivy provoked aggregation. These studies suggest the utility of this marine model for analysis of stimulus-response coupling in cells of higher species that also respond to secretagogues in the absence of external Ca.

An aggregation receptor from the siliceous sponge Geodia cydonium has been isolated and purified in an almost pure form. It sediments at about 2-6s, has a buoyant density of 1-51 g/ml in CsCl and elutes from Sephadex G-50 at a Ve/V0 value of 1-311. Chemical analysis revealed that the receptor consists of 81% neutral carbohydrate and 7-5% protein. The activity of the receptor is rapidly destroyed by Na-periodate. The receptor is released from the cell surface after removal of Ca2+ from the medium or after incubation of the cells with trypsin. The depleted cells can be charged again with isolated receptor molecules. The binding of the receptor molecules on the cell surface is prevented in the presence of trypsin. For optimal binding, physiological salt concentrations with respect to NaCl (540 mM NaCl) and Ca2+ ions are necessary. The receptor whose isolation is described in this report, is involved in secondary aggregation processes, which are initiated by a soluble aggregation factor. The primary aggregation of the cells is not influenced by the receptor. Time-course studies with receptor-depleted cells revealed that new aggregation receptor molecules are formed during the aggregation process. By competition experiments it could be shown that high concentrations of soluble aggregation receptor molecules inhibit secondary aggregation. The soluble receptor molecules can complete with surface-bound receptor molecules only if these are not linked with the aggregation factor.

A sponge extract that produced specific aggregation of dissociated cells was treated with various enzyme preparations to determine which enzymes would destroy its aggregating properties. The results indicate that proteins play a key role in the aggregating effect of the extract on dissociated, glutaraldehyde-fixed sponge cells. Further studies confirm the necessity of calcium for the aggregation and indicate the necessity of intact disulfide groups.