Phagocytic efficiency and cytotoxic responses of Indian freshwater sponge (Eunapius carteri) cells isolated by density gradient centrifugation and flow cytometry: a morphofunctional analysis

The freshwater sponge Eunapius carteri (Porifera: Demospongiae: Spongillidae), a resident of Indian freshwater ecosystems, has pharmaceutical and ecological potential, but there is inadequate information on its cellular spectrum and cell-mediated immune responses. Microscopical analysis revealed the existence of eight distinct cellular variants, i.e. blast-like cells, choanocytes, small amoebocytes, granular cells, pinacocytes, large amoebocytes, archaeocytes and sclerocytes. The cells were isolated by density gradient centrifugation and flow cytometry and used for a morphofunctional analysis. We investigated the phagocytic efficiency of E. carteri cells under the challenge of yeast particles in vitro and spectrophotometrically quantified the generation of cytotoxic molecules (superoxide anions and nitric oxide) in different isolated cellular fractions. The two cell separating technologies did not yield any significant differences in the major findings on morphology, phagocytic response and generation of superoxide anions and nitric oxide. Archaeocytes, granular cells and large amoebocytes were identified as chief phagocytes with a high phagocytic potential as recorded by light microscopy. Archaeocytes were the principal generators of superoxide anions, whereas nitric oxide was recorded in the fractions rich in archaeocytes and large amoebocytes. The present investigation thus provides useful information regarding cellular variation, cytotoxic status and innate phagocytic response of the cells of E. carteri, a common but less studied sponge of India.     

Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Ascidiacea)

A morpho‐functional study of the colonial ascidian Botrylloides leachi haemocytes was carried out to propose their classification, relationships and specializations. This characterization was obtained by (i) investigations of both living and aldehyde‐fixed cells by light and electron microscopy; (ii) cytochemical and cytoenzymatic assays; (iii) lectin‐affinity assays; (iv) phagocytosis and haemagglutination assays; and (v) anti‐CD34 immunocytochemical assay for vertebrate haematopoietic stem cells. Results indicate that the haemoblast is a circulating stem cell and there are at least five haemocyte differentiation pathways, the last two of which have never been described in botryllids: (i) phagocytic line (hyaline amoebocytes and macrophage‐like cells) share ultrastructural features, the same hydrolytic enzymes and WGA lectin binding, and are involved in yeast phagocytosis and erythrocyte rosette formation; (ii) cytotoxic line (granular amoebocytes and morula cells) with vacuoles containing oxidative enzymes and polyphenolic substrates; (iii) vacuolated cell line (pigment cells and nephrocytes) involved in catabolite storage; (iv) compartment cell line (compartment amoebocytes and compartment cells) able to agglutinate erythrocytes and characterized by vacuoles with a moderately electron‐dense content, positive to arylsulphatase activity and binding DBA, UEA‐I, HPA lectins; and (v) granular cell line includes trophic cells, able to infiltrate the gut epithelium, showing a cytoplasm filled of PAS‐positive vacuoles with arylsulphatase, chloroacetylesterase and β‐glucuronidase activities.     

Biomphalaria glabrata amoebocytes: Effect of Schistosoma mansoni infection on in vitro phagocytosis

The rate of phagocytosis by amoebocytes obtained from hemolymph of the pulmonate Biomphalaria glabrata infected with the trematode Schistosoma mansoni for 24 hr and 2, 4, and 6 weeks has been determined using the monolayer assay system. Amoebocyte preparations from snails infected for 4 and 6 weeks showed a gradual decrease in the phagocytic rates compared to those from uninfected controls. Snails harboring the parasite for 4 and 6 weeks also showed a significant increase in the number of amoebocytes in the hemolymph. No significant changes were detected in the rate of phagocytosis or number of amoebocytes in snails infected for 2 weeks or less. Alterations in the morphology and behavior of amoebocytes from infected snails were also noted.     

Induced in vitro phagocytosis of the Antarctic starfish Odontaster validus (Koehler 1906) at 0°C

Phagocytosis was studied in vitro using coelomic fluid of the Antarctic starfish Odontaster validus at 0°C. The number of coelomocytes present was determined and the phagocytic activity of the phagocytic amoebocytes (PA) was quantified with yeast during incubations of 1 and 2 h. The percentage of PA phagocytosing increased significantly from 42.29 ± 10.50% (SD) at 1 h to 52.57 ± 13.96% at 2 h. Numbers of yeast per PA also rose significantly from 2.27 to 2.45 cells per amoebocyte, indicating that phagocytic activity was maintained. In vitro phagocytosis of an Antarctic invertebrate at 0°C is shown for the first time, and the types of amoebocytes involved identified. Rates of phagocytosis were similar to, or higher than, reported data for temperate starfish, although this conclusion must be treated cautiously because of scarcity of data and differences in methods used. However, the data suggest that phagocytosis in O. validus is well adapted to low temperature. Accepted: 27 September 1999     

Ultrastructural changes in the gastrodermal phagocytic cells of the planarian Dugesia gonocephala s.l. during food digestion (Plathelminthes)

Summary In the present report the functional morphology of the planarian gastrodermal phagocytic cells is examined in feeding animals. A functional interpretation of some of the morphological findings is given. The events in the fine-structure modifications of the phagocytic cells in the course of phagocytosis and intracellular digestion of food particles were followed through five post-feeding stages in the planarian Dugesia gonocephala. Light and electron microscopical observations demonstrate that there is preliminary intraluminal digestion of food particles; their phagocytosis takes place quickly.Beef hepatocytes that served as food are found engulfed at first in food vacuoles near the apical border of the phagocytic cells, and are clearly recognizable. The vacuoles increase in number to occupy most of the cytoplasm of these cells. Progressive breakdown and disappearance of phagocytosed hepatocytes occurs. In time the vacuoles move deeper into the cells, their contents lose their identity, and condense to homogeneous or heterogeneous residual bodies. These are returned to the distal surface of the cells, and then voided into the intestinal lumen. At the same time, synthesis and accumulation of numerous lipid droplets occurs, probably as a final product resulting from metabolism of the digested material. When feeding is over, the phagocytic cells are filled with lipid droplets, acquiring their typical appearance.It is suggested that disintegration of phagocytic cells during starvation is balanced by proliferation and differentiation of neoblasts into new phagocytic cells during the feeding-starvation cycle.     

Recognition of foreignness by blood cells of the freshwater snail Lymnaea stagnalis, with special reference to the role and structure of the cell coat

The structure and function of the cell coat of the blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were studied with ultrahistochemical tests, including concanavalin A (Con A) labeling, and with in vitro phagocytosis experiments. The cell coat is intensely stained by ruthenium red and tannic acid. The cells possess binding sites for Con A. Proteolytic enzymes destroy the receptors for Con A and totally inhibit the phagocytic activity of amoebocytes. Incubation experiments with proteases, carbohydrases, and inhibition sugars revealed that (1) the Con A binding sites are anchored in the plasma membrane by proteins, and (2) glucose, fructose, mannose, and to a lesser extent N-acetylglucosamine and N-acetylgalactosamine, inhibit the binding of Con A to amoebocytes, suggesting that these carbohydrates might form part of these binding sites.     

A comparative morphological and enzyme histochemical study on blood cells of the freshwater snails Lymnaea stagnalis,Biomphalaria glabrata,and Bulinus truncatus

The morphology and ultrastructure of the blood cells of the freshwater snails Lymnaea stagnalis, Biomphalaria glabrata, and Bulinus truncatus were studied. By performing in vitro experiments and enzyme histochemical studies, special attention was paid to the role of the blood cells in phagocytosis of foreign particles. No fundamental differences were found in the ultrastructure, lysosomal enzyme contents, and phagocytic capacities of the blood cells of these species. It is concluded that only one type of blood cell, the amoebocyte, exists in the freshwater snails. Amoebocytes constitute a morphologically and functionally heterogeneous population of cells, ranging from round (electron-dense) cells with the morphological characteristics of young cells to highly phagocytic spreading cells with a prominent lysosomal system. In addition to acid phosphatase, nonspecific esterase and peroxidase were found within the lysosomes. The presence of enzyme activity in the RER and the Golgi bodies indicates that amoebocytes are able to synthesize lysosomal enzymes continuously.     

Morphological and phagocytic characteristics of peritoneal exudate cells in tilapia, Oreochromis niloticus (Trewavas), and carp, Cyprinus carpio L.

The morphology and phagocytic activity of peritoneal exudate cells (PEC) obtained by an intraperitoneal injection of liquid paraffin into tilapia, Oreochromis niloticus , and carp, Cyprinus carpio , were studied with light and electron microscopy. PEC consisted of monocyte-macrophage series cells (M-Mø), neutrophils, eosinophils (granular cells) and others. Cells exhibiting the same morphology as mammalian macrophages but different from monocytes of the same species were identified with light and electron microscopy and designated as peritoneal macrophages. Light and electron microscopy revealed that M-Mø, neutrophils and eosinophils (granular cells) phagocytozed foreign materials added in vivo and in vitro. Eosinophils appeared later in the peritoneal exudate and less actively phagocytic as compared with M-Mø and neutrophils. Small and large phagosomes were formed in M-Mø, neutrophils and eosinophils (granular cells). Large phagosomes were common in neutrophils. Fusion of cytoplasmic granules with the phagosome membrane was observed. The in vitro experiment on phagocytosis revealed that the phagocytic rates in M-Mø and neutrophils were positively correlated with the doses of foreign materials. The results indicated that these two cell types have the highest capacity of phagocytosis.     

The blood cells of the sea squirt, Ciona intestinalis: Morphology,differential counts,and in vitro phagocytic activity

The sea squirt, Ciona intestinalis, contains several types of blood cells: stem cells, hyaline, granular, and refractile amoebocytes, signet ring cells, morula cells, small and large compartment cells, and orange cells. Of these cell types, only the hyaline and granular amoebocytes are capable of phagocytosing formalized sheep erythrocytes in vitro. After the addition of erythrocytes to blood cell monolayers, the attachment and ingestion of these particles occurs rapidly. The interrelationships of the various blood cell types are discussed.     

Effects of infection of EGFP-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage 1 h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians.     

Comparative study of in vivo and in vitro phagocytosis including germicidal capacity in Odontaster validus (Koehler, 1906) at 0 degree C

The phagocytosis and germicidal capacity of Saccharomyces cerevisiae by phagocytic amoebocytes (PA) of the Antarctic starfish Odontaster validus were studied in vivo (after incubation periods of 1, 2, and 4 h) and in vitro (after incubation periods of 1, 2, 4, 8, and 12 h) at 0 degree C. The total number of PA and the phagocytic capacity (PC), phagocytic index (PI), and germicidal capacity (GC) of the PA were calculated. Results showed significant variability of the total PA number in different animals. There was a significant increase in PC and no significant differences in PI and GC for different in vitro incubation times. In vivo, experiments showed no significant difference of PC and PI, but there was a significant increase in GC as incubation periods increased. Comparison between in vitro and in vivo results revealed that PI and PC were significantly higher in vitro and that GC was significantly higher in vivo. The present study shows for the first time the phagocytosis and GC of an Antarctic invertebrate in vivo at low temperature (0 degree C), and the results are comparing with the available literature for echinoderms.     

Apoptotic DNA alterations in pig leukocytes after phagocytosis of bacteria are linked to maturation of the immune system

The effect of phagocytosis of living bacteria on apoptotic DNA changes was examined in pig leukocytes in relation to immune system maturation. Blood samples of pigs (aged 6, 12 and 18 weeks) were cultivated with a suspension of bacterial cells Salmonella typhimurium LB 5000 at 37 (o)C. In the experimental groups, killed bacteria and microspheric particles were used to detect the influence of the phagocytic process. Phagocytic activity and index were determined in each sample by means of microspheric particles. The ability to kill engulfed microbes (bactericidal capacity) was estimated from the decrease in bacterial colony-forming units (CFU). Samples of cultured cells were taken for DNA analysis at given intervals. DNA ladder assay was used for qualitative apoptotic DNA break detection and the TUNEL AP test was employed for quantification of apoptosis. In 18-week-old animals, spontaneous DNA degradation was observed in the control group without phagocytosis after 8 h. In contrast, cells cultivated with microspheric particles or killed bacteria became apoptotic after 4 h. The rate of apoptotic DNA degradation was decreased in the group exposed to living bacteria. This prolonged survival of phagocytes was also detected in 12-week-old animals, but not at 6 weeks of age. These findings were supported by the ability of phagocytes in 6-week-old animals to engulf microbes, but their killing (bactericidal) ability was significantly decreased in comparison with other stages of immune system maturation. These results suggest that the process of phagocytosis itself is accompanied by activation of the apoptotic program in phagocytic cells of the pig immune system, but the presence of phagocyted living bacteria can delay this activation. The prolonged survival of short-lived cells was only observed in later phases of immune system maturation.     

Cellular defense reactions of insect hemocytes in vitro: phagocytosis in a new suspension culture system.

In a newly developed short-term culture system the plasmatocytes of Galleria mellonella, Pieris brassicae, Calliphora erythrocephala, and Periplaneta americana are the most active cell types in the phagocytosis of latex, chick erythrocytes, and certain bacteria. The granular cells of G. mellonella and the spherule cells of P. brassicae also phagocytose these test particles to a limited extent. This culture system is described, together with the appearance of ingested particles in preparations of living cells, and in fixed and stained monolayers. In culture, the hemocytes clump together due to cell instability in vitro and to the presence of uningested particles. This clumping reaction may be similar to nodule formation observed in vivo in these insects.     

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.     

Histology and ultrastructure of the coenenchyme of the octocoral Swiftia exserta,a model organism for innate immunity/graft rejection

The octocoral Swiftia exserta has been utilized extensively in our laboratory to study innate immune reactions in Cnidaria such as wound healing, auto- and allo-graft reactions, and for some classical “foreign body” phagocytosis experiments. All of these reactions occur in the coenenchyme of the animal, the colonial tissue surrounding the axial skeleton in which the polyps are embedded, and do not rely on nematocysts or directly involve the polyps. In order to better understand some of the cellular reactions occurring in the coenenchyme, the present study employed several cytochemical methods (periodic acid–Schiff reaction, Mallory's aniline blue collagen stain, and Gomori's trichrome stain) and correlated the observed structures with electron microscopy (both scanning and transmission). Eight types of cells were apparent in the coenenchyme of S. exserta, exclusive of gastrodermal tissue: (i) epithelial ectoderm cells, (ii) oblong granular cells, (iii) granular amoebocytes, (iv) morula-like cells, (v) mesogleal cells, (vi) sclerocytes, (vii) axial epithelial cells, and (viii) cnidocytes with mostly atrichous isorhiza nematocysts. Several novel organizational features are now apparent from transmission electron micrographs: the ectoderm consists of a single layer of flat epithelial cells, the cell types of the mesoglea extend from beneath the thin ectoderm throughout the mesogleal cell cords, the organization of the solenia gastroderm consists of a single layer of cells, and two nematocyst types have been found. A new interpretation of the cellular architecture of S. exserta, and more broadly, octocoral biology is now possible.     

Cell Death and Acid Phosphatase Activity in the Regenerating Planarian Polycelis tenuis Iijima

A combination of microscopical, cytochemical, and biochemical techniques have been employed to study the changes occurring during the first seven days of blastema formation and regeneration after decapitation in adult Polycelis tenuis worms. Fine structural data reveal evidence of cell fragmentation, selective cell deletion, and phagocytosis at and below the wound surface. Initially, (0–12 h regeneration) cell debris is phagocytosed by intact parenchymal and gastrodermal cells near the cut surface which is later sealed (24 h) by a stretching of marginal epidermal cells. Wound sealing is followed by a migration of newly differentiated rhabdite cells into the epithelium. Morphological evidence of a selective cell autolysis precedes evidence of an accumuluation of lipid and glycogen reserves in the parenchymal and gastrodermal cells and the later (48 h regeneration time) aggregation of undifferentiated mitotically active neoblasts beneath the wound.
Biochemical data reveal an early period of high acid phosphatase (p-nitrophenyl phosphatase and sodium-β-glycerophosphatase) activity 3–12 h after injury, followed by a further intense period of activity at 44–48 h after decapitation. The coincident cytochemical data show an increased level of acid phosphatase activity associated with cell lysis and death in the wound and blastema zone and also with the digestion of phagocytosed cell debris.     

Phagocytic amoebocyte sub populations in the perivisceral coelom of the sea urchin Lytechinus variegatus (Lamarck, 1816)

The echinoderms are deuterostomic animals with a nonspecific immune system similar to that of vertebrates. Among coelomocytes, phagocytic amoebocytes have a key role in the nonspecific immune response in sea urchin, being responsible for microorganisms elimination through phagocytosis and also for humoral secretions of a wide spectrum. Sub-populations of phagocytic amoebocytes (PA) have been previously described and two distinct sub populations in the oral (OR) and aboral (AB) regions of the perivisceral coelom of L.variegatus in the present study were found. In the OR there is a higher number of PA with higher phagocytic capacity after 30 minutes of incubation with yeast and higher percentage of intranuclear iron crystalloids. The germicide capacity under the fluorescence technique did not show any difference. SDS-PAGE analysis showed different protein patterns between coelomocytes of OR and AB. Gravitational force had no effect in PA distribution and no physical barrier was found in the perivisceral coelom. The other coelomocyte (vibratile cells, red spherulocytes and white spherulocytes) populations were not different in OR compared with AB in their distribution. Some aspects of the possible causes of the differences found for PA are discussed in the paper.     

The characterization of granular amoebocytes and their possible roles in the asexual reproduction of the polystyelid ascidian,Polyzoa vesiculiphora

The blood cells in the bud and the zooid of the polystyelid ascidian, Polyzoa vesiculiphora, were examined by means of light and electron microscopy to identify the cells that have been named trophocytes. The large blood cells were abundant in the mesenchymal space of the bud, but not in that of the functional zooid. They contained glycogen particles, lipid droplets, large protein granules and autophagosomes in their cytoplasm and were identified as granular amoebocytes. The majority of these cells were specifically phagocytized by phagocytes during bud development and disappeared. These results indicate that the granular amoebocytes virtually represent trophocytes in Polyzoa and may participate in bud development via nutrient supply to the developing tissues.     

Vertebrate cytokines interleukin 12 and gamma interferon, but not interleukin 10, enhance phagocytosis in the annelid Eisenia hortensis

Phagocytosis assays employing class I [interleukin 12 (IL-12)], and class II [gamma interferon (gIFN) and IL-10] human recombinant cytokines were carried out to determine the biological effects of these molecules on innate immune responses in the earthworm Eisenia hortensis. Coelomocytes from E. hortensis were pre-incubated with the cytokines for 16-20 h in vitro followed by introduction of Escherichia coli expressing green fluorescent protein (E. coli/GFP). The pro-inflammatory cytokines IL-12 and gIFN stimulated statistically significant (p ? 0.05) enhanced phagocytosis of E. coli/GFP by hyaline amoebocytes as determined by flow cytometry; 10 out of 21 earthworms (48%) responded to IL-12, while eight out of 21 (38%) responded to gIFN. In contrast, the anti-inflammatory cytokine IL-10 neither stimulated nor inhibited phagocytosis in nine earthworms tested. These results demonstrate that vertebrate pro-inflammatory cytokines influence invertebrate cellular responses of immune cells causing enhanced phagocytic activity in earthworm coelomocytes.     

Ultrastructural observations on gastrodermal regeneration in the planarian Dugesia japonica (Turbellaria)

The earliest detectable change during regeneration of the gastrodermis in Dugesia japonica was an aggregation of regenerative cells underneath the gastrodermis remaining at the wound margin. The gastrodermal cells in experimental regenerates retained some of their original characters and presented no indication of cell dedifferentiation. The regenerative cells came into contact with the basal surface of gastrodermal cells, forming stratified cell layers. Differentiation of these cells into gastrodermal cells was initiated by the development of synthetic organelles within their cytoplasm. These differentiating cells gave rise to two different types of gastrodermal cells, namely phagocytic cells and sphere cells. In later stages, there was an apparent movement of differentiated gastrodermal cells towards the parenchyma.