Cellular clot formation in a sipunculan worm: entrapment of foreign particles, cell death and identification of a PGRP-related protein

Clotting in animals having open or closed circulatory system comprises humoral and cellular mechanisms. Sipunculans are a small phylum of non-segmented marine worms that do not have a true circulatory system. These worms can form a cellular clot without transforming cell-free coelomic fluid into an insoluble mass. The clot may also contribute to immune response by entrapping foreign particles. We evaluated the formation of a cellular clot ex vivo in the sipunculan Themiste petricola after activation through glass contact and sea water, the ability to entrap magnetic beads and non-pathogen cyanobacteria particles within the clot, and the presence of a peptidoglycan recognition protein S (PGRP-S) antigen in cells forming the clot. Our results showed that the clot was formed by homotypic aggregation of large granular leukocytes (LGLs), a subtype of cells found in the coelomic fluid. Aggregated LGLs served to entrap magnetic beads and non-pathogen cyanobacteria particles, and PGRP-S antigen was detected both in non-activated LGLs and in activated homotypic aggregates through immunofluorescence, Western blot and flow cytometry. Cellular clots were found to be positive to Annexin V-FITC labelling. Complete disintegration of cytoplasm with shedding of microparticles, nuclear isolation and degradation were also observed by light microscopy and flow cytometry. In conclusion, cellular clot formation in Themiste petricola may serve both haemostatic and immune functions entailing rapid activation changes in LGLs, entrapment of foreign particles within a homotypic aggregate, and further cell death.     

The primary structure of myohemerythrin.

The complete amino acid sequence of muscle hemerythrin (myohemerythrin) from the sipunculid Themiste (syn. Dendrostomum) pyroides has been determined by analysis of tryptic, chymotryptic, and cyanogen bromide peptides. The primary structure of myohemerythrin differs substantially from that of coelomic hemerythrins of Phascolopsis (syn. Golfingia) gouldii and Themiste pyroides, the amino acid sequence of the muscle protein being only 46 and 45% homologous with the respective coelomic hemerythrins. The most extensive regions of homology between muscle and coelomic proteins occur near the terminii. These and other shorter regions of homology are interpreted in terms of the essential iron ligand residues of the active center.     

Histopathology of the disease causing mass mortality of sea urchins (Strongylocentrotus droebachiensis) in Nova Scotia

The disease causing mass mortalities of Strongylocentrotus droebachiensis off Nova Scotia, Canada, from 1980 to 1983 is described. Diseased urchins were characterized by loss of preipheral muscle function in tube feet, spines, and mouth. Signs occurred primarily in the body wall and associated tissues (water vascular system, nerves, spine bases) and coelomic fluid. These symptoms were diffuse and included a general infiltration of tissues with amoebocytes. The coelomic fluid often contained reduced numbers of red and white spherule cells, and clotting was incomplete. Progressive breakdown and fragmentation of muscle cells in tube feet and spine bases resulted in destruction of coherent muscle layers and their replacement by numerous spindle-shaped fibrillar muscle remnants. Coelomic lining cells in the tube feet sloughed off into the lumen, but remained in clumps and phagocytosed muscle remnants.     

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.     

Ultrastructural insights into pathogen clearance by autophagy

Autophagy defends cells against proliferation of bacteria such as Salmonella in the cytosol. After escape from a damaged Salmonella‐containing vacuole (SCV) exposing luminal glycans that bind to Galectin‐8, the host cell ubiquitination machinery deposits a dense layer of ubiquitin around the cytosolic bacteria. The nature and spatial distribution of this ubiquitin coat in relation to other autophagy‐related membranes are unknown. Using transmission electron microscopy, we determined the exact localisation of ubiquitin, the ruptured SCV membrane and phagophores around cytosolic Salmonella. Ubiquitin was not predominantly present on the Salmonella surface, but enriched on the fragmented SCV. Cytosolic bacteria without SCVs were less efficiently targeted by phagophores. Single bacteria were contained in single phagophores but multiple bacteria could be within large autophagic vacuoles reaching 30 μm in circumference. These large phagophores followed the contour of the engulfed bacteria, they were frequently in close association with endoplasmic reticulum membranes and, within them, remnants of the SCV were seen associated with each engulfed particle. Our data suggest that the Salmonella SCV has a major role in the formation of autophagic phagophores and highlight evolutionary conserved parallel mechanisms between xenophagy and mitophagy with the fragmented SCV and the damaged outer mitochondrial membrane serving similar functions.     

The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Isolation and characterization of hemolymph clotting factors in Drosophila melanogaster by a pullout method

Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly. Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads. The validity of the assay was demonstrated by characterization of mutants. We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria. We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris. Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins. Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects.     

Capture of Lipopolysaccharide (Endotoxin) by the Blood Clot: A Comparative Study

In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot) and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse) operate to capture lipopolysaccharide (LPS, endotoxin). The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL) test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.     

Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga

Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of coagulin. The coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.     

Defective interleukin-1 production by natural killer cells of patients with cancer

Summary Large granular lymphocytes (LGLs) from patients with malignant disease and from controls were activated by endotoxin or K562 cells, and the supernatants assayed for interleukin-1 (IL-1) activity. Normal LGLs produced significant amounts of IL-1, the activity of which could be neutralized by anti-human IL-1 antiserum. In patients with advanced cancer depressed IL-1 production was observed, which generally correlated with the degree of cytotoxicity produced by the LGLs. Prior treatment of the LGLs with interferon increased production of IL-1 by both control and patient cells. It is suggested that LGLs coming into contact with K562 cells produce IL-1, which is important in the effector-target cell interaction. The decreased cytotoxic activity of LGLs from cancer patients could be related to a defect in IL-1 production, an effect which can be partially corrected by in vitro interferon treatment.Abbreviations IL-1 Interleukin-1 - LGLs large granular lymphocytes - NK cells natural killer cells - IFN interferon - IL-2 interleukin-2 - HCC hepatocellular carcinoma - MN mononuclear cells - LPS lipopolysaccharide Supported in part by grants from the South African Medical Research Council, the National Cancer Association of South Africa, and the South African Chamber of Mines     

PLATELET DISINTEGRATION DURING CLOTTING

Sequential alterations in the ultrastructure of blood platelets observed during the clotting of human platelet-rich plasma are described, with emphasis on disintegration of the platelet. As the clotting reaction proceeds, aliquots of citrated and recalcified citrated plasma are fixed by adding buffered OsO₄. After recalcification a lag period of about 10 minutes is followed by an interval of rapidly occurring changes which include reorientation of cytoplasmic contents, progressive central degeneration, and disruption of the platelet limiting membrane. Shortly thereafter vesicular platelet remains are seen at the peripheries of loosely arranged and widely spaced masses of granular material. As clot retraction proceeds, these masses gradually come closer together, become more and more compact, and finally disappear. At the same time the vesicles undergo progressive disintegration until at the end of the experiment, 2 hours after recalcification, only a few are found randomly distributed in a dense clot. The significance of progressive disintegration and the origin of the vesicles observed in the later stages of the experiment are discussed in relation to clot formation and clot retraction.     

Insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade

Here we describe a novel approach to isolate proteins involved in insect hemolymph coagulation. In order to avoid problems in purifying clot proteins after they had been crosslinked, we performed an in vitro coagulation reaction with cell-free hemolymph from the lepidopteran Galleria mellonella and used the resulting complexes to produce a specific antiserum. The antiserum reacted with a subset of hemolymph proteins as well as with granular cells, but not with other hemocyte types of Galleria. Screening expression libraries identified some positive clones, which turned out to code for some previously characterized components of immune cascades, as well as some novel candidates for clotting factors. Known components include members of both the coagulation system and the prophenol-activating cascade, lending support to the idea that both systems work together during the formation of a hemolymph clot. Novel candidates for insect clotting factors include a mucin-like protein, a glutathione-S-transferase, and a distant member of the alpha-crystallin/small heat shock protein family. Using assays measuring the activity of transglutaminase, a key enzyme in clotting reactions in both vertebrates and invertebrates, we found a partial overlap between transglutaminase substrates and proteins recognized by the antiserum against the in vitro-induced clot.     

Ultrastructure of the tentacles of Themiste lageniformis (Sipuncula)

Summary An ultrastructural study of the tentacles of Themiste lageniformis (Sipuncula) was conducted as part of a larger study of head metamorphosis in the species.The oral surface of the tentacles is constructed of a multiciliated, pseudostratified, columnar epithelium while the aboral surface is an unciliated, cuboidal epithelium. Intraepidermal mucous cells lie near the junction of the oral and aboral regions. The basal portion of the epidermal cells is embedded in a thick, collagenous extracellular matrix which contains outer circular muscles, inner longitudinal muscles, the main tentacular nerve and its branches. Three tentacular canals are present and are lined by peritoneum. Hemerythrocytes and coelomocytes flow through the lumen of the canals in a regular pattern.Justification for the designation of the tentacular canals as coelomic rather than vascular is discussed.     

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.     

The effect of RU 41.740 (Biostim) on the production of interleukin-1 by monocytes and enriched large granular lymphocytes in normals and patients with hepatocellular carcinoma

Summary The effect of RU 41.740 on natural killer (NK) cell cytotoxicity from normals and patients with advanced hepatocellular carcinoma (HCC) was studied. RU 41.740-treated normal enriched large granular lymphocytes (LGLs) showed increased cytotoxic activity against ⁵¹Cr-labelled K562 cells. Although the drug increased the cytotoxic activity of LGLs from HCC patients, these results were not statistically significant. As LGLs have been shown to release interleukin-1 (IL-1) after suitable stimulation the effects of RU 41.740 on production of this cytokine from both monocytes and LGLs was studied. The drug stimulated IL-1 release from normal cells but monocyte and LGL IL-1 production from HCC patients was not influenced by RU 41.740 by RU 41.740. It is concluded, therefore, that although RU 41.740 can stimulate natural cytotoxicity and IL-1 production by normal LGLs, it is unable to correct the defect which exists in these functions in patients with large mass tumours.     

The leukocytic organ of the megascolecid earthworm,Amynthas diffringens (annelida,oligochaeta)

Leukocytic organs of Amynthas diffringens are aggregations of leukocytes contained within a smooth muscle and stromal cell framework suspended in the coelom. Elongate processes of stromal cells subdivide each organ into numerous cell-filled compartments and are perforated by 130-nm pores that may permit the exchange of humoral substances between compartments, or between the organ and the surrounding coelomic fluid. We divide leukocytes within the organs into four morphotypes. Phagocytic leukocytes have many lysosomelike vesicles and may possess phagosomes. Mature types I, II, and III granulocytic leukocytes share certain features but are readily distinguished by cell shape and by the size, shape, and electron density of the cytoplasmic inclusions. Immature as well as mature phagocytes and granulocytes occur within these organs, suggesting that they are sites of leukocyte maturation and storage. Concentrations of leukocytes within the organs result in extensive cell to cell contact, especially within islets and tightly packed cords. Phagocytosis of cell debris occurs throughout the organs. Immature stages of the four morphotypes are difficult to distinguish even at high magnification, raising the possibility that they may originate from a common precursor. Our inability to observe mitoses or to detect lymphocytelike stem cells suggests that immature leukocytes migrate to the organs via coelomic fluid from as yet unidentified primary sites of production.     

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2–activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2–activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.     

Neisseria gonorrhoeae survives within and modulates apoptosis and inflammatory cytokine production of human macrophages

The human‐adapted organism Neisseria gonorrhoeae is the causative agent of gonorrhoea, a sexually transmitted infection. It readily colonizes the genital, rectal and nasalpharyngeal mucosa during infection. While it is well established that N. gonorrhoeae recruits and modulates the functions of polymorphonuclear leukocytes during infection, how N. gonorrhoeae interacts with macrophages present in infected tissue is not fully defined. We studied the interactions of N. gonorrhoeae with two human monocytic cell lines, THP‐1 and U937, and primary monocytes, all differentiated into macrophages. Most engulfed bacteria were killed in the phagolysosome, but a subset of bacteria was able to survive and replicate inside the macrophages suggesting that those cells may be an unexplored cellular reservoir for N. gonorrhoeae during infection. N. gonorrhoeae was able to modulate macrophage apoptosis: N. gonorrhoeae induced apoptosis in THP‐1 cells whereas it inhibited induced apoptosis in U937 cells and primary human macrophages. Furthermore, N. gonorrhoeae induced expression of inflammatory cytokines in macrophages, suggesting a role for macrophages in recruiting polymorphonuclear leukocytes to the site of infection. These results indicate macrophages may serve as a significant replicative niche for N. gonorrhoeae and play an important role in gonorrheal pathogenesis.     

Ultrastructure of the Coelomocytes in the Tentacular Coelom of Thysanocardia nigra Ikeda, 1904 (Sipuncula)

Free-floating coelomocytes in the tentacular coelomic cavity of the sipunculan Thysanocardia nigra Ikeda, 1904, were studied using light interference contrast microscopy and scanning and transmission electron microscopy. The following coelomocyte types were distinguished: hemerythrocytes, amoebocytes, and two morphological types of granular cells. No clusters of specialized cells that had been reported to occur in the trunk coelom of Th. nigra were found in the tentacular coelom. The corresponding types of coelomocytes from the tentacular and trunk coelomic cavities were shown to differ in size. These two coeloms are completely separated in sipunculans.     

Ultrastructural observations on spermiogenesis in the peanut worm,Themiste pyroides (Chamberlin, 1920) (Sipuncula; Sipunculidea)

Summary

The process of spermiogenesis and the ultrastructure of the spermatozoa in the peanut worm, Themiste pyroides, from the Sea of Japan were observed with electron microscopy (SEM and TEM). The testes are composed of groups of spermatogonia and are covered by peritoneal cells. Clusters of spermatocytes are released from the testes into the coelomic fluid. Connected by intercellular bridges, the spermatocytes within a given cluster develop asynchronously. Proacrosomal vesicles and a flagellum appear in spermatocytes. Spermatids in the clusters retain the intercellular connections. During spermiogenesis, the acrosomal vesicle, formed by coalescence of small proacrosomal vesicles in the basal part of the spermatid, migrates to the apical part of the cell to form a conical-shaped acrosome. The basal concavity lying above the nucleus is filled with subacrosomal substance. The midpiece contains four mitochondria, two centrioles, and some residual cytoplasm with dark glycogen-like granules. A peculiar annulus structure develops around the base of the flagellum. The distal centriole has a pericentriolar complex consisting of radially oriented elements. Before the spawning process, the spermatozoa are filtered throughout the ciliary nephrostomal funnel into the excretory sac of paired nephridia where they are stored for a short time. The sperm are released into the sea water via nephridiopores. Spermatozoa remaining in the coelomic fluid after spawning are resorbed by amoebocytes. This species from Vostok Bay is characterized by a prolonged spawning period from June to early October. The reproductive strategy of T. pyroides is discussed in comparison with that of Thysanocardia nigra, the latter having a unique pattern of packaging of the spermatozoa, resulting in the formation of spermatozeugmata, as a reproductive adaptation to the very short spawning period.