Erythrocyte membrane structural features that are critical for the lytic reaction of Spirograhis spallanzani coelomic fluid hemolysin.

1. Hemolytic activity of Spirographis spallanzani coelomic fluid depends on factor(s) strongly influenced by calcium but not by sulfhydril or disulfide reagents. 2. The lytic reaction was suppressed by low zinc ion concentrations but it was not influenced by the presence of proteinase inhibitors. 3. These data indicate that S. spallanzani hemolysin is a non-enzymatic, calcium-dependent, zinc-inhibitable factor that occurs naturally in the coelomic fluid. 4. In the absence of calcium, enzymatic desialization converted sheep erythrocytes into susceptible targets, suggesting the involvement of erythrocyte surface sialic acid. 5. However, the inhibitory effect of the sugar on anti-rabbit lysis was partially removed by addition of calcium. 6. Attempts to characterize membrane components that are critical for hemolysis were performed by inhibition experiments. 7. We found that saccharides, glycoproteins, mucosubstances as well as rabbit erythrocyte soluble tryptic fragments were ineffective in inhibiting hemolysis. 8. Sonicated dispersion of phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanol, sphingomyelin and cholesterol did not influence the hemolytic reaction. 9. Rabbit erythrocyte extracted from membrane lipids (chloroform phase) did not modify the lytic activity against rabbit red blood cells. 10. Conversely, the methanol phase consistently reduced the lytic capacity of the fluid. 11. The heat-stable, trypsin-resistant inhibitory factor was most probably a small molecule, since dialysis removed the inhibitory effect.

Isolation of cytolytic granules from sea urchin amoebocytes.

Calcium-dependent, heat-labile lytic activity was detected in whole Paracentrotus lividus coelomocytes. The molecules responsible for this activity were mainly expressed by an enriched amoebocyte population and were contained in cytoplasmic granules which could be isolated by Percoll density gradient centrifugation. The isolated cytoplasmic granules were small (0.1-0.25 micron) organelles containing the lysosomal marker arylsulfatase. The functional implication of our results is that P. lividus amoebocytes represent cytotoxic cells which mediate the killing event by a secretory phenomenon involving the release of cytolytic material from cytoplasmic granules.

Sea urchin coelomic fluid agglutinin mediates coelomocyte adhesion.

The sea urchin Paracentrotus lividus coelomic fluid was found to contain agglutinin which agglutinates animal erythrocytes and promotes adhesion of autologous coelomocytes. Hemagglutinating activity depended upon the presence of calcium ions and was relatively heat-stable. Through a combination of methods including ammonium sulfate precipitation and both size exclusion and ion exchange chromatographies, we purified the anti-rabbit agglutinating factor. The intact agglutinin migrates as a single band with an apparent M(r) of over 200,000. Three distinct protein bands with a calculated M(r) of 174,000, 137,000, and 76,000, respectively were observed under reducing conditions. The purified agglutinin strongly promoted the in vitro adhesion of autologous coelomocytes.

A 220 kDa coelomocyte aggregating factor involved in Holothuria polii cellular clotting.

Agglutinating molecules are released by Holothuria polii coelomocytes. In our in vitro system we observe that release depends on the number of coelomocytes but it seems not to be time- and temperature-dependent. The factor responsible for agglutination was isolated from an Edds isotonic solution coelomocyte suspension medium and purified by ammonium sulfate precipitation, gel filtration and ion exchange chromatography; it had a molecular mass of about 220 kDa on an sodium dodecyl sulfate polyacrylamide gel. The purified factor agglutinates sea cucumber coelomocytes suggesting that it could be involved in the first phase of clotting events.

Binding properties of Paracentrotus lividus (Echinoidea) hemolysin.

1. Paracentrotus lividus hemolysin binds erythrocytes, zymosan particles, lipopolysaccharide and laminarin surfaces but not auto and allogeneic cell membranes. 2. The binding could, at least for erythrocytes, involve phospholipids and cholesterol. 3. The protease activity of the coelomic fluid is not related to hemolysis. 4. The finding that very low concentrations of Zn2+ inactivate the hemolysin suggests a possible regulative function of the ion in the hemolytic reaction. 5. Ultrastructural observations on rabbit erythrocyte membranes indicate that most likely the transmembrane pores are induced by the lytic molecules.

Hemolysins: pore-forming proteins in invertebrates.

Invertebrates possess lytic molecules which lyse vertebrate erythrocytes. In all the species studied so far, hemolytic activity depends on proteins which possess a wide range of reactivity. It is generally calcium-dependent and heat-labile, although calcium-independent and heat-stable hemolysins have also been detected. The molecules interact with sugars or lipids which could represent the membrane receptors by which circular lesions on target membranes are produced. On the basis of some analogies with vertebrate lytic molecules it is conceivable that the hemolysins evolved from a common ancestral gene which also led to vertebrate pore-forming proteins.

B-lymphocyte populations in Xenopus laevis.

Two-color immunofluorescence technique was used to show the development and distribution of surface mu- cytoplasmic mu+ (s mu- c mu+) pre-B, s mu+ B- and s mu+ cIg+ plasma cells in metamorphic, postmetamorphic, and adult Xenopus. Generation of pre-B cells was evident in hematopoietic liver and spleen, but not in bone marrow, thymus, and duodenal mucosa. Surface immunoglobulin positive small lymphocytes were the most abundant in the spleen while plasma cells were detected in the thymus, duodenal mucosa, spleen, and liver. We had shown previously the appearance of s mu- c mu+ pre-B cells in the liver of Xenopus larvae at developmental stage 46 and later at stage 49 in the spleen. The frequency of pre-B cells dropped to zero at stage 58, the climax of metamorphosis. Pre-B cells start to reappear slowly as a second wave, at stage 60 through early postmetamorphic life in the liver and spleen. The percentage of surface Ig+ (sIg+) cells in the spleen of developing animals from stage 60 onward is comparable to that observed in adult life. In adult animals, the periphery of the liver continues to be active in hematopoiesis and contains some IgM producing plasma cells and rare sIg+ small lymphocytes while the pre-B cells are almost nonexistent in this region. The spleen, which is also active in some hematopoiesis, constitutes the main site of B-cell differentiation. Three ontogenic stages of pre-B, B-, and plasma cells are present in this organ. Pre-B and plasma cells are of low density and heterogeneous in size while small sIg+ B lymphocytes are of high density and much more homogeneous in size. The bone marrow in these lower anuran amphibia is rudimentary and is not a lymphopoietic tissue; in adult animals it is active only in differentiation of neutrophilic granulocytes.

The coelomocytes of Holothuria polii (Echinodermata). II. Cytochemical staining properties.

Histochemical studies by traditional staining methods for lipids, mucopolysaccharides and proteins and histoenzymatic reactions typical of lysosomal activity were carried out on the principal coelomocyte types of Holothuria polii. These are considered to be the cellular contigent of the immune response, and in Holothuria polii, they probably take part also in clotting events.

Engagement of Polian vesicles during Holothuria polii response to erythrocyte injection.

The Holothuria polii Polian vesicles are organs of inflammatory responsiveness. Under antigenic provocation, they respond with remarkable structural modifications. They are also able to produce brown bodies that represent a survival strategy, used by the holothuroid, to rid the body of non-self material presented to it.

Interactions between earthworm hemolysins and sheep red blood cell membranes.

The hemolytic activity exhibited by the coelomic fluid of the Annelid Eisenia fetida andrei is mediated by two lipoproteins of mass 40 and 45 kDa, each of them capable of hemolysis. Such an activity is not inhibited by zymosan, inulin or lipopolysaccharide (LPS), nor by hydrazine or methylamine, suggesting that earthworm hemolysins are not related to C3 or C3b complement components. Among the membrane lipids tested (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin and cholesterol) only sphingomyelin inhibited hemolysis. The analysis of E.f. andrei proteins bound to sphingomyelin microvesicles, as well as to sheep red blood cell (SRBC) membranes, revealed a polymerization of E.f. andrei 40 kDa and/or 45 kDa hemolysins. Consequently, sphingomyelin appears a likely candidate for hemolytic complex receptor. Electron microscopy observations suggested that the polymerization causes an open channel through the lipid bilayer. As demonstrated using metal ions, heparin, chondroitin sulfate, poly(L-lysine) and protamine chloride, the mode of action of earthworm hemolytic complex is not analogous to that of C9 or perforine.

Arylsulphatase in echinoderm immunocompetent cells.

Two peaks of arylsulphatase activity were detected biochemically in coelomocyte lysate preparations of seven different Echinodermata species. Both peaks were inactivated by sulphite and sulphate ions, indicating that Type II arylsulphatase is present in the coelomocytes of the species tested. Arylsulphatase was localized histochemically in the granules of spherula cells, suggesting that in echinoderms a common cell type with granulocyte-like functions is present. The enzyme was also localized in the amoebocytes of echinoid species.

Arylsulfatase in coelomocytes of Holothuria polii.

Holothuria polii coelomocytes possess arylsulfatase enzymes. Two pH optima were found for arylsulfatase activity in cell lysate preparations, one at pH 5.0 and the other at pH 5.8. Both increased after injection of zymosan particles or formalinized sheep red blood cells (fSR-BC), indicating an active role of the enzymes during phagocytosis of particulate substances. Under a light microscope, the acid hydrolase arylsulfatase were localized in the granules of spherula cells, and therefore considered lysosomal in nature.

The hemolysin-producer coelomocytes in Holothuria polii.

Using sodium metrizoate discontinuous gradients, two hemolysin-producer amebocyte populations have been separated from total circulating Holothuria polii coelomocytes. The amebocytes of population 1 are responsible for the production of the calcium-dependent and temperature-labile hemolysin, whereas those of population 2 produce the calcium-independent and temperature-stable one. The intracytoplasmic hemolysins were evidenced also by immunofluorescence. Petaloid and filipodial amebocytes were the only positive cell types.

Studies on Holothuria polii (Echinodermata) coelomocyte lysate. II. Isolation of coelomocyte hemolysins.

The lytic activity of the Holothuria polii coelomocyte lysate resides in two electrophoretically distinct hemolysins identified as He1 and He2. He1 represents the calcium dependent, heat-labile component whereas He2 is calcium independent and heat-stable. The two hemolysins share serological identity. Both hemolysins appear as single protein molecules of 80KDa molecular weight by SDS-PAGE and transblotting analysis under non-reducing conditions. However under reducing conditions, they are doublets of 76 and 80KDa molecular weight. The hypothesis that the two hemolysins could be isoforms is discussed.

Membrane damage by coelomic fluid from Holothuria polii (Echinodermata).

Rabbit erythrocyte membranes lyzed by Holothuria polii coelomic fluid, observed under the electron microscope, present lesions consisting of irregular holes which are heterogeneous in size (ranging from 50 A to 250 A) and ultrastructurally different from the ring-like structure produced by human complement. The protein pattern associated with the lyzed membrane was also examined.

The membrane attack complex of Xenopus laevis complement.

Rabbit erythrocyte membranes lyzed by Xenopus laevis serum exhibited a typical ultrastructural complement lesion with an inner diameter of 80 +/- 9 A. The protein pattern associated with lyzed membrane is compared to a similar human preparation.

Inhibitory activity of sphingomyelin on hemolytic activity of coelomic fluid of Holothuria polii (Echinodermata).

The hemolytic activity of coelomic fluid from Holothuria polii is specifically inhibited by sphingomyelin. This phospholipid is the constituent of the membrane which probably interacts with the hemolysin thereby leading to the lysis.

Studies on Holothuria polii (Echinodermata) coelomocyte lysate. I. Hemolytic activity of coelomocyte hemolysins.

The Holothuria polii coelomocyte lysate contains two trypsin-resistant lytic proteins having different chemico-physical properties: a calcium dependent and heat-labile hemolysin that is probably a constitutive component of the coelomic fluid, and another calcium independent and heat-stable one that is released after immunological stimulation; it is therefore not detectable in natural conditions. The sphingomyelin seems to be the membrane receptor with which both hemolysins interact producing lysis.