Biochemical characteristics of Eiseniapore, a pore-forming protein in the coelomic fluid of earthworms.

The cytolytic protein Eiseniapore (38 kDa) from coelomic fluid of the earthworm Eisenia fetida functionally requires sphingomyelin as revealed by using mammalian erythrocytes and phospholipid vesicles. The effects of ions, glycoproteins and phospholipids were investigated for the two-step Eiseniapore action mode, binding and pore formation in different assays. Eiseniapore lysis is activated by thiol groups but inhibited by metal ions. Eiseniapore binding to target membranes is inhibited by Eiseniapore-regulating factor, vitronectin, heparin and lysophosphatidylcholine. Ca2+ and Mg2+ were found to be not necessary for membrane binding or lytic activity. Sphingomyelin was essential for Eiseniapore-induced leakage of liposomes. We describe a cytolytic protein/toxin in Eiseniapore which differs from the established classification; it can be activated by thiol groups and is inhibited by sphingomyelin. Electron microscopy of erythrocyte membranes confirmed ring-shaped structures (pores) with a central channel with outer (10 nm) and inner (3 nm) diameters as shown previously [Lange, S., Nüssler, F., Kauschke, E., Lutsch, G., Cooper, E.L. & Herrmann, A. (1997) J. Biol. Chem. 272, 20 884-20 892] using artificial membranes. Functional evidence of pore formation by Eiseniapore was revealed as protection of lysis by carbohydrates occurred at an effective diameter above 3 nm. From these results, we suggest a plausible explanation for the mechanism by which components of the earthworm's immune system destroy non-self components.

Isolation and characterization of earthworm hemolysins and agglutinins.

Hemolytic activity in coelomic fluid of Eisenia fetida (ECF) is due to three proteins H1, H2, H3 with molecular weights of 46, 43 and 40 kD, respectively. These proteins were isolated by preparative PAGE. H1 and H2 were shown to be stable in SDS and alpha-2-ME whereas H3 splits into two fragments with molecular weights of 18 and 21 kD after SDS treatment. IEF indicates that each protein consists of different isoforms with pIs between 5.1 and 6.2 H3 was demonstrated to be a bifunctional protein that can lyse and agglutinate erythrocytes. At 56 degrees C hemolytic activity of all three proteins was inactivated, but the agglutination activity of H3 was stable. Intracoelomic injection of erythrocytes reduced the number of hemolysins from three to two. Monospecific antisera were raised against the isolated hemolysins H1,2 and 3. The use of these antibodies and of carbohydrates as inhibitors of the biological activity of the molecules demonstrates the close structural relationship of agglutinins and hemolysins in the CF of E. fetida.

Crossreactivity of hemolytic and hemagglutinating proteins in the coelomic fluid of lumbricidae (Annelida).

Oligospecific antisera against the hemolytic and hemagglutinating compounds of the coelomic fluid (CF) of the earthworm species Eisenia fetida, Lumbricus terrestris and Aporrectodea caliginosa were prepared. The proteins were bound to the membranes of erythrocytes and injected into rabbits. By the use of these antisera the following results were obtained: 1) The antisera RaL T and RaAC reached a titer of 1:64,000, the detection limit of RaEF was 1:512,000. RaEF was demonstrated to be oligospecific only against three hemolytic proteins by Western blotting. 2) RaEF is able to inhibit the biological activity not only of hemolysins (E. fetida, A. caliginosa) but also of hemagglutinins (L. terrestris, L. rubellus, D. rubidus). 3) Complex carbohydrates, but not simple sugar compounds, are able to inhibit hemolytic and agglutinating activities in the CF of the investigated species. Fet. and alpha 1ac. GP were demonstrated to be strong inhibitors both of the hemolytic and of the hemagglutinating activity, whereas N-acetylglucosamine was only able to inhibit the hemagglutinating activity. 4) The investigated compounds were shown to crossreact in ELISA experiments. 5) By the use of Western blotting, the crossreacting molecules in the CF of the investigated lumbricid species were identified.

Comparative studies on the adhesiveness of granulocytes of guinea pig and man.

The high affinity of granulocytes of guinea pig and man to glass surfaces is modified by serum. Native serum contains both an adherence-promoting activity, which is related to complement, and components which reduce the adhesiveness of granulocytes. These components are stable at 56 degrees C for 30 min and are tightly bound to the glass surface. beta-Lipoproteins are candidates for this adherence reducing ability of serum. Adherence promotion by native serum is mediated by coating the glass surface with C3b/C3bi. Human granulocytes from the peripheral blood adhered pig serum with C3b/C3bi to almost the same extent as in the presence of native serum, but on guinea pig granulocytes elicited in the peritoneal cavity, a cell surface metalloproteinase degraded the C3b/C3bi, thus reducing the adhesiveness of these cells. This proteinase was inhibited by MgEDTA, DTT, and 1,10-phenanthroline, whereby the high adhesiveness of granulocytes was restored to C3b/C3bi-coated glass.

Cytotoxic activity in the coelomic fluid of the annelid Eisenia foetida Sav.

The annelid Eisenia foetida not only causes hemolysis of red blood cells of several vertebrate species, but also has a toxic effect on a variety of cell types, such as chicken fibroblasts, guinea-pig polymorphonuclear leukocytes and insect hemocytes. However, it has no influence on the vitality of the coelomocytes of Lumbricus terrestris and other lumbricides, nor on the hemocytes of the snail Helix pomatia, the mussels Anodonta cygnea and Unio tumidus, free cells of the turbellarian Euplanaria sp. or whole Rhabditis oxycerca (nematode) and the protozoons Paramaecium caudatum and an amoeba of the Proteus-type. By electrofocussing the hemolytic activity of pooled coelomic fluid was separated into 7 hemolytic bands. Three of them are cytotoxic. The cytotoxic effect is a result of the destruction of the cell membrane, as shown by measuring the release of lactate dehydrogenase (LDH). The bacteriostatic and bacteriocidal action of the coelomic fluid of E. foetida against a broad spectrum of gram positive and gram negative bacteria was tested. An antibacterial activity could be observed only against Proteus vulgaris and Bacillus megaterium. It was noted that the so-called Eisenia foetida-factor acts on an antigenic structure at the cell surface when anti-sheep-E-antibody was used under competitive conditions. The binding between the Eisenia foetida-factor and this membrane structure is relatively strong as it cannot be removed by subsequent treatment with anti-sheep-E-antibody or 2 M KCl.

Comparative analysis of hemolytic and hemagglutinating activities in the coelomic fluid of Eisenia foetida and Lumbricus terrestris (Annelida, Lumbricidae).

The CF of Lumbricus terrestris and Eisenia foetida was analyzed with regard to the protein patterns by isoelectric focusing. The CF of E. foetida results in a variety of protein bands; after separation: seven of these with isoelectric points at pH 5.1, 5.2, 5.4, 5.6, 5.8, 6.0 and 6.2 cause the hemolysis of red blood cells. The protein pattern of normal CF of L. terrestris is relatively poor. Most of the protein bands are located at the top of the separation (pH less than 5.1) near the anode. However, the number of protein bands is drastically enhanced within 24 hours after an intracoelomic injection of foreign material. Some of the induced proteins show a similar pattern at comparable pH ranges to the CF of E. foetida. The induced proteins with isoelectric points at pH 4.7, 5.0, 5.1, 5.2, 5.4, 5.8, 6.0 and 6.2 effect the hemolysis of vertebrate erythrocytes. After stimulation with rabbit erythrocytes an enhanced bactericidal and bacteriostatic activity against Proteus vulgaris can be proven and seems to be related to the induced hemolytic proteins. The antibacterial activity can be completely adsorbed by incubation of the CF with rabbit erythrocytes, which indicates its binding ability to marker molecules on the erythrocytes surface.

Further studies on the application of the micromethod of phagocytosis using cadmium microcrystals as phagocytosis particles.

The use of Cd microcrystals together with a monolayer technique using small quantities of whole blood offers several advantages for routine studies of phagocytosis. The original method by Miler was especially minimized with regard to the necessary quantity of serum. Homologous serum was replaced by heterologous guinea-pig serum and the method was simplified by the omission of opsonization. A comparison of the stimulatory effect of inactivated and native serum as well as the inhibition of phagocytosis by trypan blue allows us to characterize Cd-phagocytosis as complement-dependent.

Bilirubin-induced cytomorphological changes in guinea-pig leucocytes.

The in vitro influence of bilirubin on guinea-pig peritoneal exudate cells was investigated at concentrations comparable to the conditions of neonatal hyperbilirubinaemia. In in vitro cultures of granulocytes and macrophages bilirubin manifested itself macroscopically by yellow colouring and microscopically by bilirubin aggregation near the nucleus. The intracellular bilirubin content was determined to be 40 nmol per 3 . 10(7) cells. The deposit of bilirubin was irreversible even after subsequent incubation with albumin. Subsequent incubation in heat-inactivated serum reverses binding of bilirubin to cells. Moreover, adding serum to the bilirubin sample prevents the influx of bilirubin. Simultaneously with the deposition of bilirubin there were conspicuous cytomorphological changes in the granulocytes and macrophages observed. The changes consist in a large number of rigid plasma extensions on the whole cell surface. The morphological changes were also reversible by subsequent incubation with serum and their formation was prevented by addition of serum. Since albumin as a well known bilirubin binding protein could not prevent the deposition of bilirubin or the morphological changes, it is suggested that serum factors can regulate the transport of bilirubin across cell membranes.

Phagocytosis-stimulating mediators in insects.

The ability of insect blood cells to ingest all kinds of synthetic particles and also a wide range of microorganisms in a very short time after injection has up to now been regarded as a phagocytic function without any humoral mediators. In a phagocytosis model with latex beads and nonhagocytosable cells of Bacillus thuringiensis subtoxicus, we are able to demonstrate the existence of lymphokine-like factors, which intervene in cellular defence reactions of insect. The following results were obtained: 1. Immediately after injection of latex beads, normally non-phagocytosable cells of Bacillus thuringiensis subtoxicus are phagocytosed. 2. Cell-free haemolymph of larvae of Galleria mellonella previously injected with latex beads, stimulates in new larvae phagocytosis of Bacillus thuringiensis subtoxicus after transfusion. 3. The fractionation of homogenates of latex-treated larvae on Sephadex G 50 shows two fractions which stimulate phagocytosis. We suppose that the appearance of these phagocytosis-stimulating factors is the result of a successful recognition of foreign material.