Pierre Valembois

Invertebrate Immunity: Another Viewpoint

All vertebrates and invertebrates manifest self/non‐self recognition. Any attempt to answer the question of adaptive significance of recognition must take into account the universality of receptor‐mediated responses. These may lake two forms: (1) rearranging, clonally distributed antigen‐specific receptors that distinguish in the broadest sense between self and non‐self, and non‐self A from non‐self B, latecomers on the evolutionary scene; (2) pattern recognition receptors, the earliest to evolve and still around, necessitating the requirement for induced second signals in T‐ and B‐cell activation. Either strategy need not force upon invertebrates the organization, structure and adaptive functions of vertebrate immune systems. Thus, we can freely delve into the unique aspects of the primitive immune mechanisms of invertebrates. In contrast, using the opposite strategy which is still problematic, i.e. linking invertebrate and vertebrate defence, seems to give us an approach to universality that might eventually reveal homologous kinship.

Antibacterial activity of the hemolytic system from the earthworm Eisenia fetida andrei

Abstract The coelomic fluid and the cocoon albumen of the earthworm Eisenia fetida andrei are demonstrated to possess an antibacterial activity. The antibacterial activity and the already known hemolytic activity are due, in fact, to the same lipoproteic molecules. The antibacterial activity (bacteriostatic effect) is only directed against the highly pathogenic soil bacteria. Only these pathogenic bacteria strains possess at least one common antigen with the sheep red blood cells.

Purification, characterization and activities of two hemolytic and antibacterial proteins from coelomic fluid of the annelid Eisenia fetida andrei

The coelomic fluid of the earthworm Eisenia fetida andrei exhibits antibacterial, hemolytic and hemagglutinating activities. These activities are mainly mediated by two proteins, named fetidins, of apparent molecular mass 40 kDa and 45 kDa, respectively. For the first time, the two proteins have been purified to homogeneity from dialysed coelomic fluid by means of anion-exchange chromatography. Three peaks had hemolytic activity. The first fraction was found to correspond to the 40 kDa fetidin, the second to mixed 40 and 45 kDa fetidins, the last one to the 45 kDa fetidin. Both purified proteins still exhibited their hemolytic and antibacterial activities as dialysed coelomic fluid did. In this study, the amino-acid sequence of purified proteins is compared to the amino-acid sequence predicted by cDNA. This cDNA was isolated by screening an expression cDNA library from earthworm total tissues (unpublished data).

Protein analysis of earthworm coelomic fluid—II. Isolation and biochemical characterization of the Eisenia fetida andrei factor (EFAF)

Abstract The hemolytic/bacteriostatic factor naturally present in the fluid of all the Eisenia fetida andrei is: 1. 1. Constituted by 2 different molecules with apparent mol. wt of 40,000 and 45,000, 2. 2. Of proteinic composition including an associated glycolipid, 3. 3. Floculable by 55°C heating, 4. 4. Isolable by preparative gel filtration. The EFAF hemolytic activity is mediated by the binding of the factor molecules on the erythrocyte membrane. This binding, which does not concern the Forssman antigens, is inhibited by the N- acetyl- d -glucosamine and the α-methyl- d -mannopyranoside (α-MM).

Formation of brown bodies in the coelomic cavity of the earthworm Eisenia fetida andrei and attendant changes in shape and adhesive capacity of constitutive cells

The formation of brown bodies in the coelomic cavity may result from an aggregation of coelomocytes around offending foreign cells such as bacteria, gregarines, incompatible graft fragments, and altered self structures such as setae or necrotic muscle cells. The initial nodule rapidly increases in volume by aggregation of new coelomocytes and various waste particles. When a brown body has reached a diameter of 1-2 mm, its external cells flatten and lose their adhesiveness toward free coelomocytes or waste particles and its pigment rapidly darkens. Brown bodies play an important role in homeostasis.

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.

Natural cytotoxic activity of sipunculid leukocytes on allogenic and xenogenic erythrocytes.

Abstract A natural cytotoxic reaction, analogous to that known in rodents, is described in sipunculid worms (coelomata invertebrates). A cytotoxic effect always occured when killer leukocytes reacted on xenogenic erythrocytes. A cytotoxic activity on allogenic erythrocytes occured in all cases when donors of leukocytes and donors of erythrocytes arose from remote stations. Between stations near each other (6 km apart), a cytotoxic effect was noticed in only one case out of three. No allogenic reaction was noticed between Sipunculus nudus from the same station but it occured in one case out of two between Siphonosoma arcassonense from the same station. Cytotoxicity could be specifically reduced following several injections of target cells in vivo . The specificity of lysis suggests that prior contact with antigen induces a tolerance in sipunculid worms.

Evidence of lipofuscin and melanin in the brown body of the earthworm Eisenia fetida andrei

By means of histochemical and electron-microscopic techniques, two pigments were identified in the brown body of the earthworm Eisenia fetida andrei. Lipofuscin, often called the aging pigment, was the more abundant pigment and the first to be synthesized. Melanin was also found but was only present in the mature brown body. A computer-assisted image analysis was used to quantify the relative abundance of the two pigments at different stages of the formation of the brown body. A previous study showing phenoloxidase activity in the coelomocytes and the present demonstration of the ability of these cells to produce reactive oxygen systems when agglutinated can be correlated with the simultaneous presence of the two pigments in the brown body. The physiological significance of pigment synthesis by brown bodies is discussed with respect to the immune response in earthworms.

Evidence for concanavalin a-receptors and their redistribution on lumbricid leukocytes

Abstract Fluorescent and radioactive concanavalin A (Con A) were used to demonstrate the existence of Con A-receptors on earthworm leukocyte membranes. Inhibition of binding by methyl-α- d -mannose, methyl-α- d - glucose and d -fructose suggested that Con A can bind to specific sites. Differences of labelling between two types of leukocytes support the existence of physiological heterogeneity among the leukocytic population. Native distribution of Con A-receptors was uniform on the whole cell membrane. After 30 minutes at room temperature, patches of Con A-binding site complexes were formed. Capping occurred in 30 % of the small-spheric-cells which represented 20 % of the non-adherent leukocytes, after 1 hour at room temperature. The con A molecules were then internalized by endocytosis of the cap membrane.

Antibacterial activity of Eisenia fetida andrei coelomic fluid: III--Relationship within the polymorphic hemolysins.

The antibacterial activity exhibited by 10 different hemolytic, genetic families was established by measuring the inhibition of spontaneous in vitro growth by cell-free coelomic fluid toward 2 bacteria which are pathogenic for the earthworm: Bacillus megaterium (Gram +) and Aeromonas hydrophila (Gram -). Only two families (B and K) displayed potent inhibitory activities. This finding is consistent with the fact that the B family occurs most frequently in both natural as well as in industrial breedings. Nevertheless, evidence of a poor antibacterial defense in some frequent families suggests the existence of alternative antibacterial mechanisms.