Antimicrobial defense of the earthworm

REVIEW

Folia Microbiol. 45 (4), 283-300 (2000) http ://www. biomed, cas. cz/mbu/folia/

Antimicrobial Defense of the Earthworm

M. BILEJ a, P. DE BAETSELIER b, A. BESCHIN b

aDepartment of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czechia

bDepartment of Immunology, Parasitology, and Ultrastructure, Flemish lnteruniversity Institute for Biotechnology, V]B-VUB, 1640 St-Genesius-Rode, Belgium

Received 18 October 2000

ABSTRACT. Discrimination of self and nonself is one of the features of all animal species but the ways of elimination of nonself are different. Defense strategies of invertebrates, which lack antibodies and lymphocytes, are based on innate defense mechanisms. The study of such, undoubtedly less complex,

defense mechanisms in invertebrates may shed,a new light on the more sophisticated immunity of vertebrates. The main aim of this review is to show on one experimental model - an oligo- chaete annelid - cellular and humoral defense pathways protecting against microbial infection.

CONTENTS

1 Introduction 283 2 General armamentarium of earthworms 3 Cellular defense mechanisms 285 4 Humoral defense mechanisms 285

4.1 Cytolytic system in E.foetida 285 4.1.1 Fetidins 286 4.1.2 Lysenin 286 4.1.3 Eiseniapore 288 4.1.4 HI, H2 and H3 haemolysins 288 4.1.5 What are the relationships between E. foetida cytolysins

4.2 C0elomic cytolytic factor as earthworm defense molecule 290 4.3 Ceelomic cytolytic factor as a TNF analogue 293 4.4 Lysozyme 295 4.5 Protein-antigen recognition 295

References 296

284

288

1 I N T R O D U C T I O N

Of the total number of extant animal species probably surpassing 2 million 95 % are included in invertebrate taxa, encompassing a diversity o f organisms ranging from unicellular protozoans to the complex protochordates. Invertebrates have evolved for hundreds of millions o f years, often surviving in very hostile environments. Their survival strategies surely involve short life span combined with numerous offspring. Moreover, all invertebrate species have evolved a variety o f active defense pathways efficiently recognizing and responding to non-self substances despite the absence o f an adaptive immune system based on antibodies or lymphocytes. Rather, invertebrates rely on short-term inducible innate mechanisms based on "pattern recognition receptors" (Janeway 1989) that in most cases do not discriminate between individual pathogens. The success o f invertebrate taxa in evolution buttresses arguments about the potential value o f gaining deep insights in their diverse recognition and/or effector defense mechanisms. In this respect, knowledge of the less complex invertebrate defense strategies may contribute to understanding the sophisticated vertebrate immune system, as well as lead to the identification of new factors with possible therapeutic use.

Since the pioneering works of Elie Metchnikoff at the end o f the 19th century (Metchnikoff 1887), invertebrate immunology has become a proper topic of study. However, it is only in the last two decades that detailed analyses o f invertebrate immune reactions and their molecular basis have begun to emerge. More- over, the data available so far are still scarce and restricted to a few animal models, in particular arthropods, mollusks, and annelids.

Earthworms belonging to oligocha~te annelids became a model for comparative immunologists in the early 1960s with the publication of results from transplantation experiments. American and French scien- tists reported that autologous transplants of earthworm body wall pieces were accepted but not transplants between individuals of another or even the same earthworm species (for review see Cooper and Roch 1994). Moreover, a cell-mediated short-term memory was noticed (Valembois 1971 b; Bailey et al. 1971). These trans-

284 M BILEJ etal. Vol 45

plantation experiments proved the existence of self and/or non-self recognition in earthworms, paving the way for extensive studies on the earthworm immune mechanisms that evolved to prevent the invasion of pathogens. The choice of earthworm for comparative immunology studies becomes more and more pertinent since they represent an inexpensive, noncontroversial, and socially acceptable model for experimentation. Because of their crucial role in soil fertilization, the Organization for Economic Cooperation and Development (OECD) and the American Environmental Protection Agency accepted an official protocol to follow immunological parameters in earthworms to monitor environmental pollution (OECD Guidelines for Testing o f Chemicals 1984; Green et al. 1989; for review see Goven and Kennedy 1996). Moreover, earthworms might be considered as a source of biologically active compounds with potential industrial or medical use. Earthworm powder has been used in traditional medicine as a drug for treatment of various diseases in the Far East since several thousands years ago. Currently, the therapeutic effect of earthworm active factors is re-evaluated by a mo- dern scientific approach. For example, a potent fibrinolytic enzyme extracted from Lumbricus rubellus is in a clinical-trial phase as a possible antithrombotic drug (Mihara et al. 1991, 1992; Nakajima et al. 1996, 1999).

[n this review we list the defense pathways protecting the earthworm, as a model invertebrate, against pathogen overload.

GENERAL A R M A M E N T A R I U M OF EARTHWORMS

Earthworms are protostomian animals endowed with a true ceelom of mesenchymal origin. The ceelomic cavity is filled with coelomic fluid containing flee wandering ccelomocytes derived from the mesenchymal lining of the cavity. The coelomic cavity is metameric and the segments are separated by transversal septa. Regulated transport of the ccelomic fluid and ceelomocytes between neighboring segments is ensured by channels comprised of sphincters within the septa. Each segment of the coelomic cavity is opened to the outer environment by paired nephridia and by one dorsal pore through which soluble metabolites and cor- puscular material, respectively, can be excreted or expelled. More detailed description of annelid physiology is available in, e.g., Laverack (1963), Mill (1978), and V~,tvi~,ka (1994).

The first protective barrier of earthworms is represented by skin. The skin of earthworms consists of the epidermis with a thin cuticle and it covers the entire body. The cuticle contains mucopolysaccharides that serve not only as the matrix for collagen fibers, but also as an antimicrobial barrier (Rahemtulla and Lovtrup 1974, 1975). The epidermis is formed by a single-layer epithelium of supporting cells, basal cells, and secretory cells. Basal cells play an important role in wound healing and graft rejection, often exerting phagocytotic activity (Chapron 1970a; Valembois I971 a). Sometimes these basal cells are not considered to be of truly epidermal origin, but rather homologous to ceelomocytes (Burke 1974a-c). Secretory cells of different types form epidermal glands secreting mucus containing the so-called mucopolysaccharide-lipid-protein complex (Dall Pai et al. 1981; Alves et al. 1984; Bernaldo de Quiros and Benito 1986). Mucus serves as a lubricant during locomotion and contains several antibacterial factors (Valembois et al. 1984, 1986, 1988).

Microorganisms passing through the epidermal barrier enter the ceelomic cavity mainly via dorsal pores. Therefore, the ccelomic fluid is not aseptic and always contains bacteria, protozoans, and fungi invading from the outer environment. Efficient mechanisms keep the growth of microorganisms under control. Dales and Kala~: (1992) reported that the ceelomic fluid contains approximately 600/pL of naturally occurring bacteria, while the number of potentially phagocytotic cells is more than ten times higher. The excess of phagocytes combined with the humoral factors can easily prevent the coelomic bacteria from multiplying. tn general, invading microorganisms are eliminated in different ways. First, they can be excreted by nephridia (Cameron 1932) or they can be engulfed by cells of the nephrostome or middle tube (Villaro et al. 1985). Second, bacteria can be phagocytosed by free coelomocytes (see below) and the phagocytes that become "exhausted" for further uptake are expelled through dorsal pores. Dorsal pores are equipped with muscular sphincters controlling intraceelomic pressure and continuous exchange of material between the outer environment and the coelom (Cameron 1932). Third, large foreign bodies or agglutinated bacteria can be eliminated by a process known as encapsulation (Ratcliffe et al. 1985). The cellular and fibrous capsule is sometimes called "brown body" because of its melanin content, which results from the activation of the prophenol-oxidase cascade (see below). The encapsulation in earthworms Eiseniafoetida was detailed by Valembois et al. (1992) who showed that most brown bodies contain tissue wastes such as necrotic muscle cells or setae, agglutinated bacteria, gregarines, and nematodes. Encapsulation begins like phagocytosis with recognition of nonself, but the engulfment cannot occur because of the size of the particle. Foreign body is within the first day surrounded by free ccelomocytes so that after several days a dense capsule composed of flattened cells encloses the implant. When the capsule reaches 1-2 mm in diameter, its external ceils lose their adhesiveness. The capsule starts to migrate to posterior segments of the ceelomic cavity where it can be

2000 ANTIMICROBIAL DEFENSE OF THE EARTHWORM--review 2115

eliminated by autotomy (Keilin 1925), Autotomy of caudal segments followed by wound healing is well developed in earthworms and seems to be under neurohormonal control. The role of neurosecretory cells of the ventral cord and particularly of subesophageal ganglia was established and some mediators secreted by these cells were described (Hubl 1956; Herlan-Meewis 1965; Chapron and Chapron 1972; Alonso-Bedate and Sequeros 1983, 1985). There is no evidence that wounding in annelids is accompanied by "leuko- poiesis" as it is generally accepted in vertebrates, since increased mitotic activity of free ccelomocytes was not recorded (Burke 1974a-c). Coelomocytes have probably a "plugging" function and the lost tissue layers regenerate from surrounding differentiated tissues (Clark and Clark 1962).

3 CELLULAR DEFENSE MECHANISMS

As mentioned above, coelomic fluid contains coelomocytes of different types. The nomenclature of coelomocytes is based mainly on morphological and cytochemical criteria (for rev iew see Stein et al. 1977; ~ima 1994). In general, there are eleocytes (free chloragogen cells) having mainly accessory and nutritive functions and hyaline or granular amoebocytes representing effectory immunocytes. All types of ameebocytes are phagocytotic although their activity differs. Similarly, the phagocytotic index, i.e. the amount of engulfed particles per single cell, varies widely among the cells of a given type and from one cell type to another. While granular ceelomocytes never accumulate more than average amounts of ingested particles, the cytoplasma of hyaline ameebocytes is occasionally full of engulfed material (Stein et al. 1977). It was shown that amcebocytes engulf all kinds of material including inert particles, microbial cell wall components, and foreign cells. It should be mentioned that in the case of phagocytosis of foreign eukaryotic cells recognition of non-self plays a crucial role. In this regard, Cameron (1932) performed an important experiment with phagocytosis of different spermatozoa. When earthworms were injected with a mammalian spermatozoon or with xenologous spermatozoa of different earthworm species a marked phagocytosis commenced within 1 d. On the other hand, allologous spermatozoa survived in the coelomic cavity for 3-4 d without any defi- nite evidence of their phagocytosis.

Humoral components represent an important factor affecting phagocytosis. The first attempts to determine the effect of the ccelomic fluid opsonins were undertaken by Stein and Cooper (1981). Although in vitro phagocytosis of yeast was not increased by the presence of the c0elomic fluid, preincubation of yeast in the ccelomic fluid prior to the test resulted in significantly enhanced phagocytotic activity and index of neutrophilic amcebocytes. Similar evidence for opsonizing properties of the c0elomic fluid was given in the case of synthetic 2-hydroxyethylmethacrylate copolymer (HEMA) particles (Bilej et al. 1990a,b, 1991a). Flow-cytometric analysis revealed that both the phagocytotic activity and the index of coelomocytes as well as the number of adhered non-engulfed particles were considerably higher in the case of opsonized particles.

Laulan et al. (1988) followed the effect of two main mammalian opsonins, immunoglobulins and complement C3 fragments on the phagocytotic activity of ceelomocytes toward sheep erythrocytes. Phago- cytosis was enhanced by vertebrate IgG and C3b complement fragments but not by IgM and C3d fragments. These data suggest the presence of some molecules on the surface of ccelomocytes that interact with vertebrate opsonins. One of the possible candidates could be the IgG-binding protein present both in the ccelomic fluid and on the surface of about 20 % of the coelomocytes (Rejnek et al. 1986). The interaction of the lgG- binding protein with immunoglobulins is not mediated by antibody binding site but by some other parts of the lgG molecule.

4 HUMORAL DEFENSE MECHANISMS

4.1 Cytolyt ic sys tem in E. foetida

The ccelomic fluid of annelids exerts numerous biological activities. Among the factors involved in E. foetida humoral immunity, particular attention has been devoted to cytolytic components secreted by ccetomocytes into the c(elomic cavity. The cytolytic activity of the ccelomic fluid was generally demonstrated on vertebrate erythrocytes, and the resulting effect was referred to as haemolysis. The majority of the h~emolysins identified so far show h~emagglutination activity and, more interestingly, a spectrum of antibacterial and/or bacteriostatic activities against pathogenic soil bacteria (Roch 1979; Valembois et al. 1982, 1986; Roch et al. 1991 a). Therefore, the biological relevance of the E. fcetida cytolytic and/or agglutinating system consists in part in growth inhibition of the worm's potential pathogens that live in manure and possess antigen(s) common with red blood cells. However, a possible contribution of the cytolytic molecules to the destruction of membranes from self-transformed cells cannot be ruled out.

286 M, BILEJ etal. Vol. 45

Current data on E. faetida cytolysins, namely the fetidins, the lysenins, eiseniapore and the HI, H2, and H3 heemolysins will be summarized below.

4.1.1 Fetidins

Fetidins are two glycoproteins of 40 and 45 kDa, secreted by chloragocytes and eleocytes that were originally described as the E. foetida haemolytic factor (Du Pasquier and Duprat 1968; Roch 1979; Roch et al. 1981, 1984). In addition to their h~emolytic activity, fetidins also agglutinate red blood cells (Valembois et al. 1984) and may participate in the cytotoxic activity of the coelomic fluid (Kauschke and Mohrig 1987). The 45-kDa protein, encoded by one nonpolymorphic gene, shows a pl of 6.0, while the 40-kDa protein is encoded by a gene possessing four alleles, giving rise to four protein isoforms with a pl of 6.3, 6.2, 5.95, and 5.9. Besides the protein isoform of pl 6.0 present in all earthworms, each individual expresses one or two active proteins (Roch 1979; Roch et al. 1987). The global amino acid sequence of the 40- and 45-kDa fetidins suggests homology between them (Roch et al. 1986), and the different molar mass of these two proteins is mainly due to the size of the saccharide component (Lass6gues et al. 1997). By screening a cDNA library with rabbit anti-45-kDa antiserum cross-reacting with the 40-kDa protein, a clone encoding a protein of 34.14 kDa, which corresponds to the size of the deglycosylated fetidins, was isolated (Hirigoyenberry et al.

1992; Lass~gues et al. 1997). The amino acid sequence presents an N-glycosylation site (position 250-252), and homologies with peroxidase (position 52-62). Accordingly, fetidins display peroxidase activity. Fetidins lyse erythrocytes in absence of Ca 2+ and Mg 2+ and do not seem to act as enzymes since the kinetic curves of h~emolysis are identical between 4 and 45 ~ (Valembois et al. 1986; Lass~gues eta/ . 1984). They polyme r- ize upon binding to sphingomyelin generating 10-nm open channels through the lipid bilayer in sphingomyelin microvesicles or red blood cell membranes (Roch et al. 1981, 1989).

Fetidin synthesis in vivo increases following an injection of pathogenic bacteria in the worm ccelom (Lass~gues et al. 1989). Moreover, these proteins exhibit antibacterial activity on Gram-negative and Gram- positive bacteria (Valembois et al. 1982; Lass+gues et al. 1981; Hirigoyenberry et al. 1992), particularly on strains that are pathogenic for earthworms (Roch et al. 1987; Valembois et al. 1986; Roch et al. 1991a). Interestingly, it was shown that the expression of a particular combination of the 45- and 40-kDa proteins in a worm individual influences the antibacterial capacity of the ccelomic fluid.

In addition to their bacteriolytic/static activity, fetidins may also mediate opsonization (Sinkora et

al. 1993). Moreover, they contribute to the clotting of the ceelomic fluid (Valembois et al. 1988). The coagulation cascade is suspected to be triggered by a serine proteinase since c~elomic fluid coagulation is enhanced by thrombin but inhibited by PMSF. In this case, the polymerization of the 40- and 45-kDa h~emolysins gives rise to a network of globular units of about 4 nm appearing as a dense lattice of intricate insoluble fila- ments. Earthworm clots that never lead to gelation of the whole c~elomic fluid are apparently not involved in wound closure (Chapron 1970b), but could spontaneously occur on the epidermal surface, particularly near the dorsal pores. Such clotting would entrap bacteria present in the earthworm biotope preventing invasion of the coelomic fluid. Since E. foetida clottable proteins also exhibit antibacterial activities, it was hypothe- sized that depending on the physiological conditions of earthworms, fetidins polymerize, leading to a dis- sociable structure, the clot fibers, or to a more stable system, the pores.

4.1.2 Lysenin

Lysenin was first identified as a 4 l-kDa protein produced by c~elomocytes that cause contraction of rat vascular smooth muscles (Sekizawa et al. 1996). Simultaneously, a lysenin-related protein with a molar mass of 42 kDa and a weak contractive activity was found. A eDNA encoding lysenin was isolated (Sekiza- wa et al. 1997). The 297 amino acid sequence with a molar mass of 33.44 kDa displays one potential N-glycosylation site (position 248). Two eDNA clones encoding the lysenin-related proteins of 300 amino acids were isolated (Sekizawa et al. 1997). The latter have, respectively, 76 and 90 % amino acid residues identical with those of lysenin. In addition, lysenin and lysenin-related protein reveal a high homology with fetidins, suggesting a close relationship between these lytie molecules (Fig. 1, Table I). Accordingly, as fetidins, lysenin does not show sphingomyelinase activity, but induces erythrocyte lysis by binding sphingomyelin to membranes (Yamaji et al. 1998). Moreover, the presence of cholesterol in membranes increases the accessibility of sphingomyelin to lysenin and fetidins, facilitating haemolysis. It is therefore likely that lysenin causes membrane damage by forming aqueous pores in the membrane.

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4.1.3 Eiseniapore

Eiseniapore cytolytic protein is a 38-kDa protein from E. foetida c~elomic fluid that requires sphingomyelin or galactosylceramide to bind to red blood cell membranes to induce lysis (Lange et aL 1997). Like with fetidins and lysenins, the eiseniapore lytic activity toward sphingomyelin-containing vesicles is enhanced by cholesterol. The protein seems to be associated in the coelomic fluid with a natural inhibitor named eiseniapore-regulating protein (Mohrig et al. 1997). Electron-microscopic analysis of ultra- structural membrane lesions, as well as protection experiments of colloid osmotic lysis by sugars, such as dextran and inulin, or polyethyl- ene glycol, have revealed that six eiseniapore molecules form ring-shaped hydrophilic pores with an outer diameter of 10 nm and an inner diameter of 3 nm on the target membrane (Fig. 2).

4. l. 4 HI, H2 and/43 hcemolysins

Eue et al. (1998) described 3 haemolytic proteins in the coelomic fluid of E. foetida, HI, H2, and H3, with respective molar mass of 46, 43 and 40 kDa. It was observed that, in contrast to HI and H2, H3 splits into two fragments of 18 and 21 kDa after SDS treatment. Moreover, the 3 haemolysins consist of several isoforms (with pl of 6.2, 6.0, 5.8 and 5.4 for Il l , 6.0 and 5.2 for 1-12, and 6.1 and 5.1 for H3) that share epitope and secondary structural similarities. However, despite the cross-reactivity of mono- specific antisera, HI, H2 and H3 show functional differences. While HI and H2 are only h~emolytic, H3 is bifunctional, showing both lytic and agglutinating properties. The ha~molytic activity of Ill and I-t2 is lost by heating to 56 ~ while both the lytic and agglutinating activity of H3 are maintained. The binding site of HI, H2 and H3 on the erythrocyte membrane during lytic events is supposedly a N-acetylneuraminic acid receptor. H3 may have an addit- ional N-acetyl-D-glucosamine-binding site invoI- red in its agglutinating activity. Recent data based on MALDI analysis suggest that H1, H2, and H3 h~emolysins differ from lysenin and fetidins (Kauschke et al. 2000).

4.1.5 What are the relationships between E. foetida cytolysins?

In invertebrate immunology, the strong natural occurrence of lyric activity in E. fcetida ccelomic fluid, probably has to do with main- raining homeostasis against infection; it has been intensely investigated for more than 30 years. Cytolytic properties were also reported in other


Fig. 2. Ulrastructural study of h~emolysis. Above left: transmission electron micrograph of control sheep erythrocytes; consistent tri- laminar cell membranes and dense finely granular stroma; above right: defects of surface membrane after a 30-min ex vivo treatment with the coelomic fluid; rarefied flocculent structure ofstroma; both parts magnified 80 000x. Below: high-resolution scanning electron micrograph of sheep erythrocytes; left: finely granular surface of a serum-exposed cell; right: multiple dense depressions and more translucent humps following incubation in the coelomic fluid; both parts magnified 47 000x. Reproduced by courtesy of Dr. P. Ross- mann (Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague) from Rossmann et al. (1997).

290 M. BILEJ etal. Vol. 45

annelids, such as Lumbricus terrestris or polych~etes, but this activity is weaker than in E. Jbetida and varies from individual to individual (Valembois et al. 1986). The E. fcetida effector molecules identified so far share similar lytic function but display interindividual heterogeneity. Accordingly, six different h~emolytic and/or antibacterial phenotypes, corresponding to the expression of particular ha~molysin isoforms, have been identified. There is suggestive evidence that all the lysins are related proteins (see Table I). They share biochemical analogies, presenting isoforms with similar molar mass of around 40 kDa and similar pl. Comparison o f the available cDNA sequences clearly show that fetidins and lysenin display strong amino acid homologies (Fig. 1). However, H3 is different from all the other lysins since it is the only one that is dimeric. On addition, MALDI analysis revealed that HI, H2, and 1-t3 haemolysins differ from lysenin and fetidins. Concerning lipid specificity, eiseniapore is quite different from lysenin and possibly from fetidins, since eiseniapore does not only bind sphingomyelin but also galactosylceramide. Inhibition experiments revealed that the h~emolytic activity of the caelomic fluid is partially inhibited by acetylated or methylated saccharides such as N-acetyl-D-glucosamine, methyl C~-D-mannopyranoside, N-acetyl-D-galactosamine and N-acetylneuraminic acid (Roch et al. 1981; Eue et al. 1998). However, neither zymosan, inulin or LPS, nor hydrazine or methylamine inhibit the coelomic fluid lyric activity, suggesting that E. fcetida h~emolysins are not related to the C3 or the C3b complement component (Roch et al. 1989). The mode of action o f the eiseniapore may be analogous to that o f the membrane attack complex of complement or perforin since heparin, vitronectin and lysophosphatidylcholine prevent its binding to erythrocytes but do not affect the membrane insertion and pore formation (Lange et al. 1999). Therefore, a relationship between the complement cascade and E. fcetida ha~molytic activity is conceivable.

It is clear that there is an urgent need to characterize the lytic components of E fcet ida by identical methods to reach a consensus in the denomination ofh~emolysins. Hence cloning of eiseniapore and 1,1t-H3 is anticipated.

4. 2 The coelomic cytolytic f ac tor as ear thworm defense molecule

We have identified and cloned a 42-kDa protein named CCF (ccelomic cytolytic factor) in the ccelomic fluid of the earthworm Eiseniafoet ida (Bilej et al. 1995a; Beschin et al. 1998). CCF is localized in the cells o f chloragogenous tissue adjacent to the gut wall and in the translucent free large coelomocytes, i,e.

in cells with macrophage-like function (Bilej et al. 1998; Fig, 3). CCF shows homology with the saccharide-

Fig. 3. Detection of CCF in a cross-section of the earthworm; survey picture of gut with strong cytoplasmic positivity in chloragogenous cells (CC) and in typhlosolis (I); endogenous peroxidase reaction in the gut (G). Processed as described in Bilej et al. 1998, probed with anti-CCF monoclonal antibody; magnified 470• Reproduced by courtesy of Dr. P. Rossmann (Institute of Micro- biology, Academy of Sciences of the Czech Republic, Prague).

binding motif o f bacterial and animal 13-1,3-g/ucanases, with: Gram-negative bacterial binding .protein and 1,3-!3-D-glucan-recognition proteins.of arthropods, and with glucan-sensitive factor G from the horseshoe crab Limulus po lyphemus (Fig. 4, Table II), Interestingly, although these proteins show high homology in the

~ " Fig. 4. Sequence alignment of E. foetida CCF with recently described homolognes; sequences were retrieved in the GenBank TM

database; the proteins are (accession no. in parentheses): Ef CCF - CCF from E. fcetida earthworm (AAC35887); Pl 5GBP - LPS and 1,3-[3-D-glucan binding protein from Pacifastacus leniusculus crayfish (CAB65353); Bra bGRP - 1,3-13-D-glucan recognition protein from Bombyx mori (BAA92243); Ms GRP - 1,3-13-D-glucan recognition protein from Manduca sexta (AAF44011); Ag GNBP - putative Gram-negative-bacteria-binding protein from Anopheles gambiae (CAA04496); Dm DGNBP - Gram-negative-bacteria-binding protein 3 from Drosophila melanogaster (AAF33851); Hc GNBP - Gram-negative-bacteria-binding protein from Hyphantria cunea (AAD09290); numbers on the left and the right indicate residue numbers of the amino acid sequence of each proteim amino acid residues including the putative potysaccharide-binding domain of bacterial glucanase (Lee et al. 1996; Yahata et aL 1990) are underhned. The catalytic active residues of bacterial glucan endo-l,3(4)-l~-glucosidase (Juncosa et al. 1994) are indicated by I '; amino acids conserved in at least 5 other sequences besides CCF are marked with o; amino acids conserved in all homologues are marked with *

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292 M~ BILEJ etal. Vol. 45

putative polysaccharide-binding domain and the catalytic sites of the bacterial glucanases, neither CCF nor its invertebrate homologues exhibit glucanase activity. It was proposed that these glucan-binding proteins developed from a primitive glucanase that evolved to proteins without enzymic activity but that instead bind glucans and operate as elicitors of defense reactions through the recognition of non-self (S6derh~ill and Cerenius 1998). CCF binds efficiently the O-antigen of LPS, 1,3-13-D-glucan and N,N'-diacetylchitobiose.

Table II. CCF and homologues proteins

Identity Homology Accession References Species Protein % % no,

Eiseniafoetida CCF AAC35887 13eschin etal. 1998

Pacifastacus leniusculus LPS and 1,3-13-D-glucan- 40 58 CAB6353 Lee et al. 2000 binding protein

AnophelesgambJae putative Gram-negative- 40 55 AAD29854 Dimopoulos et aL 1997 bacteria-binding protein

Strongylocentrotuspurpuratus 13-1,3-glucanase 43 56 AAC47235 Bachman and McClay 1996

Bombyx mori 1,3-13-D-glucan- 29 44 BAA92243 Ochiai and Ashida 2000 recognition protein

Manduca sexta 1,3-13-D-glucan- 28 43 AAF44011 Ma and Kanost 2000 recognition protein

Hyphantria cunea Gram-negative-bacteria- 28 42 AAD09290 Shin et al. 1998 binding protein

Drosophila melanogaster Gram-negative-bacteria- 27 42 AAF33851 Kim et al. 2000 binding-protein 3

Bombyx mori Gram-negative-bacteria- 26 43 JC6150 Lee et al. 1996 binding-protein precursor

Tachypleus tridentatus factor G subunit ct 25 61 A49878 Seki et al. 1994

Bacillus circulans A1 glucan endo-1,3(4)-13- 29 42 AAC60453 Yamamoto et aL 1993 glucosidase

Upon recognition of these cell wall components of Gram-negative bacteria or yeast, CCF triggers the activation of the prophenol oxidase (proPO) cascade, an important invertebrate defense mechanism. Generally, upon recognition of polysaccharides of microbial cell walls, serine proteinases cleave by limited proteolysis inactive proPO to its active state phenol okidase. Subsequently, the active enzyme(s) catalyse the o-hydroxyl- ation of monophenols (monophenol monooxygenase - EC 1.14.18.1) and oxidation of diphenols (o-diphenol oxidase - E C 1.10.3.1) to quinones, which are subsequently, polymerized to melanin which exerts cytotoxic and antibacterial properties (Smith 1996; Johansson and S6derhfill 1996). ProPO activity was evidenced both in protostomians and deuterostomians. However, suggestive evidence for the existence of the proPO activation cascade in annelids was limited to description of melanization reactions and formation of brown bodies or nodules in polych~etes and oligoch~etes (Poinar and Hess 1977; Valembois et al. 1992; Porchet- Henner6 and Vernet 1992; Porchet-Henner6 and M'Berri 1987), In addition, biochemical detection of PO activity was restricted to a few species with rather controversial results. Whereas the proPO system was not detected in the Aphrodite aculeate and Arenicola marina polych~etes (Smith and S6derh~ill 1991), PO activity was documented to occur in Lumbricus terrestris, Eisenia foetida andrei and Nereis diversicolor (Porchet-Henner6 and Vernet 1992; Fischer 1978; Valembois et al. 1991; Seymour et al. 1993).

The importance of CCF in the induction of proPO in E, foetida was confirmed showing that when CCF is removed from the coelomic fluid, the activation cascade is blocked. However, exogenous supply of recombinant CCF restores the L-Dop (formerly DOPA) oxidation (substrate for proPO) of CCF-depleted ceelomic fluid. These data point out for the existence of the proPO cascade in annelids.

Melanin and its precursors involved in the prophenol oxidase activating system stimulate, besides having antimicrobial properties, a wide range of other biological activities including phagocytosis and opsonization, capsule and/or nodule formation, and wound healing. In the earthworm cytotoxic and antimicrobial activities are tightly connected to the aggregating, h~emolytic and opsonizing activities present in the ceelomic fluid (Roch 1996; Valembois et aL 1982; Milochau et al. 1997; Lass~gues et al. 1997). In this regard, we observed that the cytotoxicity ofE. fcetida coelomocytes against coelomocytes from L. terrestris is blocked


by anti-CCF monoclonal antibody, suggesting that CCF is involved in the cell-mediated cytotoxic reaction in earthworms (Bilej et al. 1998). Moreover, CCF agglutinates smooth but not rough Gram-negative bacteria or Gram-positive bacteria (Beschin et al. 1998). In addition, CCF was reported to be involved in the opsonizing properties of the coelomic fluid providing an efficient mechanism for phagocytosis during earthworm defense reactions (Bilej et al. 1995a). More recently, CCF was shown to potentiate the lytic activity of the ceelomic fluid on rat, mouse and guinea-pig red blood cells (Bilej et al. 2000). CCF is not haemolytic by itself but present data suggest that CCF interacts with the H3 h~emolysin present in E. foetida ceelomic fluid (Eue et al. 1998). The h~emolytic activity of the ceelomic fluid is impaired by removing CCF from the ceelomic fluid or by preincubating the ceelomic fluid with insoluble 1,3-[3-D-glucans. We hypothesize that CCF, by binding to saccharide moieties on red blood cells, favors the interaction of haemolysins with erythrocyte membranes.

The pleiotropic activities of CCF suggest that this pattern recognition molecule plays a key role in innate defense mechanisms of E. foetida earthworm.

4.3 Ccelomic cytolytic factor as a TNF analogue

Earthworms have provided for more than 30 years a useful model for comparative immunology. As mentioned in the preceding sections, their ceelomic fluid exerts a large variety of biological effects including bacteriostatic, h~emolytic, proteolytic and cytolytic activities that are involved in effective defense mechanisms against invaders (for review see Bilej 1994). It was suggested that invertebrate molecules with tumori- lytic activity might be analogous to the vertebrate cytokine TNF. CCF was originally identified in experiments aimed at characterizing novel cytolytic factors from the ceelomic fluid of E. fa~tida. We observed that the coelomic fluid lyses YNF-sensitive tumor L929 cells in a proteinase-independent way, and subsequent isolation of the lytic proteins led to the identification of the 42-kDa protein CCF (Bilej et al. 1995a). The activity of CCF is not inhibited by anti-TNF neutralizing monoclonal antibodies suggesting that the mechanisms of TNF- and CCF-mediated lysis differ. In addition to the TNF-like lytic activity, CCF showed other similarities with this mammalian cytokine. CCF is secreted by macrophage-like ceelomocytes upon LPS stimulation while TNF is produced by macrophages (Bilej et al. 1998; Aggarwal 1985). Moreover, CCF is involved in the opsonizing properties of the earthworm's c~elomic fluid (see above). Similarly, TNF was reported to provide opsonin-like signals that mediate the attachment of bacteria to macrophages (Luo et al. 1993). Finally, CCF and TNF proteins bind 1,3-13-D-glucans via a lectin-like interaction (Beschin et al. 1998; Olson et al. 1996). Furthermore, it was found that CCF and TNF proteins bind immobilized N,N'-diacetylchitobiose (a N-acetyl-l,4-[3-D-glucosidic link) (Beschin et al. 1998, 1999). The binding of CCF or TNF to 1,3-!3-D-glucan is inhibited by N,N'-diacetylchitobiose, and vice versa, the binding of CCF or TNF to N,N'-diacetylchitobiose is impaired by 1,3-13-D-glucan (Fig. 5). In addition, murine monoclonal antibodies elicited against the lectin-like domain of TNF (TIP domain), that is spatially distinct from the TNF-receptor binding site (Lucas et al. 1994), cross-react with CCF. Finally a monoclonal antibody elicited against CCF reacts with TNF. Taken together these data suggest that the CCF and TNF proteins share similar 1,3-[3-D-glucan and N,N'-diacetylchitobiose lectin-like activities and/or domains.

The lectin-like domain of TNF was shown to be involved in the killing of African trypanosomes by TNF (Lucas et al. 1994; Magez et al. 1997). So, in view of the similar N,N'-diacetylchitobiose lectin-like activity of CCF and TNF, the possible trypanolytic activity of CCF was investigated (Beschin et al. 1999). The ccelomic fluid of E. fcetida as well as purified CCF display potent trypanolytic activity that can be inhibited not only by anti-CCF monoclonal antibodies but also by N,N'-diacetylchitobiose and anti-TIP TNF antibodies. Vice versa, anti-CCF antibodies neutralize TNF-mediated trypanolysis. In addition, using the ability of CCF to trigger the prophenol-oxidase cascade upon saccharide recognition in E. foetida ceelomic fluid, the N-linked N,N'-diacetylchitobiose core of the variant-specific glycoprotein (VSG) that acts as a protective coat on bloodstream forms of T. brucei, was identified as a possible target for CCF on the trypano- some surface during trypanolytic events. Moreover, pre-incubating VSG with TNF impairs the activation of the proPO cascade, confirming that the interaction with a N,N'-diacetylchitobiose saccharide moiety on VSG lies at the basis of CCF and TNF trypanolytic activity.

More recently TNF was reported to play an important role in the inflammatory process by increas- ing the membrane conductance in endothelial cells and peritoneal macrophages (Hribar et al. 1999; Van der Goot et al. 1999). This effect is independent of the TNF receptor since it occurs in cells isolated from mice deficient in both types of TNF receptors. The ion channel-gating effect of TNF was found to be mediated by the N,N'-diacetylchitobiose lectin-like domain of molecule. The increased ion permeability induced by TNF is also inhibited by amiloride, an inhibitor of sodium transport (Hribar et al. 1999). Therefore, the increase in the in- and outward current in mammalian endothelial cells was suggested to result from the binding of TNF

294 M BILEJ e ta l Vol 45

to endogenous ion channels or to proteins coupled to ion channels. The TNF-induced change in ion permeability may play an important role in the resorption of ~edema during acute inflammatory responses (Rezaiguia

et al. 1997), point ing to a contribution by the N,N'-diacetylchi tobiose lectin-like activity (and/or domain) of TNF in innate defense mechanism in vertebrate. Similarly, we documented that CCF activates an amiloride-

sensitive cationic channel in murine endothelial cells and macrophages via its N,N' -diacetylchi tobiose lectin-like domain (Bloc et al. 2000). CCF, like TNF treatment, increases the outward current in macrophages from mice lacking both types o f TNF receptors excluding the interaction o f the CCF with the TNF receptor.

A kDa 1 2 3 4 5 6 7 8

102 ,-4. :~:~, ~=~

78 --~. ' : :::

~ ,~ . : , 4 ,

49.5 --t,

34.2 ~

19.9 - * ~ ;:,/:i

W ~

kDa

102

78 --~

B 2 3 4

C 5 1 2 3

49.5 -4,

34.2 --~,

19.9 -4' i

Fig. 5. Detection of a similar lectin-like domain in CCF and TNF. A: cross-reactivity of monoclonal antibody recognizing the TIP lectin-like domain of murine TNF with CCF from Efoetida; lines 1, 2 - TNF or ccelomic fluid of E. fcetida (silver staining); 3 -5 - TNF, coelomic fluid or recombinant CCF revealed with anti-TIP monoclonal antibody (Western blot), 6, 7 - ccelomic fluid or recombinant CCF revealed with anti-TIP monoclonal antibody preincubated with TNF (Wes- tern blot); 8 - c~elomic fluid revealed with anti-TiP monoclonal antibody preincubated with recombinant CCF (Western blot). B: isolation of CCF or TNF on immobilized N,N'-diacetylchitobiose; lines 1, 2 - elution from N,N'-diacetylchitobiose-agarose incubated with TNF or E foetida ceelomic fluid (silver staining); 3, 4 - elution from cellobiose-agarose incubated with TNF or E. foetida coelomic fluid (silver staining); 5 - elution from N,N'-diacetylchitobiose-agarose incubated with E. foetida cGelomic fluid revealed with anti-CCF monoclonal antibody (Western blot). C: cross-reactivity of anti-CCF monoclonal antibody with TNF; lines 1 - 3 - TNF, ceelomic fluid or recombinant CCF revealed with anti-CCF monoclonal antibody (Western blot).

2000 ANTIMICROBIAL DEFENSE OF THE EARTHWORM--rewew 295

Importantly, despite their functional analogies, CCF and TNF do not show gene homology, indicat- ing a lack of common evolutionary origin. Therefore, it was suggested'that CCF and TNF share [3-1,3-glucan-N,N'-diacetylchitobiose lectin-like activities that may have been functionally conserved as a recognition mechanism in innate defense reactions in invertebrates and vertebrates, respectively (Beschin et al. 1999). The functional analogies between cytokines and their presumed invertebrate counterparts do not merely reflect homology but may result from convergent evolution and a structural homology in the 3D-structure of their lectin-like recognition domain.

4.4 Lysozyme

Lysozyme is a ubiquitous enzyme hydrolyzing 1,4-!3-D-linked glycosidic bond of the peptidoglycan in the bacterial cell wall and thus efficiently protecting the host against Gram-positive bacteria infections. Lysozyme activity was evidenced in ceelomocyte extracts and to lesser extent in the ceelomic fluid of E. f~e- tida earthworms ((~otuk and Dales 1984). More recently, the active protein was isolated as a 13-kDa protein, characterized, and partially sequenced (Ito et al. 1999). The N-terminal sequence of E. fcetida lysozyme revealed a considerable homology with lysozyme from mollusks, echinoderms, and the nematode Cceno-

rhabditis elegans, while the homology to other known types of lysozymes was negligible. This suggests that earthworm enzyme belongs to a distinct family of invertebrate lysozymes.

4.5 Pro te in-an t igen recognit ion

There is no real anticipatory immunity in invertebrates and their defense strategies are undoubtedly based on innate immune factors (Klein 1989). However current data suggest that inducible factors with a certain degree of specificity exist in some invertebrates. For instance, inducible antimicrobial peptides were described in numerous insect species (for rev iew see Kannost and Zhao 1996; Karp 1996). Regarding earthworms, inducible agglutinins recognizing non-self saccharide moieties were reported (Wojdani et al.

1982; Stein et al. 1982). These proteins exert a very broad specificity based probably on "pattern recognition receptors". On the other hand, there are some indications that earthworms can mount more specific defense reactions.

This idea is, supported first of all by the existence of a short-term immunological memory after xeno- and allografting (Valembois 1971b; Bailey et al. 1971). Secondly, there is evidence of humoral defense factors synthesized in response to foreign substances. Laulan et al. (1985) showed that the ccelomic fluid of L. terrestris earthworms injected with synthetic haptens coupled to a protein carrier contains, 5 to 8 d after stimulation, a substance specifically binding the relevant hapten. The binding of a different hapten even coupled to the same carrier does not exceed background levels detected in the caelomic fluid of non- stimulated earthworms. Tu~kov~ and co-workers obtained similar results, although they were never able to show such a restricted degree of specificity of the hapten-binding protein (Tu~kov~ et al. 1988, 1991a). They isolated from the ceelomic fluid of L. terrestris earthworms an adaptively formed 56-kDa molecule designated as antigen-binding protein (ABP). ABP consists of two disulfide-linked polypeptide chains (31 and 33 kDa in L. terrestris, 30-kDa homodimer in E. fcetida), both of which contribute to the formation of antigen-binding site (Tu~kov~ et al. 1991a). The binding capacity was determined in a ligand blot assay with labeled proteins and in ELISA using a panel ofmonoclonal antibodies against ABP that do not interfere with the antigen-binding site of ABP (Tu~kov~. et al. 1991b). The maximum level of ABP was observed between the 4th and 8th days after stimulation. ABP bound efficiently the proteins used for in vivo stimulation but also other proteins though to considerably lesser extent (Bilej et al. 1995b; K6hlerov~ et al. 1999). At the cellular level, the presence of ABP in neutrophilic ceelomocytes and their antigen-binding capacity was demonstrated (Bilej et al. 1990c, 1991b).

Moreover, it was shown that the proteolytic degradation of administered antigen is required to trigger the ABP synthesis and/or its release (Tu~kov~ and Bilej 1994; Bilej et al. 1994). Proteolytic enzymes of earthworm coelomic fluid are very potent, often thermoresistant and display a remarkable interindividual heterogeneity of activity (Valembois et al. 1973; Tu~kov~ et al. 1986; Mohrig et al. 1989; Roch et al.

1991b; Leipner et al. 1993). Most of the administered protein antigen is degraded within first day, proteo- iysis occurring in the ccelomic fluid and in free ccelomocytes as well (Rejnek et al. 1993; Bilej et al. 1993). The proteolysis is limited, since it results in the formation of peptides of 700-1100 Da (Hanu~ov~ et al.

1999). When such peptides are injected to the earthworms, the ABP response is faster than in individuals injected with a nondegraded entire protein. Furthermore, when the protein antigen is administered together with nontoxic proteinase inhibitor, the ABP formation does not occur. The response to already degraded peptides appears irrespective of the presence of proteinase inhibitor (Tu~kov~ and Bilej 1994; Bilej et al. 1994; Hanugov~ et al. 1999).

296 M. BILEJ etaL Vol. 45

The available data allow us to draw a tentative hypothesis on protein-antigen recogni t ion in earth-

worms. (i) The administered protein is rapidly degraded by extracellular and intracellular proteinases. (ii) Anti- gen fragments are then bound by precursor cells in the mesenchymal tissue (Rejnek et al. 1993), inducing their proliferation, differentiation, and migrat ion into the ccelomic cavity (Bilej et al. 1992). (iii) These pre-

determined ceelomocytes can undergo further mitotic cycles after the antigenic challenge. However, numerous questions remain to be answered. First, what is the biological role o f ABP. We have so far no direct evidence for any defense activity o f ABP. One can speculate that ABP can complement pat tern-recognit ion

receptors based on lect in-saccharide interaction with non-se l f and "f ine- tune" the response to particular anti- gens. Second, it should be clarified whether recognit ion by ABP is based on prote in-prote in interaction or whether lect in-sacchar ide activity takes part in this interaction. Since an ABP response was detected irrespective o f the size and extent o f glycosylat ion of administered proteins (KOhlerov~ et aL 1999), the recognition o f protein moiety of a peptide by ABP seems to be more plausible. Third, it is o f crucial importance to determine the primary structure o f ABP. Prel iminary N-terminal analyses did not reveal any homology with known proteins (unpublished results). In any case, ABP represents an interest ing invertebrate

molecule, which should be studied in more details.

This study was supported by research grants no. 310/99/1385 and no. 310/00/1372 from the Grant Agency of the Czech Republic, by NATO Collaborative Linkage Grant LST CLG974974, by Fund for Scientific Research Flanders (FWO) and the Inter- university Attraction Pole Program. We wish to thank our collaborators and colleagues for their contributions and discussions.

REFERENCES

AGGARWAL B. B.: Human tumor necrosis factor. Production, purification and characterization. ,LBiol.Chem. 260, 2345-2354 (1985). ALONSO-BEDATE M., SEQUEROS E.: Neurosecretory phenomena in the cerebral ganglia of clitellated Allolobophora caliginosa. Acta

EmbryoZ Morphol.Exp. 4, 93-103 (1983). ALONSO-BEDATE M., SEQUEROS E.: Suggested regulatory mechanisms for caudal regeneration in Allolobophora molleri (Annelida:

Oligochceta). Comp.Biochem.Physiol. 81A, 225-228 (1985). ALVES C.E, MACHA N., DALE PAl V.: Fine structure of the epidermal cuticle of some Brazilian earthworms (Oligochceta: Annelida).

Anat.Anz. 155, 1-9 (1984). BACI-IMAN E.S., MCCLAY D.R.: Molecular cloning of the first metazoan 13-1,3-glucanase from eggs of the sea urchin Strongylo-

centrotus purpuratus. Proc.Nat.AcadSci. USA 93, 6808-6813 (1996). BAILEY S., MILLER B.J., COOPER E.L.: Transplantation immunity in annelids. II. Adoptive transfer of the xenograR reaction. Immuno-

logy 21, 81-86 (1971). BERNALDO DE QUIROS I.F., BENITO J.; Ultrastructure of gland cells associated with the ch~etal follicles in the clitellar region of Lum-

bricusfriendi COGNETTI, 1904 (Ohgochaeta). Arch.Anat.Histol.Embryol. 69, 91-99 (1986). BESCHIN A., BILEJ M., HANSSENS F., RAYMAKERS J., VAN DYCK E., REVETS H., BRYS L., GOMEZ J., DE BAETSELIER P., TIMMER-

MANS M.: Identification and cloning ofa glucan- and LPS-binding protein from Eiseniafcetida earthworms involved in the activation of prophenoloxidase cascade. J.Biol. Chem. 273, 24948-24954 (1998).

BESCHIN A., BILEJ M., BRYS L., TORREELE E., LUCAS R., MAGEZ S., DE BAETSELIER P.: Convergent evolution of cytokines. Nature 400, 627--628 (1999).

BILEJ M.: Humoral defense mechanisms, pp. 245-258 in V. V~,tvi~ka, P. ~ima, E.L. Cooper, M Bilej, P. Roch (Eds): Immunology of Annelids. CRC Press, Boca Raton 1994.

BILEJ M., SCHEERLINCK J.P., VAN DEN DRIESSCHE T., DE BAETSELIER P., VI~TVI(~KA V.: The flow cytometric analysis of in vitro phagocytic activity of earthworm ceelomocytes (Eiseniafaetida, Annelida). Cell BioLlnternat.Rep. 14, 831-837 (1990a).

BILEJ M., V[~TVI(~KA V., TU~KOVA L., TREBICHAVSKY I., KOUKAL M., ~fMA P.: Phagocytosis of synthetic particles in earthworms: effect of antigenic stimulation and opsonization. Folia Biol. 36, 273-280 (1990b).

BILEJ M., TU~KOV,~ L., REJNEK J., VI~TVI(~KA V.: 11"1 vitro antigen-binding properties of c~elomocytes of Eiseniafoetida (Annelida). lmmunol.Lett. 26, 183-188 (1990c).

BILEJ M., DE BAETSELIER P., TREBICHAVSK~" I., VI~TVI~KA V.: Phagocytosis of synthetic particles in earthworms: absence of oxidative burst and possible role of lytic enzymes. Folia Biol. 37, 227-233 (1991a).

BILEJ M., ROSSMANN P., VAN DEN DRIESSCHE T., SCHEERLINCK J.P., DE BAETSEL1ER P., TU(~KOvA L., VI~TVI(~KA V., REJNEK J.: Detection of antigen in the ceelomocytes of the earthworm, Eiseniafaetida (Annelida). lmmunol.Lett. 29, 241-246 (1991b).

BILEJ M, 5~tMA P., SL|PKA J.: Repeated antigenic challenge induces earthworm c0elomocytes proliferation. Immunol.Lett. 32, 181-184 (1992).

BILEJ M., TU~KOVA L., REJNEK J.: The fate of protein antigen in earthworms: study in vitro, lmmunoLLett. 35, 1-6 (1993). BILEJ M., TU~KOVA L., ROSSMANN P.: A new approach to m vitro studies of antigenic response in earthworms. Dev.Comp.lmmunol.

18, 363-367 (1994). BILEJ M., BRYS L., BESCHIN A., LUCAS R., VERCAUTEREN E., HANU~OVA R., DE BAETSELIER P.: Identification ofa cytolytic protein in

the ccelomic fluid of Eisentafoetida earthworms, lmmunol.Lett. 45, 123-128 (1995a). BILEJ M., TU(~KOVA L., ROMANOVSK~" A.: Characterization of the limited specificity of antigen recognition in earthworms. Folia

MicrobioL 40, 436---440 (1995b). BILEJ M., ROSSMANN P., ~INKORA M., HANU~;OVA R., BESCHIN A., RA~S G., DE BAETSELIER P.: Cellular expression of the cytolytic

factor in earthworms Eiseniafoetida. Immunol. Lett. 60, 23-29 (1998). BILEJ M., BESCHIN A., KOHLEROVA P., DE BAETSELIER P., MOHP-dG W., KAUSCHKE E.: Interaction of h~emolytic and cytolytic mole-

cules in Eiseniafetida earthworms. Dev.Comp.lmmunol. 24 (Suppl. 1), $98 (2000). BLOC A., LUCAS R., DE BAETSELIER P., BILEJ M., BESCHIN A.: Earthworm functional analog of TNF increases membrane conductance

in mammalian cells. Dev.Comp.lmmunol. 24 (Suppl. 1), $95 (2000).

2000 ANTIM1CROBIAL DEFENSE OF THE EARTHWORM--review 297

BURKE J.M.: Wound healing in Eiseniafoetida (Oligochteta). I. Histology and 3H-thymidine radioautography of the epidermis. ZExp. Zool. 188, 49-63 (1974a).

BURKE J.M.: Wound healing in Eiseniafoetida (Oligochceta). II. A fine structural study of the role of the epidermis. Cell Tissue Res. 154, 61-82 (1974b).

BURKE J.M.: Wound healing of Eiseniafoetida (Oligochceta). III. A fine structural study of the role of non-epidermal tissues. Cell Tissue Res. 154, 83-112 (1974c).

CAMERON G.R.: Inflammation in earthworms. J. Pathol. 35, 933-972 (1932). CHAPRON C.: Etude histologique, infrastructurale et experimentale de la regeneration cephalique chez le Iombriciens, Eiseniafcetida.

Ann.Embr.Morphol. 3, 235-239 (1970a). CHAPRON C.: R6g6n6ration c@halique chez le lombricien Eiseniafetida unicolor: structure, origine et role du bouchon cicatriciel.

Arch.Zool.Exp. Gen. 111, 217-227 (1970b). CHAPRON C., CHAPRON J.: Influence des amines biogenes et de leurs inhibiteurs sur la regeneration. Etude chez l'annelide Eisenia

foetida. C.R.AcadSci.Paris 274, 412-414 (1972). CLARK M., CLARK R.: Growth and regeneration in Nephthys. Zool.J.PhysioL 70, 24-90 (1962). COOPER EL., ROCH P.: Immunological profile of annelids: transplantation immunity, pp. 201-243 in V. V~,tvi~ka, P. ~in~a, E L Cooper,

M. Bilej, P. Roch (Eds): Immunology ofAnnelids. CRC Press, Boca Raton 1994. ~OTUK A., DALES R.P.: Lysozyme activity in the ccelomic fluid and coelomocytes of the earthworm Eiseniafoetida SAV. in relation to

bacterial infection. Comp.Biochem.Physiol. 78A, 469-474 (1984). DALES R.P., KALA(~ Y.: Phagocytic defence by the earthworm Eiseniafaetida against certain pathogenic bacteria. Comp.Biochem

Physiol. 101A, 487-490 (1992). DALE PAI V.D., SANTOS COSTA I.R., PAEHECO A.C., ALVES C.E., MACHA N.: Histochemical study of mucopolysaccharides in epider-

mal mucous cells and subjacent granular cells of Glossoscolex uruguayensis L. (R1GHI 1978). Folia Histochem.Cytochem (Krak6w) 19, 107-113 (1981).

D1MOPOULOS G., RICHMAN A., MI~ILLER H.-M, KAFATOS F.C.: Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites. ProcNatAcadSci. USA 94, 11508-11513 (1997).

Du PASQU1ER L., DUPRAT P.: Aspects humoraux et cellulaires d'une immunit4 naturelle non sp6cifique chez l'oligoch6te Eiseniafoetida (Lumbricidae). C.R.AcadSci. Paris 266, 538-542 (I 968).

CUE 1., KAUSCHKE E. MOHRIG W., COOPER E.: Isolation and characterization of earthworm h~emolysins and agglutinins. Dev.Comp. lmmunol. 22, 13-25 (1998).

FISCHER E.: DOPA peroxidase activity in the chloragogen cells of the earthworm Lumbricus terrestris. Acta Histochem. 63, 210-223 (1978).

GOVEN A.J., KENNEDY J.: Environmental pollution and toxicity in invertebrates: an earthworm model for immunotoxicology. Adv. Comp.Environ.Physiol. 24, 169-211 (1996).

GREEN J.C., BARTELS C.L., WARREN-HICKS W.J., PARKHURST B.R., LINDER G.L., PETERSON S.A., MILLER W.E.: Protocols of Short- Term Toxicity Screening of Hazardous Waste Sites. US Environmental Protection Agency, EPA/600/3-88/029. ERLC, Corvallis (OR) 1989.

HANU~OVA R., TU(~KOVA L., HAEADA P., BEZOU~KA K., BILEJ M.: Peptide fragments induce a more rapid immune response than intact proteins in earthworms. Dev.Comp.lmmunoL 23, 113-121 (1999).

HERLAN-MEEWlS H.: Regeneration in annelids, pp. 155-215 in M. Abercrombie, J. Brachet (Eds.): Advances in Morphogenesis, Vol. 4. Academic Press, New York 1965.

HIRIGOYENBERRY F., LASSI~GUES M., ROCH P.: Antibacterial activity ofEiseniafaetida andrei coelomic fluid: immunological study of the two major antibacterial proteins. J.lnvert.Pathol. 59, 69-74 (1992).

HRIBAR M., BLOC A., VAN DER GOOT G., FRANSEN L., DE BAETSELIER P., GRAU G. E., BLUETHMANN H., MATTAY M. A., DUNANT Y., LUCAS R.: The lectin-like domain of TNF-~t increases membrane conductance in microvascular endothelial cells and peritoneal macrophages. Eur.d Immunol. 29, 3105-3111 (1999).

HUBE H.: l~lber die Beziehungen der Neurosekretion zum Regenerations geschehen bei Lumbriciden nebst Beschreibung eines neu- artigen neurosekretorischen Zelltypen in Unterschlundganglion. Arch.Entwicklungsmech.Organismen 149, 73-87 (1956).

ITO Y., YOSHIKAWA A., HOTAN1 T., FUKUDA S., SUGIMURA K., IMOTO T.: Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family. Eur.J.Biochem. 259, 456--461 (1999).

JANEWAY C.A.: Approaching the asymptote? Evolution and revolution in immunology. Cold Sprmg Harb.Symp.QuantBiol. 54, 1-13 (1989).

JOHANSSON M. W., SODERHALL KA The prophenoloxidase activating system and associated proteins in invertebrates. Progr.Mol.Sub- cell.Biol. 15, 46--66 (1996).

JUNCOSA M., PONS J., DOT T., QUEROL E., PLANAS A.: Identification of active site carboxylic residues in Bacillus lichemformis 1,3-1,4-13-D-glucan 4-glucanohydrolase by site-directed mutagenesis. J.Biol.Chem. 269, 14530-14535 (1994).

KANNOST M.R., ZHAO L.: Insect h~emolymph proteins from the lg superfamily, pp. 185-197 in E.L. Cooper (Ed.): Advances in Compa- rative and Environmental Physiology, Vol. 23. Invertebrate Immune Responses: Cells and Molecular Products. Springer- Verlag, Berlin-Heidelberg-New York 1996.

KARP RD.: Inducible humoral immune defense responses in insects, pp. 67-87 in B. Rinkevich, W.E.G. Mtiller (Eds): Progress in Molecular and Subcellular Biology, Vol. 15. Invertebrate Immunology. Springer-Verlag, Berlin-Heidelberg-New York 1996.

KAUSCHKE E., MOHRIG W.: Cytotoxic activity in the coelomic fluid of the annelid Eiseniaf~etida SAV. J.Comp.Physiol. B 157, 77--83 (1987).

KAUSCHKE E., WAGNER F., KONIG S., EUE I., MOHRIG W., COOPER E.L.: Innate immune factors in earthworms. Dev.Comp.lmmunol. 24 (Suppl. 1), $96 (2000).

KEILIN ND.: Parasitic autotomy of the host as a mode of liberation of c~elomic parasites from the body of the earthworm. Parasitology 17, 170-172 (1925).


KIM Y.S., HAN S.J., RYU J.H., CHOI K.H., HONG Y.S., CHtmG Y.H., PERROT S., RAIBAUD A., BREY P.T., LEE W.J.: Lipopolysac- charide-activated kinase, an essential component for the induction of the antimicrobial peptide genes in Drosophila melanogaster cells..ZBioI.Chem 275, 2071-2079 (2000).

KLEIN J.: Are invertebrates capable of anticipatory immune responses? ScandJ ImmunoL 29, 499-505 (1989). KOHLEROVA P., TUt~KovA L., BILEJ M.: Characterization of the limited specificity of antigen recognition in earthworms. Folia Micro-

biol. 44, 435-440 (1999). LANGE S., NOSSLER F., KAUSCHKE E LUTSCH G., COOPER E., HERRMANN A.: Interaction of eathworm hlemolysin with lipid mem-

branes requires sphingolipids. J.Biol.Chera. 272, 20884--20892 (1997). LANGE S., KAUSCHKE E. MOHRIG W., COOPER E.: Biochemical characteristics of eiseniapore, a pore-forming protein in the ccelomic

fluid of earthworms. Eur.s 262, 547-556 (1999). LASSI~GUES M., ROCH P., VALEMBOIS P., DAVANT N.; Action de quelques souches bact~riennes telluriques sur le Iombricien Etsenia

foetida andrei. C.R.Acad.ScL Paris 292, 731-734 (1981). LASSEGUES M., ROCH P., CADORET M.A., VALEMBOIS P.: Mise en ~vidence des prot~ines h~molytiques et h~magglutinantes sp~ci-

flques de I'albumen des cocoons du Iombricien Eiseniaf~etida andrei. C.R.AcadSci.Paris 299, 691-696 (I 984). LASSEGUES M., ROCH P., VALEMBOIS P.: Antibacterial activity ofEiseniafo~tida andrei coelomic fluid: evidence, induction and animal

protection. ,L.Invert.PathoL 53, 1-6 (1989). LASSEOUES M., MILOCHAU A., DOIGNON F., DU PASQUIER L., VALEMBOIS P.: Sequence and expression of an Eiseniafetida-derived

eDNA clone that encodes the 40-kDa fetidin antibacterial protein. Eur.J.Biochem. 246, 756--762 (1997). LAULAN A., MOREL A., LESTAOE J., DELAAGE M., CHATEAUREYNAUD-DUPRAT P.: Evidence of synthesis by Lumbricus terrestris of

specific substances in response to an immunization with a synthetic hapten. Immunology 56, 751-758 (1985). LAULAN A., LESTAGE J., BOUC A.M., CHATEAUREYNAUD-DUPRAT P.: The phagocytic activity of Lumbricus terrestris leukocytes is

enhanced by the vertebrate opsonins: IgG and complement C3b fragment. Dev.Comp.Immunol. 12, 269--277 (1988). LAVERACK M.S.: The Physiology of Earthworms. Pergamon Press, Oxford 1963. LEE W.J., LEE J.D., KRAVCHENKO V.V., ULEVITCH R.J., BREY P.T.: Purification and molecular cloning of an inducible Gram-negative

bacteria-binding protein from the silkworm, Bombyx morL Proc.Nat.AcadSci. USA 93, 7888-7893 (1996). LEE S.Y., WANG R., SODERHALL K.: A lipopolysaccharide- and ~3-1,3-glucan-binding protein from h~mocytes of the freshwater cray-

fish Pacifastacus leniusculus. Purification, characterization, and cDNA cloning, d.BioLChem. 275, 1337-1343 (2000). LEIPNER C., TUt~KOVA L., REJNEK J., LANGNER J.: Serine proteinases in ccelomic fluids of annelids Eiseniafcetida and Lumbricus term-

stris. Comp.Biochem.PhysioL 105B, 637-641 (1993). LUCAS R., MAGEZ S., DE LEYS R., FRANSEN L., SCHEERLINCK J.P., RAMPELBERG M., SABLON E., DE BAETSELIER P.: Mapping the

lectin-like activity of tumor necrosis factor. Science 263, 814-817 (1994). LUG G., NIESEL D.W., SHABAN R.A., GRIMM E.A., KLIMPEL G.R.: Tumor necrosis factor ct binding bacteria: evidence for a high-affi-

nity receptor and alteration of bacterial virulence properties, lnfect.lmmun. 61,830-835 (1993). MAC., KANOST M.M.: A [3-1,3-glucan recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates

the phenoloxidase cascade. JBiol. Chem. 275, 7505--7514 (2000). MAGEZ S., GEUSKENS M., BESCHIN A., DEL FAVERO H., VERSCHUEREN R., LUCAS R., PAYS E., DE BAETSELIER P.: Specific uptake of

tumor necrosis factor-ct is involved in growth control of Trypanosoma brucei. ,Z Cell Biol. 137, 715-727 (1997). METCHNIKOFF E.E.: Sur la lutte des cellules de l'organisme contrr I'invasion des microbes. Ann.lnst.Pasteur i, 322-340 (1887). MIHARA H., SUM! H., YONETA T., MIZUMOTO H., IKEDA R., SEIKI M., MARUYAMA M.: A novel fibrinolytic enzyme extracted from the

earthworm, Lumbricus rubellus. Japan ,Z Physiol. 41, 461-472 (1991 ). MIHARA H., MARUYAMA M., SUMI H.: Novel thrombolytic therapy discovered from traditional oriental medicine using the earthworm.

SoutheastAsian,~ Trop.MedPublic Health 23 (Suppl. 2), 131-140 (1992). MILL P.J.: Physiology of Annelids. Academic Press, London 1978. MILOCHAU A., LASSI~GUES M., VALEMBO1S P.: Purification, characterization and activities of two Memolytic and antibacterial proteins

from coelomic fluid of the annelid Eiseniafaetida andrei. Biochem.Biophys.Acta 1337, 123-132 (1997). MOHRIG W., EUE I., KAUSCHKE E.: Proteolytic activities in the coelomic fluid of earthworms (Annelida, Lumbricidae). Jb.Zool Phy-

siol. 93, 303-317 (1989). MOHRIG W., LANGE S., KAUSCHKE E., PREUSSE K., COOPER E.: Inhibitor controlled hemolytic activity in the coelomic fluid of earth-

worms. Dev.Comp.lmmunol. 21, 116 (1997). NAKAJIMA N., ISHIHARA K., SUGIMOTO M., SUMI H., MIKUNI K., HAMADA [q.; Chemical modification of earthworm fibrinolytic enzy-

me with human serum albumin fragment and characterization of the protease as a therapeutic enzyme. Biosci.Biotechnol. Biochem. 60, 293-300 (1996).

NAKAJIMA N., SUGIMOTO M., ISHIHARA K., NAKAMURA K., HAMADA H.: Further characterization of earthworm serine proteinases: cleavage specificity against peptide substrates and on autolysis. Biosci. Biotechnol.Biochem. 63, 2031-2033 (1999).

OCHIA1 M., ASHIDA M.: A pattern-recognition protein for 1~-l,3-glucan: the binding domain and the cDNA cloning of 13-1,3-glucan- recognition protein from the silkworm, Bombyx mori. J.BioLChem. 275, 4995-5002 (2000).

OECD Guidelines for Testing of Chemicals. Section 2. Effects of Biotic Systems: Earthworms Acute Toxicity Tests. Paris 1984. OLSON E.J., STANDING J.E, GRIEGO-HARPER N., HOFFMAN O,A., LIMPER A.H: Fungal [3-glucan interacts with vitronectin and stimu-

lates TNF-ct release from macrophages, lnJ~ct.lmmun. 64, 3548-3554 (1996). POINAR G.O. Jr, HESS R.T.: Immune responses in the earthworm Aporrectodea trapezoides (Annelida) against Rhabditis pellio

(Nematoda), pp. 69-84 in L.A. Bulla, T.C. Cheng (Eds): Comparative Pathobiology, VoL 3. Plenum Publ. Corp., New York 1977.

PORCHET-HENNERE E., M'BERRI M.: Cellular reaction of the polych~ete annelid Nereis diversicolor against coelomic parasites. J.lnvertebr.Pathol. 50, 58-66 (1987).

PORCHET-HENNER# E., VERNET G.: Cellular immunity in annelid (Nereis diversicolor, Polychceta): production of melanin by a sub- population ofgranu)ocytes. Celt Tissue Res. 269, 167-174 (1992).

RAHEMTULLA F., LOVTRUP S.: The comparative biochemistry of invertebrate mucopolysaccharides. II. Nematoda; Annelida. Comp. Biochem.Physiol. 49B, 639-646 (1974).


RAHEMTULLA F., LOVTRUP S.: The comparative biochemistry of invertebrate mucopolysaccharides. III. Oligocheta and Hirudinea. Comp Biochem. Physiol. 50B, 627-629 (1975).

RATCLIFFE N.A., ROWLEY A.F., FITZGERALD S.W., RHODES C.P.: Invertebrate immunity: basic concepts and recent advances. Inter- nat.Rev.Cytol. 97, 183-349 (1985).

REJNEK J., TUCKOVA L., LiMA P., KOSTKA J.: The proteins in Lumbricus terrestris and Eiseniafoetida coelomic fluids and on ceelomocytes reacting with sheep and goat IgG molecules. Dev.Comp.lmmunoL 10, 467--475 (1986).

REJNEK L., TUCKOVA L., ~tMA P., BILEJ M.: The fate of protein antigen in earthworms: study in vivo. lmmunoZLett. 36, 131-136 (1993).

REZAIGUIA S., GARAT C., DELCLAUX C., MEIGNAN M., FLEURY J., LEGRAND P., MATTHAY M.A., JAYR C.: Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by tumor-necrosis factor-~t dependent mechanism. J.Clin.lnvest. 99, 325- 335 (1997).

ROCH P.: Protein analysis of earthworm coelomic fluid. I. Polymorphic system of the natural h~emolysins of Eiseniafaetida andrei. Dev.Comp.lmmunol. 3, 599-608 (1979).

ROCH P., VALEMBOIS P., DAVANT N., LASS#GUES M.: Protein analysis of earthworm coelomic fluid. II. Isolation and biochemical characterization of the Eiseniafeetida andrei factor (EFAF). Comp.Biochem.Physiol. 69B, 829-836 (1981).

ROCH P., DAVANT N, LASSI~GUES M.: Isolation of agglutinins and lysins from earthworm coelomic fluid by gel filtration followed by chromatofocussing. J.Chromatogr. 290, 231-235 (1984).

ROCH P., VALEMBOIS P., VAILLER J.: Amino-acid compositions and relationships of five earthworm defense proteins. Comp.Biochem. Physiol. 85B, 747-751 (1986).

ROCH P., VALEMBOIS P., LASSEGUES M.: Genetic and biochemical polymorphism of earthworm humoral defenses. Dev.Comp.lmmunol. 11, 91-102 (1987).

ROCH P, CANICATTI C, VALEMBOIS P.: Interactions between earthworm h~emolysins and sheep red blood cell membranes. Biochim. Biophys.Acta 983, 193-198 (1989).

ROEH P, LASSI~GUES M., VALEMBOIS P.: Antibacterial activity of Eiseniaf~etida andrei coelomic fluid: III. Relationship within the polymorphic h~emolysins. Dev.Comp.lmmunol. 15, 27-32 (1991a).

ROCH P., STABILI L., PAGLIARA P.: Purification of three serine proteinases from the coelomic cells of earthworms (Eiseniafaetida). Comp.Biochem.Physiol. 98B, 597-602 (1991b).

ROCH P.: A definition of cytolytic response in invertebrates. Adv.Comp.Environ. Physiol. 23, 115-150 (1996). ROSSMANN P., BILEJ M., TU(~KOVA L., STAR~" V., KOFRO/qOVA O.: Lesion of leukocytes, erythrocytes, and mesothelial cells by the c~e-

Iomic fluid of Eiseniafoetida earthworms. Folia Microbiol. 42, 409--416 (1997). SEKI N., MUTA T., ODA T., IWAKI D., KUMA K., MIYATA T., IWANAGA S.: Horseshoe crab (l,3)-13-D-glucan-sensitive coagulation fac-

tor G. A serine proteinase zymogen heterodimer with similarities to 13-glucan-binding proteins. 3Biol.Chem. 269, 1370-- 1374 (1994).

SEKIZAWA Y., HAGIWARA K. NAKAJIMA T., KOBAYASHI H.: A novel protein, lysenin, that causes contraction of the isolated rat aorta: its purification from the coelomic fluid of the earthworm, Eiseniafaetida. BiomedRes. 17, 197-203 (1996).

SEKIZAWA Y., KUBO T., KOBAYASHI H., NAKAJIMA T., NATORI S.: Molecular cloning of cDNA for lysenin, a novel protein in the earthworm Eiseniafoetida that causes contraction of rat vascular smooth muscle. Gene 191, 97-102 (1997).

SEYMOUR J., NAPPI A., VALEMBOIS P.: Characterization of a phenoloxidase of the coelomic fluid of the earthworm Eiseniafoetida andrei. Anim.Biol. 2, 1-6 (1993).

SHIN S.W., PARK S.S., PARK D.S., KIM M.G., KIM S.C., BREY P.T., PARK H.Y.: Isolation and characterization of immune-related genes from the fall webworm, Hyphantria cunea, using PCR-based differential display and subtractive cloning, lnsect Biochem. Mol.Biol. 28, 827-837 (1998).

~iMA P.: Annelid coelomocytes and h~emocytes: roles in cellular immune reactions, pp. 115-165 in V. Vetvieka, P. ~;ima, E.L. Cooper, M. Bilej, P. Roch (Eds.): Immunology ofAnnelids. CRC Press, Boca Raton-Ann Arbor-London-Tokyo 1994.

~INKORA M., B1LEJ M., TUCKOVA L., ROMANOVSK~" A.: H~emolytic function of opsonizing proteins of the earthworm's coelomic fluid. Cell Biol.lnternat.Rep. 14, 832-837 (1993).

SMITH V.J.: The prophenoloxidase activating system: a common defence pathway for deuterostomes and protostomes? Adv.Comp. Environ.Physiol. 23, 75-114 (1996).

SMITH V.J., SODERHALL K.: A comparison of phenoloxidase activity in the blood of marine invertebrates. Dev.Comp.lmmunol. 15, 251-261 (1991).

SODERH,~LL K., CERENIUS L.; Role of prophenoloxidase-activating system in invertebrate immunity. Curr.Opin.lmmunol. 10, 23-28 (1998).

STEIN E.A., COOPER E.L.: The role of opsonins in phagocytosis by coelomocytes by the earthworm, Lumbricus terrestris. Dev.Comp. Immunol. 5, 415-425 (1981).

STEIN E.A., AVTALION R.R., COOPER E.L.: The ccelomocytes of the earthworm Lumbricus terrestris. Morphology and phagocytic properties. JMorphol. 153, 467-476 (1977).

STEIN EA., WOJDAN1 A., COOPER E.L.: Agglutinins in the earthworm Lumbricus terrestris: naturally occurring and induced. Dev. Comp.lmmunol. 6, 407-421 (1982).

TUCKOVA L., REJNEK J., ~iMA P., OND~EJOV,/~ Rd Lytic activities in c0elomic fluid of Eiseniafoetida and Lumbricus terrestris. Dev. Comp.lmmunol. 10, 181 - 189 (1986).

TU(~KOVA L., BILEJ M.: Antigen processing in earthworm, lmmunol.Lett. 41,273-277 (1994). TU~?KOVA L., REJNEK J., ~iMA P.: Response to parenteral stimulation in earthworms L. terrestris and E. foetida. Dev.Comp.lmmunol.

12, 287-296 (1988). TU~KOVA L., REJNEK J., BILEJ M., POSPI~IL R.: Characterization of antigen-binding protein in earthworms Lumbricus terrestris and

Eiseniafcetida. Dev.Comp.lmmunol. 15, 263-268 (1991a). TU(~KOVA L., REJNEK J., BILEJ M., HAJKOVA H., ROMANOVSK~" A.: Monoclonal antibodies to antigen binding protein of annelids

(Lumbricus terrestris). Comp.Biochem.Physiol. 100B, 19-23 (1991b). VALEMBOIS P.: Degenerescence et regeneration de I'epiderme a la suite d'une xenogreffe de paroi du corps entre Iombriciens.

C.R.AcadSci.Paris 96, 59-64 (1971a).


VALEMBOIS P.: Etude ultrastructurale des coelomocytes du lombricien Eiseniafcetida SAV. Bull.Soc, Zool.Fr, 96, 59-72 (1971b), VALEMBOIS P., ROCH P., DU PASQUIER L.: Ddgradation in vitro de protdine dtrangdre par les macrophages du Lombricien Eisenia

fetida SAY. C.R.AcadScLParis S~r.111277, 57-60 (1973). VALEMBOIS P., ROCH P., LASSI~GUES M., CASSAND P.; Antibacterial activity of the haemolytic system from the earthworm Eisenia

fetida andreL ~lnvert.Pathol. 40~ 21-27 (1982). VALEMBOIS P., ROCH P., LASSI~GUES M.: Simultaneous existence of h~emolysins and hemagglutinins in the ccelomic fluid and in the

cocoon albumen of the earthworm Eiseniafetida andrei. Comp. Biochem. Physiol. 78A, 14 I - 145 (1984). VALEMBOIS P., ROCH P., LASSI~GUES M.: Antibacterial molecules in annelids, pp. 74--93 in M. Brehdlin (Ed.): lmmunity in Inverte-

brates. Springer-Verlag, Berlin-Heidelberg-New York 1986. VALEMBOIS P., ROCH P., LASSEOUES M.: Evidence of plasma clotting system in earthworms, g lnvertebr.PathoL 51, 221-228 (1988). VALEMBOIS P., SEYMOUR J., ROCH P.: Evidence and cellular localization of an oxidative activity in the ccelomic fluid of the earthworm

Eisenia foetida andreL ~]nvertebr.Pathol. 57, 177-183 (1991). VALEMBOIS P., LASSI~GUES M., ROCH P.: Formation of brown bodies in the coelomic cavity of the earthworm Eisentafcetida andrei and

attendant changes in shape and adhesive capacity of constitutive cells. Dev. Comp. lmmunol. 16, 95-101 (1992). VAN DER GOOT G., PUGIN J.~ HRIBAR M.~ FRANSEN L., DUNANT Y., DE BAETSELIER P.~ BLOC A., LUCAS R.: Membrane interaction of

TNF is not sufficient to trigger increase in membrane conductance in mammalian ceils. FEBSLett. 460, 107-111 (1999). VI~TVlCKA V.: Physiology ofannelids, pp. 41-113 in V. Vetvi~ka, P. ~ima, E.L. Cooper, M. Bilej, P. Roch (Eds.): lmmunolog~ of Annefids.

CRC Press, Boca Raton-Ann Arbor-London-Tokyo 1994. VILLARO A.C., SESMA P., ALEGRiA D., VAZQUEZ J.J., LOPEZ J.: Relationship of symbiotic microorganisms to metanephridium: phago-

cytic activity in the metanephridial epithelium of two species of Oligochceta. J.Morphol. 186, 307-314 (1985). WOJDAN! A., STEIN E.A., LEMMI C.A., COOPER E.L.: Agglutinins and proteins in the earthworm, Lumbricus terrestris, before and after

i nj ection of erythrocytes, carbohydrates, and other materials. Dev. Comp. lmmunol. 6, 613-624 (1982). YAMAJI A., SEK1ZAWA Y., EMOTO K., SAKURABA H., INOUE K. KOBAYASHI H., UMEDA M.: Lysenin, a novel sphingomyelin-specific

binding protein. J.Biol.Chem 273, 5300-5306 (1998). YAHATA N., WATANABE T., NAKAMURA Y., YAMAMOTO Y., KAMIMIYA S., TANAKA H.; Structure of the gene encoding 13-t,3-glucan-

ase A 1 of Bacillus circuZans WL- 12 Gene 86, 113- I 17 (1990). YAMAMOTO M., AONO R., HORIKOSHI K : Structure of the 87-kDa 13-1,3-glucanase gene of Bacillus ctrculans IAM1165 and properties

of the enzyme accumulated in the periplasm of Eschertchia coli carrying the gene. Btosci.Blotechnol.Biochem 57, 1518- 1525 (1993).

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Antimicrobial defense of the earthworm

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