Cellular aspects of allorecognition in the compound ascidian Botryllus schlosseri

Developmental and Comparative Immunology 28 (2004) 881–889 www.elsevier.com/locate/devcompimm

Cellular aspects of allorecognition in the compound ascidian Botryllus schlosseri Francesca Cima, Armando Sabbadin, Loriano Ballarin* Dipartimento di Biologia, Universita` di Padova, Via U. Bassi 58/B, 35100 Padova, Italy Received 9 December 2003; revised 11 January 2004; accepted 6 February 2004

Abstract We studied changes in the morphology of morula cells, a common haemocyte type in botryllid ascidians, during both the rejection reaction (occurring between contacting, genetically incompatible colonies) and fusion (occurring between compatible colonies), and in short-term cultures of haemocytes incubated with heterologous or autologous blood plasma. In both the rejection reaction and haemocyte cultures in the presence of heterologous blood plasma, we observed alterations in morula cells, consistent with a degranulation event, and their expression of molecules recognised by anti-IL-1-a- and antiTNF-a-antibodies. Anti-cytokine-antibodies markedly reduced the extent of the in vitro cytotoxicity, when haemocytes were exposed to heterologous blood plasma. In addition, the increase in the production of nitrite ions and the decrease of the in vitro cytotoxicity by the nitric oxide synthase inhibitor N v-nitro-L -arginine methyl ester, suggest the role of nitric oxide in cell death. These results provide new clues to understand the process of rejection reaction in botryllid ascidians. q 2004 Elsevier Ltd. All rights reserved. Keywords: Ascidian; Botryllus; Allorecognition; Haemocytes; Morula cells

1. Introduction Colonies of the cosmopolitan ascidian species Botryllus schlosseri are formed of several zooids grouped in star-shaped systems, which share a vascular network, branching out in the common tunic, from which many blind, sausage-like ampullae Abbreviations: BA, Botryllus agglutinin; BP, blood plasma; N v-nitro-L -arginine methyl ester; FSW, filtered sea water; TEM, transmission electron microscope. * Corresponding author. Tel.: þ39-049-827-6197; fax: þ 39-049827-6199. E-mail address: [email protected] (L. Ballarin). L -NAME,

0145-305X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2004.02.001

depart. When the leading edges of different colonies contact each other, either fusion of genetically compatible colonies, with the consequent sharing of tunic and circulation, or a rejection reaction between genetically incompatible colonies occurs. The latter is characterised by the appearance of a series of dark brown, necrotic spots along the touching borders [1 –4]. According to Sabbadin et al. [3], the rejection reaction is preceded by partial fusion of the contacting tunics, in front of the opposite, facing marginal ampullae, after local disappearance of the respective cuticles. This allows the diffusion of soluble factors from the circulation of one colony to the blood vessels

882

F. Cima et al. / Developmental and Comparative Immunology 28 (2004) 881–889

of the other, which triggers the following events: (i) selective crowding of morula cells, a common haemocyte type in botryllid ascidians, representing the most abundant circulating haemocyte-type in B. schlosseri, inside the facing ampullae [5 – 8]; (ii) morula cell crossing of the ampullar epithelium [2 – 4,8]; (iii) degranulation of morula cells and release of their vacuolar contents, mainly phenoloxidase and its polyphenol substrate, which are directly responsible for the formation of the cytotoxic foci (points of rejection) in front of opposite contacting ampullae [5,10,11]. A similar series of events and the presence of allogeneic factors in blood plasma (BP) from incompatible colonies was also demonstrated in Japanese species Botrylloides simodensis [9,12]. Cell crowding, migration and degranulation are features commonly found in vertebrate inflammation which is induced and modulated by cytokines, mainly IL-1-a and TNF-a, released by activated leucocytes, upon the recognition of foreign molecules [13]. The production of soluble molecules by activated immunocytes mimicking the action of vertebrate cytokines has been reported in various invertebrates, both protostomes and deuterostomes [14 – 23]. As far as ascidians are concerned, the induction of chemotaxis and the stimulation of both phagocytosis and cell proliferation by IL-1-like molecules released by haemocytes have been described in both solitary and colonial species [24 – 28]. In addition, we recently demonstrated, in the colonial species B. schlosseri, the synthesis, by morula cells, of molecules recognised by antibodies raised against mammalian IL-1-a and TNF-a in response to the presence of non-self molecules in short-term haemocyte cultures [29]. Considering the role of morula cells in rejection reaction, in the present paper, we carried out a detailed investigation of morula cell morphology in the course of both rejection reaction and fusion, and in shortterm cultures of haemocytes incubated with either heterologous or autologous BP. In addition, we studied the expression of the above-reported molecules by morula cells upon the recognition, both in vivo and in vitro, of humoral factors in blood from genetically incompatible colonies as compared to that from subclones of the donor colony. Results are discussed in terms of a possible role of molecules

recognised by anti-cytokine antibodies in rejection reaction and immunomodulation.

2. Materials and methods 2.1. Animals Colonies of B. schlosseri from the lagoon of Venice and selected colonies of defined fusibility genotype from our laboratory were used. They were kept in aerated aquaria, attached to glass slides and fed with Liquifry Marine (Liquifry Co., Dorking, England). 2.2. Colony specificity assay Pairs of genetically incompatible and compatible colonies were juxtaposed on a supporting glass slide at a distance of 1 – 2 mm, their leading edges facing each other, in a moist chamber for 30 min before being returned to the aquarium. Within 24 –48 h, the colonies extended towards each other, the facing ampullae contacted, and a series of necrotic spots appeared along the contact border in the case of incompatibility (rejection reaction), whereas vessel anastomosis occurs in the case of compatibility (fusion). 2.3. Immunohistochemistry on paraffin sections The following combinations of contacting colonies were used for immunohistochemical studies: (i) rejecting colonies in early stages of rejection reaction, before the appearance of any necrotic spot; (ii) rejecting colonies in a more advanced stage, at the beginning of cell migration from the circulation to the tunic through the ampullar tip epithelium; (iii) fusible colonies, before the appearance of any trace of fusion. Fusing or rejecting colonies were fixed for 2 h at 4 8C in 4% paraformaldehyde plus 0.1% glutaraldehyde in saline buffer (0.2 M Na-cacodylate buffer, pH 7.4, plus 1.7% NaCl and 1% sucrose) containing 1% caffeine to preserve morula cell integrity [5], rinsed in saline buffer, dehydrated and embedded in Paraplast X-TRA (Oxford Labware). Sections (7 mm thick) were obtained with a Leitz 1212 microtome. Immunoreactivity against rabbit polyclonal

F. Cima et al. / Developmental and Comparative Immunology 28 (2004) 881–889

anti-human-IL-1-a (Santa Cruz Biotech) and goat polyclonal anti-human TNF-a (Santa Cruz Biotech) (1 mg/ml, according to manufacturer’s suggestions) was revealed by immunoperoxidase staining using the avidin – biotin – peroxidase complex method for signal enhancement [30] and 3,30 -diaminobenzidine (Fluka) as substrate. Sections were examined under a Leitz Dialux 22 light microscope. Endogenous peroxidase was blocked by incubation for 30 min in a solution of 6% H2O2 in methanol. In control preparations, primary antibodies were either substituted with non-immune sera or absorbed with homologous antigen, i.e. recombinant human IL-1-a and TNF-a (Peprotech). Moreover, a rabbit polyclonal anti-Botryllus agglutinin (BA) antibody [31], was used for specificity control. 2.4. Electron microscopy Fusing and rejecting colonies were fixed for 2 h in 2% glutaraldehyde in saline buffer containing 1% caffeine, post-fixed for 1 h in 1% OsO4 in saline buffer, dehydrated and embedded in Epon. Thick sections (1 mm), obtained with an LKB Ultratome, were stained with a hot solution of 1% toluidine blue and 1% borax in distilled water and observed under the light microscope. Thin (60 nm) sections, collected on copper grids, were stained with uranyl acetate and lead citrate, and then examined under a Hitachi H 600 transmission electron microscope (TEM). 2.5. Blood plasma and blood cell preparation Blood was collected with a glass micropipette by puncturing, with a fine tungsten needle, the tunic marginal vessels of colonies previously blotted dry. It was centrifuged at 780g and the supernatant was referred as BP. Haemocytes were obtained from blood harvested from colonies previously rinsed in 0.38% Na citrate in filtered sea water (FSW), pH 7.5, as anticlotting agent, and then centrifuged at 780g for 10 min; pellets were finally resuspended in FSW to give a concentration of 107 cells/ml. The term ‘heterologous’ and ‘autologous’ refer to non-fusible colonies and to subclones of the same colony, respectively.

883

2.6. Morula cell morphology and immunocytochemistry on haemocyte monolayers Sixty microlitres of haemocyte suspension were placed in the centre of culture chambers prepared as described elsewhere [32] and left to adhere to the coverslips for 30 min at 20 8C. Haemocytes were then incubated for 15, 30 and 60 min with 60 ml of heterologous or autologous BP and the morphology of morula cells was observed. The effects of the addition of anti-IL-1-a-, anti-TNF-a- and anti-BA antibodies to BP were also evaluated. In another series of experiments, after the incubation in autologous or heterologous BP, haemocyte monolayers were fixed for 30 min at 4 8C in 4% paraformaldehyde plus 0.1% glutaraldehyde in saline buffer, rinsed in phosphatebuffered saline (1.37 M NaCl, 0.03 M KCl, 0.015 M KH2PO4, 0.065 M Na2HPO4, pH 7.2) and permeabilised with 0.1% Triton X-100 in phosphate-buffered saline for 5 min. Anti-IL-1-a, anti-TNF-a, and anti-BA were used as primary antibodies in immunocytochemical studies, as described above. Immunoreactivity was revealed by immunoperoxidase as already reported. 2.7. Nitrite ion content of incubation medium Haemocytes were incubated for 60 min in the presence of either heterologous or autologous BP, as described above. Fifty microlitres of the incubation medium were then collected from each culture chamber and transferred into the wells of a 96-well microplate containing 50 ml of Griess reagent (equal volumes of 0.1% N-1-naphtylethyldiamine (Sigma) and 1% sulphanilamide (Sigma) in 5% H3PO4). After 15 min, the absorbance was read at 550 nm with a microplate reader. A standard curve obtained with serial dilution of a NaNO2 stock solution (1 mM) was used to estimate the concentration of nitrite in the incubation medium [33,34]. 2.8. Cytotoxicity assay Haemocytes were incubated for 60 min in heterologous BP. The incubation medium was then discarded and substituted with an equal volume of 0.25% trypan blue in FSW [35]. After 5 min, the cytotoxicity index, i.e. the percentage of dying

884

F. Cima et al. / Developmental and Comparative Immunology 28 (2004) 881–889

haemocytes acquiring a blue colour, was evaluated under the light microscope. Autologous BP was used in controls. The effects of the addition of anti-TNF-a-, anti-IL-1-a- and anti-BA antibodies (final concentration: 1 mg/ml), and of the nitric oxide synthase inhibitor N v-nitro-L -arginine methyl ester (L -NAME, Fluka; final concentration: 0.1 mM) to heterologous BP were also evaluated. 2.9. Statistical analysis At least 300 cells, in 10 optical fields at a magnification of 1250 £ , were counted in experiments aiming to determine the frequencies of positive cells. They were compared with the x2 test. Variations in nitrite ion concentrations were compared with Duncan’s test. Each experiment was carried out in triplicate. Data are expressed as mean ^ SD.

3. Results 3.1. Morula cells change their morphology upon the recognition of heterologous BP In the presence of autologous BP, living morula cells appeared as a mulberry-like cells, 15 mm in diameter, their volume being almost completely occupied by various vacuoles (2 mm in diameter) of uniform size (Fig. 1a). After aldehyde fixation, morula cells stressed their spotty outline and their vacuoles turned to a yellowish colour, which concentrates near the vacuolar membrane and appears as a dark halo in black and white pictures (Fig. 1b). This morphology was observed in cells inside facing ampullae of fusible and non-fusible colonies, in which morula cells selectively crowd in early stages of both the reactions (Fig. 1f –h), much more cells were observed inside the contacting ampullae in the case of rejection than of

Fig. 1. Morula cell morphology in various experimental conditions. (a) Living morula cell from haemocyte cultures incubated with autologous BP; (b) fixed morula cell from haemocyte cultures incubated with autologous BP showing the yellowish (dark in the picture) halo associated with vacuolar membrane which typically occurs after aldehyde fixation; (c and d) living morula cell from haemocyte cultures incubated with heterologous BP. Dark vacuoles are due to residual phenoloxidase activity; (e) fixed morula cell immunopositive to anti-TNF-a-antibody; (f–h) lumen of a facing marginal ampulla in early stages of the rejection reaction (f and g) and of fusion (h). Crowded morula cells contain regular, uniform vacuoles (arrowheads); few of them show positivity to anti-TNF-a- (f) and anti-IL-1-a- (g) antibodies (asterisks) during rejection, whereas no positive cells are visible during fusion; (i) lumen of a facing marginal ampulla in advanced stages of the rejection reaction showing changes in morula cell morphology: their vacuoles appear larger and reduced in number (arrows). Dark vacuolar halos or contents are artifacts due to the yellowish colour assumed by vacuoles, mainly concentrated near the vacuolar membranes, after aldehyde fixation. Scale bar: 15 mm.

F. Cima et al. / Developmental and Comparative Immunology 28 (2004) 881–889

885

Fig. 2. Electron microscopy of morula cells during rejection reaction. (a) Unaltered morula cell at very beginning of rejection reaction. Vacuoles are of similar size, with an almost homogeneous content; (b–d) Altered morula cells in advanced rejection reaction, showing vacuolar confluence (arrows) and flaking of the electron-dense vacuolar contents. Scale bar: 3 mm.

fusion. TEM images of morula cells obtained at this stage revealed the presence of regular, round vacuoles with highly electron-dense and almost homogeneous contents (Fig. 2a). When living haemocytes were incubated in heterologous BP, morula cells changed to a spheroidal morphology with greatly altered vacuoles: the latter reduced their size, decreased in number; frequently, vacuoles assumed a brownish colour due to the activity of phenoloxidase on residual substrate [10] and only a few, large vacuoles were often seen devoid of their content (Fig. 1c and d). After aldehyde fixation, morula cells did not change their shape and this morphology was shared by morula cells inside facing ampullae in advanced stages of rejection reaction (Fig. 1i). TEM micrographs of these cells showed the disorganisation of vacuolar content which

progressively flaked off and the confluence of vacuoles consequent to the fusion of their limiting membranes (Fig. 2b– d). The addition of the anti-cytokine and anti-BA antibodies to haemocytes incubated with autologous or heterologous BP did not affect the reported responses in living cells. 3.2. Morula cells express molecules recognised by anti-cytokine antibodies upon the recognition of heterologous BP Morula cells were the only cell-type immunopositive to anti-cytokine antibodies and positivity was located in the cytoplasm (Fig. 1e – g). In haemocytes cultured in the presence of autologous BP, the frequency of cells immunopositive to anti-TNF-a

886

F. Cima et al. / Developmental and Comparative Immunology 28 (2004) 881–889

3.3. In vitro nitrite production and cytotoxicity assay

Fig. 3. (a) Time-course of the percentage of morula cells immunopositive to anti-IL-1-a- (dashed lines) and anti-TNF-a(solid lines) antibodies, expressed as percentage of total haemocytes, after in vitro incubation of haemocytes with heterologous and autologous BP (Het BP and Aut BP, respectively); (b) effects of anti-cytokine and anti-BA antibodies and L -NAME on cytotoxicity induced by heterologous BP. Horizontal lines indicate significant comparisons with the cytotoxicity index observed in the presence of heterologous BP (Het BP). Significant differences with respect to controls (unstimulated haemocytes) are marked by asterisks. * p , 0:05; ** p , 0:01; *** p , 0:001:

and anti-IL-1-a reached about 17 and 5%, respectively, after 60 min of incubation. In the case of heterologous BP, the fractions of morula cells showing positivity for anti-TNF-a and anti-IL-1-a increased progressively to 94 and 30%, respectively, after 60 min of incubation. Significant differences in the percentage of immunopositive cells, with respect to autologous BP, were already observed after 15 min ðp , 0:001Þ for anti-TNF-a and 30 min ðp , 0:05Þ for anti-IL-1-a) (Fig. 3a). Immunopositive cells were never observed inside facing ampullae in the case of fusible colonies (Fig. 1h); a few morula cells resulted positive to anti-IL-1a- and anti-TNF-a-antibodies in early stages of the rejection reaction, before any alteration of their morphology (Fig. 1f and g). No labelling was observed in controls.

When BP was incubated with heterologous haemocytes, a significant (* p , 0:05; *** p , 0:001) increase in nitrite ions in the culture medium was measured as compared to autologous ones 7.97 ^ 0.29***, (D[NO 2 2 ] ¼ 23.48 ^ 1.16***, 2.32 ^ 0.07* mM in three different experiments). A significant ðp , 0:001Þ increase in cytotoxicity was observed when haemocytes were incubated for 60 min in heterologous BP, with respect to autologous BP. In the presence of heterologous BP and anti-TNFa- and anti-IL-1-a-antibodies, the cytotoxicity index, although significantly ðp , 0:01Þ higher than in the presence of autologous BP, was significantly ðp , 0:001Þ lower than that observed in the absence of the antibodies. A similar result was obtained with heterologous BP plus 0.1 mM L -NAME. No significant reduction in the cytotoxicity index was observed with the anti-BA antibody (Fig. 3b).

4. Discussion The rejection reaction which occurs between contacting, genetically incompatible colonies of compound ascidians of the genus Botryllus is characterised by a series of processes, such as chemotaxis, extravasation, degranulation of effector cells and cell death by necrosis [1 – 5,10,36] which also occur in vertebrate inflammation. In B. schlosseri, in the course of this reaction, morula cells crowd inside the facing ampullae and migrate, through the ampullar tip epithelium, into the fused tunic, where they contribute to the formation of the cytotoxic foci, distributed along the contact border, through degranulation and release of phenoloxidase, the key enzyme in the induction of the cytotoxicity [3,10]. Although morula cell crowding also occurs in stages preceding fusion [8, this paper], in the present work, we demonstrate that morula cells undergo remarkable changes in morphology upon the recognition of allogeneic, soluble factors either during the rejection reaction or when incubated in vitro with BP from incompatible colonies. These results agree with the previous reports, indicating degranulation as the typical response of morula cells to the recognition of allogeneic factors [9 – 11]. In addition, they show that

F. Cima et al. / Developmental and Comparative Immunology 28 (2004) 881–889

degranulation occurs, in most cases, already inside the ampullae, before morula cell migration from the circulation to the tunic, so that most of the cytotoxicity observed in the rejection reaction must probably be ascribed to phenoloxidase diffusing through the ampullar tips into the tunic. The observation that, in our species as well as in some Japanese botryllid ascidians, phenoloxidase activity is observable, well before the appearance of any necrotic spot, as a thick, coloured band along the contact borders, clearly exceeding the area where cytotoxicity will appear and including most of the ampullar region [5,6], is consistent with this view. We also demonstrate that morula cells selectively acquire immunopositivity to anti-IL-1-a- and antiTNF-a-antibodies when incubated in vitro with heterologous BP. The reported result fits our previous results indicating morula cells as the main haemocyte type able to respond to the recognition of foreign molecules with the synthesis of molecules recognised by antibodies raised against mammalian inflammatory cytokines [29]. Therefore, the expression of such molecules seems to represent a marker of morula cell activation. The limited increase in immunopositive cells observable in the presence of autologous BP can be the result of morula cell activation following mechanical disturbance during haemocyte collection and centrifugation. The presence of few immunopositive morula cells in early stages of rejection reaction and the gradual increase in the fraction of immunopositive cells in cultures suggest the occurrence of some sort of regulation of the morula cell response. Since, morula cells are the main effector cells of the rejection reaction [5,10], in our opinion, the molecules in question could represent cytokine-like molecules required for the modulation of the reaction which, analogously to vertebrate cytokines [13], are quickly synthesised and released. The released molecules may either be responsible for the progressive recruitment and activation of the circulating morula cells or directly modulate the rejection reaction itself. The reported significant decrease of the in vitro cytotoxicity, induced by heterologous BP, in the presence of anti-cytokine antibodies suggests that secreted anti-TNF-a- and anti-IL1-a-immunopositive molecules do exert a role in the rejection reaction. The fact that the percentage of morula cells

887

showing immunopositivity to the anti-TNF-a-antibody is much higher that that of cells with positivity to anti-IL-1-a-antibody may mean that, as for vertebrate cytokines [13], Botryllus IL-1-a-immunopositive molecules are synthesised and secreted upon the induction by TNF-a-immunopositive molecules. The absence of effects of anti-cytokine antibodies on morula cell degranulation indicates that, in the series of events leading to cytotoxicity, these molecules exert their role on a step subsequent to morula cell degranulation, for instance as modulators of phenoloxidase activity, as suggested recently by Olivares et al. [37], who demonstrated the activation of phenoloxidase by TNF-a-like molecules in the annelid Eisenia foetida, or as inducers of NO synthesis. As far as the last point is concerned, mammalian inflammatory cytokines are known to induce activated leukocytes to synthesise NO, which is believed to play a role in tissue destruction [38, 39]. Our experiments, showing a significant increase in nitrite ions in the incubation medium in the presence of heterologous BP, indicate the production of NO by Botryllus haemocytes, which may contribute to the induction of cytotoxicity since NO readily reacts with the superoxide anions, produced by phenoloxidase activity, to form strong oxidising agents such as peroxinitrites [40]. This assumption is corroborated by the significant inhibition of in vitro cytotoxicity by L -NAME, an inhibitor of NO synthase [41]. There is general agreement that invertebrate cytokine-like molecules are not homologous to vertebrate cytokines [42]. According to this view, recent efforts to find cytokine genes in the ascidian genome [43] have led to poor results, indicating the absence of any IL-1 genes and the probable presence of some putative members of the TNF family. Nevertheless, functional studies by various authors have reported the presence of cytokine-like molecules in invertebrates [14 –23]. Although the ligands of anti-cytokine antibodies on Botryllus haemocytes remain unknown, it is our opinion that some of the ascidian cytokine-like molecules, although different from vertebrate cytokines in the nucleotide sequence of their genes, should share a common structure with their vertebrate counterparts in, at least, some of the domains or determinants which are essential for biological activity and this can explain their

888

F. Cima et al. / Developmental and Comparative Immunology 28 (2004) 881–889

recognition by anti-mammalian cytokine antibodies. The observation that, in simpler eukaryotic organisms such as the ciliate protozoan Euplotes raikovi, mammalian cytokines are interchangeable with the Er-1 pheromone supports the above hypothesis: Er-1 can bind to the mammalian IL-2 receptor and vice versa, and both EGF and IL-2 can inhibit the binding of Er-1 to its receptor. In such organism, a similarity in the tridimensional structure of Er-1 and mammalian cytokines and the presence of short shared aminoacid sequences were demonstrated [44, 45]. In addition, in molluscs, recombinant mammalian cytokines can modulate immune responses, and stimulated immunocytes acquire immunopositivity to the antibodies raised against the same cytokines [18,19,23,46]. Future studies will try to better elucidate the nature and role of anti-TNF-a- and anti-IL1-a-immunopositive molecules in Botryllus immunobiology.

Acknowledgements The authors wish to thank Mr M. Del Favero for technical help. This work was supported by the Italian M.I.U.R.

References [1] Rinkevich B. Aspects of the incompatibility nature in botryllid ascidians. Anim Biol 1992;1:17– 28. [2] Scofield VL, Nagashima LS. Morphology and genetics of rejection reactions between oozooids from the tunicate Botryllus schlosseri. Biol Bull 1983;165:733–44. [3] Sabbadin A, Zaniolo G, Ballarin L. Genetic and cytological aspects of histocompatibility in ascidians. Boll Zool 1992;59: 167–73. [4] Saito Y, Hirose E, Watanabe H. Allorecognition in compound ascidians. Int J Dev Biol 1994;38:237–47. [5] Ballarin L, Cima F, Sabbadin A. Morula cells and histocompatibility in the colonial ascidian Botryllus schlosseri. Zool Sci 1995;12:757– 64. [6] Shirae M, Saito Y. A comparison of hemocytes and their phenoloxidase activity among botryllid ascidians. Zool Sci 2000;17:881– 91. [7] Shirae M, Ballarin L, Frizzo A, Saito Y, Hirose E. Involvement of quinones and phenoloxidase in the allorejection reaction in a colonial ascidian Botrylloides simodensis: histochemical and immunohistochemical study. Mar Biol 2002;141:659–65.

[8] Rinkevich B, Tartakover S, Gershon H. Contribution of morula cells to allogeneic responses in the colonial urochordate Botryllus schlosseri. Mar Biol 1998;131:227–36. [9] Hirose E, Saito Y, Watanabe H. Allogeneic rejection induced by cut surfaces contact in the compound ascidian, Botrylloides simodensis. Invertebr Reprod Dev 1990;17:159–64. [10] Ballarin L, Cima F, Sabbadin A. Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri. Dev Comp Immunol 1998;22:479 –92. [11] Ballarin L, Cima F, Floreani M, Sabbadin A. Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri. Comp Biochem Physiol 2002; 133C:411–8. [12] Saito Y, Watanabe H. Partial biochemical characterisation of humoral factors involved in the nonfusion reaction of a botryllid ascidian, Botrylloides simodensis. Zool Sci 1984;1: 229 –35. [13] Abbas AK, Lichtman AH, Pober JS. Cellular and molecular immunology. Philadelphia: WB Saunders Company; 2000. [14] Hughes TK, Smith EM, Chin R, Cadet P, Sinisterra J, Leung MK, Shipp MA, Scharrer B, Stefano GB. Interactions of immunoreactive monokines (interleukin-1 and tumor necrosis factor) in the bivalve mollusc Mytilus edulis. Proc Natl Acad Sci USA 1990;87:4426–9. [15] Hughes TK, Smith EM, Stefano GB. Detection of immunoreactive interleukin-6 in invertebrate hemolymph and nervous tissue. Prog Neurol Endocrin Immunol 1991;4:234 –9. [16] Beck G, Habicht GS. Primitive cytokines: harbingers of vertebrate defense. Immunol Today 1991;12:180–3. [17] Ouwe-Missi-Oukem-Boyer O, Porchet E, Capron A, Dissous C. Characterization of immunoreactive TNFa molecules in the gastropod Biomphalaria glabrata. Dev Comp Immunol 1994;18:211–8. [18] Cooper EL, Franchini A, Ottaviani E. Earthworm coelomocytes possess immunoreactive cytokines and POMC-derived peptides. Anim Biol 1995;4:25–9. [19] Ottaviani E, Franchini A, Cassanelli S, Genedani S. Cytokines and invertebrate immune responses. Biol Cell 1995;85:87–91. [20] Ottaviani E, Franchini A, Kletsas D, Franceschi C. Presence and role of cytokines and growth factors in invertebrates. Ital J Zool 1996;63:317–23. [21] Franchini A, Miyan JA, Ottaviani E. Induction of ACTH- and TNF-a-like molecules in the hemocytes of Calliphora vomitoria (Insecta, Diptera). Tissue Cell 1996;28:587–92. [22] Beck G. Macrokines: invertebrate cytokine-like molecules? Front Biosci 1998;d559 –69. [23] Granath WO, Connors VA, Tarleton RL. Interleukin 1 activity in haemolymph from strains of the snail Biomphalaria glabrata varying in susceptibility to the human blood fluke Schistosoma mansoni: presence, differential expression, and biological function. Cytokine 1994;6:21–7. [24] Beck G, Vasta GR, Marchalonis JJ, Habicht GS. Characterization of interleukin-1 activity in tunicates. Comp Biochem Physiol 1989;92B:93–8. [25] Beck G, O’Brian RF, Habicht GS, Stillman DL, Cooper EL, Raftos DA. Invertebrate cytokines III: invertebrate interleukin-1-like molecules stimulate phagocytosis by

F. Cima et al. / Developmental and Comparative Immunology 28 (2004) 881–889

[26]

[27]

[28] [29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

tunicate and echinoderm cells. Cell Immunol 1993;146: 284–99. Raftos DA, Cooper EL, Habicht GS, Beck G. Invertebrate cytokines: tunicate cell proliferation stimulated by an interleukin-1-like molecule. Proc Natl Acad Sci USA 1991;88: 9518–22. Raftos DA, Cooper EL, Stillman DL, Habicht GS, Beck G. Invertebrate cytokines II: release of interleukin-1-like molecule from hemocytes stimulated with zymosan. Lymphokine Cytokine Res 1992;11:235 –40. Beck G, Habicht GS. Invertebrate cytokines. Ann NY Acad Sci 1994;712:206–12. Ballarin L, Franchini A, Ottaviani E, Sabbadin A. Morula cells as the main immunomodulatory haemocytes in ascidians: evidences from the colonial species Botryllus schlosseri. Biol Bull 2001;201:59–64. Hsu SM, Raine L, Fanger H. Use of avidin–biotin–peroxidase complex (ABC) immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577–80. Ballarin L, Tonello C, Sabbadin A. Humoral opsonin from the colonial ascidian Botryllus schlosseri as a member of the galectin family. Mar Biol 2000;136:813–22. Ballarin L, Cima F, Sabbadin A. Phagocytosis in the colonial ascidian Botryllus schlosseri. Dev Comp Immunol 1994;18: 467–81. Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. J Immunol 1988;141:2407–12. Shen HM, Sha LX, Kennedy JL, Ou DW. Adrenergic receptors regulate macrophage secretion. Int J Immunopharmacol 1994;16:905 –10. Gorman A, McCarthy J, Finucane D, Reville W, Cotter T. Morphological assessment of apoptosis. In: Cotter TG, Martin SJ, editors. Techniques in apoptosis. A user’s guide. London: Portland Press; 1997. p. 1 –20. Taneda Y, Watanabe H. Studies on colony specificity in the compound ascidian Botryllus primigenus Oka. I. Initiation of nonfusion reaction with special reference to blood cell infiltration. Dev Comp Immunol 1982;6:243 –52. Olivares Fontt E, Beschin A, Van Dijck E, Vercruysse V, Bilej M, Lucas R, De Baetselier P, Vray B. Trypanosoma cruzi is lysed by coelomic cytolytic factor-1, an invertebrate analogue of tumor necrosis factor, and induces phenoloxidase activity in the coelomic fluid of Eisenia foetida foetida. Dev Comp Immunol 2002;26:27–34. Szabo` C, Thiemermann C. Regulation of the expression of the inducible isoform of nitric oxide synthase. In: Ignarro L, Murad F, editors. Nitric Oxide. Biochemistry, molecular

[39]

[40]

[41] [42]

[43]

[44]

[45]

[46]

889

biology and therapeutic implications, New York: Academic Press; 1995. p. 113–53. Kolb H, Kolb-Bachofen V. Nitric oxide in autoimmune disease: cytotoxic or regulatory mediator? Immunol Today 1998;19:556–61. Fukuto JM. Chemistry of nitric oxide: biologically relevant aspects. In: Ignarro L, Murad F, editors. Nitric oxide. Biochemistry, molecular biology and therapeutic implications, New York: Academic Press; 1995. p. 1–15. Whittle BRJ. Nitric oxide in physiology and pathology. Histochem J 1995;27:727–37. Beschin A, Bilej M, Torreele E, De Baetselier P. On the existence of cytokines in invertebrates. Cell Mol Life Sci 2001;58:801–14. Dehal P, Satou Y, Campbell RK, Chapman J, Degnan B, De Tomaso A, Davidson B, Di Gregorio A, Gelpke M, Goodstein D, Harafuji N, Hastings KE, Ho I, Hotta K, Huang W, Kawashima T, Lemaire P, Martinez D, Meinertzhagen IA, Necula S, Nonaka M, Putnam N, Rash S, Saiga H, Satake M, Terry A, Yamada L, Wang H-G, Awazu S, Azumi K, Boore J, Branno M, Chin-bow S, De Santis R, Doyle S, Francino P, Keys DN, Haga S, Hayashi H, Hino K, Imai KS, Inaba K, Kano S, Kobayashi K, Kobayashi M, Lee B, Makabe KW, Manohar C, Matassi G, Medina M, Mochizuki Y, Mount S, Morishita T, Miura S, Nakayama A, Nishizaka S, Nomoto H, Ohta F, Oishi K, Rigoutsos I, Sano M, Sasaki A, Sasakura Y, Shoguchi E, Shin-i T, Spagnuolo A, Stainier D, Suzuki MM, Tassy O, Takatori N, Tokuoka M, Yagi K, Yoshizaki F, Wada S, Zhang C, Hyatt PD, Larimer F, Detter C, Doggett N, Glavina T, Hawkins T, Richardson P, Lucas S, Kohara Y, Levine M, Satoh N, Rokhsar DS. The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 2002;298:2157– 67. Luporini P, Vallesi A, Miceli C, Bradshaw RA. Ciliate pheromones: potential forerunners of self-non-self cell markers. In: So¨derha¨ll K, Iwanaga S, Vasta GR, editors. New directions in invertebrate immunology. Fair Haven: SOS Publications; 1996. p. 343–54. Vallesi A, Giuli G, Ghiara P, Scapigliati G, Luporini P. Structure–function relationships of pheromones of the ciliate Euplotes raikovi with mammalian growth factors: cross reactivity between Er-1 and interleukin-2 systems. Exp Cell Res 1998;241:253– 9. Granath WO, Connors VA, Raines AE. Effects of exogenous interleukin-1b on primary sporocysts of Schistosoma mansoni (Trematoda) incubated with plasma and hemocytes from schistosome-susceptible and resistant Biomphalaria glabrata (Gastropoda). Invertebr Biol 2001;120:365 –71.