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The starfish axial organ: An ancestral lymphoid organ
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, VOI. 4, pp. 605-615, 1980 0145-305X/80/040605-11502.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.
THE STARFISH AXIAL ORGAN : AN ANCESTRAL LYMPHOIDORGAN M. Leclerc, C. Brillouet and G. Luquet D~partement de Biologie, U.E.R. Sciences, 45046 ORLEANSCEDEX, France.
INTRODUCTION In an important study of the morphology and histochemistry of the echinoid axial organ (AO), M i l l o t t and Vevers (1) observed that i t remained the most enigmatic structure in echinoderms. Several distinct functions were proposed but l i t t l e experimental analysis supported various investigators contributed important observations (2-3-5), Farmanfarmaian (personal communication). On the one hand, morphological investigations revealed that the organ is pervaded by an elaborate contractile structure representing a central point of confluence of the perivisceral coelom, the so-called "haemal system", and the water vascular system. On the other hand, the organ may not be considered excretory, genital, vestigial, a source of amoebocytes, pigment, as a site for the destruction of effete amoebocytes, nor as a heart. According to M i l l o t t (6) i t was concerned "in an intimate mechanism of defence against injury and invading organisms".
I . - MORPHOLOGYOF THE AXIAL ORGAN OF A s t ~
r~b~!~_.
The axial complex of echinoderms especially the axial organ i t s e l f has been described for a long time, from the viewpoint of embryology and anatomy but its physiological functions are unclear. In four of the five classes of echinoderms (echino~ds, ophiurids, crinoTds, and asteroTds) the axial complex is located in the interradius n° 2 or "CD interradius" below the madreporic plate. In certain species of asteroTds, a madreporal vesicle or dorsal 605
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sac is also present, but, only in the f i f t h class, the holothurids i t s location is s t i l l controversial. Some authors have described extensively the axial organ of various asteroids. In Aste~Y~ rubens, some p a r t i c u l a r points should be emphasized. The axial organ lies along the stone canal and includes - an oral region surrounded by the l e f t axial sinus and is stretched between the aboral and the oral poles ( f i g . I ) .
M.p J
A.p L,a O.p
S.c W.v.ssH.s
FIG. i - The axial complex of Ast~rias rubens. M.p., madreporic plate. A.p., aboral part. L.a., lateral appendix. O.p., oral part. S.c., stone canal. W.v.s., water vascular system ; and H.s., haemal system.
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An aboral region includes the aboral region i t s e l f and corresponds to the genital bud of young animals (Mac Bride, 1896) (7) and two mammillated lateral appendices (Pl. I, fig. l) which overflow out from the right axial sinus into the coelomic cavity corresponding to gastric hemal tufts. In Aste~na gibbosa, the aboral end of the axial organ is not composed of a madreporic vesicle with its contents, i.e. the head process, thought to be contracrubens, but its role remains unknown. t i l e . Such an organ is found in A s t ~ The so-called "oral part" bounds entirely the stone canal (fig. l) (Pl. I, fig. 2).
PLATE I -
Fig. i : The aboral part. Fig. 2 : The oral part. The scale is the same in figures E.a.s., right axial sinus. S.c., aboral part. L.a.s., left axial appendix. C.c., coelomic cavity.
1 and stone sinus. 0.p.,
5. canal. A.p., L.a., lateral oral part.
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I I . - MORPHOGENESISOF THE AXIAL ORGAN. In general, asteroid larvae have a free-swimming l i f e . This differs from A s t ~ g i b b o s a in which i t extends an adhesive apparatus on the third day of its development. In A s t ~ a gibbosa, the localisation of the stone canal is revealed by histology on the eighth day of larval l i f e . Development of the axial organ has been examined by implanting at about the 12~n day. Mesenchyme cells gather near the stone canal and determine the axial organ (8) whose origin would thus be mesodermic. In older animals (from 8 to lO months), our studies confirm the aboral origin of the axial organ : an excrescence of mesenchyme-cells is followed by an evagination towards the right axial sinus. The two lateral appendices, in the aboral plane bind the aboral portion s ~ u s ~ t o for the upper one, and the oral portion for the lower one. Apparently the formations described by M i l l o t t in echinoTds and called "axial organ" correspond to lateral appendices.
I I I . - HISTOLOGY. The structure of the axial organ is glandular, spongy and crossed by connective tissue and muscular fibers. Many f o l l i c l e s of heterogeneous size with clear zones are lined by cellular cords (Pl. I I , f i g . l e t 2).
PLATE II - Fig. 1 and 2 : The oral part at various magnifications.
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No nervous fibers have been observed but rather a g l i o - i n t e r s t i t i a l system is present. Three cellular types were observed by electron microscopy, two of which are reminiscent of small "ljnnphocyte-like" cells (shape of the nucleus few intracytoplasmic elements : mitochondriae (Pl. I l l , fig. l and 2), while the third (Pl. I l l , fig. 3) is similar to phagocytic cells.
IV.- FUNCTIONAL ANALOGYBETWEENAXIAL ORGAN(AO) CELLS AND VERTEBRATE LYMPHO-
CYTES.
As, indicated in the introduction, among the various roles envisaged for the axial organ, the most interesting suggestion was proposed by M i l l o t t (6) who strongly supported its involvment in defense mechanisms. Moreover, recent studies of Hildemann and coworkers have clearly established the existence, in echinoderms, of mechanisms of cellular immunity (9, lO). In Asteroidae (Protoreaster nodosus) and Holothuroidae (Cucuma~iatricolor) (9) f i r s t set intertegumentary allografts were completely rejected after 4-6 months. Orthotopic control autografts were not affected and were f u l l y viable in species of both classes under the same conditions. In Cucumap~/a, second-set grafts performed on animals after f i r s t rejection resulted in intensified and accelerated rejection indicating the existence of an immunological response with, at least, short term memory. A similar result was obtained in Protor~ter nodosus. Coelomocytes of various types, including hemocytes and phagocytic cells, i n f i l t r a t e d skin allografts undergoing rejection. More specifically coeIomocytes were interpreted as leukocytes, hemocytes as 13nnphocytes and phagocytic cells as macrophages. Further studies of the same research group (lO) with the starfish Dermast~as i m b r i ~ a revealed rejection with loss of pigmentation, edematous swelling an necrosis of allografts with accompanying heavy i n f i l t r a t i o n by hemocytes and phagocytic cells. From these studies, its appears that certain starfishes possess a discriminating adaptive cell-mediated immune response analogous to that found in vertebrates. The existence of cell-mediated immunity in high invertebrates such as echinoids could be expected since, in lower invertebrates (earthworms) this kind of mechanism was clearly established by the pioneer experiments of Chateaureynaud-Duprat ( l l ) and Cooper (12, 13). Finally, no clear data are available demonstrating that graft rejection in echinoids resulted from activity of cells comparable to vertebrates lymphocytes. By contrast, the situation in earthworms is different, since recent work shows that circulating cells of these invertebrate (the coelomocytes) (14) show properties of l.~imphocytes. In particular, addition of PHA seems to stimulate these cells to incorporate thymidine, just as in vertebrate T lymphocytes. Thus, morphological and structural observations are in this case supported by at least one physiological experiment involving membrane lectin receptors and activation of DNA
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:/
PLATE I I I -
Fig. 1 and 2 : "Lymphocyte-like" AO cells. Fig. 3 : A phagocytic cell. The scale is the same in figures i, 2 and 3.
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synthesis. We have performed a number of similar experiments to examine whether some correlation exists between axial organ cells and properties considered as related to the immunocompetence of vertebrate ljnnphocytes, " i n vivo" and "in v~t~o". We systematically investigated such properties by comparing the behaviour of AO cells and of vertebrate lymphocytes to that of other echinoderm c e l l s , p a r t i c u l a r l y coelomocytes. However, since the usual conditions prevailing in a marine laboratory were not available to us, we attempted to use new methods by exploring the eventual interactions between AO cells and vertebrate tissues. In a f i r s t series of experiments, we observed that a f t e r implantation of the oral part of the axial organ under the skin of a nude mouse (congenitally athymic mouse unable to effect g r a f t r e j e c t i o n ) , AO cells appeared to be able to survive for two or three weeks (15). By contrast, cells of the brachial digestive organ did not survive under the same conditions. This important f i n ding led us to investigate whether AO cells would be capable of inducing certain c h a r a c t e r i s t i c reactions in mice or other vertebrates. Two d i f f e r e n t effects of i n j e c t i o n of AO cells were studied : t h e i r a b i l i t y to induce angiogenesis reactions in i r r a d i a t e d mice and t h e i r a b i l i t y to provoke splenomegaly in neonatal chickens.
V.- In vivo DATA SUGGESTING THE EXISTENCE OF T-LIKE CELLS AMONGAO CELLS.
I. Angiogenesis. This reaction results in development in the internal surface of the skin a network of supplemental c a p i l l a r i e s (extra-vessels) (16, 17), as in certain tumors (18) or in hypersensitivity reactions (19). Recently, Sidky and Auerbach (20) showed that the reaction could also r e s u l t from the i n j e c t i o n of lymphocytes from allogenic strains of mice. No reaction was observed when isogenic lymphocytes were used or when allogenic l~nnphocytes were previously treated with mitomycin C which impairs DNA r e p l i c a t i o n . In addition, Sidky and Auerbach showed that the extent of the reaction could be estimated by the number of extra vessels and was dependent upon the number of injected allogenic lymphocytes. Using this procedure, we injected AO cells into mice and observed angiogenesis with similar c h a r a c t e r i s t i c s . The extent of the angiogenesis reaction depends on the number of AO cells injected and no reaction occurs when AO cells were treated with mitomycin C nor when other cells i . e coelomocytes of s t a r f i s h were injected.
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2. Splenomegaly. Since interpretation of the angiogenesis reaction as being equivalent to a cell-mediated immune reaction is s t i l l open to discussion, we tested the capacity of AO cells to provoke splenomegaly in neo-natal chickens (16), Chick embryos were inoculated on day 16 with AO cells and, six days after, the one day-old chicks were sacrificed. Once again the indices of splenomegaly were clearly related to the number of AO cells injected. Obviously the previous results may be correlated with cell-mediated immunity reactions and noted above, i t is not surprising to find a sort of "T-like" cell response in higher invertebrates. However, our technique shows two important aspects. F i r s t l y , these experiments do not require any special installation, secondly, the experimental period is short : 2-6 days, whereas experiments on graft rejection require several months. The most important point is however that AO cells provided from a particular organ which could be an ancestral model of primary lymphoTd organ.
VI.- In ux~t~o EFFECTSOF LECTINS ON AO CELLS.
I. Agglutination of AO cells. In further experiments, effects of various lectins were investigated. Binding of lectins to axial organ cells was f i r s t studied (agglutination t i t e r s and cell specificity). Using fluorescein isothiocarbamyl derivatives of lectins ConA binds a large number of cells while WGA, SBA and PNA bound only a small number.
2. Use of lectin to separate AO cells. Separation of axial organ cell subpopulations by the use of lectins may be compared to the separation of mouse lymphocytes obtained by Schnebli and Dukor (21) : Mouse B lymphocytes and T cells resistant to cortisone were easily agglutinated by WGA or SBA but other T lymphocytes were not. AO cells bearing SBA or WGA receptors do not induce angiogenesis while AO cells bearing ConA receptors give a positive GVH reaction inducing angiogenesis like T lymphocytes of vertebrates. On the basis of sugar specificities of lectins, two subpopulations appeared : one which presented the characteristic of a T-like subpopulation and one which might be implicated in "humoral type" reactions (22).
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3. In ux]t~to detection of a "B-like" cell subpopulation. Starfishes were injected with two different proteins (bovine serum albumin and whale myoglobin). Cell suspensions were prepared from the axial organ of injected and untreated animals. After exposure of these suspensions to SBA, agglutinable and non agglutinable cell subpopulations were obtained and compared as to their binding to fluorescent conjugates of the same proteins : FITC-serum albumin and FITC-myoqlobin. The SBA aqqlutinable subpopulation from injected animals showed a significantly higher number of membrane fluorescent cells than the non-agglutinable subpopulation. No significant membrane fluorescence was observed in the cell subpopulations obtained from untreated animals. The membrane fluorescence appeared mainly when the protein of the fluorescent conjugate was the same as that used for injecting the animal (Leclerc et a l . , 1979) (23).
4. Stimulation of AO cells, of AO subpopulations by lectins and LPS (Lipopolysa~ch'aride of S~mone~Zat x l p h ~ ) : Mitogenic effect. The PWM (Pokeweed Mitogen) lectin of PhgtoZac~ a m ~ n a stimulates AO cells after a short time of contact with the lectin (incorporation of thymidine 3H maximum after 24 hours of culture). The ConA lectin (Canav~ e~iformic) stimulates specifically the "T-like" subpopulation obtained after separation of the whole population into two subpopulations on the nylon wool column (a "T-like" and a "B-like" subpopulation). The LPS stimulates specifically the "B-like" subpopulation.
CONCLUSIONS
From the results reported in this brief review, i t seems likely that a highly sophisticated system of immunity took place in the evolution before the emergence of fishes, i.e in echinoderms which emerged during the Precambrian period. This system involves not only defense against injury or aggressions by non specific processes, but also specific responses to external agents. One aspect of this response clearly resembles the T response of vertebrates, whereas another could be a B response, suggesting the presence of cell subpopulations the axial organ : a "T-like" subpopulation, a "B-like" one. Obviously the existence of the l a t t e r is less clearly established than that of the former. The reason is that i t has not yet been possible to isolate and to analyse specific products secreted in the internal medium of these animals, such as immunoglobulins. However, from a general point of view, i t is d i f f i c u l t to imagine that two different kinds of proteins have been "invented" through phylogenesis : immunoglobulins, at the step when the hagfish appears which would have replaced another kind of protein, completely different in structure, and which would have existed in previous steps and then disappeared.
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This "dichotomy" cannot be excluded, but would seem unreasonable. Thus, the f u t u r e of t h i s work c e r t a i n l y implies more research with more refined procedures to characterize immunoglobulin l i k e p r o t e i n s . On the other hand, the f a c t that no primary lymphoid organ such as the thymus or bursa has been clearly defined in hagfish does not prove t h a t other lymphoid organs with an unexpected structure do not replace them in these vertebrates. I t is clear t h a t the way of evolution is not l i n e a r . I t is possible, t h a t c e r t a i n b i o l o g i c a l systems of great i n t e r e s t are l o s t in c e r t a i n special cases : f o r example the hagfish is a p a r a s i t i c animal and t h i s s i t u a t i o n perhaps r e s u l t s in extensive genetic deletions. F i n a l l y , i f one admits t h a t our immunofluorescence data are compatible with the neo-synthesis of s p e c i f i c products in response to s p e c i f i c antigen, i t is possible t h a t the biochemical system of excretion does not e x i s t in these c e l l s . In t h i s case, the products would remain linked to the cell membrane. Preliminary observations suggest t h a t , at l e a s t , a part of the complexes formed between antigen and fluorescent products leave the c e l l s in the form of small vesicles of 0.8~ diameter. I t is possible t h a t such formations are most e f f i c i e n t and represent, in some way, a b i o l o g i c a l advantage f o r these marine i n vertebrates. The authors are g r e a t l y indebted to Pr R. Delavault f o r s k i l l e d a s s i s tance, Pr J. Panijel f o r c o l l a b o r a t i o n and to Pr E.L. Cooper and Mrs J. Cerveau in the preparation of the manuscript. This work was supported by grants from INSERM (contrat l i b r e M. Leclerc n° 79.1.113.1).
REFERENCES ,
MILLOTT, N. and VEVERS, H.G. The morphology and histochemistry of the Echinoid axial organ. Phil. Trans. London, 253, 201, 1968.
. HAMANN, O. Beitr~ge zur H i s t o l o g i e der Echinodermen 2 : Die Asteriden, anatomish und h i s t o l o g i s h Untersuch, 119 p. FisheJt V~lag, lena, 1885. . LEIPOLDT, F. Das anglebliche Exketionsorgan der Seeigel, untersucht an Sphaerechin~ g r a n u l ~ und Dorocida~is papZllata. Z. Wiss. Zool., 55, 585, 1893. 4. BOOLOOTIAN, R.A. and CAMPBELL, J.L. A p r i m i t i v e heart in the EchinoTd Strongylocent~otus p ~ p u r a t ~ . Science, 145, 173, 1964. 5. MILLOTT, N. and VEVERS, H.G. Axial organ and f l u i d c i r c u l a t i o n in Echinoids. NatuAe, 204, 1216, 1964. . MILLOTT, N. The axial organ of Echinoids. R e - i n t e r p r e t a t i o n of i t s s t r u c t u r e and f u n c t i o n . Symp. Zool. Soc. Lond., 20, 53, 1967. . MAC BRIDE, E.W. The development of Asterina gibbosa. Quart. J. A~e~. S ~ . , 38, 339, 1896.
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8. LECLERC, M. L'organe axial et ses relations avec la sexualit@ et l'immunit~ chez les Ast~rides. Th~se Doctorat ks-Sciences, Orl@ans, 1974. 9. HILDEMANN, W.A. and DIX, T.G. Transplantation reactions of tropical australian echinoderms. Transplantation, 14, 624, 1972. IO.KARP, R.D.and HILDEMANN, W.H. Specific a l l o g r a f t r e a c t i v i t y in the sea star D~masterias imbricata. Transplantation, 22, 434, 1976. II.DUPRAT, P. Mise en ~vidence de r~actions immunitaires dans les homogreffes de paroi du corps chez le Lombricien Eisenia fo~tida. Ann. Inst. Paste~% 113, 867, 1967. 12.COOPER, E.L. Rejection of xenografts exchanged between Lumbric~ t e r r ~ t ~ and Eisenia fo~tida. Transplantation, 6, 322, 1968. 13.COOPER, E.L. Transplantation immunity in helminth and annelids. Transplant. Proc., 2, 216, 1970. 14.ROCH, Ph. R6activit~ in v i t r o des leucocytes du Lombricien EX~enia foetida. C.R. Acad. So. Paris, 284, 705, 1977. 15.LECLERC, M., PANIJEL, J. and TOYAMA, K. Heterotransplantation of invertebrate organs to congenitally athymic (nude) mice. T~uznsplavut., 19, 74, 1974. 16.LECLERC, M., REDZINIAK, G., PANIJEL, J. and El LABABIDI, M. Reactions induced in Vertebrates by Invertebrate cell suspensions. I. Specific effects of sea star axial organ cells i n j e c t i o n . Dev. and Comp. Immunol., I , 299, 1977. 17.LECLERC, M., REDZlNIAK, G., PANIJEL, J. and El LABABIDI, M. Reactions induced in Vertebrate by Invertebrate cell suspensions. I I . Non adherent axial organ cells as effector c e l l s . Dev. and Comp. Immunol., I , 311, 1977. 18. FOLKMAN, J. Tumor angiogenesis. Aduan. Canc~. Res., 19, 331, 1974. 19. POLVERINI, P.J., COTRAN, R.S. and SHOLLEY, M.M. Endothelial p r o l i f e r a t i o n in the delayed hypersensitivity reaction : an autoradiographic study. J. Im~unol., 118, 529, 1977. 20. SlDKY, Y.A. and AUERBACH, R. Lymphocyte induced angiogenesis : a quantitative and sensitive assay of the graft-versus host reaction. J. Exp. Med., 141, 1084, 1975. 21. SCHNEBLI, H.P. and DUKOR, P. Plant agglutinins used to distinguish between d i f f e r e n t classes of mouse lymphocytes. Eu~. J. Immunol., 2, 607, 1972. 22. REDZINIAK, G., LECLERC, M., PANIJEL, J. and MONSIGNY, M. Separation of two d i f f e r e n t populations of axial organ cells of A s t ~ ru6ens by the use of l e c t i n s . Biocf~6mie, 60, 525, 1978. 23. LECLERC, M., PANIJEL, J . , REDZINIAK, G., BRILLOUET, C. and BINAGHI, R. Correlation between the i n j e c t i o n of specific proteins in starfishes and the appearance of binding sites for the same proteins in cell populations of t h e i r axial organ. Cell. Immunol. in press.
THE STARFISH AXIAL ORGAN : AN ANCESTRAL LYMPHOIDORGAN M. Leclerc, C. Brillouet and G. Luquet D~partement de Biologie, U.E.R. Sciences, 45046 ORLEANSCEDEX, France.
INTRODUCTION In an important study of the morphology and histochemistry of the echinoid axial organ (AO), M i l l o t t and Vevers (1) observed that i t remained the most enigmatic structure in echinoderms. Several distinct functions were proposed but l i t t l e experimental analysis supported various investigators contributed important observations (2-3-5), Farmanfarmaian (personal communication). On the one hand, morphological investigations revealed that the organ is pervaded by an elaborate contractile structure representing a central point of confluence of the perivisceral coelom, the so-called "haemal system", and the water vascular system. On the other hand, the organ may not be considered excretory, genital, vestigial, a source of amoebocytes, pigment, as a site for the destruction of effete amoebocytes, nor as a heart. According to M i l l o t t (6) i t was concerned "in an intimate mechanism of defence against injury and invading organisms".
I . - MORPHOLOGYOF THE AXIAL ORGAN OF A s t ~
r~b~!~_.
The axial complex of echinoderms especially the axial organ i t s e l f has been described for a long time, from the viewpoint of embryology and anatomy but its physiological functions are unclear. In four of the five classes of echinoderms (echino~ds, ophiurids, crinoTds, and asteroTds) the axial complex is located in the interradius n° 2 or "CD interradius" below the madreporic plate. In certain species of asteroTds, a madreporal vesicle or dorsal 605
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sac is also present, but, only in the f i f t h class, the holothurids i t s location is s t i l l controversial. Some authors have described extensively the axial organ of various asteroids. In Aste~Y~ rubens, some p a r t i c u l a r points should be emphasized. The axial organ lies along the stone canal and includes - an oral region surrounded by the l e f t axial sinus and is stretched between the aboral and the oral poles ( f i g . I ) .
M.p J
A.p L,a O.p
S.c W.v.ssH.s
FIG. i - The axial complex of Ast~rias rubens. M.p., madreporic plate. A.p., aboral part. L.a., lateral appendix. O.p., oral part. S.c., stone canal. W.v.s., water vascular system ; and H.s., haemal system.
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An aboral region includes the aboral region i t s e l f and corresponds to the genital bud of young animals (Mac Bride, 1896) (7) and two mammillated lateral appendices (Pl. I, fig. l) which overflow out from the right axial sinus into the coelomic cavity corresponding to gastric hemal tufts. In Aste~na gibbosa, the aboral end of the axial organ is not composed of a madreporic vesicle with its contents, i.e. the head process, thought to be contracrubens, but its role remains unknown. t i l e . Such an organ is found in A s t ~ The so-called "oral part" bounds entirely the stone canal (fig. l) (Pl. I, fig. 2).
PLATE I -
Fig. i : The aboral part. Fig. 2 : The oral part. The scale is the same in figures E.a.s., right axial sinus. S.c., aboral part. L.a.s., left axial appendix. C.c., coelomic cavity.
1 and stone sinus. 0.p.,
5. canal. A.p., L.a., lateral oral part.
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I I . - MORPHOGENESISOF THE AXIAL ORGAN. In general, asteroid larvae have a free-swimming l i f e . This differs from A s t ~ g i b b o s a in which i t extends an adhesive apparatus on the third day of its development. In A s t ~ a gibbosa, the localisation of the stone canal is revealed by histology on the eighth day of larval l i f e . Development of the axial organ has been examined by implanting at about the 12~n day. Mesenchyme cells gather near the stone canal and determine the axial organ (8) whose origin would thus be mesodermic. In older animals (from 8 to lO months), our studies confirm the aboral origin of the axial organ : an excrescence of mesenchyme-cells is followed by an evagination towards the right axial sinus. The two lateral appendices, in the aboral plane bind the aboral portion s ~ u s ~ t o for the upper one, and the oral portion for the lower one. Apparently the formations described by M i l l o t t in echinoTds and called "axial organ" correspond to lateral appendices.
I I I . - HISTOLOGY. The structure of the axial organ is glandular, spongy and crossed by connective tissue and muscular fibers. Many f o l l i c l e s of heterogeneous size with clear zones are lined by cellular cords (Pl. I I , f i g . l e t 2).
PLATE II - Fig. 1 and 2 : The oral part at various magnifications.
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No nervous fibers have been observed but rather a g l i o - i n t e r s t i t i a l system is present. Three cellular types were observed by electron microscopy, two of which are reminiscent of small "ljnnphocyte-like" cells (shape of the nucleus few intracytoplasmic elements : mitochondriae (Pl. I l l , fig. l and 2), while the third (Pl. I l l , fig. 3) is similar to phagocytic cells.
IV.- FUNCTIONAL ANALOGYBETWEENAXIAL ORGAN(AO) CELLS AND VERTEBRATE LYMPHO-
CYTES.
As, indicated in the introduction, among the various roles envisaged for the axial organ, the most interesting suggestion was proposed by M i l l o t t (6) who strongly supported its involvment in defense mechanisms. Moreover, recent studies of Hildemann and coworkers have clearly established the existence, in echinoderms, of mechanisms of cellular immunity (9, lO). In Asteroidae (Protoreaster nodosus) and Holothuroidae (Cucuma~iatricolor) (9) f i r s t set intertegumentary allografts were completely rejected after 4-6 months. Orthotopic control autografts were not affected and were f u l l y viable in species of both classes under the same conditions. In Cucumap~/a, second-set grafts performed on animals after f i r s t rejection resulted in intensified and accelerated rejection indicating the existence of an immunological response with, at least, short term memory. A similar result was obtained in Protor~ter nodosus. Coelomocytes of various types, including hemocytes and phagocytic cells, i n f i l t r a t e d skin allografts undergoing rejection. More specifically coeIomocytes were interpreted as leukocytes, hemocytes as 13nnphocytes and phagocytic cells as macrophages. Further studies of the same research group (lO) with the starfish Dermast~as i m b r i ~ a revealed rejection with loss of pigmentation, edematous swelling an necrosis of allografts with accompanying heavy i n f i l t r a t i o n by hemocytes and phagocytic cells. From these studies, its appears that certain starfishes possess a discriminating adaptive cell-mediated immune response analogous to that found in vertebrates. The existence of cell-mediated immunity in high invertebrates such as echinoids could be expected since, in lower invertebrates (earthworms) this kind of mechanism was clearly established by the pioneer experiments of Chateaureynaud-Duprat ( l l ) and Cooper (12, 13). Finally, no clear data are available demonstrating that graft rejection in echinoids resulted from activity of cells comparable to vertebrates lymphocytes. By contrast, the situation in earthworms is different, since recent work shows that circulating cells of these invertebrate (the coelomocytes) (14) show properties of l.~imphocytes. In particular, addition of PHA seems to stimulate these cells to incorporate thymidine, just as in vertebrate T lymphocytes. Thus, morphological and structural observations are in this case supported by at least one physiological experiment involving membrane lectin receptors and activation of DNA
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:/
PLATE I I I -
Fig. 1 and 2 : "Lymphocyte-like" AO cells. Fig. 3 : A phagocytic cell. The scale is the same in figures i, 2 and 3.
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synthesis. We have performed a number of similar experiments to examine whether some correlation exists between axial organ cells and properties considered as related to the immunocompetence of vertebrate ljnnphocytes, " i n vivo" and "in v~t~o". We systematically investigated such properties by comparing the behaviour of AO cells and of vertebrate lymphocytes to that of other echinoderm c e l l s , p a r t i c u l a r l y coelomocytes. However, since the usual conditions prevailing in a marine laboratory were not available to us, we attempted to use new methods by exploring the eventual interactions between AO cells and vertebrate tissues. In a f i r s t series of experiments, we observed that a f t e r implantation of the oral part of the axial organ under the skin of a nude mouse (congenitally athymic mouse unable to effect g r a f t r e j e c t i o n ) , AO cells appeared to be able to survive for two or three weeks (15). By contrast, cells of the brachial digestive organ did not survive under the same conditions. This important f i n ding led us to investigate whether AO cells would be capable of inducing certain c h a r a c t e r i s t i c reactions in mice or other vertebrates. Two d i f f e r e n t effects of i n j e c t i o n of AO cells were studied : t h e i r a b i l i t y to induce angiogenesis reactions in i r r a d i a t e d mice and t h e i r a b i l i t y to provoke splenomegaly in neonatal chickens.
V.- In vivo DATA SUGGESTING THE EXISTENCE OF T-LIKE CELLS AMONGAO CELLS.
I. Angiogenesis. This reaction results in development in the internal surface of the skin a network of supplemental c a p i l l a r i e s (extra-vessels) (16, 17), as in certain tumors (18) or in hypersensitivity reactions (19). Recently, Sidky and Auerbach (20) showed that the reaction could also r e s u l t from the i n j e c t i o n of lymphocytes from allogenic strains of mice. No reaction was observed when isogenic lymphocytes were used or when allogenic l~nnphocytes were previously treated with mitomycin C which impairs DNA r e p l i c a t i o n . In addition, Sidky and Auerbach showed that the extent of the reaction could be estimated by the number of extra vessels and was dependent upon the number of injected allogenic lymphocytes. Using this procedure, we injected AO cells into mice and observed angiogenesis with similar c h a r a c t e r i s t i c s . The extent of the angiogenesis reaction depends on the number of AO cells injected and no reaction occurs when AO cells were treated with mitomycin C nor when other cells i . e coelomocytes of s t a r f i s h were injected.
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2. Splenomegaly. Since interpretation of the angiogenesis reaction as being equivalent to a cell-mediated immune reaction is s t i l l open to discussion, we tested the capacity of AO cells to provoke splenomegaly in neo-natal chickens (16), Chick embryos were inoculated on day 16 with AO cells and, six days after, the one day-old chicks were sacrificed. Once again the indices of splenomegaly were clearly related to the number of AO cells injected. Obviously the previous results may be correlated with cell-mediated immunity reactions and noted above, i t is not surprising to find a sort of "T-like" cell response in higher invertebrates. However, our technique shows two important aspects. F i r s t l y , these experiments do not require any special installation, secondly, the experimental period is short : 2-6 days, whereas experiments on graft rejection require several months. The most important point is however that AO cells provided from a particular organ which could be an ancestral model of primary lymphoTd organ.
VI.- In ux~t~o EFFECTSOF LECTINS ON AO CELLS.
I. Agglutination of AO cells. In further experiments, effects of various lectins were investigated. Binding of lectins to axial organ cells was f i r s t studied (agglutination t i t e r s and cell specificity). Using fluorescein isothiocarbamyl derivatives of lectins ConA binds a large number of cells while WGA, SBA and PNA bound only a small number.
2. Use of lectin to separate AO cells. Separation of axial organ cell subpopulations by the use of lectins may be compared to the separation of mouse lymphocytes obtained by Schnebli and Dukor (21) : Mouse B lymphocytes and T cells resistant to cortisone were easily agglutinated by WGA or SBA but other T lymphocytes were not. AO cells bearing SBA or WGA receptors do not induce angiogenesis while AO cells bearing ConA receptors give a positive GVH reaction inducing angiogenesis like T lymphocytes of vertebrates. On the basis of sugar specificities of lectins, two subpopulations appeared : one which presented the characteristic of a T-like subpopulation and one which might be implicated in "humoral type" reactions (22).
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3. In ux]t~to detection of a "B-like" cell subpopulation. Starfishes were injected with two different proteins (bovine serum albumin and whale myoglobin). Cell suspensions were prepared from the axial organ of injected and untreated animals. After exposure of these suspensions to SBA, agglutinable and non agglutinable cell subpopulations were obtained and compared as to their binding to fluorescent conjugates of the same proteins : FITC-serum albumin and FITC-myoqlobin. The SBA aqqlutinable subpopulation from injected animals showed a significantly higher number of membrane fluorescent cells than the non-agglutinable subpopulation. No significant membrane fluorescence was observed in the cell subpopulations obtained from untreated animals. The membrane fluorescence appeared mainly when the protein of the fluorescent conjugate was the same as that used for injecting the animal (Leclerc et a l . , 1979) (23).
4. Stimulation of AO cells, of AO subpopulations by lectins and LPS (Lipopolysa~ch'aride of S~mone~Zat x l p h ~ ) : Mitogenic effect. The PWM (Pokeweed Mitogen) lectin of PhgtoZac~ a m ~ n a stimulates AO cells after a short time of contact with the lectin (incorporation of thymidine 3H maximum after 24 hours of culture). The ConA lectin (Canav~ e~iformic) stimulates specifically the "T-like" subpopulation obtained after separation of the whole population into two subpopulations on the nylon wool column (a "T-like" and a "B-like" subpopulation). The LPS stimulates specifically the "B-like" subpopulation.
CONCLUSIONS
From the results reported in this brief review, i t seems likely that a highly sophisticated system of immunity took place in the evolution before the emergence of fishes, i.e in echinoderms which emerged during the Precambrian period. This system involves not only defense against injury or aggressions by non specific processes, but also specific responses to external agents. One aspect of this response clearly resembles the T response of vertebrates, whereas another could be a B response, suggesting the presence of cell subpopulations the axial organ : a "T-like" subpopulation, a "B-like" one. Obviously the existence of the l a t t e r is less clearly established than that of the former. The reason is that i t has not yet been possible to isolate and to analyse specific products secreted in the internal medium of these animals, such as immunoglobulins. However, from a general point of view, i t is d i f f i c u l t to imagine that two different kinds of proteins have been "invented" through phylogenesis : immunoglobulins, at the step when the hagfish appears which would have replaced another kind of protein, completely different in structure, and which would have existed in previous steps and then disappeared.
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This "dichotomy" cannot be excluded, but would seem unreasonable. Thus, the f u t u r e of t h i s work c e r t a i n l y implies more research with more refined procedures to characterize immunoglobulin l i k e p r o t e i n s . On the other hand, the f a c t that no primary lymphoid organ such as the thymus or bursa has been clearly defined in hagfish does not prove t h a t other lymphoid organs with an unexpected structure do not replace them in these vertebrates. I t is clear t h a t the way of evolution is not l i n e a r . I t is possible, t h a t c e r t a i n b i o l o g i c a l systems of great i n t e r e s t are l o s t in c e r t a i n special cases : f o r example the hagfish is a p a r a s i t i c animal and t h i s s i t u a t i o n perhaps r e s u l t s in extensive genetic deletions. F i n a l l y , i f one admits t h a t our immunofluorescence data are compatible with the neo-synthesis of s p e c i f i c products in response to s p e c i f i c antigen, i t is possible t h a t the biochemical system of excretion does not e x i s t in these c e l l s . In t h i s case, the products would remain linked to the cell membrane. Preliminary observations suggest t h a t , at l e a s t , a part of the complexes formed between antigen and fluorescent products leave the c e l l s in the form of small vesicles of 0.8~ diameter. I t is possible t h a t such formations are most e f f i c i e n t and represent, in some way, a b i o l o g i c a l advantage f o r these marine i n vertebrates. The authors are g r e a t l y indebted to Pr R. Delavault f o r s k i l l e d a s s i s tance, Pr J. Panijel f o r c o l l a b o r a t i o n and to Pr E.L. Cooper and Mrs J. Cerveau in the preparation of the manuscript. This work was supported by grants from INSERM (contrat l i b r e M. Leclerc n° 79.1.113.1).
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8. LECLERC, M. L'organe axial et ses relations avec la sexualit@ et l'immunit~ chez les Ast~rides. Th~se Doctorat ks-Sciences, Orl@ans, 1974. 9. HILDEMANN, W.A. and DIX, T.G. Transplantation reactions of tropical australian echinoderms. Transplantation, 14, 624, 1972. IO.KARP, R.D.and HILDEMANN, W.H. Specific a l l o g r a f t r e a c t i v i t y in the sea star D~masterias imbricata. Transplantation, 22, 434, 1976. II.DUPRAT, P. Mise en ~vidence de r~actions immunitaires dans les homogreffes de paroi du corps chez le Lombricien Eisenia fo~tida. Ann. Inst. Paste~% 113, 867, 1967. 12.COOPER, E.L. Rejection of xenografts exchanged between Lumbric~ t e r r ~ t ~ and Eisenia fo~tida. Transplantation, 6, 322, 1968. 13.COOPER, E.L. Transplantation immunity in helminth and annelids. Transplant. Proc., 2, 216, 1970. 14.ROCH, Ph. R6activit~ in v i t r o des leucocytes du Lombricien EX~enia foetida. C.R. Acad. So. Paris, 284, 705, 1977. 15.LECLERC, M., PANIJEL, J. and TOYAMA, K. Heterotransplantation of invertebrate organs to congenitally athymic (nude) mice. T~uznsplavut., 19, 74, 1974. 16.LECLERC, M., REDZINIAK, G., PANIJEL, J. and El LABABIDI, M. Reactions induced in Vertebrates by Invertebrate cell suspensions. I. Specific effects of sea star axial organ cells i n j e c t i o n . Dev. and Comp. Immunol., I , 299, 1977. 17.LECLERC, M., REDZlNIAK, G., PANIJEL, J. and El LABABIDI, M. Reactions induced in Vertebrate by Invertebrate cell suspensions. I I . Non adherent axial organ cells as effector c e l l s . Dev. and Comp. Immunol., I , 311, 1977. 18. FOLKMAN, J. Tumor angiogenesis. Aduan. Canc~. Res., 19, 331, 1974. 19. POLVERINI, P.J., COTRAN, R.S. and SHOLLEY, M.M. Endothelial p r o l i f e r a t i o n in the delayed hypersensitivity reaction : an autoradiographic study. J. Im~unol., 118, 529, 1977. 20. SlDKY, Y.A. and AUERBACH, R. Lymphocyte induced angiogenesis : a quantitative and sensitive assay of the graft-versus host reaction. J. Exp. Med., 141, 1084, 1975. 21. SCHNEBLI, H.P. and DUKOR, P. Plant agglutinins used to distinguish between d i f f e r e n t classes of mouse lymphocytes. Eu~. J. Immunol., 2, 607, 1972. 22. REDZINIAK, G., LECLERC, M., PANIJEL, J. and MONSIGNY, M. Separation of two d i f f e r e n t populations of axial organ cells of A s t ~ ru6ens by the use of l e c t i n s . Biocf~6mie, 60, 525, 1978. 23. LECLERC, M., PANIJEL, J . , REDZINIAK, G., BRILLOUET, C. and BINAGHI, R. Correlation between the i n j e c t i o n of specific proteins in starfishes and the appearance of binding sites for the same proteins in cell populations of t h e i r axial organ. Cell. Immunol. in press.