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AMER. ZOOL., 15:63-71 (1975). The Phylogeny of Thymic Dependence MARGARET J. MANNING Department of Zoology, University of Hull, Hull, HU6 7RX, England SYNOPSIS. Thymic dependence has been studied in the clawed toad,Xenopus laevis. Examination of toadlets thymectomized as larvae at 8 days post-fertilization shows that: (i) The body weights are normal; (ii) The spleen is smaller than normal and there are fewer lymphocytes in the red pulp; (iii) There is an overall suppression of specific antibody, including all IgM, following immunization with human gamma globulin (HGG) in adjuvant or with sheep erythrocytes (SRBC); (iv) Stimulation using HGG elicits some splenic pyroninophilia but the spleens are less reactive than in controls; (v) The normal pattern of HGG localization in the splenic white pulp is absent; (vi) There is an increased susceptibility to Mycobacterium marinum; (vii) Serum immunoglobulin levels and the amounts of surface-associated immunoglobulin on splenic cells are increased; (viii) First-set rejection of skin allografts is prolonged but eventually goes to completion. These findings are discussed both in relation to the role of the thymus in the dichotomy of cell-mediated and humoral immune responses and in a phylogenetic context. INTRODUCTION The use of amphibians in studies on the role of the thymus in immune responses avoids some of the problems inherent in mammalian investigations. Free-living larvae are readily accessible for experimentation and the superficially located thymus can be removed at a rudimentary stage of lymphoid tissue differentiation. Thus, thymectomy can be performed earlier than is technically feasible in mammals and the possibility of maternal thymic influences in utero is absent. The immune system of amphibians thymectomized as larvae has been studied in a number of laboratories, both in anurans (Hildemann and Cooper, 1963; Cooper and Hildemann, 1965 a,b; Du Pasquier, 1965, 1968; Curtis and Volpe, 1971; Manning, 1971; Horton and Manning, 1972; Baculi and Cooper, 1973) and in urodeles (Charlemagne and Houillon, 1968; Tournefier, 1973). We have used the clawed toad,Xenopus laevis, whose larva has a single pair of thymic buds, readily visible through the transparent skin. These buds can be destroyed by microcautery at stage 48 of Nieuwkoop and Faber (1967), at 8 days post-fertilization, when they measure some 100 /u.m in diameter (Horton and Manning, 1972). The results of early thymectomy on the immune capabilities of Xenopus are brought together here in order to build up a picture of thymic dependence in the clawed toad and to discuss the phylogenetic implications. RESULTS OF EARLY THYMECTOMY IN XENOPUS Growth and development Growth rates of thymectomized Xenopus are comparable with those of shamthymectomized and intact control animals; metamorphosis occurs over the same period of time. The majority of toadlets remained healthy throughout the period of investigation, which lasted for over a year in some experiments. Histogenesis of lymphoid organs The work was supported by research grants from the Medical Research Council. 63 The lymphoid tissues of the thymectomized Xenopus undergo relatively normal histogenesis during the larval stages of development with the exception of the splenic red pulp and the pharyngeal lymphoid organs (ventral cavity bodies); these show some evidence of lymphocytic depletion MARGARET J. MANNING (Manning, 1971). In the normal postmetamorphic Xenopus, accumulations of lymphocytes occur in the spleen, kidney, liver, gut wall, and bone marrow. Of these, early thymectomy has its greatest effect on the spleen which is reduced in size and has fewer lymphocytes in the red pulp. The splenic region which shows the greatest reduction of lymphocytes is the penfollicular area of the red pulp that surrounds the white pulp. This is also the region which first receives carbon after injection via the dorsal lymph sac (Turner, 1969) (Fig. 1) and where soluble antigens administered by the same route first arrive (Fig. 2) (M. H. Collie, personal communication from this laboratory). From histological sections, it would appear that branches from the central arteriole enter the penfollicular region and empty blood into it. Thus, the thymus-dependent area of the spleen in Xenopus seems to have more in common with the marginal zone of the mammalian spleen (see Weiss, 1972) than with the periarteriolar sheath which forms the thymus-dependent area in the mammal (Parrott et al., 1966). These differences in the territories of thymus-dependent lymphocytes perhaps reflect phylogenetic differences in their circulatory pathway but, as yet, there is little information. Serum antibody production Serum antibody responses following larval thymectomy have been studied in toadlets aged between 20 and 40 weeks. Intact Xenopus are capable of producing antibody in two distinct molecular classes which are similar to mammalian IgM and IgG (Lykakis, 1969; Marchalonis et al., 1970; Hadji-Azimi, 1971). In our laboratory we have used human gammaglobulin (HGG) and sheep erythrocytes (SRBC) as antigens. Comparison of the elution profiles obtained after Sephadex G-200 filtration of normal and immune sera (Fig. 3a,b) shows that the peak corresponding with IgG is usually absent from non-immunized toadlets of our colony. After administration of HGG in Freund's complete adjuvant to control animals, specific antibody of both high and low molecular weight is produced FIG. 1. Xenopus spleen from an animal killed 24 hr after injection of carbon into the dorsal lymph sac: stained with haematoxylin and eosin. A ring of heavily aggregated carbon can be seen surrounding the white pulp. Central arteriole of white pulp (A); elongated nucleus of a boundary layer cell (B); carbon in red pulp (C); red pulp (R); white pulp (W). Arrows indicate cells of the boundary layer; these clearly delineate the white pulp area. FIG. 2. Immunofluorescence seen in a cryostat section of the spleen of a toadlet killed 6 hr after injection of HGG in saline into the dorsal lymph sac. The HGG is traced by applying a fluorescein-labeled antiserum. Two white pulp regions are seen in the field with part of a third in the bottom of the picture. There is a ring of antigen (HGG) around each. Note that the antigen occupies a penfollicular position similar to that seen for carbon in Figure 1. Red pulp (R); white pulp (W); arrows to boundary layer. and a new distinct peak appears in the IgG position (Fig. 3b). In contrast, thymectomized animals immunized with HGG failed to show this peak and there was no specific antibody detectable in any fraction. Similar studies using SRBC as the antigen revealed anti-SRBC activity in the controls but only in the IgM moiety. Again, there was no specific antibody in the serum from 65 PHYLOGENY OF THYMIC DEPENDENCE (b) (a) 1-5- o CO CN u O 10 < 0-5 0 15 20 25 30 35 40 45 0 15 25 30 35 40 45 Fraction No. FIG. 3. Gel filtration on Sephadex G-200 of Xenopus serum: a, Serum from a normal animal. In Xenopus, the first protein peak (containing IgM macroglobulin) is higher than the other peak (the albumin), b, Pooled serum from 5 control (sham-thymectomized) toadlets. These received three injections, at weekly intervals, of a mixture containing equal parts of a 10 mg/ml HGG solution and Freund's complete adjuvant (dose 5 /xl/g body weight). They were killed at week 8. Note that specific antibody of both high and low molecular weight is produced and a new, distinct peak appears in the IgG position. Thymectomized animals, in contrast, failed to produce this peak and all fractions were negative for anti-HGG activity. • • distribution of serum proteins; o o distribution of anti-HGG antibodies ( -Iog2 passive haemagglutination titres). thymectomized animals. These experiments (Turner and Manning, unpublished) demonstrate an inhibition of specific antibody production following early thymectomy in Xenopus which is of greater severity than that in thymusdepleted mammals since the latter can still produce IgM antibodies in normal or subnormal amounts (Taylor and Wortis, 1968; Mitchell et al., 1971; Manning et al., 1972; Pantelouris and Flisch, 1972). Discussion of this thymic dependence of antibody formation inXenopus must remain speculative until more antigens have been studied, in particular those which are thymus-independent in the mammal (see Basten and Howard, 1973). Possible expla- nations include those based on phylogenetic differences. Unlike the mammal, Xenopus fails to shift antibody production from IgM to IgG during the time course of a response (Hadji-Azimi, 1971), and this may imply a different relationship in the requirements for thymus-derived cells in IgM and IgG production. Another possibility is that the larval thymus may be a source of lymphocytes with B-cell functions in Xenopus. Alternatively, there may be an ontogenetic explanation: Thus, if there are thymus-derived cells which are involved in any way with IgM production and if these differentiate and peripheralize early in ontogeny, they may fail to occur in our animals because of the early timing of the 66 MARGARET J. MANNING thymectomy operation (which may perhaps render Xenopus more completely athymic than the thymus-depleted mammal). A different kind of explanation is that, in the amphibian, exhaustive overstimulation intervenes. Thus, if the thymectomized Xenopus is combatting environmental antigens with an incomplete immune system, some non-specific component may become in short supply, resulting in an overall reduction of specific antibody formation. Ruben et al. (1973) have shown that cellular co-operation in antibody production occurs in the newt Triturus viridescens. It may be that, in Xenopus also, co-operation is required and is perhaps of over-riding importance. However, it is not yet known whether one (or more than one) of the cooperating populations described by Ruben et al. (1973) is dependent on the presence of an intact thymus and this remains a crucial question. Splenic responses to immunization Turner and Manning (1973) described the cellular changes which occur in the spleen of intactXenopus after immunization with SRBC or with HGG. These changes include intense proliferative activity with the production of large pyroninophilic cells (Figs. 5, 6). Immunofluorescence studies following administration of HGG in adjuvant (Horton and Manning, 1974) reveal a distinct peripheral zone of antigen retention within the splenic white pulp (Fig. 4). The picture closely resembles the dendritic localization of antigen which occurs in the lymphoid follicles of both birds and mammals (White et al., 1967; Balfour and Humphrey, 1967). We do not yet know whether it is antigen or immune complexes which are being trapped in the Xenopus spleen, but we have found the reaction to be thymus-dependent. This may well be related to the failure to produce specific antibody after thymectomy, but whether as a cause, due to inefficient handling of the antigen, or as an effect, due to failure to form antigen-antibody complexes, is uncertain. The increase in numbers of '*< FIG. 4. Immunofluorescence picture in a cryostat section from the spleen of a control toadlet immunized with HGG (injection schedule as described in the legend to Fig. 3); killed at week 3. The application of a fluorescein-labeled antiserum to HGG shows that the antigen is now concentrated within the white pulp where it is localized in a distinct zone towards the periphery. Red pulp (R); white pulp (W); arrows to boundary layer. FIG. 5. Spleen from a toadlet injected with HGG (injection schedule as described in the legend to Fig. 3): killed at week 3, 4 hr after injection of tritiated thymidine. It can be seen from this autoradiograph that there are a number of labeled nuclei (black) indicating high proliferative activity. Red pulp (R); white pulp (W); arrows to boundary layer. Methyl-green pyronin stain. pyroninophilic cells and in the size of the white pulp regions which occurs in the spleen of intact, control Xenopus in response to administration of HGG in adjuvant is less marked in the thymectomized animal. Nevertheless, some increase in pyroninophilia occurs and this may suggest a certain level of heightened reactivity (Horton and Manning, 1974). Susceptibility to infection Clothier (1972) has shown that Mycobac- PHYLOGENY OF THYMIC DEPENDENCE sponses to micro-organisms can occur in thymectomized Xenopus. If they do form part of the residual defense mechanism, this may account for the results of Weiss et al. (1972) whose semi-quantitative studies using an antiserum to Xenopus immunoglobulin revealed an increased level of serum immunoglobulin in thymectomized animals from this colony. It is not known, however, whether the excess immunoglobulins have any functional specificities. An alternative explanation is that high imFIG. 6. Methyl-green pyronin stained section of a munoglobulin levels may result from the Xenopus spleen showing two large pyroninophilic cells withdrawal of a normal regulatory influamongst the lymphocytes of the white pulp. The pyroninophilia which develops in the stimulated ence of the thymus. This latter suggestion spleen is largely due to cells of this type. Large has been put forward by Weiss et al. (1972) pyroninophilic cell (LPC). and is used by them to account for the occurrence in the spleens of thymectomized terium marinum, an acid-fast bacillum, fre- Xenopus of an increased percentage of cells quently present in laboratory aquaria, is expressing large amounts of surface aspathogenic in Xenopus when injected in sociated immunoglobulin. large doses. His preliminary results suggest that thymectomized animals from our colony are more susceptible than their sham- Alloimmune responses operated controls. A decreased resistance to infection by Experiments in which skin allografts are micro-organisms may account for our find- applied to early thymectomized Xenopus are ing that non-immunized thymectomized described in another contribution to this toadlets display more variability than nor- symposium (Horton and Horton, 1975). mal animals in their splenic histology and They reveal severe deficiencies in alloimoccasionally show large white pulp areas mune reactivity. The rejection of first-set with a level of pyroninophilia suggestive of grafts is often a very prolonged process, stimulation (Horton and Manning, 1974). nevertheless it usually goes to completion Such stimulation could well be due to an- (Horton and Manning, 1972). Second-set tigenic challenge from the environment, the allografts from the same donor applied to intact animals being able to deal with these thymectomized animals after the first-set challenges quickly and more effectively. A grafts have been rejected are destroyed few thymectomized Xenopus in our colony more rapidly and within times comparable have developed hydrops, a disease of un- to the secondary response of control aniknown aetiology (Reichenbach-Klinke and mals (Horton and Horton, 1975). It would Elkan, 1965). Since most animals remain in seem that the population of lymphocytes apparent good health, presumably they capable of invading a graft is reduced by must retain some part of their defense po- thymectomy, but that some capabilities retential. A decreased resistance to infection main. Possibly, when the residual small may also account for the occasional runting population of reactive cells is expanded— described after thymectomy in other am- either by a slow build-up of numbers in phibians (reviewed by Du Pasquier, 1973) first-set responses or through second-set as well as wasting in mammals (Azar, 1964). stimulation—graft rejection can occur. Presumably, in the normal animal, the process is amplified and accelerated through a Immunoglobulin levels thymus-dependent system. It is not known whether the residual cells with alloimmune We have yet to discover whether reactivities acquired their immunocompethymus-independent humoral antibody re- 68 MARGARET J. MANNING status or they may be the result of varying amounts of residual thymic factors. Thus, none of the experimental systems so far employed necessarily ensures the complete lack of a thymic influence. If surgery is used, questions arise concerning the amount of peripheralization of thymusderived cells which had already occurred before the thymectomy was performed. GENERAL CONSIDERATIONS Thymus regeneration is a hazard, particularly in amphibian experiments, but the use of cautery reduces the possibility of thymus Thymic depletion re-growth from the pharyngeal epithelium Many of the experiments in which or the chance of accidental self-grafting of thymectomized amphibians have been live thymic cells during the operation itself. studied were primarily designed to investi- Experiments using antilymphocytic serum gate the role of the thymus in the process of raise problems about the target cells which allograft rejection. This work has proved are reached, particularly if the antiserum is very rewarding and is well documented (see administered in vivo. Studies of mammals Cooper, 1973; Du Pasquier, 1973; Horton thymectomized in utero encounter the and Horton, 1975, for reviews). There is question of a possible transplacental pasless information about antibody produc- sage of thymic factors from the mother, tion by thymectomized amphibians and no while congenitally athymic animals may clear picture has yet emerged. In the exper- have some residual, albeit dysplastic, iments on Xenopus described above, specific thymic tissue and some peripheral thetaantibody production was suppressed, while bearing lymphocytes. They also have an larval thymectomy in the midwife toad, underlying genetic defect which affects Alytes obstetricans, has been shown to reduce more than the lymphoid tissue alone (see the response to foreign erythrocytes (see Pantelouris, 1968). These unavoidable difDu Pasquier, 1973). In contrast, in the ficulties in achieving the perfect athymic bullfrog, Rana catesbeiana, where lymph condition make it highly desirable to study glands occur, the lymph glands are thought models with minimal thymic influence in as to be of greater importance than the many different systems as possible. Amthymus in directing antibody production phibians have proved to be excellent (Cooper et al., 1971) and partial thymec- laboratory animals for use as one such systomy increases the humoral antibody re- tem, but in order to assess the results their sponse to haemocyanin (Baculi and phylogenetic status must be evaluated. Cooper, 1973). This may indicate an increasingly important role of lymph glands, starting in the higher anurans. On the Phylogenetic status of amphibian immune sysother hand, it is becoming apparent, within tems the mammals as well as in amphibians, that Table 1 shows some of the evolutionary the outcome of thymic depletion can be pressures which may have led to increased manifested in different ways. Thus, the de- sophistication of the immune system in ficiencies in the immune system of the lamb progressive stages of vertebrate phylogeny. following thymectomy in utero differ sigOne change which has occurred in vernificantly from those of the thymustebrates with the emergence from aquatic depleted mouse (Morris, 1973). to terrestrial life is the acquisition of bone This variability may be due to different marrow as a site of stem cell hemopoiesis. environmental or developmental condi- The first regular appearance of this tissue is tions which may place pressure on different in adult anurans. It is presumably related to components of the immune system. Alter- the possession of the type of hollow bone natively, they may be due to phylogenetic structure evolved in relation to locomotion tence in tissues other than the thymus or whether they were already established before the thymus was removed. The former is perhaps more likely since the early thymectomized Xenopus can have received, at most, only minimal thymic influence (see Horton and Manning, 1972). PHYLOGENY OF THYMIC DEPENDENCE 69 TABLE 1. Steps in vertebrate evolution with possible immunological implications. Evolutionary step 1) Increased body size and longer life span 2) Loss of free-living larval stages 3) More efficient circulation of body fluids 4) Homoiothermy 5) Placentation on land. Although some fish bear hemopoietic tissue in association with the cranial skeleton, fish bones are not in general suitable for this purpose. At first, bone marrow may simply represent a re-housing of stem cell populations previously situated elsewhere in the body. Nevertheless, the tissue becomes increasingly important in adult tetrapods and thymus-bone marrow interactions have been reported in leopard frogs (Cooper, 1973). In the more primitive tetrapods the peripheral lymphoid organs are relatively simple structures. In Xenopus, only the spleen shows any complexity of structural organization, and Turner (1973) has shown that this organ is not essential for antibody production. Much of the amphibian lymphoid tissue occurs in organs with sinusoidal blood flow such as the kidney and liver and the lymph glands possessed by some anurans (see Baculi and Cooper, 1967; Horton, 1971). These may provide sites where lymphoid cells can cluster and respond to the presence of antigen. Moreover, in tissues where the blood flow is slow, this is possibly all that is required. The development of elaborate lymphocytic migratory pathways is perhaps related to a more efficient circulation of the body fluids. Thus, in the high pressure vascular systems of the more advanced vertebrates, architectural modifications within the lymphoid organs may be necessary to ensure that cells can move in and out of the circulation at appropriate sites. Some of the apparent simplicity of the amphibian lymphoid organs may, therefore, be related to general features of the animal's anatomy and physiology rather than to deficiencies in Significance to immune system Requirement for efficient self-surveillance Amniotic protection from environmental antigens; this may permit prolongation of immune differentiation Lymphocytic circulatory pathways may become more specialized Affects internal environment for growth of micro-organisms Maternal-foetal interactions immune potential. Nevertheless, a certain lack of sophistication has been demonstrated in the immune system of Xenopus; for example, there is a high requirement for adjuvants in antibody production to soluble antigens (Manning and Turner, 1972). At the amphibian stage of evolution the free-living larva is retained. In these circumstances foreign antigens may be encountered when the lymphoid system is still very immature. This contrasts with the condition in amniote embryos; it may necessitate a rapid maturation of the pathways which lead to positive immune responses perhaps at the expense of more advanced differentiation. DuPasquier etal. (1972) showed that the thymus inXenopus is the first source, during development, of lymphocytes bearing detectable surfaceassociated immunoglobulin. They suggest that there may be a basic larval type of immune response which originates from the thymus and which precedes the later adult specializations. In the amniote embryo the need to produce such immunocompetent cells would be less urgent and this may allow a further build-up of cellular populations before functional commitments need be made. This, together with homoiothermy, may have been further factors leading to increasing sophistication of the vertebrate immune system. T-cell and B-cell origins The clear-cut dichotomy in the origin of lymphocytes concerned with T-cell and B-cell functions, as exemplified in the chicken (see Warner and Szenberg, 1964; 70 MARGARET J. MANNING Good et al., 1966), has become somewhat blurred now that further species have been studied. This is not to deny that a functional distinction exists between lymphocytes concerned with cell-mediated reactions and those which are precursors of antibody forming cells, nor that the thymus has an extremely important influence on lymphocyte development. One simply questions whether, under some circumstances, differentiation towards T-cell competence can occur elsewhere than in the thymus, and also, whether development within the thymus is exclusively that of T-cells or whether B-cells may sometimes be spawned there as well. The latter seems a distinct possibility in the larvalXenopus (see Cooper, 1973) and would be consonant with the results from our laboratory reported above. The case has been clearly argued by Morris (1973). Morris suggests, from his experiments on in utero thymectomy in the lamb, that the thymus is not a unique source of T-cells but that, on the contrary, under some conditions T-cell immune capabilities can be endowed elsewhere. A better understanding of the phylogenetic origins of T-cell and B-cell differentiation would obviously help to clarify the situation and the need for more comparative studies, both, within the Mammalia and in other vertebrate classes, has now become urgent. REFERENCES Azar, H. A. 1964. Bacterial infection and wasting in neonatally thymectomized rats. Proc. Soc. Exp. Biol. Med. 116:817-823. Baculi, B. S., and E. L. Cooper. 1967. Lymphomyeloid organs of Amphibia. II. Vasculature in larval and adult Rana catesbeiana. J. Morphol. 123:463-480. Baculi, B. 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