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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.
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