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Lymphocyte proliferation in neonatally thymectomized rats.

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THE ANATOMICAL RECORD 208:427-433 (1984)
Lymphocyte Proliferation in Neonatally Thymectomized Rats
ANNA LUISA GRANATA, C. VECCHI, R. BUDASSI, AND A. CORSI
Laboratory of General Pathology, University of Anconq 60100 Anconq Italy
ABSTRACT
Autoradiography has been used to evaluate lymphocyte proliferation in the neonatally thymectomized rat in comparison with the normal
animal. The data obtained show that the proliferative activity of lymphocytes
is greatly increased in the thymus-dependent areas 4-6 weeks after thymectomy, whereas it is normal or slightly increased 3 months later. It seems
plausible to assume that a thymus factor or chalones in situ produced by
specific cells normally regulate the proliferation of thymus-derived cells. The
increase of the proliferative activity accounts for the repopulation of the thymus-dependent areas, which are completely replenished in the older animals.
Recirculation of the thymus cells is not confined to the thymus-dependent
areas.
It is generally recognized that the thymus
generates lymphocytes which migrate to specific areas in the peripheral lymphoid tissues, particularly in the neonatal life (Parrott
et al., 1966; Weissman, 1967; Laissue et al.,
1976). The main sites of the thymus cell migration are identical with the areas of lymphocyte depletion appearing in the spleen
and lymph nodes after neonatal thymectomy.
These areas were delineated in the rat by
Goldschneider and McGregor (1973), in basic
agreement with observations in the mouse
(Parrott et al., 1966). Thymus cells also migrate to the internodular areas of the Peyer's
patches (Eikelenboom et al., 1979).The available data do not allow us to define the relevance of cell migration from the thymus or
the role of the proliferation of the migrated
cells in the development and maintenance of
the thymus-dependent pool. It was suggested
that the thymus affects the lymphocyte proliferation in the peripheral lymphoid organs
by a stimulating factor (Metcalf, 1956, 1964).
This possibility was not ruled out by more
recent experiments with thymus grafts (Papiernik and Bach, 1979) or with isolated thymus factors (Trainin and Small, 1970; Basch
and Goldstein, 1974; Bach et al., 1975).
A markedly reduced lymphocytopoiesis in
young thymectomized rats was reported by
Rieke and Schwarz (1966). Their observations in the lymph and blood do not seem to
be entirely congruent with their data in the
lymph nodes: the labeling pattern of the
lymph and blood lymphocytes as reported by
them points to increased proliferative po0 1984 ALAN R. LISS, INC.
tential of lymphocytes after thymectomy,
whereas the reduced labeling in their radioautographs of sections of lymph nodes is
evidence to the contrary. However, they did
not quantify the labeling pattern in the thymus-dependent areas.
The purpose of this study was to evaluate
the proliferative activity of lymphocytes both
in the thymus-dependent and in the thymusindependent areas of the peripheral lymphatic organs and in bone marrow after neonatal thymectomy.
MATERIALS AND METHODS
Animals
All experiments were carried out with Wistar albino rats that were bred in our animal
colony.
Thymectomy
Thymectomy was performed by suction according to the method of Miller (19621, mostly
from 24 to 36 hours after birth. The time
interval never exceeded 38 hours. The efficacy of the procedure was checked in each
animal by dissection after killing. Small residues were found only in one of five operated
animals, those with residues over 20 mg
being rejected. Twenty milligrams was chosen as a safe limit both for theoretical rea-
Received June 17,1982; accepted October 3,1983.
Address reprint requests to Dr. A.L. Granata, Laboratorio di
Patologia Generale, Facolta di Medicina dell'universita, 60100
Ancona, Italy.
428
A.L. GRANATA, C. VECCHI, R. BUDASSI, AND A. CORSI
sons (the weight of thymus at 6 weeks of age
is over 300 mg) and for experimental data: in
our experience a residue of 30 mg a t 6 weeks
does not affect appreciably the histologic
changes caused in the secondary lymphoid
organs by early thymectomy.
Labeling Procedure
The animals were divided into three
groups. Animals in the first group, including
thymectomized and control unoperated rats
6 weeks of age, were injected intravenously
with a single dose of 1 pCilg body weight of
[meth~l-~Hlthymidine
2 Cilmmol (the Radiochemical Center, Amersham, England) and
were killed 15 min after the injection. Animals in the second group, including thymectomized and unoperated rats 4-5 weeks of
age, were injected intraperitoneally with 3
doses of 1 pCilg body weight of tritiated thymidine every 2 hours and were killed 4 days
later. Animals in the third group, including thymectomized and unoperated rats 4
months of age, were treated in the same way
as the first group and were killed 15 min
after the injection.
Histology
Smears of blood and lymph from the thoracic duct were made immediately prior to
killing and fixed in methanol. Samples of the
spleen, mesenteric, and axillary lymph nodes
and Peyer’s patches were fixed in a modified
Carnoy’s solution (65% ethanol absolute, 30%
chloroform, 5% acetic acid) and embedded in
paraffin. Femoral bone marrow samples were
diluted with homologous serum and fixed in
methanol. A morphometric study of Malpighia n bodies was carried out in six normal and
five thymectomized animals, 4 months of age,
by a point-counting method (Rohr et al.,
1976). Counts of the intersection points of a
reticulum ocular within the profile of each
Malpighian body were done on random sections of 50 bodieslanimal with a magnification of ~ 1 2 5 The
.
significance of the data
was tested by analysis of variance.
A u toradiography
The tissue sections and the smears were
processed for autoradiography by dipping the
slides in the Eastman Kodak NTB-2 emulsion. Exposure times of 26 and 23 days were
employed for smears and tissues, respectively. The tissue sections were stained with
haematoxylin and eosin. The smears of blood,
lymph, and marrow cells were stained with
haematoxylin, eosin, and Azur B.
At least 200 lymphoid cells were counted
in each area of the organs and in each sampie
of blood, lymph, and bone marrow, and the
percentage of labeled cells was determined.
Three or more radioautographic grains in the
nuclei were assumed to indicate positive labeling by comparison with counts of background grains. The significance of experimental results was determined by the parametric Student’st-test.
RESULTS
Histology
Our findings 4-6 weeks after thymectomy
are in accordance with the observations of
Parrott et al. (1966)in the mouse and of Goldschneider and McGregor (1973) and Eikelenboom et al. (1979) in the rat. Lymphocytes
were sparsely present around the central arteriole of the spleen nodules (Fig. 1). The
lymph nodes were smaller than in normal
rats of the same age: the cortex was thinner
and often depleted of lymphocytes in the mid
and deep zone. On the whole, the histologic
differences from the normal were not a s constant and obvious in the lymph nodes as in
the Malpighian bodies.
In older thymectomized animals several
observations consistently showed that the
morphology of the lymphoid organs progressively approached normality. After 4 months
a slight reduction of size was seen only in
occasional lymph nodes. The appearance of
the Malpighian bodies was almost always
normal (Fig. 2); morphometric analysis excluded a significant difference in size of the
Malpighian bodies between normal and
thymectomized animals [F = 1.625, 10, and
539 degrees of freedom (DF); P > 0.11.
Labeling Pattern of the Lymphoid Cells 15
Minutes After the Administration of the
Isotope 6 Weeks After Thymectomy
A greater percentage of lymphoid cells were
labeled in the periarteriolar sheaths of the
spleen and in the deep cortex of the mesenteric lymph nodes of thymectomized animals
than in controls. The labeled cells were on an
average 12.2% in the periarteriolar sheaths
of the spleen and 7.4% in the deep cortex of
the mesenteric lymph nodes of thymectomized rats. By contrast, the average numbers of labeled cells in controls were 2.6%
and 2.2%, respectively (Table 1).The differences were highly significant (P < 0.001). In
the thymectomized animals a percentage
greater than normal of lymphoid cells were
labeled also in the internodular zones of the
LYMPHOCYTE PROLIFERATION AFTER THYMECTOMY
Fig. 1. A. Spleen of a normal 42-day old rat. The periarteriolar area is densely populated by
lymphocytes. B. Spleen of a neonatally thymectomized 42-day old rat. Lymphocytes are sparsely
present around the central arteriole. Magnification, x 150.
429
430
A.L. GRANATA, C. VECCHI, R. BUDASSI, AND A. CORSI
Fig. 2. A. Spleen of a normal 4-month old rat. Note the large secondary nodule. B. Spleen
of a neonatally thymectomized 4-month old rat. Note the large secondary nodule. Magnification, ~ 1 5 0 .
431
LYMPHOCYTE PROLIFERATION AFTER THYMECTOMY
TABLE 1. Percentage o f 9 m p h o i d cells labeled 15 m i n after a single dose of [methylHlthymidine in rats 6 weeks o f age
Normal
Spleen
Periarteriolar sheaths
Nodules
Marginal zone
Mesenteric lymph nodes
Outer cortex
Deep cortex (paracortex)
Medullary cords
Germinal centers
Axillary lymph nodes
Outer cortex
Deep cortex (paracortex)
Medullary cords
Peyer’s patches
Internodular areas
Nodules
Germinal centers
Bone marrow
Thymectomized
+
2.6 f 0.7 (7)
2.1 f 0.3 (6)
1.7 f 0.5 (7)
12.2 2.1 (7)
2.1 k 0.6 (6)
1.9 i 0.2 (7)
2.4 f 0.7 (6)
2.2 k 0.4 (6)
7.1 0.4 (6)
12.0 1.9 (6)
3.1 _+ 0.9 (8)
7.4 f 1.0 (8)
9.6 k 1.1(8)
13.9 ? 2.1 (8)
2.8 _+ 0.8 (7)
5.7 f 0.7 (7)
5.6 f 1.9 (6)
2.3 f 0.4 (7)
6.3 k 1.0 (7)
10.6 _+ 1.4 (7)
3.7 f 1.1(6)
8.3 f 3.3 (6)
12.4 k 2.6 (6)
2.4 + 0.5 (4)
8.1 i 1.7 (7)
13.9 f 2.5 (7)
11.4 k 2.0 (7)
2.5 0.6 (4)
*+
+
Results are the means & SEM of (n) animals. The areas in the spleen and in the lymph nodes were
delineated according to Goldschneider and McGregor (1973).
Peyer’s patches and in the medullary cords
of the axillary lymph nodes; the differences
from the normal values were near to significant (P < 0.1) (Table 1).
Few lymphoid cells were labeled in bone
marrow (2.5%) and in blood (0.5% or less),
and no change was seen after thymectomy.
Labeling Pattern of Lymphoid Cells 4 Days
After the Administration of the Isotope 4-5
Weeks After Thymectomy
The results are summarized in Table 2.
The labeling pattern was altered throughout
the white pulp of the spleen: the percentage
of labeled lymphoid cells was greater in the
thymectomized animals than in controls, the
difference being significant (P < 0.01) in the
area immediately surrounding the central
arteriole and in the marginal zone. The mean
values were 32.4% and 12.9%, respectively,
after thymectomy, whereas only 7.5% and
4.2%, respectively, were labeled in control
animals.
A significant increase of labeled cells was
found in the medullary cords of the axillary
lymph nodes after thymectomy (P < 0.05);
the mean percentage of labeled cells was 39.6
in the experimental animals and 23.0 in
controls.
A highly significant difference (P < 0.001
and P < 0.01, respectively) was seen in blood
and lymph. The percentage of labeled lymphocytes in blood was on a n average 42.5
after thymectomy and 23.7 in controls. The
mean values in lymph were 22.1 and 11.8,
respectively.
No appreciable difference was seen in the
labeling pattern of lymphoid cells in the bone
marrow (39.5% after thymectomy and 42.0%
in controls).
Labeling Pattern of Lymphoid Cells 15
Minutes After the Administration of the
Isotope 4 Months After Thymectomy
The labeling pattern of lymphoid cells was
investigated in the spleen and the mesenteric lymph nodes. The results are shown in
Table 3. The labeled cells were on a n average
2.8% in the periarteriolar sheaths of the
spleen and 3.0% in the deep cortex of the
mesenteric lymph nodes of thymectomized
animals. The average numbers in the control
animals were 1.7% and 1.5%, respectively.
The difference was significant (P < 0.01) only
in the deep cortex of the mesenteric lymph
nodes. The percentage of labeled cells 4
months after thymectomy was greatly reduced in the spleen and in the deep cortex of
the lymph nodes, if compared with the data
in younger animals 6 weeks after thymectomy: the difference was significant. The reduction in the percentage of labeled cells was
432
A.L. GRANATA, C. VECCHI, R. BUDASSI, AND A. CORSI
TABLE 2. Percentage of lymphoid cells labeled 4 days after administration of [methyl3H]thymidine in rats 4-5 weeks of age
Spleen
Periarteriolar sheaths
Nodules
Marginal zone
Axillary lymph nodes
Outer cortex
Deep cortex (paracortex)
Medullary cords
Germinal centers
Peyer’s patches
Internodular areas
Nodules
Germinal centers
Bone marrow
Blood
Lymph
Normal
Thymectomized
7.5 f 2.3 (6)
10.2 f 2.2 (6)
4.2 f 1.0 (6)
32.4 f 5.2 (6)
18.6 f 3.7 (6)
12.9 f 1.8 (6)
11.1k 0.6 (6)
14.6 ? 2.0 (6)
16.4 f 3.4 (6)
39.6 f 6.1 (6)
19.0 f 2.9 (3)
12.6 k 0.8 (6)
23.0 k 3.3 (6)
39.0 f 6.5 (5)
17.6
16.3
31.6
42.0
23.7
11.8
5 3.0 (5)
f 1.9 (5)
f 3.4 (5)
k 3.7 (5)
f 3.3 (6)
f 1.9 (5)
19.2
17.6
25.6
39.5
42.5
22.1
f 1.1(5)
f 2.7 (5)
f 6.8 (6)
f 5.4 (5)
5 5.3 (7)
& 1.7 (5)
Results are the means SEM of (n) animals. The areas in the spleen and in the lymph nodes were
delineated according to Goldschneider and McGregor (1973).
TABLE 3. Percentage
lymphoid cells labeled 15 m i n after a single dose of [methyl3H]thymidine in rats 4 months of age
of
Spleen
Periarteriolar sheaths
Nodules
Marginal zone
Mesenteric lymph nodes
Outer cortex
Deep cortex (paracortex)
Medullary cords
Germinal centers
Normal
Thymectomized
1.7 f 0.2 (4)
0.6 0.1 (4)
0.5 f 0.1 (4)
2.8 i 0.4 (5)
0.7 f 0.1 (5)
0.5 f 0.1 (5)
0.9 k 0.2 (4)
1.5 + 0.1 (4)
1.5 f 0.7 (5)
3.0
0.3 (5)
5.9 k 2.6 (5)
9.8 f 1.0 (5)
*
5.6 f 2.0 (4)
8.6 f 1.4 (4)
+
Resuts are the means 5 SEM of (n) animals The areas in the spleen and in the lymph nodes were
delineated accordmg to Goldschneider and McGregor (1973).
small in normal older animals: the difference
in comparison with normal younger animals
was not significant.
though the actual number of dividing cells
may be smaller than normal because the total thymus-dependent pool is smaller, the
increased proliferative activity soon after
DISCUSSION
thymectomy affords a satisfactory explanaThe labeling pattern demonstrates that the tion of why the thymus-dependent areas are
percentage of dividing cells is generally in- replenished a few months later.
Our results show that a thymus factor is
creased in the thymus-dependent areas of the
rat 4-6 weeks after neonatal thymectomy: 4 not required for the proliferation of the thymonths after thymectomy the labeling pat- mus-derived cells. By contrast, our results
tern approaches normality and the thymus- are compatible with the view that a circulatdependent areas are replenished. It appears ing thymus factor normally inhibits the prothat the proliferative activity of the early- liferation of specific cells. In fact, a thymic
migrating thymus cells is markedly in- inhibitor of lymphocyte proliferation was isocreased 4-6 weeks after thymectomy. Al- lated by Rijke and Balliew (1979). It also
LYMPHOCYTE PROLIFERATION AFTER THYMECTOMY
seems plausible that in situ-produced chalones play a role in the regulation of the thymus-dependent pool. A soluble fraction capable of inhibiting proliferation of lymphocytes was obtained by Moorhead et al. (1969).
The repopulation of the thymus-dependent
areas was reported by Parrott et al. (1966) in
occasional thymectomized mice surviving 67 weeks: the authors had no satisfactory explanation for their finding, which seems to
have been ignored by the other research
workers in this field. One might speculate
that a difference in the regulation of development and maintenance of the lymphatic
pools may account for the much greater
proneness of mice to runt disease after thymectomy in comparison with rats.
It is worth noting that in the thymectomized animals 4-5 weeks of age, examined
a t 4-day intervals after administration of tritiated thymidine, the difference in the labeling pattern is not confined to the thymusdependent areas. This is most likely the consequence of further cell migration and
recirculation.
ACKNOWLEDGMENTS
The authors are indebted to Professor G.
Doria and Professor Z. Ovary for their helpful comments on this manuscript.
The technical assistance of Mr. V. Rossi is
gratefully acknowledged.
LITERATURE CITED
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