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The influence of thyroid and testicular hormones on globule leucocytes in the rat duodenal crypt epithelium.

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The Influence of Thyroid and Testicular Hormones on
Globule Leucocytes in the Rat Duodenal
Crypt Epithelium
R. CARRIERE AND M. BUSCHKE
Department of Anatomy and Cell Biology, Downstate Medical Center,
Brooklyn, New York 11203
ABSTRACT
The globule leucocyte is a cell with a wide distribution in the
digestive, respiratory, biliary, urinary and genital tract epithelia of homeotherms. I t occurs in the crypts of the rat small intestine and within the epithelium on the villous bases but not on the remainder of the villi. A characteristic feature of this cell is the presence of acidophilic cytoplasmic granules, 0.5
to a few pm in diameter. The nucleus resembles that of intraepithelial intestinal lymphocytes. In this investigation, a quantitative study was made of the effect of thyroidectomy andlor castration on the number of globule leucocytes in
the duodenal crypts of immature and adult rats. In sham-operated rats, globule
leucocytes were rare, occurring with a frequency of 1 or 2 per 1,000 epithelial
cells. After thyroidectomy, they increased to 14-20 per 1,000 epithelial cells.
Castration did not influence the number of globule leucocytes but resulted in
a n increase in the number, size and acidophilia of their cytoplasmic granules.
In immature rats, but not in adults, castration combined with thyroidectomy
enhanced the effect of thyroidectomy, increasing globule leucocyte number to
32 per 1,000 epithelial cells.
Treatment of thyroidectomized-castrates with thyroxine, initiated 38 days
after operation, reduced the crypt globule leucocyte population t o normal,
whereas treatment with testosterone did not. Growth hormone failed to influence the elevated number of globule leucocytes in thyroidectomized rats, suggesting that this action of thyroxine was not mediated via an influence on
growth hormone release.
Because of their similar nuclear morphology, intraepithelial crypt lymphocytes were also counted and there was no obvious relationship in the fluctuations of these two cell populations.
It is suggested t h a t the greater number of crypt globule leucocytes in thyroid
deficiency may reflect changes in the intestinal secretory immunoglobulin
system and this is being investigated, beginning with a study of the distribution of IgE.
In 1919, Weill described a cell with a small,
dark, “lymphoid” nucleus and an unstained
cytoplasm containing eosinophilic bodies and
he called i t the “Schollenleukozyt.” He observed i t among epithelial cells of the gastrointestinal mucosa in several mammalian
species (Weill, ’19) and also in the human decidual mucosa (Weill, ’20). The term “Schollenleukozyt” was translated to globule leucocyte by Keasbey (’23) who studied i t in sheep
abomasum. Since then, several studies were
ANAT. REC. (19781 192: 407-422.
published describing its distribution, morphology, cytochemistry and their species variations a t the light microscopic level (Clara, ‘26;
A. B. Dawson, ’27; Toro, ’31; H. L. Dawson, ’43;
von Numers, ’42, ’53; Kirkman, ’50; Kent, ’52;
Tiltbach, ’54; Astaldi and Strosselli, ’60;
Kellas, ’61; Rootes, ’61; Whur and Gracie, ’67;
Murray et al., ’68; Takeuchi et al., ’69; Cantin
Received Aug. 10, ‘77. Accepted June 23, ’78.
I Present address. Department of Anatomy, Albert Einstein College of Medicine, Bronx, New York 10461.
407
408
R. CARRIERE AND M. BUSCHKE
and Veilleux, '72; Veilleux and Cantin, '72).
As a result, the presence of intraepithelial
globule leucocytes in the respiratory, digestive, urinary, biliary and genital tracts of a
variety of homeothermic species is well established. In addition, an ileal tumour composed
of globule leucocytes has been described in the
cat (Finn and Schwartz, '72).
The cytoplasmic granules of globule leucocytes range from 0.5 to a few pm in diameter
and contain basic protein, acid mucopolysaccharide and histamine (Murray e t al., '68;
Takeuchi et al., '69; Veilleux and Cantin, '72).
Ultrastructural investigations have revealed
that the granules are membrane-bound structures with a granular content (Toner, '65;
Toner and Ferguson, '71; Kent, '66; Carr, '67;
Whur and Johnson, '67; Carr and Whur, '68;
Murray et al., '68; Holman, '68, '70a,b, '73;
Takeuchi et al., '69; Cantin and Veilleux, '72;
Collan, '72; Rahko, '73).
The function of globule leukocytes is unknown. They become abundant during parasitic infestations (Taliafero and Sarles, '39;
Sprent, '46; Kirkman, '47, '50; Sommerville,
'56; Ahlqvist and Kohonen, '59; Pierce et al.,
'62; Pierce and Long, '65; Armour et al., '66;
Asdrubali and Mughetti, '66; Dobson, '66a,b,c;
Zipper, '66; Whur, '66a,b, '67; Whur and Johnston, '67; Rahko, '70, '72a,b, '73; Miller and
Jarrett, '71). Several investigators therefore
proposed a function related t o the immune
process, and recently, globule leucocytes of
rats experimentally infected with Nippostrongylus brasiliensis were shown by immunocytochemistry to contain IgE (Mayrhofer et al.,
'76, Mayrhofer, '77).
The origin of globule leucocytes is also a
subject for further investigation. They appear
to enter epithelia from the lamina propria and
investigators tentatively proposed lymphocytes (Weill, '19; Keasbey, '23; A. B. Dawson,
'27; von Numers, '42; H. L. Dawson, '43; Kent,
'52; Kent et al., '54, '56; Toner, '65; Holman,
'70a,b) and plasma cells (Kirkman, '50; Dobson, '66a; Whur, '66a; Whur and Johnston,
'67) as their source, largely on the basis of
morphological similarities. On the other hand,
Miller et al., ('671, Jarrett et al. ('67a,b) and
Murray et al. ('68) suggested that intestinal
globule leucocytes may be derived from
subepithelial mast cells. In the rat, mast cells
of the intestinal mucosa are smaller and differ
from the usual connective tissue mast cells in
their fixation and staining properties and in
their response to the histamine liberator, 48/
80 (Maximow, '06; Taliafero and Sarles, '39;
Dalgaard and Dalgaard, '48; Riley, '59; Enerback, '66a,b,c,d; Enerback and Haggendal,
'70a,b; Enerback and Lunden, '74). During the
response to local parasitic infestation, mast
cells in the sheep, cow and rat intestinal lamina propria increased in number and this
change was followed by the appearance of
numerous globule leucocytes in the crypt epithelium (Jarrett et al., '67b; Murray et al., '68,
'70). Second infections were followed by second sequential rises in both cell populations,
but the administration of cortisone suppressed the increase of mast cells and no
globule leucocyte formation was seen (Jarrett
et al., '67b; Murray et al., '71). These studies
and the fact that their ultrastructure appeared identical led to the hypothesis that the
intestinal globule leucocytes were derived
from subepithelial mast cells (Jarrett et al.,
'67a,b; Miller et al., '67; Murray et al., '68;
Miller, '71; Miller and Jarrett, '71; Miller and
Walshaw, '72; Murray, '72). Ultrastructural
and population studies of globule leucocytes
and mast cells in the biliary tract of parasitized goat and cattle gave similar results
(Rahko, '70, '73). Magnesium deficiency augmented the number of globule leucocytes in
the transitional epithelium of the rat urinary
bladder and this increment was preceded by a
rise in the number of subepithelial mast cells;
the administration of cortisone suppressed
these reactions and, here also, the two cell
t y p e s displayed s i m i l a r u l t r a s t r u c t u r e
(Veilleux and Cantin, '72; Cantin and
Veilleux, '72, '73). Finally, in an immunocytochemical study of the localization of
IgE, Mayrhofer ('77) found that, in the intestines of rats infested with Nippostrongylus
brasiliensis, the mucosal mast cells, and not
plasma cells, bound the fluorescent anti-IgE.
As mentioned above, t h e intraepithelial
globule leucocytes also bound anti-IgE. In
summary, there are experimental observations compatible with the suggestion that
globule leucocytes may be derived from mast
cells, but this postulate requires further investigation.
Globule leucocytes have been shown to be
influenced by hormones. Kent et al. ('54) reported decreases of globule leucocyte number
in the tracheal mucosa in response to ACTH or
cortisone. Other reports of the effect of cortisone were mentioned above. Hypophysectomy caused a reduction in intestinal globule
leucocytes together with "a shift in position of
THYROID EFFECT ON INTESTINAL GLOBULE LEUCOCYTES
a considerable portion of the globule leucocytes from the tunica propria to the crypt epithelium" (Kent et al., '56). During an investigation of the influence of thyroid and testicular hormones on the growth of duodenal crypts
in the rat (Carriere, '661, quantitative differences were seen in the globule leucocyte
populations of t h e various experimental
groups. Globule leucocytes and other non-epithelial crypt cells, mainly lymphocytes, were
therefore counted and the results reported
here show t h a t thyroid deficiency leads to an
increase in the number of globule leucocytes
in the duodenal crypt epithelium and that
castration enhances this effect in sexually immature rats but not in adults.
MATERIALS AND METHODS
Treatment of animals and tissue processing
Experiment 1: (Experiment 1in Carriere, '66)
Male Sherman rats weighing 49 gm were
either sham-operated, or thyroidectomized
andlor castrated and kept 90 days, except for 8
sham-operated rats which were killed on day
1. Beginning on the thirty-eighth post-operative day, the thyroidectomized-castrates were
divided into three groups and given two daily
injections of 1.5 p g dl-thyroxine, or 125 p g
testosterone, or saline solution. All other animals were injected twice daily with saline
solution. Tissues were fixed in Bouin's fluid,
embedded in paraffin, sectioned a t 5 pm and
stained with hematoxylin and eosin.
Experiment 2 :
Weanling male Sherman rats weighing 35
gm were either sham-operated or thyroidec-
tomized and kept 90 days, except 5 sham-operated rats which were killed on day 1. The tissues were fixed in Carnoy's fluid, embedded in
paraffin, cut a t 5 pm and stained with hematoxylin and eosin.
Experiment 3 :
Adult male Sherman rats weighing 180 gm
were thyroidectomized, or thyroidectomized
and castrated, or sham-operated and kept 136
days except five sham-operated rats which
were killed on day 1. The tissue preparation
was the same as in Experiment 2.
Experiment 4: (Experiment 1in Carriere, '62)
Male hooded rats weighing 50 gm were
sham-operated or thyroidectomized and kept
90 days. Beginning the fifty-fourth day after
409
operation, the thyroidectomized rats received
two daily injections of either 1.5 pg dl-thyroxine, or 50 pg growth hormone or saline solution. Sham-operated controls received two
daily saline injections. Tissues were fixed in
Orth's fluid, embedded in paraffin, cut a t 6
pm and stained with hematoxylin and eosin.
Experiment 5 : (Experiment 2 in Carriere, '62)
Male Sherman rats, 68 gm body weight,
were sham-operated or thyroidectomized and
kept 147 days. Hormone injections were begun
on the hundred-third day, following the same
procedure as in Experiment 4. Sham-operated
controls corresponding t o the initial (68 gm)
and final (106 gm)body weights of the salineinjected thyroidectomized rats were added a t
the end of the experiment. Tissues were processed as in Experiment 4.
In all experiments, the rats were given 4%
Ca lactate in distilled water to drink for the
first three post-operative weeks and distilled
water thereafter. They were fed an iodine-deficient diet throughout the experiment.
Histometric procedures
Experiments 1, 2 and 3
Epithelial cell nuclei, globule leucocytes
and intraepithelial lymphocytes were counted
in 100 longitudinally sectioned crypts per
animal, a t a magnification of 930 X using
apochromatic oil-immersion objective and
condenser lenses on a Leitz Dialux microscope.
Globule leucocytes with only a few faintly
stained granules were readily identified under
these conditions. Since the number of epithelial cells per crypt differed with the various
endocrine treatments (Carriere, '661, the results were expressed in terms of the numbers
of globule leucocytes and of lymphocytes per
1,000 epithelial cells for each animal, and the
average value for each experimental group
was then calculated. The accuracy of this procedure was verified as follows: in repeated
counts of the same slide from a sham-operated
rat, there were 0.13 and 0.16 globule leucocytes per crypt; in two different slides from
another sham-operated animal, there were
0.225 and 0.206; in counts of a given slide by
two different people, there were 1.245 and
1.26. All of these differences were far smaller
than the variations between individual animals within the experimental group.
Experiments 4 and 5
Only one and two sections per animal were
410
R. CARRIERE AND M. BUSCHKE
available and this did not provide 100 crypts
in full longitudinal section. In order to increase sample size, crypts in oblique section
were included. In animals with few globule
leucocytes, a large sample was necessary t o
obtain reproducible counts. Since the number
of epithelial cells per crypt section could not
be determined in obliquely sectioned crypts, a
point sampling technique was more suitable
than cell counts. A grid with 121 cross-points
was placed in the microscope ocular and the
sections were studied a t a magnification of
430 X , using a Leitz Dialux microscope with
apochromatic lenses. The cytoplasmic granules stained brightly following Orth fixation
and there was no gain in accuracy when the
magnification was increased. The number of
cross-points falling over crypt epithelium and
those over globule leucocytes were counted.
Lymphocytes were not counted. In changing
fields, the tissue was kept out of focus t o avoid
deliberately placing cross-points over globule
leucocytes. From 700 to 3,000 points per animal were recoded over 25 to 60 crypts per animal. The percentages of points over globule
leucocytes for each animal and the average for
each experimental group were then calculated. The accuracy of this technique was verified by repeating some counts and the results
were nearly identical.
In all experiments, slide identifications
were masked and the slides were randomly recoded for the histometric work. The results
were subjected to analysis of variance and the
Neumann-Keuls multiple comparison “t” test
(Winer, ’71). Differences with a probability
greater than 99% (P < 0.01) were considered
significant.
RESULTS
Experiment 1
The results for Experiment 1are reported in
table 1. Body weights have been included because growth changes provide a rough evaluation of the experimental conditions, especially
where thyroid function is concerned.
The number of globule leucocytes in the
crypts of the initial and final sham-operated
controls was very low, of the order of 1 and 2
per 1,000 epithelial cells, and there was no significant change with age over the 80 days of
this experiment. As a rule, the cytoplasmic
granules were few in number, they often
stained only faintly and clustered near the nucleus (fig. 1).However, some well-granulated
cells were also seen. Eighty days following re-
moval of the testes, there was no change in the
number of globule leucocytes but there was a
striking morphological difference, for cytoplasmic granules were large and well-stained
and a t least half of the cells were heavily
granulated (fig. 2).
Eighty days after thyroidectomy, there
4.3 globule leucocytes per 1,000
were 17.4
epithelial cells and they were heavily granulated with brightly stained globules. Thus,
prolonged thyroid deficiency beginning a t a
young age significantly increased the number
of crypt globule leucocytes as well as their
granule content.
Despite the limited influence of castration
alone, castration combined with thyroidectomy enhanced the effect of thyroidectomy on
globule leucocyte number. There were 31.9 &
7.4 per 1,000 epithelial cells in the crypts of
the thyroidectomized-castrates and this represented a statistically significant increase (P
< 0.01) when compared with the effect of
thyroidectomy alone. The granules were abundant and stained brightly (fig. 3). Following
treatment of thyroidectomized-castrates with
testosterone during the last 42 days, there was
no reduction of globule leucocyte number in
comparison with the untreated thyroidectomized-castrates. However, the globules were
of strikingly large size, often up t o 2 or 3 Fm in
diameter (fig. 4).
Treatment of thyroidectomized-castrates
with thyroxine reduced the globule leucocyte
population to a value similar to that of the
sham-operated controls (table 1) and the
abundance and staining of the granules also
diminished.
The number of lymphocytes within the
crypt epithelium appeared to be independent
of thyroid and testicular function as well as
unaffected by changes in the number of
globule leucocytes. No statistically significant change was seen in any of the groups
(table 1).
An increase of globule leucocytes was seen
in tracheal epithelium following thyroidectomy and thyroidectomy plus castration (figs.
5, 61, but these sections were not subjected to
cell counting procedures.
*
Experiment 2
This experiment confirmed the observation
that thyroidectomy shortly after weaning significantly augments the number of globule
leucocytes in duodenal crypt epithelium. This
was clearly seen in animals killed 90 days
411
THYROID EFFECT ON INTESTINAL GLOBULE LEUCOCYTES
TABLE 1
The influence of thyroid and testis on the number ofglobule leucocytes in duodenal crypts ofjuvenile rats
Experimental groups
Globule leucocytes
per 1,000 epithelial
cells 2 S.E.
Intraepithelial
lymphocytes
per 1,000 epithelial
cells 2 S.E.
2.120.3
2.620.2
I'
2.320.5
1.720.5
17.424.3
''
1.150.3
1.820.6
2.020.4
83.0a 6.2
"
31.92 7.4
I
*
1.720.4
7
94.4'2 2.7
"
29.527.3
'
4
191.52 14.3 I *
Number
of
animals
Body weight
2 S.E.
8
48.82 1.7
4
7
7
269.22 10.3
241.62 7.7
91.72 4.4
8
Sham-operated
initial controls
Sham-operated
final controls
Castrated
Thyroidectomized
Thyroidectomized and
castrated
Thyroidectomized and
castrated
testosterone
Thyroidectomized and
castrated
thyroxine
'
'
"
+
+
1.92 0.4
1.620.5
1.020.3
The average body weight for this group differs from t h a t reported by Carriere. '66, because sections were no longer available for one of the
animals and i t s body weight was omitted here.
' Value significantly different (p < 0.01) from initial sham-operated controls.
Value significantly different (p < 0.011 from final sham-operated controls. See text for other significant differences.
'
TABLE 2
The influence ofthyroidectomyon the globule leucocytepopulation ofduodenal crypts in weanling and adult rats
Experimental groups
Experiment 2: weanling rats
Killed day 90
Initial controls
Sham-operated
(final controls)
Thyroidectomized
Experiment 3: adult rats
Killed day 136
Initial controls
Sham-operated
(final controls)
Thyroidectomized
Thyroidectomizedcastrated
Globule leucocytes
per 1.000
epithelial cells
2 S.E.
Lymphocytes
per 1,000
epithelial cells
2 S.E.
Number
of
animals
Body weight
2 S.E.
5
37.22 1.2
'
7
7
204.6e8.6
66.42 3.3
'
'
5
182.222.6'
81.92 3.8
1.420.5
7
7
344.627.2 I
176.628.0
78.22 1.8
78.52 2.4
1220.3
18.62 3.2
'*
2.6*0.5
3.82 0.3
7
189.726.6
71.422.5
16.02 3.7
I
'
2.820.5
'
Epithelial cells
per crypt
'
90.52 2.4 '
71.02 1.9 '
65.52 1.6
I
2.620.5
4.32 1.1
0.420.1
20.62 3.3
*
2.020.1
3.82 0.7
1.82 0.2
' Value significantly different (p < 0.01) from initial controls
Value significantly different (p
< 0.01) from final controls.
after operation, when growth had ceased for
some time (table 2). The increase appears to
begin in the early post-operative period, when
growth retardation first becomes evident, because in three animals studied 15 days after
thyroidectomy, there were 4.2, 5.6 and 6.0
globule leucocytes per 1,000 epithelial cells,
respectively, and these values were higher
than those seen in the initial sham-operated
controls.
The number of epithelial cells per crypt has
not been reported previously for this experiment and, as seen in table 2, a reduced crypt
growth followed thyroidectomy in weanling
rats, confirming results reported previously in
the slightly older rats of experiments 1,4and
5 (Carriere, '66).
Again, there was no significant change in
the number of intraepithelial lymphocytes in
the crypts (table 2).
412
R. CARRIERE AND M. BUSCHKE
TABLE 3
The influence of thyroxine and growth hormone on the increased frequency ofglobule leucocytes in the
duodenal crypts of thyroid deficient rats
Experimental groups
Experiment 4:
killed 90 days after operation,
injected last 26 days
Sham operated (final controls)
Thyroidectomized
Thyroidectomized growth hormone
Thyroidectomized + thyroxine
Experiment 5:
killed 147 days after operation
injected last 144 days
Sham-operated (initial controls)
Sham-operated (final controls)
Thyroidectomized
Thyroidectomized + growth hormone
Thyroidectomized thyroxine
+
+
' Value significantly different
Value significantly different
YU points on
globule leucocytes
Number of
animals
Body weight
8
248.1*6.0*
66.0a4.4 '
97.326.8 I *
145.72 7.4 I *
0.27-tO.06 '
4.45'- 1.12 I
5 . 5 4 2 1.70
1.5920.58 *
63.3'- 1.4
381.42 12.1
104.3a5.0
157.528.2
209.82 7.9 I . z
0.09'-0.02
0.11a0.13 *
2.50T0.36 I
3.41a0.73 '
0 . 2 7 2 0.07 *
7
5
7
6
7
7
8
4
f
S.E.
f
'
S.E.
(p < 0.01) from sham-operated final controls.
(< 0.01) from untreated thyroidectomized rats
The urinary bladders were examined, and
the increase of globule leucocytes in the transitional epithelium of the thyroidectomized
rats was striking. There were rare globule
leucocytes in the bladder epithelium of shamoperated rats, except in two where parasites
were also seen. Figures 7 and 8 show the presence of globule leucocytes in two different
thyroidectomized rats. The bladder sections
were not subjected to cell counting procedures.
Experiment 3
In rats thyroidectomized after sexual maturity, the number of globule leucocytes increased significantly (table 2). However, when
castration was combined with thyroidectomy,
there was no further increment, unlike the result observed in Experiment 1.Removal of the
testis did not enhance the effect of thyroidectomy on globule leucocyte number if thyroid
deficiency was initiated in adult rats.
Crypt growth, in terms of the number of epithelial cells per crypt section, was not altered
significantly by thyroidectomy in adult rats
(table 2) and this is in contrast t o its effect in
growing rats (Experiment 2, and Carriere,
'66).
In this experiment, thyroidectomy significantly augmented crypt lymphoid cell number
in comparison with the initial controls, but
the value was not significantly different from
that seen in the final sham-operated rats.
Experiments 4 and 5
These experiments confirmed repeatedly
the observation that thyroidectomy in sexually immature rats leads to an increase in the
crypt globule leucocyte population. Treatment
of thyroidectomized rats with thyroxine again
diminished the globule leucocyte number to
normal levels (table 3).
Treatment with growth hormone, begun
after growth had ceased, was effective in restoring body weight gains in the absence of
the thyroid, but i t did not reduce the elevated
number of globule leucocytes (table 3). The effect of thyroid hormone on globule leucocytes
in crypt epithelium is therefore not mediated
through its influence on the release of pituitary growth hormone.
General observations
The globule leucocytes were usually distributed throughout the upper two-thirds of
the crypts. Some were situated near the basal
lamina, among the basal portions of the epithelial cells (fig. l),but most frequently they
were slightly apical to the epithelial cell nuclei (figs. 2-41. None was found between the
apical portions of the epithelial cells nor in the
crypt lumen. At the junction of the crypts and
villi, heavily granulated globule leucocytes occurred a t the mouths of the crypts and on the
bases of the villi; the latter were not included
in the counts reported here. No well-granu-
THYROID EFFECT ON INTESTINAL GLOBULE LEUCOCYTES
lated globule leucocyte was seen in the villous
epithelium above the base.
The agranular crypt leucocytes appeared
mostly as lymphocytes and were distributed
primarily in the lower half of the crypts, particularly a t the bottom. They were usually
among the basal poles of the epithelial cells, at
the basal lamina. Occasional agranular leucocytes were larger than typical lymphocytes
and had irregularly shaped, larger and more
euchromatic nuclei; they may have been
larger lymphoid cells or potentially phagocytic cells.
No mitosis of globule leucocytes was seen in
the material reported here. More than 8,000
globule leucocytes were counted in all experiments combined. Several mitoses of villous
lymphocytes were seen within the villous epithelium.
DISCUSSION
The occurrence ofglobule leucocytes
in the crypts of normal rats
The intestines of 57 sham-operated rats
were examined and globule leucocytes were
always present. No intestinal parasite was
seen grossly or microscopically, nor any inflammatory reaction. In experiments 1, 2 and
3, the values ranged mostly between 0.3 and 2
globule leucocytes per 1,000 epithelial cells
and their detection often required the careful
scrutiny of cell counting procedures in a large
number of crypts. There was no significant
difference between the initial and final controls (Experiments 1 , 2 and 51, suggesting that
the relative globule leucocyte number remains fairly constant after weaning. Globule
leucocytes have been seen in the crypts of
germ-free rats (Kent, personal communication; Carriere, unpublished), and so they do occur without parasitic infestation, a fact which
must be considered in any speculation concerning their function.
In contrast to their low number in intestinal
crypts and in the transitional epithelium of
the urinary bladder, there were many globule
leucocytes in the tracheal epithelium of normal rats. This has been reported by Greulich
('49) in chicken trachea and by Kent et al.
('54) and Kent ('66) in r a t trachea. At this
site, their function may be related to that of
the pseudostratified ciliated epithelium, for
globule leucocytes were not seen in regions
where the epithelium lost ciliated and goblet
cells and became stratified (Carriere, unpublished).
413
The influence of the thyroid and testis
on globule leucocytes
Thyroidectomy always resulted in a significant increase of the crypt globule leucocyte
population, whether or not the animals were
adult a t the time of gland extirpation. The increase was not limited to intestinal crypt epithelium, for the subjective estimation of
globule leucocyte number in tracheal and
urinary bladder epithelia of thyroidectomized
rats revealed clear increases.
Treatment of thyroidectomized rats with
thyroid hormone restored the crypt globule
leucocyte population to its normal size and
this was not due to an influence on growth
hormone release, since the injection of growth
hormone to thyroidectomized rats was without effect. A synergistic action of the two hormones is not ruled out, but growth hormone
alone produced no change. Growth hormone
treatment was effective in terms of body
growth, since the animals weighed significantly more than thyroidectomized controls
and showed daily weight gains. They were
smaller than the sham-operated controls because treatment was begun long after growth
had ceased in response to thyroidectomy.
The influence of the testis was not as pronounced as that of the thyroid. Following
castration, the globule leucocytes remained
few in number but there were increases of
granule size and staining intensity. Furthermore, in sexually immature rats, castration
enhanced the effect of thyroid lack on crypt
globule leucocyte number. Such observations
point to an action of testicular hormone upon
these cells but its nature is not clear. Following treatment of thyroidectomized-castrates
with testosterone, there was no decrease of
globule leucocyte number. The injected testosterone was active since it induced prostate
and seminal vesicle growth (Carriere, '66).
Under similar conditions, thyroid hormone diminished the globule leucocyte number to normal, thus demonstrating the far clearer influence of the thyroid gl.and.
Lymphocytes in crypt epithelium
Non-granulated leucocytes in the crypts
were counted primarily to see whether
changes in globule leucocyte numbers would
be reflected in complementary changes of this
cell population; their nuclear morphology was
similar and they could represent a stage in
globule leucocyte degranulation. However,
414
R. CARRIERE AND M. BUSCHKE
these cells, largely lymphocytes, were located
primarily in the bottom of the crypts while the
globule leucocytes were mostly in the upper
two-thirds. The number of non-granular
lymphoid cells showed no consistent significant change in response to thyroid or testis removal and no obvious relationship between
the crypt lymphocyte and globule leucocyte
populations was indicated. Globule leucocytes
were not present in villous epithelium beyond
the villous base. Preliminary counts of lymphocytes within the villous epithelium are
compatible with the suggestion that globule
leucocytes move with the epithelial cells, are
degranulated a t the crypt-villous junction and
thus become indistinguishable from true intraepithelial villous lymphocytes. This suggestion is now under further investigation.
Origin and function of globule leucocytes
in crypts of thyroid-deficient rats
Numerous studies have shown that both
mast cells and globule leucocytes of the gut
mucosa increase in number in local parasitic
infestations and these cells have been assumed t o play a role in the immune process
(Dobson, '66d; Miller et al., '67; Jarrett et al.,
'67a,b; Murray et al., '68, '71). As mentioned
above, Mayrhofer ('77) has detected the presence of IgE in globule leucocytes and mast
cells of parasitized rats, using an immunofluorescent procedure. The results reported
here suggest t h a t the secretory immunoglobulin system of the thyroid-deficient rat should
be investigated, beginning with observations
on the localization of IgE. It is possible that
thyroid deficiency alters the animal's reaction
to antigens within the intestinal lumen. Such
a study is in progress, using the immunoperoxidase technique.
There was no indication of the source of the
increased number of globule leucocytes in the
crypts of thyroidectomized rats. It has been
postulated that globule leucocytes are degranulating mast cells in the reaction to
parasitic infestation (Miller et al., '67; Jarrett
et al., '67a,b; Murray et al., '68) and also in the
response of the urinary bladder to magnesium
deficiency (Cantin and Veilleux, '72, '73;
Veilleux and Cantin, '72). Our current electron microscopic observations of globule leucocytes and mast cells in the duodenum of
thyroidectomized rats show that their ultrastructure is almost identical. The possibility
that these cells are related is being investigated by radioautography, counting globule
leucocytes and mast cells following the injection of tritiated-thymidine a t various intervals after thyroidectomy, using histochemical
procedures which allow accurate detection of
the mucosal mast cells.
ACKNOWLEDGMENTS
This work was supported by a grant from
the N.I.H. National Institute of Arthritis and
Metabolic Diseases (AM04976) and by a
grant-in-aid from t h e University Awards
Committee for the Research Foundation of the
State University of New York (012-7123).The
hospitality of the Marine Biological Laboratory and its library, a t Woods Hole, Massachusetts, is gratefully acknowledged.
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PLATE 1
EXPLANATION OF FIGURES
1 Globule leucocyte in a duodenal crypt of a sham-operated r a t . The granules a r e few
and relatively small. In some cases. they were too faint for photographic representation. The tips of t h e arrows a r e a t t h e globule leucocyte plasmalemma. Bouin fixation, H a n d E s t a i n . X 900.
2
Globule leucocyte (arrows) in duodenal crypt of a castrated r a t . The granules a r e
larger and more brightly stained t h a n in normal rats, although t h e cells do not increase in number. Bouin fixation. H and E stain. x 900.
3
Globule leucocyte (arrows) in duodenal crypt of thyroidectomized-castrated r a t . The
cells increased in number and contained abundant, well-stained granules. Bouin fixation, H and E stain. X 900.
4 Globule leucocyte (arrows) in duodenal crypt of thyroidectomized-castrate treated
with testosterone after a long post-operative interval. The cells remained increased
in number and displayed t h e largest granules seen in any of t h e experimental
groups. Bouin fixation, H and E stain. x 900.
THYROID EFFECT ON INTESTINAL GLOBULE LEUCOCYTES
R ('arnere and M Buschke
PLATE 1
417
PLATE 2
EXPLANATION OF FIGURES
5
Tracheal epithelium of normal rat. Although globule leucocytes do occur in the
pseudostratified ciliated epithelium of normal rats, areas devoid of these cells are
easily located. Bouin fixation, H and E stain, X 1.000.
6 Tracheal epithelium of thyroidectomized rat. Globule leucocytes (arrows) are increased in number and some appear to cross into the tracheal lumen. Bouin fixation,
H and E stain, X 1,000.
418
THYROID EFFECT ON INTESTINAL GLOBULE LEUCOCYTES
R. Carriere and M. Buschke
PLATE 2
419
PLATE 3
EXPLANATION OF FIGIJRES
7 . 8 Transitional epithelium of urinary bladders from two r a t s thyroidectomized at 35
g n 1 body weight (weanlings). Globule leucocytes a r e rare in t h e normal parasitefree bladder epithelium but they increase in number due to thyroid lack, to t h e
extent t h a t one can see several per field (arrows). Carnoy fixation, H and E stain,
x 1.000.
420
THYROID EFFECT ON INTESTINAL GLOBULE LEUCOCYTES
R. Carriere and M. Buschke
PLAIE 3
42 1
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