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Studies on the fate of lymphocytes III. The migration and metamorphosis of in situ labeled thymic lymphocytes

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Studies on the Fate of Lymphocytes
111. THE MIGRATION AND METAMORPHOSIS OF IN SITU
LABELED THYMIC LYMPHOCYTES
'
RAYMOND G . MURRAY AND PHYLLIS A. WOODS
Department of Anatomy and Physiology, Indiana University,
Bloomington, Indiana
ABSTRACT
The in situ thymus of the guinea pig was labeled locally with
H3-thymidine and sections of various tissues analyzed by radioautography for the
location of labeled cells which might have left the thymus from 30 minutes to three
days after labeling. Labeled lymphocytes were located especially in the mesenteric
lymph nodes and spleen, giving direct evidence for the migration of thymocytes under
near-physiological conditions. Only a few cells were found in the bone marrow, and
none in the liver. The identified cells were not seen to be undergoing subsequent
proliferation, but about one-fourth of them were transforming to other cell types plasma cells, heterophil granulocytes, reticular cells, and macrophages. The fate of
another portion of the cells seemed to be loss to the gut lumen through the mucosa.
These data suggest that a normal function of the thymus of the adult guinea pig is
the proliferation of a population of pluripotential stem cells which, after migration
to other tissues, may further differentiate under appropriate stimulus. Possible applications of the local labeling technique are indicated.
In the last few yeaxs there has been a
heightened interest in the thymus with the
work of Miller ('63) and others in establishing the thymus as a critical organ in
the initiation of immunological competency. The part played by the thymus as
a highly productive lymphatic tissue,
acting as a contributor of cells, has also
been stressed by many (Walker and
Leblond, '58; Fichtelius, '60; Yoffey, '60;
and many others). However, all the evidence in support of the thesis that the thymus acts as a supplier of cells for other
tissues has been indirect from the following principal methods of study: ( 1 ) thyectomy of both newborn and adult animals, noting subsequent cellular changes
(Gyllensten, '53; Beirring, '60; Metcalf,
'60; Jankovic et al., '62), ( 2 ) cell population kinetics, studied by mitotic index
(Kindred, '42; Leblond, '59; Sainte-Marie
and Leblond, '64), ( 3 ) morphologic evidence of migration within the lymphatics
and blood vessels of the thymus (SainteMarie and Leblond, '58, '64; Clark, '63;
Weiss, '63) (4) transfusion of labeled cell
suspensions from the thymus (Keohane
and Metcalf, '58; Fichtelius, '60; Rankin,
'60; Murray and Murray, '61; Diderholm,
'61 ; Mims, '62), and (5) ontogenetic develANAT.REC., 150: 113-128.
opment of the thymus and other lymphatic
tissues (Miller, Marshall and White, '62;
Papermaster et al., '64).
In a recent review, Arnason et al. ('62)
concluded, "The thymus appears to be a
principle source of the small lymphocyte
population of blood, spleen, and lymph
nodes." Nevertheless, the critical demonstration that significant numbers of thymus
cells actually migrate from the intact organ
to other parts of the body has not been
made (Yoffey, '62; Miller, '63). We propose
to supply this demonstration, avoiding
some of the objections to the transfusion
experiments cited above, by providing
more nearly physiological conditions. To
do so we have: (1) left the thymus
in situ, with its attachments to the circulation and lymphatics of the animal, and
(2) injected directly, with minimum
trauma, minute volumes of a label which
is relatively innocuous and essentially
permanent once acquired. The guinea pig
was used because of the accessibility of the
thymus.
1
This investigation was supported in part by Grant
no. HE-06257 from the National Institutes of Health.
N.S.F. Summer Graduate Teaching Fellow 1963
In partial fulfillment of the requirements for the
dexree Master of Arts in Anatomy and Physiology,
Indiana University.
2
113
114
RAYMOND G. MURRAY AND PHYLLIS A. WOODS
Preliminary reports of this technique
were given by Murray ('62) and Woods
('64).
MATERIALS AND METHODS
Twenty-seven guinea pigs from a colony
maintained in OUT laboratory, with an average weight of about 400 gm, ranging from
293 to 598, were used for the study. For
the injection of H3-thymidine (Schwarz
Laboratories, specific activity, 6.0 C/mM,
isotonic) into the left lobe of the thymus,
the animals were anesthetized with Nembutal, and under sterile conditions a midline incision was made in the neck. Blunt
dissection was used to carefully detach
the lower part of the left lobe from the
surrounding connective tissue, leaving the
major vessels to the lobe intact. A ligature
was placed around the vascular pole and
was constricted just prior to the injection
of the thymidine. Table 1 lists the animals,
body weights, dose of thymidine administered, treatment of the circulation to the
lobe during the injections, and intervals
allowed before autopsy. The dose ranged
from 4 yc to 83 yc, and was administered
usually in about six small injections spread
over a five minute interval in various portions of the lobe. The total volume injected
was approximately 0.05 ml, and was injected with a 30 gauge needle in a microsyringe. Tyrode-moistened gauze was
placed around the lobe during the injection
and before loosening the ligature to absorb
any H3-thymidine which may have come
to the surface of the tissue from the injected point. Usually five minutes were
allowed after the injection before the circulation to the lobe was restored, but sometimes the interval allowed was 15, and
in seven animals the circulation was left
open and no dissection around the lobe
was performed for the injection of the
label. After the injections the animals
received Penicillin intraperitoneally, were
sutured, and placed in recovery cages.
The intervals between injection of thymidine and autopsy ranged from 30 minutes to five days (see table 1 for details).
The animals were killed with ether. Duodenum, spleen, liver, kidney, Peyer's patch,
mesenteric lymph node, lung, cervical
lymph nodes from the right and left sides,
both lobes of the thymus, and femoral bone
marrow were examined. The procedures
for radioautography are discussed in the
previous paper (Murray and Murray, '64),
as well as the conditions relating to the
significance of grain counts. We have
again used the value four grains or more
to designate a positively labeled cell.
OBSERVATIONS
Labeling in the thymus
Labeling efticiency. The injected lobe
was adequately labeled (i.e. 20-40% of
the cells labeled) when 25-50 I.IC were
employed with brief ligation. Labeling was
inadequate with 4 yc, and leakage was
excessive without ligation, whether 50 or
83 LLCwere used. Although the local concentration of Ha-thymidine was probably
higher than that following the customary
systemic labeling procedures, the technique did not result in obvious damage to
the cells. A comparison of the animals
which received 4 yc with those receiving
83 yc showed no significant differences
in cell death in the thymus. Patches of
dead cells were occasionally noted, but
these appeared to have resulted from handling rather than from irradiation damage.
Although there are obvious drawbacks to
occluding the circulation to the thymus,
even for only five minutes, attempts to
achieve a strictly local label were unsuccessful without this precaution. Care was
taken that the occlusion was made only
immediately preceding the injection and
released immediately thereafter.
Pattern of thymus label. The heaviest
and most thorough label was found at the
periphery of the cortex. At least 50% of
the cells in the cortex were labeled with
six or more grains two days after 50 yc of
thymidine, and some of these cells were so
densely labeled that their nuclei were completely obscured (see fig. 1 ) . Usually about
20% of the labeled cells found in the cortex had this very heavy label. Grain counts
were not made in the thymus because of
the large errors involved in the counting
of these heavily-labeled cells, but it appeared that the average intensity of label
was not strikingly reduced at three days
over earlier intervals.
Both lymphocytes and epithelial cells
were labeled, as well as occasional devel-
115
FATE O F LOCALLY LABELED THYMOCYTES
TABLE 1
Details of faCtOTS involved in treatment of the animals
Dose of
ThH3
Treatment of
circulation to
thymus during
injections
Interval
before
autopsy
Animal
number
Weight of
animal
4
30 min
30 min
1day
2 days
31
32
29
30
372
296
367
405
ligated for 5 min
ligated for 5 min
ligated for 5 min
ligated for 5 min
25
1day
3 days
5 days
56
51
54
371
289
336
ligated for 15 min
ligated for 15 min
ligated for 15 min
41
1day
2 days
2 days
50
49
52
458
516
459
ligated for 15 min
ligated for 15 min
ligated for 15 min
50
30 min
1hr
1day
1day
2 days
2 days
3 days
3 days
3 days
3 days
38
47
34
44
33
35
36
37
48
53
406
264
501
267
345
395
298
511
238
251
ligated for 5 min
free circulation
ligated for 5 min
free circulation
ligated for 5 rnin
ligated for 5 min
ligated for 5 min
ligated for 5 min
free circulation
ligated for 15 rnin
46
39
40
43
41
42
45
413
560
598
478
407
413
388
free circulation
free circulation
free circulation
free circulation
free circulation
free circulation
free circulation
gm
CC
83
Notes on
special
treatment
1hr
12 hr
12 hr
1day
2 days
2 days
3 days
~
~~
both lobes injected
partially thymectomized
both lobes injected
~~
1 All animals designated as “free circulation” showed thymidine leakage, and were discarded for the principal analysis of the fate of thymocytes.
oping eosinophil myelocytes. Within the
Hassall’s bodies, label was faund in immature eosinophil leukocytes and dead
cells as well as in epithelial cells of the
outer layers. The medulla was, in general,
much less heavily labeled than the cortex,
but with time, labeled small lymphocytes
in the medulla increased, which would be
consistent with the migration of cells from
the cortex to medulla, as observed by
Ernstrom (’63), and Sainte-Marie and
Leblond (’64).
Based on measurement of 100 labeled
cells in each animal, a high inverse correlation between nuclear diameter and the
length of time after injection of H3-thymidine is illustrated (figs. 2 and 3). The rise
with time in the population of small
lymphocytes was presumably by mitosis of
the larger cells. This finding is in agreement with the evidence of Leblond and
Sainte-Marie (’60), and Yoffey et al. (’61)
that in the thymus the cellular proliferation is from the larger lymphocytes to
small lymphocytes.
Not only did the average size of labeled
cells decrease, but the total per cent of
labeled cells increased with time. Figure
4 illustrates the per cent of cells labeled
in the thymus at the various intervals, and
indicates an early labeling efficiency of
about 3% at 30 minutes, which rises with
time. By three days, in one instance, about
half of the cells of the thymus were
labeled. These percentages were calculated
from counting in each animal about 500
cells selected from five different fields,
including one area of dense cortex, one of
medulla, and three intermediate fields.
It was concluded from these measurements that a good labeling efficiency is
attained by an injection of 25-50 I.IC of the
116
RAYMOND G. MURRAY AND PHYLLIS A. WOODS
label, with the vasculature occluded for
five minutes, and allowing an interval of
two or three days before autopsy.
Degree of confinement of label
to the thymus
The degree of label in the epithelial cells
of the duodenal mucosa was chosen as a
measure of the extent of systemic distribution of the H3-thymidine injected. This
choice was based on the known ability of
this tissue to acquire the label (Quastler
and Sherman, ’59; and others) and on our
observations in those cases where a ligature was not employed and some leakage
occurred. These animals were readily distinguishable from those in which a ligature was used (figs. 5 and 6 ) . The duodenal epithelial cells were well labeled,
and although label was also found in
mesenteric lymph nodes, spleen and bone
marrow, the grain counts over duodenal
epithelial cells were as high as, and in
most cases higher than, those seen elsewhere. Exceptions to this were occasionally noted in cervical lymph nodes and
uninjected thymus lobe, but in both these
sites there may have been some direct contamination with label at the time of injection.
Each animal was evaluated for the extent of label in the duodenal epithelium
and if these cells were labeled above the
critical value (four grains/nucleus), that
animal was not included in the analysis.
If the critical value was not exceeded in
the duodenal epithelium, isolated cells
found elsewhere with a label above this
value were considered to represent cells
which had been labeled in the thymus and
migrated therefrom.
Labeled cells identified in
various tissues
The following is a description of the
tissues analyzed and the cells identified.
In order not to be repetitive in the use of
the term “labeled cell,” this designation is
implied wherever an identified cell is mentioned. The criteria for the identification
of the several types seen is as follows:
1. Lymphocytes were characterized by
their heavily-massed chromatin, distinct
nucleoli, and limited basophilic cytoplasm,
the latter being relatively more extensive
the larger the cell. The distinction between
small lymphocytes and the category medium and large was based on nuclear diameter, all cells 5 and below classed as
small, and above this as medium and
large.
2. The plasma cell series differed in the
greater condensation of the nuclear material and increased area and basophilia of
the cytoplasm.
3. Macrophages were designated on the
basis of engulfed particulate matter, or
extensive, usually vacuolated cytoplasm.
4. Reticular cells were distinguished
from lymphoid by their delicate chromatin
pattern and in their faintly stained cytoplasm.
5. Heterophil myelocytes had polymorphic nuclei and specific granulation.
Mesenteric lymph node. The mesenteric lymph node contained the highest
number of migrated cells in nearly all animals studied. As seen in table 2, the average number of cells identified in these
nodes per 100 oil fields was 13. Although
the number varied widely among the several animals, a few labeled cells could always be identified here. At one day a
mitotic figure was observed, which was the
only such instance in any of the animals
of the study. Also seen at this interval
were several large, medium, and small
lymphocytes, as well as a few macrophages, reticular cells, and one pre-plasma
cell (figs. 7, 8, 9). At the two day interval,
besides the cell types previously mentioned, early heterophils, with the typical
horse-shoe nucleus of the metamyelocyte,
and two dead cells engulfed in the cytoplasm of unlabeled macrophages were
seen. There was some indication of an
increase of cells with time, and in one
node at three days as many as 38 cells/
100 fields were indentified. There was no
consistent pattern of distribution, cells
Fig. 1 Radioautograph of thymus cortex of
a guinea pig which two days previously received
50 pc H3-thymidine locally. Exposure 14 days.
Note the extent and intensity of label in small
lymphocytes. x 1,800.
Fig. 2 Radioautograph of thymus cortex of a
guinea pig which 30 minutes previously received 4 pc of H3-thymidine locally. Arrows indicate three large labeled lymphocytes. “a” focused
at nuclear level and “b” focused on the silver
grains. Exposure 14 days. X 2,100.
FATE OF LOCALLY LABELED THYMOCYTES
Figures 1 and 2
117
118
RAYMOND G. MURRAY AND PHYLLIS A. WOODS
..
1
(’61) who found after autologous thymus
cell transfusion in the guinea pig the opposite trend with time. As in the mesenteric Iymph node, the average grain counts
showed no pattern with time, and the cell
types observed here were similar to those
in the nodes. See figure 10 for a preplasma cell in the spleen.
Ceruical l y m p h nodes. The number of
cells in the cervical lymph nodes was third
compared to other tissues, with an average
of 3/100 fields. Both left and right nodes
were examined, and although one might
expect the node adjacent to the labeled
lobe of the thymus to contain more, no
significant differences were seen. Here, as
in other tissues, more medium than small
lymphocytes were identified, and were seen
usually in the sinuses and cortex.
Bone marrow. Occasionally the bone
marrow contained a few labeled cells
(fig. 11) and in one case as many as six
were recorded in 100 fields. These included a reticular cell and three heterophil
myelocytes. See figure 12 for an illustration of a heterophil myelocyte in the bone
marrow at one day. Another animal after
three days had one heterophil myelocyte
and one reticular cell, but most of the
remaining animals had no labeled cells.
Lung. Labeled cells were rarely observed in the lung. A few medium and
small lymphocytes, macrophages, dead
cells, and one plasma cell were seen. The
lung sometimes had large accumulations
of hemosiderin which made the analysis
somewhat difficult. This material, however could always be distinguished from
the silver grains by its brownish color and
tendency to occur in large aggregates.
Peyer’s patch. Occasional cells were
seen at the one and two day intervals,
but none was identified at three days.
One animal examined after a one day
interval had nine positive cells/100 oil
4
30
T
.
MIN.
1 DAY
2 DAYS
3 DAYS
Fig. 3 Change i n average diameter of labeled
nuclei i n locally labeled thymus with time after
injection of HJ-thymidine. Each point represents
100 measurements in one animal.
44
lot
30 MIN.
1 DAY
2 DAYS
3 DAYS
Fig. 4 Change in percentage of labeled cells
i n locally labeled thymus with time after injection of H3-thymidine. Each point represents an
animal.
being found in the cortex, medullary cords,
and sinuses at all intervals. The average
grain count over cells varied slightly but
showed no clear trend with time.
Spleen. The spleen usually was second
to the mesenteric lymph node with respect
to numbers per 100 fields, but sometimes
there were more here than in any other tissue. Usually the great majority was seen
in the white pulp, and in one animal at
one day they were exclusively in this area,
but there was some suggestion of a shift
toward the red pulp with time. At three
days they were approximately evenly
divided between red and white pulp. This
differs with the findings of Diderhdm
Fig. 5 Radioautograph of basal portions of
duodenal crypts in a guinea pig which one day
previously received 50 pc of H”-thymidine locally
i n the thymus while the vessels were occluded.
Note absence of label. Exposure 14 days. X 1,700.
Fig. 6 Radioautograph of basal portions of
duodenal crypts in a guinea pig which one day
previously received 83 pc of H3-thymidine locally
in the thymus without vascular occlusion. Note
numerous labeled epithelial cells. Exposure 14
days. x 1,700.
FATE OF LOCALLY LABELED THYMOCYTES
Figures 5 and 6
119
120
RAYMOND G . MURRAY AND
PHYLLIS A . WOODS
TABLE 2
Quantitation of labeled cells found outside t h e t h y m u s in six representative animals
Animal number
interval
and dose
Tissue
analyzed
1
Number of
labeled
cells/
100 oi!
immersion
fields
Average
grain
count
Range of
grain
counts
50
Id
41pc
mesenteric lymph node
spleen
bone marrow
Peyer’s patch
duodenal mucosa
7
8
6
9
14
8
10
9
7
5
5-15
4-30
4-20
4-18
5-8
56
Id
25pc
mesenteric lymph node
spleen
bone marrow
2
2
1
12
13
5
4-20
4-25
5
49
2d
41pc
mesenteric lymph node
spleen
cervical lymph node
kidney
duodenal mucosa
10
9
8
5
3
5
6
7
5
5
4-6
4-20
4-12
5-6
4-7
52
2d
41uc
mesenteric lymph node
spleen
cervical lymph node
duodenal mucosa
15
9
2
4
13
18
20
5
5-10
7-30
20
4-7
51
3d
25pc
mesenteric lymph node
spleen
cervical lymph node
10
8
5
12
12
15
7-25
5-25
5-30
53
3d
50pc
mesenteric lymph node
spleen
cervical lymph node
duodenal mucosa
38
2
2
4
9
6
20
4-25
6
20
4-7
1
5
In those tissues not mentioned, no labeled cells were located.
fields, including dead cells, lymphocytes,
and pre-plasma cells.
Lamina propria and submucosa of the
duodenum. Several labeled cells were
located in the lamina propria and submucosa of the gut, representing 8% of all
the identified cells outside the thymus.
Cells were seen in the lamina propria of
the villi, and occasionally were found between the epithelial cells in the wall of
the villus (see fig. 13). More dead cells
were identified in the lamina propria than
in any other tissue examined. The material
suggests that cells were being lost into the
gut lumen, although the content of the gut
was not examined for a confirmation of
this point.
Liver. I n spite of extensive examination, no cell was ever identified in the liver
of any experimental animal. The absence
of cells here does not correlate well with
current theories as to the liver’s possible
function in recirculation of lymphocytes
(Fichtelius, ’63). Moreover, thymus cell
transfusions have usually resulted in some
cells lodging in the liver, usually at the
early intervals (see Fichtelius, ’60, for a
review of these studies). It is possible that
the finding of transfused thymic cells in
the liver is a function of the method of
study, reflecting the reticulo-endothelial
capacity of this organ. Since this capacity
seems not to have been exercised in our
experiments, it seems likely that our conditions are more nearly physiological than
those obtaining in transfusion experiments.
Fig. 7-9 Radioautographs of mesenteric lymph
nodes from guinea pigs which had been injected
one day before autopsy with 50 pc of H3-thymidine directly into, and confined to, the thymus.
In each case “a” is focused on the nucleus and
“b” on the grains and exposure was 14 days.
Fig. 7
Fig. 8
Fig. 9
Medium lymphocyte.
Macrophage. x 2,000.
Plasma cell. X 3,000.
x
3,000.
FATE OF LOCALLY LABELED THYMOCYTES
Figures 7-9
121
122
RAYMOND G. MURRAY AND PHYLLIS A. WOODS
Kidney. Most of the animals examined
revealed no cells in the kidney, with the
exception of one at two days and one at
three days, which had a few lightly-labeled
lymphocytes.
Unlabeled thymus. The unlabeled right
lobe of the thymus was always examined
for labeled cells, and positive cells were
occasionally identified within it. There is
the possibility of local spreading of thymidine into this tissue, and in some instances, a very light general labeling was
noted on the periphery of the tissue, confirming the probability that the label had
spread into the lobe. However, in other
instances, single, well-labeled cells were
identified deep within the tissue, and in
these instances were assumed to have
migrated from the other lobe.
Summary of labeled cells in tissues outside the thymus. Figure 14 summarizes
the relative distribution of recovered cells
among the several tissues examined. Although the absolute numbers seen were
low, a rough calculation is possible to
estimate the proportion of the total presumed output of the thymus which is
represented by the cells identified. Using
Yoffey's figure of 4.7 x 10' cells produced
/day in the thymus of the 400 gm guinea
pig, divided by two since we injected only
one lobe, and assuming a 25% labeling
efficiency in the thymus itself, one would
expect from 1 to 2 labeled cells/100 oil
fields if these cells were evenly distributed
throughout the body and remained for 24
hours after migration. A s can be seen
from table 2, certain tissues contained
much higher concentrations than this figure. Since many areas were not examined,
we cannot be certain that the remainder
of the cells do not lie in the unsampled
regions. Although the selection of tissues €or examination may have introduced
some bias, it seems clear that the lymphatic tissues contained the bulk of the
cells, which suggests a predilection for
these sites. Although we cannot account
for all of the presumed daily production
of the thymus, neither is it necessary to
postulate massive cell death within the
thymus to explain our results.
Summary of cell types identified outside the thymus. Figure 15 compares the
percentages of each cell type among the
total of labeled cells found. It can be seen
that about 60% of the cells identified were
lymphoid, with more cells falling in the
medium and large than in the small category. This finding could mean either (1)
that small lymphocytes leave the thymus
and settle in other tissues where they
undergo hypertrophy to medium and large
lymphocytes, or ( 2 ) that under normal
conditions more medium and large lymphocytes actually leave the thymus. All the
transforming cells, that is, those identified
as reticular cells, macrophages, plasma
cells, or heterophil myelocytes, accounted
for 24% of the labeled cells. This lends
support to the thesis of Maximow ('09)
and others, who have considered the
lymphocytes as pluripotential stem cells.
It is suggested that because of the more
nearly physiological treatment of the cells
in this study, as contrasted to transfusion
studies, they were allowed to display more
fully their potentials for development.
DISCUSSION
Possible limitations of the technique
Reutilization. Use of H3-thymidine as
a marker is widely accepted (Cronkite,
et al., '60), but there remains the uncertainty as to the degree to which the label
may be transferred to other cells, that
is, reutilized within the organism. Evidence for reutilization has been reported
(Trowell, '57; Speirs et al., '62; Bryant, '63;
Rieke, '63). It is necessary, then, to consider whether the labeled cells which we
found in various tissues might have acquired their label by this means. Since the
Figs. 10-13 Radioautographs of tissues from
guinea pigs which had been injected one day
before autopsy with 40-50 pc H3-thymidine directly into, and confined to, the thymus. When
paired, "a" was focused on the nucleus and "b"
on the silver grains. Exposure was 14 days in
each case.
Fig, 10 Pre-plasma cell in the spleen. X 3,000.
Fig. 11 Cell with nucleus resembling a reticular cell, in the bone marrow. X 3,000.
Fig. 12 Heterophil myelocyte in the bone
marrow. x 3,000.
Fig. 13 Part of a villus in the duodenal
mucosa. Lower arrow indicates a labeled lymphocyte in the epithelium and the upper arrow
a labeled dead cell i n the lamina propria.
x 1,300.
FATE OF LOCALLY LABELED THYMOCYTES
Figures 10-13
123
124
RAYMOND G. MURRAY AND PHYLLIS A. WOODS
L A M I N A PROPRI
CERVICAL L N
MESENTERIC L N
LIVER
41 6
0
MEDIUM AND L A R G E
LYMPHOCYTE
SMALL LYMPHOCYTE
U NlDENTlFlED
10
20
30%
Fig. 15 The distribution of labeled cells
among the several types identified outside the
thymus in all animals in which the label was
originally confined t o the thymus, and at all
intervals from 30 minutes to three days.
label is thought to be firmly bound in the
nucleus, it presumably cannot be transferred to other cells, except by nuclear
breakdown. Bryant postulates such a
mechanism in his experiments, and Rieke's
results in ascites tumors could be explained
on the same basis. In order that such transfer occur in sufficient quantities to label
other cells, it would seem that either a
massive disintegration supplying large
amounts to an area, or intimate association
of more limited numbers of dead labeled
cells with the recipient nucleus would be
required. Even when substantial numbers
of labeled dead lymphocytes were injected
by Diderholm ('61), no transfer of label
to host cells was observed. No evidence
of substantial numbers of dying labeled
cells was found in our study other than in
the injected thymus. Thus, there might
have been reutilization within the thymus,
and some suggestion that this occurred is
found in the relatively slow decrease in
grain counts with time. This cell death
within the thymus might also result in the
release of some marker to the general circulation. That this cannot have occurred
to a degree sufficient to account for our
finding of labeled cells is shown by the
same controls (that is, the absence of
label in duodenal epithelial cells) as were
utilized to assess the degree of leakage of
the original label. The possibility remains
that labeled cells may have been engulfed
by other cells, thus providing an intimate
contact whereby the engulfing cell might
be labeled. We have, however, seen only
two instances of phagocytosis of labeled
cells outside the thymus, and no evidence
that the nucleus of the macrophage had
thus acquired the label. Mims ('62) introduced H3-thymidine labeled thvmus lymphocytes into cultures and although he
noted phagocytosis of labeled cells, none
of the phagocytes, even though subsequently undergoing mitosis, acquired the
label. It seems highly unlikely, therefore.
that any significant portion of the labeled
cells we identified were labeled in this
manner.
Other limitations. In the course of
handling and ligating the thymus, and
the injection of thymidine, some damage
to thymic cells was inevitable. The extent
of the effect on the cells, however, must
FATE OF LOCALLY LABELED THYMOCYTES
certainly be less than occurs in the course
of extirpation of the thymus and extraction
of the cells for preparation of suspensions
for reinjection. This is further indicated
by the fact that the spleen and the liver,
organs which normally acquire particulate
matter injected into the blood stream,
acquire the major part of transfused cells
(Fichtelius, ’60). In our experiments, on
the other hand, labeled cells were never
found in the liver, and the concentration
was usually higher in mesenteric lymph
nodes than in the spleen.
The objection can be raised that such
high concentrations of H3-thymidine as we
used may cause cell damage and death by
irradiation. This may, in fact, have contributed to the moderate degree of cell damage
noted. However, a large portion of the cells
were only moderately labeled, and the
death of some cells should not essentially
affect the fate of less heavily labeled and
presumably undamaged cells. This might
explain the fact that cells outside the
thymus were, on the whole, less heavily
labeled than those within the thymus. It is
possible that the extent of migration, and
possibly even the degree of subsequent
differentiation of thymus cells, might have
been reduced by local irradiation damage.
This does not affect the significance of
those migrated cells we observed, but may
mean that the numbers we found represent
minimum values.
Since the thymus is thought by some to
be separated from the general circulation
by a “hemato-thymic barrier” (Clark, ’63;
Weiss, ’63), the direct introduction of the
label may have stimulated the thymus cells
in a manner alien to the normal activity
of the thymus. Thus the finding of plasma
cells of thymic origin might have resulted
from such an abnormal stimulation. This
does not, however, negate the demonstration that thymus lymphocytes are capable
of such transformations if sufficiently
stimulated.
We must allow also for the possibility
that not all the cells labeled in, and migrating from, the thymus are lymphocytes.
A few developing myelocytes are always
seen in and around the thymus, which
might account for our finding of labeled
myelocytes outside the thymus. We noted,
however, that myelocytes mature within
125
the thymus, accumulating and dying in
the Hassd’s bodies, and there is no other
evidence, such as circulating immature
myelocytes, to indicate that they may be
migrating as such. Moreover, the myelocytes identified in the thymus were nearly
all of the eosinophil series, while those
found outside were invariably heterophil
myelocytes.
Bearing of our observations on current
hypotheses concerning lymphocytes
and the thymus
The immune reaction is believed to be
fundamentally a cellular one (Damashek,
’62), and the lymphocyte is widely conceded to be a key cell in this system. Moreover, the role of the thymus in initiating
certain of the immunological capabilities
of the organism is under extensive investigation (Good and Gabrielson, ’64). The
thymus might exert its influence by supplying iymphoid cells directly, by “seeding”
other lymphatic tissues resulting in their
further proliferation, or by a humoral factor which stimulates lymphocyte production in lymphatic tissue (see review by
Arnason et al., ’62). Migration of cells
from the thymus to other sites, although
demonstrated indirectly by numerous techniques (see introduction) and directly for
a few cells by Nossal (’64), has not been
clearly proved for significant numbers of
cells. We believe that our results supply
this proof, and indicate that lymphatic
tissues are the preferred sites for this
migration. There is almost no evidence,
however, in our material, of the migrating
cells proliferating in these locations. Moreover, we do not find them in the germinal
centers, where proliferation is most intense. On the contrary, we find transformations, which may be significant in
an understanding of the fate of thymic
cells and possibly of lymphocytes in general. This lack of proliferation of the
migrated cells suggests that at least in the
adult guinea pig, seeding of other lymphatic tissues for further proliferation does
not play a major role. The existence of a
humoral factor which stimulates lymphopoiesis, as proposed by Metcalf (’56) and
supported by Osoba and Miller (’63 and
’64), is not precluded by our findings.
126
RAYMOND G . MURRAY AND PHYLLIS A . WOODS
That the Iymphocyte may act as a stem
cell for other types in the hemopoietic tissues was suggested by Maximow ('09)
and supported by numerous authors since
(see review by Yoffey and Courtice, '56).
Yoffey ('62) has recently emphasized the
need in the guinea pig of a source, for
example the thymus, to supply large numbers of small stem cells to the bone marrow, which transform there into myeloid
cells. We find few labeled cells in the
marrow, and although we did note transformations to myelocytes, the extent of
this process does not accord with the massive seeding postulated by Yoffey. However, as indicated above, our technique
may be reducing appreciably the extent
of the normal migration.
Transformations of lymphocytes to macrophages and to plasma cells have been
frequently reported (macrophages-Bloom,
'27; Murray, '47; Fichtelius and Diderholm,
'61; and others; and plasma cells -Nossal
and Makela, '62; and many others). Although Nossal ('64) saw no transformations in experiments similar to ours, we
believe we have demonstrated that thymic
lymphocytes do change into other cell
types. These are not isolated phenomena,
but represent about 24% of the cells identified. These data thus support the direct
cellular role of the thymus in the immune
response of the organism, but the finding
of myelocytes and of many unidentifiable
cells (no more than 60% could be reliably
classified as typical lymphocytes) indicates
that the thymic lymphocytes may have a
variety of roles, rather than one limited
entirely to immune reactions. Most studies
using transfusion of thymocyte suspensions (see Diderholm, '61) report the failure to find transformations to other celI
types. Murray and Murray ('64), however, found evidence of transformation
toward erythroblasts, myelocytes, and
macrophages, after both homologous and
isologous injections. The portion of cells
transforming was low but this may have
been due to the more unphysiological conditions relating to the handling of the cells.
Bunting and Huston ('21) believed that
a major fraction of lymphocytes is lost
from the body into the lumen of the intestine. We have recovered a small number
of labeled cells in the duodenal mucosa,
but it is not possible from our data to
establish how significant these are quantitatively, as we have no measure of the time
consumed in such migrations. Since the
vast majority of lymphocytes in the mucosa remain unlabeled, it must be that
either they remain there for many days,
or that the majority of migrating cells
come from sources other than the thymus.
Further possible applications of the
local labeling technique
Since it has been shown that cellular
migration from the thymus may be studied
directly in the young adult animal under
nearly physiological conditions, the possibilities of the application of this technique
merit attention. In order to compare the
normal cell furnishing capacity of the
thymus at different stages of maturity, it
would be well to study the fetal, neonatal,
and older animal with this technique. Since
it is known that the guinea pig thymus
produces fewer cells with respect to the
total lymphoid complex than other animals, such as the rat (Yoffey, 'SO), the
technique might be adapted for other
species.
The possible cellular role of the thymus
in immunological mechanisms might be
studied by this technique, in order to determine the extent of participation of thymic
cells in graft rejections, inflammatory response, and antibody production.
Although thymus cells have not proved
effective in repopulation of tissues depleted
by irradiation, the probability that under
some conditions they may act as hemopoietic stem cells suggests the need for
additional work in this area. Experiments
employing the local labeling technique are
in progress in our laboratory to explore
further the possibility of a protective roIe
of the thymus in irradiation damage.
It is suggested that the local labeling
technique might be adapted to study the
cellular kinetics of other tissues, such as
spleen, lymph nodes, appendix, and bursa
of Fabricius. With proper attention to
technique and controls, this method may
well prove to be a useful and important
tool for exploring cellular migrations and
transformations within the organism.
FATE OF LOCALLY LABELED THYMOCYTES
ACKNOWLEDGMENT
The authors wish to acknowledge the
technical assistance of Josephine Grosch.
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