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Radioautographic studies of reticular and blast cells in the hemopoietic tissues of the rat.

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Radioautographic
Studies of Reticular and Blast Cells
- in the Hemopoietic Tissues of the Rat '
RUTH W. CAFFREY, N. B. EVERETT AND W. 0. RIEKE
Department of Biological Structure, University of Washington
School of Medicine, Seattle, Washington
ABSTRACT
The rates of proliferation and developmental capabilities of reticular
cells in the hemopoietic tissues of the rat were studied after administering single and
multiple injections of tritiated thymidine. Radioautographic analyses were made using
both smears and sections of the various hemopoietic tissues. Liquid scintillation
counting of tissues was also employed. Sublethal gamma irradiation was administered
to animals previously labeled with H3-thymidine in ordcr to evaluate the role of reticular cells during the repopulation o f the marrow.
The labeling patterns (per cent label and average grain count) of reticular cells,
both phagocytic and non-phagocytic types, are compared with the labeling patterns of
blast cells. It is shown that reticular cells are proliferating at a slow rate and do not
demonstrate the labeling patterns expected of stem cells for the rapidly proliferating
myeloblasts, pronormoblasts, lymphoblasts and megakaryoblasts. The labeling patterns
of cells which are generally believed to precede the committed blast cells, the hemocytoblasts, are also discussed and are contrasted with the labeling patterns of the
reticular cells. During recovery from sublethal irradiation there was no evidence of
differentiation or transformation of labeled reticular cells into blast cells of the various blood cell lines. The labeling patterns of blast cells suggest that the great majority
are derived from members of their own group and that any proposed precursors of
these blasts would need to be relatively few in number, rapidly proliferating and
highly radiosensitive.
In a previous report from this laboratory, proliferative rates of the various cell
lines in the mesenteric lymph node of the
rat were reported as determined by the
radioautographic analysis of tissue sections and smears following H3-thymidine
administration (Rieke et al., '63). From
these studies it was concluded that lymph
node reticular cells were proliferating at a
slow rate and showed no evidence of serving as stem cells for lymphoblasts or plasmoblasts.
This view is in conflict with the long
standing hypothesis first put forth by
Downey and Weidenreich ('12) and later
developed by Maximow ('27) that a reticular cell can separate from the stroma of
the hemopoietic tissues and can give rise
to the free cells therein. The concept that
reticular cells are stem cells for the myeloid and lymphoid cell lines has, in fact,
been widely accepted (Rebuck, '60; Bessis,
'56; Rebuck and LoGrippo, '61). The major
evidence in support of this concept is the
occurrence of cells which are intermediate
in morphological characteristics between
the fixed reticular cell and the free blast
cells.
ANAT. REC., 155: 41-58.
While our earlier radioautographic studies are in accord with the classic concepts
of cell derivation following the hemocytoblast stage, they are in conflict with the
view that the blasts are derived from reticular cells. A more extensive study concerning the life span of cells of the reticular network and the possibility that
reticular cells give rise to the free blast
cells has been made using both control
and irradiated rats. The results of this
study, again employing H3-thymidine and
radioautography, provide the basis for the
present report.
MORPHOLOGY AND TERMINOLOGY
Since the present study is restricted to
examining the classic concept that cells of
the fixed reticular network in the adult
hemopoietic tissues are the stem cells for
the free cells therein, no attempts are
made to characterize the stem cell or cells
for any particular cell line, nor to elucidate the derivation or possible relationship
among the various cell lines. In order to
1 Supported by the U. S. Atomic Energy Commission contract AT (45-1)-1377and by Public Health
Service Research grant GM-06309.
41
42
RUTH W. CAFFREY, N. B. C V L n L T T A N D --- RIEKE
W . V.
facilitate adequate communication, it is
necessary to define cells in terms of their
morphological characteristics and to draw
boundaries between the cell categories.
The definitions used here are believed to
be in accord with those used by the majority of other investigators. They are not
meant to favor either a monophyletic or
polyphyletic theory of blood cell development.
In this paper the term reticular cell is
restricted to those cells which form a part
of the fixed reticular network of the hemopoietic tissues. Some of the reticular cells
are phagocytic and have often been referred to as fixed macrophages. Others
are apparently nonphagocytic and have
been called undifferentiated reticular cells
(Maximow, '27). The category includes
those cells pictured and described by Maximow as undifferentiated reticulum cells
and as phagocytic reticular cells ('27), by
Rebuck and LoGrippo as inactive reticulum cells ('61), by Bessis as reticuloendothelial cells ('56) and by Sundberg and
Downey as undifferentiated reticulum cells
('42). In smear preparations stained by
Romanowsky stains, these cells usually
have the following morphological characteristics: ( a ) a very delicate network of
chromatin, ( b ) a definite nuclear outline
formed by a continuous thin band of chromatin which is subjacent to the nuclear
membrane, (c) a small clear blue nucleolus surrounded by a chromatin band and
(d) if cytoplasm is still attached, it is a
pale gray with indistinct limits (figs. 6, 7).
In tissue sections these cells have round,
oval, or irregular shaped nuclei with a pale
staining stellate chromatin network and indistinct cytoplasmic boundaries (fig. 18).
In this paper the term blast cell will be
used as a category to include all large free
cells with definite cytoplasmic boundaries
which are usually pictured as preceding
the committed blast cells (hemocytoblasts
as well as the lymphoblast, plasmoblast,
myeloblast, megakaryoblast and pronormoblast) in the developmental sequence (figs.
8-12). This category is believed to include
those free cells pictured as the hematopoietic reticulum ceUs by Rebuck ('60) and
Sundberg ('47), as hemocytoblasts and
histioblasts by Bessis ('56), and as hemocytoblasts by Maximow and Bloom ('42)
and probably also the "basket cells" by
Cronkite et al. ('59).
METHODS
Injection schedules
The rats used in this study were males
of the Lewis or Sprague-Dawley strain and
five groups of animals were injected with
Hs-thymidine (TTH) 1.9 c/mM as follows:
Group I (single injection series). Rats
in this group received an injection of TTH
and were sacrificed at varying intervals
thereafter. This series allowed for following the proliferative and maturation rates
of the various cells types. The group included 24 animals weighing 140-150 gm
each. The TTH was administered intravenously (1 w/gm body weight) and the
animals were sacrificed at intervals ranging from 15 minutes to 14 days post-TTH.
Group I1 (intensive injection series).
Rats of this group received a large amount
of TTH administered over a short period of
time which labeled 100% of the rapidly
proliferating cells. Six rats (150-250 gm)
were in this group and each was injected
intraperitoneally with 1 w/gm body weight
of TTH every four hours for 36 hours. All
were sacrificed four hours after the last
injection.
Group 111 (cumulative injection series).
Animals in this group received enough
TTH to label every cell entering DNA synthesis during an extended period of time
(11 days) and were sacrificed during the
course of the injections. The experiment
was designed to measure the rate at which
slowly proliferating cells become labeled.
Eleven rats, each with an initial weight of
65 gm, were injected intraperitoneally
with 0.75 w/gm body weight of TTH
every six hours until sacrificed. Beginning
24 hours after the initial injection, an
animal was sacrificed daily six hours after
the last injection. During the time of injection the rats gained weight at the normal rate of approximately 3% per day.
Group IV (multiple injection, interval
sacrifice series). Animals in this group
received a series of TTH injections over an
extended period of time and were sacrificed serially at prolonged intervals after
the last injection. This series was designed to label a major percentage of
RETICULAR AND BLAST CELLS OF THE RAT
slowly proliferating cells and to study the
rates at which label is lost from the population. Such a series also allows for investigating the relationship of the slowly
proliferating to the rapidly proliferating
cells. Each animal in this group (13 rats
weighing 60 gm each) was given 12 intraperitoneal injections of TTH (0.5 w/gm
body weight) during a 16-day period.
Three animals (140 gm each) were sacrificed four hours after the last injection
and the remainder were sacrificed at one,
two and five days; at one, two, four, six
and eight weeks and at 4 and 12 months
post-TTH.
Group V (irradiation series). This group,
consisting of 12 rats, received TTH by the
same injection schedule as group IV. At
12 days after the last injection, each rat
was given 300 r of total body irradiation
from a Cosasource and the animals were
sacrificed at intervals ranging from four
hours to 1 4 days post-irradiation. These
animals provided for investigating the effect of irradiation on the slowly proliferating cells and for evaluating the relationship of the slowly proliferating to the
rapidly proliferating cells during the irradiation recovery period.
43
ing system. Radioactivity present was converted to total disintegrations per minute
in the sample by use of internal standards.
A second aliquot of the TCA extract was
used for DNA determinations by the indole
method (Ceriotti, '52). The amount of
DNA per milligram wet weight of tissue
was calculated and the radioactivity was
expressed as disintegrations per minute
(dpm) per gamma of DNA.
RESULTS
Evidence that cells of the
blast cell category are
rapidly proliferating
Group I (single injection series). Fifteen
minutes after a single injection of TTH
50% of the blasts of mesenteric lymph
nodes were labeled with an average grain
count of 90. This percentage remained
constant between 15 minutes and 36 hours
but the average grain count per cell decreased as a logarithmic function of the
post-TTH interval with a half-time of approximately 12 hours. Thus the average
generation time of the blast cells of the
MN is estimated to be 12 hours (Rieke
et al., '63).
General techniques
In bone marrow of these same animals
A sample of spleen, mesenteric lymph approximately 60% of the blasts were
node ( M N ) , and bone marrow from each labeled at 15 minutes post-TTH with an
animal was teased in a small amount of average grain count of 90. By four and
homologous serum and smears were made eight hours post-TTH the average grain
for radioautography a s previously de- count had decreased from the initial 90 to
scribed (Everett et al., '60). A sample of 60 and 40 respectively. Thus the average
each tissue was also used for tissue sec- blast labeled at eight hours post-TTH had
tions; they were fixed in Zenker's fluid, either divided once or had arisen through
embedded in methacrylate and sectioned the division of a labeled stem cell. By 24
at 1 CI. The sections were processed for hours post-TTH, 87% of the blasts of the
radioautography as previously described bone marrow were labeled with an average
(Rieke et al., ' 6 3 ) . The exposure time was grain count of 30, and at 48, 72 and 120
eight weeks for all slides except for those hours, 100% of them were labeled with
of the cumulative and intensive injection average grain counts of 23, 16 and 9 reseries which were exposed for 35 days.
spectively (fig. 1 ). Reutilization of labeled
Liquid scintillation counting and DNA DNA precursors seems to be the most
determinations were made in some cases. reasonable explanation for 100% of these
A sample of each tissue was weighed and blast cells being labeled between two and
extracted with 5% trichloracetic acid at five days post-TTH. The slower rate of de90°C for 15 minutes (100 mg of tissue per cline in average grain count of the blast
ml of TCA). After extraction, 0.1 ml of of the bone marrow as compared to mesthe TCA extract was mixed in p-dioxane enteric node during the 24- to 120-hour
naphthalene solvent for radioassay with a period is also attributed to reutilization of
Packard Tricarb liquid scintillation count- labeled DNA precursors.
44
RUTH W . CAFFREY, N. B. EVERETT AND W. 0. RIEKE
Ioc
9c
8C
7c
0 BM
6C
*MN
5c
4c
3c
2c
.s
8 IC
.s
8
24
48
i2
96
I i.0
Hours Post-TTH
Fig. 1 Graph showing the decrease in average grain count of the blast population in
mesenteric lymph nodes ( M N ) and bone marrow ( B M ) following a single TTH injection.
The difference in grain counts apparent after 16 hours is interpreted as due to reutilization
of labeled DNA precursors in marrow.
Fifty per cent of the blasts of the spleen
were also labeled at 15 minutes post-TTH
and these like those in bone marrow and
lymph node evidenced a rapid decrease
in grain count through division. By 10
to 14 days after a single injection, no
blast was seen in any hemopoietic organ
which evidenced more than two or three
grains above background. Thus it may be
assumed that the specific activity of the
“reutilizable DNA precursor pool” in all
tissues had, by two weeks post-TTH, fallen
to a level below radioautographic detection.
Group I1 and group 111. One-hundred
per cent of the blasts of the node, spleen
and bone marrow were labeled in rats
which received intensive or cumulative
TTH injections for 36 or more hours.
Thus the maximum turnover time of any
blast cell does not exceed 36 hours. Animals in group I11 which were sacrificed
at intervals between 2 and 11 days showed
no unlabeled blasts and thus any precursors must have entered DNA synthesis before developing the morphological characteristics which would place them in the
blast cell category.
Group IV. In rats which received 12
injections in 16 days, the majority of blasts
were labeled at four hours after the last
injection (average grain count of 30).
Thereafter, the average grain count of
these cells decreased at a logarithmic rate
with a half-time of approximately two and
one-half days. By 14 days post-TTH no
labeled blasts were observed in any of the
hemopoietic tissues. Since no labeled
blasts were found in any of the animals
sacrificed after two weeks post-TTH, any
RETICULAR AND BLAST CELLS OF THE RAT
45
thelial cells and mature plasma cells (figs.
13, 1 4 ) . After 11 days of TTH administration, approximately 35% of the reticuEvidence that the reticular cells are
lar cells were labeled and the grain count
slowly proliferating cells
of these varied from 5 to more than 100
Group I . Less than 1% of the reticular grains. In contrast, all blast cells had
cells of the bone marrow were labeled in more than 30 grains (fig. 13). The mean
animals sacrificed between 15 minutes and grain count of the reticular cells was coneight hours after a single injection of siderably less than that of the other cell
TTH. This small percentage was heavily types and suggests that the majority of
labeled (more than 100 grains). The per- reticular cells were labeled through recentage of labeled reticular cells increased utilization of labeled material rather than
significantly between 12 hours and two through the direct uptake of TTH.
The rate at which reticular cells were
days post-TTH, and the marrow from animals sacrificed between two and five days labeled in the thymus, spleen and MN is
post-TTH showed approximately 12% of shown in figure 2. As observed in the
the reticular cells labeled with an average marrow, the average labeled reticular cell
grain count of 20. The heavily labeled re- of these tissues had a grain count conticular cells persisted and were easily dis- siderably less than that of the average
tinguished by grain count from the more blast cell. In considering the rate at which
weakly labeled cells. Since the heavily la- slowly proliferating cells become labeled,
beled reticular cells remained, it does not body weight gain must be considered.
appear that the weakly labeled reticular During the period of the injections the
cells were products of reticular cell divi- rats were gaining weight at a rate of apsion. Phagocytized labeled material was proximately 3% per day and thus it is
observed in many of the reticular cells be- reasonable to assume that from 3-6% of
tween 12 hours and five days post-single the cells could become labeled daily due
injection suggesting that these cells ob- to an increase in cell number related to
tained their label through reutilization. growth rather than to cell renewal.
Group IV (multiple injection -internal
Although no label was found in the blasts
or any erythroid, granulocytic, megakaryo- sacrifice series). In this series of rats a
cytic or lymphocytic element of the mar- large percentage of slowly proliferating
row at 10 to 14 days post-single injection, cells were labeled by giving the TTH at a
between 15 and 20% of the reticular cells slow rate to growing animals. The life
were labeled. The heavily labeled reticu- span of the cells was then estimated by
lar cells were also found at these later following the rate at which labeled cells
post-TTH intervals.
disappeared. This same approach could
Approximately 5% of the reticular cells not be applied to estimating the life span
of the MN and spleen were labeled at 15 of rapidly proliferating cells because it was
minutes post-TTH. Like the marrow the found that the specific activity of the repercentage of reticular cells in these tis- utilizable DNA precursors was within the
sues with a low grain count increased dur- limits of radioautographic detection for
ing the 2- to 3-day period. At 10 to 14 days the first few days after the last injection
post-single injection approximately 10% of TTH. However, by two weeks post-TTH
of those in the M N and spleen were labeled. no labeled blast cells were observed and
Group I l l (cumulative injections). After thus it may be assumed that cells which
24 hours of TTH injections approximately remained labeled for prolonged periods,
3% of the reticular cells of the bone mar- after two weeks, have a long life span and
row were labeled. Thereafter, the per- do not arise as a consequence of reutilizacentage of labeled reticular cells continued tion of labeled material.
to increase in proportion to the time of
At four hours after the last TTH injecTTH injection (fig. 2). The only cell types tion approximately 40% of the reticular
in marrow which were not 100% labeled cells in the bone marrow were labeled.
by five days of injections were reticular The percentage of labeled reticular cells
cells, mast cells, fat cells, capillary endo- increased during the first two-week post-
precursors to the blasts must also have
been rapidly proliferating.
46
RUTH W. CAFFREY,
N. B. EVERETT AND
90.
W. 0 . RIEKE
/
80.
70-
60.
Spleen0
MNa
Thymus
‘b
22
P,
8 50.
\
d
\
2 40
BM
x
30.
20
10
2
4
6
8
10
Days of TTH-injections
12
Fig. 2 Graph to show the rates at which the reticular cells of mesenteric lymph nodes,
spleen, thymus and bone marrow are labeled in animals receiving TTH injections every
six hours. Note that the percentage of labeled reticular cells increased at a relatively slow
rate and is proportional to the time of injections.
TTH period. As an example, at four hours
post-TTH, 42% of the reticular cells in
erythroblastic islets were labeled and at
two weeks post-TTII, 68% were labeled
(fig. 3). (This increase in percentage of
labeled cells is again attributed to reutilization of labeled DNA precursors.) The labeling intensity varied greatly; some nuclei were obscured by reduced silver, while
others had only a few grains (figs. 16,
17). The average labeled reticular cell
had a grain count of approximately 30.
The intensity of label appeared to increase
during the first two weeks post-TTH although an accurate evaluation was difficult because of the great variation in
grain density. At eight weeks post-TTH,
65% of the reticular cells were labeled
with no apparent decrease in grain count.
Approximately 65% of the reticular cells
in node and spleen were labeled at four
hours post-multiple injection. At the end
of two weeks post-TTH, 65% were still
labeled. This same high percentage of
labeled reticular cells was seen in all the
hemopoietic tissues of rats sacrificed a t
two, four, six and eight weeks post-TTH,
although no labeled blast cells were ever
encountered in any tissue from animals
sacrificed after two weeks post-TTH. Tissue sections of bone marrow revealed a
high percentage of labeled reticular cells
and no labeled free cells (fig. 21). The
labeled reticular cells were distributed
throughout the tissue and were morphologically indistinguishable from the nonlabeled reticular cells. The only free cells
of the hemopoietic organs which showed
47
RETICULAR AND BLAST CELLS OF THE RAT
label at 2 to 8 weeks post-TTH were the
long-lived small lymphocytes of the spleen,
M N and Peyer's patches, an occasional free
macrophage or mast cell and a few mature
plasma cells in bone marrow.
One animal was given TTH injections
for a period of two weeks during early life
and sacrificed one year after the last injection. Many labeled reticular cells were
observed in the bone marrow, spleen and
mesenteric node, figure 19. These studies
clearly indicate that the majority of reticular cells have a very long life span,
perhaps comparable to the life span of the
rat.
Additional evidence that the reticular
cells are not stern cells for
the blasts
The striking feature in the above experiments at long post-TTH intervals, was the
observation that no label was observed in
any hemocytoblast, pronormoblast, myeloblast, megakaryoblast, lymphoblast or plasmoblast in the same organs which showed
between 60 and 70% of the reticular cells
labeled. Tissue sections revealed that the
labeled reticular cells were an integral
part of the reticular network. If the reticular cells give rise to blast cells either
by modulation or division as classic concepts proposed, some labeled blasts or their
counterparts should have been encountered.
Group V (irradiation series). In view
of the many observations supporting the
reticular cell as a stem cell, it seemed
important to investigate the potential of
the reticular cells when exposed to conditions of stress or stimulation. To accomplish this, rats which had a large percentage (60-70% ) of reticular cells labeled
and no labeled blast cells were given
300 r of total body irradiation. The animals were then sacrificed at intervals
throughout the irradiation recovery period
in order to determine if the labeled reticular cells would transform and give
rise to blast cells.
The loss of cells and the recovery process in the hemopoietic organs followed
the same patterns as reported by other
TTH kpc/gm.
uii IUI
f ,/-
70;~
60
I
//
-b
d 50-
s!2
.
\
\
O\
'\.
\O
1
/
4
P
I
40-
9,
L
Y
I
I
I
30-
I
I
20-
I
I
10-
I
I
01
f
I
c
48
RUTH W. CAFFREY, N. B. EVERETT AND W. 0. RIEKE
investigators using sublethal doses of irradiation (Harris, '56; Hulse, '57, '61, '63;
Everett et al., '62). The cell content of the
hemopoietic organs decreased rapidly
reaching a minimum by 2 to 3 days postirradiation. At this time both smears and
tissue sections showed that the number
of free cells of the organs had been greatly
reduced leaving the reticular network with
60 to 70% of the reticular cells labeled.
The number of blasts in relation to the
number of reticular cells was greatly decreased during this early post-irradiation
period, indicating that the blasts (or their
precursors) were radiosensitive. Between
4 and 14 days post-irradiation erythropoiesis, granulopoiesis, and lymphopoiesis recovered rapidly. By 14 days postirradiation the bone marrow had returned
to a normal cell content. The M N , spleen
and thoracic duct lymph, however, showed
a deficiency in long-lived small lymphocytes for prolonged post-irradiation periods
as previously reported (Everett et al., '62).
During the early recovery period, at ap-
proximately 3 to 4 days post-irradiation, a
relatively large number of blasts were seen
in the hemopoietic tissues just prior to the
regeneration of more mature cells. None
of the blasts were labeled, however, at this
time nor at any interval prior to or after
this critical period. At 14 days post-irradiation between 60 and 70% of the reticular
cells were labeled with no apparent decrease in grain count.
Liquid scintillation counting and DNA
determinations were also made on the tissues of these irradiated animals. The DNA
per milligram wet weight of bone marrow
declined rapidly, reaching a minimum at
approximately three days post-irradiation,
reflecting the reduced cellularity of the
marrow (fig. 4 ) . As the rats recovered, the
DNA per milligram approached a normal
level. Between 2 to 5 days post-irradiation
as the DNA content decreased, and the
percentage of reticular cells encountered
in smears increased, the specific activity
(disintegrations per minute per Mgm of
DNA in the marrow} increased to approxi-
EFFECT OF IRRADIATION ON DNA CONTENT OF
BONE MARROW
--------
Controls
-- ---
---4
0
/--
0
0
0
'1
2
4
6
8
I0
Days Post -irradiation
I2
I4
Fig. 4 Rats received 300 r total body irradiation and were sacrificed at intervals from
15 minutes to 14 days post-irradiation. The decrease of DNA per mg of marrow, minimum
at 2 to 4 days post-irradiation, is interpreted to reflect the loss of nucleated free cells. At
this time, the marrow is composed of a high percentage of erythrocytes and stromal elements
(reticular cells). As the marrow recovers, the DNA per mg and cell content return to a
normal Ievel.
49
RETICULAR AND BLAST CELLS O F THE RAT
EFFECT OF IRRADIATION ON SPECIFIC ACTIVITY
OF BONE MARROW DNA
I
I
2
4
6
I
I
I
1
8
10
12
14
Days Post-irradiation
Fig. 5 Multiple injection interval (MII) rats received 300 r total body irradiation a t 12
days post-TTH when, with minor exception (see text), only the reticular cells were labelcd.
As the percentage of free nucleated cells reached a minimum a t 3 to 4 days post-irradiation
(fig. 4), the specific activity of the marrow is increased to approximately four times that of
non-irradiated control rats, indicating that the labeled reticular cells are highly radioresistant. As the marrow recovered to a normal cell content, the specific activity approached
that of controls with no apparent loss (over that of controls) in radioactivity from the
reticular cell population.
mately four times the value of the controls
(fig. 5). As the DNA content returned to
a normal level the specific activity of the
marrow DNA in irradiated animals approached that of the controls and was approximately the same as that of the controls by 14 days post-irradiation. These
results clearly indicate that the labeled reticular cells were not destroyed by irradiation as were the rapidly proliferating nonlabeled cells, and that radioactivity of the
reticular cell population remained essentially static during the irradiation recovery
process.
As further tests to determine the possible capacity of the labeled reticular cells
to serve as stem cells, hemopoiesis was
stimulated in three additional series of
rats (multiple injection, interval sacrifice)
at two weeks post-TTH by bleeding or by
administering bacterial vaccines (Salmonella and Pertussis). No labeled blasts
were observed in any of these animals
during the periods of hemocytopoietic
response.
DISCUSSION
Since this paper is not concerned with
the cytogenetic relationships of the various
blast cells, the term blast has been used
to represent all those cells which are USUally pictured as hemocytoblast, lymphoblast, myeloblast, megakaryoblast and pronormoblast in the developmental sequence.
The labeling patterns of the cells in the
blast category were so similar with the
types of TTH injections which were used
that it would seem unwarranted to draw
distinctions between them. In each experimental series the hemocytoblasts of
comparable hemopoietic organs showed
essentially the same per cent label and
average grain count as the committed blast
cells. In this respect the radioautographic
evidence is in accord with the classic concepts of cell dcrivation which places the
hemocytoblasts as precursors of the committed blast cells but it is in conflict with
the hypothesis that hemocytoblasts are derived from cells of the fixed reticular network.
50
RUTH W. CAFFREY, N. B. EVERETT AND W. 0. RIEKE
Previous radioautographic studies €ram
several laboratories have shown that more
than 50% of the pronormoblasts, myeloblasts, megakaryoblasts and lymphoblasts
(Cronkite et al., '59; Everett et al., '60;
Bond et al., '59) are in DNA synthesis at
any one time. The observation that 100%
of the blast cells are labeled by 36 hours
of multiple TTH injections sets this period
as their maximum turnover time in the
rat and shows that they are not derived
from nonlabeled precursors by modulation
before entering DNA synthesis. It is to be
recalled that more than 95% of the reticular cells remain unlabeled at 36 hours
in this series, and if reticular cells were
giving rise to blasts through modulation,
some of the latter would also be unlabeled.
The observation that the reticular cells
are slowly proliferating is also well documented although most of the previous
work has been based on mitotic counts
rather than radioautography. Yoffey noted
that the reticular cells of the bone marrow
of guinea pigs were not only few in number but rarely showed signs of mitosis or
differentiation ('60). Harris et al. ('63)
reported that in bone marrow of guinea
pigs recovering from sublethal irradiation
mitosis of reticulum cells was rarely seen
and that there was little evidence that
they served as stem cells. Kindred's earlier
studies of the hemopoietic organs of the
young adult rat ('42) showed the mitotic
rate for reticular cells to be low: E M =
0.07%, M N = 0.2% and spleen = 0.3%.
He found no evidence that reticular cells
gave rise to myeloid elements.
Maximow's hypothesis that reticular
cells can give rise to the blast cells rests
on the observation that there are cells
which show a gradual transition in morphological characteristics ranging from
the fixed reticular cell to the free blast
cells. I n the present study a n apparent
continuum of cells was recognized extending from the reticular cell to the blast cell.
In smear preparations 90-95% of these
cells could be placed in either the reticular
cell or blast cell category using morphological criteria. The remaining 5-10% did
not have enough distinguishing morphological characteristics to be placed in
either category. With smears or tissue sections in the radioautographic studies, how-
ever, a sharp line of demarcation in the
labeling patterns of the two categories of
cells was found. Moreover, the results
from the multiple injection interval series
clearly show that the labeled reticular cells
did not transform into the blasts by modulation as proposed by classical concepts i n
spite of the observation that intermediate
morphological forms may be observed.
Another possibility which must be considered is that a small percentage of the
reticular cells is different from the majority and that these have stem cell
capacity. These, however, would need to
be rapidly proliferating. In view of the
fact that only a small percentage of reticular cells incorporate TTH after a single
injection and that they retain their label
for long post-TTH periods, this possibility
seems unlikely. Also, the reticular cells
which were labeled at 2 to 8 weeks postTTH were morphologically indistinguishable (both in smears or tissue sections)
from the nonlabeled reticular cells.
Much of the evidence i n conflict with
the reticular stem hypothesis comes from
studies using total body irradiation. Following supralethal irradiation injury, bone
marrow transplantation experiments implicate "free rather than fixed" cells
as responsible for hemopoietic recovery
(Congdon, '59). It may be noted, too, that
many studies have shown that cells of the
reticular network of the hernopoietic tissues are very radioresistant while the blast
cells are highly radiosensitive (Barrow
et al., '51; Barrow and Tullis, '52; W.
Bloom, '47; M. Bloom, '48; Latta and Waggener, '54; Tullis, '49). In spite of this
observation, some investigators have maintained that the radioresistant reticular
cells are the source of the blast cells which
reappear during the irradiation recovery
process following sublethal doses of irradiation (Downey, '48; Frenkel et al.,
'63). Although 60-70% of the reticular
cells were labeled in the irradiation series
of animals prior to their exposure to sublethal doses of total body irradiation, no
labeled blast cells were seen in any of the
animals sacrificed serially throughout the
irradiation recovery period. This same percentage of reticular cells remained labeled
during the recovery period.
RETICULAR AND BLAST CELLS OF THE RAT
With respect to the derivation of blasts
the radioautographic evidence would indicate that the cells of origin must be
rapidly proliferating (turnover time less
than two weeks) which is in conflict with
the radioautographic studies of Fliedner
et al. (’64). The labeling patterns of
blast cells are mathematically in accord
with the concept that the majority are
derived from members of their own group.
For examzle, in the spleen and M N (where
there was no evidence that the extent of
reutilization of labeled material interfered
with the labeling patterns), 50% of the
blasts were labeled at 15 minutes after a
single injection of TTH. The per cent label
remained constant thereafter and the
average grain count decreased as a logarithmic function of time with a half-life
of approximately 12 hours.
The observations that reticular cells do
not contribute to hemocytopoiesis and the
suggestion that blasts are derived only
from members of their own kind are in
accord with the view of Goss (’28). Goss
concluded from experiments in which the
primitive blood island was removed from
Amblystoma embryos that the blood island
was a specific primordium for red blood
cells and that blood vessels and blood cells
have a different origin.
LITERATURE CITED
Barrow, J., and J. L. Tullis 1952 Sequence of
cellular responses to injury in mice exposed
to 1,100r total body X-radiation. Arch. Path.,
53: 391-407.
Barrow, J., J. L. Tullis and F. W. Chambers, Jr.
1951 Effect of X-radiation and antihistamine
drugs on the reticulo-endothelial system measured with colloidal radiogold. Am. J. Physiol.,
164: 822-831.
Bessis, M. 1956 In: Cytology of the Blood and
Blood-forming Organs. Grune and Stratton,
New York and London, pp. 567-577.
Bloom, Margaret A. 1948 Bone marrow. In:
Histopathology of Irradiation from External
and Internal Sources. National Nuclear Energy
Series, Division IV, Vol. 221. 1Villiam Bloom,
ed., McCraw-Hill Book Company, Inc., New
York, Chap. 6, pp. 162-242.
Bloom, W. 1947 Histological changes following radiation exposures. Radiology, 49: 344348.
Bond, V. P., T. M. Fliedner, E. P. Cronkite, J. R
Rubini, G. Brecher and P. Schork 1959 Proliferative potentials of bone marrow and blood
cells studied by in &To uptake of H3-thymidine.
Acta Haematol., 21: 1-15.
51
Ceriotti, G. 1952 A microchemical determination of desoxyribonucleic acid. J. Biol. Chem.,
198: 297-303.
Congdon, C. C. 1959 Recovery from radiation
injury, with special consideration of the use
of bone marrow transplantation. In: Progress
in Hematology, L. M. Toncantins, ed., Grune
and Stratton, New York, Vol. 11, pp. 21-46.
Cronkite, E. P., V. P. Bond, T. M. Fliedner and
J. R. Rubini 1959 The use of tritiated thymidine in the study of DNA synthesis and cell
turnover in hemopoietic tissues. Lab. Invest.,
8: 263-277.
Downey, H. 1948 Cytology of rabbit thymus
and regeneration of its thymocytes after irradiation. Blood, 3: 1315-1341.
Downey, H., and F. Weidenreich 1912 Uber
die bildung der lymphocyten in lymphdriisen
und milz. Arch. mikr. Anat., 80: 306-395.
Everett, N. B., R. W. Caffrey and W. 0. Rieke
1962 Radioautographic studies of the effect
of irradiation on the long-lived lymphocyte of
the rat. Rad. Res., 21: 383-393.
Everett, N. B., W. 0. Rieke, W. 0. Reinhardt and
J. M. Yoffey 1960 Radioisotopes i n the study
of blood cell formation with special reference
to lymphocytopoiesis. Ciba Foundation Symposium on Haemopoiesis, G. E. W. Wolstenholme and Maeve O’Connor, eds., J. and A.
Churchill Ltd., London, pp. 43-66.
Fliedner, T., F. 0. Thomas, L. M. Meyer and E.
P. Cronkite 1964 The fate of transfused
H3-thymidine-labeled bone marrow cells i n irradiated recipients. Ann. N. Y. h a d . Sci., 114:
510-527.
Frenkel, E. P., Y. Sugino, R. C. Bishop and R. L.
Potter 1963 Effect of X-radiation on DNA
metabolism in various tissues of the rat. Rad.
Res., 19: 701-716.
Goss, Charles 11. 1928 Experimental removal
of the blood island of Amblystoma punctatum
embryos. J. Exp. Zool., 52. 45-63.
Harris, P. F. 1956 Quantitative examination of
bone marrow i n guinea pigs after gamma irradiation. Brit. Med. J., 2: 1032-1035.
Harris, P. F., G. Haigh and J. H. Kugler 1963
Observations on the accumulations of mononuclear cells and the activities 01the reticulum
cells in bone marrow of guinea pigs recoverinn
from whole body irradiation. Acta Haematol.,
29: 166-179.
Hulse, E. V. 1957 Quantitative studies of the
depletion of the erythropoietic cell i n bone marrow of the irradiated rat. Brit. J. Haemat., 3:
348-358.
1961 The recovery of myelopoietic cells
after irradiation, a quantitative study i n the
rat. Brit J. Haemat., 7: 430441.
1963 Lymphocytic recovery after irradiation and its relation to other aspects OP
haemopoiesis. Brit. J. Haemat., 9: 376-384.
Kindred, J. E. 1942 A quantitative study of
the hemopoietic organs of young albino rats.
Am. J. Anat., 71: 207-243.
Latta, J. A., and R. E. Waggener 1954 The hematological effects resulting from injection of
radioactive phosphorus (P”) into albino rats.
Anat. Rec., 119: 357-386.
52
RUTH W. CAFFREY, N. B. EVERETT AND W. 0. RIEKE
Maximow, A. 1927 Lymphoides oder reticulares gewebe. In: Handbuch der Mikroskopischen Anatomie des Menschen, W. van Mollendorff, ed., J. Springer, Berlin, Vol. I1 (Pt.
l ) , pp. 335-378.
Maximow, A. A., and W. Bloom 1942 In: A
Textbook of Histology, W. B. Saunders, Philadclphia, pp. 95-98.
Rebuck, J. W. 1960 In: The Lymphocyte and
Lymphocytic Tissue. Hoeber, New York, pp.
260-289.
Rebuck, J. W., and G. A. LoGrippo 1961 Characteristics and interrelationships of the various
cells in the RE cell, macrophage, lymphocyte
and plasma cell series in man. Lab. Invest.,
10: 1068-1093.
Rieke, W. O., R. W. Caffrey and N. B. Everett
1963 Rates of proliferation and interrelationship of ceUs in the mesenteric lymph node of
the rat. Blood, 22: 674-689.
Sundberg, R. D. 1947 Lymphocytogenesis i n
human lymph nodes. J. Lab. Clin. Med., 32:
777-792.
Sundberg, R. D., and H. Downey 1942 Comparison of lymphoid cells of bone marrow and
lymph nodes of rabbits and guinea pigs. Am.
J. Anat., 70: 455497.
Tullis, J. L. 1949 The response of tissue to
total body irradiation. Am. J. Pathol., 25: 829851.
Yoffey, J. M. 1960 In: Quantitative Crllular
Haematology. Charles C Thomas, Springfield,
pp. 83-85.
PLATE 1
E X P L A N A T I O N OF FIGURES
Figures 6 and 7 show the morphology of reticular cells i n smear
preparations of bone marrow (non-radioautographs). Figure 7, which
shows the characteristic morphology of the reticular cell nucleus, serves
to illustrate that in smear preparations it is common for the reticular
cell cytoplasm to be stripped from the nucleus. X 1,575.
Figures 8 to 12 show the morphology of the various blast cells i n bone
marrow smears (non-radioautograph). X 1,575.
8
Proerythroblast.
9
Three myeloblasts above a n erythroblastic islet.
10
Early erythroblast above myeloblast below.
11 Hemocytoblast a t the right of two granulocytic precursors.
12 Hemocytoblast above intensely stained erythroblasts.
RETICULAR AND BLAST CELLS OF THE RAT
PLATE 1
Ruth W. Caffrey, N. B. Everett and W. 0. Rieke
53
PLATE 2
EXPLANATION OF FIGURES
Radioautographs showing labeled cells i n smear preparations
of bone marruw
13
This figure shows that after ten days of cumulative TTH injection
all blast cells and all cells of the erythrocytic, granulocytic and lymphocytic series are heavily labeled in marrow. Note the unlabeled
reticular cell, center, and a weakly labeled reticular cell at left.
X 1,575.
14 A group of reticular cells, four of which are heavily labeled. Animal
received cumulative TTH injections for ten days. X 1,575.
15 A labeled reticular cell and a n unlabeled hemocytoblast three weeks
following multiple injections of TTH. X 1,575.
16 A heavily labeled reticular cell two weeks after a single TTH injection.
X 1,575.
17 A weakly labeled and a moderately labeled reticular cell two weeks
after a single TTH injection. x 1,575.
54
RETICULAR AND BLAST CELLS OF THE RAT
Ruth W. Caffrey, N. B. Everett and W. 0. Rieke
PLATE 2
55
PLATE 3
EXPLANATION O F PIGURES
Photomicrographs of 1 sections of mesenteric lymph nodes
56
18
This photomicrograph, of a non-radionutographic preparation, shows
the morphology uf the blasts ( b ) a n d reticular cells ( 7 ) . x 2,100.
19
Three labeled reticular cells i n the nicdullary cords one gear postmultiple injections of TTH. S 1,260.
20
Labeled blast cells and non-Iabclcd reticuIar cells ( r ) 15 minutes
post-single injection of TTH. Approximately 50% of the blast cclls
arc labeled. >: 1,260.
21
Numerous labeled reticular cells at three wecks post-multiple injection of TTH. There are n o labeled blast cells. X 1,260.
22
Labeled hemocytoblast at 15 minutes post-singlc injection of TTH.
Note the attenuated cytoplasm at the upper pole of the ccll. X 1:260.
RETICULAR AND BLAST CELLS O F THE RAT
Ruth W. Caffrey, N. B. Everett and W. 0.Rieke
PLATE 3
57
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