Cellular response and cell population kinetics were studied during lymphopoiesis in the thymus of the mouse under continuous gamma irradiation using autoradiographic techniques and specific labeling with tritiated thymidine. On the basis of tissue weights, it is concluded that the response of both the thymus and spleen to continuous low dose-rate irradiation is multiphasic. That is, alternating periods of steady state growth, followed by collapse, which in turn is followed by another period of homeostasis. Since there are two populations of lymphocytes - short lived and long-lived, it may be that different phases of steady state growth are mediated by different lymphocytes. The spleen is affected to a greater extent with shorter periods of steady-state growth than exhibited by the thymus

Foster, B. R.

English

NASA Technical Reports Server

The effect of continuous low dose-rate gamma irradiation on cell population kinetics of lymphoid tissue

The problem studied involved cell proliferation in mice thymus undergoing irradiation at a dose rate of 10 roetgens/day for 105 days. Specifically, the aim was to determine wheather or not a steady state of cell population can be established for the indicated period of time and what compensatory mechanisms of cell population are involved

N7 3 31999
FOURTH SEMI-ANNUAL STATUS REPORT*
SUBMITTED TO:
REFERENCE NUMBER:
INSTITUTION:
TITLE:
CASE FIL
COPY
DURATION:
SUM GRANTED BY NASA:
TYPE OF FUNDING:
DATE:
SIGNATURE:
PRINCIPAL INVESTIGATOR
NASA Scientific and Technical
Information Facility
NCR 19-011-008
Grambling College
Grambling, LA
The Effect of Continuous
Low Dose-Rate Gamma
Irradiation on Cell
Population Kinetics of
Lymphoid Tissue
Initially One (1) Year,
currently extended until
August 1, 1973
$16,500.00
Joint (NASA in cooperation
with the National Science
Foundation, $35,600.00 for
2 years)
July 13, 1973
Dr. Bessie R. Foster
Department of Physics
Grambling College
Grambling, LA 71245
*See comments on nature of report in Preface, page i.
https://ntrs.nasa.gov/search.jsp?R=19730023267 2020-03-23T01:02:37+00:00Z
PREFACE
A semi-annual status report has been prepared instead
of a final report because I have submitted a proposal to
NASA to extend the current research. The proposed starting
date for the extended research is August 1, 1973.
Although we have not been officially awarded a grant
to extend the current research, we do have indications
from NASA officials that we will be granted funds to under-
take another phase of investigation. Hence, a semi-annual
report is deemed more appropriate than a final report at
this time.
The Effect of Continuous Low Dose-Rate Gaimna Irradiation on
Cell Population Kinetics of Lymphoid Tissue*
By
Bessie Ruth Foster**
PURPOSE, METHODS, AND DISCUSSION
The following report concerns cellular response and cell
population kinetics of lymphoid tissue in mice under the stress
of continuous irradiation.
The problem being studied involves the mechanism of cell
proliferation in the thymus of mice under continuous irradiation
at a dose rate of 10 roentgens (R) per day for 105 days (15
weeks). Our aim is to determine whether or not a steady state
of cell population can be established for the indicated period
of time, and what compensatory mechanisms of cell population
are involved.
Changes in thymus weights, thymic cell counts, distri-
bution of PAS-positive cells, non-PAS-positive reticular cells,
distribution of lymphocytes and labeling among these cell types
under continuous irradiation have been discussed in previous
semi-annual status reports.
*A research project jointly funded by the National Science
Foundation (NSF Grant GB 29136) and the National Aeronautics
and Space Administration (NASA Grant NCR 19-011-008), and
initiated at the Argonne National Laboratory where the prin-
cipal investigator was a participant in the Argonne Faculty
Research Participation Program Summer, 1971.
**Currently Professor of Physics, Grabbling College, Grambling,
Louisiana, where the study is being completed.
On the basis of thymidine labeling and distribution of
various cell types in the thymus under continuous irradiation,
it was concluded that at least three compensatory mechanisms
serve to maintain a near steady state of cellular proliferation:
1) an increase in the proportion of PAS-positive cells
which stimulate mitotic activity
2) "maturation arrest" of precursor cells which tend
to replenish the cells damaged or destroyed by
radiation, and
3) increase in the "proportion of cells proliferating."
This report deals with findings as they relate to cell
cycle tiroes in the thymus under continuous irradiation, and
changes in individual classes of thymic lymphocytes by using
autoradiographic techniques and specific labeling with tri-
tiated thymidine (Td&-3H).
The Cell Cycle
On the basis of thymus weights and thymic cell counts
(discussed previously) it was demonstrated that a seemingly
steady state of cellular proliferation was achieved by about
four weeks of continuous irradiation. In order to determine
whether or not there were any changes in the generation time
of thymic cells, a group of UO, four-week old BCFj male mice
were exposed to continuous irradiation at a dose rate of 10 R
per day for about ** weeks, removed from the irradiation unit,
injected intraperitoneally with TdR-3H (1.0 p Ci/gram body
weight), and sacrificed at various time intervals from 0.5
to 30.0 hours following TdR-3!!. Thymus tissue was dissected,
fixed, processed through autoradiography, developed, stained,
examined microscopically, and labeled mitoses scored at each
sacrifice interval using routine histological and autoradio-
graphic procedures.
Forty unirradiated mice of the same age, sex, and strain
were processed similarly, these mice served as controls.
Two hundred mitotic figures were scored at each sacrifice
interval, and each data point on a given labeled mitosis
curve represents an average taken on two animals.
Figure 1 illustrates the curve of labeled mitoses.
Although there was no well defined descending limb of the first
wave of mitosis nor was there a well defined second wave in
irradiated nor in control thymuses, it was possible to roughly
approximate a cell cycle time of about 10.5 hours in both
groups as indicated in the figure.
Theoretically, all cells in DNA synthesis at the time of
TdR- H infection should have been labeled and should have
passed through mitosis to give 100% labeling (Johnson, 1961).
However, in the present study labeling reached only about 98%
on the ascending limb of the initial wave in irradiated thy-
muses and about 96% in control thymuses at 5 hours and 3
hours after TdR- H, respectively. Less than 100% labeling
during the first wave of the cell cycle has been reported in
spontaneous breast cancer (Mendelsohn, Dohan and Moore, 1960),
transplanted tumors (Steel, Adams and Barrett, 1966), and the
regenerating liver (Fabrikant, 196^ ; 1967a); this may be due,
in part, to the spread in the duration of the 62 + M/2 com-
plex (Fabrikant, 1967a) and the failure of a proportion of
labeled cells (false negatives) lying deep in a section to
give a radiographic image because of the very short range of
CO
LU
CO
O
100
80
60
40
20
Q
LU
_J
LU
CD
UJ 100
CD
80
o:LU
Q_
60
40-
0
0-IRRADIATED-
e-CONTROLS---
-? *
* I
\9
\
\
i
I
9
I 4-
/ Tc f^, 10 S HOURS
I I I I i
10 14 18 22 26 30
FIGURE I. SACRIFICE INTERVAL (HOURS)
tritium beta particles (Johnson, 1961; Fabrikant, 1967a; Fry,
personal communication).
The mean duration of the S phase in both the irradiated and
control groups was approximated to be about 6.5 hours; since the
seemingly descending limb of the initial wave of both curves is
less steep than the rise, the S phase in individual cells (reti-
cular cells, large, medium, and small lymphocytes) is of unequal
lengths. The low labeling among mitotic figures following the
peak is possibly due to cells which were in the Sj phase at the
time of labeling. The variability in the duration of the G^
phase must be extremely great, which partly accounts for the
absence of a well-defined peak for the S phase of the daughter
cells.
Curves of this type are characteristic of several tissues
such as the small intestine (Quastler and Sherman, 1959), bone
marrow (Cronkite, Bond, Fliedner, and Rubini, 1959), tumors
(Mendelsohn, et al., 1960; Mendelsohn, 1962; 1963), hair follicles
(Cattaneo, Quastler, and Sherman, 1961; McCarter and Quastler,
1962; Griem, 1966), skin (Gelfant, 1963), and regenerating liver
(Fabrikant, 1967a; 1967b, 1967c; 1968a; 1968b).
Furthermore, the radiation response of a cell system may
very well be more marked in autoradiographic studies due to
intranuclear radiation effects from incorporated TdR-3H in cells
preparing for division (Painter, Drew and Hughes, 1958; Drew and
Painter, 1959; Johnson and Cronkite, 1959; Wimber, 1959; 196«*;
Post and Hoffman, 1961).
Another factor which should be taken into account is that
the thymus is a multicompartmental cell system with at least U
major c*ll types, each with a different generation time. There-
fore, the findings reported here are, at best, a rough approxi-
mation of the cell cycle time. Nonetheless, there were no apparent
changes in the generation time of cells in irradiated thymuses
compared to controls. It was concluded, therefore, that changes
in the cell cycle time in thymuses irradiated at 10 R per day
were not contributing compensatory mechanisms.
Since there was no apparent change in the generation time
of thymus cells in irradiated mice compared to controls, the
next line of investigation was to determine the distribution of
cell types in the thymus population and the incorporation of
i at various intervals under continuous irradiation.
Distribution and Labeling Among Thvmus Cell Types
One hundred, 29-day old BCF^ male mice were irradiated at
a dose rate of 10 R per day for 105 days. Four mice were
removed from the irradiation unit and sacrificed one hour fol-
lowing an intraperitoneal injection of TdR-3H at various time
intervals until 105 days had elasped. Standard techniques were
used to process the tissue through autoradiography with subse-
quent staining and microscopic examination.
One hundred unirradiated mice of the same strain
 s
sex, and age as the irradiated group were processed in a similar
manner. These mice served as controls.
Each data point on any given graph represents an average on
4 animals, and at least 1000 thymus cells were counted and
categorized per microscopic examination per animal. Cells were
classified on the basis of morphology and size as reticular cells,
large (>11.0p in smears, > e.fyi in sections), medium (7.0 to
ll.Oyu in smears, 4.5 to 6.0^ in sections), and small (<7.0/u in
smears, <"+.5yu in sections) lymphocytes after descriptions of
Metcalf and Wiadrowski (1966) for tissue smears, and of Sainte-
Marie and Leblond (1958) for sections.
For convenience and due to their relative proportions, data
on medium and 'small lymphocytes were sometimes pooled.
Figures 2-U illustrate the distribution of the various cell
types in the thymus under continuous irradiation. There was
generally an increase in the proportion of reticular cells among
irradiated thymuses compared to controls. Since lymphopoiesis
in the thymus appears to occur as a result of asymmetric division
(Osgood, 1957; 1961) of reticular cells into lymphocytes of pro-
gressively decreasing sizes (Leblond and Sainte-Marie, 1960;
Yoffey, 1960; Yoffey, et. al., 1961), an additional compensatory
mechanism under continuous irradiation seems to be an increase
in the proportion of precursor cells.
There was generally a smaller proportion of large lympho-
cytes among irradiated thymus tissue compared to controls with
a few exceptions as indicated in Figure 3.
Data on medium and small lymphocytes were pooled. Figure U
illustrates that there was a very slight increase in the pro-
portion of medium and small lymphocytes among irradiated thymuses
compared to controls throughout most of the irradiation period.
Due to the relative numbers of medium and small Ivmphocytes,
and since the small lymphocyte is generally non-dividinp in vivo,
these findings suggest that the medium lymphocyte is contributing
an appreciable amount to the proliferative activity of the cell
8O
0)
O
ro
CO
c\j
CMCM
Si
g
O)
oo
i^ -
CD
IO
M-
ro
CM
2
O
Q
<
CC
cr
U.
O
CO
Q
OJ
UJ
o:
ID
CD
U_
IO CVJ
S1130
9S31AOOHdWAl 39VlN30d3d
QI
I
CO
_J
Q I-
< 2
<z o
E o
cfo
l\
e6\
\
99 -
i
t rr\ i t i 1 i 1 1 1 1
to
CM
"*~
o
o o o o o
o °o co ^ cj
•
r}-
>*
UJ
cr
Z)
o
u_
O10
O
ro
CJ
CVJ
CO
to
10
O^
Q
±
o
o>
CO
N-
a:
cc
LL
0
CO
>-
<
o
39VlN30d3d
11
system. The relative proliferative capacity of each cell type
will be discussed later in this report.
Because of the volume of data accumulated on the distri-
bution of each cell type at each sacrifice interval, an average
was taken over the entire 105-day irradiation period and
tabulated in Table I below.
TABLE I
PERCENTAGES OF VARIOUS THYMIC CELL TYPES
UNDER CONTINUOUS IRRADIATION
Reticular Large Medium and Small
Cells Lymphocytes Lymphocytes
Irradiated 2.0 2.7 95.3
Controls 1.3 3.8 9U.9
Table I shows that there was an overall increase in the
percentage of reticular cells in irradiated thymuses to almost
twice the value observed in controls. There was a decrease in
the overall percentage of large lymphocytes among irradiated
thymus, and a slight increase in the proportion of medium and
small lymphocytes.
Thymidine labeling among various thymic cell types is
illustrated in Figures 5-7. There was generally less labeling
in irradiated reticular cells and among large lymphocytes
compared to controls. However, among the medium and small lym-
phocyte category there was an increase in labeling in irradiated
groups during the initial and final phase of the irradiation
period.
12
S1130
Q
UJ CO
< o
— a:
2 H< z:
<* o
* oI I
o ©
13
O
lO
O
ro
Ho
CVJ
00 Q
o
— u_
o
o>
CO
o
CO
CO
lO
ro
w uj
a:
_ z>
CD
o
o
o
00
o
CO
o
CM
9Nll3aVl 39VlN30d3d
LU CO
9= oI I
o 9
o <
<M
S31AOOHd!AUn T1VI/MS
CM CO
oto
o
ro
C\J
(M
CM
00
CO
(O
«•
ro
CJ
14
o
g —
u_
C* O
CO
LU
o:
ID
o
u.
9NH38V1 39VlN3Dd3d
15
The relative increase in labeling among the medium and small
lymphocyte category further suggests that the medium lymphocyte,
in particular, is contributing appreciably to the proliferative
activity of the thymus cell population.
The next line of investigation was to determine the "pro-
liferative fraction" among the various thymus cell types to gain
some idea of the proportion of a given cell category that is
proliferating. That is to say, a cell in a population may or may
not be proliferating. Therefore, in order to determine which
cell type is contributing most to the proliferative activity, one
needs to know the proliferative fraction of that category, and
also some indication of its relative proliferative capacity.
The Proliferative Fraction of Thvmic Cell Types
The proliferative fraction of various thymic cell types
vinder continuous irradiation was determined using a modification
of the method of Lala and Patt (1966). Figure 8 illustrates the
findings. Only in the medium and small lymphocyte categories was
there an appreciable increase in the proliferative fraction during
the initial and final phase of the irradiation period. These
findings further suggest that it is the medium lymphocyte category
that is contributing most to the proliferative activity of the
thymus.
Again, because of the volume of data accumulated an average
was taken over the entire period of irradiation for each cell
type, and these data are reported here. On an average there
was about 85% of the reticular cell population proliferating, 75%
of the large lymphocytes, 20% of the medium lymphocytes and about
X-1
N
\
\
\
J_
CO
LU
O
o
•X.
CL
«i
X
a
/
H
Q K
si§
Sit
_J n a
./'.
O
10
O
ro
CVJ
CM
CM
00
00
Q i
to
^J-
ro
CM
»<
_L
O
CM
o o
CO
NOIlOVdd
O
fO
— g
3AllVd3dI10dd
o o o o o10 «o o 10
16
Q
<
o:K
LL_
O
CO
>-
<
O
oo
UJ
Q:
^>CD
u.
17
5% of the small lymphocytes.
Although only a small percentage of the medium and small
lymphocyte population was proliferating, their relative numbers
play an important role in the proliferative activity of the total
population as will be seen in the following discussion.
Relative Proliferative Capacity of Thymic Cell Types
The relative proliferative capacity of each thymic cell type
was determined using the method of Berman, Winter and Mewby (1966).
These data are shown in Figure 9. This figure illustrates that
there was generally an increase in the relative proliferative
capacity among irradiated reticular cells compared to controls
throughout most of the irradiation period. Also, there was an
\
increase in the relative proliferative capacity among medium and
small lymphocytes during the initial and final phases of the
irradiation period. Most importantly, the figure shows that the
medium lymphocyte has a relative proliferative capacity of about
U times that of reticular cells, and about 2 times that of large
and small lymphocytes.
Whether or not any of these findings are statistically signi-
ficant will be known when the computer analysis of the data is
completed.
PROGRESS REPORT
All phases of this study are nearing completion with the excep-
tion of a part of the computer analysis which is currently being
processed, and the publication of several papers in scientific
journals. The research papers are in preparation.
\CO
u
h-
>~
o
o
X
0.
Sco
Q i
LJ co
f>!< i
\
JL J_
O
o
CD
o o o
o o o
CO sfr (M
O O O
8 ° °S^ CO <fr
Al lOVdVO
19
SUMMARY AND CONCLUSIONS
On the basis of labeled mitoses, distribution of various cell
types, and relative proliferative capacities of thymus cells
under continuous irradiation, it is concluded that an additional
compensatory mechanism which serves to maintain a near-steady
state of cellular proliferation is an increase in the proportion
of precursor cells.
Also, it is the medium lymphocyte which contributes most to
the proliferative activity of the thymus.
COMMENTS
When we are granted support from NASA to extend our current
research, a similar study will be carried out using the lymphoid
component of the spleen (white pulp) of mice to make a comparison
study with findings we have observed in the thymus.
20
REFERENCES
Herman, I., Winter, S. R., and Newby, E. J., (1966).
Anat Rec, 154. 635.
Cattaneo, W. M., Quastler, H., and Sherman, F. G., (1961).
Nature (London), 190, 923.
Cronkite, E. P., Bond, V. P., Fliedner, T. M., and
Rubini, J. R., (1959). Lab Investigation, £, 263.
Drew, R. M., and Painter, R. B., (1959). Radiat Res,
11, 535.
Fabrikant, J. I., (1964). Ph.D. Thesis, University of London.
Fabrikant, J. I., (1967a). Radiat Res, 31, 304.
Fabrikant, J. I., (1967b). Brit J Radiol, 40_, 487.
Fabrikant, J. I., (1967c). Radiat Res, 32_, 804.
Fabrikant, J. I., (1968a). J Cell Biol, 36_, 551.
Fabrikant, J. I., (1968b). American J Roentgenal Radium
Therapy Nuc Med 102, 811.
Fry, R. J. M., personal communication.
Gelfant, S., (1963). Exp Cell Res 32_, 521.
Griem, M. L. (1966). Nature (London), 210, 213.
Johnson, H. A., (1961). Int J Cytol, 26_, 32.
Johnson, H. A., and Cronkite, E. P.,(1959). Radiat Res,
11, 825.
Lala, P. K., and Patt, H. M., (1966). Proc Nat Acad Sci,
56_, 1935.
Leblond, C. P., and Sainte-Marie, G., (1960). In
Haemopoiesis, 152, edited by Wolstenholme, G. E. W., and
O'Connor, M., Boston: Little Brown.
McCarter, J. A., and Quastler, H., (1962). Nature (London),
194, 873.
Mendelsohn, M. L., (1962), J Nat Cancer Inst 28, 1015.
Mendelsohn, M. L., (1963). Iin Cell Proliferation, 190
edited by Lamerton, L. F., and Fry, R. J. M.
Oxford: Blackwell.
21
Mendelsohn, M. L., Dohan, F. C., Jr., and Moore, H. A., Jr.,
(1960). J Nat Cancer Inst, 25_, 477.
Metcalf, D., and Wiadrowski, M. (1966). Cancer Res 26_, 483.
Osgood, E. E., (1957). J Nat Cancer Inst, L3, 155.
Osgood, E. E., (1961). Ann N Y Acad Sci, 95_, 828.
Painter, R. B., Drew, R. M., and Hughes, W. L., (1958).
Science, 127, 1244.
Post, J., and Hoffman, J., (1961). Radiat Res, 14, 713.
Quastler, H., and Sherman, F. C. (1959). Exp Cell Res,
17_, 420.
Sainte-Marie, G., and Leblond, C. P., (1958). Proc Soc
Exp Biol Med, 9j3, 909.
Steel, C. G., Adams, K., and Barrett, J. C. (1966). Brit
J Cancer, 20, 784.
Wimber, D. E., (1959). Proc Nat Acad Sci, 45_, 839.
Wimber, D. E., (1964). In Advances in Radiation Biology, 85,
edited by AugensteitT, L. G., Mason, R., and Quastler, H.,
New York: Academic Press.
Yoffey, J. M., (1960). Quantitative Cellular Haematology, edited
by Bethell, F. H., Springfield, 111: Charles C. Thomas.
Yoffey, J. M., Reinhardt, N. 0., and Everett, N, B., (1961).
J Anat 95_, 293.


The effect of continuous low dose-rate gamma irradiation on cell population kinetics of lymphoid tissue

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server

NASA Technical Reports Server