The mechanism of cell proliferation is studied in the lymphoid tissue of the mouse spleen under the stress of continuous irradiation at a dose-rate of 10 roentgens per day for 105 days. Autoradiography and specific labeling with tritiated thymidine were utilized. It was found that at least four compensatory mechanisms maintained a near-steady state of cellular growth:  (1) an increase in the proportion of PAS-positive cells which stimulate mitotic activity, (2) maturation arrest of proliferating and differentiating cells which tend to replenish the cells damaged or destroyed by irradiation, (3) an increase in the proportion of cells proliferating, and (4) an increase in the proportion of precursor cells. The results are compared to previous findings observed in the thymus

Foster, B. R.

English

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(NASA-CR-142068) LYMPHOID CELL KINETICS N75-16213
UNDER CONTINUOUS LOW DOSE-RATE GAMMA
IRRADIATION: A COMPARISON STUDY Semiannual
Status Report (Grambling State Univ., La.) Unclas
15 p HC $3.25 CSCL 06E G3/52 08984
SEMI-ANNUAL STATUS REPORT
SUBMITTED TO: NASA Scientific and Technical
Information Facility
REFERENCE NUMBER: NSG 9014
INSTITUTION: Grambling State University
Grambling, Louisiana
TITLE: Lymphoid Cell Kinetics Under
Continuous Low Dose-Rate Gamma
Irradiation A Comparison Study
SUM GRANTED BY NASA $19300.00
DURATION: One Year
DATE:
Of Award July 29, 1974
Of This Report February 12, 1975
SIGNATURE:
Principal Investigator
Dr. Bessie R. Foster
Department of Physics
Grambling State University
Grambling, Louisiana 71245
1314i3'~s j
U-1a
AC
https://ntrs.nasa.gov/search.jsp?R=19750008141 2020-03-23T01:26:53+00:00Z
Lymphoid Cell Kinetics Under Continuous Low Dose-Rate Gamma Irradiation
A Comparison Study
by
Bessie Ruth Foster
PURPOSE, METHODS AND FINDINGS
The objective of the current research is to make a comparison study
of the effects of continuous low dose-rate gamma irradiation on cell pop-
ulation kinetics of lymphoid tissue (white pulp) of the mouse spleen with
findings as they relate to the mouse thymus.
Experimental techniques employed include autoradiography and specific
labeling with tritiated thymidine (TdR-3H).
The problem being studied involves the mechanism of cell proliferation
of lymphoid tissue of the mouse spleen under the stress of continuous ir-
radiation at a dose-rate of 10 roentgens (R) per day for 105 days (15 weeks).
On the basis of thymidine labeling and distribution of various cell
types in the thymus under continuous irradiation, it was concluded that at
least four compensatory mechanisms served 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 proliferating and differentiating cells
which tend to replenish the cells damaged or destroyed by irradiat-
ion
3) an increase in the "proportion of cells proliferating," and
4) an increase in the proportion of precursor cells.
Data relative to thymus and spleen weights under continuous irradiation
have been presented and discussed in the Final Technical Report associated
with Grant NGR-19-011-008, submitted to NASA Scientific and Technical
2Information Facility October 4, 1974.
This report deals with findings as they relate to distribution of
cell types and thymidine labeling in the lymphoid component of the spleen
compared to previous findings observed in the thymus.
Distribution of Cell Types and TdR-3H Labeling
One hundred, 29-day old BCF1 male mice were irradiated at a dose rate
of 10OR per day for 105 days. Four mice were removed from the irradiation
unit and sacrificed one hour following an intraperitoneal injection of
TdR-3H at various time intervals until 105 days had elapsed. Standard tech-
niques were used to dissect thymuses and spleens and to process the tissue
through autoradiography with subsequent staining and microscopic examination.
One hundred unirradiated mice of the same strain, 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 or spleen cells (white pulp) were counted and
categorized per microscopic examination per animal. Cells were classified
on the basis of stain hue and morphology as PAS-positive reticular cells,
non-PAS positive reticular cells, and lymphocytes.
Figrue 1 illustrates the distribution of PAS-positive reticular cells
in the thymus and spleen under continuous irradiation. Although the total
number of PAS-positive cells observed was small, a significant increase in
their abundance occurred in the irradiated thymuses during the latter
phase of the irradiation period. There was also an increase in the pro-
portion of PAS-positive cells in the irradiated spleen, but only during
the initial phase of the irradiation period.
THYMUS
7
6 ,
*-CONTROLS- - - -
1o 5
- o- IRRADIATED
S3 1 e--*.
"\ e
3-
> 0
o SPLEEN
7a. 
l 6-a. S-CONTROLS- - ---
5m
S&-IRRADIATED
< 4-FL-
S 2
0 1 2 3 4 5 6 7 8 9 10 14 18 22 26 30 50 70 90 110
DAYS OF IRRADIATION
Figure 1
PAS-positive reticulum cells in the thymic cortex and mitoses in the
lymphoid cells in contact with the PAS cells have been observed (Goodwin,
1939; Gordon, 1955; Metcalf and Ishidate, 1962; Metcalf, 1964). These cells
are reported to be most marked in the thymuses of non-neoplastic, high-
leukemia AKR mouse strains, and to a lesser degree in the thymuses of other
mouse strains. In the thymus of high-leukemia AKR mouse strains there were
three times as many mitoses in lymphocytes surrounding these cells as in
lymphocytes elsewhere in the thymus (Metcalf and Ishidate, 1962; Metcalf,
1964). These changes were interpreted as a "triggering process" induced
by PAS-positive reticulum cells stimulating mitosis among lymphocytes com-
ing in contact with them, and thereby regulating lymphopoiesis.
It has been reported previously (Final Technical Report associated with
Grant NGR-19-011-008, October 4, 1974), that the response of both the thy-
mus 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. Further, that
the spleen is affected to a greater extent with shorter periods of steady-
state growth than exhibited by the thymus.
The absence of PAS-positive cells in the spleen from about two weeks
of irradiation throughout the remainder of the 105-day irradiation period
suggests that an increased stimulus for mitotic activity as evidenced by
the presence of PAS-positive cells was not operative in the splenic lym-
phoid tissue at a similar time interval nor to the same extent at that
exhibited in the thymus.
Data on non-PAS positive reticular cells are presented in Figure 2.
These data illustrate that the proportion of non-PAS positive reticular
16
THYMUS
-J 12 \
S \ O-- CONTROLS-----
S- o- IRRADIATED
- 8 \
I- 4 *ef
. o-
c 16
O0
a.
SPLEEN
a. A- CONTROLS- 
-
z &-IRRADIATEDOZ 8 A 6
A ' 
A 
, A "A/ie "A A- " '
z
X 
. I , p p I I I I
a, 0 2 4 6 8 10 14.18 22 26 30, 50 70 90 I10
DAYS OF IRRADIATION
Figure 2 o0
6cells was generally smaller in irradiated thymuses and spleens at the on-
set of irradiation, compared to non-irradiated controls. After the first
few weeks of irradiation, there was an increase in the proportion of non-
PAS reticular cells in the irradiated thymus. However, this occurrence
was not observed in the spleen until about 6 weeks of irradiation, and the
increase in the proportion of non-PAS reticular cells in irradiated spleens
was only slightly greater than that observed in comparable controls.
Since reticular cells of lymphoid tissue are precursor cells which
give rise to lymphocytes, these findings suggest that another compensatory
mechanism which serves to re-establish the near-steady state of cellular
proliferation in the thymus and spleen under continuous irradiation is a
state of "maturation arrest" among non-PAS positive reticular cells. That
is, a greater proportion of the lymphoid cell population remains in the
"progenitor" or "precursor" state under continuous irradiation, thereby,
producing cells which compensate for those that are damaged or destroyed
by radiation.
As with the findings on PAS cells in the thymus and spleen, the in-
crease in the proportion of non-PAS reticular cells was observed at dif-
ferent time intervals and to a lesser extent in irradiated spleen tissue
compared to the thymus,
Thymidine labeling in non-PAS reticular cells is illustrated in
Figure 3. Generally, there was a smaller percentage of labeling among
irradiated non-PAS reticular cells in both the thymus and the spleen, with
the exception of about a two-week period in the spleen (day 14 - day 28).
Since there was generally less labeling in irradiated non-PAS reticular
cells in the thymus and spleen, these data suggest that the reticular
cell population did not increase its capacity to proliferate.
(n THYMUS
-J
-1 70- e.. -- CONTROLS- -- --
o 60- o- IRRADIATED
I *, 0 *
U 30F
S20 -
4 0
I
z
0
z 70- SPLEEN
z 60 A-CONTROLS-..
w 50- A- IRRADIATED
S40-
S10-
130 A-- f., A,. A
0 I 2 3 4 5 6 7 8 9 10 14 18 22 26 30 50 70' 90 110
DAYS OF IRRADIATION
Figure 3
8The proportion of lymphocytes and labeling among lymphocytes are il-
lustrated in Figure 4 and Figure 5, respectively. The percentage of lym-
phocytes in both the thymus and spleen averaged about 95% in irradiated
and control tissue. Therefore, there was no significant change in the pro-
portion of lymphocytes under continuous irradiation.
Thymidine labeling among lymphocytes was greater in the irradiated
spleen at the onset of irradiation compared'to thymus tissue. After about
three weeks of irradiation, there was a significant increase in thymidine
labeling in the irradiated thymus, with very little labeling in comparable
spleens. Since thymidine labeling suggests DNA synthesis which in turn
suggests that cells are in preparation for division, a third compensatory
mechanism which enables the lymphoid tissue of the thymus and spleen to
maintain a near-steady state of cellular proliferation under continuous
irradiation may be an increase in the "proportion of cells proliferating."
Here, too, a similar pattern of comparison is borne out. That is, a
similar phenomenon occurs in both the thymus and spleen, but at different
time intervals and to a different degree in the spleen. For example, there
was only about 4% labeling among lymphocytes of the irradiated spleens
compared to about 26% labeling among lymphocytes in irradiated thymuses
averaged over the 105-day irradiation period.
Labeling in the total population is illustrated in Figure 6. General-
ly, there was a greater percentage of labeling in the irradiated spleen
during the first few weeks of irradiation compared to the irradiated thy-
mus. Thereafter, labeling in irradiated and control thymuses and spleens
paralleled each other, with labeling in the spleen taking on lesser
values.
Lastly, a comparison of findings observed in the thymus and spleen
under continuous irradiation is found in Table 1. An average was taken
75-
45- THYMUS
c)
W
o- CONTROLS----
O 15
00
M: o - IRRADIATED
_-0
- A A 2 4 7- 9 h
c)
15 A- IRRADI TED
F .
0 I 2 3 4 5 6 7 8 9 10 14 18 22 26 30 50 70 90 110
DAYS OF IRRADIATION
Figure 4
THYMUS
60 - * -CONTROLS-----
c,
So- IRRADIATED
40-\ /
0 0,
20
..J
w
m SPLEEN
60. A-CONTROLS-- --
w
a -IRRADIATED---
z 40
crJ
'. 20
a- - --
0 I 2 3 4 5 6 7 8 9 10 14 18 22 26 30 50 70 90 110
DAYS OF IRRADIATION
Figure 5
20 THYMUS i
I
o 15 -, - CONTROLS - -- I *-e
- 10 1
a-
5
o
c 20
z SPLEEN
<15 A -CONTROLS----
, -IR RADIATED-
0 0 -A•
. I I A I I I
0 1 2 3. 4 5 6 7 8 9 10 14 18 2 2 26 30 50 70 90 110
DAYS OF IRRADIATION
Figure 6
12
TABLE 1
COMPARISON OF FINDINGS OBSERVED IN THE THYMUS AND SPLEEN UNDER CONTINUOUS
IRRADIATION (AVERAGED OVER 105-DAY IRRADIATION PERIOD)
Thymus Spleen
Cell Percentage of Population Percentage of Population
Type or or Category or Category
Parameter
Controls Irradiated Controls Irradiated
PAS Positive
Reticular
Cells 0.11 0.12 0.00 0.05
Non-PAS
Positive 4.22 4.06 5.89 4.54
Reticular
Cells
Labeling Non-
PAS Positive 37.01 25.06 23.70 20.16
Reticular Cells
Lymphocytes 95.74 95.22 94.00 95.48
Labeling
Lymphocytes 19.97 26.30 3.93 3.96
Labeling
Total 10.00 7.59 4.86 4.76
Population
13
on each parameter studied over the entire 105-day irradiation period.
These averages suggest that there were about twice as many PAS-positive
cells in irradiated thymus tissue compared to spleens. Among non-PAS
reticular cells, labeling in non-PAS cells, and the proportion of lympho-
cytes; findings in thymus and spleen tissue were comparable. Labeling in
thymus lymphocytes was about 6 to 7 times the values observed in spleens,
and labeling in the thymus population averaged about twice that observed
in spleen tissue.
PROGRESS REPORT
Two studies pertaining to the current research are yet to be carried
out, one on the distribution of lymphocyte classes and their proliferative
capacity, and an estimation on the cell cycle time. Microscopic examinat-
ion on the former study is in progress, while tissue sections for the
latter are being prepared for autoradiography.
At our current pace, the research should be completed during the
proposed period of performance.
SUMMARY AND CONCLUSIONS
On the basis of distribution of cell types and thymidine labeling,
it is concluded that response of both the thymus and spleen to continuous
irradiation is similar, but the compensatory mechanisms are operative at
different time intervals for the two tissues, and that spleen cells are
stimulated to proliferate to a lesser extent than thymus cells.
COMMENTS
No further comments are pertinent at this time.
14
REFERENCES
Goodwin, M. C. (1939)i Amer J Anat, 64, 165.
Gordon, A. S. (1955). Ann N Y Acad Sci, 59, 907.
Metcalf, D. (1964), In The Thymus in Immunobiology, 150, edited by
Good, R. A,, and Gabrielsen, A. E. New York: Harper & Row.
Metcalf, D. and Ishidate, M., Jr. (1962). Austral J Exp Biol, 40, 57.


Lymphoid cell kinetics under continuous low dose-rate gamma irradiation: A comparison study

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