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Hemopoietic repopulating potential of subcutaneous exudate cells.

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Hemopoietic Repopulating Potential of Subcutaneous
Exudate Cells 1
PHILIP SCUDERI, CORNELIUS ROSSE AND NEWTON B. EVERETT
Department of Biological Structure, University of WashingtonSchool of Medicine,
Seattle,Washington98195
ABSTRACT Inflammatory exudate (SE) cells were collected from subcutaneous coverslips in mice and transferred into lethally irradiated (1,000 r) recipients. Eight days after transplantation 59Feincorporation in the spleen and
bone marrow was significantly greater than in controls treated with the suspending medium only. One hundred percent of mitoses were of the T6T6 karyotype in
the marrow and spleen when SE cells were obtained from CBA/T6T6 donors. The
repopulating potential of SE cells, however, lagged significantly behind that of
bone marrow cells and the failure to observe consistently macroscopic spleen colonies calls into question whether the observed regeneration was due to pluripotent stem cells. Radioautographic studies with 3H-TdRshowed that the majority
of SE cells had recently been generated, but long-lived, noncycling cells of
lymphoid and monocytoid morphology were also present in the exudate.
Cells capable of repopulating completely or
partially the hemopoietic system of lethally
irradiated animals are known to be present in
various hemopoietic tissues (Loutit, '67; Metcalf and Moore, '71). Their potential for preferentially promoting the development of certain types of hemopoietic cells after transplantation bears some relationship t o thetissue of their origin. For instance, the ratio
of erythroid versus granulocytic coloniesin the
recipient spleen has been shown to be different when bone marrow, spleen or fetal liver
cells are transplanted (Wolf et al., '72; Duplan, '72). Thus transplantable hemopoietic
stem cells obtained from different sources
may vary not only with respect to their proliferative potential (Duplan, '68, '72; Schofield,
'70; Micklem et al., '72; Gidali et al., '74) but
also with respect to commitment.
In the mouse, in addition to bone marrow,
spleen and fetal liver, hemopoietic stem cells
have been shown to be present in the peripheral blood (Goodman and Hodgson, '62;
Micklem, '66) and in peritoneal exudate (Cole,
'63; Lin, '74). Previous studies with mice in
this laboratory suggested that mononuclear
cells from sterile, subcutaneous inflammatory
exudates also possessed erythropoietic repopulating ability ('I'yler et al., '72). The mononuclear cells which were implicated in promoting erythropoiesis comprised 23% of the cells
ANAT.
REC., 189: 141-148.
present in the exudates. They were similar in
morphology to the category of cells designated
earlier, from studies on irradiated rats, as
monocytoid and were considered to include
cells having multipotential stem cell capacity for hemopoietic repopulation (Tyler and
Everett, '66).
The implications of the earlier experiments
with mice (Tyler et al., '72) were considered to
be of such importance in relation to the
hemopoietic stem cell problem that i t was
considered desirable to substantiate the observations under more stringent experimental
conditions in attempts to more precisely characterize the exudate cells which migrate to
hemopoietically depleted tissues and promote
recovery, more specifically erythroid recovery,
in the present experiments. Thus, the origin
of the proliferating cells in the spleen and
bone marrow of the recipients was examined
by chromosome markers, and hemopoietic repopulation in these tissues was measured by
59Fe incorporation. In addition, we have examined the proliferative behavior of the exudate cells prior to transplantation by sHthymidine radioautography and we have also
tested in the exudate for the presence of longlived noncycling cells.
Received Feb. 9, '77. Accepted Mar. 31, '77.
' This research supported by US ERDA Contract AT(45-1)-2225.
141
142
PHILIP SCUDERI, CORNELIUS ROSSE AND NEWTON B. EVERETT
MATERIALS AND METHODS
coverslips were implanted in subcutaneous
pockets created in the shoulder and hip
regions on either side of the animal. The incisions were closed with wound clips. Eighteen
to 20 hours later the mice were killed by cervical dislocation and the coverslips were removed and placed in Hank's. Adherent cells
were removed from the glass by a rubber
policeman and the subcutaneous pockets were
repeatedly flushed with Hank's, pooling the
washings with the cells recovered from the
coverslips. After washing twice with Hank's,
the number of viable cells was determined by
trypan blue exclusion and hemocytometric
methods.
BM cells were collected from donor mice by
flushing the femoral medullary cavity with
Hanks. The cells were washed and counted in
the same way as SE cells. Cells were always
kept in ice cold medium and the final dilutions
of both BM and SE were made up in 0.5 ml aliquots for injection.
Seven days after transplantation recipient
mice were injected IP with 0.5 pCi 59Fe(ferrous citrate, New England Nuclear, Boston,
Massachusetts) in 0.1 ml balanced sodium citrate. Eighteen hours later (i.e., on the eighth
day after transplantation) the mice were
sacrificed and their femurs and spleens were
removed. Gamma counts for each were
Eight- to ten-week-oldvirgin female mice of
CBAIJ and CBA/TGT6 strains were obtained
from Jackson Laboratories (Bar Harbor,
Maine). The data were based on three separate experiments in which both donors and recipients were CBAIJ. Karyotyping was performed in a fourth experiment on 9 CBA/J recipients which were transplanted with CBA/
T6T6 cells.
Prospective recipient mice were exposed to
1,000 r whole body irradiation (Maxitron 250
kvp, 30 mA, 0.5 mm Cu and 1m A1 filtration,
dose rate 105 R/min) using a rotating carousel
with a source to target distance of 60 cm. Dose
corrections were made for ambient temperature and pressure. All irradiated mice received 0.125 mg/gm streptomycin sulphate
(Pfizer Labs, New York, New York) and 5,000
IU penicillin G (Squibb & Sons, Inc., Princeton, New Jersey) once a day.
One to two hours after irradiation the mice
were injected via the lateral tail vein with
subcutaneous exudate cells (SE), bone marrow cells (BM) or the suspending medium
(Hank's balanced salt solution, Microbiological Associates, Bethesda, Maryland).
SE cells were obtained from normal donor
mice as described previously (Tyler et al., '72).
Under light ether anesthesia, circular glass
TABLE 1
Percentages of mononuclear cell types labeled with 3H-TdRin subcutaneous inflammatory exudate under various
protocols of 3H-TdRadministration
X
Group
Labeling procedure
~~~~~~
1
2
3
4
5
6
Total
mono.
nuclear
cells
m
Small
lymphocytes
x
Medium
and large
lymphoid
cells
91
Monwytes
and
monocytoid cells
~
3H-TdR1 hour prior to
harvesting cells
3H-TdR6 hours and 1hour
prior to harvesting
cells
3H-TdR12,6 and 1 hour
prior to harvesting
cells
3H-TdR24,18,12,6and
1 hour prior to harvesting
cells
3H-TdRat 6 hourly intervals
for 48 hours followed by 48-hour
interval before harvesting
of cells
3H-TdR1daily injection for
14 days followed by a 14day period with no 3H-TdR
injection
24
0
17
33
35
5
33
44
50
12
50
50
72
18
79
85
77
50
66
86
6
0
10
8
~~~
I
Coverslips were always inserted 18-20 hours prior to sacrifice and harvesting of cells regardless of injection protocol of WTdR.
143
HEMOPOIETIC REPOPULATION BY EXUDATE CELLS
obtained in a Nuclear-Chicago 1185 Series
automatic gamma counting system. Tissues
were counted twice for one minute together
with 0.1 ml aliquot of 59Fesolution used for
injecting the mice. 59Fe incorporation by
spleen and bone marrow was expressed as a
percentage of the activity present in the 59 Fe
sample.
Four hours prior t o sacrifice, the recipients
used for karyotyping were injected IP with
Colcemid (0.04 mg/g body weight, Ciba Laboratories, Ltd.) and chromosome spreads were
prepared according to the method previously
described (Monie and Everett, '74).
For the cytokinetic studies, 3H-TdR (New
England Nuclear, specific activity 6.7 Ci/mM,
dose 20 pCi/mouse) was injected IP according
to six different protocols outlined in table 1.
SE cells were pooled from four CBA/J mice in
each of the six groups. After the cells were
washed in Hank's and suspended in calf serum, smears were prepared, fixed in methanol,
and processed for radioautography. The slides
were coated with NTB2 nuclear emulsion
(Eastman Kodak, Rochester), exposed for four
weeks, and after development were stained
with MacNeal's tetrachrome stain. By count-
ing 100 cells, the percentage of labeling was
determined in the following morphological
types of mononuclear cells: small lymphocytes, medium and large lymphoid cells,
monocytes and monocytoid cells. Granulocytes and macrophages were not included
in this cytokinetic analysis since only cells in
the category having suspected hemopoietic
potential were considered.
RESULTS
Cytokinetic properties of SE cells
Eighteen to twenty hours after implantation of the coverslips, the subcutaneous exudate contained both cycling and noncycling
cells (table 1).One hour after a single pulse of
3H-TdR (Group 11, labeled mononuclear cells
included medium and large lymphocytes and
monocytoid cells but not small lymphocytes.
When injections of 3H-TdR were repeated a t
6-hour intervals prior to harvesting the cells
(Groups 2-41, the proportion of labeling in
each cell category, including small lymphocytes, had increased progressively over a 24hour period. Thus, around 70%of mononuclear
SE cells had either been newly generated or
had entered into S phase during 24 hours
TABLE 2
Percent 5sFe incorporation in the spleen of mice transfused with SE or BM cells
s°Fe uptake
Cells transfused
5x 105s~
1 X 10"E
5 X 106SE
1x 107s~
5 X 1OSBM
1 X 106BM
Hank's
Number of animals
Mean 'X
S.D.
t'
P'
7
7
8
7
5
6
9
0.165
0.223
0.212
0.201
1.95
1.80
0.097
0.053
0.063
0.065
0.077
0.816
0.618
0.027
3.35
4.86
4.17
3.37
7.04
8.42
< 0.01
-
< 0.001
< 0.001
< 0.01
< 0.001
< 0.001
-
' "t" and "p" values represent comparisons with control animals injected with Hank's only. A two-tailed student t test was used
in the calculations.
TABLE 3
Percent 5sFe incorporation in the femur of mice transfused with SE or BM cells
5gFe uptake
Cells transfused
Number of animals
5X
1x
5X
1x
1OSSE
106SE
10"E
107s~
5 X 1OSBM
1 x 1OEBM
Hank's
Mean 'X
S.D.
0.093
0.108
0.126
0.092
0.325
0.373
0.103
0.014
0.012
0.025
0.013
0.063
0.089
0.012
t
1.76
2.46
-
10.56
9.15
-
01
< 0.1
< 0.05
-
< 0.001
< 0.001
-
' "t" and "p" values represent comparisons with control animals injected with Hank's only. A two-tailed student t test was used
in the calculations.
144
PHILIP SCUDERI, CORNELIUS ROSSE AND NEWTON B. EVERETT
(Group 4). A similar level of labeling was observed a t the time of harvesting when prospective recipients of coverslips were injected
repeatedly with 3H-TdR for 48 hours but an
additional 48 hours were allowed to elapse,
during which no thymidine was administered
(Group 5). The SE cells had accumulated during the latter 18 to 20 hours and had acquired
their label prior to implantation of the
coverslips. Thus, over 70% of SE cells were
derived from proliferating precursor cells
located elsewhere than the exudate and their
longest period of survival in this experiment
was four days. That cells with a substantially
longer life span were also present in the exudate was demonstrated in the mice of Group 6.
The mice were injected once a day with 3HTdR for two weeks in order to label a large
number, but not all cells generated over this
time period. No isotope was administered for
the subsequent two weeks, a t the end of which
time coverslips were implanted and the SE
cells were harvested 18 to 20 hours later. In
the exudate 8%of monocytoid and 10%of medium or large lymphoid cells were labeled
with 3H-TdRwhile none of the small lymphocytes contained demonstrable radioactivity.
The labeled cells were generated during the
availability period of 3H-TdR and had survived for two to four weeks. The grain density
suggested that the number of divisions during
this period was either very few (1-3) or none
a t all. Thus, the exudate contains predominantly newly generated cells but a substantial number of mononuclear cells other than
small lymphocytes are also present, which
had been out of cell cycle for an extended period of time.
5g Fe incorporation
Spleen colonies were observed in recipients
of bone marrow but they were infrequent
after SE cell treatment and none were seen in
the controls. In the spleen of recipients of SE
cells, 59Feincorporation exceeded significant-
ly the 59Fe activity in Hank's injected controls (table 2). The difference was maximal
with lo6 and 5 X lo6 cells but it was smaller,
though still statistically significant, with
lower and higher cell doses. Comparable numbers of BM cells brought about an approximately ten times greater increase in splenic
59Feuptake.
In the femur of recipients a significant increase in 59Feincorporation was observed only
with 5 X lo6 SE cells (table 3). With smaller
and larger cell doses, femoral 59Fe activity
was comparable to that in Hank's injected
controls. Recipients of 5 X lo5 or lo6 BM cells
incorporated around five times as much "Fe
in their femoral marrow as recipients of the
optimal dose of SE cells.
Proliferating cells of donor origin
Four CBAIJ mice were transplanted with
SE cells and five with BM cells obtained from
CBA/T6T6 donors. On the eighth day after
transplantation, karyotyping of mitoses in
the spleen and femoral marrow of recipients
revealed that 100% of clearly identifiable
chromosome spreads in both tissues were derived from transplanted BM or transplanted
SE cells (table 4). Figure 1 illustrates a chromosome spread prepared from the bone marrow of a CBA/J recipient which was treated
with CBA/T6T6 SE cells.
DISCUSSION
The present experiments establish that
cells obtained from subcutaneous inflammatory exudates can initiate regeneration in
the bone marrow and spleen of lethally irradiated recipient mice. Although the repopulating ability of SE cells lags significantly
behind that of BM cells, the following findings
indicate that the modest regeneration observed resulted from the proliferating progeny of transplanted SE cells:
1. One hundred percent of mitoses in the
marrow and spleen of CBA/J recipients were
TABLE 4
Karyoww analysis in the spleen and bone marrow of CBAIJrecipents transplanted with CBA/TGTGSE or BMcells
CBAm6T6 cells transfused
Number of CBAI+
recipients
1x 107s~
4
1 x 106BM
5
+
Number of karyotypes
Tissue
studied
Spleen
Femur
Spleen
Femur
T6T6
++
227
243
520
339
0
0
0
0
HEMOPOIETIC REPOPULATION BY EXUDATE CELLS
145
Fig. 1 A chromosome spread prepared from the bone marrow of a CBA/J mowe eight daye fter it received 1,000 r total
body irradiation and was transplanted with 1 X lo7 subcutaneous cells obtained from a normal CBAM'6T6 donor.
The two marker chromosomes are indicated by arrows.
of the T6T6 karyotype when the only source of
the marker chromosomes were the transplanted SE cells.
2. Incorporation of 59Fe was significantly
greater in the spleen and bone marrow of mice
transfused with SE cells than in mice which
received the suspending medium only. Thus, a
cell line not represented a t all among the
transplanted cells, does develop in host hemopoietic tissues. The differences between BM
146
PHILIP SCUDERI, CORNELIUS ROSSE AND NEWTON B. EVERETT
and SE cell recipients suggest that cells capable of proliferation after transplantation are
fewer in subcutaneous exudate than in bone
marrow and the growth of transplanted SE
cells lags significantly behind BM cells. It is
not known whether the limited erythroid
recovery in SE cell recipients would become
manifest later by erythroid colony formation.
Although the increase in 59Feuptake suggests that some of the dividing cells were
erythroblasts, the karyotype studies cannot
provide direct confirmation for this. The presence of proliferating cells of donor origin
could also be explained by the proliferation of
the lymphocytic and mononuclear phagocyte
cell lines, neither of which form in vivo macroscopic colonies. Both cell lines are represented in the inocula by cells capable of proliferation. Furthermore, it is known that the
spleen of irradiated mice supports proliferating descendants of immunocompetent cells
sensitized by antigen. Antigenic stimulation
was not introduced in the present experiments but its existence cannot be ruled out altogether despite the sterile techniques. Such
antigen sensitized units, however, are believed to seed to secondary lymphoid organs
and not to the bone marrow. The present experiments demonstrated dividing cells of
donor origin also in the marrow (fig. 1). No
data were obtained in these studies pertaining to the regeneration of granulocytes or
megakaryocytes. Thus the evidence for the
presence of stem cells among SE cells is restricted to the increase in 59Feuptake, which
has been regarded as a measure of stem cell
function (Hodgson, '62; Smith, '64; Lajtha,
'70), and the presence of proliferating cells of
donor origin in spleen and bone marrow.
No explanation can be offered for the observed decrease in radioiron uptake of the
spleen and bone marrow of recipient mice
when the number of transplanted SE cells
exceeded 5 X lo6 (tables 2, 3). Lala ('73) suggested that certain cell fractions of mouse
bone marrow contain cells which, if transplanted in increasing numbers, inhibit the
clonal expansion of stem cells in the host. A
similar explanation may pertain to SE cells
but the phenomenon has not been specifically
tested.
Known differences between the proliferative potential of stem cells obtained from
different sources could account for the differences between SE cell and BM cell recip-
ients. Micklem et al. ('75) presented evidence
that the-proliferative potential of circulating
stem cells falls significantly below those resident in the bone marrow. The origin of cells in
subcutaneous inflammatory lesions is from
the blood and many of them have been shown
to be discharged from the bone marrow (Volkman and Gowans, '65; Everett and Tyler, '68).
Our findings indicate that while the majority
of cells which migrate into the inflammatory
exudate have recently been generated, there
are immigrant cells which - judging by their
radioautographic grain density - have either
not entered into division over a 2 to 4-week period, or have divided only once or twice. Previous studies in rats (Tyler and Everett, '66)
suggested that cells morphologically similar
to monocytes, so-called monocytoid cells, possess stem cell potential. On the other hand,
cells of lymphoid morphology, so-called transitional cells, have also been implicated in
various stem cell and various progenitor cell
functions (reviewed by Rosse, '76).Both types
of cells are present in the exudate. Recently
generated as well as long-lived cells exist in
the exudate in both cell categories. The present studies do not address the question of
stem cell identity but they do indicate that
cells are present in subcutaneous inflammatory exudate which are capable of initiating
regeneration in hemopoietic tissues of lethally irradiated mice. Whether such cells are
long lived, resting cells or have recently been
generated, and whether they are multipotential remains to be investigated.
LITERATURE CITED
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mice. Blood, 29: 460-467.
HEMOPOIETIC REPOPULATION BY EXUDATE CELLS
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