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. 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