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Haemopoietic spleen colony formation in the rat: Effect of 89Sr-induced bone marrow aplasia
j , Comp. Path. 1994 VoL 1 I0, 195-205
Haemopoietic Spleen Colony Formation in the Rat: Effect of 89Sr-Induced Bone Marrow Aplasia T. Inoue, Y. Hirabayashi, M. Kanisawa, H. Mitsui, S. Taniguchl and K. Yoshida* Department of Patholo~, YokohamaCity UniversitySchool of Medicine, 3-9 Fukuura, Kanazawaku, Yokohama-236and *Division of Pto~siologyand Pathology, National Institute of Radiological Science, 4-9-1 Anagawa, Inageku, Chiba-260,Japan
Summary Regulation o["spleen-colony [brmation, a clonal assay for haemopoietic stem cells, is very different in mice and rats. In the rat, there is an involution of the colony-fbrming ability of the spleen during infantile development, whereas in mice the ability is maintained throughout life. In our re-evaluation of endogenous spleen colony Ibrmation in rats after graded doses of total-body irradiation, colonies ceased to appear after 12 weeks of age. However, histological sections showed that there were tiny colonies growing in the spleen, even at 20 weeks. These microscopical colonies developed into visible colonies when the rats were given treatments that increased the haemopoietic requirement. Thus, the amount of 5UFeC13 incorporated into the spleen increased to compensate for a decrease in uptake by the bone marrow. Rats in wlnch bone-marrow activity was mhmbatedby "SrC1v showed extenswe colony formation in the spleen, even after 12 weeks of age, when the endogenous colonies were induced by a sublethal dose of radiation. About twice as much 59Fe acetate activity was incorporated into the spleens of the experimental animals than in those of control rats. These findings imply that spleen-colony formation responds to the haemopoietic requirement of the spleen rather than that of the bone marrow. Furthermore, the requirement of the spleen seems to be much smaller in rats than in mice, because bone-marrow capacity for haemopoiesis is relatively larger in rats. •
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Introduction Haemopoietic spleen colonies are clonal growths originating from the haemopoietic stem cells. When mice are irradiated (whole body) with a lethal dose of radiation and given an injection of syngeneic bone-marrow cells, exogenous spleen colonies develop from the haemopoietic stem cells in the transfused marrow (Till and McCulloch, 1961), whereas endogenous spleen colonies develop from the surviving haemopoietic stem cells. Comas and Byrd (1967) first described haemopoietic spleen colonies in the rat, some 6 years after the report of Till and McCulloch (1961) on mouse spleen colonies. However, the formation of haemopoietic spleen-colonies in the rat was quite different from that in the mouse. Thus, in rats there was no dose-response relationship between radiation exposure and the number of endogenous spleen colonies (Comas and Byrd, 1967); also, the size of the colonies was much smaller than 0021-9975/94/020195 + 11 $08.00/0
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that of murine spleen colonies. Furthermore, neither granuloid nor megakaryocytic colonies were found in any of the 459 colonies examined histologically (Rauchwerger et al., 1973). Vacek et al. (1976) observed a drastic decrease in the ability to form exogenous spleen colonies shortly after birth in recipient rats; they suggested that this occurred because a proportion of the erythropoietic activity is transferred to the bone marrow from the spleen at an early stage of development. The haematopoietic stem cells of the rat have also attracted special interest because of recent advances in experimental and clinical bone-marrow transplantation, not only for haematological disorders but also for inherited metabolic diseases; however, a reasonable linear relationship between the dose of injected haematopoietic cells and the resulting number of colonies has hardly ever been obtained (Goldschneider et al., 1980). In the present study, endogenous spleen-colony formation was used to study age-related changes in the ability of rats receiving increasing doses of radiation to form such colonies. Results from experimental studies on bone-marrow aplasia in which 89SrC1~ was used suggest that the essential role of haematopoietic cooperation between the spleen and bone marrow differs in the rat and mouse. M a t e r i a l s and M e t h o d s
Rats Wistar strain rats, 8-10 weeks old, were purchased from Shizuoka Laboratory Animal Center, Hamamatsu,Japan (SLC-Japan), and housed in laminar-flow cage racks in an environmentally controlled clean room with a 12h light-dark cycle. They were allowed to breed, and 10-12 rats of the same litter constituted an experimental group.
Irradiation Total body irradiation was carried out with a Cobalt-60 clinical irradiator (RFG-20D, Shimazu Co,, Ltd, Tokyo) fitted with an aluminium filter, 0'2 cm in thickness, at a dose rate of 67 cGy/min, and a fenestration/substance distance of 80 cm.
Endogenous Spleen Colony Assay Endogenous spleen colonies were observed on day 9 after various doses of irradiation. (A single dose of 7"5 Gy does not completely destroy marrow activity but induces regenerating haemopoiesis, i.e. endogenous spleen colonies, in 6-week-old rats.) No bone marrow cells were transfused. Spleens were removed immediately after killing the rat and fixed in Bouin's solution. Surface colonies larger than 0'2 mm in diameter were counted under low magnification. To induce endogenous spleen colonies, a 7.5 Gy 7-irradiation was given to rats whose bone marrow had been ablated by injection of ~gSr-chloride 6 weeks previously.
Histological Examination Spleens and femurs were fixed in Bouin's fluid for 24h and then placed in formaldehyde 4% and prepared for routine histology. Mid-saggital sections were cut and stained with haematoxylin and eosin (HE). The histological classification of colonies used was based on the classical report of Curry and Trentin (1967). The colonies in each section were counted and traced. Colonies were assumed to be
Spleen Golony Formation in the Rat
19 7
ellipsoidal. From the tracings, the size of the colonies was computed from measurements of two diameters by the following equation:
V = (2/3)~ab(a+ b), where a and b are the greatest and the least diameters on the largest cut face of each colony in the spleen.
*~Sr-Bone Marrow Aplasia agStrontium chloride (2"22MBq) was injected intravenously (i.v.) into rats at 6 weeks of age and the animals received 7'5 Gy whole body irradiation 6 weeks later (Kshirsagar, 1977). Five to seven rats were used for each datum point (see Table 1 in Results). The half-life of agSr is 50'5 days, and the emission of radiation is 100% 13-ray with 1"49 MeV of radiation energy; thus, the radiation remaining 6 weeks later is 56"2% of the initial inoculation, agSr 13 ray-activity incorporated was measured simultaneously with 3' ray-activity in a well-type GM scintillation counter.
S~Fe-Uptake According to Rauchwerger et al. (1973), the lineages of colonies in rat spleen are exclusively erythroid; thus, it is reasonable to evaluate colony-forming ability by measuring the incorporation of radio-iron into the colonies. 59Fe-uptake in the peripheral blood, spleen and femur was measured. 5"~Fe-aeetate (lmCi) was injected intraperitoneally 8 days after whole-body irradiation, i.e. 24h before killing and measurement of incorporated 3'-activity (Cavill and Ricketts, 1975).
Endogenous Spleen Colonies and the Dose of Radiation Endogenous spleen colonies were assayed in relation to a graded dose of radiation (between 7 and 10,5 Gy) for 3-week-old rats with 10-12 rats per datum point, and for 2-, 6-, 12-, 16- and 21-week-old rats with two or three rats per datum point.
Endogenous Spleen Colonies and the Effect of Age of Rats Six data points were provided by 2'5-, 3'0-, 3'5-, 4'0-, 4'5- and 5.0-week-old groups of rats. Groups of 10-12 rats were assayed for their endogenous colony-forming ability after receiving 7"5 Gy total body irradiation. For a dose of 6'0 Gy, each group consisted of only three animals.
Effect of Bone Marrow Aplasia on Splenic Colony-Forming Ability The [3 emitter, sgStrontium chloride, was used to suppress the haemopoietic activity in the bone marrow. 89Strontium chloride (2"22MBq) was injected intravenously into rats at 6 weeks of age and then, 6 weeks later, the rats received 7'5 Gy whole body irradiation for the possible induction of endogenous spleen colony formation. To measure the colony-forming activity, 59Fe-acetate (lmCi) was injected 24h before the rats were killed for measurement of 59Fe-uptake in the peripheral blood, spleen and femur. In this experiment, no control group (given neither ~UStrontium-abrasion nor 7'5 Gy-induction) was included, because no spleen colonies develop unless induced by radiation.
Results
Relationship of Number of Endogenous Spleen Colonies to Dose of Radiation The numbers of endogenous spleen colonies after various doses of g a m m a irradiation are plotted in Fig. 1. The n u m b e r of colonies in groups & r a t s aged
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D o s e of Radiation Fig, 1. Survival curves, after graded doses of radiation, of endogenous spleen colonies in the rat. Wlstar rats, of various ages (weeks), were given graded doses of radiation. Day 9 colonies were scored. The figure shows the number of spleen colonles (vertical axis) vs the graded dose of radiation (cOy) (horizontal axis). Vertical bars show standard deviation of the mean. The regression line calculated shows: Y(=log y ) = 6 ' 3 0 - 0 ' 6 7 X ; where y = n u m b e r of colonies per spleen, and X = d o s e of radiation in Gy; r~= 0.999. The data for 2 weeks of age (dosed squares) represent only two or three rats; thus, no standard deviation can be given. Asterisk at 7 Gy means confluent, uncountable colonies. Open triangles with a dashed line represent the combined data from 6-, 12-, 16-, and 21-week-old rats, which showed no visible colonies. The regression line calculated for the 2-week-old rats shows: Y(=log y ) = 3 . 7 5 - 0 . 2 6 X ; where y = n u m b e r of colonies per spleen, and X = d o s e of radiation in Gy; = 0'943.
2 and 3 weeks decreased exponentially with an increasing dose of radiation; however, in the groups over 6 weeks of age, no macroscopical colonies appeared at any radiation exposure. There were more colonies in 2-week-old than in 3-week-old rats; colonies in the spleens of the 2-week-old group exposed to 7.0 Gy were confluent and uncountable. The size of the colonies seemed to differ macroscopically in groups exposed to different doses of radiation.
Effect of Development on Endogenous Spleen Colonies The data shown in Fig. 1 suggested that colony-forming ability changed during development. Therefore, the number of colonies appearing in different age groups, ranging from 2 to 6 weeks, after the same dose of irradiation (7"5Gy) was examined. The results are shown in Fig. 2. T h e number of
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Endogenous colonies induced by total-body irradiation with 7'5 Gy in rats of various ages (weeks). The Wistar rats were irradiated with a 7.5 Gy 6~Co-gamma source, and killed on day 9, when the spleen colonies were counted. The figure shows the number of spleen colonies (vertical axis) vs increasing age at irradiation (horizontal axis). No statistical analysis was made on the data for 6week-old rats because of the limited numbers. The calculated regression line (solid) shows: Y(--log y) = 2 ' 4 5 - 0 ' 3 7 X ; where y = n u m b e r of colonies per spleen, and X represents age in weeks; ~ = 0'982. T h e data points with dashed lines for 8 to I0 Gy are plotted for reference with the same data as in Fig. 1.
colonies decreased exponentially and no macroscopical colonies were observed at and after 6 weeks. Data points for exposures over 8"0 Gy were plotted for comparison.
Histological Examination All the macroscopical colonies were seen to be erythroid upon histological examination. Taking into account small colonies that could only be seen microscopically, both megakaryocytic and granulocytic colonies were rarely to be found beneath the splenic cord or in the white pulp. The size of the colonies decreased with age, and the proportion of microscopical colonies was higher in mature than in younger rats (Fig. 3). In particular, it was noted that a few microscopical colonies were recognizable even in the 21-week-old group, in which macroscopical colonies were no longer visible. No differential counts were made; however,, undifferentiated erythroid colonies occurred with increased frequency in rats aged > 6 weeks. In contrast to the findings for spleen colonies, the bone marrow of rats over 6 weeks old
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Histology of the spleen colonies. On the left, an erythroid colony from a 2-week-old rat shows mature erythroblastic celIs in the centre (demarcated by open triangles), surrounded by a border of immature blastic cells (demarcated by arrows). On the right, a small erythroblastie cell-aggregate is seen in the centre (demarcated by arrows). HE × 165.
showed, histologically, prominent regenerating haemopoiesis, with tri-lineage haemopoiesis including erythropoiesis, granulopoiesis and marked megakaryopoiesis.
Size of Spleen Colonies Because the size appeared to be influenced both by the dosd of radiation and the age of the rat, separate evaluations were made of colony size against dose of radiation and age. Figure 4 shows the size of colonies in 2-week-old rats after graded doses of radiation. Although each d a t u m point shows a large standard deviation, the size appeared to decrease exponentially with increasing doses of radiation. Consequently, the relationship between the size of the colony and age was examined. In this experiment, each group was irradiated with 7"5 Gy. Figure 5 shows that as the age increased, the colonies decreased in size until they were no longer visible after 6 weeks of age, when the average colony diameter was less than 200 gm.
Effect of Experimental Aplasia of the Bone Marrow on Endogenous Spleen Colony Formation T h e decline in colony-forming ability in the rat spleen in the early developmental stages seemed to coincide with the development of the capacity of the
201
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bone marrow for haematopoiesis. Therefore, to attempt to produce complete aplasia of the bone-marrow, 89Sr-chloride was injected into the rats. At the sixth week after the radiostrontium treatment, endogenous spleen colonies after total body irradiation with 7'5 Gy were examined. This dose induces endogenous spleen colonies when rats are 6 weeks old. Twenty-four hours before killing the rats to observe their spleens, the animals were given intravenous injections of 59Fe-acetate for quantitative measurements and comparisons of erythropoiesis in the bone marrow and spleen. As shown in Fig. 6, strikingly well developed colonies were observed in the spleens of the rats that had been treated with radiostrontium. Histological findings, shown on the right (top) in Fig. 6, confirmed that the spleens had clearly developed colonies. In contrast, no colonies of comparable size are seen in the lower section. The bone marrow from these treated rats showed an entirely aplastic marrow, i.e. a radiation-induced pancytopaenia (data not shown), whereas the marrow from
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Volume of spleen colonies from rats at various ages given a total-body irradiation of 7'5 Gy. The vertical bars show the standard deviation of the mean. For details of the method to estimate the colony size, see Materials and Methods. The regression line shows: Y(=log y ) = 2 ' 4 2 - 0 ' 2 7 X ; r~~ 0-913; where y = average volume of colonies ( × 106 lama), and X = age in weeks.
rats without agSr-treatment showed an active haematopoiesis. Dense argyrophilic grains due to [3-rays from incorporated radiostrontium were observed along the cortical bone of the femoral shaft on autoradiographic sections (data not shown). The results of 59Fe-uptake are shown in Table 1. Since groups B and C did not receive 59Fe-acetate, radioactivities per femur in groups B and C were due to incorporated radiostrontium, whereas the activities incorporated into the spleen of groups B and C, 102 and 91 cpm, and in the peripheral blood, 95 and 88 cpm, are assumed to be background levels since 89Sr is not retained in the spleen and peripheral blood. The radioactivity in the femurs of group A, 16943 cpm, was close to that in the femurs of groups B and C, 17210 and 16259 cprn, respectively. This is considered to be the result of complete marrow ablation due to the [3-activity of agSr; the radiation activity in femurs of A minus by B or C (lines p and q in Table 1) leaves 0 or 684 cpm with large standard errors, i.e. substantially a zero value, where p and q represent 59Feactivity incorporated in each tissue. Note that group D, which did not receive 89Sr chloride, showed activity fi'om 59Fe incorporated into haemoglobin
203
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[,eli panel shows gross appearance of the spleens ( x 0'78) with prominent surface colonies fi'om rats treated with radiostrontium (upper row), as compared with spleens without visible colonies fi'om the control rats. Right panel shows histological findings of spleens corresponding to those on the left panel. Scattered dark spots in the upper spleen show developed erythroid colonies. (See text for details.) HE. x 2'7.
Table I Shift of erythropoiesis in radiostrontium (~gSr)-treated rats*
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Peripheral blood (cpm) Meauq- S.E. 15934-34 954- 2 88:t: 2 17274-45
(9) (10) (l l) (12)
14984-36 (17) 15054-36 (18)
Percent change in 5~Fe-activity by marrow aplasia '~WE-activity incorporated (p/D)x 100(%) 5SFe-activity incorporated (q/D)x 100(%)
0 (<43.2) (19) 197.84-31.2 (21) 86"74-3"0 (23) 22'04-38'7 (20) 198.34-31,3 (22) 87'1±3'0 (24)
* Both radioactivitics, [3-rays due to agSr and y-rays clue to S!~Fe,were measured simultaneously by a well-type GM-scintillation counter. Significant (P<0"001): (1)-(3) and (4), (5)-(7) and (8), (10)-(ll) and (12). Insignificant: (1) and (2) and (3), (6) and (7), (9) and (12), (10) and (11), (13) and (14), (15) and (16), (17) and (18), (19), (21) and (22), (23) and (24). "~"!'Strontium chloride (2'22 MBq) was injected (i.v.) into 6week-old rats (tL/~of "~'Sr: 50'5 days.) ~ Whole-body irradiation (7.5 Gy) was made by 'i~Co-7-irradiator 6 weeks after radiostrontium injection. §~"Fe-acetate (37 KBq) was injected (i.v.) a week after whole body irradiation; 24. h belbre killing and measurement of incorporated y-activity. IITwo out of seven rats in group A, and one out of five in group B, died after irradiation.
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synthesis in late normoblasts and in reticulocytes. When the values in p and q are divided by the value in D, the value for uptake in the femoral bone marrow is near to 0% (fl/D x 100) compared with that in the control group D. By contrast, 59Fe-uptake in the spleen increased dramatically, reaching up to 197'8 and 198.3% of the control value (p/D or q/D in the column for spleen; Table 1), when haemopoiesis in the bone marrow was ablated by radiostrontium. No change or a slight decrease of 59Fe-uptake was observed in the peripheral blood (86"7 and 87"1%). Discussion
With recent gains in knowledge of the regulation ofhaemopoiesis in the mouse by a variety of cytokines and their receptor networks, experimental trials of gene therapy for seveal haemopoietic or metabolic diseases have been conducted in this species (Sporn and Roberts, 1991). Information on the rat as an experimental disease model has also accumulated (Registry of Comparative Pathology from 1970-1990), but apart from a few earlier studies (Comas and Byrd, 1967; Vacek et al., 1976), little is known about the background of spleen colony formation in the rat as compared with data available for the mouse. The present data show that the ability to produce spleen colonies decreased rapidly and exponentially with age in the rat during its early development (Fig. 2). From this it now seems likely that the reason for Comas and Byrd's failure to find a linear dose-response relationship between the number of spleen colonies and the number of bone-marrow cells injected was their use of rats of various ages in their assay. The failure by Vacek et al. (1976) to demonstrate such linearity for exogenous spleen colony formation can also be explained by the age of the recipient rats. The continuous decline i n colonyforming ability in the spleen does not seem to reside in the colony-forming units in this organ but rather in factors such as the splenic stroma that supports spleen colony formation. This speculation is supported by the fact that stem ceils still exist in the rat spleen but never grow well after 6 weeks of age; although the colonies decrease in size with age, they never disappear during the animal's entire life (Vacek et al., 1976). Microscopical spleen colonies seen in both endogenous and exogenous colony formation were the key phenomenon in spleen colony formation in the rat (Figs 3 and 4). Experimental marrow aplasia, induced by the ~3-emitting (~gSr) radiostrontium, led to marked spleen colony formation; this finding confirmed our hypothesis of a lower haemopoietic demand on the spleen in adult rats. However, the reason for this lower demand is still not fully known. Evaluation of stromal cell activity in the spleen may resolve the question in adult rat haemopoiesis. Possibly ablation of marrow stimulates IL-3 production, which with erythropoietin stimulates erythropoiesis in the spleen; however, little is known in the rat about the species-specificity of cytokines and cytokine receptors. Figure 1 shows the different slopes of the survival curves of spleen colonies for 2- and 3-week-old rats. Ahhough experimental evidence is lacking, we suggest that the haemopoietic function of the stroma in producing spleen
Spleen Colony Formation in the Rat
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colonies, and of the receptor network in regulating positive and negative signal transduction in the haemopoietic stem cells, may change during development. A single, exponential and dramatic decrease in the size of spleen colony during early development supports the speculation outlined above, regarding either an alteration of the stromal cell function, or of the affinity of cytokine receptors for the haemopoietic cells, The decline in colony-forming ability observed from 6 to 21 weeks cannot reflect an increase in radiosensitivity, or be explained by changes in stromal radiosensitivity or in the radiosensitivity of the haemopoietic stem cells. Stromal cells in the spleen may have a regulatory receptor system responsible for releasing a variety of cytokines, which seem to be internalized under regular haemopoietic conditions after development is completed. This possible mechanism may be regulated separately from that of the stromal cells in the bone marrow tissue. Acknowledgments
The authors express their appreciation to Dr Avril Woodhead, Biology Department, and Dr E. P. Cronkite, Medical Department, Brookhaven National Laboratory, New York, for reading the manuscript and providing constructive discussion and comments. This work was supported in part by Grants-ln-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture. References
Cavill, I. and Ricketts, C. (1975). The kinetics of iron metabolism. In: Iron in Biochemistry and Medicine, A. Jacobs and M. Worwood, Eds, Academic Press, New York, pp. 613-64.7. Comas, F. V. and Byrd, B. L. (1967). Hemopoietic spleen colonies in the rat. Radiation Research, 32, 355-365. Curry, J. L. and Trentin, J. J. (1967). Hemopoietic spleen colony studies. I. Growth and differentiation. Developmental Biology, 15, 395-413. Goldschneider, I., Metcalf, D., Battye, F. and Mandel, T. (1980). Analysis of rat hemopoietic cells on the fluorescence-activated cell sorter. I. Isolation of pluripotent hemopoietic stem cells and granulocyte-macrophage progenitor cells. Journal of Experimental Medicine, 152, 419-446. Kshirsagar, S. G. (1977). Radiostrontium distribution measured in vitro between bound and free forms in the soft tissues of rat. International Journal of Radiation Biology, 32, 561-569. Rauchwerger, J. M., Gallagher, M. T. and Trentin, J. J. (1973). Role of the hemopoietic inductive microenvironments (HIM) in xenogeneic bone marrow transplantation. Transplantation, 15, 610-618. Registry of Comparative Pathology (1970-90). Animal Models of Human Disease, C. C. Capen, T. C. Jones, D. B. Ha&el and G. Migaki, Eds, Armed Forces Institute of Pathology, Washington, D.C. Sporn, M. B. and Roberts, A. B. (Eds) (1991). Peptide Growth Factors and their Receptors, Vols 1 and 2. Springer, New York. Till, J. E. and McCulloch, E. A. (1961). A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Research, 14, 213-222. Vacek, A., Bartonickova, A. and Tkadlecek, L. (1976). Age dependence of the number of the stem cells in haemopoietic tissues of rats. Cell and Tissue Kinetics, 9, 1-8.
I Received, August 1 l th, 1993 -] Accepted, .November 8th, 1993_]
Haemopoietic Spleen Colony Formation in the Rat: Effect of 89Sr-Induced Bone Marrow Aplasia T. Inoue, Y. Hirabayashi, M. Kanisawa, H. Mitsui, S. Taniguchl and K. Yoshida* Department of Patholo~, YokohamaCity UniversitySchool of Medicine, 3-9 Fukuura, Kanazawaku, Yokohama-236and *Division of Pto~siologyand Pathology, National Institute of Radiological Science, 4-9-1 Anagawa, Inageku, Chiba-260,Japan
Summary Regulation o["spleen-colony [brmation, a clonal assay for haemopoietic stem cells, is very different in mice and rats. In the rat, there is an involution of the colony-fbrming ability of the spleen during infantile development, whereas in mice the ability is maintained throughout life. In our re-evaluation of endogenous spleen colony Ibrmation in rats after graded doses of total-body irradiation, colonies ceased to appear after 12 weeks of age. However, histological sections showed that there were tiny colonies growing in the spleen, even at 20 weeks. These microscopical colonies developed into visible colonies when the rats were given treatments that increased the haemopoietic requirement. Thus, the amount of 5UFeC13 incorporated into the spleen increased to compensate for a decrease in uptake by the bone marrow. Rats in wlnch bone-marrow activity was mhmbatedby "SrC1v showed extenswe colony formation in the spleen, even after 12 weeks of age, when the endogenous colonies were induced by a sublethal dose of radiation. About twice as much 59Fe acetate activity was incorporated into the spleens of the experimental animals than in those of control rats. These findings imply that spleen-colony formation responds to the haemopoietic requirement of the spleen rather than that of the bone marrow. Furthermore, the requirement of the spleen seems to be much smaller in rats than in mice, because bone-marrow capacity for haemopoiesis is relatively larger in rats. •
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Introduction Haemopoietic spleen colonies are clonal growths originating from the haemopoietic stem cells. When mice are irradiated (whole body) with a lethal dose of radiation and given an injection of syngeneic bone-marrow cells, exogenous spleen colonies develop from the haemopoietic stem cells in the transfused marrow (Till and McCulloch, 1961), whereas endogenous spleen colonies develop from the surviving haemopoietic stem cells. Comas and Byrd (1967) first described haemopoietic spleen colonies in the rat, some 6 years after the report of Till and McCulloch (1961) on mouse spleen colonies. However, the formation of haemopoietic spleen-colonies in the rat was quite different from that in the mouse. Thus, in rats there was no dose-response relationship between radiation exposure and the number of endogenous spleen colonies (Comas and Byrd, 1967); also, the size of the colonies was much smaller than 0021-9975/94/020195 + 11 $08.00/0
© 1994 Academic Press Limited
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that of murine spleen colonies. Furthermore, neither granuloid nor megakaryocytic colonies were found in any of the 459 colonies examined histologically (Rauchwerger et al., 1973). Vacek et al. (1976) observed a drastic decrease in the ability to form exogenous spleen colonies shortly after birth in recipient rats; they suggested that this occurred because a proportion of the erythropoietic activity is transferred to the bone marrow from the spleen at an early stage of development. The haematopoietic stem cells of the rat have also attracted special interest because of recent advances in experimental and clinical bone-marrow transplantation, not only for haematological disorders but also for inherited metabolic diseases; however, a reasonable linear relationship between the dose of injected haematopoietic cells and the resulting number of colonies has hardly ever been obtained (Goldschneider et al., 1980). In the present study, endogenous spleen-colony formation was used to study age-related changes in the ability of rats receiving increasing doses of radiation to form such colonies. Results from experimental studies on bone-marrow aplasia in which 89SrC1~ was used suggest that the essential role of haematopoietic cooperation between the spleen and bone marrow differs in the rat and mouse. M a t e r i a l s and M e t h o d s
Rats Wistar strain rats, 8-10 weeks old, were purchased from Shizuoka Laboratory Animal Center, Hamamatsu,Japan (SLC-Japan), and housed in laminar-flow cage racks in an environmentally controlled clean room with a 12h light-dark cycle. They were allowed to breed, and 10-12 rats of the same litter constituted an experimental group.
Irradiation Total body irradiation was carried out with a Cobalt-60 clinical irradiator (RFG-20D, Shimazu Co,, Ltd, Tokyo) fitted with an aluminium filter, 0'2 cm in thickness, at a dose rate of 67 cGy/min, and a fenestration/substance distance of 80 cm.
Endogenous Spleen Colony Assay Endogenous spleen colonies were observed on day 9 after various doses of irradiation. (A single dose of 7"5 Gy does not completely destroy marrow activity but induces regenerating haemopoiesis, i.e. endogenous spleen colonies, in 6-week-old rats.) No bone marrow cells were transfused. Spleens were removed immediately after killing the rat and fixed in Bouin's solution. Surface colonies larger than 0'2 mm in diameter were counted under low magnification. To induce endogenous spleen colonies, a 7.5 Gy 7-irradiation was given to rats whose bone marrow had been ablated by injection of ~gSr-chloride 6 weeks previously.
Histological Examination Spleens and femurs were fixed in Bouin's fluid for 24h and then placed in formaldehyde 4% and prepared for routine histology. Mid-saggital sections were cut and stained with haematoxylin and eosin (HE). The histological classification of colonies used was based on the classical report of Curry and Trentin (1967). The colonies in each section were counted and traced. Colonies were assumed to be
Spleen Golony Formation in the Rat
19 7
ellipsoidal. From the tracings, the size of the colonies was computed from measurements of two diameters by the following equation:
V = (2/3)~ab(a+ b), where a and b are the greatest and the least diameters on the largest cut face of each colony in the spleen.
*~Sr-Bone Marrow Aplasia agStrontium chloride (2"22MBq) was injected intravenously (i.v.) into rats at 6 weeks of age and the animals received 7'5 Gy whole body irradiation 6 weeks later (Kshirsagar, 1977). Five to seven rats were used for each datum point (see Table 1 in Results). The half-life of agSr is 50'5 days, and the emission of radiation is 100% 13-ray with 1"49 MeV of radiation energy; thus, the radiation remaining 6 weeks later is 56"2% of the initial inoculation, agSr 13 ray-activity incorporated was measured simultaneously with 3' ray-activity in a well-type GM scintillation counter.
S~Fe-Uptake According to Rauchwerger et al. (1973), the lineages of colonies in rat spleen are exclusively erythroid; thus, it is reasonable to evaluate colony-forming ability by measuring the incorporation of radio-iron into the colonies. 59Fe-uptake in the peripheral blood, spleen and femur was measured. 5"~Fe-aeetate (lmCi) was injected intraperitoneally 8 days after whole-body irradiation, i.e. 24h before killing and measurement of incorporated 3'-activity (Cavill and Ricketts, 1975).
Endogenous Spleen Colonies and the Dose of Radiation Endogenous spleen colonies were assayed in relation to a graded dose of radiation (between 7 and 10,5 Gy) for 3-week-old rats with 10-12 rats per datum point, and for 2-, 6-, 12-, 16- and 21-week-old rats with two or three rats per datum point.
Endogenous Spleen Colonies and the Effect of Age of Rats Six data points were provided by 2'5-, 3'0-, 3'5-, 4'0-, 4'5- and 5.0-week-old groups of rats. Groups of 10-12 rats were assayed for their endogenous colony-forming ability after receiving 7"5 Gy total body irradiation. For a dose of 6'0 Gy, each group consisted of only three animals.
Effect of Bone Marrow Aplasia on Splenic Colony-Forming Ability The [3 emitter, sgStrontium chloride, was used to suppress the haemopoietic activity in the bone marrow. 89Strontium chloride (2"22MBq) was injected intravenously into rats at 6 weeks of age and then, 6 weeks later, the rats received 7'5 Gy whole body irradiation for the possible induction of endogenous spleen colony formation. To measure the colony-forming activity, 59Fe-acetate (lmCi) was injected 24h before the rats were killed for measurement of 59Fe-uptake in the peripheral blood, spleen and femur. In this experiment, no control group (given neither ~UStrontium-abrasion nor 7'5 Gy-induction) was included, because no spleen colonies develop unless induced by radiation.
Results
Relationship of Number of Endogenous Spleen Colonies to Dose of Radiation The numbers of endogenous spleen colonies after various doses of g a m m a irradiation are plotted in Fig. 1. The n u m b e r of colonies in groups & r a t s aged
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D o s e of Radiation Fig, 1. Survival curves, after graded doses of radiation, of endogenous spleen colonies in the rat. Wlstar rats, of various ages (weeks), were given graded doses of radiation. Day 9 colonies were scored. The figure shows the number of spleen colonles (vertical axis) vs the graded dose of radiation (cOy) (horizontal axis). Vertical bars show standard deviation of the mean. The regression line calculated shows: Y(=log y ) = 6 ' 3 0 - 0 ' 6 7 X ; where y = n u m b e r of colonies per spleen, and X = d o s e of radiation in Gy; r~= 0.999. The data for 2 weeks of age (dosed squares) represent only two or three rats; thus, no standard deviation can be given. Asterisk at 7 Gy means confluent, uncountable colonies. Open triangles with a dashed line represent the combined data from 6-, 12-, 16-, and 21-week-old rats, which showed no visible colonies. The regression line calculated for the 2-week-old rats shows: Y(=log y ) = 3 . 7 5 - 0 . 2 6 X ; where y = n u m b e r of colonies per spleen, and X = d o s e of radiation in Gy; = 0'943.
2 and 3 weeks decreased exponentially with an increasing dose of radiation; however, in the groups over 6 weeks of age, no macroscopical colonies appeared at any radiation exposure. There were more colonies in 2-week-old than in 3-week-old rats; colonies in the spleens of the 2-week-old group exposed to 7.0 Gy were confluent and uncountable. The size of the colonies seemed to differ macroscopically in groups exposed to different doses of radiation.
Effect of Development on Endogenous Spleen Colonies The data shown in Fig. 1 suggested that colony-forming ability changed during development. Therefore, the number of colonies appearing in different age groups, ranging from 2 to 6 weeks, after the same dose of irradiation (7"5Gy) was examined. The results are shown in Fig. 2. T h e number of
Spleen Colony Formation
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Endogenous colonies induced by total-body irradiation with 7'5 Gy in rats of various ages (weeks). The Wistar rats were irradiated with a 7.5 Gy 6~Co-gamma source, and killed on day 9, when the spleen colonies were counted. The figure shows the number of spleen colonies (vertical axis) vs increasing age at irradiation (horizontal axis). No statistical analysis was made on the data for 6week-old rats because of the limited numbers. The calculated regression line (solid) shows: Y(--log y) = 2 ' 4 5 - 0 ' 3 7 X ; where y = n u m b e r of colonies per spleen, and X represents age in weeks; ~ = 0'982. T h e data points with dashed lines for 8 to I0 Gy are plotted for reference with the same data as in Fig. 1.
colonies decreased exponentially and no macroscopical colonies were observed at and after 6 weeks. Data points for exposures over 8"0 Gy were plotted for comparison.
Histological Examination All the macroscopical colonies were seen to be erythroid upon histological examination. Taking into account small colonies that could only be seen microscopically, both megakaryocytic and granulocytic colonies were rarely to be found beneath the splenic cord or in the white pulp. The size of the colonies decreased with age, and the proportion of microscopical colonies was higher in mature than in younger rats (Fig. 3). In particular, it was noted that a few microscopical colonies were recognizable even in the 21-week-old group, in which macroscopical colonies were no longer visible. No differential counts were made; however,, undifferentiated erythroid colonies occurred with increased frequency in rats aged > 6 weeks. In contrast to the findings for spleen colonies, the bone marrow of rats over 6 weeks old
200
Fig. 3.
T . I n o u e e t al.
Histology of the spleen colonies. On the left, an erythroid colony from a 2-week-old rat shows mature erythroblastic celIs in the centre (demarcated by open triangles), surrounded by a border of immature blastic cells (demarcated by arrows). On the right, a small erythroblastie cell-aggregate is seen in the centre (demarcated by arrows). HE × 165.
showed, histologically, prominent regenerating haemopoiesis, with tri-lineage haemopoiesis including erythropoiesis, granulopoiesis and marked megakaryopoiesis.
Size of Spleen Colonies Because the size appeared to be influenced both by the dosd of radiation and the age of the rat, separate evaluations were made of colony size against dose of radiation and age. Figure 4 shows the size of colonies in 2-week-old rats after graded doses of radiation. Although each d a t u m point shows a large standard deviation, the size appeared to decrease exponentially with increasing doses of radiation. Consequently, the relationship between the size of the colony and age was examined. In this experiment, each group was irradiated with 7"5 Gy. Figure 5 shows that as the age increased, the colonies decreased in size until they were no longer visible after 6 weeks of age, when the average colony diameter was less than 200 gm.
Effect of Experimental Aplasia of the Bone Marrow on Endogenous Spleen Colony Formation T h e decline in colony-forming ability in the rat spleen in the early developmental stages seemed to coincide with the development of the capacity of the
201
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bone marrow for haematopoiesis. Therefore, to attempt to produce complete aplasia of the bone-marrow, 89Sr-chloride was injected into the rats. At the sixth week after the radiostrontium treatment, endogenous spleen colonies after total body irradiation with 7'5 Gy were examined. This dose induces endogenous spleen colonies when rats are 6 weeks old. Twenty-four hours before killing the rats to observe their spleens, the animals were given intravenous injections of 59Fe-acetate for quantitative measurements and comparisons of erythropoiesis in the bone marrow and spleen. As shown in Fig. 6, strikingly well developed colonies were observed in the spleens of the rats that had been treated with radiostrontium. Histological findings, shown on the right (top) in Fig. 6, confirmed that the spleens had clearly developed colonies. In contrast, no colonies of comparable size are seen in the lower section. The bone marrow from these treated rats showed an entirely aplastic marrow, i.e. a radiation-induced pancytopaenia (data not shown), whereas the marrow from
202
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Volume of spleen colonies from rats at various ages given a total-body irradiation of 7'5 Gy. The vertical bars show the standard deviation of the mean. For details of the method to estimate the colony size, see Materials and Methods. The regression line shows: Y(=log y ) = 2 ' 4 2 - 0 ' 2 7 X ; r~~ 0-913; where y = average volume of colonies ( × 106 lama), and X = age in weeks.
rats without agSr-treatment showed an active haematopoiesis. Dense argyrophilic grains due to [3-rays from incorporated radiostrontium were observed along the cortical bone of the femoral shaft on autoradiographic sections (data not shown). The results of 59Fe-uptake are shown in Table 1. Since groups B and C did not receive 59Fe-acetate, radioactivities per femur in groups B and C were due to incorporated radiostrontium, whereas the activities incorporated into the spleen of groups B and C, 102 and 91 cpm, and in the peripheral blood, 95 and 88 cpm, are assumed to be background levels since 89Sr is not retained in the spleen and peripheral blood. The radioactivity in the femurs of group A, 16943 cpm, was close to that in the femurs of groups B and C, 17210 and 16259 cprn, respectively. This is considered to be the result of complete marrow ablation due to the [3-activity of agSr; the radiation activity in femurs of A minus by B or C (lines p and q in Table 1) leaves 0 or 684 cpm with large standard errors, i.e. substantially a zero value, where p and q represent 59Feactivity incorporated in each tissue. Note that group D, which did not receive 89Sr chloride, showed activity fi'om 59Fe incorporated into haemoglobin
203
S p l e e n C o l o n y F o r m a t i o n in the R a t
Fig. 6.
[,eli panel shows gross appearance of the spleens ( x 0'78) with prominent surface colonies fi'om rats treated with radiostrontium (upper row), as compared with spleens without visible colonies fi'om the control rats. Right panel shows histological findings of spleens corresponding to those on the left panel. Scattered dark spots in the upper spleen show developed erythroid colonies. (See text for details.) HE. x 2'7.
Table I Shift of erythropoiesis in radiostrontium (~gSr)-treated rats*
Groups
Treatment
Number of rats
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Yes Yes Yes No
Yes Yes No Yes
Yes No No Yes
511 4II 5 7
p(=A-g) q(=A-C)
Femur (cpm) Mean4-S.E. 169434- 590 172104-1021 162594- 614 31104- 98
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47024-716 1024- I 9I 4- 3 23254- 60
(5) (6) (7) (8)
0(<1344) (13) 46004-717 (15) 6844-1204 (14) 46114-719 (16)
Peripheral blood (cpm) Meauq- S.E. 15934-34 954- 2 88:t: 2 17274-45
(9) (10) (l l) (12)
14984-36 (17) 15054-36 (18)
Percent change in 5~Fe-activity by marrow aplasia '~WE-activity incorporated (p/D)x 100(%) 5SFe-activity incorporated (q/D)x 100(%)
0 (<43.2) (19) 197.84-31.2 (21) 86"74-3"0 (23) 22'04-38'7 (20) 198.34-31,3 (22) 87'1±3'0 (24)
* Both radioactivitics, [3-rays due to agSr and y-rays clue to S!~Fe,were measured simultaneously by a well-type GM-scintillation counter. Significant (P<0"001): (1)-(3) and (4), (5)-(7) and (8), (10)-(ll) and (12). Insignificant: (1) and (2) and (3), (6) and (7), (9) and (12), (10) and (11), (13) and (14), (15) and (16), (17) and (18), (19), (21) and (22), (23) and (24). "~"!'Strontium chloride (2'22 MBq) was injected (i.v.) into 6week-old rats (tL/~of "~'Sr: 50'5 days.) ~ Whole-body irradiation (7.5 Gy) was made by 'i~Co-7-irradiator 6 weeks after radiostrontium injection. §~"Fe-acetate (37 KBq) was injected (i.v.) a week after whole body irradiation; 24. h belbre killing and measurement of incorporated y-activity. IITwo out of seven rats in group A, and one out of five in group B, died after irradiation.
204
T. Inoue et al.
synthesis in late normoblasts and in reticulocytes. When the values in p and q are divided by the value in D, the value for uptake in the femoral bone marrow is near to 0% (fl/D x 100) compared with that in the control group D. By contrast, 59Fe-uptake in the spleen increased dramatically, reaching up to 197'8 and 198.3% of the control value (p/D or q/D in the column for spleen; Table 1), when haemopoiesis in the bone marrow was ablated by radiostrontium. No change or a slight decrease of 59Fe-uptake was observed in the peripheral blood (86"7 and 87"1%). Discussion
With recent gains in knowledge of the regulation ofhaemopoiesis in the mouse by a variety of cytokines and their receptor networks, experimental trials of gene therapy for seveal haemopoietic or metabolic diseases have been conducted in this species (Sporn and Roberts, 1991). Information on the rat as an experimental disease model has also accumulated (Registry of Comparative Pathology from 1970-1990), but apart from a few earlier studies (Comas and Byrd, 1967; Vacek et al., 1976), little is known about the background of spleen colony formation in the rat as compared with data available for the mouse. The present data show that the ability to produce spleen colonies decreased rapidly and exponentially with age in the rat during its early development (Fig. 2). From this it now seems likely that the reason for Comas and Byrd's failure to find a linear dose-response relationship between the number of spleen colonies and the number of bone-marrow cells injected was their use of rats of various ages in their assay. The failure by Vacek et al. (1976) to demonstrate such linearity for exogenous spleen colony formation can also be explained by the age of the recipient rats. The continuous decline i n colonyforming ability in the spleen does not seem to reside in the colony-forming units in this organ but rather in factors such as the splenic stroma that supports spleen colony formation. This speculation is supported by the fact that stem ceils still exist in the rat spleen but never grow well after 6 weeks of age; although the colonies decrease in size with age, they never disappear during the animal's entire life (Vacek et al., 1976). Microscopical spleen colonies seen in both endogenous and exogenous colony formation were the key phenomenon in spleen colony formation in the rat (Figs 3 and 4). Experimental marrow aplasia, induced by the ~3-emitting (~gSr) radiostrontium, led to marked spleen colony formation; this finding confirmed our hypothesis of a lower haemopoietic demand on the spleen in adult rats. However, the reason for this lower demand is still not fully known. Evaluation of stromal cell activity in the spleen may resolve the question in adult rat haemopoiesis. Possibly ablation of marrow stimulates IL-3 production, which with erythropoietin stimulates erythropoiesis in the spleen; however, little is known in the rat about the species-specificity of cytokines and cytokine receptors. Figure 1 shows the different slopes of the survival curves of spleen colonies for 2- and 3-week-old rats. Ahhough experimental evidence is lacking, we suggest that the haemopoietic function of the stroma in producing spleen
Spleen Colony Formation in the Rat
205
colonies, and of the receptor network in regulating positive and negative signal transduction in the haemopoietic stem cells, may change during development. A single, exponential and dramatic decrease in the size of spleen colony during early development supports the speculation outlined above, regarding either an alteration of the stromal cell function, or of the affinity of cytokine receptors for the haemopoietic cells, The decline in colony-forming ability observed from 6 to 21 weeks cannot reflect an increase in radiosensitivity, or be explained by changes in stromal radiosensitivity or in the radiosensitivity of the haemopoietic stem cells. Stromal cells in the spleen may have a regulatory receptor system responsible for releasing a variety of cytokines, which seem to be internalized under regular haemopoietic conditions after development is completed. This possible mechanism may be regulated separately from that of the stromal cells in the bone marrow tissue. Acknowledgments
The authors express their appreciation to Dr Avril Woodhead, Biology Department, and Dr E. P. Cronkite, Medical Department, Brookhaven National Laboratory, New York, for reading the manuscript and providing constructive discussion and comments. This work was supported in part by Grants-ln-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture. References
Cavill, I. and Ricketts, C. (1975). The kinetics of iron metabolism. In: Iron in Biochemistry and Medicine, A. Jacobs and M. Worwood, Eds, Academic Press, New York, pp. 613-64.7. Comas, F. V. and Byrd, B. L. (1967). Hemopoietic spleen colonies in the rat. Radiation Research, 32, 355-365. Curry, J. L. and Trentin, J. J. (1967). Hemopoietic spleen colony studies. I. Growth and differentiation. Developmental Biology, 15, 395-413. Goldschneider, I., Metcalf, D., Battye, F. and Mandel, T. (1980). Analysis of rat hemopoietic cells on the fluorescence-activated cell sorter. I. Isolation of pluripotent hemopoietic stem cells and granulocyte-macrophage progenitor cells. Journal of Experimental Medicine, 152, 419-446. Kshirsagar, S. G. (1977). Radiostrontium distribution measured in vitro between bound and free forms in the soft tissues of rat. International Journal of Radiation Biology, 32, 561-569. Rauchwerger, J. M., Gallagher, M. T. and Trentin, J. J. (1973). Role of the hemopoietic inductive microenvironments (HIM) in xenogeneic bone marrow transplantation. Transplantation, 15, 610-618. Registry of Comparative Pathology (1970-90). Animal Models of Human Disease, C. C. Capen, T. C. Jones, D. B. Ha&el and G. Migaki, Eds, Armed Forces Institute of Pathology, Washington, D.C. Sporn, M. B. and Roberts, A. B. (Eds) (1991). Peptide Growth Factors and their Receptors, Vols 1 and 2. Springer, New York. Till, J. E. and McCulloch, E. A. (1961). A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Research, 14, 213-222. Vacek, A., Bartonickova, A. and Tkadlecek, L. (1976). Age dependence of the number of the stem cells in haemopoietic tissues of rats. Cell and Tissue Kinetics, 9, 1-8.
I Received, August 1 l th, 1993 -] Accepted, .November 8th, 1993_]