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Radiation effects on regeneration and T-cell-inducing function of the thymus
CELLULAR
IMMUNOLOGY
84,
Radiation
372-379
(1984)
Effects on Regeneration and T-Cell-Inducing Function of the Thymus
KATSUIKU
HIROKAWA*
AND TOSHIHIKO
SADO~
*Department of Pathology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo-l 73, and TDivision of Physiology and Pathology, National Institute of Radiological Sciences, Chiba-280, Japan Received October 14, 1983; accepted November 22, 1983 Radiation effects on regeneration and T-cell-inducing function of the thymus were studied in three sets of experiments. When TXB mice were grafted with l-week-old thymus which had been previously irradiated at various doses, an exponential decrease was observed in the morphological regeneration of the thymus grafts and in their T-cell-inducing function at doses of 600 R and over, showing about 10% that of the control at 1500 R. When in situ thymus of adult mice was locally irradiated, the radiation effect on T-cell-inducing function was less pronounced as compared with the first experiment; i.e., about 40% of the control at 1797 R. When in situ thymus of l-day-old newborn mice was locally irradiated, regeneration potential of ldayold newborn thymus was highly resistant to radiation exposure and no effect on immunological functions was observed even by local irradiation of 2000 R.
INTRODUCTION DiSerentiation of T cells is dependent upon humoral factors secreted by the epithelial tissue of the thymus (1, 2). In mice, the activity of the thymic epithelial tissue to promote T-cell differentiation was reported to decline with age (3, 4), beginning at an early phase of the life, long before the mice reach their immunological maturity (5). Nevertheless, when young adult mice are lethally irradiated and then protected with syngeneic bone marrow cells, they generally restore their immunological competency to approximately 50-60% of the level of unirradiated age-matched control within 3 months after irradiation and injection of bone marrow cells (6-8). This would suggest that (a) the T-cell-inducing activity of the thymus is fairly radioresistant and (b) the decline of the T-cell-inducing function may not be necessarily irreversible. In view of these considerations, attempts were made to assess the radiobiological parameters of the thymic epithelial tissue to promote T-cell differentiation by comparing the magnitude of restoration of T-cell function in three experimental systems. In the first system, young adult mice were thymectomized, exposed to 800 R totalbody X irradiation, and reconstituted with syngeneic bone marrow cells (TXB mice). These TXB mice were then implanted with one thymic lobe which had been exposed to graded doses of X rays immediately before grafting. In the second system, young adult mice were exposed to 1000 R of total-body X irradiation, followed by local exposure of the thymic portion to graded doses of X rays, and then reconstituted with syngeneic bone marrow cells. In the third system, the thymic portion of neonatal 372 0008~8749184 $3.00 Copyright 0 1984 by Academic PIES, Inc. AU rights of rqxcdudon in any form resewed
RADIATION
EFFECT ON THYMIC
FUNCTION
373
mice was exposed to graded doses of X rays. The regeneration of the thymic tissue as well as the immune functions were then evaluated in these three systems. The results of such analyses were reported in this paper. MATERIALS
AND
METHODS
Mice. Mice were female and male (C57BL/6JNrs X C3H/HeMsNrs)F, (hereafter referred to as B6C3Fi mice), which were bred in the colonies at the Animal Production Facility of National Institute of Radiological Sciences. Thymus grafting. Female B6C3Fi mice were thymectomized at 4 to 6 weeks of age and 2 weeks later received 800 R of total-body X irradiation (200 kVp, 20 mA, added filtration of 0.5 mm Cu + 0.5 mm Al, and 60 R/min at a target-skin distance of 50 cm in air), followed by an iv injection of 2 X lo6 syngeneic bone marrow cells from 3-month-old female donors (hereafter referred to as TXB mice). One month later, one thymic lobe from 7day-old syngeneic female mice, which had been given different doses of total-body X irradiation under the same conditions mentioned above, was grafted under the capsule of the right kidney of TXB mice. As described in the previous reports (4, 9), implanted thymic grafts undergo necrosis within a few days and then regenerate in a week. Thus, the donor-derived thymocytes are completely replaced by host-derived thymocytes within 3 weeks, and the regenerated thymic tissue is thus composed of donor-derived thymic epithelial tissue and thymocytes of host origin. In other words, progenitors of T cells of host origin proliferate and differentiate into mature T cells under influence of donor-derived thymic epithelial tissue. Accordingly, the activity of thymic epithelial tissue, treated at different doses of radiation and then implanted into T-cell deprived TXB mice, could be assessed by the weight of regenerated thymic tissue and the degree of recovery of T-cell functions of the recipient TXB mice. Assessment of T-cell function of the recipient mice was carried out 12 weeks after the implantation of irradiated thymic tissue. Local thymic irradiation. Local thymic irradiation was performed on 2-month-old adult mice and l-day-old newborn mice. Two-month-old male mice, first given totalbody X irradiation of 1000 R, were anesthetized with an ip injection of Nembutal then given local X irradiation to the thymus at doses of 797 and 2391 R, followed by an iv injection of 2 X lo6 syngeneic bone marrow cells from 2-month-old male donors. The local thymic irradiation was performed by placing the anesthetized mice on their backs and exposing the upper half of their chests to X rays through a port, 13 mm in diameter, cut in a lead plate 4 mm thick. The newborn mice were anesthetized by ice cooling and fixed with adhesive plaster on a lead plate 4 mm thick. The upper portion of their chests was exposed to X rays through a port, 6 mm in diameter, cut in the lead plate. Various doses of X-ray irradiation were delivered to the thymic portion at 200 kVp, 20 mA, with filtration of 0.5 mm Cu + 0.5 mm Al and targetskin distance of 54.8 cm. The dose rate was 47 R/min in the case of 6-mm port and 48 R/min in the case of 13-mm port. Assessment of immunologic functions were performed 12 weeks after the treatment. Since newborn mice within 24 hr after birth are still immunodeficient and contain few T cells in the peripheral lymphoid tissue, the degree of immunological development after the local thymic irradiation is considered to reflect the degree of radiation effects on the T-cell-inducing function of the thymus.
374
HIROKAWA
AND SAD0
Weight of thymus. The grafked and in situ thymic tissues were fixed with 0.1 A4 phosphate-buffered 4% formaldehyde (pH 7.2) and weighed later. They were then processed for histological observation. Mitogenic response. Assays were performed in microplate (Falcon Microtest III, 3072) as previously reported (3). Briefly, 5 X lo5 cells in 0.2 ml of RPM1 1640, supplemented with 5% fetal calf serum and kanamycin (0.06 mg/ml), were stimulated with optimum doses of either PHA (Wellcome Reagent Ltd., England, 1 rg), Con A (Sigma Chemical Co., St. Louis, MO., 1 pg) or LPS (Escherichiu coli 011 l:B4, Difco Laboratories, Detroit, Mich., 1 rg). The plates were incubated at 37°C in a 5% COz in air atmosphere for 6 hr then 0.25 PCi of [3H]thymidine (sp act 5.0 Ci/mmol) in 0.005 ml was added, and 2 hr later the cells were harvested and processed for beta scintillation counting (LS-250, Beckman). Anti-SRBC response. Five days after the mice were injected intraperitoneally with 1 ml of 1% SRBC, their spleen cells were assessed for the number of antibody-forming cells per spleen by DPFC assay of Plotz et al. ( 10). Cytolytic T cell activity. Five million spleen cells from B6C3FI (H-2bk) were cocultured in quadruplicate with an equal number of preirradiated (1500 R) spleen cells from DBA/2 (H-2d) mice in a total volume of 2 ml of RPM 1 1640 medium supplemented with 5 X lo-’ M 2-mercaptoethanol, kanamycin (0.06 mg/ml), and 10% fetal calf serum, in multiwell plates (24 wells, Coming 25820). The plates were placed at 37’C in a 5% CO2 in air atmosphere for 5 days, and the cells were harvested and processed for their cytolytic activity according to the method of Cerottini et al. ( 11) as previously described ( 12). RESULTS (1) RESTORATION OF T-CELL WITH IRRADIATED
FUNCTIONS IN TXB THYMUS GRAFT
MICE
Morphology of regenerated thymus grafs. As reported earlier (4, 8), implanted thymic tissue underwent necrosis with a few days and then regenerated in a week. The weight of the regenerated thymic tissue was measured 12 weeks after the implantation and plotted semilogarithmically as a function of dose of radiation exposure (Fig. 1). The curve showed a shoulder below 300 R, followed by an exponential decrease with increasing doses up to 1500 R. The histology of the thymus grafts also clearly showed that the regeneration ability of the thymic graft decreased with increasing doses of radiation exposure. The regenerated thymic tissues were interspersed with islet-like medullary areas and this architecture was different from that of normal thymus. Mitogenic response. The ability of thymus graft to generate PHA-responsive T cells, assessed at 12 weeks after the thymus grafting, was not affected by 300 R, but decreased exponentially at doses of 600 R and greater (Fig. 2). As expected, the responsiveness of splenic lymphocytes to a B-cell-specific mitogen, LPS, was not affected by the thymus grafting, regardless of whether they had been irradiated or not; i.e., the level of LPS response to TXB mice with or without thymus grafts was comparable to that of normal age-matched controls (Fig. 2). Anti-SRBC response. The recovery of the helper-T-cell function was assessed indirectly by determining the number of splenic DPFC 5 days after ip injection of SRBC 12 weeks after thymus grafting, and the results were summarized in Fig. 2.
“G
NEDNATAL LOCAL IRRADIATION
\
ADULT LOCAL IRRADiiTfbfi“‘%
‘ic
IN VITRO IRRAOIAlION BEFORE GRAFTING
‘\
I
I I 0 MoEa
0
II
WI 1aQ DOSES OF IRRADIATIOM (R)
FIG. 1. Wet weight of thymus 12 weeks after the irradiation treatment. Open squares, l-week-old thymus was irradiated at different doses and implanted under kidney capsule of TXB mice; solid squares with dotted line, 3-month-old mice were given 1000 R of total-body X irradiation, followed by additional local X irradiation to in situ thymus, and then protected with syngeneic bone marrow cells; solid squareswith solid line, local X irradiation of various doses was given to in situ thymus of l-day-old newborn mice. Each point is an average of 5-6 samples.
105. 8.
6. 4 2
4
102
AGE
CcNnla
I
I
I
%
0
300
600
900
DOSES OF RADIATION
I
1000 EUTIVE UlylRa (R)
FIG. 2. Immune potentials of TXB mice bearing thymus graft irradiated at different doses, 12 weeks after the implantation. DPFC (open circles), activity of antibody formation against sheep red blood cells, expressedby number of direct plaque-forming cells per spleen; PHA (solid circles) and LPS (open triangles), mitogenic activity of spleen cells by phytohemagglutinin and lipopolysaccharide, respectively, expressedby Acpm per culture (experimental cpm - control cpm); vertical bars, one standard error of mean. Each point is an average of 5-6 samples. Age control, sham-operated normal mice; negative control, sham-operated TXB mice. 375
376
20’
[ 0
HIROKAWA
AND SADO
I
I
10X
1797 DOSES OF IRRADIATION
I
3391 (R)
FIG. 3. Immune potentials of 6-month-old mice which had been treated with 1000 R of total-body X irradiation, followed by additional local X irradiation to in situ thymus and bone marrow transplantation, 12weeks earlier. DPFC(open circles), activity of antibody formation against sheep red blood cells, expressed by the number of direct plaque-forming cells per spleen; PHA (solid circles) and LP!S (open triangles), mitogenic activity of spleen cells by phytohemagglutinin and lipopolysaccharide, respectively, expressed by Acpm per culture (experimental cpm - control cpm); CTL (solid triangles), activity of cytolytic T cells against allogenenic tumor cells, expressed by lytic units per spleen; vertical bars, one standard error of mean. Each point is an average of 5-6 samples.
As in the case of the weight of the regenerated thymic tissue, a suppressive effect was not detected when the dose was below 300 R. In fact, when assessment was made in TXB mice with the 300 R-treated thymus graft, a significant enhancement of antiSRBC response was observed, suggesting that there was either an acceleration of helper-T-cell production or a deceleration of suppressor-T-cell production. After exposure to 600 R or greater, the response decreased gradually with increasing radiation exposures. After exposure to 1500 R, a marked decrease was observed in the response, but the level was still significantly higher than that of untreated TXB mice. These results indicated that the ability of thymic epithelial tissue to promote differentiation of helper T cells could not be totally abrogated by exposure to as high as 1500 R. (2) EFFECTOFLOCALX IRRADIATIONTOIN SITU THYMUSONTHE T-CELL-INDUCING FUNCTIONS
Since the thymus grafts undergo necrosis, then regenerate, and thereafter become influential on T-cell differentiation, there is a possibility that the depressed T-cellinducing function caused by the radiation treatment of the thymus grafts could be ascribed to their impaired regeneration ability. In order to test this possibility, restoration of T-cell function was assessed in adult mice as well as newborn mice whose thymuses had been locally exposed to graded doses of X irradiation.
RADIATION
EFFECT ON THYMIC
(a) Local Thymic Irradiation
FUNCTION
377
in Adult Mice
Three-month-old male mice were given 1000 R of total-body X irradiation, followed by additional local X irradiation to the in situ thymus, and then protected with syngeneic bone marrow. These mice were used for immunological assessment 12 weeks after the treatment. Histology. Thymocytes in the cortex disappeared quickly, within 24 hr after X irradiation over 1000 R, but no distinct change was histologically observed in the medulla. Repopulation of the thymus to the normal level occurred 2 weeks after the treatment. As shown in Fig. 1, the wet weight of the regenerated thymus appeared to decrease with increasing doses of X irradiation, but the magnitude of the decrease was much slighter than that observed in the thymus grting experiment. Anti-SRBC response. As shown in Fig. 3, the level of anti-SRBC response in adult mice given 1000 R total-body X irradiation and protected with syngeneic bone marrow cells was significantly lower than that of age-matched controls, showing about 42% of the control level. However, with increase of radiation exposure of the thymus over 1000 R, the level stayed constant showing about 53 and 50% at 1797 and 3391 R, respectively. It was revealed that the activity of in situ thymus to promote differentiation of helper T cells appeared to be relatively radioresistant and was well preserved even after exposure of X irradiation over 3000 R. Mitogenic response. The PHA response decreased exponentially with irradiation under 1797 R and leveled off thereafter (Fig. 3). The thymic activity to promote differentiation of PHA-responsive T cells was preserved at the 45% level of the control even after X-ray exposure over 3000 R. Contrarily, no effect was observed in LPS response.
I 0
I 1000 DOSES OF IRRADIATION CR)
I 2000
FIG. 4. Immune potentials of mice 12 weeks after local X irradiation to in situ thymus of l-day-old newborn mice. DPFC (open circles), activity of antibody formation against sheep red blood cells, expressed by number of direct plaque-forming cells per spleen; PHA (solid circles), Con A (open squares), and LP!I (open triangles), mitogenic activity of spleen cells against phytohemagglutinin, concanavalin A and lipopolysaccharide, respectively, expressed by Acpm per culture (experimental cpm - control cpm); vertical bars, one standard error of mean. Each point is an average of 5-6 samples.
378
HIROKAWA
AND SAD0
Cell-mediated cytolysis. Activity of cell-mediated cytolysis decreased slightly in the irradiated groups, but statistically there was no significant difference compared with control level. It was suggested that the activity of adult in situ thymus was highly radioresistant in terms of differentiation of killer T cells. (b) Local Thymic Irradiation
in Newborn Mice
Newborn male mice, within 24 hr after birth, were treated for local thymic irradiation in the manner mentioned under Materials and Methods. Immunological assessment was performed 12 weeks after the birth. The irradiation of thymic region did not influence the general growth of these newborn mice; i.e., body weight was 33.6 & 0.4, 34.5 + 0.9, and 33.6 + 5.3 g in control, 1000 R, and 2000 R groups, respectively. Immunologically, no significant change was observed in T-cell-dependent immunological functions, such as the anti-SRBC response, Con A response, and PHA response, and as observed in the former experiments, LPS response did not show any change (Fig. 4). Thus it was revealed that under 2000 R irradiation the newborn thymus was more radioresistant than the adult thymus, since the latter showed a decreased response to anti-SRBC and PHA, but the former did not. The only change detected was the wet weight of thymus, measured 12 weeks after the birth, i.e., 47.6 + 3.2, 44.8 + 3.9, and 37.0 & 3.0 mg in control, 1000 R, and 2000 R groups, respectively (Fig. 1). DISCUSSION The primary purpose of this study was to evaluate the radiosensitivity of thymic epithelial tissue in terms of the capacity to promote differentiation and proliferation of T cells, and three sets of experiments were conducted. The first experiment employed the use of adult TXB mice which were grafted with a thymus lobe obtained from 1-week-old syngeneic donor mice that had been exposed to graded doses of X rays immediately before grafting. The second one employed the use of adult mice which were given total-body X irradiation of 1000 R, followed by additional local irradiation in graded doses to in situ thymus, and then protected with syngeneic bone marrow cells. The third one employed newborn mice within 24 hr after birth. Since the newborn mice were used well before the maturation of immunological functions and were still immunoincompetent, the radiation effect on in situ thymus could be observed simply by measuring the level of immunological functions at a certain interval after local thymic irradiation to the in situ thymus. The results of these three sets of experiments indicated that T-cell-inducing ability of in situ thymus is more radioresistant than that of grafted thymus; e.g., in local thymic irradiation in newborn mice, no decline of immunological functions was observed by exposure of in situ thymus to 2000 R, while in the thymus-grafting experiment, the restoration of immunological functions was about 10% of the control after the implantation of 1500 R-treated thymus. The difference between these results could be considered as follows: in the case of irradiation of the in situ thymus, the regeneration of the thymus is accomplished only by repopulation of thymocytes, while in the thymus-grafting experiment, both thymic epithelial tissue and thymocytes first undergo necrosis, and then the thymic epithelial tissue has to regenerate before influencing differentiation of T cells. Under this condition, the effect of radiation exposure has an effect on not only the T-cell-inducing function of thymic epithelial
RADIATION
EFFECT ON THYMIC
FUNCTION
379
tissue, but also on its regeneration. Thus comparing the results of three experiments, it was apparent that the T-cell-inducing function of the thymic epithelial tissue was highly radioresistant, while its regeneration potential was radiosensitive. Using a similar system of irradiation of in situ thymus, Davies and Cole (13) reported that thymic function eventually recovered to normal levels after in situ exposure to as many as 2500 R, when reconstituted with syngeneic bone marrow cells. To the contrary, Miller et al. (14) reported that thymic grafts irradiated in vitro with 2000 R were deficient in their functional capacity to restore immunologic functions. The results of these reports were quite consistent with our findings reported here. Using the system of thymus grafting, we reported earlier (4) that T-cell-inducing functions of the thymic epithelial tissue declined progressively with age. We must now consider a possibility that aging could have an influence not only on T-cell-inducing functions of the thymic epithelial tissue but also on its regenerating ability after grafting into TXB mice. It was surprising that the newborn thymus, where thymocytes were rapidly proliferating, was much more radioresistant than the adult thymus, when they were irradiated in situ. In adult thymus, there appeared to be at least two subpopulations of thymic epithelial tissues, one radiosensitive and the other radioresistant, since responses of anti-SRBC and PHA declined to 50% of the control at 1000 R and leveled off thereafter. It might be interpreted that decline of the responses at 1000 R could be due to elimination of radiosensitive epithelial tissue and well-preserved responses at 3391 R due to a remnant of the radioresistant subpopulation. The difference in radiosensitivity of thymic function might be ascribed to not only radiosensitivity of thymic epithelial tissue but also difference in sensitivity of T precursor cells to thymic hormones. In other words, T precursor cells of newborn mice could be easily transformed to T cells by smaller amounts of thymic hormones than those of adult mice. Nevertheless, there are still some questions to be clarified before we can make a conclusive statement about the difference in radiosensitivity of thymic function between newborn and adult mice. ACKNOWLEDGMENTS The authors thank Ms. H. Kamisaku and E. Kubo for their expert technical assistance.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I. 12. 13. 14.
Trainin, N., Physiol. Rev. 54, 272, 1974. Cantor, H., and Boyse, E. A., Immunol. Rev. 33, 105, 1974. Hirokawa, K., and Makinodan, T., J. Immunol. 114, 1659, 1975. Hirokawa, K., and Sado, T., Mech. Age. Develop. 7, 89, 1978. Makinodan, T., and Adler, W., Fed. Proc. 34, 153, 1975. Davies, A. J. S., Carter, R. L., Leuchars, E., Wallis, V., and Koller, P. C., Immunology 16, 57, 1969. Gregory, C. J., and Lajtha, L. G., Int. J. Rudiut. Biol. 17, 117, 1970. Sado, T., and Kamisaku, H. Acta Haemtol. Japan. 40,48, 1977. Dukor, P., Miller, J. F. A. P., House, W., and Allman, V., Transplantation 3, 639, 1965. Plotz, P. H., Talal, N., and Asofsky, R., J. Immunol. 100, 744, 1968. Cerottini, J. C., Egner, H. D., MacDonald, H. R., and Brunner, K. T., J. Exp. Med. 140, 703, 1974. Hirokawa, K., Sato, K., and Makinodan, T., Clin. Immunol. Immunopathol. 24, 251, 1982. Davies, W. E., Jr., and Cole, L. J., Proc. Sot. Exp. &of. Med. 130, 1336, 1969. Miller, J. F. A. P., De Burgh, P. M., Dukor, P., Grant, G., Allman, V., and House, W., Clin. Exp. Immunol.
1, 61, 1966.
IMMUNOLOGY
84,
Radiation
372-379
(1984)
Effects on Regeneration and T-Cell-Inducing Function of the Thymus
KATSUIKU
HIROKAWA*
AND TOSHIHIKO
SADO~
*Department of Pathology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo-l 73, and TDivision of Physiology and Pathology, National Institute of Radiological Sciences, Chiba-280, Japan Received October 14, 1983; accepted November 22, 1983 Radiation effects on regeneration and T-cell-inducing function of the thymus were studied in three sets of experiments. When TXB mice were grafted with l-week-old thymus which had been previously irradiated at various doses, an exponential decrease was observed in the morphological regeneration of the thymus grafts and in their T-cell-inducing function at doses of 600 R and over, showing about 10% that of the control at 1500 R. When in situ thymus of adult mice was locally irradiated, the radiation effect on T-cell-inducing function was less pronounced as compared with the first experiment; i.e., about 40% of the control at 1797 R. When in situ thymus of l-day-old newborn mice was locally irradiated, regeneration potential of ldayold newborn thymus was highly resistant to radiation exposure and no effect on immunological functions was observed even by local irradiation of 2000 R.
INTRODUCTION DiSerentiation of T cells is dependent upon humoral factors secreted by the epithelial tissue of the thymus (1, 2). In mice, the activity of the thymic epithelial tissue to promote T-cell differentiation was reported to decline with age (3, 4), beginning at an early phase of the life, long before the mice reach their immunological maturity (5). Nevertheless, when young adult mice are lethally irradiated and then protected with syngeneic bone marrow cells, they generally restore their immunological competency to approximately 50-60% of the level of unirradiated age-matched control within 3 months after irradiation and injection of bone marrow cells (6-8). This would suggest that (a) the T-cell-inducing activity of the thymus is fairly radioresistant and (b) the decline of the T-cell-inducing function may not be necessarily irreversible. In view of these considerations, attempts were made to assess the radiobiological parameters of the thymic epithelial tissue to promote T-cell differentiation by comparing the magnitude of restoration of T-cell function in three experimental systems. In the first system, young adult mice were thymectomized, exposed to 800 R totalbody X irradiation, and reconstituted with syngeneic bone marrow cells (TXB mice). These TXB mice were then implanted with one thymic lobe which had been exposed to graded doses of X rays immediately before grafting. In the second system, young adult mice were exposed to 1000 R of total-body X irradiation, followed by local exposure of the thymic portion to graded doses of X rays, and then reconstituted with syngeneic bone marrow cells. In the third system, the thymic portion of neonatal 372 0008~8749184 $3.00 Copyright 0 1984 by Academic PIES, Inc. AU rights of rqxcdudon in any form resewed
RADIATION
EFFECT ON THYMIC
FUNCTION
373
mice was exposed to graded doses of X rays. The regeneration of the thymic tissue as well as the immune functions were then evaluated in these three systems. The results of such analyses were reported in this paper. MATERIALS
AND
METHODS
Mice. Mice were female and male (C57BL/6JNrs X C3H/HeMsNrs)F, (hereafter referred to as B6C3Fi mice), which were bred in the colonies at the Animal Production Facility of National Institute of Radiological Sciences. Thymus grafting. Female B6C3Fi mice were thymectomized at 4 to 6 weeks of age and 2 weeks later received 800 R of total-body X irradiation (200 kVp, 20 mA, added filtration of 0.5 mm Cu + 0.5 mm Al, and 60 R/min at a target-skin distance of 50 cm in air), followed by an iv injection of 2 X lo6 syngeneic bone marrow cells from 3-month-old female donors (hereafter referred to as TXB mice). One month later, one thymic lobe from 7day-old syngeneic female mice, which had been given different doses of total-body X irradiation under the same conditions mentioned above, was grafted under the capsule of the right kidney of TXB mice. As described in the previous reports (4, 9), implanted thymic grafts undergo necrosis within a few days and then regenerate in a week. Thus, the donor-derived thymocytes are completely replaced by host-derived thymocytes within 3 weeks, and the regenerated thymic tissue is thus composed of donor-derived thymic epithelial tissue and thymocytes of host origin. In other words, progenitors of T cells of host origin proliferate and differentiate into mature T cells under influence of donor-derived thymic epithelial tissue. Accordingly, the activity of thymic epithelial tissue, treated at different doses of radiation and then implanted into T-cell deprived TXB mice, could be assessed by the weight of regenerated thymic tissue and the degree of recovery of T-cell functions of the recipient TXB mice. Assessment of T-cell function of the recipient mice was carried out 12 weeks after the implantation of irradiated thymic tissue. Local thymic irradiation. Local thymic irradiation was performed on 2-month-old adult mice and l-day-old newborn mice. Two-month-old male mice, first given totalbody X irradiation of 1000 R, were anesthetized with an ip injection of Nembutal then given local X irradiation to the thymus at doses of 797 and 2391 R, followed by an iv injection of 2 X lo6 syngeneic bone marrow cells from 2-month-old male donors. The local thymic irradiation was performed by placing the anesthetized mice on their backs and exposing the upper half of their chests to X rays through a port, 13 mm in diameter, cut in a lead plate 4 mm thick. The newborn mice were anesthetized by ice cooling and fixed with adhesive plaster on a lead plate 4 mm thick. The upper portion of their chests was exposed to X rays through a port, 6 mm in diameter, cut in the lead plate. Various doses of X-ray irradiation were delivered to the thymic portion at 200 kVp, 20 mA, with filtration of 0.5 mm Cu + 0.5 mm Al and targetskin distance of 54.8 cm. The dose rate was 47 R/min in the case of 6-mm port and 48 R/min in the case of 13-mm port. Assessment of immunologic functions were performed 12 weeks after the treatment. Since newborn mice within 24 hr after birth are still immunodeficient and contain few T cells in the peripheral lymphoid tissue, the degree of immunological development after the local thymic irradiation is considered to reflect the degree of radiation effects on the T-cell-inducing function of the thymus.
374
HIROKAWA
AND SAD0
Weight of thymus. The grafked and in situ thymic tissues were fixed with 0.1 A4 phosphate-buffered 4% formaldehyde (pH 7.2) and weighed later. They were then processed for histological observation. Mitogenic response. Assays were performed in microplate (Falcon Microtest III, 3072) as previously reported (3). Briefly, 5 X lo5 cells in 0.2 ml of RPM1 1640, supplemented with 5% fetal calf serum and kanamycin (0.06 mg/ml), were stimulated with optimum doses of either PHA (Wellcome Reagent Ltd., England, 1 rg), Con A (Sigma Chemical Co., St. Louis, MO., 1 pg) or LPS (Escherichiu coli 011 l:B4, Difco Laboratories, Detroit, Mich., 1 rg). The plates were incubated at 37°C in a 5% COz in air atmosphere for 6 hr then 0.25 PCi of [3H]thymidine (sp act 5.0 Ci/mmol) in 0.005 ml was added, and 2 hr later the cells were harvested and processed for beta scintillation counting (LS-250, Beckman). Anti-SRBC response. Five days after the mice were injected intraperitoneally with 1 ml of 1% SRBC, their spleen cells were assessed for the number of antibody-forming cells per spleen by DPFC assay of Plotz et al. ( 10). Cytolytic T cell activity. Five million spleen cells from B6C3FI (H-2bk) were cocultured in quadruplicate with an equal number of preirradiated (1500 R) spleen cells from DBA/2 (H-2d) mice in a total volume of 2 ml of RPM 1 1640 medium supplemented with 5 X lo-’ M 2-mercaptoethanol, kanamycin (0.06 mg/ml), and 10% fetal calf serum, in multiwell plates (24 wells, Coming 25820). The plates were placed at 37’C in a 5% CO2 in air atmosphere for 5 days, and the cells were harvested and processed for their cytolytic activity according to the method of Cerottini et al. ( 11) as previously described ( 12). RESULTS (1) RESTORATION OF T-CELL WITH IRRADIATED
FUNCTIONS IN TXB THYMUS GRAFT
MICE
Morphology of regenerated thymus grafs. As reported earlier (4, 8), implanted thymic tissue underwent necrosis with a few days and then regenerated in a week. The weight of the regenerated thymic tissue was measured 12 weeks after the implantation and plotted semilogarithmically as a function of dose of radiation exposure (Fig. 1). The curve showed a shoulder below 300 R, followed by an exponential decrease with increasing doses up to 1500 R. The histology of the thymus grafts also clearly showed that the regeneration ability of the thymic graft decreased with increasing doses of radiation exposure. The regenerated thymic tissues were interspersed with islet-like medullary areas and this architecture was different from that of normal thymus. Mitogenic response. The ability of thymus graft to generate PHA-responsive T cells, assessed at 12 weeks after the thymus grafting, was not affected by 300 R, but decreased exponentially at doses of 600 R and greater (Fig. 2). As expected, the responsiveness of splenic lymphocytes to a B-cell-specific mitogen, LPS, was not affected by the thymus grafting, regardless of whether they had been irradiated or not; i.e., the level of LPS response to TXB mice with or without thymus grafts was comparable to that of normal age-matched controls (Fig. 2). Anti-SRBC response. The recovery of the helper-T-cell function was assessed indirectly by determining the number of splenic DPFC 5 days after ip injection of SRBC 12 weeks after thymus grafting, and the results were summarized in Fig. 2.
“G
NEDNATAL LOCAL IRRADIATION
\
ADULT LOCAL IRRADiiTfbfi“‘%
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WI 1aQ DOSES OF IRRADIATIOM (R)
FIG. 1. Wet weight of thymus 12 weeks after the irradiation treatment. Open squares, l-week-old thymus was irradiated at different doses and implanted under kidney capsule of TXB mice; solid squares with dotted line, 3-month-old mice were given 1000 R of total-body X irradiation, followed by additional local X irradiation to in situ thymus, and then protected with syngeneic bone marrow cells; solid squareswith solid line, local X irradiation of various doses was given to in situ thymus of l-day-old newborn mice. Each point is an average of 5-6 samples.
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4
102
AGE
CcNnla
I
I
I
%
0
300
600
900
DOSES OF RADIATION
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1000 EUTIVE UlylRa (R)
FIG. 2. Immune potentials of TXB mice bearing thymus graft irradiated at different doses, 12 weeks after the implantation. DPFC (open circles), activity of antibody formation against sheep red blood cells, expressedby number of direct plaque-forming cells per spleen; PHA (solid circles) and LPS (open triangles), mitogenic activity of spleen cells by phytohemagglutinin and lipopolysaccharide, respectively, expressedby Acpm per culture (experimental cpm - control cpm); vertical bars, one standard error of mean. Each point is an average of 5-6 samples. Age control, sham-operated normal mice; negative control, sham-operated TXB mice. 375
376
20’
[ 0
HIROKAWA
AND SADO
I
I
10X
1797 DOSES OF IRRADIATION
I
3391 (R)
FIG. 3. Immune potentials of 6-month-old mice which had been treated with 1000 R of total-body X irradiation, followed by additional local X irradiation to in situ thymus and bone marrow transplantation, 12weeks earlier. DPFC(open circles), activity of antibody formation against sheep red blood cells, expressed by the number of direct plaque-forming cells per spleen; PHA (solid circles) and LP!S (open triangles), mitogenic activity of spleen cells by phytohemagglutinin and lipopolysaccharide, respectively, expressed by Acpm per culture (experimental cpm - control cpm); CTL (solid triangles), activity of cytolytic T cells against allogenenic tumor cells, expressed by lytic units per spleen; vertical bars, one standard error of mean. Each point is an average of 5-6 samples.
As in the case of the weight of the regenerated thymic tissue, a suppressive effect was not detected when the dose was below 300 R. In fact, when assessment was made in TXB mice with the 300 R-treated thymus graft, a significant enhancement of antiSRBC response was observed, suggesting that there was either an acceleration of helper-T-cell production or a deceleration of suppressor-T-cell production. After exposure to 600 R or greater, the response decreased gradually with increasing radiation exposures. After exposure to 1500 R, a marked decrease was observed in the response, but the level was still significantly higher than that of untreated TXB mice. These results indicated that the ability of thymic epithelial tissue to promote differentiation of helper T cells could not be totally abrogated by exposure to as high as 1500 R. (2) EFFECTOFLOCALX IRRADIATIONTOIN SITU THYMUSONTHE T-CELL-INDUCING FUNCTIONS
Since the thymus grafts undergo necrosis, then regenerate, and thereafter become influential on T-cell differentiation, there is a possibility that the depressed T-cellinducing function caused by the radiation treatment of the thymus grafts could be ascribed to their impaired regeneration ability. In order to test this possibility, restoration of T-cell function was assessed in adult mice as well as newborn mice whose thymuses had been locally exposed to graded doses of X irradiation.
RADIATION
EFFECT ON THYMIC
(a) Local Thymic Irradiation
FUNCTION
377
in Adult Mice
Three-month-old male mice were given 1000 R of total-body X irradiation, followed by additional local X irradiation to the in situ thymus, and then protected with syngeneic bone marrow. These mice were used for immunological assessment 12 weeks after the treatment. Histology. Thymocytes in the cortex disappeared quickly, within 24 hr after X irradiation over 1000 R, but no distinct change was histologically observed in the medulla. Repopulation of the thymus to the normal level occurred 2 weeks after the treatment. As shown in Fig. 1, the wet weight of the regenerated thymus appeared to decrease with increasing doses of X irradiation, but the magnitude of the decrease was much slighter than that observed in the thymus grting experiment. Anti-SRBC response. As shown in Fig. 3, the level of anti-SRBC response in adult mice given 1000 R total-body X irradiation and protected with syngeneic bone marrow cells was significantly lower than that of age-matched controls, showing about 42% of the control level. However, with increase of radiation exposure of the thymus over 1000 R, the level stayed constant showing about 53 and 50% at 1797 and 3391 R, respectively. It was revealed that the activity of in situ thymus to promote differentiation of helper T cells appeared to be relatively radioresistant and was well preserved even after exposure of X irradiation over 3000 R. Mitogenic response. The PHA response decreased exponentially with irradiation under 1797 R and leveled off thereafter (Fig. 3). The thymic activity to promote differentiation of PHA-responsive T cells was preserved at the 45% level of the control even after X-ray exposure over 3000 R. Contrarily, no effect was observed in LPS response.
I 0
I 1000 DOSES OF IRRADIATION CR)
I 2000
FIG. 4. Immune potentials of mice 12 weeks after local X irradiation to in situ thymus of l-day-old newborn mice. DPFC (open circles), activity of antibody formation against sheep red blood cells, expressed by number of direct plaque-forming cells per spleen; PHA (solid circles), Con A (open squares), and LP!I (open triangles), mitogenic activity of spleen cells against phytohemagglutinin, concanavalin A and lipopolysaccharide, respectively, expressed by Acpm per culture (experimental cpm - control cpm); vertical bars, one standard error of mean. Each point is an average of 5-6 samples.
378
HIROKAWA
AND SAD0
Cell-mediated cytolysis. Activity of cell-mediated cytolysis decreased slightly in the irradiated groups, but statistically there was no significant difference compared with control level. It was suggested that the activity of adult in situ thymus was highly radioresistant in terms of differentiation of killer T cells. (b) Local Thymic Irradiation
in Newborn Mice
Newborn male mice, within 24 hr after birth, were treated for local thymic irradiation in the manner mentioned under Materials and Methods. Immunological assessment was performed 12 weeks after the birth. The irradiation of thymic region did not influence the general growth of these newborn mice; i.e., body weight was 33.6 & 0.4, 34.5 + 0.9, and 33.6 + 5.3 g in control, 1000 R, and 2000 R groups, respectively. Immunologically, no significant change was observed in T-cell-dependent immunological functions, such as the anti-SRBC response, Con A response, and PHA response, and as observed in the former experiments, LPS response did not show any change (Fig. 4). Thus it was revealed that under 2000 R irradiation the newborn thymus was more radioresistant than the adult thymus, since the latter showed a decreased response to anti-SRBC and PHA, but the former did not. The only change detected was the wet weight of thymus, measured 12 weeks after the birth, i.e., 47.6 + 3.2, 44.8 + 3.9, and 37.0 & 3.0 mg in control, 1000 R, and 2000 R groups, respectively (Fig. 1). DISCUSSION The primary purpose of this study was to evaluate the radiosensitivity of thymic epithelial tissue in terms of the capacity to promote differentiation and proliferation of T cells, and three sets of experiments were conducted. The first experiment employed the use of adult TXB mice which were grafted with a thymus lobe obtained from 1-week-old syngeneic donor mice that had been exposed to graded doses of X rays immediately before grafting. The second one employed the use of adult mice which were given total-body X irradiation of 1000 R, followed by additional local irradiation in graded doses to in situ thymus, and then protected with syngeneic bone marrow cells. The third one employed newborn mice within 24 hr after birth. Since the newborn mice were used well before the maturation of immunological functions and were still immunoincompetent, the radiation effect on in situ thymus could be observed simply by measuring the level of immunological functions at a certain interval after local thymic irradiation to the in situ thymus. The results of these three sets of experiments indicated that T-cell-inducing ability of in situ thymus is more radioresistant than that of grafted thymus; e.g., in local thymic irradiation in newborn mice, no decline of immunological functions was observed by exposure of in situ thymus to 2000 R, while in the thymus-grafting experiment, the restoration of immunological functions was about 10% of the control after the implantation of 1500 R-treated thymus. The difference between these results could be considered as follows: in the case of irradiation of the in situ thymus, the regeneration of the thymus is accomplished only by repopulation of thymocytes, while in the thymus-grafting experiment, both thymic epithelial tissue and thymocytes first undergo necrosis, and then the thymic epithelial tissue has to regenerate before influencing differentiation of T cells. Under this condition, the effect of radiation exposure has an effect on not only the T-cell-inducing function of thymic epithelial
RADIATION
EFFECT ON THYMIC
FUNCTION
379
tissue, but also on its regeneration. Thus comparing the results of three experiments, it was apparent that the T-cell-inducing function of the thymic epithelial tissue was highly radioresistant, while its regeneration potential was radiosensitive. Using a similar system of irradiation of in situ thymus, Davies and Cole (13) reported that thymic function eventually recovered to normal levels after in situ exposure to as many as 2500 R, when reconstituted with syngeneic bone marrow cells. To the contrary, Miller et al. (14) reported that thymic grafts irradiated in vitro with 2000 R were deficient in their functional capacity to restore immunologic functions. The results of these reports were quite consistent with our findings reported here. Using the system of thymus grafting, we reported earlier (4) that T-cell-inducing functions of the thymic epithelial tissue declined progressively with age. We must now consider a possibility that aging could have an influence not only on T-cell-inducing functions of the thymic epithelial tissue but also on its regenerating ability after grafting into TXB mice. It was surprising that the newborn thymus, where thymocytes were rapidly proliferating, was much more radioresistant than the adult thymus, when they were irradiated in situ. In adult thymus, there appeared to be at least two subpopulations of thymic epithelial tissues, one radiosensitive and the other radioresistant, since responses of anti-SRBC and PHA declined to 50% of the control at 1000 R and leveled off thereafter. It might be interpreted that decline of the responses at 1000 R could be due to elimination of radiosensitive epithelial tissue and well-preserved responses at 3391 R due to a remnant of the radioresistant subpopulation. The difference in radiosensitivity of thymic function might be ascribed to not only radiosensitivity of thymic epithelial tissue but also difference in sensitivity of T precursor cells to thymic hormones. In other words, T precursor cells of newborn mice could be easily transformed to T cells by smaller amounts of thymic hormones than those of adult mice. Nevertheless, there are still some questions to be clarified before we can make a conclusive statement about the difference in radiosensitivity of thymic function between newborn and adult mice. ACKNOWLEDGMENTS The authors thank Ms. H. Kamisaku and E. Kubo for their expert technical assistance.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I. 12. 13. 14.
Trainin, N., Physiol. Rev. 54, 272, 1974. Cantor, H., and Boyse, E. A., Immunol. Rev. 33, 105, 1974. Hirokawa, K., and Makinodan, T., J. Immunol. 114, 1659, 1975. Hirokawa, K., and Sado, T., Mech. Age. Develop. 7, 89, 1978. Makinodan, T., and Adler, W., Fed. Proc. 34, 153, 1975. Davies, A. J. S., Carter, R. L., Leuchars, E., Wallis, V., and Koller, P. C., Immunology 16, 57, 1969. Gregory, C. J., and Lajtha, L. G., Int. J. Rudiut. Biol. 17, 117, 1970. Sado, T., and Kamisaku, H. Acta Haemtol. Japan. 40,48, 1977. Dukor, P., Miller, J. F. A. P., House, W., and Allman, V., Transplantation 3, 639, 1965. Plotz, P. H., Talal, N., and Asofsky, R., J. Immunol. 100, 744, 1968. Cerottini, J. C., Egner, H. D., MacDonald, H. R., and Brunner, K. T., J. Exp. Med. 140, 703, 1974. Hirokawa, K., Sato, K., and Makinodan, T., Clin. Immunol. Immunopathol. 24, 251, 1982. Davies, W. E., Jr., and Cole, L. J., Proc. Sot. Exp. &of. Med. 130, 1336, 1969. Miller, J. F. A. P., De Burgh, P. M., Dukor, P., Grant, G., Allman, V., and House, W., Clin. Exp. Immunol.
1, 61, 1966.