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RECOVERY IN THE MOUSE SPLEEN AFTER SUBLETHAL IRRADIATION

RECOVERY IN THE MOUSE SPLEEN AFTER SUBLETHAL IRRADIATION Tlie Institute of Biophysics. The Ministry of Health, U.S.S.R., Moscow (Received 25 April I968 ; recision received I2 July 1968) ABSTRACT 1. CBA and C57BI mice were exposed to LD,,,,, doses of y-radiation and at intervals after irradiation the numbers of haemopoietic colony-forming units (CFU) and of antibody-producing cells in the spleen were determined. 2. CFU repression was observed after transfer of non-irradiated cells to hybrid recipients. However, this repression did not appear until 60 days after irradiation of the donor. 3. The recovery after irradiation of the capacity to inactivate CFUs in spleen cell mixtures was studied. The results suggested that cells which produce 19s antibody are different from those which are reactive against foreign cells. INTRODUCTION The destructive effects of ionizing radiations on the haematopoietic and lymphoid systems are well known (Kraevsky, 1957; Bond, Fliedner & Archambeau, 1965). However, the recovery processes have proved more difficult to understand. The spleen, for example, is a highly complex organ to which no single overall function can be ascribed. Equally, because of this complexity and uncertainty as to function, the depletion and recovery processes in the spleen after total body irradiation are difficult to interpret. Nevertheless, in recent years several satisfactory methods have emerged for the quantitation of antibody producing cells (Jerne & Nordin, 1963), haematopoietic stem cells (Till & McCulloch, 1961, 1964) and cells which are reactive against foreign cells (Petrov & Zaretskaja, 1965; Micklem & Loutit, 1966; Egorova, 1966). Here presented is an attempt to determine the recovery pattern of each of these three cellular components of the spleen after sub-lethal total body X-irradiation. METHODS Experimental aniinals 8-12-week-old mice of the CBA and C57Bl inbred strains of either sex and their F, hybrid were used. Correspondence: Dr R . V. Petrov, Institute of Biophysics, Zhivopisnaya 22, Moscow D-182, U.S.S.R. R . V. Petrov, V. A . Koslov and L. S. Seslavina Irradiation Mice were irradiated with 6oCogamma rays at a dose rate of about 610 r/min. Host mice (ride infru) received 850 r, donor mice either 600 r (CBA) or 530 r (C57B1) (Fig. I ) . Colony forining units The numbers of colony forming units (CFU) present in various spleen cell suspensions was determined following the method of McCulloch and Till (1963). Pooled donor cell suspensions in medium 199 were injected into the tail vein of syngeneic recipient (host) mice 24 hr after irradiation. Usually 2 x lo6 viable (as determined by the capacity to exclude trypan-blue) cells were injected but as will be seen in the table of results it was occasionally necessary to use fewer cells. Eight days after irradiation and spleen injection the recipient mice were killed and their spleens fixed in Bouin solution preparatory to counting of visible i g 2x106 &&- 8 ( C B A x C 5 7 B C ) F, 850r Standard I la=-& / CBA 2 CJ 2x 106 Experimental ( C B A x C 5 7 B C ) FI C.5781 Nor Standard I I ( C B A x C 5 7 B L ) F, C F U ( 8 t h day) FIG.1 . Scheme for the ‘stem cell inactivation’ technique. colonies. An attempt was also made to ascertain whether the phenomenon of CFU repression, shown by McCulloch & Till to follow injection of haematopoietic cells into allogeneic recipients, could be demonstrated. For this purpose either CBA or C57BI spleen cells were injected into irradiated (CBA x C57BI)F, hybrid recipients. Jerne plaque counts The numbers of 19s antibody liberating cells was determined by the Jerne method (Jerne & Nordin, 1963) 4, 5, 6 and 7 days after intravenous injection of about 1 x 108 sheep erythrocytes. Colony inhibition as a result of reactions between allogeneic spleen cells As has been shown by Petrov & Seslavina (1967), injection of mixtures of CBA and C57BI spleen cells into irradiated F, hybrid recipients engenders fewer haematopoietic spleen colonies than could be expected if the two injected cell populations were independent of Recovery itz the mouse spleeti after sublethal irradiation each other. It is reasonable to suppose, by reference t o the appropriate controls, that the reaction between the two cell populations involves immunological antagonism. A measure of this is given by the inactivation index which compares the number of cells observed with the number of cells expected assuming no interaction. The calculation of this index is given in the tables of results. Recovery of the ability t o form Jerne plaques, to potentiate spleen haematopoietic cell colonies and t o react with foreign cells in such a way as to impair their stem cell function was studied in mice 1, 7, 14, 30 and 60 days after irradiation. RESULTS Haeiiiatopoietic coloiiy prohirtioii Table I shows the effect of irradiation on the numbers of stem cells in the spleens of CBA and C57B1 mice. It can be seen that immediately after irradiation there is a sharp drop in TABLE The effect of irradiation on the number of stem cells in the spleens of CBA and C57BI mice as I. determined by transfer to irradiated syngeneic recipients The mean number of CFU per 10' donor spleen cells a t different times after irradiation Non-irradiated animals Strain 1 day 7 days 11.16 + 2.82 (25) 33.1 t 6.52 (29) I4 days 30 days 60 days 84.16 i 14.56 : (32)* C57B1 152.06 & 6.98 (29) CBA 3.78 i 1.2 (18) 8.38 1 2.56 (13) 442.5 i 34.7 (24) 232.08 i 23.44 (12) 61.08 rt 15.46 (24) 142.14 i 18.14 (14) 63.72 i 7.14 (15) 130.44 rt 17.26 (23) Values in parentheses are the numbers of recipients per group. the number of cells capable of giving rise to CFUs but that by 7 days some recovery is evident. Values above normal were seen on day 14 but thereafter approximately normal levels were demonstrable. Table 2 compares the performance of C B A and C57Bl spleen cells in syngeneic and in hybrid recipients. It can be seen that 'CFU repression' was observed after non-syngeneic TABLE The number of CFU in the spleens of CBA and C57BI mice as deduced by transplantation into 2. syngeneic and F , hybrid recipients No. of recipients studied Donor strain No. of cells injected Recipient strain Mean no. of CFU per lo7 injected nucleated spleen cells 32 58 29 236 25* CBA CBA C57BI C57BI 10b 10' 10" ' 10' CBA (CBA x C57BI)FI C57BI (CBA iC57BI)FI 84.16 f 14.56 8.58 40.76 152.06 i 6.98 91.46 + 3.68 I 03 R. V. Petrov, V. A . Koslov atid L. S. Seslavina 86E .. .. . . -.. “---I - , ’ 1-i 1 7 loo 30 Days ofter X-irradiation Fic. 2. Number of C F U in the spleen of mice of CBA strain at different intervals after irradiation. , CBA + CBA; ----, CBA (CBA x C57BI)Fl. ---f transfers. I n Fig. 2 is shown the capacity of spleen cells from irradiated CBA mice to be repressed by a foreign environment. It can be seen that the phenomenon of repression was not observed until 60 days after irradiation and it can only be concluded that in some way the repressive effect is a property of the donor cells which can be affected by irradiation. Jerne plaque production The ability of C57Bl and CBA mice to produce a response to sheep red cells can be seen to be sharply curtailed after irradiation (Table 3) and further the recovery process is slow, not being completed by the end of the experiment in the instance of CBA mice. Mixed spleen cell inactii3ation In Table 4 are revealed the results of the experiments concerned with inactivation of colony forming units in spleen cell mixtures as a function of time after irradiation. Experiments were performed in duplicate and thus a total of about ten recipients was employed for each given mean value. The coefficients of variation were about 10-20% for most of the means. Irradiation of only one component of the mixture (CBA cells) was attempted. Previous TABLE The effect of irradiation on the number of antibody-forming cells in the spleens of CBA and 3. C57B1 mice The mean no. of antibody-formingcells per Non-irradiated animals spleen cells at different times after irradiation lo7 Strain 1 day 7 days 14 days 30 days 60 days 1224 $I 21 1.1 CBA C57B1 356.7 f 41.39 40.3 i 6.65 42.1 f 7.3 23.5 i 5.85 20.6 f 5.9 293.2 i 42.96 131.1 + 28.49 566 f 39.12 268.9 I 2 6 . 0 5 714.3 f 75.99 356.3 rt 66.45 C rp TABLE The inactivation intensity of non-syngeneic stem cells at different times after irradiation of CBA mice 4. Time after irradiation of CBA cells -~ < -. % _______ Inactivation index Mean colony number ('lo) Non-irradiated animals 7 days _________ 1 day 14 days: 30 days 60 days z i Inactivation index (I1") Donor spleen Mean colony number ("") Inactivation index* Mean colony number Inactivation index Inactivation index Inactivation index Mean colony number (':,) Mean colony number ("o) Mean colon! number .n -7 4(A ) 19@) -17 --lot C BA C57B1 CBA t C57BI 5(C) -lot lot a- t Inactivation is statistically insignificant. 4 x 1 0 5 (not 2 x 106) spleen cells were taken from CBA donors as at this time after irradiation the number of C F U in spleen cell population increased sharply. e -. w 4 w R. A . Petrov, V . A . Koslov and L. S . Seslavina studies (Petrov & Seslavina, 1967) have shown that it is these cells which can be thought of as the aggressive and the C57Bl cells as the suppressed component. This is an oversimplification but it is clear that, as the two cells do not interreact on an equal footing, i t was better to irradiate the apparently more aggressive cell type. It can be seen that the capacity to inactivate C57B1 cells was negligible in the immediate postirradiation period but had substantially recovered by day 30 and was of normal magnitude by day 60 after irradiation. DISCUSSION In Fig. 3 are compared the three different post-irradiation recovery parameters with the changes in total nucleated spleen cell count. Not surprisingly in the first few days after irradiation all estimated functions of the spleen showed a dramatic decline, presumably because many cells died during this time. The haematopoietic capacity recovered first and completely within a relatively short space of time. The other two measurements made probably reflect different aspects of recovery of immunological responsiveness. Their slow and incomplete restoration until quite late after radiation could be imputed to a competition for a common stem cell precursor which favours haematopoiesis over lymphopoiesis. It seems more likely, however, that different cell populations are involved in the two processes and, further, that the cells responsible for restoring (albeit incompletely) the capacity to produce 19s antibody are somewhat different from those which are reactive against foreign cells in the stem cell inactivation phenomenon. I n these respects it will be interesting i n ''I.. -. 4- 2102- 8: 6- 4- 210' ; 8: 64- 2- Days after X-irradiation FIG.3. Dynamics of the studied characteristics in the spleen of mice of CBA strain at different interval after irradiation. -. .-, Total number of the nucleated spleen cells; -, number of CFU per lo7 of spleen cells; ----, number of antibody-forming cells per lo6 of spleen cells; -. -,inactivation index of non-syngeneic stem cells (?A). Recovery in the mouse spleen after sublethal irradiatiotz future work to see firstly whether 7s antibody production has the same pattern of restoration as that for 19s antibody and further whether any or all of the immunological recovery phenomena are thymus dependent. Investigation of these points may help to clarify which cells are engaged in the relevant immune responses. The last and perhaps most intriguing point is that concerning the 'allogeneic inhibition' type of effect observed when either C57BI or CBA spleen cells were injected into F, hybrid recipients. Theestimates ofthe repression oftheir activities as haematopoietic colonies seemed to show the same radiation recovery pattern as that of the more obviously immunologically oriented functioiis. The effects can hardly be imputed Lo the host mice which remained constant throughout. It can be supposed that some graft versus host reaction, which is radiation sensitive, i n some way affects the capacity of the injected haematopoietic cells to proliferate. The mechanism of this effect awaits elucidation. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Proliferation Wiley

RECOVERY IN THE MOUSE SPLEEN AFTER SUBLETHAL IRRADIATION

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Publisher
Wiley
Copyright
1968 Blackwell Science Limited
ISSN
0960-7722
eISSN
1365-2184
DOI
10.1111/j.1365-2184.1968.tb00965.x
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Abstract

Tlie Institute of Biophysics. The Ministry of Health, U.S.S.R., Moscow (Received 25 April I968 ; recision received I2 July 1968) ABSTRACT 1. CBA and C57BI mice were exposed to LD,,,,, doses of y-radiation and at intervals after irradiation the numbers of haemopoietic colony-forming units (CFU) and of antibody-producing cells in the spleen were determined. 2. CFU repression was observed after transfer of non-irradiated cells to hybrid recipients. However, this repression did not appear until 60 days after irradiation of the donor. 3. The recovery after irradiation of the capacity to inactivate CFUs in spleen cell mixtures was studied. The results suggested that cells which produce 19s antibody are different from those which are reactive against foreign cells. INTRODUCTION The destructive effects of ionizing radiations on the haematopoietic and lymphoid systems are well known (Kraevsky, 1957; Bond, Fliedner & Archambeau, 1965). However, the recovery processes have proved more difficult to understand. The spleen, for example, is a highly complex organ to which no single overall function can be ascribed. Equally, because of this complexity and uncertainty as to function, the depletion and recovery processes in the spleen after total body irradiation are difficult to interpret. Nevertheless, in recent years several satisfactory methods have emerged for the quantitation of antibody producing cells (Jerne & Nordin, 1963), haematopoietic stem cells (Till & McCulloch, 1961, 1964) and cells which are reactive against foreign cells (Petrov & Zaretskaja, 1965; Micklem & Loutit, 1966; Egorova, 1966). Here presented is an attempt to determine the recovery pattern of each of these three cellular components of the spleen after sub-lethal total body X-irradiation. METHODS Experimental aniinals 8-12-week-old mice of the CBA and C57Bl inbred strains of either sex and their F, hybrid were used. Correspondence: Dr R . V. Petrov, Institute of Biophysics, Zhivopisnaya 22, Moscow D-182, U.S.S.R. R . V. Petrov, V. A . Koslov and L. S. Seslavina Irradiation Mice were irradiated with 6oCogamma rays at a dose rate of about 610 r/min. Host mice (ride infru) received 850 r, donor mice either 600 r (CBA) or 530 r (C57B1) (Fig. I ) . Colony forining units The numbers of colony forming units (CFU) present in various spleen cell suspensions was determined following the method of McCulloch and Till (1963). Pooled donor cell suspensions in medium 199 were injected into the tail vein of syngeneic recipient (host) mice 24 hr after irradiation. Usually 2 x lo6 viable (as determined by the capacity to exclude trypan-blue) cells were injected but as will be seen in the table of results it was occasionally necessary to use fewer cells. Eight days after irradiation and spleen injection the recipient mice were killed and their spleens fixed in Bouin solution preparatory to counting of visible i g 2x106 &&- 8 ( C B A x C 5 7 B C ) F, 850r Standard I la=-& / CBA 2 CJ 2x 106 Experimental ( C B A x C 5 7 B C ) FI C.5781 Nor Standard I I ( C B A x C 5 7 B L ) F, C F U ( 8 t h day) FIG.1 . Scheme for the ‘stem cell inactivation’ technique. colonies. An attempt was also made to ascertain whether the phenomenon of CFU repression, shown by McCulloch & Till to follow injection of haematopoietic cells into allogeneic recipients, could be demonstrated. For this purpose either CBA or C57BI spleen cells were injected into irradiated (CBA x C57BI)F, hybrid recipients. Jerne plaque counts The numbers of 19s antibody liberating cells was determined by the Jerne method (Jerne & Nordin, 1963) 4, 5, 6 and 7 days after intravenous injection of about 1 x 108 sheep erythrocytes. Colony inhibition as a result of reactions between allogeneic spleen cells As has been shown by Petrov & Seslavina (1967), injection of mixtures of CBA and C57BI spleen cells into irradiated F, hybrid recipients engenders fewer haematopoietic spleen colonies than could be expected if the two injected cell populations were independent of Recovery itz the mouse spleeti after sublethal irradiation each other. It is reasonable to suppose, by reference t o the appropriate controls, that the reaction between the two cell populations involves immunological antagonism. A measure of this is given by the inactivation index which compares the number of cells observed with the number of cells expected assuming no interaction. The calculation of this index is given in the tables of results. Recovery of the ability t o form Jerne plaques, to potentiate spleen haematopoietic cell colonies and t o react with foreign cells in such a way as to impair their stem cell function was studied in mice 1, 7, 14, 30 and 60 days after irradiation. RESULTS Haeiiiatopoietic coloiiy prohirtioii Table I shows the effect of irradiation on the numbers of stem cells in the spleens of CBA and C57B1 mice. It can be seen that immediately after irradiation there is a sharp drop in TABLE The effect of irradiation on the number of stem cells in the spleens of CBA and C57BI mice as I. determined by transfer to irradiated syngeneic recipients The mean number of CFU per 10' donor spleen cells a t different times after irradiation Non-irradiated animals Strain 1 day 7 days 11.16 + 2.82 (25) 33.1 t 6.52 (29) I4 days 30 days 60 days 84.16 i 14.56 : (32)* C57B1 152.06 & 6.98 (29) CBA 3.78 i 1.2 (18) 8.38 1 2.56 (13) 442.5 i 34.7 (24) 232.08 i 23.44 (12) 61.08 rt 15.46 (24) 142.14 i 18.14 (14) 63.72 i 7.14 (15) 130.44 rt 17.26 (23) Values in parentheses are the numbers of recipients per group. the number of cells capable of giving rise to CFUs but that by 7 days some recovery is evident. Values above normal were seen on day 14 but thereafter approximately normal levels were demonstrable. Table 2 compares the performance of C B A and C57Bl spleen cells in syngeneic and in hybrid recipients. It can be seen that 'CFU repression' was observed after non-syngeneic TABLE The number of CFU in the spleens of CBA and C57BI mice as deduced by transplantation into 2. syngeneic and F , hybrid recipients No. of recipients studied Donor strain No. of cells injected Recipient strain Mean no. of CFU per lo7 injected nucleated spleen cells 32 58 29 236 25* CBA CBA C57BI C57BI 10b 10' 10" ' 10' CBA (CBA x C57BI)FI C57BI (CBA iC57BI)FI 84.16 f 14.56 8.58 40.76 152.06 i 6.98 91.46 + 3.68 I 03 R. V. Petrov, V. A . Koslov atid L. S. Seslavina 86E .. .. . . -.. “---I - , ’ 1-i 1 7 loo 30 Days ofter X-irradiation Fic. 2. Number of C F U in the spleen of mice of CBA strain at different intervals after irradiation. , CBA + CBA; ----, CBA (CBA x C57BI)Fl. ---f transfers. I n Fig. 2 is shown the capacity of spleen cells from irradiated CBA mice to be repressed by a foreign environment. It can be seen that the phenomenon of repression was not observed until 60 days after irradiation and it can only be concluded that in some way the repressive effect is a property of the donor cells which can be affected by irradiation. Jerne plaque production The ability of C57Bl and CBA mice to produce a response to sheep red cells can be seen to be sharply curtailed after irradiation (Table 3) and further the recovery process is slow, not being completed by the end of the experiment in the instance of CBA mice. Mixed spleen cell inactii3ation In Table 4 are revealed the results of the experiments concerned with inactivation of colony forming units in spleen cell mixtures as a function of time after irradiation. Experiments were performed in duplicate and thus a total of about ten recipients was employed for each given mean value. The coefficients of variation were about 10-20% for most of the means. Irradiation of only one component of the mixture (CBA cells) was attempted. Previous TABLE The effect of irradiation on the number of antibody-forming cells in the spleens of CBA and 3. C57B1 mice The mean no. of antibody-formingcells per Non-irradiated animals spleen cells at different times after irradiation lo7 Strain 1 day 7 days 14 days 30 days 60 days 1224 $I 21 1.1 CBA C57B1 356.7 f 41.39 40.3 i 6.65 42.1 f 7.3 23.5 i 5.85 20.6 f 5.9 293.2 i 42.96 131.1 + 28.49 566 f 39.12 268.9 I 2 6 . 0 5 714.3 f 75.99 356.3 rt 66.45 C rp TABLE The inactivation intensity of non-syngeneic stem cells at different times after irradiation of CBA mice 4. Time after irradiation of CBA cells -~ < -. % _______ Inactivation index Mean colony number ('lo) Non-irradiated animals 7 days _________ 1 day 14 days: 30 days 60 days z i Inactivation index (I1") Donor spleen Mean colony number ("") Inactivation index* Mean colony number Inactivation index Inactivation index Inactivation index Mean colony number (':,) Mean colony number ("o) Mean colon! number .n -7 4(A ) 19@) -17 --lot C BA C57B1 CBA t C57BI 5(C) -lot lot a- t Inactivation is statistically insignificant. 4 x 1 0 5 (not 2 x 106) spleen cells were taken from CBA donors as at this time after irradiation the number of C F U in spleen cell population increased sharply. e -. w 4 w R. A . Petrov, V . A . Koslov and L. S . Seslavina studies (Petrov & Seslavina, 1967) have shown that it is these cells which can be thought of as the aggressive and the C57Bl cells as the suppressed component. This is an oversimplification but it is clear that, as the two cells do not interreact on an equal footing, i t was better to irradiate the apparently more aggressive cell type. It can be seen that the capacity to inactivate C57B1 cells was negligible in the immediate postirradiation period but had substantially recovered by day 30 and was of normal magnitude by day 60 after irradiation. DISCUSSION In Fig. 3 are compared the three different post-irradiation recovery parameters with the changes in total nucleated spleen cell count. Not surprisingly in the first few days after irradiation all estimated functions of the spleen showed a dramatic decline, presumably because many cells died during this time. The haematopoietic capacity recovered first and completely within a relatively short space of time. The other two measurements made probably reflect different aspects of recovery of immunological responsiveness. Their slow and incomplete restoration until quite late after radiation could be imputed to a competition for a common stem cell precursor which favours haematopoiesis over lymphopoiesis. It seems more likely, however, that different cell populations are involved in the two processes and, further, that the cells responsible for restoring (albeit incompletely) the capacity to produce 19s antibody are somewhat different from those which are reactive against foreign cells in the stem cell inactivation phenomenon. I n these respects it will be interesting i n ''I.. -. 4- 2102- 8: 6- 4- 210' ; 8: 64- 2- Days after X-irradiation FIG.3. Dynamics of the studied characteristics in the spleen of mice of CBA strain at different interval after irradiation. -. .-, Total number of the nucleated spleen cells; -, number of CFU per lo7 of spleen cells; ----, number of antibody-forming cells per lo6 of spleen cells; -. -,inactivation index of non-syngeneic stem cells (?A). Recovery in the mouse spleen after sublethal irradiatiotz future work to see firstly whether 7s antibody production has the same pattern of restoration as that for 19s antibody and further whether any or all of the immunological recovery phenomena are thymus dependent. Investigation of these points may help to clarify which cells are engaged in the relevant immune responses. The last and perhaps most intriguing point is that concerning the 'allogeneic inhibition' type of effect observed when either C57BI or CBA spleen cells were injected into F, hybrid recipients. Theestimates ofthe repression oftheir activities as haematopoietic colonies seemed to show the same radiation recovery pattern as that of the more obviously immunologically oriented functioiis. The effects can hardly be imputed Lo the host mice which remained constant throughout. It can be supposed that some graft versus host reaction, which is radiation sensitive, i n some way affects the capacity of the injected haematopoietic cells to proliferate. The mechanism of this effect awaits elucidation.

Journal

Cell Proliferation – Wiley

Published: Oct 1, 1968

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