Abstract
Aim: To study the effects of whole-body irradiation (WBI) on lymphocyte proliferation at different times (0, 7, 15, 30, 90 and 180 days) in an animal sensitive to radiation, BALB/c-mice. Materials and Methods: Mice were irradiated (4 Gy) and euthanized at different times, and lymphocytes underwent different treatments: quiescent cells were cultured with calcium ionophore (5 min or 48 h) with/without phorbol myristate acetate (PMA). Lymphocytes were cultured with mitogens and underwent the same treatment. Cell proliferation was measured by a tritiated thymidine assay. Results: The results obtained varied at different time points: at 15 days post-irradiation, quiescent cells and PMA-treated cells showed a significantly decreased proliferation, but increased at 90 days; moreover, when cells were treated with ionophore, a significant stimulation was noted at all times. When cells were exposed to mitogens, they behaved according to its nature: thus, concanavalin A (ConA) and phytohaemaglutinin A (PHA) behaved differently with PMA, while lipopolysaccharide (LPS) had an inhibitory effect at 30 days post-irradiation, and pokeweed (PWM) stimulated proliferation at both 90 and 180 days. Accordingly, there were very few variations in the test results when mitogen concanavalin A (ConA) and calcium ionophore with/without PMA were used. Conclusion: Our model is based on BALB/c mice. Cells induced to proliferate by the PKC enzyme and calcium ionophore are more resistant to irradiation than the same cells treated with specific T- and B-cells mitogen.
In order to study the effect of ionizing radiation on the immune response, we developed a model based on the mouse strain BALB/c (1) since it is more radiosensitive than others, such as C57BL/6 and Kunming (2-4). Previous results have shown that in irradiated animals sacrificed at different times after exposure, immunological alterations occur, which affect lymphocyte subpopulations as well as the lymphoproliferative response induced by mitogens. These alterations tend to normalize with time, but can be observed up to 180 days post-exposure. In accordance with the time required to recover a normal response, it has been concluded that B-lymphocytes are regenerated before T-lymphocytes, and that T-helper lymphocytes regenerate faster than T-cytotoxic ones (1).
Lymphocytes are cells in a quiescent state (G0) which can be stimulated to enter the cell cycle (G1) when antigens/mitogens interact with specific membrane receptors, receptors which can differ according to the lymphocyte subpopulation to which they belong (5). When an antigen/mitogen receptor recognizes its receptor, the hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) occurs through the action of phospholipase C (PLC), generating diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), two second messengers that can activate protein kinase C (PKC) and increase the intracellular calcium concentration [Ca2+]i, respectively (6, 7). DAG generation leads to the activation of the RAS and PKC pathways (6, 8). IP3 opens the Ca2+ channels (IP3R) located in the endoplasmic reticulum membrane, and later it opens the calcium release channels (CRAC) located in the cytoplasmic membrane (7, 9).
Pharmacological agents which mimic the effects of IP3 and DAG can be specifically used to study each branch of bifurcated pathway activation. Ionophore A23187 can be utilized to initiate calcium flow, and phorbol myristate acetate (PMA) activates the PKC enzyme (10). Thus, a former study conducted in BALB/c mice found that the presence of ionophore A23187 over a 5-min period, and the subsequent addition of PMA, gave rise to a proliferative response (4).
The fact that very little information is available on the effects of whole-body irradiation (WBI) on these activation pathways of immune cells, demonstrates a set of disparate observations, which are sometimes contradictory, and have been generally obtained using less radiosensitive strains. Thus, exposure of Kunming mice to X-rays (75 mGy) enhanced the response of lymphocytes to mitogen (ConA) while a high dose (2 Gy) was suppresive (11, 12); additionally, WBI leads to increase in [Ca2+]i and to stimulation of PKC in lymphocytes and thymocytes (13, 14). Thus, exposure of C57BL/6J mice to different WBI doses led to decreased spleen mass, lymphocyte density and high spontaneous DNA synthesis after exposure (15). Exposure of Swiss mice to different WBI doses revealed that PKC is active in vivo on irradiation at a lower dose, and finally, that mitogen-activated protein kinase (MAP) is more active on ex vivo irradiation (16, 17). Moreover, with in vitro radiation, the presence of ionophore A23187 potentiated the modulatory effects of Ca2+ (18).
The purpose of this study was to investigate the short-, mid- and long-term effects of a single dose of ionizing irradiation (4 Gy) in vivo on lymphocytes from irradiated BALB/c mice, which were either mitogen-stimulated or not, with A23187 (5 min or 48 h) and/or PMA. We discuss the possible mechanisms of the observed effects.
Materials and Methods
Animals. Male and female BALB/c mice were used, which were free of pathogens, weighed 22-25 g and were 8 to 12 weeks old. They were bred in the research center and kept in plastic boxes (4 mice per box) at a constant temperature (24±1°C) and relative humidity 75±5%. They were fed on rodent food (Purina Mouse Chow; Valencia, Spain) and were provided water ad libitum.
In vivo whole-body gamma-irradiation procedure. Animals were exposed to gamma radiation, electromagnetic radiation by photons with energy propagation. In vivo irradiation took place in a Sagitarius (25 MV) linear accelerator at the Oncology Service of La Fe Hospital, Valencia. The dose rate was 2.4 Gy/min, with a dose error under 2%. Mice were placed individually in ventilated methacrylate boxes whose structure held the animals in a supine position.
To study the short-, mid- and long-term effects of the Iymphoproliferative response, six groups of mice were given a single-dose of WBI (4 Gy), were killed and the proliferation test was carried out after periods of 0, 7, 15, 30, 90 and 180 days. All the animals were rapidly and humanely euthanized with 100% CO2 in accordance with standard intemational animal care protocols (19). As explained in the first part of this work (1), it is necessary to use three control groups: C1 for the mice studied in the 0, 7, 15 and 30 days period, C2 for 90 days, and C3 for 180 days.
Culture system. The spleens of both the irradiated mice and the control group were removed under sterile conditions, and cell cultivations were prepared using sterile pressure sieves. Cells were washed twice in saline solution and then resuspended in RPMI-1640 (Gibco, Paisley, United Kingdom) containing 5% of fetal calf serum (Gibco), glutamine 2 mM (Flow Laboratories, lrvine, United Kingdom) and 100 μg/ml cefotaxime (Hoechst, Barcelona, Spain) at a density of 2×106 cells/ml.
Aliquots of the cell suspensions were pre-incubated at 37°C for 5 min with 200 nM A23187 (Sigma-Aldrich, Spain), then washed twice in saline solution and re-adjusted to the original cell density. Both the untreated and A23187 pre-incubated cells were distributed at 4×106 cells/well in 96-well flat-bottom microtiter plates and were cultured without mitogen or with different doses of ConA (Sigma-Aldrich), phytohaemaglutinin A (PHA; Sigma-Aldrich), lipopolysaccharide from Escherichia coli (LPS; Difco, Spain) and pokeweed (PWM; Gibco). A23187 was added directly to some of the wells containing untreated cells when culture was begun. In all experiments, parallel cultures were performed in which 10 ng/ml PMA (Sigma-Aldrich) were added.
After seeding, duplicate cultures were incubated at 37°C in a humid atmosphere with 5% CO2 for 48 h. Next, 18.5 kBq of tritiaded thymidine (185 GBq or 5 Ci/mmol, Amersham Biosciences, Spain) were added to each well; thus, each culture received 0.5 mCi. Cells were collected 18 h later by suction through fiberglass filters and the radioactivity in the cells was measured with a liquid scintillation counter.
Study of cell viability. Other than the previously described cultivations, a study of cellular viability was conducted with the irradiated and non-irradiated animals using the cytometric acridine orange method (20).
Interaction index. The effect of A23187 and PMA was studied individually and in combination, as was the interaction between these agents and mitogens. Given two chemicals, A and B, the interaction was calculated according to the following expression: the Interaction Index=[(E A × B) − (E A + E B)/(E A + E B)]×100, where E is the number of counts per minute obtained in each case. Therefore, values close to zero indicate an additive effect, if they are of a positive synergism, and a negative effect if they are antagonism.
Statistical analysis. A comparison of the proliferation tests was made of the results obtained between the irradiated and non-irradiated animals with paired Student's t-test.
Results
Effects of A23187 and/or PMA on quiescent cells. As seen in Table I, the spontaneous DNA synthesis in the non mitogen-stimulated cells was significantly inhibited at 0 and 15 days post-irradiation, but was normalized in the remaining cases.
The splenocytes of irradiated animals were not generally stimulated by PMA. However, they were inhibited by PMA treatment at 15 days post-irradiation and at 30 days, but not significantly. By 90 days post-irradiation, they were strongly stimulated by PMA.
Short pre-treatments with the calcium ionophore A23187 (5 min) did not induce proliferation in the non-stimulated splenocytes of either irradiated mice or their controls. When A23187 was present in the 48-hour culture, it did not induce lymphocyte proliferation in irradiated animals, and only slightly increased DNA synthesis in one out of the three control groups.
Effects of a single in vivo dose of ionizing irradiation (4 Gy) on spontaneous DNA synthesis according to the period of incubation with A23187 (200 nM) and/or PMA (10 ng/ml). Irradiated animals were euthanized 0 (n=10), 15 (n=10), 30 (n=30), 90 (n=8) or 180 (n=6) days post-irradiation. Data represent means±SEM, where n=20, n=8 and n=8 for controls (C1, C2 and C3) of 0-30 days, 90 days and 180 days respectively. Untreated cells were compared vs. A23187+PMA (paired Student's t-test): *p<0.05, **p<0.01, ***p<0.001. ND, Not determined.
When cells were pre-incubated with A23187 for 5 min and PMA was later added to the culture, a slight increase in DNA synthesis was observed in some cases in the splenocytes of both irradiated mice (15, 90 and 180 days post-irradiation) and controls. For this reason, when calcium ionophore and PMA simultaneously remained in the culture from the very beginning, a spectacular increase in cell proliferation in the lymphocytes of both irradiated animals and controls was observed.
To study the type of interaction between A23187 and PMA in irradiated animals and their controls, the method of Papadogiannakis and Johnnsen (21) was followed. The results are provided in Table II. When cells were pre-treated with A23187 (5 min) and then cultured with PMA, the results became variable; in some cases, a slight synergistic effect was noted, but a slight antagonistic effect in others. Moreover, the simultaneous presence of A23187 (48 h) and PMA in culture had a marked synergistic effect on the spleen of both irradiated animals and controls.
Effects of PMA on mitogen-stimulated cells. To study the effects of PMA on DNA synthesis induced by mitogens in the splenocytes of irradiated mice, a series of experiments was carried out. Thus, ConA-induced proliferation was significantly inhibited in the splenocytes of mice irradiated 0, 7, 15 or 30 days earlier (Figure 1). Furthermore in the control animals (C1, C2 and C3), PMA increased ConA-induced DNA synthesis. Similarly, PMA increased ConA-induced proliferation in the splenocytes of irradiated mice. This increase was significant in some cases (30 and 180 days post-irradiation) for both mitogen doses. It is noteworthy that at 15 days post-irradiation PMA did not increase ConA-induced synthesis.
PHA-induced proliferation (Figure 2) was significantly inhibited at 0 and 15 days post-irradiation. Hence, the effects of PMA on PHA-stimulated cells were more homogeneous than those on the ConA-stimulated cells because PMA significantly stimulated PHA-induced DNA synthesis in both the control and the irradiated animals, irrespectively of the mitogen dose and the post-irradiation time employed in the study.
Index values for interaction between ionizing radiadion (IR) treatment with A23187 and phorbol myristate acetate (PMA).
Figure 3 shows that LPS-induced proliferation was immediately inhibited after irradiation and that it was augmented 15 days later. In the control animals (C1, C2 and C3), PMA increased LPS-induced DNA synthesis, although this increase was not significant at certain times. The effect of PMA on the splenocytes of irradiated animals was variable: at times it inhibited proliferation (30 days post-irradiation), while not affected at other times (15 and 180 days post-irradiation), and it stimulated proliferation at other times (0 and 90 days post-irradiation).
Figure 4 depicts how PWM-induced proliferation was inhibited immediately after irradiation. PMA significantly augmented PWM-induced DNA synthesis in the splenocytes of the control animals. However, PMA significantly stimulated only the proliferation of the splenocytes of mice irradiated in certain cases (0, 90 and 180 days post-irradiation).
To study the type of interaction between the various mitogens and PMA in irradiated animals and their controls, the Interaction Index was calculated (Table III). The results show that the interaction between ConA and PMA was synergistic in some cases, but in others, it was additive in the cells of both irradiated animals and their controls. Thus, the interaction between PHA and PMA was synergistic in all the cases studied. Moreover, the simultaneous presence of LPS and PMA in the culture had a slightly synergistic effect on the cells of controls, although the effect was variable on the splenocytes of irradiated mice; a slightly synergistic effect was noted in some cases, whereas an antagonistic effect was seen in others. Finally, the interaction between PMW and PMA was, in general, synergistic (control and irradiated), although two cases of a discrete antagonistic effect were observed in the cultures corresponding to irradiated mice.
Lymphoproliferative response induced by 3 μg/ml of concanavalin A in control groups (C) and irradiated mice in respect to the time post-irradiation (IR) and the absence (−) or presence (+) of phorbol myristate acetate (PMA) (similar results were obtained with 1 μg/ml). Arithmetic mean±standard deviation are shown. The number of animals euthanized was 30, 15 and 12 for the control groups C1, C2 and C3 respectively, and 26, 10, 27, 15, 20 and 12 for the irradiated groups at 0, 7, 15, 30, 90 and 180 days. Statistical significance was obtained by paired Student's t-test: *p<0.05, **p<0.01, ***p<0.001.
Lymphoproliferative response induced by 10 μg/ml of phytohaemaglutinin A in control groups (C) and irradiated mice in respect to the time post-irradiation (IR) and the absence (−) or presence (+) of phorbol myristate acetate (PMA) (similar results were obtained with 5 μg/ml). Arithmetic mean±standard deviation are shown. The number of animals euthanized was 30, 15 and 12 for the control groups C1, C2 and C3 respectively, and 26, 10, 27, 15, 20 and 12 for the irradiated groups at 0, 7, 15, 30, 90 and 180 days. Statistical significance was obtained by paired Student's t-test: *p<0.05, **p<0.01, ***p<0.001.
Lymphoproliferative response induced by 20 μg/ml of lipopolysaccharide in control groups (C) and irradiated mice in respect to the time post-irradiation (IR) and absence (−) or presence (+) of phorbol myristate acetate (PMA) (similar results were obtained with 40 μg/ml). Arithmetic mean±standard deviation are shown. The number of animals euthanized was 17, 15 and 10 for the control groups C1, C2 and C3 respectively, and 26, 27, 15, 17 and 12 for the irradiated groups at 0, 15, 30, 90 and 180 days. Statistical significance was obtained by paired Student's t-test: *p<0.05, **p<0.01, ***p<0.001.
Lymphoproliferative response induced by 2% of pokeweed in control groups (C) and irradiated mice in respect to the time post-irradiation (IR) and the absence (−) or presence (+) of phorbol myristate acetate (PMA) (similar results were obtained with 1%). Arithmetic mean±standard deviation are shown. The number of animals euthanized was 17, 15 and 10 for the control groups C1, C2 and C3 respectively, and 26, 27, 15, 17 and 12 for the irradiated groups at 0,15, 30, 90 and 180 days. Statistical significance was obtained by paired Student's t-test: *p<0.05, **p<0.01, ***p<0.001.
Effects of A23187 on mitogen-stimulated cells. The objective of this experiment was to study the effects of calcium ionophore A23187 on ConA-induced DNA synthesis in the splenocytes of irradiated mice. As shown in Figure 5, ConA-induced proliferation was inhibited at 0, 15 and 90 days post-irradiation. Thus, a 5-min pre-treatment with A23187 did not affect ConA-induced proliferation in the splenocytes of either irradiated animals or their controls. However, a 48-h treatment with A23187 drastically and significantly lowered ConA-induced DNA synthesis in the lymphocytes of the control and irradiated mice, except for immediate post-irradiation, which had no effect at all. Table IV shows the Interaction Index, where the 5-min pre-treatment with A23187 and the subsequent ConA action had a variable effect on induced DNA synthesis which was, in general, synergistic, but sometimes antagonistic. In contrast, the simultaneous presence of A23187 (48 h) and ConA in the culture had a marked antagonistic effect on the proliferation of splenocytes in all the studied cases.
Lymphoproliferative response induced by 3 μg/ml of concanavalin A in control groups (C) and irradiated mice in respect to the time post irradiation (IR) and the absence (−) or presence (+) of A23187 (similar results were obtained with 1 μg/ml). Arithmetic mean±standard deviation are shown. The number of animals euthanized was 16, 8 and 6 for the control groups C1, C2 and C3 respectively, and 10, 10, 8 and 6 for the irradiated groups to 0, 15, 90 and 180 days. Statistical significance was obtained by paired Student's t-test: *p<0.05, **p<0.01, ***p<0.001.
Index values for interaction between different mitogens and phorbol myristate acetate (PMA). ND, Not determined.
Overall effect of A23187 and PMA on mitogen-stimulated cells. As seen in Figure 6, ConA-induced proliferation was inhibited at 0 and 15 days post-irradiation. In addition, the effects of A23187 on ConA-induced DNA synthesis were the same as those discussed in the previous section. Therefore, PMA increased ConA-induced proliferation in the splenocytes of non-irradiated animals, and this increase was significant for both the mitogen doses and the different control groups. Likewise in the lymphocytes of the irradiated animals, PMA also enhanced ConA-induced DNA synthesis, although significant only in certain cases (15 and 180 days post-irradiation for ConA 1 μg/ml, and 15, 90 and 180 days post-irradiation for ConA 3 μg/ml). For this reason, the A23187 pre-treatment of cells (5 min) and the subsequent culture with ConA and PMA brought about a slight increase if compared with the proliferation induced by ConA or by ConA and PMA. This increase was significant only in some cases and varied in the control and irradiated animals with variations depending on the ConA dose used.
Lymphoproliferative response induced by 3 μg/ml of concanavalin A in control groups (C) and irradiated mice in respect to the time post-irradiation (IR) and the absence (−) or presence (+) of A23187/phorbol myristate acetate (PMA) (similar results were obtained with 1 μg/ml). Arithmetic mean±standard deviation are shown. The number of animals euthanized was 16, 8 and 16 for the control groups C1, C2 and C3 respectively, and 10, 10, 8 and 6 for the irradiated groups at 0, 15, 90 and 180 days. Statistical significance was obtained by paired Student's t-paired: *p<0.05, **p<0.01, ***p<0.001.
Index values of interaction between the mitogen concanavalin A (ConA) and the two treatments with A23185.
Index values of interaction between the mitogen concanavalin A (ConA) and the combined treatment with A23187 and phorbol myristate acetate (PMA).
The presence of A23187 (48 h) and PMA in the cultures stimulated with ConA had a strong inhibitory effect on cell proliferation in the splenocytes of both the control and irradiated animals. The study of the interaction between the mitogen and the joint A23187 and PMA treatment (Table V) reveals that a 5-min pre-treatment with A23187 and the subsequent culturing with ConA and PMA had a synergistic effect on lymphocyte DNA synthesis for both the mitogen doses and in all the cases studied, except for 90 days post-irradiation. Thus, the simultaneous presence of A23187 (48 h) and PMA in the cultures stimulated with ConA had a strong antagonistic effect on proliferation in the splenocytes of both irradiated mice and controls.
Discusion
The results obtained in the first part of this work (1) showed that: a) the spleens of irradiated mice are much smaller than those of controls, with a minimum value at 7 days which gradually rises to 180 days; b) lymphocyte subpopulations are highly modified by radiation, and B-cells are practically non-existent at 7 days, whereas they are the majority at 15 and 30 days, and reach normal levels at 90 days; c) spontaneous proliferation and B-cell mitogen-induced proliferation (LPS and PWM) show a characteristic pattern, with a peak at 15 days post-irradiation, which differs from the T-lymphocytes that peak at 90 days post-WBI. Therefore, our results and those of other authors (22-24) indicate that the immune system takes much longer to return to normal, as long as 12 months for BALB/c mice (22). Furthermore, studies in humans who have survived nuclear accidents reveal that the reduction of T-cells (CD4 and CD8) and increase of memory T-cells are dose-dependent (25), and that the same may occure within an a immunosenescence process of T-cells (26) for a much longer period after exposure.
In this article, we studied the effect of radiation on BALB/c mice treated with PMA with/without A23187 on lymphocyte proliferation: a) PMA; it was shown that PMA significantly decreases spontaneous DNA synthesis at 15 days post-irradiation, as it also does at 30 days post-WBI, but not significantly. In addition, the PMA-induced increase noted at 90 days post-WBI is very significant; indeed a 180-day period must pass to normalize this increase; b) the short-(5 min) or long-term (48 h) A231887 treatment does not induce proliferation; and c) the treatment with A231887 (48 h) and PMA has a mitogenic and synergistic effect, and also an immediate post-irradiation effect, and that these data are similar to, or even superior than, mitogen-induced proliferation (1). It has also been shown that short pre-treatments (5 min) with A231887 and a subsequent culture with PMA also induce a significant increase in the proliferation of splenocytes from BALB/c mice, although it is less intense than the one obtained, when both drugs were present throughout the culture (4).
These results show that irradiation-induced alterations in lymphocyte subpopulations are far from normal. Moreover, it is possible that subpopulations display particular sensitivity to PMA. Thus, when B-cells appear in small quantities and duplicate spontaneously, we can consider that the PKC-mediated signaling pathway is partially closed, or perhaps that B-cells have become refractory to PMA because they would not need it given their duplication. However, when T-cells divide spontaneously, they are particularly sensitive to the PMA pathway, therefore, the activation of PKC occurs. Thus, the effect of PMA on irradiated quiescent cells can be explained if an increase in PKC expression occurs in murine splenocytes. Another group has shown that different kinases behave distinctly, depending on the radiation being applied in vivo or ex vivo (16, 17). Thus, when mice undergo WBI, they were sacrificed at variable time periods to obtain lymphocytes and/or thymocytes. We can observe: changes in the performance of the PMA dose (11); increased PKC activity as a result of the increased calcium ion (13, 14); changes in the protein membranes associated with the G0/G1 cell cycle, and increasing levels of radiation required to induce apoptosis (27). In addition, PMA is known to induce the expression of certain genes that have very different effects on irradiated cells, with variable effects on apoptosis mechanisms and changes in cytokine-mediated regulation (28-30).
Regarding calcium ionophore, short pre-treatments (5 min) with A231887 and a subsequent culture with PMA induce significant proliferation, especially in the later irradiation phase, at 90 and 180 days. Thus, lymphocytes are partly recovered, as is the percentage of subpopulations, during this time period (1). Therefore, the presence of Ca2+ is required for proliferation events to finish (6). Moreover, if calcium ionophore and PMA are present throughout the culture, a much more marked mitogenic effect is observed, as is a mitogenic effect of the T-cells (ConA and PHA), which is maintained at 90 and 180 days post-irradiation. These results agree with those for atomic bomb survivors (23, 31).
Moreover in irradiated animals whose cells are stimulated with mitogens and treated with A23187 and/or PMA, we can see: a) the effects of PMA, depending on the time elapsed since irradiation, mitogen, and its dose. Thus, PMA increases the proliferative response induced by ConA, PHA, PWM and LPS, sometimes it is significant, but other times it is not. However LPS has an inhibitory effect at 30 days post-irradiation; b) the effect of PMA and A23187 is markedly antagonistic upon ConA-induced proliferation.
The results obtained in the cells stimulated with mitogens and PMA lead us to believe that the responses activated by ConA and PHA can be explained by considering that these mitogens activate different cell populations. The studies by Liu et al. (11) show that after WBI (75 mGy), thymic and splenic cells undergo stimulation when treated with the mitogens ConA and PMA, which also stimulate the activation of PKC in lymphocytes, with different activation peaks seen, depending on the T- or B-cells (13). We must consider that by 15 days post-irradiation, significant spontaneous proliferation occurs and LPS-induced DNA synthesis is significantly enhanced, while the percentage of B-cells is higher than normal. Therefore, it is conceivable that ionizing irradiation can alter the PKC pathway in the mid-term.
Thus, the responses to T-cell mitogens are inhibited, if compared to their controls, by 75-88%; responses to LPS and PWM are inhibited by 92-97%, while those induced by A23187 (48 h) and PMA are inhibited by only 50%. This suggests that ionizing irradiation in vivo immediately causes alterations in the binding of mitogens to their membrane receptors and/or to the transmission of the mitogenic signal.
Furthermore, it has been demonstrated that short pre-treatments (5 min) with A23187 and the subsequent culture with PMA also bring about a significant increase in the proliferation of murine splenocytes, as in non-irradiated mice, but to a lesser extent than that obtained when both drugs are present throughout the culture (4).
Our results have been obtained in BALB/c mice, one of the most radiosensitive strains, especially to radiation-induced apoptosis in the spleen (32). This phenomenon has also been found in healthy humans who exhibit differences in sensitivity to radiation (33). Moreover, this suggests that splenocytes from previously irradiated BALB/c mice respond to treatment with A23187 and/or PMA more actively than their controls. Therefore, the effects of ionizing radiation in vivo are more intense and persistent than those described in vitro.
Acknowledgements
The Authors thank Dr. A González-Molina and the entire staff of Experimental Immunology Unit (Hospital La Fe, Valencia, Spain). This work was supported in part by a grant P.LE. 04428.
- Received September 17, 2012.
- Revision received October 22, 2012.
- Accepted October 23, 2012.
- Copyright © 2013 The Author(s). Published by the International Institute of Anticancer Research.
