Role of APCs in the activation of T cells induced by LDR
The importance of the interaction between the APCs and lymphocytes in normal immune response is well known [8]. But the role of APCs in radiation-induced immunological changes has not been fully elucidated. Figure 1 illustrates the importance of the interactions between the APCs and lymphocytes in the proliferative response induced by Con A in normal as well as irradiated states. In this figure there are 5 groups of experiments (A to E) and in each group there are 3 columns marked as (1) lymphocytes from sham-irradiated mice, (2) lymphocytes from 0.075Gy-irradiated mice and (3) lymphocytes from 2Gy-irradiated mice, respectively. In the analysis to follow, these will be designated by the group (A, B, C, D or E) followed by a column number (1, 2 or 3) for comparison of the changes. As illustrated in Group A of figure 1, the proliferation of splenocytes in response to Con A after whole-body irradiation (WBI) with 0.075 Gy was significantly up-regulated to a value more than doubling that of the sham-irradiated control (P < 0.002, A1 vs A2), while WBI with 2 Gy caused a profound depression of this reaction to about one third of the control (P < 0.01, A1 vs A3). When splenic cells were depleted of their adherent APCs, the proliferative response to Con A was significantly suppressed (P < 0.001, B1 vs A1). In this case no stimulatory effect was observed for the lymphocytes from the 0.075Gy-irradiated mice (P > 0.05, B1 vs B2; P < 0.001, A2 vs B2), while the suppression of proliferation by 2 Gy was still marked (P < 0.05, B1 vs B3), being more marked than that in the control group (P < 0.001, A3 vs B3). When APCs obtained from the control mice were added to the separated lymphocytes, the proliferation compared to group A was largely recovered for the lymphocytes from the control (P > 0.05, A1 vs C1) and 0.075Gy-irradiated mice (P > 0.05, A1 vs C2), and fully recovered for the lymphocytes from the 2Gy-irradiated mice (P > 0.05, A3 vs C3). But in group C, neither the stimulation from LDR nor the suppression from HDR was significant (P > 0.05, C1 vs C2 and C1 vs C3). When APCs from both the 0.075Gy-irradiated and 2Gy-irradiated mice were added to the separated lymphocytes (groups D and E in figure 1), the proliferative response of lymphocytes from the control mice recovered (P > 0.05, A1 vs Dl and A1 vs E1) and that of lymphocytes from the 0.075 Gy-irradiated mice was greater than the control of each group (P < 0.02, D1 vs D2; P < 0.05, E1 vs E2), and this response was also greater than the normal control (P < 0.05, A1 vs D2 and A1 vs E2). The proliferative response in groups D and E after addition of APCs from the 2Gy-irradiated mice was lower than the control of each group (P < 0.001, D1 vs D3 and El vs E3) and the magnitude of response was about the same as that in group A (P > 0.05, A3 vs D3 and A3 vs E3). A similar phenomenon was observed when secretion of IL-2 was measured as an end point (data not shown) and peritoneal macrophages were used instead of splenic adherent cells as the APCs for both the proliferative response (figure 2 ) and IL-2 secretion (data not shown).
Expression of B7 molecules on APCs in response to ionizing radiation
The time course of the expression of B7-1 (CD80) and B7-2 (CD86) on peritoneal macrophages was studied after WBI with 0.075 Gy and 2 Gy. It can be seen from the left panel of figure 3 that the expression of B7-1 was markedly up-regulated on peritoneal macrophages after WBI with both low and high doses, with the start of up-regulation being earlier after 2 Gy (8h) than after 0.075 Gy (12h), but peaking at 24 h for both doses. The expression of B7-2 on macrophages was also stimulated (right panel), reaching the peak earlier after LDR (8 h) than after HDR (12 h), and the up-regulation sustained for a longer time after LDR (P < 0.05 for the 24 h point in the 0.075 Gy experiment, but P > 0.05 for the 24 h point in the 2 Gy experiment). The dose-response relationship of the changes in the expression of these molecules showed up-regulation following doses up to 4 Gy with a distinct peak at 0.075 Gy followed by a second peak at 1.0 Gy for B7-1 and at 4 Gy for B7-2. These are illustrated in figure 4 and figure 5 in which both the response of B7-1 and B7-2 on mouse peritoneal macrophages to WBI and that of B7-1 and B7-2 on J774A.1 cells to in vitro irradiation are shown. It should be emphasized that the up-regulation of expression of these two molecules on the peritoneal macrophages after WBI with doses of 0.075 and 2 Gy was statistically significant at multiple time points (P < 0.05 or P < 0.01).
Expression of CD28/CTLA-4 on lymphocytes
The CD28 and CTLA-4 molecules were detected on the surface of splenic and thymic lymphocytes by flow cytometry (FCM) with double immunofluorescence after WBI with different doses. It is seen in figure 6 that WBI with 0.075 Gy caused up-regulation of CD28 expression with opposite changes in CTLA-4 expression on splenocytes (panel A) while WBI with 2 Gy suppressed CD28 expression with opposite changes in the expression of CTLA-4 (panel B). Dose-effect relationship of the expression of these two molecules showed opposite direction of the changes both for the splenocytes 4 h after WBI (panel C) and for the thymocytes 72 h after WBI (panel D).
Secretion of IL-12p70 by peritoneal macrophages and synthesis of IL-10 in lymphocytes
As shown in figure 7, the secretion of IL-12p70 as measured by ELISA was stimulated by both low and higher doses (left panel) with an abrupt increase 4 h after WBI with 0.075 Gy followed by its rapid return to the basal level and a sustained increase after WBI with 2 Gy during the observation period of 48 h. The synthesis of IL-10 in the splenocytes detected by FCM, and expressed as a percentage of fluorescence stained positive cells, was significantly suppressed after WBI with 0.075 Gy and markedly up-regulated after WBI with 2 Gy. (right panel).