Radiation-induced Bystander Effect in Immune Response 1

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1 BIOMEDICAL AND ENVIRONMENTAL SCIENCES 17, (2004) Radiation-induced Bystander Effect in Immune Response 1 SHU-ZHENG LIU 2, SHUN-ZI JIN, AND XIAO-DONG LIU Department of Radiation Biology, Jilin University School of Public Health, 8 Xinmin Street, Changchun , Jilin, China Objective Since most reports on bystander effect have been only concerned with radiation-induced damage, the present paper aimed at disclosing whether low dose radiation could induce a stimulatory or beneficial bystander effect. Methods A co-culture system containing irradiated antigen presenting cells (J774A.1) and unirradiated T lymphocytes (EL-4) was established to observe the effect of J774A.1 cells exposed to both low and high doses of X-rays on the unirradiated EL-4 cells. Incorporation of 3 H-TdR was used to assess the proliferation of the EL-4 cells, expression of CD80/86 and CD48 on J774A.1 cells was measured with immunohistochemistry and flow cytometry, respectively. NO release from J774A.1 cells was estimated with nitrate reduction method. Results Low dose-irradiated J774A.1 cells could stimulate the proliferation of the unirradiated EL-4 cells while the high dose-irradiated J774A.1 cells exerted an inhibitory effect on the proliferation of the unirradiated EL-4 cells. Preliminary mechanistic studies illustrated that the differential changes in CD48 expression and NO production by the irradiated J774A.1 cells after high and low dose radiation might be important factors underlying the differential bystander effect elicited by different doses of radiation. Conclusion Stimulatory bystander effect can be induced in immune cells by low dose radiation. Key words: Bystander effect; Radiation; Antigen presenting cells; T lymphocytes; CD48; NO INTRODUCTION Bystander effect was first noticed with radiation-induced genetic alterations evoked by α particles traversing the cytoplasm of the cell without hitting the nucleus and observations were later extended to cellular interactions in different model systems [1-5]. It is now agreed that both high and low LET radiation can produce significant effects on cells which are not directly hit by the radiation. Several methods have been used to explore the effect including very low fluence of alpha particles, microbeam technology and simple medium transfer. Now the phenomenon is widely accepted, but very little is known about the exact mechanisms involved. Several experimental studies performed in the past decade have suggested the occurrence of low-dose-specific phenomena such as the bystander effect (viewed as damage induction in cells not directly hit by radiation) and adaptive response (induction of resistance to subsequent irradiation with higher doses). Cellular communication has been widely regarded as a key factor in the mechanism of induction of such effects. 1 This work was supported by grants from NSFC (No , No ). 2 Correspondence should be addressed to Shu-Zheng LIU. Biographical note of the first author: Shu-Zheng LIU, male, born in 1925, Professor of Radiation Biology, Chairman of Steering Committee of Key Laboratory of Radiobiolgy, Ministry of Health, PRC & Honorary Chairman of Department of Radiation Biology, Jilin University. Research field: biological effects of low level radiation /2004 CN Copyright 2004 by China CDC

2 RADIATION-INDUCED BYSTANDER EFFECT IN IMMUNE RESPONSE 41 Bystander effect and adaptive response may have a non-negligible role in modulating low dose radiation effects not only in cells, but also in tissues and organs [6]. Previous observations reported were mostly concerned with radiation-induced damage. To the knowledge of the present authors little has been documented on stimulatory or beneficial responses possibly evoked via bystander effect, especially when the immune system is concerned. Here we report the observations of interactions between antigen presenting cells (APCs) and T lymphocytes (TLCs) in a co-culture system to demonstrate a bystander effect from the irradiated APCs on the unirradiated TLCs and the differential effects with low versus higher doses. Cells MATERIALS AND METHODS J774A.1 cells (mouse macrophage cell line) and EL-4 cells (mouse T lymphocyte cell line) from ATCC were used as the APCs and TLCs, respectively. The cells were cultured in DMEM (Gibco BRL) containing 10% NBS, 50 μg/ml kanamycin and 8 μg/ml tylosin. Cells were incubated at 37 under humidified air with 5% CO 2. Irradiation J774A.1 cells were irradiated with X-rays from a deep therapy apparatus (Model X.S.S.205 FZ) with 200 kv/10 ma and filters of 1 mm Cu/0.5 mm Al. Two doses were used, i.e., Gy and 2.0 Gy. The dose rate for the former was 12.5 mgy/min and that for the latter was 287 mgy/min. Co-culture of J774A.1 and EL-4 Cells The irradiated J774A.1 cells ( cells) and unirradiated EL-4 cells ( cells) were co-cultured for 12 h after 2 Gy or 24 h after Gy irradiation of the J774A.1 cells in DMEM medium at 37 under humidified air with 5% CO 2 for 1, 3, 6, 12 and 24 h, respectively, before separation of the EL-4 cells from the J774A.1 cells. Co-culture of unirradiated J774A.1 and EL-4 cells was set up as control. Measurement of Proliferation of EL-4 Cells The concentration of EL-4 cells separated from the J774A.1 cells was adjusted to cells/ml and 180 μl was dispensed to each well of a 96-well microplate in triplicate followed by addition of 18.5 kbq 3 H-TdR in 20 μl. The cells were collected on glass fiber filter disks with a cell harvester after incubation for 6 h. The radioactivity was measured with an LKB 1214 scintillation counter (Sweden). The results were expressed as cpm/ cells. Detection of Expression of Surface Molecules Expression of CD80 and CD86 on J774A.1 cells was assessed with immunohistochemistry as previously reported [7]. Flow cytometry with direct immunofluorescence using PE-antimouse CD48 (Pharmingin, USA) was used for detecting CD48 molecules with FACScan (Becton-Dickinson, Mountain View, CA) using FACS software in collection of cells (10 4 cells for each sample) and LYSIS software for data analysis.

3 42 LIU, JIN, AND LIU Measurement of Nitric Oxide Production by J774A.1 Cells Supernatants of irradiated J774A.1 cells were collected after incubation for 24 h and stored at -70 before estimation of NO according to reference [8]. Statistics Student t test was used for statistical analysis. RESULTS It can be seen from Fig. 1 that the effect of J774A.1 cells exposed to low (0.075 Gy) versus high (2.0 Gy) doses on the unirradiated EL-4 cells after co-culture for different time intervals showed entirely different results. The APCs exposed to low dose radiation exerted a stimulatory effect on the co-cultured TLCs as shown by the increased proliferation, while the APCs exposed to a higher dose resulted in an opposite effect. Both the stimulatory and suppressive effects of the irradiated J774A.1 cells on the unirradiated EL-4 cells became significant beginning from the 3rd hour after interaction of the 2 types of cells and persisted for at least 24 h. FIG. 1. Opposite effects of irradiated J774A.1 cells exposed to low and high doses of radiation on proliferation of co-cultured EL-4 cells. Both high and low doses of X-rays exerted stimulatory effect on CD80 expression, but with different time courses. In other words, high dose caused an early significant up-regulation followed by rapid return to its original level (slightly, but not significantly, below control 24 h after irradiation, see the open circles of Fig. 2), and low dose radiation elicited an up-regulation only 24 h after irradiation (see closed circles of Fig. 2). For CD86, low dose radiation caused stimulation of its expression from 8 to 24 h, while high dose exerted its stimulatory effect only at 12 to 24 h (Fig. 3). The expression of CD48 reacted differentially to low and high doses of radiation. As shown in Fig. 4, low dose radiation caused a prompt increase in expression of this molecule followed by its decreased expression, while a sustained decrease in its expression was observed after high dose radiation remaining significantly at a level lower than at the control one at the end of the 24 h-observation period.

4 RADIATION-INDUCED BYSTANDER EFFECT IN IMMUNE RESPONSE 43 FIG. 2. Effect of irradiation with low versus high dose X-rays on CD80 expression of J774A.1 cells. FIG. 3. Effect of irradiation with low versus high dose X-rays on CD86 expression of J774A.1 cells. FIG. 4. Effect of irradiation with low versus high dose X-rays on CD48 expression on J774A.1 cells.

5 44 LIU, JIN, AND LIU As an active humoral factor produced by macrophages in response to various stress stimuli, NO production was measured in 24 h after different doses of radiation. Fig. 5 shows the dose-response curve of the amount of NO released into the supernatant of the J774A.1 cells in 24 h after in vitro irradiation. It can be seen from Fig. 5 that NO production by J774A.1 cells was significantly stimulated after 1 and 2 Gy X-rays, but in the low dose region, including Gy, its secretion was only slightly, but not significantly, increased. FIG. 5. Dose-effect relationship of NO production by J774A.1 cells in 24 h after X-irradiation. DISCUSSION It is demonstrated for the first time in the present paper that low dose-irradiated J774A.1 cells exerted a stimulatory effect on the proliferation of the co-cultured unirradiated EL-4 cells beginning from the 3rd hour of interaction and high dose-irradiated J774A.1 cells showed an opposite effect (Fig. 1). The mechanism of such a bystander effect remains to be elucidated. As important co-stimulatory molecules on APCs, CD80 (B7-1) and CD86 (B7-2) interact with CD28/CTLA-4 on TLCs, determining the outcome of immune reactions. About 20%-30% of resting TLCs in mouse spleen express CD28 and only 1%-3% of them express CTLA-4 [9]. Therefore, increased expression of CD80/86 would augment the interaction between these molecules with CD28 excluding the inhibitory action of CTLA-4 with the result of enhanced TLC proliferation. Now the expression of CD80/86 was up-regulated after exposure of the J774A.1 cells to both low and high doses of radiation (Figs. 2 and 3), so the stimulatory effect of low dose-irradiated J774A.1 cells on the proliferation of unirradiated EL-4 cells and the inhibitory effect of high dose-irradiated J774A.1 cells on the proliferation of unirradiated EL-4 cells could not be explained by the changes in CD80/86 expression. CD48 on mouse APCs (CD58 for human cells) and CD2 on mouse TLCs are another pair of co-stimulatory molecules in the immunologic synapse [10]. The increased expression of CD48 on J774A.1 cells after low dose irradiation and its decreased expression after high dose irradiation (Fig. 4) might be one of the mechanisms of the differential effect in the co-culture experiment with low versus high dose-irradiated J774A.1 cells exerting stimulatory and inhibitory effects, respectively, on the proliferation of unirradiated EL-4

6 RADIATION-INDUCED BYSTANDER EFFECT IN IMMUNE RESPONSE 45 cells. There have been experiments designed to determine whether signaling pathways arising in the cell membrane might mediate the bystander effect [11]. Cells were irradiated in the presence of Filipin, an agent that disrupts lipid rafts, effectively inhibiting membrane signaling, and the induction of sister chromatid exchange and HPRT mutations by very low fluences of alpha particles (mean doses cgy) were measured. Filipin completely suppressed the induction of both genetic effects in bystander cells. These results suggested that membrane signaling might play an important role in the bystander effect of radiation. In our experiment with low LET radiation the suppressed expression of CD48 after high dose radiation might not be related to membrane disruption, since the expression of other surface molecules, such as CD80/86, was even up-regulated after a dose as high as 2 Gy. The increased and decreased interactions of CD48 of the APCs with CD2 of the TLCs after low and high doses of radiation might be one of the factors influencing the nature of the bystander effect. Another possible mechanism is the differential changes in NO production by the cultured J774A.1 cells after low versus high dose of radiation as shown by the dose-response curve after different doses of X-rays in the range of Gy (Fig. 5). We found that exposure to 1-2 Gy X-rays caused marked increase in NO production by these cells while lower doses had no significant effect. An understanding of radiation-induced bystander effects is emerging that places them in the broader context of the interaction of cells with their microenvironment [12,13]. From this perspective, radiation-induced bystander effects are part of a coordinated multicellular response in irradiated tissue with the objective of modulating cellular programs. As proposed by Barcellos-Hoff and Brooks, this is a paracrine phenomenon whereby hit cells release factors into the extracellular medium that modify the local environment of neighboring cells and influence their behaviors. Endogenous NO is the product of nitric oxide synthases, which catalyzes a five-electron oxidation of L-arginine (with the aid of NADPH and tetrahydrobiopterin) to yield L-citrulline and NO. Our previous work showed that whole-body X-irradiation of Kunming mice caused a significant increase of NO production by the peritoneal macrophages after exposure to 2 and 4 Gy, but not after exposure to doses of 1 Gy or lower and inos in the peritoneal macrophages as estimated by immunohistochemistry showed a similar doseresponse relationship [14]. However, the dose-response relationship of other humoral factors, including IL-12, IL-1β and TNFα, produced by peritoneal macrophages after whole-body X-irradiation is quite different in that exposure of both high and low doses of radiation resulted in increased secretion of these cytokines by the macrophages. Therefore, the changes in secretion of these factors following irradiation may not participate in the differential changes evoked by low versus high doses of radiation on the bystander effect of APCs on TLCs [9,15,16]. It was reported that reactive nitrogen oxides could inhibit a variety of enzymes, initiate lipid peroxidation, and directly damage DNA. Moreover, certain thiol groups on the surface of endothelial cells and/or polymorphonuclear neutrophils are thought to be required for normal leukocyte-endothelial cell adhesion, and the reaction of dinitrogen trioxide with these thiol groups can form S-nitrosothiol adducts that may inhibit the adhesion process and consequently decrease leukocyte infiltration during the resolution phase of the inflammatory response. It is currently not known if such changes also affect the interaction between co-stimulatory molecules of APCs and TLCs. The full understanding of the mechanisms underlying the differential bystander effect in immune cells induced by low versus high dose radiation deserves further study.

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