Experimental immunology
Immune mechanism of the retarded growth of tumor nodules in mice exposed to single low-level irradiations with X-rays

Introduction
As we have been led to believe and according to the doctrine underlying the current radiation protection regulations each exposure to ionizing radiation may lead to the induction of cancer [1]. In fact, solid tumours and leukemias have been detected in people acutely exposed to intermediate to high doses1 of radiation during nuclear detonations in Hiroshima and Nagasaki or radiotherapy [3-5]. On the other hand, a number of recent epidemiological studies indicate that cancer incidence and mortality are not elevated among inhabitants of the high- versus low-background radiation areas [6, 7]. Moreover, results of the emerging animal studies have demonstrated that whole-body exposures to low doses of X- or g-rays are associated with the reduced cancer rate and increased latency of spontaneous lymphomas and leukemias in the irradiated subjects [8, 9]. It has been suggested that the anti-neoplastic effects of ionizing radiation may result from stimulation of the tumour surveillance immune mechanisms [10-15] related to the activity of natural killer (NK) lymphocytes, cytotoxic T lymphocytes (CTL), and activated macrophages whose functions are mediated by a variety of cytolytic factors and cytokines [16-27]. In the present investigation we estimated the development of syngeneic pulmonary tumour nodules in mice exposed to single low-level irradiations with X-rays and aimed at the assessment of the possible involvement of NK cells and macrophages in this effect.
Material and methods

Animals and irradiation
Male BALB/c mice aged 6-8 weeks were used throughout. The animals, obtained from the Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland, were whole-body irradiated (WBI) from the HS320 Pantak X-ray generator (230 kV, 20 mA) supplied with the 1-mm Al and Cu filters, at 2.2 Gy/h dose rate to obtain the absorbed doses of 0.1, 0.2 or 1.0 Gy per mouse (the absorbed doses were verified using thermoluminescent dosimeters implanted subcutaneously (s.c.) in the middle abdominal region). Control mice were sham-exposed (generator at the off-mode) in identical conditions. All the studies were carried out by permission of the Local Ethical Committee for Experimentation on Animals at the National Institute of Public Health in Warsaw.
Tumour cells
L1 sarcoma cells were obtained from the Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland. These cells developed spontaneously in the lungs of a BALB/c mouse and have since been propagated in the in vitro culture [28]. YAC-1, a murine lymphoma cell line, was obtained from the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. The cells were grown in a culture medium (CM) composed of the RPMI-1640 medium (Sigma, Poznan, Poland), 10% FBS (GIBCO BRL, Karlsruhe, Germany), 100 U/ml penicillin, 100 µg/ml streptomycin (Polfa, Warsaw, Poland) and 2 mM L-glutamine (Sigma), and stabilized with Na₂CO₃ (Sigma).
Immunogenicity assay
Mice were intraperitoneally (i.p.) injected with 10⁶ L1 sarcoma cells, 10⁶ splenocytes obtained from allogeneic C57Bl/6 mice, or pure CM. Four and seven days later the animals were sacrificed and cells from the mesenteric lymph nodes were collected and pulled. After washing, the cells were resuspended in wells of the 96-well culture plate (Corning, Warsaw, Poland) at 3x105 cells per well and incubated for 24 hours at 37°C in a humidified atmosphere of 95% air and 5% CO₂ with 14.8 KBq of [3H]-thymidine (Polatom, Otwock-Œwierk, Poland). After that, the cells were washed and their radioactivity was measured in the Tri-Carb 2100TR Counter (Canberra-Packard, Warsaw, Poland). For each experimental group three mice were used.
Lung tumour colony assay
The assay utilizing L1 sarcoma cells was used as a mouse model of experimental tumour metastases. To obtain the cells for the assay, 14 days after s.c. transplantation of 10⁶ L1 cells, the developed tumours were removed, minced, and incubated for 30 min. at room temperature with 0.25% trypsin-EDTA (GIBCO BRL) and standard DNase I enzyme solution (Sigma). After that, the cells were washed and resuspended in CM to the final concentration of 10⁶ cells/ml. The lung tumour colony assay was performed as described previously [29]. Briefly, two hours after the irradiation mice were i.v. injected with 0.2 ml of the L1-cell suspension per mouse. Fourteen days later, the animals were sacrificed, their lungs injected with India ink and total numbers of superficial macroscopic colonies per lung were counted using a magnifying glass. Each experimental group consisted of 12 mice.
Preparation of the NK cell suspension
NK cells were purified as previously described [30]. Briefly, single cell-suspensions in CM were prepared from the spleens of both irradiated and sham-irradiated mice and incubated on glass Petri dishes for 40 min. at 37°C in a humidified atmosphere of 95% air and 5% CO₂; in each case the cells were collected and pulled from at least three mice. The non-adherent cells were then collected, washed, and incubated for 30 s at room temperature in the ammonium chloride solution to lyse the erythrocytes. After washing and resuspending in CM the cells were passed through a nylon wool column and the wool-nonadherent cells were used for the NK cell-mediated cytotoxicity assay. Preparation of the macrophage-enriched cell suspension Two days before the collection of cells, mice were i.p. injected with 10% Sephadex G-25 (Pharmacia, Uppsala, Sweden). Peritoneal macrophages were collected on the third day post-irradiation, pulled from at least four mice per each experimental group, resuspended in CM, and incubated on glass Petri dishes for 2 h at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The glass-adherent macrophages were then harvested and resuspended in CM. NK cell-mediated cytotoxicity assay Cytotoxic activity of NK cells was measured on the second day post-irradiation using the standard in vitro ⁵¹Cr-release assay [31]. In brief, 10⁶ YAC-1 target cells suspended in 0.1 ml CM were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂ for one hour with 5.55 MBq of Na₂⁵¹CrO₄ (Polatom). After the incubation, the cells were washed with PBS and 100-µl aliquots containing 10⁴ cells were placed in wells of the microtiter plates (Corning, Warsaw, Poland). The NK-enriched cell populations were then added at the 100:1 effector-to-target (E:T) cell ratio; five samples were performed for each in vitro experimental group. After the four-hour incubation at 37°C in humidified atmosphere of 95% air and 5% CO₂, aliquots of the cell-free supernatants were harvested and radioactivity of ⁵¹Cr released from the target cells was measured in a γ-counter (Auto-Gamma Cobra II gamma counter; Canberra-Packard). The rate of cytotoxic activity was calculated using the formula: 100% x [(experimental release – spontaneous release)/(maximum release – spontaneous release)]; the release of ⁵¹Cr from the target cells cultured in the medium alone was taken as the spontaneous release, while ⁵¹Cr release from the target cells lysed with 1% Trition X 100 (Sigma) was regarded as the maximum release.
Macrophage-mediated cytotoxicity assay
Cytotoxic activity of the macrophages was measured on the third day post-irradiation, as described previously [32], using the L1 sarcoma cells as targets. Briefly, 4x10⁶ L1 cells were suspended in 2.5 ml CM supplemented with 0.3 MBq [3H] thymidine and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂ for 20h. The macrophages were then added at the 20:1 effector-to-target (E:T) cell ratio and the CM was supplemented or not with 50 U/ml IFN-γ (Sigma) and 100 ng/ml LPS (Sigma). After the 48-hour incubation viable adherent cells were lysed, harvested, and their radioactivity was monitored in a b-counter (Tri-Carb 2100TR Counter, Canberra-Packard). The rate of the cytotoxicity was calculated using the formula: [(A – B)/A] x 100%, where A indicates isotope counts taken by target cells when they were cultured alone, and B does those when cultured with effector cells.
Suppression of the NK-cell-mediated activity
To suppress the NK-cell-mediated cytotoxicity in vivo the rabbit anti-asialo GM₁ antibody (GM₁Ab; Wako Chemicals, Neuss, Germany) was used as a classical blocker of the activity of murine NK cells [33, 34]. For this purpose, one day before the irradiation and injection of the L1 cells mice were treated i.p. with GM₁Ab (20 µl Ab in 0.5 ml PBS) or 0.5 ml PBS and two or 14 days later assayed for the activity of NK splenocytes and the number of the developed pulmonary tumour colonies, respectively. For each experimental group four (the NK cell-mediated cytotoxicity assay) and 12 (the tumour lung colonies assay) mice were used.
Suppression of the macrophage-mediated activity
To suppress macrophage functions in vivo carrageenan (CGN; Sigma) was used as a classical blocker of the activity of these cells [35]. Briefly, one day before the irradiation and four days before the collection of macrophages mice were i.p. injected with CGN (4 mg in 0.4 ml PBS per mice) or 0.4 ml PBS. The animals were then assayed for the number of pulmonary tumour colonies and the collected peritoneal cells were assessed for their cytotoxic activity.
Statistical analysis
Mann-Whitney U test for non-parametric trials was used for statistical analysis of the differences between the results obtained for each of the irradiated vs. sham-exposed groups and p values lower than 0.05 were regarded as significant.
Results
Immunogenic characteristics of the syngeneic L1 sarcoma cells and allogeneic C57Bl/6 cells are summarized in Table 1. Based on the study by Ryżewska et al [36], the examined cells can be regarded as immunogenic if the index of stimulation, i.e. the ratio of the activity of [3H]-thymidine incorporated into the mesenteric lymph nodes obtained from mice injected with the cells to the activity of the nodes dissected from mice given only culture medium, exceeds 3.0. Thus, the results shown in Table 1 clearly indicate that L1 sarcoma cells are not immunogenic for the BALB/c mice. Figure 1 shows rates of the pulmonary tumour colonies (expressed as percentages of the control values obtained in the sham-exposed animals) that grown in mice after the single WBI with various doses of X-rays. As indicated in all the four separate experiments irradiation with 0.1 or 0.2 Gy led to the significant inhibition of the development of the colonies. In contrast, in most of the trials, no statistically significant reduction in the number of pulmonary tumour nodules could be detected when mice were pre-exposed to 1.0 Gy X-rays. Figure 2 shows the results of the assessments of the in vitro cytolytic activity of NK lymphocytes obtained from the spleens of mice two days after exposure to 0.1, 0.2, or 1.0 Gy X-rays compared to the activity of NK splenocytes obtained from the control, sham-irradiated mice. As indicated, irradiation with each of the applied doses of X-rays resulted in the significant boosting of the cytotoxic function of the NK-type splenocytes. When mice were injected with GM₁Ab the activity of these cells tested two days later was totally abrogated and this inhibition could not be reversed by WBI with 0.1, 0.2 or 1.0 Gy X-rays. As shown in figure 3, a single WBI of mice with either 0.1 or 0.2 Gy X-rays led to the significant elevation of the cytotoxic activity of the IFN-γ- and LPS-stimulated peritoneal macrophages against the L1 tumour targets on the third day post-exposure to X-rays compared to the activity of these cells obtained from both the sham-irradiated and 1.0 Gy exposed mice. Macrophages collected from mice pre-injected with CGN were significantly less cytotoxic against the L1 cells in vitro than macrophages obtained from the CGN-untreated animals in all the examined groups. Figure 4 shows the relative numbers (expressed as percent of the control values measured in the sham-exposed animals) of the pulmonary tumour colonies developed in mice pre-treated with GM₁Ab or CGN. Injection of the NK cell- or macrophage-blocker almost totally eliminated the differences in the numbers of tumour colonies between the irradiated and control groups. This effect was markedly more pronounced in the CGN- than in the GM₁Ab-treated mice.
Discussion
The results of the present study indicate that development of the pulmonary tumour colonies is significantly retarded in mice pre-injected with L1 sarcoma cells and whole body-irradiated with 0.1 or 0.2 Gy of X-rays as compared to the sham-exposed as well as 1.0 Gy-irradiated mice. This observation corroborates the findings of Hosoi and Sakamoto [11] who detected a marked inhibition of both artificial and spontaneous pulmonary metastases in mice inoculated with tumour cells a few hours before or after the exposure to 0.15, 0.2 and 0.5 Gy X-rays. Likewise, significant reduction in the number of pulmonary tumour nodules was reported by Ju et al [12] who irradiated mice with single doses of X-rays ranging from 0.05 to 0.15 Gy 24 hours before the i.v. injection of B16 melanoma or Lewis lung cancer cells. Decreased incidence of lung and lymph node metastases was also reported by Hashimoto et al [14] who exposed rats to 0.2 Gy of g-rays 14 days after s.c. implantation of hepatoma cells; the same dose, however, did not reduce the number of metastases after local irradiation of the primary tumour nor did it affect the in vitro growth of the tumour cells irradiated in the culture medium. Recently, Sakai et al [37] reported that protracted irradiation of mice with g-rays for over 250 days inhibited the growth of the 20-methylocholantrene-induced tumours. These results collectively suggest that the inhibitory effect of low doses of low-LET radiation on the development of metastases may result from the stimulation of anti-cancer immune mechanisms of the host rather than from the direct reduction of proliferation and/or viability of cancer cells. NK cells, CTL and activated macrophages are primary effectors of the anti-tumour surveillance system [16, 17, 21, 24, 25, 38]. Since the L1 cells used in the present investigation to induce sarcoma colonies in the lungs are not immunogenic for BALB/c mice specific activity of CTL is not likely to be involved in the described inhibition of the tumour growth by 0.1 and 0.2 Gy X-rays. Hence, it was interesting to note in the present study that the activity of NK cells obtained from mice pre-exposed two days earlier to 0.1 or 0.2 Gy of X-rays was significantly elevated compared the counterpart cells collected from the control, sham-irradiated animals. Similar stimulation was also described by Liu et al [21] 2-6 days after the single exposures of mice to 0.075 Gy and 0.5 Gy X-rays. Interestingly, in our study the activity of splenic NK cells was also markedly stimulated by irradiation with 1.0 Gy X-rays, the dose that did not lead to inhibition of the growth of the pulmonary tumour nodules. However, stimulatory effect of this dose of X-rays on the activity of NK splenocytes could be partially explained by the possible elimination of radiosensitive T and B cells from the spleen and hence the relative increase in the percentage of the NK effectors in the cytotoxic assay (data not shown). In fact, as indicated by Lin et al [39] and Harrington et al [40] NK cells exhibit the highest radioresistance among the splenic lymphoid cells. Moreover, in the present investigation peritoneal macrophages collected on the third day post-irradiation of mice with 0.1 or 0.2 Gy X-rays and incubated in the presence of IFN-γ and LPS exhibited significantly elevated cytotoxic activity against the L1 sarcoma cells. Similar stimulation of the cytotoxicity of the IFNg- and LPS-treated peritoneal macrophages derived from mice exposed to 0.04 Gy of g-rays was reported by Ibuki & Goto [18, 19] who used the P815 tumour cells as targets and assayed the effector macrophages already on the day of the exposure. In the present study i.p. injection of both the anti-asialo GM₁ antibody and CGN suppressed the cytolytic function of NK lymphocytes and macrophages, and abrogated the differences between the numbers of the lung tumour colonies developed in mice exposed to 0.1, 0.2 Gy and 1.0 Gy. These results suggest that stimulation of the NK cell- and macrophage-mediated activities was responsible for the retardation of the development of tumour metastases by the low doses of X-rays. Notably, injection of CGN appeared to be a more potent suppressor of the anti-neoplastic effect of the low-level exposures to X-rays than the GM₁Ab. This observation may be explained by the possible suppression by CGN of the cytotoxic functions of both macrophages and NK cells. Indeed, Minarovits et al [41] demonstrated that concurrent application of the two inhibitors promoted tumor growth in mice transplanted with the SP94 adenocarcinoma and BaF1 fibrosarcoma cells to the same extent as did the sole injection of CGN. Moreover, several cytokines produced by macrophages (e.g., IL-12 and IL-18) are potent modulators of the activity of NK lymphocytes [42] and suppression of the activity of the former may compromise the function of the latter cells. In conclusion, our present results indicate that suppression of artificial metastases by single low-level irradiations with X-rays may be causatively related to stimulation by such exposures of the cytotoxic functions of NK cells and macrophages. It remains to be explored in future studies whether other immune cells and/or reactions are also involved in the tumour-suppressory effect of the low-dose irradiations with low-LET radiation.
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