To address the direct effect of LDR on NK cells in vitro
, NK cells were enriched up to 80% purity using MACS isolation kits prior to irradiation. Since IL-2 is widely used to assess NK cell function in vitro
] and also produced by DCs early after sensing microbial maturation-promoting stimuli or influenza viruses in vivo
], we recapitulated this cytokine milieu in vitro
by stimulating NK cells with a suboptimal dose (100 U/ml) of IL-2 as ‘activation’ regimen (Fig. A). When culturing primary NK cells, the usual loss of 50% after 3 days of culture in low-dose IL-2 at 100 U/ml is not uncommon. Under ideal culture conditions, using high-dose IL-2 (1000 U/ml or more), greater viability is observed than low-dose IL-2, but still cells undergo apoptosis as shown [22
]. This likely resulted from the combination of activation-induced cell death as well as death from IL-2 unresponsiveness. We performed a chromium release assay to demonstrate the cytolytic effect of NK cells activated with varying doses of IL-2 in Fig. B. We chose a dose that was relatively suboptimal in order to be able to detect possible synergy between IL-2 and LDR. Since a high dose of IL-2 is less physiologic and the effect of LDR on high-dose IL-2-induced cytotoxicity was found to be minimal, we did not pursue the remainder of experiments with high-dose IL-2.
Fig. 1. The effect of suboptimal dose of IL-2 on NK killing activity. (A) Purified NK cells cultured with 100, 500 and 1000 U/ml of IL-2 after 6 days were shown as forward and side scatter plots. (B) Cytotoxicity against RMA/S tumor targets by NK cells (more ...)
When purified NK cells were cultivated with 100 U/ml of IL-2 and exposed to LDR simultaneously for 2 days, no significant change in cell numbers was observed (Fig. A). The FSChiSSClo-gated live population was approximately ~52% for the Sham and ~49% for the LDR group after culture in IL-2 for 3 days (Top panels, Fig. B). The fraction of cells undergoing apoptosis/necrosis, as defined by Annexin V/PI staining, was similar between the two groups in the total ungated population (no gate, middle panels). The gated live population also demonstrated no significant difference in the late apoptotic/necrotic cells between the Sham and LDR groups (R1 gate, bottom panels). To further confirm the rate of cell death in these cultures, we analyzed cells for expression of active caspase 3. As shown in Fig. C, the percentage of active caspase 3+ cells was also comparable in the Sham and LDR groups among ungated total (middle panels) or gated live populations (bottom panels). These data demonstrate that LDR did not significantly affect the viability or apoptosis/necrosis processes of purified NK cells in cultures with low dose IL-2.
Fig. 2. The effect of LDR on NK cell viability. (A) Relative cell numbers at the end of culture were depicted as bar graphs. (B) Top, The rates of early and late apoptosis/necrosis of NK cells at the end of culture are shown as Annexin V+ PI– and Annexin (more ...)
The rate of cell proliferation, measured at the end of incubation period using 3H-thymidine incorporation assay, demonstrated significantly elevated levels of DNA synthesis in response to IL-2 in both Sham (19.76 ± 14.52 × 104 dpm) and LDR (14.93 ± 15.38 × 104 dpm) groups (Fig. A). However, LDR did not appear to alter NK cell proliferation since little or no statistical difference was observed between the Sham and LDR group (P > 0.05). Upon IL-2 activation, NK cells develop natural cytotoxicity against tumor targets, such as class I-negative RMA/S T cell lymphoma lines, as seen in Fig. B. Thus, we examined whether LDR could influence NK natural cytotoxicity. Anti-tumor effector function of NK cells, as measured by a standard 51Cr release assay against RMA/S cells, was found to be similar in both the Sham and LDR group (Fig. B, at the E:T ratio of 10:1 and 3:1, P > 0.05). Taken together, these data corroborate the fact that direct exposure of LDR to NK cells was not sufficient to trigger signaling pathways associated with apoptosis, proliferation or cytotoxic function.
Fig. 3. Neither effector function nor proliferation in response to IL-2 was altered in NK cells exposed to LDR. (A) NK cells were subjected to 3H-thymidine incorporation assay to monitor the level of DNA synthesis. The data shown are averaged from three independent (more ...)
The results presented above demonstrate that neither LDR alone nor LDR with suboptimal levels of IL-2 were sufficient to augment NK cell activation in vitro. Since healthy individuals often experience prior exposure to low levels of viral or bacterial infections that lead to cytokine activation and generally rapid resolution through NK cell activity, we next questioned if LDR exposure enhances the function of previously cytokine-activated NK cells. For this, purified NK cells were pre-cultured with 100 U/ml of IL-2 for 3 days prior to LDR exposure and NK cell proliferation was assessed 2 days following LDR (Fig. A). The FSChiSSClo-gated live population was comparable between the Sham and LDR groups. When the rate of cell proliferation was measured at the end of incubation, significant levels of DNA synthesis in activated NK cells was observed in response to IL-2 (Fig. B). However, exposure of LDR on activated NK cells did not alter the level of proliferation in NK cells (17.79 ± 7.59 × 104 dpm in the Sham vs. 18.80 ± 4.42 × 104 dpm in the LDR group, P > 0.05, Fig. B), similar to those observed in cultures simultaneously treated with LDR + IL-2 (Fig. A). In the absence of IL-2 or LDR, NK cells did not undergo proliferation. Strikingly, NK cells activated with IL-2 demonstrated significant enhancement of cytotoxicity, up to 2.1-fold higher (P = 0.014 at 10:1 E:T ratio), against RMA/S tumor targets. At an E:T ratio of 10:1, the percentage of lysis in the Sham group was 8.57 ± 3.40%, while that in the LDR group was 17.94 ± 2.81%. Therefore, LDR appeared to specifically enhance anti-tumor cytotoxic function of NK cells but only when NK cells were activated with IL-2. Since increased cytotoxicity by LDR could be partially attributed to the up-regulation of NK cell surface receptors, we next examined the level of NK activation receptors, CD69, and 2B4, NK1.1, NKG2D by flow cytometry. As can be seen in Fig. D, all NK cells expressed comparable levels of NK1.1, NKG2D, CD69, 2B4, in both the Sham and LDR groups. Therefore, the enhancement of NK cytotoxicity by LDR was not found to be associated with alteration of surface activating receptors. Nevertheless, regulation of NK cytotoxicity by LDR, occurring independently of cell proliferation, apoptosis, or surface receptor expression, might provide insights into the functional benefits of LDR at the cellular level.
Fig. 4. Exposure to LDR significantly enhances anti-tumor cytotoxicity of NK cells when cells were activated by IL-2. (A) Purified NK cells were incubated with 100 U/ml of IL-2 for 3 days prior to the exposure with LDR. Cells were analyzed 1 day (more ...)
While it is well established that exposure to sufficiently high doses of radiation can lead to DNA damage, necrotic and apoptotic cell death, and alteration of immune functions [4
], very little is known regarding the physiological consequences of low-dose or low-dose-rate ionizing radiation. One of the main obstacles to studying the direct effect of LDR comes from the fact that irradiation alone did not generally induce visible changes both at the cellular or whole body levels. Consistent with this, we found no substantial effect of LDR on innate immune NK cells in their viability, apoptosis, proliferation or cytotoxic functions when LDR was given alone or simultaneously with IL-2. To circumvent this hurdle, we attempted to recapitulate in vivo
conditions following minimal pathogen exposure prior to LDR exposure and decided to prime NK cells with low-dose cytokine, i.e. IL-2. In a series of preliminary experiments, we activated NK cells with 100 U/ml of IL-2 from 1 to 3 days in vitro
and found that 3-day activation of NK cells ensured sub-optimal cytokine-mediated NK cell activation. In this setting, we found that significant augmentation of anti-tumor cytotoxicity was observed in IL-2-primed NK cells upon irradiation, compared with those not irradiated. However, the non-NK cell population did not contribute significantly to the killing of RMA/S tumor targets as depletion of NK cells from the culture abrogated any detectable tumor target lysis in both groups (data not shown). Therefore, LDR was capable of synergizing NK cytotoxicity among NK cells previously exposed to cytokines or foreign pathogens. This effect was most profound when cells were activated with the suboptimal, and not maximal [23
], level of IL-2, demonstrating the co-stimulatory feature of LDR in the presence of weak activating signals. Therefore, the synergism seen between LDR and cytokines in NK cells may explain, in part, the functional basis underlying diverse beneficial effects seen in the disease animals chronically exposed to LDR.