Natural Killer Cell Viability After Hyperthermia Alone or Combined with Radiotherapy with or without Cytokines.

BACKGROUND: The effects of hyperthermia and irradiation, alone and in combination, on natural killer (NK) cell viability were investigated in vitro. The roles of interleukin-2 (IL-2) and interferon (IFN) α, ß and γ in rescuing NK cells from hyperthermia and irradiation were studied. MATERIALS AND METHODS: Non-selected NK cells were used as effector cells and K-562 cells as target cells. NK and K-562 cells were treated at 37 to 45°C for 0 to 180 min. The cells were irradiated at room temperature using single doses from 0 to 60 Gy. Recombinant IL-2 at 100 to 450 U/ml and recombinant IFNα, ß and γ at 1,000 U/ml were used for different periods of time. NK cell viability was measured by intracellular adenosine tri-, and diphosphate (ATP, ADP) levels via luminometer, trypan blue exclusion and propidium iodide (PI) staining. Binding capacity of NK effector cells to target K-562 cells was also microscopically assessed. RESULTS: Thermal treatments between 37 and 41°C did not significantly affect the ATP levels of NK cells. Between 41°C and 42°C, ATP levels significantly decreased, whilst there was an insignificant reduction up to 45°C. At 42°C or higher, no recovery was detectable. At 42°C, the ATP level of NK cells rescued by IL-2 were significantly higher than those of controls at 37°C. IFNα, ß and γ had no significant effects. A combination of heating at 42°C and irradiation at 20 Gy significantly reduced the ATP levels (p<0.001) more than heating and irradiation alone. At 42°C, IL-2 abolished the reduction of ATP levels by heating and irradiation. This effect was dependent on heating time and irradiation dose. The ATP/ADP ratio did not significantly change when NK cells were heated for different times at 42°C. Thermal treatment of target K-562 cells at temperatures from 37 to 45°C reduced the number of NK cells binding K-652 cells. CONCLUSION: In vitro, NK cell viability was strongly reduced between 41°C and 42°C. At 42°C, the combination of irradiation and thermal treatment reduced the ATP levels in NK cells. However, IL-2 restored cell viability depending on thermal and radiation doses.

Restoring Natural Killer Cell Cytotoxicity After Hyperthermia Alone or Combined with Radiotherapy.

BACKGROUND: The aim of the present study was to investigate in vitro the effect of hypo- and hyperthermia alone or in combination with irradiation on natural killer cell (NK) cytotoxicity, recovery of this function and the possibility of preventing damage to or enhancing cytotoxicity recovery using interferons (IFNs) α, ß, and γ and interleukin-2 (IL-2). MATERIALS AND METHODS: We used non-selected NK cells and measured their cytotoxicity using the (51)Cr release assay. Temperatures ranging from 31-45°C and thermal treatment times from 0-180 min were assessed. IFNs were applied at concentrations from 0-1,000 IU/ml and IL-2 from 0-450 IU/ml. The range of irradiation dose was from 0-30 Gy. RESULTS: We detected no significant differences in cytotoxicity at temperatures from 31-37°C. The most significant decrease in cytotoxicity was observed between 41 and 42°C (p=0.0010), and heating NK cells at 42°C for 180 min almost completely abolished this function. NK cell cytotoxicity largely recovered during the first 24 h, depending on the heating time. IFN-α, ß, and γ demonstrated no concentration-dependent ability to aid in recovery when used before or after the thermal treatment. In contrast, IL-2 restored cytotoxicity in a concentration- and incubation time-dependent manner and was equally active when used before, during or after heating. NK cells were heated at 42°C for various times and then irradiated with a single dose or first irradiated and then heated; however, no statistically significant differences were observed (p=0.520). An approach of IL-2 treatment followed by radiation and heating was the most effective in restoring NK cytotoxicity (p=0.000). CONCLUSION: NK cell cytotoxicity is impaired in vitro at 42°C and above, with possible partial recovery. IL-2, but not IFNs, was able to restore NK cell cytotoxicity in a concentration-dependent manner. IL-2 can also reverse the damage caused by combined hyperthermia and irradiation.

Post-irradiation viability and cytotoxicity of natural killer cells isolated from human peripheral blood using different methods.

Purpose We compared the pre- and post-irradiation viability and cytotoxicity of human peripheral natural killer cell (NK) populations obtained using different isolation methods. Material and methods Three methods were used to enrich total NK cells from buffy coats: (I) a Ficoll-Paque gradient, plastic adherence and a nylon wool column; (II) a discontinuous Percoll gradient; or (III) the Dynal NK cell isolation kit. Subsequently, CD16(+) and CD56(+) NK cell subsets were collected using (IV) flow cytometry or (V) magnetic-activated cell sorting (MACS) NK cell isolation kits. The yield, viability, purity and cytotoxicity of the NK cell populations were measured using trypan blue exclusion, flow cytometry using propidium iodide and (51)Cr release assays after enrichments as well as viability and cytotoxicity after a single radiation dose. Results The purity of the preparations, as measured by the CD16(+) and CD56(+) cell content, was equally good between methods I-III (p = 0.323), but the content of CD16(+) and CD56(+) cells using these methods was significantly lower than that using methods IV and V (p = 0.005). The viability of the cell population enriched via flow cytometry (85.5%) was significantly lower than that enriched via other methods (99.4-98.0%, p = 0.003). The cytotoxicity of NK cells enriched using methods I-III was significantly higher than that of NK cells enriched using methods IV and V (p = 0.000). In vitro the NK cells did not recover cytotoxic activity following irradiation. In addition, we detected considerable inter-individual variation in yield, cytotoxicity and radiation sensitivity between the NK cells collected from different human donors. Conclusions The selection of the appropriate NK cell enrichment method is very important for NK cell irradiation studies. According to our results, the Dynal and MACS NK isolation kits best retained the killing capacity and the viability of irradiated NK cells.

Effects of Single and Fractionated Irradiation on Natural Killer Cell Populations: Radiobiological Characteristics of Viability and Cytotoxicity In Vitro.

BACKGROUND: Natural killer (NK) cells are important in destroying tumor cells. However, they are damaged by radiation therapy. We studied the effects of single and fractionated irradiation on the viability and cytotoxicity of human non-selected NK cells and sub-groups with cluster of differentiation (CD) CD16(+) and CD56(+) in vitro. Only very few studies dealing with the standard radiobiological parameters for characterizing NK cells exist in the literature. MATERIALS AND METHODS: NK cell populations were isolated from buffy coats using different methods and irradiated with single doses up to 80 Gy and fractionated doses of 10 or 30 Gy with different numbers of applications and at different intervals. The study end-points were viability using propidium iodide (PI), trypan blue and intracellular adenosine triphosphate (ATP) assays, and cytotoxicity using the (51)Cr-release assay. The standard radiobiological parameters α and ß of the linear-quadratic (L-Q) model and the mean inactivation dose D̅ taken as the area under the curve (AUC) were calculated to characterize the radiosensitivity of different NK cell populations. RESULTS: The AUC values of the 51Cr release data in the dose range of 0-40 Gy were as follows: for non-selected NK cells, 23.6-20.9 Gy; for CD16(+) and CD56(+) cells, 14.5-13.2 Gy. The AUC values of ATP, trypan blue and propidium iodide methods equally well described the viability of irradiated NK cells. The α/ß ratio for cytotoxicity and viability data in the L-Q model corresponded to the acutely responding tissues. Splitting a 30-Gy dose into two fractions applied at different intervals caused a significant rise in ATP levels and cytotoxicity. Dividing the total dose into four doses applied at fixed intervals also resulted in significant elevations of ATP content and cytotoxicity of NK cells at 10 Gy. CONCLUSION: According to the L-Q method, irradiated NK cells behaved similarly to acutely responding human tissues with respect to cytotoxicity and viability. The AUC proved very useful for comparing the effects of irradiation on NK cells.