Interferon-Beta Combined with Interleukin-2 Restores Human Natural Cytotoxicity Impaired In Vitro by Ionizing Radiations

Abstract

It is well known that ionizing radiations induce a marked downregulation of antigen-dependent and natural immunity for a prolonged period of time. This is due, at least in part, to radiation-induced apoptosis of different lymphocyte subpopulations, including natural killer (NK) cells. Aim of this study was to investigate the capability of Beta Interferon (β-IFN) and Interleukin-2 (IL2), alone or in combination, to restore the functional activity of the natural immune system. Mononuclear cells (MNCs) obtained from intact or in vitro irradiated human peripheral blood were treated in vitro with β-IFN immediately before or at the end of the 4-day treatment with IL2. Time-course analysis was performed on the NK activity, the total number and the apoptotic fraction of CD16+ and CD56+ cells, the 2 main NK effector cell subpopulations. The results indicate that radiation-induced impairment of natural cytotoxicity of MNC could be successfully antagonized by the β-IFN+IL2 combination, mainly when exposure to β-IFN preceded IL2 treatment. This radioprotective effect is paralleled by lower levels of radiation-induced apoptosis and increased expression of the antiapoptotic Bcl-2 protein. Since natural immunity can play a significant role in antitumor host's resistance, these results could provide the rational basis for a cytokine-based pharmacological strategy able to restore immune responsiveness and to afford possible therapeutic benefits in cancer patients undergoing radiotherapy.

Introduction

R adiotherapy plays an important role in cancer treatment although its application is limited by acute and delayed side effects on normal cells (Zaider and Hanin 2011). A wide range of susceptibility of normal tissues to ionizing radiations (IR), has been detected although the mechanisms underlying the variable responses to IR remain unclear and comprise both endogenous and exogenous factors, including hereditary factors (Broval and Schacter 1981; Liu 2010; Shiao and Coussens 2010; Mayer and others 2011).

Different processes appear to be involved in the cellular response to IR, for example, generation and scavenging of free radicals, DNA duplication and repair, apoptosis, and inflammation (Sharma and others 2010; Thoms and Bristow 2010). In particular, repair of DNA damage after IR is believed to be a major protective mechanism since nuclear DNA is the most susceptible target in living cells (Wu and others 2011).

Two main compartments of the immune system, that is, antigen-dependent (adaptive) and antigen-independent (innate or natural) immunity are endowed with different, but frequently integrated functions in host's defenses against foreign invaders or malignant cells. Natural Killer (NK) cells play a major role in innate immunity by killing target cells directly or indirectly by releasing a variety of cytokines and chemokines. NK cells lyse a wide range of tumor cell types without presensitization and restriction by the major histocompatibility complex, do not express T-cell antigen receptors, CD3 T cell markers, or surface immunoglobulin B cell receptors. They are phenotypically characterized as CD56+ (a surface antigen present at low or at high levels) and CD16+ (FcγRIII receptor), and constitutively express p75 chain of the Interleukin-2 (IL2) receptor (De Maria and others 2011; Vivier and others 2011). The incubation of peripheral blood lymphocytes with IL2 induces rapidly a selective proliferation and increase of NK activity (Dunne and others 2001) followed by generation and proliferation of an activated NK cell population [i.e., lymphokine-activated killer (LAK) cells]. For their immunological characteristics, NK and LAK cells appear to play a notable role in antiviral response and tumor control (Sinkovics and Horwath 2005; Fuggetta and others 2008; Suck and Koh 2010). Radiotherapy, which is widely utilized in cancer treatment causes severe damage to hematopoietic and lymphoid tissues and results in a marked immunodepression for a prolonged period of time (Ashwell and others 1988; Field and Becker 1989; Park and others 2011). In particular, it is well documented that γ-radiations impair host's NK activity in a dose-dependent manner (Hochman and others 1978; Blomgren and others 1980; Blomgren and others 1982; Zarcone and others 1989). In addition, decline of NK activity and decrease in the percentage of NK cells in the peripheral blood has been found in cancer patients receiving radiotherapy (McGinnes and others 1987).

Previous reports have shown that the exposure of mononuclear cells (MNCs) to IR is followed by a time-dependent decrease of NK activity in vitro (Zarcone and others 1989) and a marked induction of apoptosis (Delic and others 1993). The decline of NK activity was found to be correlated with a reduction of the number of CD16- and CD56-positive cells and of the percentage of the binding of effector cells to their targets (Fuggetta and others 1998).

Moreover, it has been recently demonstrated that exposure to IR induces long-term expression of senescence markers in vivo, thus showing that irradiation can be followed by persistent damage to different host's tissues, including components of the immune system (Le and others 2010). This is consistent with the commonly accepted opinion that radiotherapy downregulates immune responses. Surprisingly however, little information is presently available on possible pharmacological strategies able to restore immune responsiveness in irradiated subjects.

Among biological response modifiers, interferons (IFNs) represent a group of cytokines with a long history as immunotherapeutic agents employed in the treatment of various solid tumors and hematological malignancies. The use of IFNs in cancer therapy was based on their antiproliferative and immunomodulatory effects. More recently, IFNs have been found to possess cytotoxic and antiangiogenic properties (Bracarda and others 2010; Commins and others 2010; Moschella and others 2010). Therefore, combined use of radiotherapy and IFNs could be considered of potential value in the management of various types of neoplastic diseases. Actually, preclinical studies in vivo and in vitro have demonstrated that IFN modifies the radiation effects, enhancing the killing activity of IR on tumor cells and increasing immune responsiveness (Ortaldo and Mc Coy 1980). In particular, it is well known that IFNs are potent activators of NK cell function in vitro and in vivo (Silva and others 1980).

In previous studies, we have demonstrated that in vitro treatment with beta IFN (β-IFN) antagonizes the detrimental effect of γ-rays on the NK activity of MNC obtained from healthy donors, although this effect was of short duration (Fuggetta and others 1989; Fuggetta and others 1998). Moreover, Mor and Cohen (Mor and Cohen 1996) showed that IL2 is able to protect antigen-specific T cells from radiation injury. On the basis of these observations and to obtain a more potent and prolonged antagonistic effect, we investigated the immune-restorative activity of β-IFN in combination with IL2 on irradiated MNCs. In particular, in this study, we have examined the effect of γ-rays on cell-mediated natural immunity function against 3 human hematological malignancies. The results provided evidence that IR-induced impairment of natural cytotoxicity of MNCs could be antagonized, at least in part, by IL2 and particularly by the β-IFN+IL2 combination, mainly, when exposure to β-IFN precedes IL2 treatment. This protective effect is paralleled by lower levels of IR-induced apoptosis and increased expression of the antiapoptotic Bcl-2 protein in treated cells.

Materials and Methods

Cell lines

The NK- and LAK-susceptible erythroleukemia cell line, K562, and the NK-resistant and LAK-sensitive Burkitt lymphoma cell line, DAUDI, were obtained from ATCC. The EBV-transformed NK-resistant and LAK-sensitive B lymphoblastoid cell line BSM was described by Christmas and Moore (Christmas and Moore 1987). All cell lines were maintained in suspension culture in RPMI-1640 containing 10% fetal calf serum (FCS; Gibco Lab.) and 2 mM l-Glutamine (Flow Laboratories) hereafter referred to as the “Complete Medium” (CM) and subcultured 3 times weekly.

Irradiation of peripheral blood and separation of MNCs

Peripheral blood obtained from healthy adult volunteers after signed consent, and admixed with ethylenediaminetetraacetic acid to prevent coagulation, was exposed in vitro to 20 Gy using a Cesium-137 irradiator (Gamma Cell 1000; mod A, AECL).

MNCs from intact or irradiated peripheral blood were separated on a Ficoll-Hypaque gradient (Pharmacia) and hereafter called MNC and MNC/ir, respectively, as illustrated in Table 1. MNC and MNC/ir collected from the interface were washed twice with RPMI-1640 (Gibco Lab.), resuspended, and cultured in the CM.

Table 1.

Treatment Schedules of Mononuclear Cells Utilized in All Experiments

		Irradiation (20 Gy)
Treatment Code (TC)	Abbreviations used in the text	Cell type	Day ^a	β - IFN IU/mL	IL2 IU/mL	Incubation time	Day of collection
Control	MNC	—	—	—	—	0–4 days	1–4
TC-1	MNC/ir	WB^b	0	—	—	0–4 days	1–4
TC-2	LAK	—	—	—	500	4 days	4
TC-3	MNC/ir/IL2	WB^b	0	—	500	4 days	4
TC-4	LAK/ir^c	LAK	4	—	500	4 days	4
TC-5	MNC/IFN/IL2	—	—	200^d	500	4 days	4
TC-6	MNC/IL2/IFN	—	—	200^e	500	4 days	4
TC-7	MNC/ir/IFN/IL2	WB^b	0	200^d	500	4 days	4
TC-8	MNC/ir/IL2/IFN	WB^b	0	200^e	500	4 days	4

Day 0 is considered the day of experiment start.

WB, whole blood. Anticoagulated whole blood was irradiated before MNC separation.

LAK/ir (treatment code TC-4) indicates LAK cells from nonirradiated MNCs, subjected to 20 Gy γ-irradiation. These cells were utilized for cytotoxicity assay within 2 h after irradiation.

Test cells were treated with β-IFN (IFN in the abbreviation) for 2 h on day 0, washed, and then incubated with IL2 for additional 4 days. When MNC/ir (TC-7) were used, test cells were incubated with β-IFN shortly after blood exposure to ionizing radiations and cell separation.

Test cells were treated with IL2 from day 0 through day 4, and then treated with β-IFN for 2 h, and washed. When MNC/ir (TC-8) were used, test cells were incubated with IL2 shortly after blood exposure to ionizing radiations and cell separation.

LAK, lymphokine-activated killer; MNC, mononuclear cell.

Cytokines

Recombinant human IL2 was kindly provided by Roche, and natural fibroblast β-IFN, was kindly provided by Serono Pharma.

Treatment of MNC with cytokines alone or in combination

All in vitro treatment schedules utilized in the present study are summarized in Table 1.

On day 0, MNC and MNC/ir were suspended at the final concentration of 2×10⁶ cells/ml and cultured in the CM alone or in the presence of 500 UI/mL of IL2 at 37°C in a 5% CO2 humidified atmosphere for further 4 days. IL2-stimulated effector cells (called hereafter LAK and MNC/ir/IL2, respectively, as reported in Table 1, under TC-2 and TC-3 code, respectively) were recovered, counted, washed twice, and used for cytotoxicity assays and cytofluorimetric analysis.

Moreover, LAK cells obtained from intact MNCs were suspended in the CM at the concentration of 2×10⁶ cells/ml and irradiated with 20 Gy. Shortly after irradiation, LAK cells, called hereafter as LAK/ir (Table 1, TC-4) were used as effector cells against DAUDI, BSM, and K562 cell lines in a standard ⁵¹Cr-release assay.

Combined treatments with β-IFN and IL2 were performed on MNCs or MNC/ir at a final concentration of 2×10⁶ cells/mL on day 0. Test cells were incubated in the CM with 200 IU/mL of β-IFN for 2 h, washed, and treated with IL2 for further 4 days (i.e., Table 1, schedule TC-5 and TC-7, respectively). On the other hand, MNCs or MNC/ir at a final concentration of 2×10⁶ cells/ml were exposed to IL2 from day 0 through day 4 and then treated with 200 IU/mL of β-IFN for 2 h (i.e., Table 1, schedule TC-6 and TC-8, respectively). At the end of treatment, all test cells, control or subjected to cytokine treatment, were washed with the CM and assayed for cytolytic activity against DAUDI and BSM cell lines in a standard ⁵¹Cr-release assay.

Cell enumeration and viability of all MNCs treated (see Table 1) has been performed by trypan blue dye exclusion.

Cytotoxicity assay

The cytotoxicity activity of effector cells was determined using a standard ⁵¹Cr-release assay (Alvino and others 1999). Experimental results were expressed in terms of % specific lysis, or in terms of number of target cells lysed (i.e., killed) by a fixed number of effector cells, as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}\hbox{KC ( m )} = {\rm m} \times {\rm T} \times {\rm n} {\rm \%} / {\rm En} \times 100\end{align*} \end{document}

were KC(m) is the number of target cells killed by m effector cells; T is the number of target cells present in each well (i.e., 2×10³ cells); En is the number of effector cells present in each well at the selected E:T ratio, and n% is the specific lysis produced theoretically by En effector cells. The n% is extrapolated from the best fit curve obtained by plotting the different percentages of specific lysis versus ln of the number effector cell/well. In the present study, KC(m) was calculated for m=10⁶ [i.e., KC (10⁶)] and En=2×10⁵ cells (i.e., E:T cell ratio of 100:1) (Alvino and others 1999).

Immunofluorescence staining and cytometric analysis

The surface phenotypes of MNC and MNC/ir, nontreated or incubated with β-IFN and IL2 were determined by binding of the fluorescein isotiocianate-conjugated (FITC) monoclonal antibodies (mAb) recognizing the CD16 or CD56 marker (Becton Dickinson). In both cases, nonspecific Ig G binding was used as a negative control. Staining was performed at 4°C with optimal concentrations of mAbs for 30 min. After treatment, the cells were washed twice in phosphate buffer saline (PBS) and 0.02% sodium azide (Sigma) and used immediately.

MNC/irr exposed to the 2 cytokines were also tested for Bcl-2 expression. Cultured cells were washed twice in PBS, supplemented with 0.1% bovine serum albumin, and 0.02% sodium azide (PBS-A). Cells (1×10⁶) were then fixed in 1% formaldehyde in PBS at 4°C for 15 min and permeabilized in 70% ethanol at −20°C for 1 h. Cells were then suspended in 50 μL of PBS-A containing mAb recognizing Bcl-2, clone 124 (DAKO A/S, Denmark), washed, and labeled with rabbit anti-mouse immunoglobulins, F(ab')2- FITC (DAKO, code F0313). FITC-conjugated mouse IgG₂ was used as a negative control. Analysis was performed using a FACScan flow cytometer (Becton-Dickinson).

Flow cytometry was also used to detect apoptosis in MNCs and MNC/ir (Nicoletti and others 1991), nontreated or incubated with the cytokines. Cells were then washed in PBS and fixed with 50% acetone:methanol 1:4 in PBS at 4°C for at least 18 h. After 2 washes in PBS, the cells were suspended in PBS (1×10⁶cells/mL) containing 50 μg/mL of propidium iodide (PI; Sigma) and 100 KU/mL of RNase A (Sigma) and incubated in the dark at room temperature for 30 min. The DNA content per cell was evaluated by PI fluorescence measured on a linear scale using the FACScan flow cytometer (Becton-Dickinson) with an argon ion laser emitting at 488 nm. Data collection of 10,000 cells/sample was gated utilizing forward light scatter and side light scatter to exclude cell debris and aggregates. All data were recorded using CellQuest software (Becton-Dickinson). Apoptotic cells were determined by their hypochromic, sub-G1 staining profiles (Nicoletti and others 1991).

The effect of the 2 cytokines on the production of γ-IFN by CD56+ cells was analyzed by intracellular cytokine staining. Control MNC and MNC/ir were exposed to IL2 at 37°C for 16 h. In the meantime, the cells were incubated with 1 μg of Brefeldin A (Sigma-Aldrich). Thereafter, all cells were washed, stained with the FITC-conjugated anti-CD56 antibody and then permeabilized and fixed using Cytofix/Cytoperm (PharMingen) according to the manufacturer's instructions. PE-conjugated anti-γ-IFN antibody or isotype-matched control was added (4°C, 30 min) to the cells that were then washed twice, and analyzed by flow cytometry.

Western blot analysis

MNCs obtained from whole blood irradiated with 20 Gy (i.e., MNC/ir) were exposed to the cytokines under investigation for 24 h, washed in PBS, centrifuged, and pelleted. The cell pellets were suspended in the lysis buffer (50 mM Tris-HCl ph 7.5, 1 mM phenyl methylsulfoniyl peroxide-2 mM EGTA-10mMDTT-5 mg/mL of aprotinin, 200 mg/mL of leupeptin-0.1%-Triton X-100) kept on ice for 30 min, sonicated 5 s at 4°C, and centrifuged for 5 min at 15,000 g at 4°C in an Eppendorf microcentrifuge. All reagents were obtained from Sigma. Protein concentrations were determined using the Bio-Rad protein reagent with bovine serum albumin as the standard protein. Thereafter, proteins were separated on 12% SDS(w/v) polyacrylamide gel and transferred to a nitrocellulose filter, using a Biorad electrophoretic blotting apparatus (BioRad). Transfer was carried out at 25 V, at 4°C for 14 h. After transfer, membranes were incubated with 3% (w/v) dry milk (Bio Rad) in TBS (20 mM Tris -HCl ph7.5; 0.9 NaCl) with gentle agitation for 1 h. The membrane was then incubated at room temperature with mouse anti-human Bcl-2 (DAKO, 1:1000) in TBS containing 0.05% Tween 20 (TBST) for 2 h. Thereafter, the membrane was washed with TBST and incubated with the alkaline phosphatase-coupled secondary antibody diluted 1:7500 in TBST for 1 h. The band was visualized using the Photobed (Promega Biotec) reagents, according to the procedures provided by the manufacturer.

Statistical analysis

Differences in cytolytic effects produced by effector cells in various experimental conditions were evaluated taking into account the percentage of specific cytotoxicity at all E/T ratios. Therefore, p-values were calculated using covariance analysis performed on the regression of the percentage of specific ⁵¹Cr release over the ln of the number of effector cells/well. All data relative to cell-mediated cytolysis are expressed in terms of mean KC(10⁶) values without conventional standard error (SE) or standard deviation of the mean. Actually, no statistical analysis can be performed using SE or standard deviation, which are not suitable for covariance analysis of regression lines (Alvino and others 1999).

Where the results are expressed in terms of means±SE, statistical significance was determined using the Student's t-test analysis.

Results

Effect of IL-2 and β-IFN on the functional activity and number of cells exposed to γ-rays

Control MNCs or MNC/ir obtained from peripheral blood exposed to γ-rays, were treated with 500 IU/mL of IL-2 for 4 days (LAK generation) and tested for cytotoxicity against DAUDI, BSM, or K562 cell lines. The results of a representative experiment (Fig. 1) expressed in terms of percent-specific lysis or of KC(10⁶) (see legend of Fig. 1), show that pre-exposure to γ-rays reduces significantly the cytotoxic activity of effector cells. This inhibition, ranging from 40% to 75%, was detectable both against a NK-resistant DAUDI and BSM target cell lines, and against NK-susceptible K562 target cells. We also examined whether similar effects could be obtained when the γ-ray exposure was performed after LAK generation, that is, after IL2 treatment. When MNCs were first treated with IL2 for 4 days and then irradiated with γ-rays (i.e., LAK/ir), they displayed a reduced cytotoxic activity against DAUDI and BSM, but retained almost entirely the lytic activity against the K562 cell line (Fig. 1B).

FIG. 1.

Cytotoxic activity of lymphokine-activated killer (LAK) cells against 3 different target cells. All results are expressed in terms of % specific lysis at different effector/target ratios. Bars represent the SE of the mean. The data relative to A and B refer to different representative experiments that have been repeated 2 times each. (A) Cytotoxic activity of LAK cells generated from mononuclear cells (MNCs) isolated from untreated (LAK, ▪) or irradiated (MNC/ir/IL2, •) peripheral blood, tested against DAUDI, BSM, and K562 target tumor cells (see the treatment schedule described in Table 1). KC(10⁶) calculated for all effector versus target cell combinations and the relative P evaluation, are as follows: LAK versus DAUDI and MNC/ir/IL2 versus DAUDI=8500 and 2200 (P<0.01); LAK versus BSM and MNC/ir/IL2 versus BSM=5300 and 1950 (P<0.01); LAK versus K562 and MNC/ir/IL2 versus K562=850 and 4600 (P<0.01). (B) Cytotoxic activity of LAK cells generated from MNC isolated from untreated peripheral blood, control (LAK, □) or irradiated (LAK/ir, ○) and tested within 2 h against DAUDI, BSM, and K562 tumor cells (see the treatment schedule described in Table 1). KC(10⁶) calculated for all effector versus target cell combinations, are as follows: LAK versus DAUDI and LAK/ir versus DAUDI=7700 and 4600 (P<0.01); LAK versus BSM and LAK/ir versus BSM=7100 and 3700 (P<0.01); LAK versus K562 and LAK/ir versus K562=6700 and 6150 (P<0.05).

To determine the ability of β-IFN to reverse the inhibitory effect of γ-rays on functional LAK cell generation, 2 alternative protocols of treatment were tested. According to the schedules illustrated in Table 1, intact MNCs were exposed to the sequence β-IFN→IL2 (TC-5 schedule), or to IL2→β-IFN (TC-6 schedule), respectively. In parallel, irradiated MNCs were exposed to the same treatment schedules (i.e., TC-7 and TC-8). In both cases, the cytotoxic activity was assayed against DAUDI and BSM target cells on day +4 of culture. The results of a representative experiment concerning DAUDI target cells, show that the treatment of MNC/ir with β-IFN after radiation and before IL2 stimulation (i.e., TC-7), completely reversed the impairment induced by γ–rays on IL2-activated cytotoxic function (Fig. 2). On the contrary, when β-IFN treatment was performed after IL2 stimulation (i.e., TC-8), the lytic activity was found to be comparable to that obtained with irradiated MNCs treated with IL2 alone. Opposite results were obtained when nonirradiated MNCs were treated with β-IFN and IL2 combination. In this case, the sequence IL2→β-IFN (TC-6) was found to be superior to β-IFN→IL2 (TC-5) in increasing the cytolytic activity, in line with previous observations (Fuggetta and others 1993). Similar results were obtained using BSM target cells (data not shown).

FIG. 2.

Effect of β-IFN and IL2 alone or in combination on the cytotoxic activity of MNCs isolated from intact or irradiated (20 Gy) blood, tested against DAUDI tumor cells (see Table 1 for treatment schedules). The results concern a representative experiment and are expressed in terms of KC×10⁶EC/10^–2 *P<0.01, cytotoxic activity of MNC/ir/βIFN/IL2 versus other irradiated cytokine-treated groups (MNC/ir/IL2 and MNC/ir/IL2/βIFN, respectively).

Secretion of γ-IFN has been shown to correlate with the cytolytic activity of NK cells (Bryceson and others 2011). Therefore, studies were performed to establish whether the increase of the lytic response observed in MNC/ir treated with β-IFN/IL2 could be associated with the production of γ-IFN. In particular, the percentage of CD56+ effector cells producing the cytokine was evaluated by flow cytometry determination. The results illustrated in Fig. 3A indicate that the number of γ-IFN-producing CD56+ cells was significantly higher than that detectable in nontreated cells. Therefore, β-IFN-mediated increase of the lytic response of MNC/ir activated by IL2 (expressed as number of killed cells and percentage of specific lysis) clearly correlated with an increased amount of γ-IFN-producing cells (Fig. 3A, B).

FIG. 3.

(A) CD56+ cells producing γ-IFN evaluated by flow cytometric analysis. Data are expressed as number of CD56+/γIFN double-positive cells per μL. (B) βIFN-mediated increase of the lytic response activated by IL2 in irradiated MNCs. The data are expressed in terms of number of killed cells KC×10⁶ effector cells (left) and % of specific lysis at different attacker/target ratios (right). Bars represent the SE of the mean. The data relative to Fig. 3A and B refer to different representative experiments that have been repeated 3 times each. (*) P<0.01 i MNC/ir/βIFN/IL2 versus MNC/ir and MNC/ir/IL2.

Viable (trypan blue exclusion) cell count of intact or irradiated MNCs, nontreated or subjected to cytokine treatment, was performed on day 1 and 4 of culture (Fig. 4). It was found that: a) IR induce a step and progressive decline in the number of MNCs; b) treatment of MNC/ir with IL2 alone did not antagonize the effects of γ-rays on the total number of cells tested on day 1; c) treatment with β-IFN before IL2 stimulation reduced significantly the detrimental effect of γ-rays on the number of cells assessed on day 1; d) treatment with IL2 alone or combined with β-IFN antagonized equally well the effect of radiations on the number of cells on day 4. It should be mentioned also that treatment of intact MNCs with IL2 alone or in combination with β-IFN induced a modest increase of the number of viable cells on day 4 (data not shown).

FIG. 4.

Effect of IFN and IL2, alone or in combination, on the cell number of MNCs collected from intact or irradiated (20 Gy) peripheral blood (treatment schedules are described in Table 1). Results of a representative experiment, that are expressed in terms of geometric mean (each group in triplicate) of viable cell counts. Bars represent the SE of the geometric mean. (*) P<0.05, number of viable cells of MNC/ir versus MNC/ir/IFN/IL2 on day 1. (**) P<0.05, number of viable cells of MNC/ir versus MNC/ir/IL2 and MNC/ir versus on day 4.

Effect of IL2 and β-IFN on γ-ray induced apoptosis and Bcl-2 expression

It is well known that γ-rays can induce apoptotic death in human lymphocytes (Belloni and others 2005; Sharma and others 2010). Therefore, MNC/ir were treated with IL2 for 4 days alone or in combination with β-IFN 2 h before IL2 exposure (Table 1, TC-7); induction of apoptosis was evaluated by flow cytometry on day 1, 2, and 3 after irradiation. The results shown in Fig. 5 reveal that only a 0%–5% of apoptotic cells were detected in intact MNCs treated or not with IL2 alone or in combination with β-IFN. In untreated MNC/ir, a time-dependent increase of apoptosis from less than 10% to close to 30% was observed. However, a considerable and statistically significant decrease of apoptotic fraction was observed when MNC/ir were treated with IL2 alone. Treatment of MNC/ir with β-IFN before IL2 stimulation did not significantly influence IL2-mediated protection from apoptosis as demonstrated by data obtained from 4 pooled different donors (Fig. 5).

FIG. 5.

Time-course of apoptosis induction in cultured MNCs collected from intact or from irradiated (20 Gy) blood, and treated with β-IFN and IL2 alone or in combination (See Table 1 for treatment schedules). The percentage of apoptotic cells was evaluated by flow cytometry from day 1 through day 3 of culture. Data are expressed in terms of mean values obtained from MNCs of 4 different donors, and bars represent the standard error of the mean. (*)P<0.05, percentage of apoptosis in MNC/ir/βIFN/IL2 and MNC/ir/IL2 versus MNC/ir on day 3.

Large experimental evidence is now available showing that overexpression of the oncogene Bcl-2 is able to inhibit apoptosis induced by a variety of stimuli. Therefore, Bcl-2 expression was examined to establish whether it could be involved in the rescue effect produced by the combination of β-IFN+IL2 on radiation-induced apoptosis.

The levels of the Bcl-2 protein were analyzed in irradiated cells after 24 h of exposure to β-IFN and IL2 alone or in combination. The results obtained by Western Blot analysis (Fig. 6A) showed that the treatment with IL2 resulted in an appreciable increase of the protein, whereas the treatment with β-IFN induced a marked decrease of the same protein. The combination of β-IFN and IL2 produces a protective effect similar to that obtainable with IL2 alone, suggesting that IL2 counteracts, in part, the depressive effect of β-IFN. The endogenous Bcl-2 expression was enhanced by IL2 and reduced by β-IFN also in intact MNCs (data not shown). The results obtained after 24-h culture were consistently confirmed by flow cytometric analysis as illustrated in Fig. 5B.

FIG. 6.

MNC obtained from irradiated (20 Gy) blood, were cultured with medium alone (MNC/ir), with β-IFN (200 IU/mL) alone (MNC/ir/IFN), with IL2 (500 IU/mL) alone (MNC/ir/IL2), or with β-IFN+IL2 (MNC/ir/IFN/IL2), for 24 h and tested for Bcl-2 expression. (A) Western Blot analysis. β-actin has been used as a loading control. (B) Cytofluorimetric analysis. Results are expressed in terms of percentage of Bcl-2-positive cells.

Time-course of the number of CD56+ and CD16+ lymphocyte subpopulations after exposure to γ-rays and incubation with the cytokines under investigation

The phenotypes of MNC, untreated or incubated with IL2 (i.e., LAK cells), of MNC/ir, untreated or exposed to IL-2 alone (Table 1, TC-3), or incubated with β-IFN followed by IL2 (TC-7) were evaluated by flow cytometric analysis. The fraction of CD16⁺ and CD56⁺ cells was measured on day 0, +1, and +4, after irradiation. The results of a representative experiment (Table 2) show that a marked reduction of the number of CD16⁺ and CD56⁺ cells occurred in irradiated cells. This decrease was time dependent and was more pronounced for CD16+ cells compared to that relative to CD56⁺ cells, especially on day +4, after irradiation (82% loss for CD56+ lymphocytes and 97% loss for CD16+ lymphocytes). Treatment of MNC/ir with IL2 alone or combined with β-IFN afforded significant protective effects on both CD16⁺ and CD56⁺ cells. This is demonstrated by the substantial cytokine-mediated reduction of CD16+ or CD56+ cell loss occurring in irradiated MNCs from 1 to 4 days after exposure to γ-rays.

Table 2.

Time-Course of the Total Number of Viable CD56⁺ and CD16⁺ Cells (Expressed in Terms of Means×10³ Cells/mL) Found in Mononuclear Cells That Were Obtained from Nontreated or from Irradiated (20 Gy) Blood and Treated with Cytokines

	DAY 0				DAY 1				DAY 4
	CD56		CD16		CD56		CD16		CD56		CD16
MNC ^a treatment	M (SE)	%	M (SE)	%	M (SE)	%	M (SE)	%	M (SE)	%	M (SE)	%
MNC (control)	266 (32)	13.3	310 (37)	15.4	306 (23)	16.1	190 (14)	10	306 (81)	18	131 (34.6)	7.7
MNC/ir	280 (34)	14	300 (36)	15	66 (1.6)	5.3	47.5 (1.1)	3.8	50.4 (5.9)	8.4	9 (1.1)	1.5
LAK	NT		NT		290 (13)	14.5	124 (5.6)	6.2	453.6 (25.7)	16.8	165 (9.3)	6.1
MNC/ir/IL2^b	NT		NT		192 (23.2)	16	80 (9.7)	6.7	254 (27.7)	23.1	108 (11.8)	9.8
MNC/ir/IFN/IL2	NT		NT		233.6 (23.2)	14.6	123.2 (12.2)	7.7	262 (24)	23.8	89 (8.2)	8.1

On day 0, MNC were seeded in 24-well plates at the concentration of 2×10⁶ cells/mL, 2 mL/well.

Isolated MNCs or MNC/ir (2×10⁶ cells/mL) were cultured in vitro from day 0 through day 4. Treatment with IL2 alone or combined with β-IFN reduced significantly (P<0.01) CD56+ as well CD16+ cell loss produced by irradiation, either on day 1 or on day 4 after exposure to γ-rays.

M (SE), mean±standard error (3 observations/group).

For abbreviation identification, see Table 1.

No statistically significant difference was detected between the mean number of CD56+ cells relative to MNC/ir/IL2 group and that relative to MNC/ir/IFN/IL2 group on day 1 or on day 4. Similar results were obtained with CD16+ cells.

%, percent of MNC-positive for the relative marker evaluated by flow cytometry; NT, not tested.

Discussion

The present study has been designed to investigate the potential role of β-IFN and IL2 in contrasting the depressive effects of IR on human natural cell-mediated immunity. The results illustrated in this article show that γ-irradiated lymphocytes can be still stimulated with IL2 and acquire cytotoxic effector functions that are characteristic of LAK cells. Actually, MNC/ir treated with IL2 express the ability to lyse NK-resistant or NK-susceptible tumor cells, although this cytotoxic activity is lower than that expressed by their nonirradiated counterparts.(Fig. 1A). Moreover, we have confirmed that irradiation of mature human LAK cells (Table 1, TC-4) induces a rapid and marked decrease of their cytotoxic activity (Fig. 1B) as previously described by other authors in different experimental conditions (Zarcone and others 1989; Chong and others 1991).

In earlier studies (Fuggetta and others 1989, 1998), we found that β-IFN can successfully antagonize the depressive effect of IR on the NK activity when the cytokine was given after radiation. α-IFN and γ-IFN showed similar effects, although at a lesser extent (Fuggetta and others 1998). However, this antagonism declined rapidly on day 5 after irradiation, when the NK activity of irradiated MNCs treated with β-IFN did not differ from that detectable in irradiated, nontreated controls.

To achieve a more prolonged protective effect against γ-rays, we investigated the effects of β-IFN in combination with IL2 after blood irradiation. Two different protocols of treatment were considered, that is, β-IFN added to cell culture before (Table 1, TC-7) or after IL2 stimulation (TC-8). The results relative to the cytotoxic effects of cytokine-treated MNC/ir against DAUDI target cells (Fig. 2) suggest that the treatment with β-IFN immediately after radiation and before IL2 stimulation antagonizes efficiently the detrimental effect of γ rays. In addition, the cytokine-dependent recovery of the cytotoxic activity of effector cells was detectable against both target cells used (data for BSM not shown). On the other hand, this was not true when MNC/ir were exposed to β-IFN after treatment with IL2.

Several molecules have been involved in the effector function of activated NK cells. Target cell killing is achieved through directed release of lytic granules comprising Granzyme B and perforin [Bryceson and others 2011). Moreover, recognition of target cells is accompanied by the production of chemokines and cytokines, including γ-IFN that can coordinate the overall immune response (Bryceson and others 2011). Actually, the inhibition of NK activity by some antitumor agents was found to be correlated with suppression of Granzyme B expression (Allavena and others 1998). In this case, restoration of cytotoxic function by β-IFN was coincident with reappearance of Granzyme B gene transcription. In the present study, we found a rapid and marked decline of the number of MNCs and of their natural cytotoxic activity following exposure to IR. Treatment of irradiated cells with β-IFN and IL2 produced an increase of the number of γ-IFN-producing CD56+ cells, which strongly correlated with upregulation of the cytotoxic effector function in terms of number of killed tumor target cells and % of specific lysis (Fig. 3A, B).

As mentioned before, time-course analysis of cultured MNCs obtained from irradiated blood shows that rapid decay of the mean number of cells occurred within 4 days of cell culture (Fig. 4). A substantial rescue of cell numbers was found on day 4 in groups treated with IL2 or β-IFN+IL2 (Fig. 4). In line with these findings, the results detailed in Table 2, show that a marked decline of the total number of the 2 main subpopulations of NK effector cells (i.e., CD56+ and CD16+ lymphocytes) occurred after irradiation during the 4-day observation period. Treatment of irradiated MNCs with IL2 prevented almost entirely this effect. However, exposure of MNC/ir to β-IFN before treatment with IL2 did not show a noticeable influence on the survival of CD56 and CD16 effector cells and on the percentage of NK cells in the whole MNC population, especially on day 4 of culture in the presence of the IL2 (see footnote of Table 2). Therefore, it is reasonable to hypothesize that the mechanism by which MNC/ir/βIFN/IL2 cells are significantly more cytotoxic than MNC/ir/IL2 cells is not based on β-IFN-induced enrichment of natural immunity effector cells in the total MNC population. It could be rather suggested that β-IFN increased the intrinsic cytotoxic activity of irradiated MNCs, and that IL2 expanded an effector cell population endowed with cytolytic function higher compared with cells not treated with β-IFN.

The current literature concerning the effect of IL2 and IFNs in combination with IR on the natural immune system is very limited. Hietanen and others (1995) evaluated in vitro the modulating effect of IFN alpha, beta, and gamma as well as of IL2 on the radiosensitivity of large granular lymphocytes endowed with the NK cell activity. These authors showed that no combination of any type of IFN and IL2 was more radioprotective than IL2 used alone (Hietanen and others 1990, 1995). The reason of the discrepancy between our results and Hietanen's data may be due to the different time intervals elapsing between cell irradiation and cytotoxicity assay as well as to different IL2 and β-IFN concentration and treatment schedules used. Moreover, in the studies performed by Hietanen, purified large granular lymphocytes were exposed to ionizing irradiation in vitro. Instead, most of the experiments illustrated in the present investigation have been conducted on MNCs obtained from whole peripheral blood that was irradiated before lymphocyte separation. With this treatment schedule indeed, we intended to utilize an in vitro experimental model to mimic as closely as possible the clinical setting in radiotherapy.

In human blood, NK cells can be subdivided into different populations based mainly on the relative expression of the surface markers, CD16 and CD56, which show different functional properties. The CD56+ subset is more immunoregulatory, has little cytolytic activity, but releases high levels of cytokines, whereas the CD16+ subset, which predominates in peripheral blood and inflamed tissues, is characterized by a lower cytokine production, but displays high cytotoxic effector function (Moretta and others 2008; Maghazachi 2010). It has been demonstrated that NK cells belong to highly radiosensitive lymphocyte subpopulation. On the contrary, B and CD8+ T cells show a weaker susceptibility to IR, and CD4+ T cells are relatively radioresistant (Rana and others 1990). However, the radiosensibility of NK cells remains still a matter of debate (Philippé and others 1997; Wilkins and others 2002; Vokurková and others 2006).

In a previous study, we confirmed that decrease of the NK activity is time dependent and could be associated to a significant reduction of the number of CD16⁺ cells that appeared to be the most sensitive subpopulation to radiation damage (Fuggetta and others 1998). This has been confirmed by the results of the present investigation showing that in case of irradiated blood the number of CD16+ cells decreased severely, whereas a less pronounced cell loss occurred at the level of CD56+ lymphocyte subpopulation on day 4 of culture (Table 2). It must be pointed out that a minimal CD8+/CD56+ NKT cell population has been also indicated as a lymphocyte subset particularly susceptible to low doses of γ-rays (Vokurková and others 2010). In any case, the effects of irradiation on specific NK subpopulations are still an open question and the literature is controversial, and in most cases, available information has been obtained from clinical experimentation (Santin and others 2002; Eric and others 2009).

The mechanisms underlying radiation damage of NK cells are not entirely understood. Numerous studies showed that radiations compromise the integrity and the cytotoxic activity of NK cells both in vivo and in vitro and induce programmed cell death (Philippé and others 1997; Louagie and others 1998; Wilkins and others 2002; Sharma and others 2010). In line with these general observations, the results illustrated in Fig. 5 show that marked and progressive increase in the percentages of apoptotic cells can be found in MNC/ir up to 3 days postirradiation. Moreover, the same figure provides evidence that the treatment with IL2 alone or combined with β-IFN induces a statistically significant reduction of apoptosis. These results are consistent with previous observations showing that high concentration of IL2 is able to rescue T cells from radiation-induced apoptosis and that this phenomenon is independent from the growth-promoting effect of IL2 (Mor and Cohen 1996). Interestingly, β-IFN induces an increase of the lytic function without significantly affecting IL2-mediated protection from apoptosis (see legend of Fig. 5).

Several authors have shown that IL2 antagonizes the detrimental effects of IR on viability and cytotoxicity of effector lymphocytes, depending on the IL2 concentration, radiation dose, and intervals between irradiation and observation. (Chong and others 1991; Seki and others 1995; Mor and Cohen 1996). This cytokine may play an important role in the regulation of lymphocyte survival in the control of the cell cycle during the immune response both in health and disease. In particular, expression of the Bcl-2 gene promotes cell survival by counteracting apoptosis stimuli, and the mechanism by which IL2 downregulates apoptosis of lymphocytes can be reasonably linked to the induction of Bcl-2 (Mor and Cohen 1996; Adachi and others 1998). Actually, evasion from DNA damage-induced cell death, via overexpression of prosurvival antiapoptotic Bcl-2 family proteins (Wyllie and others 2010) could be considered a key step toward radiation resistance. The results illustrated in Fig. 6 are in line with this hypothesis since Western Blotting and flow cytometry analysis show that enhanced expression of the Bcl-2 protein occurs in IL2-treated MNC/ir. When β-IFN was associated with IL2 treatment, it should be noted that both cytokines steered target effector cells toward increased functional cytotoxic activity. However, considering the MNC population in toto, the antiapoptotic effects of IL2 could have been neutralized by the proapoptotic activity of IFN against T lymphocytes and, in particular, against helper T cells (Durelli and others 2009). Nonetheless, the Bcl-2 inducing activity and the resultant protective effects of IL2 on MNC survival after irradiation prevailed over the potentially negative influence of β-IFN on lymphocyte viability. In addition, several data from the literature indicate that β-IFN does not induce apoptotic death in cells participating in the natural immune system, as recently confirmed by Sanvito and others (2011), Shiao and Coussens 2010. Therefore, the 2-cytokine association presented in this report guaranteed high cytolytic function and lymphocyte survival even in the biologically adverse situation created by exposure to a relatively high dose of γ-rays.

Of particular interest are the results of a preliminary study performed in our laboratory showing that treatment of human melanoma and breast cancer cells with β-IFN+IL-2 shortly after exposure to γ-rays did not increase Bcl-2 expression, as evidenced by Western Blot analysis (data not shown). If this will be confirmed by further studies, it would indicate a possible differential effect of cytokine treatment on host and certain types of malignant cells, favoring tumor death and survival of natural host immunity under the influence of IR.

In conclusion, we have demonstrated that β-IFN followed by IL2 affords a noticeable radioprotective effect in terms of cell survival and function, leading to an almost complete recovery of natural cytotoxicity in MNCs isolated from blood exposed to γ-rays. No direct prosurvival effects seem to be mediated by IL2 in various malignancies, including melanoma (Rosemberg 2005), and a marked proapoptotic activity against tumors appears to be obtainable with β-IFN (Caraglia M and others 2009). Therefore, the results of the present study may have important therapeutic implication for patients receiving radiotherapy and possibly subjected to further cytokine-mediated immunotherapy.

Footnotes

Acknowledgment

This work was partially supported by MIUR Italy, PRIN 2008 n°20089E83YR_005 to MP Fuggetta.

Author Disclosure Statement

No competing financial interests exist.

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