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RADIATION RESEARCH 158, 464–474 (2002)0033-7587/02 $5.00 2002 by Radiation Research Society.All rights of reproduction in any form reserved.

Clearance of Radiation-Induced Apoptotic Lymphocytes: Ex Vivo Studiesand an In Vitro Co-culture Model

M. Benderitter,1 V. Durand, C. Caux and P. Voisin

Institut de Protection et de Surete Nucleaire, Departement de Protection de la sante de l’Homme et de Dosimetrie, Section Autonome de

Radiobiologie Appliquee a la Medecine, Laboratoire de Dosimetrie Biologique Multiparametrique, IPSN, B.P. no. 6,

F-92265 Fontenay-aux-Roses Cedex, France

Benderitter, M., Durand, V., Caux, C. and Voisin, P. Clear-

ance of Radiation-Induced Apoptotic Lymphocytes: Ex Vivo

Studies and an In Vitro Co-culture Model. Radiat. Res. 158,

464–474 (2002).

Lymphocytes are very sensitive to radiation. Our aim was

to test the possibility of detecting apoptosis in lymphocytes as

a potential short-term biomarker of ionizing radiation expo-

sure. Our in vitro data confirmed the dose–time–effect rela-

tionships involved in radiation-induced apoptosis. The detec-

tion of in vivo induction of apoptosis in circulating lympho-

cytes after exposure of animals to radiation appears to depend

critically on the technique used to measure apoptosis. Among

the different techniques we investigated, mitochondrial mod-

ification was the most appropriate; they allowed establishment

of dose–time–effect relationships when animals were observed

for 72 h. A model of in vitro phagocytosis of apoptotic lym-

phocytes by macrophages was developed to mimic clearance

of apoptotic cells occurring in vivo. Together, our data showthat mitochondrial labeling may make it possible to detect ex

vivo radiation-induced apoptosis of lymphocytes before mac-

rophage ingestion occurs. We propose the measurement of

apoptosis in lymphocytes as a potential short-term biomarker

of ionizing radiation exposure. 2002 by Radiation Research Society

INTRODUCTION

Apoptosis is a physiological mode of cell death in manycell systems. It involves active cellular processes and oc-curs as a controlled biological response of the cell to slightmodifications of its external environment or internal ho-meostasis. In particular, apoptosis has been widely studiedas a cellular response to radiation exposure (1, 2). Necrosis,on the other hand, has been described as an accidental modeof cell death that occurs as a response to extreme externaldamage during which cell integrity cannot be maintained.

1 Address for correspondence: Institut de Protection et de Surete Nu-cleaire, Departement de Protection de la sante de l’Homme et de Dosi-metrie, Section Autonome de Radiobiologie Appliquee a la Medecine, La-boratoire de Dosimetrie Biologique Multiparametrique, IPSN, B.P. no. 6,F-92265 Fontenay-aux-Roses Cedex, France; e-mail: [email protected].

High-dose radiation exposure is one example. The initiationof radiation-induced apoptosis is a ubiquitous process.

An emerging consensus considers that the process of ap-optosis involves a three-step sequence of events (3, 4). The

first step, termed initiation, results mainly from cellular le-sions, transmembrane signals, or modifications of intracel-lular homeostasis. A different intracellular signaling path-way leads to the commitment step, during which the cellmakes an irreversible choice between destruction and sur-vival. The final step is the execution of apoptosis; it ischaracterized by DNA fragmentation, membrane modifi-cations, and specific proteolytic cleavages. There is generalagreement about the characterization of the two last steps,but the definition of initiation remains a subject of debate.Because energy is deposited through the entire volume of the cell after radiation exposure, different targets may beaffected simultaneously (5). Cregan et al. (6 ) described two

main processes by which radiation exposure initiates apo-ptosis in human lymphocytes: The first, slower process isinitiated by DNA lesions, the other, more rapid process bymodification of membrane oxidation. The relative propor-tions of these processes and the probable interconnectionbetween the cellular targets remains unclear, however (7 ).The specific radiation-induced lesions and the cell’s capac-ity to repair them determine the process by which damaged,unrepaired cells are eliminated. In contrast to necrosis,which leads to uncontrolled swelling, rupture of cellularmembranes, and the consequent release of active enzymaticcompounds, apoptosis leaves the disassembled subcellularstructures packaged in membrane-bound vesicles, thereby

preventing toxic intracellular components from leaking andcausing tissue damage (8 ). In vivo, phagocytes can thenengulf the apoptotic bodies without eliciting an inflamma-tory response.

To determine what role apoptosis can play in assessingradiation injury, we need to know how long after irradiationapoptotic cells remain detectable in vivo, how reliable in

vitro studies are in assessing in vivo radiation injury, andwhat dose–response relationship exists between apoptosisand radiation in the tissue being sampled. The aim of thisstudy was to evaluate radiation-induced apoptosis in lym-



465RADIATION-INDUCED APOPTOSIS

phocytes as a potential biomarker of radiation exposure.This investigation took place in three stages. We first as-sessed the kinetics of lymphocyte apoptosis after exposureof human blood samples to radiation and then comparedit to the in vitro kinetics of radiation-induced apoptosis inanimal lymphocytes exposed in vivo to radiation. Finally,

an in vitro co-culture model was used to verify the clear-ance of apoptotic lymphocyte after radiation exposure.

MATERIALS AND METHODS

Investigation of In Vitro Radiation-Induced Lymphocyte Apoptosis

1. Irradiation and in vitro lymphocyte culture

Human blood samples were collected from healthy volunteers into hep-arinized tubes and were exposed to radiation from a 60Co source (ICO4000, IPSN, France). The irradiation (0 to 6 Gy) was performed in a37C thermostated water bath at a dose rate of 0.5 Gy min1. The bloodwas then layered onto Ficoll-Hypaque gradient (Lymphoprep, Nyegaard,Denmark) to separate the lymphocytes, which were then washed twice inRPMI 1640 medium (without Ca2 and Mg2) at room temperature. The

washed cells were resuspended at 2 106 lymphocyte ml1 in culturemedium (RPMI 1640 medium supplemented with L-glutamate, Hepesbuffer, penicillin, streptomycin and 20% SVF) and kept there up to 6days after irradiation in a fully humidified incubator with 95% air and5% CO2. All chemicals were purchased from Sigma Aldrich Co. (St Lou-is, MO). Cells were counted daily with an automated blood analysis sys-tem (Advia 120, Bayer Diagnostic, Puteaux, France). Lymphocyte apo-ptosis was detected as described below.

2. Quantification of apoptotic lymphocytes

First, cells were discriminated by flow cytometry. The FSC/SSC dotplot allows separation of lymphocyte populations (R 1). This region wasinvestigated in terms of the lymphocyte subpopulation and cell viability.Briefly, cells (2 105) were incubated at 4C in the dark for 20 minwith a combination of two antibodies (anti-CD2-PE, anti CD3-FITC oranti CD11b-FITC, Beckman-Coulter, Marseille, France). After centrifu-

gation at 4C, cells were resuspended in 0.5% PBS-BSA and 7-AAD (7-amino actinomycin, Sigma-Aldrich) and immediately analyzed by flowcytometry.

Second, radiation-induced lymphocyte apoptosis was detected by sev-eral parameters that were based on membrane, mitochondrial and nuclearlabeling.

Viable and dead cells were separated after fluorescein-conjugated an-nexin V binding to membrane phosphatidylserine. The loss of phospho-lipid membrane asymmetry results in externalization of phosphatidylser-ine during cell death (22). Compromised cell membrane permeability,which determines whether or not PI can bind to the cellular DNA, al-lowed necrotic and apoptotic cells to be distinguished. Briefly, cells (5 105) were resuspended in binding buffer and subsequently incubatedwith annexinV-FITC (0.25 g ml1 final concentration) and PI (0.25 gml1 final concentration) (all three products from R&D Systems Europe

Ltd, Abingdon, UK) for 15 min at room temperature in the dark. Labeledcells were immediately analyzed with a Facsvantage flow cytometer (Bec-ton Dickinson, Franklin Lakes, NJ). As Fig. 2b shows, three different cellsubpopulations were distinguished by the following combinations: viable(annexin V /PI), necrotic (annexin V /PI) and apoptotic cells (annexinV /PI). Markers and compensation were set with unlabeled cells andsimple labeled cells.

The cationic dye DiOC6 (3, 3dihexyloxacarbocyanine iodide, SigmaChemical Co.) binds strongly to cell mitochondria and is commonly usedto label and measure mitochondrial transmembrane potential. In the earlystages of the process leading to apoptosis, mitochondrial transmembranepotential is reduced (23). Cells with compromised cell membrane per-meability allow PI to bind to the cellular DNA and thus discriminatebetween apoptotic and necrotic cells. DiOC6 was stored in DMSO at a

concentration of 1 m M. It was further diluted in ethanol and added (finaconcentration 40 n M ) to 5 105 lymphocytes. Cells were incubated a37C in the dark for 15 min and then, after addition of PI (0.25 g ml

final concentration), immediately analyzed by flow cytometry. This technique discriminates three different lymphocyte subpopulations: viablecells (DiOC6high /PI), necrotic cells (DiOC6low /PI), and apoptotic cell(DiOC6low /PI) (Fig. 2c). Markers and compensation were set with unlabeled cells and simple labeled cells.

DNA fragmentation was assayed by fluorometric analysis of DNA un-winding (FADU) as described previously by Birnboim and Jevcak (24)This test is a fast and reliable method to detect DNA strand breaks, basedon the destabilization of short duplex regions by unwinding from singlestrand breaks after alkaline denaturation. The remaining double-strandregions are selectively bound to ethidium bromide (2,7-diamino-10-ethyl9-phynel-phenanthrridium bromide, Sigma Chemical Co.). The morstrands that are broken, the more DNA is unwound, and the less fluorescence is present. Fluorescence intensity is read at 25C with an SFM 25spectrofluorometer (Bio-Tek Kontron, Saint Quentin Yvelines, France) a520 nm excitation and 590 nm emission. The lymphocytes were dividedinto three sets: a ‘‘total’’ sample (T) in which the reaction was blockedbefore a 60-min alkalization treatment, thereby yielding total fluorescenceincluding possible contaminants; a ‘‘blank’’ sample (B) obtained by celsonification (20 W cm1 for 30 s); and an ‘‘experimental’’ sample (P)

DNA extraction and 60 min of alkalization were conducted separately foeach set, and the means of six fluorescence readings were calculated forT, B and P. The percentage of double-strand DNA was measured for eachsample with the following formula: %D (P B)/(T B) 100.

Quantification of Ex Vivo Radiation-Induced Lymphocyte Apoptosis

Male Wistar rats weighing 250 g were exposed to radiation with a60Co source (ICO 4000). TBI (0.5 and 1.5 Gy) was performed at a doserate of 0.75 Gy min1. After irradiation, they were housed four per cageand received commercial rodent chow and water ad libitum. At differentimes postirradiation (0, 24, 48, 72, 96, 120 and 144 h), animals wereanesthetized (i.p., 0.1 ml Imalgene 1000/100 g animal weight), a needlepuncture of the abdominal aorta performed, and blood was collected inheparinized tubes. Blood cells were counted daily with an automated

blood analysis system (Advia 120, Bayer Diagnostic, Puteaux, France)All these experiments were conducted in accordance with the Frenchregulations for animal experimentation (Ministry of Agriculture, decreeno. 87-848, 19 October 1987). The blood was layered onto Ficoll-Hy-paque gradient (Lymphoprep, Nyegaard, Denmark) to separate the lymphocytes, which were washed twice in RPMI 1640 medium (without Ca2

and Mg2) at room temperature. The washed cells were resuspended a2 106 lymphocytes ml1 in RPMI 1640 medium supplemented with L

glutamate, Hepes buffer, penicillin, streptomycin and 20% FCS. Radiation-induced apoptosis of lymphocytes was assessed as described above

Quantification of Clearance of In Vitro Apoptotic Lymphocytes

1. Irradiation and co-culture

Isolated lymphocytes were obtained after layering human blood sam

ples (previously collected into heparinized tubes) onto Ficoll-Hypaquegradient (Lymphoprep). A total of 20 l MACS CD 14 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) per 10 7 cells ml1 were incubated for 15 min at 6C. After cell washing, the magnetic cells weresorted with positive selection columns (MS /VS). A 70–75% yieldwas obtained with a purity of 90%.

The positive and negative fractions were suspended at 2 106 cellml1 in RPMI 1640 medium supplemented with L-glutamate, Hepes buffer, penicillin, streptomycin and 20% FCS. The negative fraction, supplemented with a 20% BSA/10% DMSO mixture (1:1; v:v), was graduallyfrozen and stored for 1 week in liquid nitrogen. Monocytes (2 106 cellml1) were further incubated for 1 week in culture medium supplementedwith 800 IU ml1 GM-CSF (R&D Systems Europe Ltd.) and conditionedin 100-ml Teflon culture pockets (Poly Labo, Strasbourg, France). Incu



466 BENDERITTER ET AL.

FIG. 1. Flow cytometric discrimination of the lymphocyte subpopulation (panel a). Specific immunological staining allows discriminating of Tlymphocytes, B lymphocytes, natural killer cells, and monocytes by using combination of CD2, CD3 and CD11b (panel b). CD2-CD3 dot plot (panelb.1) allows discrimination of B lymphocytes (upper-left quadrant: UL), NK cells and T lymphocytes (upper right quadrant: UR). CD2-CD11b dot plot(panel b.2) allows discrimination of T and B lymphocytes (upper left quadrant: UL), NK cells (upper right quadrant: UR), and monocytes (lower rightquadrant: LR). Panel c shows how simultaneous 7-AAD labeling enables the discrimination, in each quadrant, as defined in panel b, viable cells (7-AAD-negative cells) from dead cells (7-AAD-positive cells). The percentages of cells in each quadrant are indicated.

bation took place in a fully humidified incubator with 95% air and 5%

CO2. Maturation of monocyte into macrophages was checked every 2days with May-Grunwald Giemsa labeling. All other chemicals were pur-chased from Sigma Chemical Co.2. Quantification of the basal phagocytosis capacity of macrophages

The basal phagocytosis capacity of macrophages was checked with thetechnique described by Patterson-Delafield and Leher (25), based on in-gestion of inactivated yeast, i.e. Saccharomyces cerevisiae. Adherentmacrophages were obtained after seeding 5 104 cells into a Lab-Tek chamber slide system (Nalgene Nunc International, Naperville, IL). Mac-rophages were then exposed to radiation from a 60 Co source (ICO 4000,IPSN), at doses of 2, 15 or 30 Gy (0.8 Gy min1). The phagocytosiscapacity of the adherent macrophages was determined 48 h postirradia-tion, by adding heat-inactivated yeast (Saccharomyces cerevisiae, SigmaChemical Co.) at a 1:3 ratio. After 3 h of co-incubation, wells were

washed with PBS and slides were stained with May-Grunwald Giemsa.

The percentage of phagocytic cells was determined by counting thosethat had ingested at least one yeast. In all, 400 macrophages were countedon each slide, with four counts in different areas.3. Quantification of macrophage capacity to phagocytose apoptotic lym-

phocytes

Forty-eight hours postirradiation, lymphocytes (30 106 cells) wereincubated with 50 g of a viable long-lived dye, TAMRA [5-(5&6)-carboxytetramethylrhodamine, succinimidyl ester; Molecular Probes Eu-rope BV, Leiden, The Netherlands] in 2 ml DMEM plus 2 ml PBS for15 min at 37C, following the method described by Hess et al. (16 ). Cellswere washed and the TAMRA-labeled lymphocyte solution was exposedto radiation from a 60Co source (ICO 4000, IPSN) at doses of 1 and 2Gy (0.5 Gy min1). Two main points were verified in our experimentalconditions. We ensured that TAMRA labeling did not interfere with ra-




FIG. 2. Flow cytometry plots of a 48-h culture of nonirradiated human cells (left column) and human cellsirradiated with 2 Gy (right column). In the FSC/SSC plot, an R 1 gate is drawn around the lymphocyte (line a). AFl1:annexin V-FITC/Fl2:PI plot was obtained on the R1 region (line b). The lower left quadrants represent the viablePBL, the lower right quadrant the early apoptotic lymphocyte, and the upper right quadrant the late apoptoticlymphocyte. A Fl1:DiOC6 /Fl2:PI plot was obtained of the R1 region (line c). The R2 region represents the viable

lymphocyte, the R3 region the early apoptotic lymphocyte subpopulation, and the R4 region the late apoptotic lym-phocyte subpopulation. The percentages of cells in each quadrant are indicated.

diation-induced apoptosis by comparing the percentage of radiation-in-duced apoptotic lymphocytes when TAMRA labeling did and did not takeplace before irradiation. We also ensured that the label was not acquiredby or transmitted to unlabeled cells during the experiment.

Simultaneously with the lymphocyte preparation, we seeded 2 ml of 1 105 macrophages ml1 into 12-well microplates (Nalgene Nunc Inter-national). Nonadherent macrophages were eliminated just before irradi-ation by washing the wells. Adherent macrophages were exposed to

radiation from a 60Co source (ICO 4000, IPSN) at doses of 2, 15 and 30Gy (0.8 Gy min1). Just after irradiation, the supernatant was removed

and 2 ml of lymphocyte suspension (4 105 lymphocytes ml1) waadded and co-cultured for 48 h with the adherent macrophages, for a finallymphocyte:macrophage ratio of 5. After 48 h, macrophages were labeledwith 40 n M DiOC6 (3,3dihexyloxacarbocyanine iodide) at 37C for 15min. The co-cultures were then examined with an MRC 1024 ES confocaimaging system equipped with a 488-nm argon/krypton laser (both fromBio-Rad Microscopy Division, Hemel Hempstead, UK, as are the filtermentioned hereafter). The dyes were excited at a wavelength of 488 nmEmissions were collected with an LP565 filter for the TAMRA and anRSP580 filter for the DiOC6. The TAMRA-labeled lymphocytes that were




FIG. 3. Quantification of apoptotic lymphocytes after in vitro exposure of human blood to 60Co rays. Dosesranged from 0.5 to 6 Gy (0.5 Gy min1). Black circles, 0 Gy; gray circles, 0.5 Gy; dark gray circles, 2 Gy; lightgray circles, 6 Gy. Detection of apoptosis was done during a 6-day culture of lymphocytes. Data obtained aftermembrane labeling (annexin V-FITC/PI) and flow cytometric detection are shown in panel d. Data obtained aftermitochondrial labeling (DiOC6 /PI) and flow cytometry are shown in panel c. Data obtained after nuclear labelingand spectrofluorometric detection (fluorometric analysis of DNA unwinding) are shown in panel b. Panel a showsthe lymphocyte count. Data are expressed as the means SEM of six different experiments. Statistical analyseswere carried out by a two-way analysis of variance. When appropriate, all pairwise multiple comparisons wereperformed.

adherent to the surface of the DiOC6-labeled macrophages were distin-guished from those ingested by macrophages with Z-series microphotog-raphy.

Statistical Analysis

Data are presented as the means standard errors of the mean (SEM)for six experiments. Statistical analyses were carried out by a two-wayanalysis of variance (ANOVA). When appropriate, all pairwise multiplecomparisons (Tukey test) were performed.

RESULTS Investigation of in Vitro Radiation-Induced Lymphocyte Apoptosis

Human blood samples were irradiated in vitro; the sep-arated lymphocytes were then maintained in culture for 6days. Figure 3a shows a slight but nonsignificant decreaseof 10% in the sham-exposed lymphocyte count during the6-day cell culture. As expected, the decrease was muchmore marked after irradiation. Significant 35 and 55%drops were observed 6 days after irradiation with 0.5 and6 Gy, respectively. The kinetics of radiation-induced apo-

ptosis was determined by labeling the membranes (Fig. 3d),the mitochondria (Fig. 3c), and the nuclei (Fig. 3b).

First, cells of interest were gated in region R1 and gran-ulocytes, platelets and cell debris were excluded (Fig. 1a).It must be noted that this region is modified during thecourse of the experiment. Cells gated in region R1 becomemore condensed and granular with time, as shown by thedecreased forward scatter (FSC) and increased side scatter(SSC) values (Fig. 1a), respectively. Two regions were de-fined R1a and R1b (Fig. 1a). We used immunolabeling to

ensure that the cells undergoing these modifications wereactually lymphocytes and accordingly must be included inthe R1 region (R1 R1a R1b). Lymphocyte subpopula-tions and monocytes were differentiated with anti-CD2,anti-CD3 and anti-CD11b (Fig. 1b). With this approach, weconfirmed that the distribution of the lymphocyte subpop-ulation in R1 was preserved during the cell shift we ob-served there. Additional 7-AAD labeling clearly showedthat the cells that shifted were lymphocytes undergoing celldeath. These cells are characterized by high 7-AAD fluo-rescence intensity (Fig. 1c).




Second, we ensured that under our experimental condi-tions, changing the medium during the 6-day culture didnot significantly modify the induction of lymphocyte apo-ptosis after irradiation. A basal and constant level of lym-phocyte apoptosis was detected regardless of the techniqueused (5 to 10% for membrane labeling and 10 to 25% for

mitochondrial labeling). This basal level did not changesignificantly during the 6 days of culture (Figs. 3b and c).A two-way analysis of variance of our in vitro data revealeda significant dose–effect relationship in the dose range of 0 to 6 Gy for the late apoptotic lymphocyte population formembrane (Fig. 3d) and mitochondrial (Fig. 3c) labeling.The curve flattened toward the highest doses. This in vitro

dose–effect relationship persisted through the sixth day of postirradiation culture. Regardless of the dose, the percent-age of apoptotic cells was highest at 72 h after irradiation.This observation remained constant for all detection tech-niques. For example, with membrane labeling, we notedapoptosis indices of 19.2% 0.6% and 44.7% 0.8% 72

h after doses of 0.5 Gy and 6 Gy, respectively, while withmitochondrial labeling, the apoptosis indices in the sameconditions were 37.9% 0.9% and 76.5% 0.4%. Be-yond this interval, these indices reached a plateau. Our find-ings with membrane and mitochondrial labeling were com-parable: The principal difference between the two tech-niques is that mitochondrial labeling detects more apoptoticlymphocytes.

Radiation-induced DNA fragmentation, as measured bythe FADU assay and expressed as Qd values, was normal-ized to zero and therefore represented a positive increaseabove the control level (Fig. 3b). A significant (P 0.01)dose-dependent increase in DNA fragmentation was ob-

served in the minutes after irradiation. This effect disap-peared 24 h later. Subsequent culturing revealed a gradualincrease in DNA fragmentation. A significant (P 0.001)dose–effect relationship was observed for all postirradiationintervals. The percentage of apoptotic cells peaked at 96 hpostirradiation. Beyond this delay, this value reached a pla-teau.

Investigation of Ex Vivo Radiation-Induced Lymphocyte Apoptosis

After animals were exposed to radiation, blood sampleswere collected at different times; lymphocytes were sepa-rated, labeled immediately, and analyzed with fluorometry.In our experimental conditions, the lymphocyte countreached its nadir 2 days after the animal’s exposure to dosesof 0.5 or 1.5 Gy (Fig. 4a). Dot plots of rat blood samples,as obtained by flow cytometry, were similar to those ob-tained from human blood samples.

According to our ex vivo data from the mitochondriallabeling, in the first hours after irradiation, the radiation-induced apoptosis increased in a dose- and time-dependentmanner (Fig. 4c). The percentage of apoptotic lymphocytesreached a maximum 48 h after irradiation, regardless of

dose, and remained detectable for up to 24 h. This maxi-mum was 29.5 3.4% (P 0.001) for animals exposedto 1.5 Gy. With membrane labeling (Fig. 4b), however, themaximum percentage of detectable apoptotic cells for thesame animals was only 9.8 2.1% (P 0.05) 4 h post-irradiation and was no longer detectable after that. The

FADU investigation (Fig. 3d) of these ex vivo experimentsshows no statistically significant variation in the percentageof radiation-induced DNA fragmentation.

Moreover, both membrane and mitochondrial labelingshowed two-wave kinetics in the induction of apoptosisThe first wave reached its maximum within 4 and 7 hrespectively (Fig. 4b and c), and the second wave betweenday 2 and day 3 when using membrane labeling and at day2 when using mitochondrial labeling.

Clearance of Apoptotic Lymphocytes and In VitroCo-culture

Lymphocytes and adherent macrophages were irradiatedseparately and then co-incubated for another 48 h to mimicthe physiological clearance of apoptotic lymphocytes. A ra-tio of four lymphocytes per macrophage was fixed in ourexperimental conditions (Fig. 5a). In the first step of thisexperiment, we compared the proportion of apoptotic lymphocytes present in the supernatant of co-cultures with andwithout macrophages. The basal phagocytosis capacity ofthe macrophages was assessed in the second step by mea-suring their capacity to phagocytose inactivated yeast. Fi-nally, confocal microscopy and flow cytometry were usedto quantify the specific phagocytosis of apoptotic lympho-cytes by macrophages.

The first step showed that under our experimental con-ditions, without any macrophage co-incubation, there wasa basal level ranging from 5 to 15% of spontaneously ap-optotic lymphocytes (Fig. 5). As described above, this wasthe case regardless of the technique used to detect apopto-sis. The 48-h co-incubation of nonirradiated lymphocyteswith macrophages did not modify this basal level significantly (Fig. 5b). More precisely, the basal percentage ofapoptotic lymphocytes decreased slightly but not significantly when nonirradiated lymphocytes were co-incubatedwith macrophages, compared to the basal level withoumacrophage co-incubation. Moreover, membrane labelingshowed that without such co-incubation, the apoptotic lym-phocyte subpopulation increased 32.5% (not significantand 64.3% (P 0.01), respectively, 48 h after doses of 1and 2 Gy (Fig. 5b, left panel), while the comparable figureswith mitochondrial labeling were 49.8% (P 0.001) and78.2% (P 0.001) (Fig. 5b, right panel). These percentages are consistent with those described above.

Our data show clearly that apoptotic lymphocytes induced by radiation disappeared in the presence of macrophages. Specifically, the percentage of apoptotic lympho-cytes measured with mitochondrial labeling decreased63.6% (P 0.001) and 74.5% (P 0.001) after doses of






FIG. 5. Quantification of apoptotic lymphocytes after a 48-h lympho-

cyte and nonirradiated macrophage co-culture. Lymphocyte were isolatedfrom human blood and exposed to doses of 1 and 2 Gy (0.5 Gy min1)60Co rays. Panel a: Macrophages were obtained after in vitro differ-entiation of human blood monocytes and co-cultured with lymphocytes(macrophage:lymphocyte ratio was 1:4). Panel b: Apoptosis of lympho-cytes was detected after membrane labeling (left panel) and after mito-chondrial labeling (right panel). Data are expressed as the means SEMof six different experiments. Statistical analyses were carried out by atwo-way analysis of variance. When appropriate, all pairwise multiplecomparisons were performed. *P 0.05; **0.05 P 0.01; ***0.01 P 0.001.

panel). Similarly, phagocytotic activity decreased 43% and

48% 48 h after identical exposures but when the co-incu-bation was with lymphocytes that had received 2 Gy of radiation.

The basal level of apoptotic lymphocytes remaining inthe co-culture supernatant was 5 to 15% when nonirradiatedmacrophages were co-incubated with irradiated lympho-cytes (Fig. 6b, right panel). These percentages remain un-affected when macrophages irradiated with 2 Gy were co-incubated with irradiated lymphocytes. When we comparedthese percentages with the basal level of apoptotic lympho-

cytes without macrophage co-incubation (Fig. 5b, right pan-

el), we noted a decrease of 72.1% for lymphocytes irradi-ated with 1 Gy and 76.7% for those irradiated with 2 Gy.

Macrophages exposed to 15 Gy rays retained a capacity for phagocytosis (Fig. 6b, right panel). Apoptotic lym-phocytes decreased by 42% after macrophages exposed to15 Gy were co-incubated with lymphocytes exposed to 2

Gy (Fig. 6b, right panel) compared to the level withoumacrophage co-incubation (Fig. 5b, right panel). Whenthese lymphocytes were co-incubated with macrophages ir-radiated with 30 Gy, there was no longer any differencebetween the percentages of apoptotic lymphocytes. Whenthese lymphocytes were co-incubated with macrophages ir-radiated with 30 Gy (Fig. 6b, right panel), the percentageof apoptotic lymphocytes no longer differed significantlyfrom the basal level without macrophage co-incubation(Fig. 5b, right panel). Thus the exposure of the macrophages to 30 Gy of rays totally inhibited the eliminationof apoptotic lymphocytes.

DISCUSSION

To determine what role apoptosis can play in assessingradiation injury, we need to know how long after irradiationapoptotic cells remain detectable ex vivo, how reliable in

vitro studies are in assessing radiation injury ex vivo, andwhat dose–response relationship exists between apoptosisand radiation in the tissue sampled. The results reportedhere contribute to a better understanding of these biologicaprocesses. In particular, this study characterized the discrepancy between in vitro and ex vivo apoptosis in lymphocytes after radiation exposure. We were thus able todevelop a model of in vitro phagocytosis of apoptotic lym-

phocytes that partially mimics commitment to apoptosiand clearance of these apoptotic cells.

We used a variety of biochemical assays that allow sen-sitive and rapid detection of apoptotic cells. The emergingconsensus is that optimal investigation of the processes thatlead to apoptosis can occur only with a multiparametricapproach. In the present study, we used three different as-says to quantify lymphocyte apoptosis. Regardless of thetechnique, dose–effect relationships were observed in vitro

after radiation exposure. Our data corroborate those reported elsewhere (9). Moreover, our data and those of Borehamet al. (10) point out the possibility of using FADU assaysto visualize the repair of initial radiation-induced DNAdamage; most of this damage is repaired within hours ofexposure. The reappearance of DNA damage is believed toresult from DNA fragmentation within the cell that is undergoing apoptosis. The techniques we used were particu-larly sensitive to relatively low doses of radiation (0 to 2Gy). Hertveldt et al. (9) demonstrated that doses of radia-tion as low as 0.1 Gy induce detectable apoptosis, a findingthat suggests the absence of any threshold level of ionizingradiation with regard to the induction of apoptosis. Dose–response curves for apoptosis were characterized by a flattening of the curve for doses greater than 3 Gy, which




FIG. 6. Evaluation of in vitro clearance of apoptotic cells in a lymphocyte and macrophage co-culture model.Panel a shows the phagocytotic capacity of macrophages after radiation exposure, checked by opsonization of inactivated yeast (macrophage:yeast ratio was 1:3). Panel b, left frame, shows the confocal microscopy quantificationof phagocytosis of TAMRA-labeled apoptotic lymphocytes by macrophages after radiation exposure. Panel b, rightframe, shows flow cytometry quantification of apoptotic lymphocytes present in the supernatant of the lymphocyteand macrophage co-culture (macrophage:lymphocyte ratio was 1:4). M0, M2, M15 and M30 correspond respectivelyto nonirradiated macrophages and macrophages irradiated with 2, 15 and 30 Gy (0.8 Gy min1, 60Co rays). Ly0,Ly1 and Ly 2 correspond respectively to lymphocytes irradiated with 0, 1 and 2 Gy (1 Gy min 1, 60Co rays). Dataare expressed as the means SEM of six different experiments. Statistical analyses were carried out by a two-wayanalysis of variance. When appropriate, all pairwise multiple comparisons were performed. * P 0.05; ***0.01

P 0.001.

suggests that above this dose level, the damage to the cellis so great that necrosis becomes the predominant type of cell death. The effect we observed was noted at 24 h post-irradiation and persisted for the 6 days of the experiment.This too is consistent with the observations of Hertveldt et

al. (9), who also found that the process leading to apoptosisin vitro became apparent only 24 h after cell incubation.They did not observe radiation-induced lymphocyte apo-ptosis during the first 12 h of culture. A plateau value wasreached 72 to 96 h after irradiation. The likely explanation

is that apoptotic lymphocytes may remain in the culturemedium without being physiologically eliminated and thusmay still be detectable for a long time after beginning theprocess. These cells ultimately become secondary necroticcells.

The ex vivo occurrence of radiation-induced apoptosisafter irradiation of animals was established previously, butthe ability to detect it remains critical to any possible useof apoptosis for assessing radiation injury. Currently, theapoptotic lymphocytes detected by studies of membrane




markers can be quantified only during the few hours afterthe animal’s exposure. Detection was best 4 h after 0.5 or1.5 Gy irradiation. With DiOC6, a fluorescent probe lo-cated on the mitochondria, apoptotic lymphocytes could bedetected ex vivo for up to 72 h postirradiation, with detec-tion optimal 48 h postirradiation. The literature concerning

ex vivo detection of apoptotic lymphocytes after radiationexposure is very sparse. Nevertheless, Castedo et al. (11)noted that detection was improved in HIV patients by usingmitochondrial rather than nuclear labeling techniques.Moreover, our ex vivo data show that induction of lympho-cyte apoptosis has two-wave kinetics, perhaps attributableto different lymphocyte subpopulations. Philippe et al. (12)showed that apoptosis in immune cell subsets of in vitro

cultured lymphocytes is correlated with differences in theradiation sensitivity of the subpopulations. Subsets with ashorter ex vivo life span are more susceptible to in vitro

apoptosis. B lymphocytes are known to be more radiosen-sitive than T lymphocytes or NK cells (13). Therefore, B

lymphocytes probably enter the process leading to apopto-sis first and correspond to the first wave we observed, whileT lymphocytes and NK cells, which are more resistant toradiation, may therefore enter the process later and contrib-ute to the second wave. The apparent discrepancy betweenin vitro and ex vivo data on the apoptosis of peripherallymphocytes may be resolved by assuming the migrationof pre-apoptotic cells or the efficient engulfment and rapiddegradation of dying cells. The 4-h delay we observedprobably corresponds to the ex vivo mobilization of phago-cytic systems after radiation exposure, as described in theliterature (14).

Phagocytosis of apoptotic cells plays an important role

in clearing nonfunctional dying cells and thereby prevent-ing the release of toxic cellular enzymes and other by-prod-ucts that might stimulate a potent inflammatory response.The ability to recognize and phagocytose apoptotic cellshas evolved as a protective mechanism to prevent dyingcells from disrupting general homeostasis (8 ). The phago-cytic cells known to be involved in this process includemacrophages, fibroblasts (15) and endothelial cells (16 ).Here we describe a human cell model of in vitro phago-cytosis of apoptotic lymphocytes by monocyte-derivedmacrophages. First an inactivated yeast opsonization assayenabled us to verify the phagocytic capacity of adherentmacrophages under our experimental conditions. After 48h culture without radiation exposure, the basal proportionof active macrophages was 40–50%. Forty-eight hours afterirradiation of macrophages with a dose of 2, 15 or 30 Gy,the phagocytotic capacity decreased progressively, evenwhen macrophage mortality was unaffected in our experi-mental conditions. Macrophages are described in the liter-ature as very radioresistant. As Buescher and Gallin (17 )pointed out, their in vitro survival for the first 3 weeks of culture decreases significantly only at doses of 25 to 50 Gy.As our data showed, however, their physiological phago-cytotic properties may be altered by lower doses, at least

10 Gy, as shown our data. Because this decrease in phago-cytotic capacity after radiation exposure is controversia(18, 19), we verified by confocal microscopy after TAMRAlabeling that apoptotic lymphocytes were really phagocy-tosed. Current assays to verify cell phagocytosis by mac-rophages are unable to distinguish apoptotic cells clearly

from the adherent mechanisms involved in other cell-to-cellinteractions, but the technique proposed by Hess et al. (16

overcomes this technical limitation. Thus, after 48 h of culture with cells not exposed to radiation, we observed abasal proportion of active macrophages ranging from 70 to95%. The difference in the phagocytotic capacity of mac-rophages co-cultured with lymphocytes or co-incubatedwith inactivated yeast may be explained by the ability ofapoptotic lymphocytes to release biological factors thastimulate macrophage activity (18 ). Our data show thaphagocytotic activity increases when macrophages are coincubated with radiation-induced apoptotic lymphocytesthe number of which also increases, dose dependently. The

relatively small changes in the fraction of macrophagehaving ingested at least one lymphocyte (from 75 to 95%)after lymphocyte irradiation are presumably due to the highratios of lymphocytes to macrophages in the phagocytosisassays, and might have been made more dramatic by reducing this ratio. The increased phagocytosis by the mac-rophages in the co-cultures was corroborated by the decreased number of apoptotic lymphocytes in the supernatants as measured by flow cytometry. These data must beconsidered a mirror image of the data described above. Taken together, they provide new information about the efficiency of clearance of apoptotic lymphocytes by phagocytesystems after radiation exposure. Our data suggest that ra

diation affects macrophage phagocytosis by modifying themechanical properties of the phagocytotic process rathethan by altering sensitivity of macrophages to the biologicalfactors released by lymphocytes after irradiation. The dis-crepancy in the change in the phagocytotic capacity, observed after in vitro and ex vivo irradiation, must also beborne in mind. In particular, the ex vivo presence of different cell types interacting and controlling macrophagephagocytosis may be modified—positively or negatively—after ex vivo radiation exposure (18 ).

The detection of apoptotic lymphocytes ex vivo requiresthat it be possible to verify early events of the process leading to apoptosis—before phagocytosis becomes efficientOur model of in vitro phagocytosis was used to evaluatewhether any possible markers of apoptosis were expressedbefore phagocytic recognition of apoptotic cells. Mitochon-drial labeling appears to be a more valuable probe thanmembrane labeling for detecting early apoptotic cells be-fore their ingestion by macrophages. Other markers mayalso be promising for quantifying this process ex vivo. Durrieu et al. (20) observed measurable amounts of caspase 3activity in cells committed to apoptosis. Moreover, Hug e

al. (21) have described a new substrate that makes it pos-sible to measure caspase 3 activity directly in intact cells




In our opinion, this type of assay is a prerequisite for thedevelopment of a diagnostic method for detecting apoptosisex vivo in the pathophysiological situations where this pro-cess plays a role. Complementary studies are necessary todetermine the useful dose range and the sensitivity thresh-old of this technique. We are currently investigating the

application of this method to blood samples from patientsundergoing radiotherapy. Those findings should make itpossible to determine finally whether radiation-induced ap-optosis can serve a biomarker for ionizing radiation.

ACKNOWLEDGMENTS

This research was supported by Electricite France (EDF).

Received: November 20, 2001; accepted: May 2, 2002

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