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The use of non-radioactive chromium as an alternative to 51Cr in NK assay
JOURNAL OF IWJNO&OGICAL ELSEVIER
Journal of Immunological Methods 186 (1995) 101-110
The use of non-radioactive
chromium as an alternative to ?r NK assay
Paola Borella a,*, Annalisa Bargellini a, Stefano Salvioli b, Cristina Incerti Medici Andrea Cossarizza b
in a ,
a Dipartimento di Scienze Biomediche, Sezione di Igiene e Microbiologia, Uniuersit6 degli Studi, Via Campi 287, I-41 100 Modena, Italy h Diparfimento di Scienze Biomediche, Sezione di Patologia Generale, Uniuersitci degli Studi, Modena, ltalv
Received 25 October 1994; revised 26 January 1995; accepted 1 June 1995
Abstract A novel method to measure target cell cytolysis based on the use of ‘cold’, non-radioactive chromium and on the determination of metal release by graphite furnace atomic absorption spectroscopy (FAAS) is proposed. Natural killer (NK) assays were performed by labelling target cells with chromium as Na,CrO,, and results were compared with those obtained by conventional overnight labelling with “Cr of targets killed by the same effecters. The cytotoxic capacity of peripheral blood lymphocytes from healthy subjects was evaluated, and NK activity measured with both methods showed a good agreement at each of the tested effector to target cell ratios (between 1OO:l and l:l), with a high and significant coefficient of correlation (I = 0.931, p < 0.0001). The selection of the appropriate Cr concentrations for Iabeliing target cells took into account both the sensitivity of our instrumentation and the possible toxic effects of the metal. A study of the effects of Cr on the cell line (K562) which is usually employed as a target in NK tests showed that Cr could have a detrimental effect on cellular function, with significant numbers of cells with depolarised mitochondria and reduced DNA synthesis after 24 h incubation using Cr levels higher than 15 pmol/l (780 pg/l). The method proposed here has a number of advantages, including the use of a non-radioactive tracer, limited costs, high sensitivity and reproducibility, and the possibility of storing samples. In addition, the technique uses a fixed Cr concentration which is known to be non toxic. Keywords:
Non-radioactive labeling; Chromium cell toxicity; Mitochondrial
Atomic absorption membrane potential
spectroscopy;
Cytotoxicity
assay; Natural
killer
cell;
1. Introduction
Abbreviations: %Zr, chromium-51; Cr, chromium; NK, natural killer; K562, human erythroleukaemic cell line; E:T, effector to target cell, FAAS, flameless atomic absorption spectroscopy; JC-1, 5,5’,6,6’-tetrachloro-1,1’.3,3-tetraethylbenzimidazolcarbocyanine iodide. * Corresoonding author. Tel.: + 39 59 360084: Fax: + 3959363057; e-mail: [email protected]. 0022-1759/95/$09.50 0 1995 Elsevier SSDI 0022.1759(95)00140-9
Science
The chromium-51 (51Cr) release assay has been extensively used in the evaluation of natural killer (NK) cell activity for the direct detection of target cell lysis (Sanderson, 1964; Wigzell, 1965; Brunner et al., 1968). However, the method suffers from a number of well known disadvantages, in-
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eluding the use of a radioactive tracer that emits gamma rays with health hazards for workers, limited storage time due to the short half-life of 51Cr and the high costs associated both with the tracer and the facilities required for waste disposal. For these reasons, a number of new methods have been proposed for the detection of cytolysis, such as the use of fluorescent markers (Bruning et al., 1980; Brenan and Parish, 1988), the measurement of endogenous enzymes released from the cells (Szekeres et al., 1981; Korzeniewski and Callewaert, 1983), and automated calorimetric assays (Parish and Miillbacher, 1983). However, none of these techniques has been widely adopted. The use of the non-radioactive element, europium (Blomberg et al., 1986; Bouma et ai., 1992), as well as radioactive 75Se (Leibold and Bridge, 1979; Heymer and Leibold, 1993) have recently been suggested on the basis of their sensitivity and rapidity, but, at present, neither tracer is available at a reasonable cost. In previous papers, we have reported that a number of metals accumulate in metabolising human cells such as lymphocytes, and that their uptake can be followed and measured using the flameless atomic spectroscopy absorption technique (FAAS) (Borella et al., 1990; Borella and Giardino, 1991). The very high sensitivity and accuracy of this technique led to the suggestion of adopting a non-radioactive metal such as chromium to label target cells in NK assays, and to evaluate its release due to cell lysis in terms of absolute metal concentrations. In this paper, we present the results obtained using chromium as Na,CrO,, and we compare the uptake and release of ‘cold’ Cr with that of “Cr. In previous studies on the biological effects of trivalent and hexavalent chromium, we demonstrated that, even at very low doses, the hexavalent form of Cr was able to interfere with the functional characteristics of lymphocytes (Borella and Bargellini, 1993). Such effects were related to the capability of Cr to accumulate in cells. Although radioactive Cr is considered a non-toxic substance for targets, a detrimental action towards K562 cells, an erythroleukaemic human tumor line, cannot be excluded. Thus, we thought it worthwhile to add Cr during the period cur-
rently used to label target cells with 51Cr and we subsequently studied both Cr uptake by target cells and the effects on a number of cellular parameters, focusing on DNA synthesis, mitochondrial activity and cell viability. The use of the same metal in the identical chemical form has the advantage of being as specific as the radioactive assay. Finally, the optimal labelling conditions and the sensitivity of our method were analysed and these findings are described in detail.
2. Materials and methods 2.1. Preparation of NK effector cells
Lymphocytes were separated from 20 ml peripheral blood of healthy volunteers, aged 25-42 years, using a modified Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient centrifugation procedure. Briefly, the heparinized blood was diluted with phosphate buffer solution (PBS pH 7.4) l:l, layered on the gradient and centrifuged 400 xg for 30 min at room temperature. Mononuclear cells from the interface were washed 3 times with Hanks’ balanced salts solution without Ca*+ and Mg2+ (HBSS) (Gibco BRL, Paisley, Scotland, UK) and resuspended in complete medium, i.e. RPM1 1640 supplemented with 10% heat-inactivated foetal calf serum (FCS, from Gibco Europe, Glasgow, Scotland), penicillin (950 IU/ml), and streptomycin (50 mg/l). Lower concentrations of cells were prepared by serial Z-fold dilutions in complete medium. 2.2. Target cells The human erythroleukaemic cell line K562 was used as the target (Lozzio and Lozzio, 19X>, and these cells were maintained in complete medium using tissue culture flasks (Greiner, Frickenhausen, Germany), with half medium changed every second day.
2.3. Labelling of target cells with jlCr Target cells (3 X 106) were incubated with 100 PCi of “chromium (sodium chromate in aqueous
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solution, from The Radiochemical Centre, Amersham, England; specific activity 250-500 mCi/mg of Cr) in 1.5 ml of complete medium for 18 h a 37°C in a humidified 5% CO4 atmosphere. Taking into account the decay of Cr, the volumes of radioactive solution added were SO-250 ~1, depending on the reference date. The Cr added varied between 320 and 1000 pg/l. At the end of the incubation, cells were washed three times with complete medium and adjusted to a concentration of 5 x lo4 cells/ml. In each experiment, 0.1 ml (5000 cells) of this suspension was used. 2.4. Labelling of target cells with ‘cold’ non-radioactive chromium
Target cells were labelled with Na,CrO, (BDH Limited Poole, England). A standard solution (1 mmol Cr/l) was daily prepared in sterile physiologic solution and 100 ~1 were added in the culture flasks containing 10 ml medium and approximately 3-5 x lo6 cells, to obtain a final solution of 10 pmol Cr/l, corresponding to 520 pg Cr/l. This concentration was selected after preliminary experiments carried out to define the optimum labelling conditions and did not change during the course of the experiments. After an 18 h incubation, we followed an identical procedure as for the ‘lCr assay, except that cells were adjusted to a concentration of lo5 cells/ml. This number of cells was selected on the basis of the detection limit of non-radioactive Cr by FAAS. 2.5. NK cytolytic assay Assays were carried out in parallel by dividing effector cells into two aliquots, one for ‘cold’ Cr and the other for 51Cr. 100 ,ul of labelled target cells (Cr or 51Cr) were pipetted into 96-well round bottom microplates (Corning Glass Work, New York). An equal volume (100 ~1) of different concentrations of effector cells was added to give effector to target (E:T) ratios from 1OO:l to 1:l. Each test was set up in quadruplicate. The plates were centrifuged for 2-5 min at 100 x g and incubated for 4 h in a humidified 5% CO, atmosphere at 37°C. After the incubation period, the plates were newly centrifuged and 100 ~1 of
Methods 186 (1995) 101-l 10
103
supernatant were immediately collected for measuring ‘cold’ Cr and 51Cr release. In both methods, the spontaneous release was determined by incubating (in quadruplicate) target cells with complete medium only (0.2 ml), and the maximum release was determined by lysing cells with a detergent (Nonidet P40, BDH, final concentration 2% v/v>, as previously described (Cossarizza et al., 1991). 2.6. Measurement of 5’Cr The radioactivity from the 51Cr release assay was measured in a CompuGamma counter (Beckman 5500). The percentage of specific marker release was calculated according to the formula: % of specific lysis experimental release - spontaneous release maximum release - spontaneous release x 100 2.7. Measurement of non-radioactive Cr Levels of chromium released into the supernatants were measured by an atomic absorption spectrophotometer (Perkin-Elmer mod. 5000) equipped with a graphite furnace system (HGA mod. 400), a tungsten-halogen arc background corrector and an autosampler (P.E. mod. 40). As a light source, hallow cathode lamp for chromium was used. The furnace heating procedure and the instrumental conditions were appropriately chosen for our material; the total time of specimen processing was less than 2 min. Before analysis, 10 ~1 of detergent solution (Nonidet P40) were added to the supernatant, and 20 ~1 of the sample were then automatically introduced in the furnace. A calibration curve was prepared daily in complete medium plus 2% detergent. Standard solutions of 1 mmol/l of Cr were prepared using Na,CrO,, and working solutions were obtained in the range 1-15 pg/l (approximately 0.02-0.3 pmol/l) within the linearity section of the curve. The analytical sensitivity of the method was 3.5 Pg.
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2.8. Chromium uptake by K562 cells
In order to evaluate the trend of Cr accumulation in target cells, sodium chromate was added to the culture medium over the range 5-25 pmol/l (260-1300 pg/l), and cells were incubated for 24 h. Controls consisted of tubes incubated for the same time, without the addition of metal. For each concentration, experiments were carried out in duplicate, and repeated three times. At the end of the incubation, cells were washed three times with complete medium and counted. Aliquots of lo4 cells in 0.2 ml were collected and lysed with 2% Nonidet P40. Then, the Cr content was measured by FAAS. 2.9. Thymidine incoporation
into K562 cells
K562 cells, resuspended in complete medium, were adjusted at 3.5 x lo5 cells/ml, and added with eight different Cr concentrations, over the range 2-200 pmol/l (104-10400 pg/l). Each experiment was performed at least in duplicate. Cells were incubated for 24 h, washed three times to remove chromium in the solution and added with 1.0 ~1 of [3H]thymidine c3H-TdR, 15 Ci/mmol, New England Nuclear). After a 4 h incubation stage, cells were disrupted with digitonin (3 g/l in PBS). 3H-TdR incorporation was determined by measurement of trichloro-acetic acid (TCA)-precipitable radioactivity contained in the homogenates which were recovered on Whatman GF/C filters. Radioactivity was determined in a Packard liquid scintillation spectrometer with a toluene-based liquid scintillation fluid. The counting efficiency was about 40%. The values were expressed in dpm, and represented the mean of duplicate tubes. Control tubes, i.e. cultures without the addition of metal, were tested in parallel.
2.10. Analysis of mitochondrial membrane potential
Mitochondrial membrane potential was measured by a new cytofluorimetric method which we have recently described, and based on the use of the lipophilic cationic probe 5,5’,6,6’-tetrachloro-
1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide (JC-1, from Molecular Probes, Eugene, OR, USA) (Cossarizza et al., 1993, 1994). This molecule, able to selectively enter into mitochondria (Smiley et al., 1991) exists in a monomeric form emitting at 527 nm after excitation at 490 nm. However, depending on the membrane potential, JC-1 is able to form J aggregates that are associated with a large shift in emission (590 nm) (Hada et al., 1977; Reers et al., 1991). Thus, the colour of the dye changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarised (Smiley et al., 1991). Both colours can be detected using the filters commonly mounted in flow cytometers, so that green emission can be analyzed in fluorescence channel 1 (FLl) and greenish orange emission in channel 2 (FL2). Cell suspension was adjusted to a density of 0.5 X lo6 cells/ml and incubated in complete medium for 10 min at room temperature in the dark with 10 pg/ml JC-1. JC-1 was dissolved and stored according to the manufacturer’s instruction. At the end of the incubation period cells were washed twice in cold PBS, resuspended in a total volume of 400 ~1 and analyzed. 2.11. Cell viability Viability of K562 cells was studied by the method of propidium iodide (PI) exclusion, as previously described (Cook and Mitchel, 1989). Briefly, samples were incubated with 10 pg/ml PI in isotonic solution for 5 min at room temperature in the dark, and immediately analysed by flow cytometry. 2.12. Flow cytometry A FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a single 488 nm argon laser was used. In this instrument, the filter in front of the FL1 photomultiplier (PMT) transmits at 530 nm and has a bandwidth of 30 nm, the filter used in the FL2 channel transmits at 585 nm and has a bandwidth of 42 nm. For the analysis of cells stained with JC-1, the PMT value of the detector in FL1 was set at
P. Borella et al. /Journal
of Immunological Methods 186 (1995) 101-110
105
360 V, FL2 PMT at 310 V; FLl-FL2 compensation was 4.0%, FL2-FL1 compensation was 12.0%. A minimum of lo5 cells per sample were acquired in list mode, and analysed with Lysys II software (Becton Dickinson).
Table 1 NK activity in comparative assays where the same target cells (K562) were labelled either with ‘cold’ non-radioactive Cr or with 51Cr, and killed by the same NK effector cells
E:T ratio
‘Cold’ Cr
+r
2.13. Statistical analysis
1OO:l 5O:l 25:l 12:l 6:l 3:l 1:l
52.63 k 15.39 43.41+ 14.63 31.46 f 10.95 25.33 + 9.59 18.13 * 8.84 12.26 + 8.52 7.61+ 6.60
54.14 * 15.80 43.84+ 16.18 31.46+ 12.23 24.04 f 11.22 16.16k9.89 9.81 k 6.27 6.40 f 4.22
A paired Student’s t test was used to compare the percentages of lysis in the NK assays run simultaneously at each of the E:T ratios. Linear regression analysis was performed to correlate results obtained with the same effecters and targets labelled either with 51Cr or cold Cr. Analysis of variance for repeated measures and the Newman-Keuls t test were applied to analyse differences in 3H-TdR incorporation (expressed as dpm), cell viability (expressed as the percentage of viable cells) and mitochondrial activity (expressed as the percentage of cells with depolarised mitochondria) with increasing Cr concentrations. Some figures are shown as boxplots, obtained by SPSS for Windows software. Boxplots are formed from ‘boxes’ which delineate the 50% of values falling between the 25th and 75th percentiles, and the ‘whiskers’ which represent lines that extend from the box to the highest and lowest values, excluding outliers, which are identified with a black circle. A line inside the box indicates the median.
%specific marker release (mean f SD)
No significance by paired Student’s t test.
3. Results
K562 target cells at an effector to target cells ratio of 1OO:l was 52.63 k 15.39% with our method, compared to 54.14 k 15.80% in simultaneously run ‘*Cr release assays. Furthermore, in both cases it was possible to appreciate individual NK responsiveness. In a low responder, the percentages were 26.0 versus 19.7, respectively, and in a high responder they were 70.8 versus 60.5 (E:T = 1OO:l). The spontaneous release was 15.7 + 1.8% (five experiments) using ‘cold’ Cr and slightly higher using 51Cr (17.9 f 2.5%). Re-examination of the samples after storage in either freezer or refrigerator up to one week did not change the results obtained by FAAS (not shown). The relationship between the results obtained using the two methods and the relative regression analysis are presented in Fig. 1, which shows a highly significant correlation between the non-radioactive and radioactive Cr release assays.
3.1. NK cell cytotoxic assays
3.2. Eualuation of Cr uptake
The comparison of NK cytolysis from cells labelled with non-radioactive Cr and “Cr showed a very good agreement when the mean percentages measured in seven healthy subjects were calculated (Table 1). Indeed, at effector to target cell ratios ranging from 1OO:l to l:l, no statistical difference was observed using Student’s t test for paired data, and this tendency was the same at all the E:T ratios. The standard deviations in both assay systems were also comparable. In the 4 h assay, the specific release from the NK sensitive
In the experiments carried out to define the trend of Cr uptake in K.562 cells, and optimal Cr labelling, a progressive increase in intracellular Cr was noted with increasing metal concentrations. After 24 h incubation, the amount of Cr incorporated was directly proportional to the concentration of Cr used over the range 5-10 kmol/l and corresponded to 1.60 f 0.22 nmol/106 cells and 3.23 f 0.50 nmol/106 cells, respectively. Interestingly, a non linear accumulation appeared to be associated with metal levels
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P. Borella et al. /Journal
of Immunological
in the percentage of cells with depolarised mitochondria was observed with increasing Cr concentrations (Fig. 2 lower). Also, in this case, the results were significant for Cr levels corresponding to 15 kmol/l with a mean of 24.2% of cells with depolarised mitochondria. For higher Cr levels, the number of cells with altered mitochondrial membrane potential ranged between 30.7%
0.937 pco.ooo1 r =
IdcT O&
13
15 0
‘8
0’0
0
_._.~..-.
bo
0
,;
Methods 186 (1995) IO1 -110
r, 0
15
30
45
._.~_ 60
75
“Cold” Chromium Release
Fig. 1. Correlation between ‘cold’ Cr and ‘rCr release in NK assays run simultaneously. Each point represents the value of paired observations for all the E:T ratios tested in the subjects examined.
? z‘ *OI 0
1
50
.
I
x
E
a z
higher than 10 pmol/l; thus, with a double dose, intracellular Cr was only 40% higher (4.51 f 0.67 nmol/106 cells). The levels of Cr found in these experiments were in line with values obtained in the NK assays. Overnight incubation (18-20 h) with 10 ~mol/l Cr was associated with a maximum release of 2.87 k 0.61 nmolCr/106 cells, and a minimum (spontaneous) release of 0.53 _+ 0.20 nmolCr/106 cells (mean of five different experiments).
40
-7
r-l
I
o!_
3.3. Evaluation of Cr cytotoxicity When we studied the effects of Cr over the range 2-200 pmol/l (104-10400 pg/l) on some functional characteristics of K562 cells, Cr had a detrimental effect on both DNA synthesis and mitochondrial activity. A progressive reduction in 3H-TdR incorporation was noted with increasing Cr concentrations in the culture medium (Fig. 2 upper). For Cr levels of 15 pmol/l, the decrease reached a mean of 41% and was significantly lower than that of untreated cells. Incorporation was less than 30% at the highest tested concentration (200 ~mol/O. Cr toxicity was also confirmed by the analysis of mitochondrial function. A progressive increase
7 0
nl “:
0
2
5
10
15
25
50
.A
100
I
200
Chromium added (umollL)
Fig. 2. Modifications in K.562 cells after 24 h of incubation with increasing chromium concentrations. Upper: DNA synthesis expressed as tritiated thymidine (3H-TdR) incorporation (five experiments). Analysis of variance shows an F index = 8.19, with p < 0.001. Laoer: percentage of K562 celb with depolarized mitochondria (four experiments). Analysis of variance shows an F index = 13.81, with p < 0.001. Data are shown in form of box plots (see text for explanation). The box plots marked with an asterisk have a significance of p < 0.05 compared to untreated cells according to Student-NewmanKeuls test.
P. Borella et al. /Journal
4. Discussion
and 40.7%. A representative example is shown in Fig. 3. Under our experimental conditions (24 h incubation), cell viability was slightly, but progressively reduced by the presence of Cr (Fig. 4); thus, when the dose of Cr added was 200 pmol/l, cell viability was 88.0 f 2.2%. 98%
7
-
3
107
of Immunological Methods 186 (1995) 101-110
Lo
In this paper we describe a new NK cell assay performed by labelling cells with non-radioactive chromium, and evaluating the samples by flameless atomic absorption spectroscopy (FAAS). This method allowed us to analyse the possible toxic r
88::
415
CONTROL
Lc
uM
I* i
FLI-HVLl-Height
FLl-WFLI-Height
--->
-->
88% Co
r
1
25
a
UN
/
FLl-HYFLI-Height
--->
FLl-H\FLl-Height
--->
Fig. 3. Cytofluorimetric analysis of cells treated with different doses of cold chromium (15, 25 and 100 FM) and stained Numbers indicate the percentage of cells with depolarized mitochondria. Abscissa: FLI; ordinate: FL2.
with JC-1.
P. Borella et al. /Journal
108
of Immunological Methods 186 (1995) 101-110
100
s?
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90 m =
b m
4
80
5 6 -? 0
70I,
6CI__ 0
2
5
10
15
25
50
100
200
Chromiumadded (i.rmol/L) Fig. 4. Cell viability measured by propidium iodide exclusion test in K562 cells after 24 h of incubation with increasing chromium concentrations. Data are shown in form of box plots (see text for explanation), and represent four experiments. Analysis of variance shows an F index = 5.77, with p < 0.001. No statistical significance exists between treated and untreated cells according to Student-Newman-Keuls test, except for those treated with 100 pmol Cr (asterisk).
effects of Cr on cell viability, DNA synthesis and mitochondrial function, as well as permitting us to demonstrate that the concentrations commonly used in radioactive assays can have detrimental effects on some target cell functions. The comparison of per cent specific release from cells labelled with either Cr or ‘?r showed a good correlation at each of the effector to target ratios tested (between 1OO:l and 1:l). The sensitivity and precision of our method was comparable to the radioactive system. Due to the use of the same marker (Cr as Na,CrO,), the relatively slow release from the target cells does not reduce the assay time, whereas shorter incubation times are possible using europium (Blomberg et al., 1986). The time required to measure Cr by FAAS would be a disadvantage, especially with large assays. However, the length of time required for sample reading is largely counterbalanced by the use of a safe, non-radioactive material, and by the possibility of long storage without any modification of the Cr. Furthermore, the limited volume required for FAAS readings means that the analysis can be repeated on the same sample. Studies are underway to analyse
the reiiability of a faster furnace program able to reduce the sample reading time to about 1 min. To evaluate optimal Cr addition to the K562 cultures, we studied Cr uptake in lo4 cells after 24 h incubation. For levels up to 10 pmol/l, we found a good linear Cr uptake corresponding to about 14.5% of Cr added to the culture medium. We selected 10 pmol/l (520 pg/l) overnight (about 20 h) as the optimal conditions for Cr labelling to guarantee good linearity, reproducibility and sufficient Cr levels. Under those conditions, the minimum release assay gave values corresponding to about 7-fold the detection limit of our instrumentation, a level sufficiently high to assure good reproducibility. The selected optimal concentration of Cr also took into account the possible loss of function caused by the metal. It is interesting that significant modifications in K562 cells occurred with increasing Cr concentrations (Figs. 2 and 3). The cut-off level appears to be 15 kmol/l (780 pg/l), above which Cr-induced functional loss significantly increased. The existence of parallel trends for these two phenomena was suggested in previous studies carried out on human lymphocytes (Borella and Bargellini, 1993). After 24 h incubation, cell viability was only slightly reduced even at the highest tested concentration, but clear signs of functional impairment were observed. With the addition of 15 pmol/l of Cr, there was a significant decrease in DNA synthesis (over 40%) and a marked increase in cells with depolarised mitochondria (24%). The fact that the percentage of cells with altered mitochondrial function was much lower than that of dead cells suggests that mitochondria are an important early target of the toxic effects of chromium. Moreover, as described for other damaging agents (Cossarizza et al., 1994, Tropea et al., 19951, cells with depolarised mitochondria, i.e. with a dramatic loss of energy charge, seem to be those undergoing cell death. Although we could not prove that such a detrimental effect would affect the cytolytic assay, we suggest that functionally impaired cells might respond differently to the lytic effects of NK cells. With the present method, it is possible to verify day by day cell sensitivity to Cr uptake and
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of Immunological Methods 186 (1995) 101-110
release, since the selected dose does not vary among different experiments. Moreover, a detectable level of Cr can be obtained using a non-toxic concentration of Cr. With the traditional method, the very fast decay time of 51Cr dictates the volume of radioactive tracer to be used for labelling (usually from 100 to 300 ~1) and in many laboratories the radioactive substance is added in 100-300 ~1 medium (Press and Baines, 1982; Strayer et al., 1984; Blomberg et al., 1986; Barone et al., 1989; Kusaka et al., 1992). Under such conditions, we calculate that if in the initial solution Cr content is 5 pg/ml (or 5 pg/mCiI, the metal added can easily reach levels as high as 2000-6000 pug/l (approximately 40-100 ~mol/l), which are toxic for target cells. Thus, caution is required to avoid an excess Cr concentration in the labelling step, and the use of a well defined controlled Cr addition substantially improves the quality of the cytotoxic assay. In general, we suggest using no more than lpg Cr/ml cell solution for no more than 18-20 h incubation. The radiotoxic effects of 51Cr were not considered in this study but normally must be taken into account. As far as the incubation time is concerned, preliminary experiments showed that a good labelling efficiency, with negligible toxicity, can be obtained by treating cells with l-2.5 pg Cr/ml for 1 h (manuscript in preparation). This new labelling technique based on the use of non-radioactive metal and FAAS analysis is promising and produces good results in the detection of NK cell activity. The method has a number of definite advantages: 1. the use of a non-radioactive tracer with no hazards for the health of workers; 2. a significant reduction in costs as the required chromium salt (Na,CrO,) is inexpensive, and the only equipment change required is the graphite furnace; 3. the possibility of storing samples (and perhaps labelled cells) and repeating the readings; 4. the possibility of working with a well-defined Cr concentration. This assures a non-toxic addition of the metal and a strict control of cell sensitivity to Cr uptake and release; 5. a very high sensitivity and accuracy, which is similar to that of ‘lCr.
109
We are well aware that FAAS is not a cheap instrument; it requires specialised technical staff and is not standard equipment in most immunology laboratories which commonly use radioactivity assays. The real advantage of this method goes far beyond the substitution of ‘iCr. Indeed, at present, the method described here could be adopted by laboratories usually equipped with FAAS, and which may not be authorised to use radioactive reagents. They include those dealing with environmental and basic sciences, epidemiology, preventive medicine, toxicology, occupational and forensic medicine, where there is a growing interest in the relationships between stressful events, exposure to toxic substances, and unhealthy life styles on the one side, and immunological cytotoxic responses on the other (Cossarizza et al., 1990a; Cossarizza et al., 1990b; Sansoni et al., 1993; Franceschi et al., 1992, 1995). Lastly, our method could be extended to other metal tracers and could be applied to study other cytotoxicity models.
Acknowledgements
This work was partially supported by grants from the Minister0 Ricerca Scientifica e Tecnologica 60%. The authors thank Professor Claudio Franceschi for critically reading of the manuscript. They also acknowledge all volunteers who donated blood that was used in this study (donors and staff of the local AVIS).
References Barone, J., Hebert, J.R. and Reddy, M.M. (1989) Dietary fat and natural-killer-cell activity. Am. J. Clin. Nutr. 50, 861. Blomberg, K., Granberg, C., Hemmilae. I. and Liivgren, T. (1986) Europium-labelled target cells in an assay of natural killer cell activity. II. A novel non-radioactive methods based on time-resolved fluorescence. Significance and specificity of the method. J. Immunol. Methods 92, 117. Borella, P. and Bargellini, A. (1993) Effects of trace elements on immune system: results in cultured human lymphocytes. J. Trace Elem. Electrolytes Health Dis. 7, 231. Borella, P. and Giardino, A. (1991) Lead and cadmium at very low doses affect in vitro immune response of human lymphocytes. Environ. Res. 55, 165.
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Borella, P., Manni, S. and Giardino, A. (1990) Cadmium, nickel, chromium and lead accumulate in human lymphocytes and interfere with PHA-induced proliferation. J. Trace Elem. Electrolytes Health Dis. 4, 87. Bouma, G.J., Van der Meer-Prins, P.M.W., Van Bree, F.P.M.J., Van Rood, J.J. and Class, F.H.J. (1992) Determination of cytotoxic T-lymphocyte precursor frequencies using europium labelling as a nonradioactive alternative to labelling with chromium-51. Hum. Immunol. 35, 85. Brenan, M. and Parish, CR. (1988) Automated fluorometric assay for T cell cytotoxicity. J. Immunol. Methods 112, 121. Bruning, J.W., Kardol, M.J. and Arentzen, R. (1980) Carboxyfluorescein fluorochromasia assays. I. Non-radioactively labelled cell mediated lympholysis. J. Immunol. Methods 33, 33. Brunner, K.T., Mauel, J., Cerottini, J.-C. and Chapuis, B. (1968) Quantitative assay of the lytic action of immune Iymphoid cells on %r-Iabelled allogeneic target cells in vitro: inhibition by isoantibody and drugs. Immunology 14, 181. Cook, A. and Mitchel, J.B. (1989) Viability measurements in mammalian cell systems. Anal. Biochem. 179, 1. Cossarizza, A., Monti, D., Dagna-Bricarelli, F., Montagnani, G. and Franceschi, C. (1990a) LAK activity is inducible in blood mononuclear cells from human fetus. Immunol. Lett. 24, 137. Cossarizza, A., Monti, D., Montagnani, G., Ortolani, C., Masi, M., Zannotti, M. and Franceschi, C. (1990b) Precocious aging of the immune system in Down’s syndrome: alterations of B-lymphocytes, T-lymphocyte subsets and of cells with NK markers. Am. J. Med. Genet. 7 (suppl.), 213. Cossarizza, A., Ortolani, C., Forti, E., Montagnani, G., Paganelli, R., Zannotti, M., Marini, M., Monti, D. and Franceschi, C. (1991) Age-related expansion of functionally inefficient cells with markers of NK activity in Down’s syndrome. Blood 77, 1263. Cossarizza, A., Baccarani Contri, M., Kalashnikova, G. and Franceschi, C. (1993) A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5’,6,6’-tetrachloro1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem. Biophys. Res. Commun. 197, 40. Cossarizza, A., Kalashnikova, G., Grassilli, E., Chiappelli, F., Salvioli, S., Capri, M., Barbieri, D., Troiano, L., Monti, D. and Franceschi, C. (1994) Mitochondrial modifications during rat thymocyte apoptosis: a study at the single cell level. Exp. Cell Res. 214, 323. Franceschi, C., Cossarizza, A., Monti, D. and Ottaviani, E. (1991) Cytotoxicity and immunocyte markers in cells from the freshwater snail Planorbarius comeus (L.) (Gastropoda, Pulmonata): implications for the evolution of NK cells. Eur. J. Immunol. 21, 489. Franceschi, C., Monti, D., Sansoni, P. and Cossarizza, A. (1995) The immunology of exceptional individuals: the lesson of centenarians. Immunol. Today 16, 12. Hada, H., Honda, C. and Tanemura, H. (1977) Spectroscopic study on the J-aggregate of cyanine dyes. I. Spectral
Methods 186 (1995) 101-I 10
changes of UV bands concerned with J-aggregate formation. Photogr. Sci. Eng. 21, 83. Heymer, J., Leibold, W. (1993) Single and dual labelling of cytotoxic target cell: comparison of three radioactive tracers, [35S]methionine, [75Se]seleniomethionine, and chromium-51. J. Immunol. Methods 161, 217. Korzeniewski, C. and Callewaert, C.M. (1983) An enzyme-release assay for natural cytotoxicity. J. Immunol. Methods 64, 313. Kusaka, Y., Kondou, H. and Morimoto, K. (1992) Healthy lifestyles are associated with higher natural killer cell activity. Prev. Med. 21, 602. Leibold, W. and Bridge, S. (1979) 75 Se-release: a short and long term assay system for cellular cytotoxicity. Immunobiol., Z. Immun.-Forsch. 155, 287. Lozzio, C.B. and Lozzio, B.B. (1975) Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome. Blood 45,321. Parish, C.R. and Miillbacher, A. (1983) Automated colorimetric assay for T cell cytotoxicity. J. Immunol. Methods 58, 225. Pross, H.F. and Baines, M.G. (1982) Studies of human killer cells. I. In vivo parameters affecting normal cytotoxic function. Int. J. Cancer 29, 383. Reers, M., Smith, T.W. and Chen, L.B. (1991) J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480. Sanderson, A.R. (1964) Application of isoimmune cytolysis using radio-labelled target cells. Nature 204, 2.50. Sansoni, P., Cossarizza, A., Brianti, V., Fagnoni, F., Snelli, G., Monti, D., Marcato, A., Passeri, G., Ortolani, C., Forti, E., Fagiolo, U., Passeri, M. and Franceschi, C. (1993) Lymphocyte subsets and natural killer cell activity in healthy old people and centenarians. BLood 80, 2767. Smiley, ST., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, M., Smith, T.W., Steele, G.D. and Chen, L.B. (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA 88, 3671 Strayer, D.R., Carter, W.A., Mayberry, SD., Pequinot, E. and Brodsky, I. (1984) Low natural cytotoxicity of peripheral blood mononuclear cells in individuals with high familial incidences of cancer. Cancer Res. 44, 370. Szekeres, J., Pacsa, A.S. and Pejtsik, B. (1981) Measurement of lymphocyte cytotoxicity by assessing endogenous alkaline phosphatase activity of the target cells. J. Immunol. Methods 40, 151. Tropea, F., Troiano, L., Monti, D., Lovato, E., Malorni, W., Rainaldi, G., Mattana, P., Viscomi, G., Portolani, M., Cermelli, C., Cossarizza, A. and Franceschi, C. (1995) Sendai virus and Herpes virus type I induce apoptosis in human peripheral blood mononuclear cells. Exp. Cell Res., in press. Wigzell, H. (1965) Quantitative titration of mouse H-2 antibodies using ‘ICr labelled target cells. Transplantation 3, 423.
Journal of Immunological Methods 186 (1995) 101-110
The use of non-radioactive
chromium as an alternative to ?r NK assay
Paola Borella a,*, Annalisa Bargellini a, Stefano Salvioli b, Cristina Incerti Medici Andrea Cossarizza b
in a ,
a Dipartimento di Scienze Biomediche, Sezione di Igiene e Microbiologia, Uniuersit6 degli Studi, Via Campi 287, I-41 100 Modena, Italy h Diparfimento di Scienze Biomediche, Sezione di Patologia Generale, Uniuersitci degli Studi, Modena, ltalv
Received 25 October 1994; revised 26 January 1995; accepted 1 June 1995
Abstract A novel method to measure target cell cytolysis based on the use of ‘cold’, non-radioactive chromium and on the determination of metal release by graphite furnace atomic absorption spectroscopy (FAAS) is proposed. Natural killer (NK) assays were performed by labelling target cells with chromium as Na,CrO,, and results were compared with those obtained by conventional overnight labelling with “Cr of targets killed by the same effecters. The cytotoxic capacity of peripheral blood lymphocytes from healthy subjects was evaluated, and NK activity measured with both methods showed a good agreement at each of the tested effector to target cell ratios (between 1OO:l and l:l), with a high and significant coefficient of correlation (I = 0.931, p < 0.0001). The selection of the appropriate Cr concentrations for Iabeliing target cells took into account both the sensitivity of our instrumentation and the possible toxic effects of the metal. A study of the effects of Cr on the cell line (K562) which is usually employed as a target in NK tests showed that Cr could have a detrimental effect on cellular function, with significant numbers of cells with depolarised mitochondria and reduced DNA synthesis after 24 h incubation using Cr levels higher than 15 pmol/l (780 pg/l). The method proposed here has a number of advantages, including the use of a non-radioactive tracer, limited costs, high sensitivity and reproducibility, and the possibility of storing samples. In addition, the technique uses a fixed Cr concentration which is known to be non toxic. Keywords:
Non-radioactive labeling; Chromium cell toxicity; Mitochondrial
Atomic absorption membrane potential
spectroscopy;
Cytotoxicity
assay; Natural
killer
cell;
1. Introduction
Abbreviations: %Zr, chromium-51; Cr, chromium; NK, natural killer; K562, human erythroleukaemic cell line; E:T, effector to target cell, FAAS, flameless atomic absorption spectroscopy; JC-1, 5,5’,6,6’-tetrachloro-1,1’.3,3-tetraethylbenzimidazolcarbocyanine iodide. * Corresoonding author. Tel.: + 39 59 360084: Fax: + 3959363057; e-mail: [email protected]. 0022-1759/95/$09.50 0 1995 Elsevier SSDI 0022.1759(95)00140-9
Science
The chromium-51 (51Cr) release assay has been extensively used in the evaluation of natural killer (NK) cell activity for the direct detection of target cell lysis (Sanderson, 1964; Wigzell, 1965; Brunner et al., 1968). However, the method suffers from a number of well known disadvantages, in-
B.V. All rights reserved
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eluding the use of a radioactive tracer that emits gamma rays with health hazards for workers, limited storage time due to the short half-life of 51Cr and the high costs associated both with the tracer and the facilities required for waste disposal. For these reasons, a number of new methods have been proposed for the detection of cytolysis, such as the use of fluorescent markers (Bruning et al., 1980; Brenan and Parish, 1988), the measurement of endogenous enzymes released from the cells (Szekeres et al., 1981; Korzeniewski and Callewaert, 1983), and automated calorimetric assays (Parish and Miillbacher, 1983). However, none of these techniques has been widely adopted. The use of the non-radioactive element, europium (Blomberg et al., 1986; Bouma et ai., 1992), as well as radioactive 75Se (Leibold and Bridge, 1979; Heymer and Leibold, 1993) have recently been suggested on the basis of their sensitivity and rapidity, but, at present, neither tracer is available at a reasonable cost. In previous papers, we have reported that a number of metals accumulate in metabolising human cells such as lymphocytes, and that their uptake can be followed and measured using the flameless atomic spectroscopy absorption technique (FAAS) (Borella et al., 1990; Borella and Giardino, 1991). The very high sensitivity and accuracy of this technique led to the suggestion of adopting a non-radioactive metal such as chromium to label target cells in NK assays, and to evaluate its release due to cell lysis in terms of absolute metal concentrations. In this paper, we present the results obtained using chromium as Na,CrO,, and we compare the uptake and release of ‘cold’ Cr with that of “Cr. In previous studies on the biological effects of trivalent and hexavalent chromium, we demonstrated that, even at very low doses, the hexavalent form of Cr was able to interfere with the functional characteristics of lymphocytes (Borella and Bargellini, 1993). Such effects were related to the capability of Cr to accumulate in cells. Although radioactive Cr is considered a non-toxic substance for targets, a detrimental action towards K562 cells, an erythroleukaemic human tumor line, cannot be excluded. Thus, we thought it worthwhile to add Cr during the period cur-
rently used to label target cells with 51Cr and we subsequently studied both Cr uptake by target cells and the effects on a number of cellular parameters, focusing on DNA synthesis, mitochondrial activity and cell viability. The use of the same metal in the identical chemical form has the advantage of being as specific as the radioactive assay. Finally, the optimal labelling conditions and the sensitivity of our method were analysed and these findings are described in detail.
2. Materials and methods 2.1. Preparation of NK effector cells
Lymphocytes were separated from 20 ml peripheral blood of healthy volunteers, aged 25-42 years, using a modified Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient centrifugation procedure. Briefly, the heparinized blood was diluted with phosphate buffer solution (PBS pH 7.4) l:l, layered on the gradient and centrifuged 400 xg for 30 min at room temperature. Mononuclear cells from the interface were washed 3 times with Hanks’ balanced salts solution without Ca*+ and Mg2+ (HBSS) (Gibco BRL, Paisley, Scotland, UK) and resuspended in complete medium, i.e. RPM1 1640 supplemented with 10% heat-inactivated foetal calf serum (FCS, from Gibco Europe, Glasgow, Scotland), penicillin (950 IU/ml), and streptomycin (50 mg/l). Lower concentrations of cells were prepared by serial Z-fold dilutions in complete medium. 2.2. Target cells The human erythroleukaemic cell line K562 was used as the target (Lozzio and Lozzio, 19X>, and these cells were maintained in complete medium using tissue culture flasks (Greiner, Frickenhausen, Germany), with half medium changed every second day.
2.3. Labelling of target cells with jlCr Target cells (3 X 106) were incubated with 100 PCi of “chromium (sodium chromate in aqueous
P. Borella et al. /Journal
of Immunological
solution, from The Radiochemical Centre, Amersham, England; specific activity 250-500 mCi/mg of Cr) in 1.5 ml of complete medium for 18 h a 37°C in a humidified 5% CO4 atmosphere. Taking into account the decay of Cr, the volumes of radioactive solution added were SO-250 ~1, depending on the reference date. The Cr added varied between 320 and 1000 pg/l. At the end of the incubation, cells were washed three times with complete medium and adjusted to a concentration of 5 x lo4 cells/ml. In each experiment, 0.1 ml (5000 cells) of this suspension was used. 2.4. Labelling of target cells with ‘cold’ non-radioactive chromium
Target cells were labelled with Na,CrO, (BDH Limited Poole, England). A standard solution (1 mmol Cr/l) was daily prepared in sterile physiologic solution and 100 ~1 were added in the culture flasks containing 10 ml medium and approximately 3-5 x lo6 cells, to obtain a final solution of 10 pmol Cr/l, corresponding to 520 pg Cr/l. This concentration was selected after preliminary experiments carried out to define the optimum labelling conditions and did not change during the course of the experiments. After an 18 h incubation, we followed an identical procedure as for the ‘lCr assay, except that cells were adjusted to a concentration of lo5 cells/ml. This number of cells was selected on the basis of the detection limit of non-radioactive Cr by FAAS. 2.5. NK cytolytic assay Assays were carried out in parallel by dividing effector cells into two aliquots, one for ‘cold’ Cr and the other for 51Cr. 100 ,ul of labelled target cells (Cr or 51Cr) were pipetted into 96-well round bottom microplates (Corning Glass Work, New York). An equal volume (100 ~1) of different concentrations of effector cells was added to give effector to target (E:T) ratios from 1OO:l to 1:l. Each test was set up in quadruplicate. The plates were centrifuged for 2-5 min at 100 x g and incubated for 4 h in a humidified 5% CO, atmosphere at 37°C. After the incubation period, the plates were newly centrifuged and 100 ~1 of
Methods 186 (1995) 101-l 10
103
supernatant were immediately collected for measuring ‘cold’ Cr and 51Cr release. In both methods, the spontaneous release was determined by incubating (in quadruplicate) target cells with complete medium only (0.2 ml), and the maximum release was determined by lysing cells with a detergent (Nonidet P40, BDH, final concentration 2% v/v>, as previously described (Cossarizza et al., 1991). 2.6. Measurement of 5’Cr The radioactivity from the 51Cr release assay was measured in a CompuGamma counter (Beckman 5500). The percentage of specific marker release was calculated according to the formula: % of specific lysis experimental release - spontaneous release maximum release - spontaneous release x 100 2.7. Measurement of non-radioactive Cr Levels of chromium released into the supernatants were measured by an atomic absorption spectrophotometer (Perkin-Elmer mod. 5000) equipped with a graphite furnace system (HGA mod. 400), a tungsten-halogen arc background corrector and an autosampler (P.E. mod. 40). As a light source, hallow cathode lamp for chromium was used. The furnace heating procedure and the instrumental conditions were appropriately chosen for our material; the total time of specimen processing was less than 2 min. Before analysis, 10 ~1 of detergent solution (Nonidet P40) were added to the supernatant, and 20 ~1 of the sample were then automatically introduced in the furnace. A calibration curve was prepared daily in complete medium plus 2% detergent. Standard solutions of 1 mmol/l of Cr were prepared using Na,CrO,, and working solutions were obtained in the range 1-15 pg/l (approximately 0.02-0.3 pmol/l) within the linearity section of the curve. The analytical sensitivity of the method was 3.5 Pg.
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2.8. Chromium uptake by K562 cells
In order to evaluate the trend of Cr accumulation in target cells, sodium chromate was added to the culture medium over the range 5-25 pmol/l (260-1300 pg/l), and cells were incubated for 24 h. Controls consisted of tubes incubated for the same time, without the addition of metal. For each concentration, experiments were carried out in duplicate, and repeated three times. At the end of the incubation, cells were washed three times with complete medium and counted. Aliquots of lo4 cells in 0.2 ml were collected and lysed with 2% Nonidet P40. Then, the Cr content was measured by FAAS. 2.9. Thymidine incoporation
into K562 cells
K562 cells, resuspended in complete medium, were adjusted at 3.5 x lo5 cells/ml, and added with eight different Cr concentrations, over the range 2-200 pmol/l (104-10400 pg/l). Each experiment was performed at least in duplicate. Cells were incubated for 24 h, washed three times to remove chromium in the solution and added with 1.0 ~1 of [3H]thymidine c3H-TdR, 15 Ci/mmol, New England Nuclear). After a 4 h incubation stage, cells were disrupted with digitonin (3 g/l in PBS). 3H-TdR incorporation was determined by measurement of trichloro-acetic acid (TCA)-precipitable radioactivity contained in the homogenates which were recovered on Whatman GF/C filters. Radioactivity was determined in a Packard liquid scintillation spectrometer with a toluene-based liquid scintillation fluid. The counting efficiency was about 40%. The values were expressed in dpm, and represented the mean of duplicate tubes. Control tubes, i.e. cultures without the addition of metal, were tested in parallel.
2.10. Analysis of mitochondrial membrane potential
Mitochondrial membrane potential was measured by a new cytofluorimetric method which we have recently described, and based on the use of the lipophilic cationic probe 5,5’,6,6’-tetrachloro-
1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide (JC-1, from Molecular Probes, Eugene, OR, USA) (Cossarizza et al., 1993, 1994). This molecule, able to selectively enter into mitochondria (Smiley et al., 1991) exists in a monomeric form emitting at 527 nm after excitation at 490 nm. However, depending on the membrane potential, JC-1 is able to form J aggregates that are associated with a large shift in emission (590 nm) (Hada et al., 1977; Reers et al., 1991). Thus, the colour of the dye changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarised (Smiley et al., 1991). Both colours can be detected using the filters commonly mounted in flow cytometers, so that green emission can be analyzed in fluorescence channel 1 (FLl) and greenish orange emission in channel 2 (FL2). Cell suspension was adjusted to a density of 0.5 X lo6 cells/ml and incubated in complete medium for 10 min at room temperature in the dark with 10 pg/ml JC-1. JC-1 was dissolved and stored according to the manufacturer’s instruction. At the end of the incubation period cells were washed twice in cold PBS, resuspended in a total volume of 400 ~1 and analyzed. 2.11. Cell viability Viability of K562 cells was studied by the method of propidium iodide (PI) exclusion, as previously described (Cook and Mitchel, 1989). Briefly, samples were incubated with 10 pg/ml PI in isotonic solution for 5 min at room temperature in the dark, and immediately analysed by flow cytometry. 2.12. Flow cytometry A FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a single 488 nm argon laser was used. In this instrument, the filter in front of the FL1 photomultiplier (PMT) transmits at 530 nm and has a bandwidth of 30 nm, the filter used in the FL2 channel transmits at 585 nm and has a bandwidth of 42 nm. For the analysis of cells stained with JC-1, the PMT value of the detector in FL1 was set at
P. Borella et al. /Journal
of Immunological Methods 186 (1995) 101-110
105
360 V, FL2 PMT at 310 V; FLl-FL2 compensation was 4.0%, FL2-FL1 compensation was 12.0%. A minimum of lo5 cells per sample were acquired in list mode, and analysed with Lysys II software (Becton Dickinson).
Table 1 NK activity in comparative assays where the same target cells (K562) were labelled either with ‘cold’ non-radioactive Cr or with 51Cr, and killed by the same NK effector cells
E:T ratio
‘Cold’ Cr
+r
2.13. Statistical analysis
1OO:l 5O:l 25:l 12:l 6:l 3:l 1:l
52.63 k 15.39 43.41+ 14.63 31.46 f 10.95 25.33 + 9.59 18.13 * 8.84 12.26 + 8.52 7.61+ 6.60
54.14 * 15.80 43.84+ 16.18 31.46+ 12.23 24.04 f 11.22 16.16k9.89 9.81 k 6.27 6.40 f 4.22
A paired Student’s t test was used to compare the percentages of lysis in the NK assays run simultaneously at each of the E:T ratios. Linear regression analysis was performed to correlate results obtained with the same effecters and targets labelled either with 51Cr or cold Cr. Analysis of variance for repeated measures and the Newman-Keuls t test were applied to analyse differences in 3H-TdR incorporation (expressed as dpm), cell viability (expressed as the percentage of viable cells) and mitochondrial activity (expressed as the percentage of cells with depolarised mitochondria) with increasing Cr concentrations. Some figures are shown as boxplots, obtained by SPSS for Windows software. Boxplots are formed from ‘boxes’ which delineate the 50% of values falling between the 25th and 75th percentiles, and the ‘whiskers’ which represent lines that extend from the box to the highest and lowest values, excluding outliers, which are identified with a black circle. A line inside the box indicates the median.
%specific marker release (mean f SD)
No significance by paired Student’s t test.
3. Results
K562 target cells at an effector to target cells ratio of 1OO:l was 52.63 k 15.39% with our method, compared to 54.14 k 15.80% in simultaneously run ‘*Cr release assays. Furthermore, in both cases it was possible to appreciate individual NK responsiveness. In a low responder, the percentages were 26.0 versus 19.7, respectively, and in a high responder they were 70.8 versus 60.5 (E:T = 1OO:l). The spontaneous release was 15.7 + 1.8% (five experiments) using ‘cold’ Cr and slightly higher using 51Cr (17.9 f 2.5%). Re-examination of the samples after storage in either freezer or refrigerator up to one week did not change the results obtained by FAAS (not shown). The relationship between the results obtained using the two methods and the relative regression analysis are presented in Fig. 1, which shows a highly significant correlation between the non-radioactive and radioactive Cr release assays.
3.1. NK cell cytotoxic assays
3.2. Eualuation of Cr uptake
The comparison of NK cytolysis from cells labelled with non-radioactive Cr and “Cr showed a very good agreement when the mean percentages measured in seven healthy subjects were calculated (Table 1). Indeed, at effector to target cell ratios ranging from 1OO:l to l:l, no statistical difference was observed using Student’s t test for paired data, and this tendency was the same at all the E:T ratios. The standard deviations in both assay systems were also comparable. In the 4 h assay, the specific release from the NK sensitive
In the experiments carried out to define the trend of Cr uptake in K.562 cells, and optimal Cr labelling, a progressive increase in intracellular Cr was noted with increasing metal concentrations. After 24 h incubation, the amount of Cr incorporated was directly proportional to the concentration of Cr used over the range 5-10 kmol/l and corresponded to 1.60 f 0.22 nmol/106 cells and 3.23 f 0.50 nmol/106 cells, respectively. Interestingly, a non linear accumulation appeared to be associated with metal levels
106
P. Borella et al. /Journal
of Immunological
in the percentage of cells with depolarised mitochondria was observed with increasing Cr concentrations (Fig. 2 lower). Also, in this case, the results were significant for Cr levels corresponding to 15 kmol/l with a mean of 24.2% of cells with depolarised mitochondria. For higher Cr levels, the number of cells with altered mitochondrial membrane potential ranged between 30.7%
0.937 pco.ooo1 r =
IdcT O&
13
15 0
‘8
0’0
0
_._.~..-.
bo
0
,;
Methods 186 (1995) IO1 -110
r, 0
15
30
45
._.~_ 60
75
“Cold” Chromium Release
Fig. 1. Correlation between ‘cold’ Cr and ‘rCr release in NK assays run simultaneously. Each point represents the value of paired observations for all the E:T ratios tested in the subjects examined.
? z‘ *OI 0
1
50
.
I
x
E
a z
higher than 10 pmol/l; thus, with a double dose, intracellular Cr was only 40% higher (4.51 f 0.67 nmol/106 cells). The levels of Cr found in these experiments were in line with values obtained in the NK assays. Overnight incubation (18-20 h) with 10 ~mol/l Cr was associated with a maximum release of 2.87 k 0.61 nmolCr/106 cells, and a minimum (spontaneous) release of 0.53 _+ 0.20 nmolCr/106 cells (mean of five different experiments).
40
-7
r-l
I
o!_
3.3. Evaluation of Cr cytotoxicity When we studied the effects of Cr over the range 2-200 pmol/l (104-10400 pg/l) on some functional characteristics of K562 cells, Cr had a detrimental effect on both DNA synthesis and mitochondrial activity. A progressive reduction in 3H-TdR incorporation was noted with increasing Cr concentrations in the culture medium (Fig. 2 upper). For Cr levels of 15 pmol/l, the decrease reached a mean of 41% and was significantly lower than that of untreated cells. Incorporation was less than 30% at the highest tested concentration (200 ~mol/O. Cr toxicity was also confirmed by the analysis of mitochondrial function. A progressive increase
7 0
nl “:
0
2
5
10
15
25
50
.A
100
I
200
Chromium added (umollL)
Fig. 2. Modifications in K.562 cells after 24 h of incubation with increasing chromium concentrations. Upper: DNA synthesis expressed as tritiated thymidine (3H-TdR) incorporation (five experiments). Analysis of variance shows an F index = 8.19, with p < 0.001. Laoer: percentage of K562 celb with depolarized mitochondria (four experiments). Analysis of variance shows an F index = 13.81, with p < 0.001. Data are shown in form of box plots (see text for explanation). The box plots marked with an asterisk have a significance of p < 0.05 compared to untreated cells according to Student-NewmanKeuls test.
P. Borella et al. /Journal
4. Discussion
and 40.7%. A representative example is shown in Fig. 3. Under our experimental conditions (24 h incubation), cell viability was slightly, but progressively reduced by the presence of Cr (Fig. 4); thus, when the dose of Cr added was 200 pmol/l, cell viability was 88.0 f 2.2%. 98%
7
-
3
107
of Immunological Methods 186 (1995) 101-110
Lo
In this paper we describe a new NK cell assay performed by labelling cells with non-radioactive chromium, and evaluating the samples by flameless atomic absorption spectroscopy (FAAS). This method allowed us to analyse the possible toxic r
88::
415
CONTROL
Lc
uM
I* i
FLI-HVLl-Height
FLl-WFLI-Height
--->
-->
88% Co
r
1
25
a
UN
/
FLl-HYFLI-Height
--->
FLl-H\FLl-Height
--->
Fig. 3. Cytofluorimetric analysis of cells treated with different doses of cold chromium (15, 25 and 100 FM) and stained Numbers indicate the percentage of cells with depolarized mitochondria. Abscissa: FLI; ordinate: FL2.
with JC-1.
P. Borella et al. /Journal
108
of Immunological Methods 186 (1995) 101-110
100
s?
!*
90 m =
b m
4
80
5 6 -? 0
70I,
6CI__ 0
2
5
10
15
25
50
100
200
Chromiumadded (i.rmol/L) Fig. 4. Cell viability measured by propidium iodide exclusion test in K562 cells after 24 h of incubation with increasing chromium concentrations. Data are shown in form of box plots (see text for explanation), and represent four experiments. Analysis of variance shows an F index = 5.77, with p < 0.001. No statistical significance exists between treated and untreated cells according to Student-Newman-Keuls test, except for those treated with 100 pmol Cr (asterisk).
effects of Cr on cell viability, DNA synthesis and mitochondrial function, as well as permitting us to demonstrate that the concentrations commonly used in radioactive assays can have detrimental effects on some target cell functions. The comparison of per cent specific release from cells labelled with either Cr or ‘?r showed a good correlation at each of the effector to target ratios tested (between 1OO:l and 1:l). The sensitivity and precision of our method was comparable to the radioactive system. Due to the use of the same marker (Cr as Na,CrO,), the relatively slow release from the target cells does not reduce the assay time, whereas shorter incubation times are possible using europium (Blomberg et al., 1986). The time required to measure Cr by FAAS would be a disadvantage, especially with large assays. However, the length of time required for sample reading is largely counterbalanced by the use of a safe, non-radioactive material, and by the possibility of long storage without any modification of the Cr. Furthermore, the limited volume required for FAAS readings means that the analysis can be repeated on the same sample. Studies are underway to analyse
the reiiability of a faster furnace program able to reduce the sample reading time to about 1 min. To evaluate optimal Cr addition to the K562 cultures, we studied Cr uptake in lo4 cells after 24 h incubation. For levels up to 10 pmol/l, we found a good linear Cr uptake corresponding to about 14.5% of Cr added to the culture medium. We selected 10 pmol/l (520 pg/l) overnight (about 20 h) as the optimal conditions for Cr labelling to guarantee good linearity, reproducibility and sufficient Cr levels. Under those conditions, the minimum release assay gave values corresponding to about 7-fold the detection limit of our instrumentation, a level sufficiently high to assure good reproducibility. The selected optimal concentration of Cr also took into account the possible loss of function caused by the metal. It is interesting that significant modifications in K562 cells occurred with increasing Cr concentrations (Figs. 2 and 3). The cut-off level appears to be 15 kmol/l (780 pg/l), above which Cr-induced functional loss significantly increased. The existence of parallel trends for these two phenomena was suggested in previous studies carried out on human lymphocytes (Borella and Bargellini, 1993). After 24 h incubation, cell viability was only slightly reduced even at the highest tested concentration, but clear signs of functional impairment were observed. With the addition of 15 pmol/l of Cr, there was a significant decrease in DNA synthesis (over 40%) and a marked increase in cells with depolarised mitochondria (24%). The fact that the percentage of cells with altered mitochondrial function was much lower than that of dead cells suggests that mitochondria are an important early target of the toxic effects of chromium. Moreover, as described for other damaging agents (Cossarizza et al., 1994, Tropea et al., 19951, cells with depolarised mitochondria, i.e. with a dramatic loss of energy charge, seem to be those undergoing cell death. Although we could not prove that such a detrimental effect would affect the cytolytic assay, we suggest that functionally impaired cells might respond differently to the lytic effects of NK cells. With the present method, it is possible to verify day by day cell sensitivity to Cr uptake and
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release, since the selected dose does not vary among different experiments. Moreover, a detectable level of Cr can be obtained using a non-toxic concentration of Cr. With the traditional method, the very fast decay time of 51Cr dictates the volume of radioactive tracer to be used for labelling (usually from 100 to 300 ~1) and in many laboratories the radioactive substance is added in 100-300 ~1 medium (Press and Baines, 1982; Strayer et al., 1984; Blomberg et al., 1986; Barone et al., 1989; Kusaka et al., 1992). Under such conditions, we calculate that if in the initial solution Cr content is 5 pg/ml (or 5 pg/mCiI, the metal added can easily reach levels as high as 2000-6000 pug/l (approximately 40-100 ~mol/l), which are toxic for target cells. Thus, caution is required to avoid an excess Cr concentration in the labelling step, and the use of a well defined controlled Cr addition substantially improves the quality of the cytotoxic assay. In general, we suggest using no more than lpg Cr/ml cell solution for no more than 18-20 h incubation. The radiotoxic effects of 51Cr were not considered in this study but normally must be taken into account. As far as the incubation time is concerned, preliminary experiments showed that a good labelling efficiency, with negligible toxicity, can be obtained by treating cells with l-2.5 pg Cr/ml for 1 h (manuscript in preparation). This new labelling technique based on the use of non-radioactive metal and FAAS analysis is promising and produces good results in the detection of NK cell activity. The method has a number of definite advantages: 1. the use of a non-radioactive tracer with no hazards for the health of workers; 2. a significant reduction in costs as the required chromium salt (Na,CrO,) is inexpensive, and the only equipment change required is the graphite furnace; 3. the possibility of storing samples (and perhaps labelled cells) and repeating the readings; 4. the possibility of working with a well-defined Cr concentration. This assures a non-toxic addition of the metal and a strict control of cell sensitivity to Cr uptake and release; 5. a very high sensitivity and accuracy, which is similar to that of ‘lCr.
109
We are well aware that FAAS is not a cheap instrument; it requires specialised technical staff and is not standard equipment in most immunology laboratories which commonly use radioactivity assays. The real advantage of this method goes far beyond the substitution of ‘iCr. Indeed, at present, the method described here could be adopted by laboratories usually equipped with FAAS, and which may not be authorised to use radioactive reagents. They include those dealing with environmental and basic sciences, epidemiology, preventive medicine, toxicology, occupational and forensic medicine, where there is a growing interest in the relationships between stressful events, exposure to toxic substances, and unhealthy life styles on the one side, and immunological cytotoxic responses on the other (Cossarizza et al., 1990a; Cossarizza et al., 1990b; Sansoni et al., 1993; Franceschi et al., 1992, 1995). Lastly, our method could be extended to other metal tracers and could be applied to study other cytotoxicity models.
Acknowledgements
This work was partially supported by grants from the Minister0 Ricerca Scientifica e Tecnologica 60%. The authors thank Professor Claudio Franceschi for critically reading of the manuscript. They also acknowledge all volunteers who donated blood that was used in this study (donors and staff of the local AVIS).
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