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A modified short-term cytotoxicity test: Assessment of natural cell-mediated cytotoxicity in whole blood
Journal of Immunological Methods, 62 (1983) 79-85 Elsevier
79
JIM 2707
A Modified Short-Term Cytotoxicity Test: Assessment of Natural Cell-Mediated Cytotoxicity in Whole Blood R o b e r t C. R e e s a n d A n d r e w A. P l a t t s Department of Virology, The Academic Division of Pathology, The University of Sheffield Medical School, Beech Hill Road, Sheffield $10 2RX, U.K. (Received 1 October 1982, accepted 17 February 1983)
Using whole blood from normal subjects, we have observed natural killing of K562 cells in a 4 h 51Cr_release assay comparable with that shown by separated PBMC and whole blood depleted of serum components. Separated plasma was not toxic towards K562 targets, and failed to potentiate the level of PBMC cytotoxicity through ADCC. The presence of red blood cells did not influence natural killing. The natural cytotoxicity of whole blood was augmented by interferon and depressed by prostaglandins E1 and E2. Studies with appropriate control blood fractions show that cytotoxicity tests with whole blood provide results reflecting natural cell-mediated cytotoxicity. Key words: natural cytotoxicity - N K cell assay
Introduction
Short-term (4-6 h) cytotoxicity (isotope release) assays, mostly based on the 5~Cr-release assay of Brunner et al. (1968), are widely used to establish the presence of peripheral blood effector cells mediating natural killer (NK) activity (Herberman and Holden, 1978) in mononuclear cell fractions separated from whole blood. Although the precise relevance of this form of innate cytotoxicity is unclear, experimental evidence indicates a role for N K cells in immune surveillance against tumours (Kasai et al., 1979; Hanna, 1982) and as a primary defence mechanism against viral infections (Ault and Weiner, 1979; Welsh and Hallenbeck, 1980; Biron and Welsh, 1982). Fractionated peripheral blood mononuclear cells have also been used to further characterise the effector cells mediating natural cytotoxicity, and to establish patterns of natural killing in cancer patients (Pross and Baines, 1976; Takasugi et al., 1977; Ferson et al., 1979; Platsoucas et al., 1980; Hersey et al., 1980, 1982; Ziegler et al., 1981; Britten et al., 1982; Hawrylowicz et al., 1982) and other disease entities (Svedmyr and Jondal, 1975; Koren et al., 1978; Pross et al., 1979; Benczur et al., 1980; Oshimi et al., 1980). In such studies it may be preferable to determine the 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
80 cytolytic capacity of unfractionated blood, rather than the activity of separated PBMC, and we describe here a modified short-term (4 h) cytotoxicity test for assessing the natural cytotoxic potential of whole blood samples.
Materials and Methods
Target cells The leukaemic cell line K562 was used in tests for natural cytotoxicity, and was maintained in stationary suspension culture in RPMI supplemented with antibiotics and 10% newborn calf serum (RPMI-NBCS) (Lozzio and Lozzio, 1975).
Interferon Freeze dried preparations of lymphoblastoid (Namaiva) interferon ( H I F N ) were kindly provided by Drs. Fantes and Johnson (Wellcome Research Laboratories, Beckenham, Kent). Reconstituted H I F N was aliquoted and stored at - 8 0 ° C and used in assays at a final concentration of 100 U H I F N / m l , diluted in RPMI-NBCS medium.
Prostaglandins Prostaglandins El and E2 were obtained from Upjohn, (Kalamazoo, MI) and stored at 4°C. Before use, these reagents were diluted in RPMI-NBCS and used in tests to give a final concentration of 200 and 1000 ~ g / m l .
Peripheral blood and preparation of effector cells. Peripheral blood from healthy individuals was collected in heparin (10 U / m l ) and processed immediately. It was considered important to compare the activities of whole blood, of whole blood free from plasma, of PBMC recovered from the interface fraction and of the red blood cell (RBC) pellet obtained from Ficoll-triosil separation. The following cell fractions were prepared. (1) Whole blood (WH.BL). Collected, heparinised (10 U / m l ) from normal subjects. (2) Washed whole blood (WA.BL). Aliquots (2.0 ml) of whole blood were centrifuged at 500 x g for 10 min, and the plasma removed. The pellet was subsequently washed ( x 3) in 10 ml of RPMI-NBCS and resuspended to the original volume in RPMI-NBCS. (3) Ficoll-triosil fractions. Peripheral blood (usually 2 ml) was layered onto Ficoll-triosil ( f / t ) and centrifuged at 550 x g for 30 rain. The PBMC interface fraction and the pellet containing red blood cells (RBC) were recovered, washed ( x 3) in R P M I - N B C S and resuspended to the original blood volume. This technique has been described fully by B6yum (1968). (4) Plasma. This was recovered from step 2 and included in all tests to control for serum toxicity.
4 h 51Cr-release assay K562 target cells in 0.2 ml R P M I - N B C S were labelled with Na 2 s l CrOa (100/~Ci)
81 (Radiochemical Centre, Amersham, Bucks) for 1 h at 37°C, and the cells washed ( x 3) and resuspended in RPMI-NBCS, and incubated for a further hour at 37°C. The cells were then washed ( x 3), counted and resuspended at 1 x l0 s cells/ml in RPMI-NBCS. Dilutions (1:2, 1:4, and 1:8) of WH.BL or cell fractions were prepared in RPMI-NBCS and 0.1 ml volumes added in triplicate to round-bottomed microtest plate wells (Falcon microtest III flexible assay plates; Becton Dickinson). K562 target cells (0.1 ml/well) were then added to each well, and the plates incubated at 37°C for 4 h in a 5% CO2/95% air humidified atmosphere. Control wells containing 0.1 ml target cells plus 0.1 ml RPMI-NBCS medium were incubated in parallel. Following incubation the plates were centrifuged at 200 x g for 5 min, and 0.1 ml of supernatant (SN) removed to clean wells. The plates were then dried, sealed with parafilm and counted in a 3,-spectrophotometer. The per cent 5~Cr-release was determined according to the formula: % 51Cr_release =
( 1 / 2 SN) x 2 x 100. ( 1 / 2 SN) + (cell pellet + 1/2 SN)
The results can be expressed as the per cent cytotoxicity: (test release) - (spontaneous release) × 100. 1O0 -- (spontaneous release) The spontaneous release from target cells incubated in RPMI-NBCS alone was within the range of 3-10% for all experiments.
Results
Table I shows the results obtained with blood from 3 separate donors, where significant ( P = < 0 . 0 5 - < 0 . 0 0 1 ) cytotoxicity was observed using WH.BL at all dilutions, although individuals displayed variable levels of activity. PBMC cytolytic activity was comparable with that of WH.BL, while WA.BL in most experiments appeared to possess slightly reduced levels of killing. Cells recovered from the RBC pellet and plasma, were both relatively non-toxic for K562 targets. Fig. l compares the cytotoxicity of WH.BL with that of WA.BL and plasma assayed at a 1:2 dilution. Although the c3/totoxicity of WA.BL compared with WH.BL was reduced from 17.7 to 11.4 (mean cytotoxic value), this was not a statistically valid reduction. Further experiments (results not given) showed that the addition of autologous plasma or separated red blood cells (obtained from the f / t pellet) did not influence the cytotoxic potential of PBMC. WH.BL and PBMC were tested in parallel to determine the effect of 2 immunomodulators (human interferon and E series prostaglandins) known to affect human N K cell activity in vitro. Human lymphoblastoid interferon (HIFN) was added to WH.BL or PBMC overnight and the cells then assayed for natural killing, against K562 targets. Incubation with H I F N (100 U / m l ) caused a significant increase
82
TABLE I N A T U R A L C Y T O T O X I C I T Y O F W H O L E B L O O D A N D B L O O D F R A C T I O N S A G A I N S T K562 TARGET CELLS Exp. no. a
1
2
3
Dilution b
% C y t o t o x i c i t y of: Whole blood (WH.BL.)
Washed blood (WA.BL.)
PBMC ( f / t interface)
RBC ( f / t pellet)
Plasma
1:2
17 **
1 0 . 9 **
1 4 . 7 ***
3.3 *
0
1:4
10 ~'**
4 . 5 **
8.4 *
1.0
-
3.5 *
1.0
0
-
1:8
4 . 5 **
1:2
35.4 ***
23.1 ***
26 ***
6.1
0
1:4
18.8 ***
14.8 ***
18.5 **'~
0
0
1:8
10.2 ***
10.8 * * *
13.1 * * *
0
0
1:2
10.8 *** 5.2 ***
10.1 **
15.4 ***
0.6
0.2
6.3 *
7.8 ***
0.8
0.8
1.5
3.6
0.4
0.3
1:4 1:3
3.1 *
Three of 8 e x p e r i m e n t s giving similar results are shown. b Samples diluted in R P M I - N B C S . Statistical significance of cytotoxicity was d e t e r m i n e d with S t u d e n t ' s t-test: *** P < 0.001; ** P < 0.01; * P < 0 . 0 5 .
( P = < 0.05- < 0.001) in the cytotoxic capability of WH.BL and PBMC compared with control cell cultures incubated without H I F N (Fig. 2). Enhanced cytolytic activity was observed whether or not H I F N was removed prior to assay, although removal of the cultured medium caused a significant increase in cytotoxicity of both H I F N and control WH.BL and PBMC. The influence of prostaglandins El and E2 on the natural cytotoxicity of WH.BL and PBMC was also determined. Fig. 3 shows that the addition of prostaglandins E1 and E2 to effector cells immediately prior to assay significantly ( P = < 0.05- < 0,001) inhibited WH.BL and PBMC cytotoxicity.
40
20
WH.BL
WA. BL
Plasma
Fig. 1. C o m p a r i s o n of n a t u r a l cytotoxicity of whole blood (WH.BL), w a s h e d blood (WA.BL), a n d p l a s m a from 8 n o r m a l subjects. Bar lines indicate m e a n + S.D.
83 P3MC
WH.BL 30
20
I0 O-,
O
~ i
,
~
,
,
Dilution
Dilution
Fig. 2. Potentiation of natural cytotoxicity of whole blood (WH.BL) and separated PBMC by human interferon (HIFN). H I F N was added to WH.BL and PBMC to a final concentration of 100 U / m l , and incubated at 37°C overnight. Effector cells incubated with (A) or without (A) H I F N and washed prior to assay; effector cells incubated with ( © ) or without (e) H I F N and assayed without prior washing.
WH.BL
40
PBMC
~
._~
C
'5 ~-~- 20
Dilution
Dilution
Fig. 3. Effect of prostaglandins El and E2 (at final concentrations of 2 0 0 / t g / m l ) on natural cytotoxicity mediated by effector cells presented in whole blood (WH.BL) or separated PBMC. C, no prostaglandin.
84
Discussion Lymphocytes mediating natural cytotoxicity, and in particular N K cells, are thought to play a prominent role in surveillance against newly arising cancer cells (Herberman and Holden, 1978; Kasai et al., 1979; Karre et al., 1980), and prevent secondary blood-borne tumour metastases (Talmadge et al., 1980; Gorelik et al., 1982). It may be important, therefore, to determine natural cytotoxicity in different disease states and relate the results as closely as possible to the patient's clinical status. Separation of PBMC prior to assay may assist in the removal of factors influencing natural cytotoxicity, for example endogenous interferon(s) (Trinchieri and Santoli, 1978; Herberman et al., 1979; Zarling et al., 1979) which potentiates cytolytic activity, and prostaglandins (Droller et al., 1978; Kendall and Targon, 1980) which are immunosuppressive. The reflection in vitro of in vivo cytolytic potential may therefore only be apparent in whole blood. The present study shows that whole blood gives comparable natural cytotoxicity to that of washed blood cells and separated PBMC resuspended to the original volume. Plasma from normal subjects proved to be non-toxic for K562 target cells, but this control should be included in all tests to distinguish between cell-mediated activity and soluble cytotoxins. Plasma products do not appear to contribute significantly to PBMC natural cytotoxicity, and cells recovered from the RBC pellet failed to influence the natural cytotoxicity of PBMC. This is an important consideration, since the number of RBC as well as other cell types may vary between individual patients, or with the stage of the disease. Whole blood natural cytotoxicity may be augmented by H I F N and decreased by the presence of prostaglandins of the E series. This modulation is comparable with that seen with separated PBMC effectors, and is consistent with properties already ascribed to N K cells. The assay procedure described here appears to be applicable to studies where it may be important to determine cytolytic activity in individuals, or groups of patients. Tests may be easily carried out with small volumes (1.0 ml) of blood, which increases their practical value.
Acknowledgements This work was supported by the Yorkshire Cancer Research Campaign. We are grateful to Professor C.W. Potter and Dr. A. Clark for their helpful comments on this work, and to Mrs. C. Mullan and Miss T. Johnson for typing the manuscript.
References Ault, K.A. and H.L. Weiner, 1979, J. Irnmunol. 122, 2611. Benczur, P., G.Gy. Petranyi, Gy. Palfrey, M. Varga, M. Talas, B. Kotsy, I. Foldes and S.R. Hollan, 1980, Clin. Exp. lmmunol. 39, 657. Biron, C.A. and R.M. Welsh, 1982, Med. Microbiol. Immunol. 170, 155. B6yurn, A., 1968, Scand. J. Clin. Lab. Invest. 21, (Suppl. 97), 77.
85 Britten, V., R.C. Rees, A. Clegg, G.T. Smith, C.W. Potter, M. Fox and J.L.W. Williams, 1982, Brit. J. Urol. 54, 261. Brunner, K.T., J. Manel, M.C. Cerottini and B. Chapius, 1968, Immunology 14, 181. Droller, M.J., M.U. Schneider and P. Perlmann, 1978, Cell Immunol. 39, 165. Ferson, M., A. Edwards, A. Lind, G.W. Milton and P. Hersey, 1979, Int. J. Cancer 23, 603. Gorelik, E., R.H. Wiltrout, K. Okumura, S. Habu and R.B. Herberman, 1982, Int. J. Cancer 30, 107. Hanna, N., 1982, Cancer Met. Revs. 1, 45. Hawrylowicz, C.M., R.C. Rees, B.W. Hancock and C.W. Potter, 1982, Eur. J. Cancer Clin. Oncol. 108, 1081. Herberman, R.B. and H.T. Holden, 1978, Adv. Cancer Res. 27, 305. Herberman, R.B., J.R. Ortaldo and G.D. Bonnard, 1979, Nature (London) 277, 221. Hersey, P., A. Edwards and W.H. McCarthy, 1980, Int. J. Cancer 25, 187. Hersey, P., A. Edwards, R. Lewis, A. Kemp and J. McInnes, 1982, Clin. Exp. Immunol. 48, 205. Karre, K., G.O. Klein, R. Kiessling, G. Klein and J. Roder, 1980, Nature (London) 284, 624. Kasai, M., J.C. Leclerc, L. McVay-Boubrean, F.W. Shen and H. Cantor, 1979, J. Exp. Med. 149, 1260. Kendall, R.A. and S. Targon, 1980, J. Immunol. 125, 2770. Koren, H.S., D.B. Amos and R.H. Buckley, 1978, J. lmmunol. 120, 796. Lozzio, C.B. and B.B. Lozzio, 1975, Blood 45, 321. Oshimi, K., N. Gonda, M. Sumiya and S. Kano, 1980, Clin. Exp. Immunol. 40, 83. Platsoucas, C.D., G. Fernandes, S.L. Gupta, S. Kempin, B. Clarkson, R.A. Good and S. Gupta, 1980, J. Immunol. 125, 1216. Pross, H.F. and M.G. Baines, 1976, Int. J. Cancer 18, 593. Pross, H.F., S. Gupta, R.A. Good and M.G. Baines, 1979, Cell Immunol. 43, 160. Svedmyr, E. and M. Jondal, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 1622. Takasugi, M., A. Ramseyer and J. Takasugi, 1977, Cancer Res. 37, 413. Talmadge, J.E., K.M. Meyers, D.J. Prleur and J.R. Starkey, 1980, J. Natl. Cancer Inst. 65, 929. Trinchieri, G. and D. Santoli, 1978, J. Exp. Med. 147, 1314. Welsh, R.M. and L.A. Hallenbeck, 1980, J. Immunol. 124, 2491. Zarling, J.M., L. Eskra, E.C. Borden, J. Horoszewicz and W.A. Carter, 1979, J. Immunol. 123, 63. Ziegler, H.W., N.E. Kay and J.M. Zarling, 1981, Int. J. Cancer 27, 321.
79
JIM 2707
A Modified Short-Term Cytotoxicity Test: Assessment of Natural Cell-Mediated Cytotoxicity in Whole Blood R o b e r t C. R e e s a n d A n d r e w A. P l a t t s Department of Virology, The Academic Division of Pathology, The University of Sheffield Medical School, Beech Hill Road, Sheffield $10 2RX, U.K. (Received 1 October 1982, accepted 17 February 1983)
Using whole blood from normal subjects, we have observed natural killing of K562 cells in a 4 h 51Cr_release assay comparable with that shown by separated PBMC and whole blood depleted of serum components. Separated plasma was not toxic towards K562 targets, and failed to potentiate the level of PBMC cytotoxicity through ADCC. The presence of red blood cells did not influence natural killing. The natural cytotoxicity of whole blood was augmented by interferon and depressed by prostaglandins E1 and E2. Studies with appropriate control blood fractions show that cytotoxicity tests with whole blood provide results reflecting natural cell-mediated cytotoxicity. Key words: natural cytotoxicity - N K cell assay
Introduction
Short-term (4-6 h) cytotoxicity (isotope release) assays, mostly based on the 5~Cr-release assay of Brunner et al. (1968), are widely used to establish the presence of peripheral blood effector cells mediating natural killer (NK) activity (Herberman and Holden, 1978) in mononuclear cell fractions separated from whole blood. Although the precise relevance of this form of innate cytotoxicity is unclear, experimental evidence indicates a role for N K cells in immune surveillance against tumours (Kasai et al., 1979; Hanna, 1982) and as a primary defence mechanism against viral infections (Ault and Weiner, 1979; Welsh and Hallenbeck, 1980; Biron and Welsh, 1982). Fractionated peripheral blood mononuclear cells have also been used to further characterise the effector cells mediating natural cytotoxicity, and to establish patterns of natural killing in cancer patients (Pross and Baines, 1976; Takasugi et al., 1977; Ferson et al., 1979; Platsoucas et al., 1980; Hersey et al., 1980, 1982; Ziegler et al., 1981; Britten et al., 1982; Hawrylowicz et al., 1982) and other disease entities (Svedmyr and Jondal, 1975; Koren et al., 1978; Pross et al., 1979; Benczur et al., 1980; Oshimi et al., 1980). In such studies it may be preferable to determine the 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
80 cytolytic capacity of unfractionated blood, rather than the activity of separated PBMC, and we describe here a modified short-term (4 h) cytotoxicity test for assessing the natural cytotoxic potential of whole blood samples.
Materials and Methods
Target cells The leukaemic cell line K562 was used in tests for natural cytotoxicity, and was maintained in stationary suspension culture in RPMI supplemented with antibiotics and 10% newborn calf serum (RPMI-NBCS) (Lozzio and Lozzio, 1975).
Interferon Freeze dried preparations of lymphoblastoid (Namaiva) interferon ( H I F N ) were kindly provided by Drs. Fantes and Johnson (Wellcome Research Laboratories, Beckenham, Kent). Reconstituted H I F N was aliquoted and stored at - 8 0 ° C and used in assays at a final concentration of 100 U H I F N / m l , diluted in RPMI-NBCS medium.
Prostaglandins Prostaglandins El and E2 were obtained from Upjohn, (Kalamazoo, MI) and stored at 4°C. Before use, these reagents were diluted in RPMI-NBCS and used in tests to give a final concentration of 200 and 1000 ~ g / m l .
Peripheral blood and preparation of effector cells. Peripheral blood from healthy individuals was collected in heparin (10 U / m l ) and processed immediately. It was considered important to compare the activities of whole blood, of whole blood free from plasma, of PBMC recovered from the interface fraction and of the red blood cell (RBC) pellet obtained from Ficoll-triosil separation. The following cell fractions were prepared. (1) Whole blood (WH.BL). Collected, heparinised (10 U / m l ) from normal subjects. (2) Washed whole blood (WA.BL). Aliquots (2.0 ml) of whole blood were centrifuged at 500 x g for 10 min, and the plasma removed. The pellet was subsequently washed ( x 3) in 10 ml of RPMI-NBCS and resuspended to the original volume in RPMI-NBCS. (3) Ficoll-triosil fractions. Peripheral blood (usually 2 ml) was layered onto Ficoll-triosil ( f / t ) and centrifuged at 550 x g for 30 rain. The PBMC interface fraction and the pellet containing red blood cells (RBC) were recovered, washed ( x 3) in R P M I - N B C S and resuspended to the original blood volume. This technique has been described fully by B6yum (1968). (4) Plasma. This was recovered from step 2 and included in all tests to control for serum toxicity.
4 h 51Cr-release assay K562 target cells in 0.2 ml R P M I - N B C S were labelled with Na 2 s l CrOa (100/~Ci)
81 (Radiochemical Centre, Amersham, Bucks) for 1 h at 37°C, and the cells washed ( x 3) and resuspended in RPMI-NBCS, and incubated for a further hour at 37°C. The cells were then washed ( x 3), counted and resuspended at 1 x l0 s cells/ml in RPMI-NBCS. Dilutions (1:2, 1:4, and 1:8) of WH.BL or cell fractions were prepared in RPMI-NBCS and 0.1 ml volumes added in triplicate to round-bottomed microtest plate wells (Falcon microtest III flexible assay plates; Becton Dickinson). K562 target cells (0.1 ml/well) were then added to each well, and the plates incubated at 37°C for 4 h in a 5% CO2/95% air humidified atmosphere. Control wells containing 0.1 ml target cells plus 0.1 ml RPMI-NBCS medium were incubated in parallel. Following incubation the plates were centrifuged at 200 x g for 5 min, and 0.1 ml of supernatant (SN) removed to clean wells. The plates were then dried, sealed with parafilm and counted in a 3,-spectrophotometer. The per cent 5~Cr-release was determined according to the formula: % 51Cr_release =
( 1 / 2 SN) x 2 x 100. ( 1 / 2 SN) + (cell pellet + 1/2 SN)
The results can be expressed as the per cent cytotoxicity: (test release) - (spontaneous release) × 100. 1O0 -- (spontaneous release) The spontaneous release from target cells incubated in RPMI-NBCS alone was within the range of 3-10% for all experiments.
Results
Table I shows the results obtained with blood from 3 separate donors, where significant ( P = < 0 . 0 5 - < 0 . 0 0 1 ) cytotoxicity was observed using WH.BL at all dilutions, although individuals displayed variable levels of activity. PBMC cytolytic activity was comparable with that of WH.BL, while WA.BL in most experiments appeared to possess slightly reduced levels of killing. Cells recovered from the RBC pellet and plasma, were both relatively non-toxic for K562 targets. Fig. l compares the cytotoxicity of WH.BL with that of WA.BL and plasma assayed at a 1:2 dilution. Although the c3/totoxicity of WA.BL compared with WH.BL was reduced from 17.7 to 11.4 (mean cytotoxic value), this was not a statistically valid reduction. Further experiments (results not given) showed that the addition of autologous plasma or separated red blood cells (obtained from the f / t pellet) did not influence the cytotoxic potential of PBMC. WH.BL and PBMC were tested in parallel to determine the effect of 2 immunomodulators (human interferon and E series prostaglandins) known to affect human N K cell activity in vitro. Human lymphoblastoid interferon (HIFN) was added to WH.BL or PBMC overnight and the cells then assayed for natural killing, against K562 targets. Incubation with H I F N (100 U / m l ) caused a significant increase
82
TABLE I N A T U R A L C Y T O T O X I C I T Y O F W H O L E B L O O D A N D B L O O D F R A C T I O N S A G A I N S T K562 TARGET CELLS Exp. no. a
1
2
3
Dilution b
% C y t o t o x i c i t y of: Whole blood (WH.BL.)
Washed blood (WA.BL.)
PBMC ( f / t interface)
RBC ( f / t pellet)
Plasma
1:2
17 **
1 0 . 9 **
1 4 . 7 ***
3.3 *
0
1:4
10 ~'**
4 . 5 **
8.4 *
1.0
-
3.5 *
1.0
0
-
1:8
4 . 5 **
1:2
35.4 ***
23.1 ***
26 ***
6.1
0
1:4
18.8 ***
14.8 ***
18.5 **'~
0
0
1:8
10.2 ***
10.8 * * *
13.1 * * *
0
0
1:2
10.8 *** 5.2 ***
10.1 **
15.4 ***
0.6
0.2
6.3 *
7.8 ***
0.8
0.8
1.5
3.6
0.4
0.3
1:4 1:3
3.1 *
Three of 8 e x p e r i m e n t s giving similar results are shown. b Samples diluted in R P M I - N B C S . Statistical significance of cytotoxicity was d e t e r m i n e d with S t u d e n t ' s t-test: *** P < 0.001; ** P < 0.01; * P < 0 . 0 5 .
( P = < 0.05- < 0.001) in the cytotoxic capability of WH.BL and PBMC compared with control cell cultures incubated without H I F N (Fig. 2). Enhanced cytolytic activity was observed whether or not H I F N was removed prior to assay, although removal of the cultured medium caused a significant increase in cytotoxicity of both H I F N and control WH.BL and PBMC. The influence of prostaglandins El and E2 on the natural cytotoxicity of WH.BL and PBMC was also determined. Fig. 3 shows that the addition of prostaglandins E1 and E2 to effector cells immediately prior to assay significantly ( P = < 0.05- < 0,001) inhibited WH.BL and PBMC cytotoxicity.
40
20
WH.BL
WA. BL
Plasma
Fig. 1. C o m p a r i s o n of n a t u r a l cytotoxicity of whole blood (WH.BL), w a s h e d blood (WA.BL), a n d p l a s m a from 8 n o r m a l subjects. Bar lines indicate m e a n + S.D.
83 P3MC
WH.BL 30
20
I0 O-,
O
~ i
,
~
,
,
Dilution
Dilution
Fig. 2. Potentiation of natural cytotoxicity of whole blood (WH.BL) and separated PBMC by human interferon (HIFN). H I F N was added to WH.BL and PBMC to a final concentration of 100 U / m l , and incubated at 37°C overnight. Effector cells incubated with (A) or without (A) H I F N and washed prior to assay; effector cells incubated with ( © ) or without (e) H I F N and assayed without prior washing.
WH.BL
40
PBMC
~
._~
C
'5 ~-~- 20
Dilution
Dilution
Fig. 3. Effect of prostaglandins El and E2 (at final concentrations of 2 0 0 / t g / m l ) on natural cytotoxicity mediated by effector cells presented in whole blood (WH.BL) or separated PBMC. C, no prostaglandin.
84
Discussion Lymphocytes mediating natural cytotoxicity, and in particular N K cells, are thought to play a prominent role in surveillance against newly arising cancer cells (Herberman and Holden, 1978; Kasai et al., 1979; Karre et al., 1980), and prevent secondary blood-borne tumour metastases (Talmadge et al., 1980; Gorelik et al., 1982). It may be important, therefore, to determine natural cytotoxicity in different disease states and relate the results as closely as possible to the patient's clinical status. Separation of PBMC prior to assay may assist in the removal of factors influencing natural cytotoxicity, for example endogenous interferon(s) (Trinchieri and Santoli, 1978; Herberman et al., 1979; Zarling et al., 1979) which potentiates cytolytic activity, and prostaglandins (Droller et al., 1978; Kendall and Targon, 1980) which are immunosuppressive. The reflection in vitro of in vivo cytolytic potential may therefore only be apparent in whole blood. The present study shows that whole blood gives comparable natural cytotoxicity to that of washed blood cells and separated PBMC resuspended to the original volume. Plasma from normal subjects proved to be non-toxic for K562 target cells, but this control should be included in all tests to distinguish between cell-mediated activity and soluble cytotoxins. Plasma products do not appear to contribute significantly to PBMC natural cytotoxicity, and cells recovered from the RBC pellet failed to influence the natural cytotoxicity of PBMC. This is an important consideration, since the number of RBC as well as other cell types may vary between individual patients, or with the stage of the disease. Whole blood natural cytotoxicity may be augmented by H I F N and decreased by the presence of prostaglandins of the E series. This modulation is comparable with that seen with separated PBMC effectors, and is consistent with properties already ascribed to N K cells. The assay procedure described here appears to be applicable to studies where it may be important to determine cytolytic activity in individuals, or groups of patients. Tests may be easily carried out with small volumes (1.0 ml) of blood, which increases their practical value.
Acknowledgements This work was supported by the Yorkshire Cancer Research Campaign. We are grateful to Professor C.W. Potter and Dr. A. Clark for their helpful comments on this work, and to Mrs. C. Mullan and Miss T. Johnson for typing the manuscript.
References Ault, K.A. and H.L. Weiner, 1979, J. Irnmunol. 122, 2611. Benczur, P., G.Gy. Petranyi, Gy. Palfrey, M. Varga, M. Talas, B. Kotsy, I. Foldes and S.R. Hollan, 1980, Clin. Exp. lmmunol. 39, 657. Biron, C.A. and R.M. Welsh, 1982, Med. Microbiol. Immunol. 170, 155. B6yurn, A., 1968, Scand. J. Clin. Lab. Invest. 21, (Suppl. 97), 77.
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