191
Journal of lmmunologzcal Methods, 139 (1991) 191-195 © 1991 Elsevaer Science Pubhshers B.V. 0022-1759/91/$03.50 ADONIS 00221759001748
JIM05928
Evaluation of a test system for measuring cytokine production in human whole blood cell cultures U. Elsiisser-Beile 1, S. V o n Kleist 1 and H. Gallati 2 1 lnstztute of Immunoblology, Unwers:ty of Fretburg D-7800 Fretburg, F.R. G, and ~ Hoffmann-La Roche A G, Central Research Umts, CH-4002 Basel, Swztzerland
(Received 5 September 1990, revised received 2 January 1991, accepted 5 February 1991)
A simple and reproducible method is described for the measurement of mitogen-induced cytokine production in cultures of both human peripheral blood mononuclear cells (PBMC) and whole blood. In the culture supernatants the cytokines intedeukin-la (IL-la), interleukin-2 (IL-2), interferon-y (IFN-y) and tumor necrosis factor a (TNF-a) were determined by a rapid and sensitive immunoassay using various monoclonal and polyclonal antibodies. Comparing the PBMC cultures with the whole blood system a good correlation was obtained if the cell number was taken into account. In the post-induction supernatants the cytokine values were found to follow typical kinetic curves. The protocol was evaluated by screening 60 cancer patients with primary disease and 60 healthy controls. A markedly reduced secretion of IFN-7 and I L - l a was found in the cancer patients compared to controls, although leukocyte and lymphocyte counts were almost identical in both groups. Key words" Whole-blood mononuclear cell culture; Cytokine production; ELISA; Immunodlagnosls
Introduction It is well known that lymphocyte activation by stimulation with mitogens leads to cell proliferation as well as to secretion of cytokines. Whilst methods for the determination of proliferative responses are well established, few approaches exist for the assessment of mitogen induced cytokine
Correspondence to: U. Els~isser-Beale, Institute of Immunobiology, Stefan-Meier-Str. 8, D-7800 Freiburg, F.R.G. Abbrematlons. PBMC, peripheral blood mononuclear ceils; PHA, phytohemagglutinin; PWM, pokeweed mitogen; ELISA, enzyme-linked immunoassay; IL, mterleukin; IFN, interferon; TNF, tumor necrosis factor; mab, monoclonal antibody; pod, peroxidase.
production as a parameter of cellular immunological activity in man. Nevertheless a small number of studies have been published describing impaired cytokine production of peripheral blood mononuclear cells following stimulation with mitogens in individuals with cancer or clinical immunosuppression (Paganelli et al., 1984; Vilcek et al., 1986; Bubenik et al., 1988; H o et al., 1988). Such stimulation experiments are usually done in cultures of separated peripheral blood mononuclear cells. However, the separation procedure can give rise to selective depletion or enrichment of certain subpopulations of lymphocytes or monocytes, or can even lead to preactivation. Therefore simpler methods using whole blood have been advocated (Kirclmer et al., 1981). By combining a whole blood stimulation method with a
192 rapid and sensitive immunoassay for the detection of cytokines in supernatants, we have established a test system which permits the screening of large numbers of cell samples from patients.
flat bottom microtiter plates (Nunc, Kamstrup, Denmark) using the above conditions. Proliferative responses were determined by measuring the incorporation of [3H]thyrnidlne (0.4 /zCi/well), added 24 h before the harvest of the culture.
Materials and methods
Specific and quantitative determmatton of cytokmes Enzyme-linked immunoassays (ELISA) were used for qualitative and quantitative determinations of the different cytokines. These tests were developed by Hoffmann-La Roche to monitor the production, purification and clinical trials of the various recombinant cytokines. In addition to establishing their sensitivity and specificity, a correlation was shown between assay performance of the naturally occurring cytokines and their recombinant counterparts. A correlation was also found between the ELISA measurements and the biological tests (AVA).
Blood donors Healthy female individuals (n = 60), aged between 18 and 65 years, served as controls for the 60 patients with neoplasias. The latter had primary disease and comprised the following carcinomas: breast (n = 26), cervix (n = 20), ovary (n = 14). 10 ml of heparinized blood were taken from the healthy donors and preoperatively from the patients. The samples, kept at room temperature, were used within 3 h. A 0.5 ml aliquot of blood was removed for total and differential leukocyte counts, from which the total lymphocyte count was determined. Whole blood cell culture and tsolated mononuclear cell culture Cultures were performed in loosely capped 12 mm polystyrol test tubes (Becton Dickinson, Heidelberg, F.R.G.) and the total culture volume was adjusted to 5 0 0 / d / t u b e . Heparinized venous blood was diluted 1 / 1 0 with RPMI 1640 (Gibco, Eggenstein, F.R.G.) supplemented with penicillin (Gibco) 50 U / m l , streptomycin (Gibco) 50/~g/ml. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density layer centrifugation and cultured at 200,000 cells per 500/~1 RPMI 1640 with antibiotics and 6% autologous plasma. For stimulation 1-10 /~g/ml phytohemagglutinin (PHA, Wellcome, Burgwedel, F.R.G.) and 1 - 5 / ~ g / m l pokeweed mitogen (PWM, Sigma, Disenhofen, F.R.G.) were used. Incubation of the cultures was performed at 3 7 ° C in a humidified atmosphere of 5% CO 2. After 1-7 days of culture without change of medium 320 /~1 of supernatant were removed from each tube to be assayed for cytokine levels. Proliferatwe response For the measurement of proliferative activity whole blood and isolated PBMC were cultured in
lnterferon-'y ELISA IFN-'/ levels in the supernatants were determined as previously described (Gallati et al., 1986). Briefly, the supernatants or a standard IFN-~, solution was added to a microtiter plate coated with a murine monoclonal antibody (mab) to IFN-~, (clone 69). Following the addition of a second mab to IFN-7, (clone 123), which was coupled to peroxidase (pod), the plate was incubated at room temperature for 16-24 h. Unbound material was removed by a washing step and the amount of bound peroxidase was determined by a short incubation with tetramethylbenzidine. On stopping the reaction with sulfuric acid the color changed to yellow and its intensity was determined at 450 nm by a computerized multichannel photometer. The amount of human IFN-7, was calculated from a standard curve. This ELISA had an assay range of 0-1000 p g / m l and a detection limit of about 50 p g / m l IFN-3,. lnterleukm-1 a-ELISA This test was based on the same principle as the IFN-~, test. Microtiter plates (Nunc) were coated with a polyclonal goat anti-human-IL-la antibody. For the detection of protein bound I L - l a the pod-linked Fab fragment of a polyclonal goat anti-human-IL-la antibody was used. (assay range: 5-100 pg/ml).
193
TNF-a-ELISA In this test, the first immobilized antibody was a monoclonal mouse a n t i - h u m a n - T N F - a antibody and the second antibody was a pod-coupled polyclonal rabbit a n t i - h u m a n - T N F - a antibody. This test had an assay range of 10-500 p g / m l . lnterleukm-2-ELISA For this test microtiter plates were coated with two monoclonal mouse anti-human IL-2 antibodies, and a third pod-linked m a b was used for detection of bound IL-2 (assay range: 50-1000 pg/ml). Statistwal analysts The results in the tumor and control groups were statistically evaluated using the Kruskal-Wallis test (X 2 approximation).
Results
Comparison of tsolated PBMC cultures and whole blood cell cultures In both systems there was an evident dependence of cytokine production on mononuclear cell concentrations. Fig. 1 shows a typical curve of I F N - 7 production in cultures of isolated PBMC and whole blood of the same donor at various cell concentrations. At equal cell numbers the cytokine values compared very well (except for high blood
IF N - g a m m a 70
(ng/ml)
60 50
~
40
30 20 10 0
m i
2
3
4
I
I
I
I
5
6
7
8 X i00000
PBMC/500 ~1
Fig. 1. Correlation of mononuclear cell count and mitogen-induced IFN-7 production m whole blood cell cultures and isolated PBMC cultures. In whole blood cell cultures the absolute number of monoeytes and lymphoeytes was calculated from the differential cell count and adjusted by dilution Mltogen. PWM 5 pg/ml; incubation time' 4 days.
concentrations). Ttus was not true for the T N F values, wtuch were always higher in the P B M C cultures. The effect of adding heparin, which was necessary to prevent clotting in the whole blood cultures, was also tested. In the range up to 400 U / m l heparin had no influence either on cytokine production or on the ELISA. In contrast, when using E D T A blood, only minimal stimulation was obtained. The reproducibility of the whole blood culture system was very good. Ten independent cultures of one blood sample gave very reproducible cytokine values under equal stimulation conditions, the coefficients of variation ranging between 5% and 20%. In contrast, the reproducibility of the stimulation experiments with isolated P B M C was less satisfactory.
Kinetics of cytokine productzon and the prohferatwe response Fig. 2 shows cumulative time-response curves of cytokine production in whole blood cell cultures upon stimulation with PHA. The curves for the four cytokines differed: I F N - 7 and I L - l a production steadily increased until day 4 and then reached a plateau. For T N F there was already a m a x i m u m after 24 h of incubation and often a second peak between day 4 and day 6. IL-2 secreuon reached a m a x i m u m between day 2 and day 3 and then declined. The corresponding kinetics of mitogen induced proliferation of the same cultures is also shown. The kinetics of the isolated P B M C cultures were similar. Cytokme values in whole blood cultures of healthy donors and tumor pattents In a first trial we used the whole blood system for the screening of 60 cancer patients and 60 healthy controls. We chose two of the four cytokines and measured the values of I F N - y and I L - l a in the 4 day post-induction supernatants following stimulation with P H A and PWM. In the controls I F N - 7 levels ranged from 10 to 250 n g / m l , and I L - l a values were between 50 and 500 p g / m l regardless of which mitogen was studied. Cell samples from the tumor patients had a significantly lower capacity to produce I F N - 7 and I L - l a at all mitogen concentrations used, although
194
leukocyte and lymphocyte counts were similar in both groups of individuals (Fig. 3).
IFN-gamma
(ng/=l)
250 .
MAX
Discussion We have described a test system for the measurement of four different cytokines using whole blood cell cultures and simple and reproducible enzymoimmunological tests. Comparing the whole blood method with isolated PBMC cultures we found a direct correlation for IFN-7 and IL-la concentrations if the cell numbers were adjusted in both systems. From these results we conclude that, in spite of the complexity of the cellular events taking place in these cultures, whole blood tests do indeed reflect the performance of T lymphocytes and monocytes as already postulated by other authors (Luquetti et al., 1976). In con-
200
150
~3
100
...........
ME]
50 ng/ml x6o
CPM
x
i000
14o
~=o 0 xoo controls
tumor
controls
tumor
Fig. 3. IVhtogen-mduced IFN-7 productton m whole blood cell
so
cultures of 60 tumor patients and 60 healthy controls. Incubation time: 4 days. Mltogens: 10 /~g/ml PHA (left); 5 / t g / m l PWM (right).
6o
4o
2o
• .......
Pg/~oo
p~.
laoo ~ooo
aoo
~ 2oo 6oo
x
2
3
•
(dsy)
~
7
Fig. 2. T ~ e course of cytoMne pr~uctmn m whole bkKxt ce~ cultures and the ~ i a t ~
pro~rative r ~ p o n ~ .
trast, TNF-a values were generally higher in the PBMC cultures than in the corresponding whole blood tests. We suggest that the reason for this is prestimulation of cells either through the separation procedure or cell death or both. The whole blood method is based on an optimal dilution of the blood cells in medium and has great advantages over isolated PBMC cultures. It requires only small amounts of blood and no unphysiological cell separation is involved. Another advantage is that this method is easy and rapid, making it suitable for testing large populations of patients. This was also true for the enzymoimmunological tests for the determination of the cytokines. Comparing the time course of the four cytokine values in the culture superuatants we decided to take measurements on day 4 of incubation.
195
With this test system a normal range of mitogen responsiveness could be defined for the controls and it was possible to show that the tumor patients had a demonstrable decrease in IFN--/ and IL-1 production by their mononuclear cells. We were also able to show that these reduced cytokine levels were not due to a reduced lymphocyte count in the cancer patients. This finding is in accordance with the results of Fritze et al. (1984), who showed a reduced lymphocyte activity, as measured by lymphocyte proliferation, in cultures of whole blood from cancer patients. Further investigations wdl be needed to show whether this method provides appropriate information about the state of cellular immunity and whether or not it will be useful for prognostic purposes.
Acknowledgements The authors wish to thank B. Koch and I. Pracht for their excellent technical assistance.
References Bubenik, J., Kleler, J., Tromholt, V., Herman, G and Jandlova, T. (1988) Defect m lectm-mduced mterleukin 2 production
by peripheral blood lymphocytes of pauents with lnvaslve urinary bladder carcinoma. Immunol. Lett. 18, 115 Fntze, D. and Dystant, P. (1984) Detection of impaired rmtogen responses m autologous whole blood of cancer pauents using an o p ~ d method of lymphocyte sUmulatton. Immunol. Lett. 8, 243 Gailata, H., Pracht, I., Schnudt, J., HRnng, P. and Garotta, G. (1987) A simple, rapid and large capacity ELISA for btologically active and recombinant human IFN-gamma. J. Biol. Regul. Homeost. Agents 1, 109. Ho, A.D., Moritz, T., Rensch, K., Hunstem, W and Kirchner, H. (1988) Defloency m interferon producUon of peripheral blood leukocytes from patients with non-Hodgkm lymphoma. J. Interferon Res. 8, 405 Karchner, H., Klemicke, Ch. and Ehgel, W. (1981) A whole blood techmque for testing producUon of human interferon by leukocytes. J. Immunol. Methods 48, 213. Luquetti, A. and Janossy, G. (1976) Lymphocyte acUvaUon VIII. The apphcatlon of a whole blood test to the quanUtauve analysis of PHA responsive T cells. J. Immunol. Methods 10, 7. Paganelh, R., Capoblanclu, M.R., Matncardl, P.M., Cloc, L., Sermnara, R. Dlanzam, F. and AinU, F. (1984) Defecave interferon-gamma production m Atayaa-Telang~cctasia Chn. Immunol. Immunopathol. 32, 387. Vdcec, J., Klion, A , Henrikscn-DeStefano, D., Zemtsov, A., Dawdson, D.M, Dawdson, M., Frledman-I(den, A.E. and Le, J. (1986) Defective gamma-interferon producUon m peripheral blood leukocytes of paUents with acute tuberculosls J Chn. Immunol. 6, 146.
Journal of lmmunologzcal Methods, 139 (1991) 191-195 © 1991 Elsevaer Science Pubhshers B.V. 0022-1759/91/$03.50 ADONIS 00221759001748
JIM05928
Evaluation of a test system for measuring cytokine production in human whole blood cell cultures U. Elsiisser-Beile 1, S. V o n Kleist 1 and H. Gallati 2 1 lnstztute of Immunoblology, Unwers:ty of Fretburg D-7800 Fretburg, F.R. G, and ~ Hoffmann-La Roche A G, Central Research Umts, CH-4002 Basel, Swztzerland
(Received 5 September 1990, revised received 2 January 1991, accepted 5 February 1991)
A simple and reproducible method is described for the measurement of mitogen-induced cytokine production in cultures of both human peripheral blood mononuclear cells (PBMC) and whole blood. In the culture supernatants the cytokines intedeukin-la (IL-la), interleukin-2 (IL-2), interferon-y (IFN-y) and tumor necrosis factor a (TNF-a) were determined by a rapid and sensitive immunoassay using various monoclonal and polyclonal antibodies. Comparing the PBMC cultures with the whole blood system a good correlation was obtained if the cell number was taken into account. In the post-induction supernatants the cytokine values were found to follow typical kinetic curves. The protocol was evaluated by screening 60 cancer patients with primary disease and 60 healthy controls. A markedly reduced secretion of IFN-7 and I L - l a was found in the cancer patients compared to controls, although leukocyte and lymphocyte counts were almost identical in both groups. Key words" Whole-blood mononuclear cell culture; Cytokine production; ELISA; Immunodlagnosls
Introduction It is well known that lymphocyte activation by stimulation with mitogens leads to cell proliferation as well as to secretion of cytokines. Whilst methods for the determination of proliferative responses are well established, few approaches exist for the assessment of mitogen induced cytokine
Correspondence to: U. Els~isser-Beale, Institute of Immunobiology, Stefan-Meier-Str. 8, D-7800 Freiburg, F.R.G. Abbrematlons. PBMC, peripheral blood mononuclear ceils; PHA, phytohemagglutinin; PWM, pokeweed mitogen; ELISA, enzyme-linked immunoassay; IL, mterleukin; IFN, interferon; TNF, tumor necrosis factor; mab, monoclonal antibody; pod, peroxidase.
production as a parameter of cellular immunological activity in man. Nevertheless a small number of studies have been published describing impaired cytokine production of peripheral blood mononuclear cells following stimulation with mitogens in individuals with cancer or clinical immunosuppression (Paganelli et al., 1984; Vilcek et al., 1986; Bubenik et al., 1988; H o et al., 1988). Such stimulation experiments are usually done in cultures of separated peripheral blood mononuclear cells. However, the separation procedure can give rise to selective depletion or enrichment of certain subpopulations of lymphocytes or monocytes, or can even lead to preactivation. Therefore simpler methods using whole blood have been advocated (Kirclmer et al., 1981). By combining a whole blood stimulation method with a
192 rapid and sensitive immunoassay for the detection of cytokines in supernatants, we have established a test system which permits the screening of large numbers of cell samples from patients.
flat bottom microtiter plates (Nunc, Kamstrup, Denmark) using the above conditions. Proliferative responses were determined by measuring the incorporation of [3H]thyrnidlne (0.4 /zCi/well), added 24 h before the harvest of the culture.
Materials and methods
Specific and quantitative determmatton of cytokmes Enzyme-linked immunoassays (ELISA) were used for qualitative and quantitative determinations of the different cytokines. These tests were developed by Hoffmann-La Roche to monitor the production, purification and clinical trials of the various recombinant cytokines. In addition to establishing their sensitivity and specificity, a correlation was shown between assay performance of the naturally occurring cytokines and their recombinant counterparts. A correlation was also found between the ELISA measurements and the biological tests (AVA).
Blood donors Healthy female individuals (n = 60), aged between 18 and 65 years, served as controls for the 60 patients with neoplasias. The latter had primary disease and comprised the following carcinomas: breast (n = 26), cervix (n = 20), ovary (n = 14). 10 ml of heparinized blood were taken from the healthy donors and preoperatively from the patients. The samples, kept at room temperature, were used within 3 h. A 0.5 ml aliquot of blood was removed for total and differential leukocyte counts, from which the total lymphocyte count was determined. Whole blood cell culture and tsolated mononuclear cell culture Cultures were performed in loosely capped 12 mm polystyrol test tubes (Becton Dickinson, Heidelberg, F.R.G.) and the total culture volume was adjusted to 5 0 0 / d / t u b e . Heparinized venous blood was diluted 1 / 1 0 with RPMI 1640 (Gibco, Eggenstein, F.R.G.) supplemented with penicillin (Gibco) 50 U / m l , streptomycin (Gibco) 50/~g/ml. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density layer centrifugation and cultured at 200,000 cells per 500/~1 RPMI 1640 with antibiotics and 6% autologous plasma. For stimulation 1-10 /~g/ml phytohemagglutinin (PHA, Wellcome, Burgwedel, F.R.G.) and 1 - 5 / ~ g / m l pokeweed mitogen (PWM, Sigma, Disenhofen, F.R.G.) were used. Incubation of the cultures was performed at 3 7 ° C in a humidified atmosphere of 5% CO 2. After 1-7 days of culture without change of medium 320 /~1 of supernatant were removed from each tube to be assayed for cytokine levels. Proliferatwe response For the measurement of proliferative activity whole blood and isolated PBMC were cultured in
lnterferon-'y ELISA IFN-'/ levels in the supernatants were determined as previously described (Gallati et al., 1986). Briefly, the supernatants or a standard IFN-~, solution was added to a microtiter plate coated with a murine monoclonal antibody (mab) to IFN-~, (clone 69). Following the addition of a second mab to IFN-7, (clone 123), which was coupled to peroxidase (pod), the plate was incubated at room temperature for 16-24 h. Unbound material was removed by a washing step and the amount of bound peroxidase was determined by a short incubation with tetramethylbenzidine. On stopping the reaction with sulfuric acid the color changed to yellow and its intensity was determined at 450 nm by a computerized multichannel photometer. The amount of human IFN-7, was calculated from a standard curve. This ELISA had an assay range of 0-1000 p g / m l and a detection limit of about 50 p g / m l IFN-3,. lnterleukm-1 a-ELISA This test was based on the same principle as the IFN-~, test. Microtiter plates (Nunc) were coated with a polyclonal goat anti-human-IL-la antibody. For the detection of protein bound I L - l a the pod-linked Fab fragment of a polyclonal goat anti-human-IL-la antibody was used. (assay range: 5-100 pg/ml).
193
TNF-a-ELISA In this test, the first immobilized antibody was a monoclonal mouse a n t i - h u m a n - T N F - a antibody and the second antibody was a pod-coupled polyclonal rabbit a n t i - h u m a n - T N F - a antibody. This test had an assay range of 10-500 p g / m l . lnterleukm-2-ELISA For this test microtiter plates were coated with two monoclonal mouse anti-human IL-2 antibodies, and a third pod-linked m a b was used for detection of bound IL-2 (assay range: 50-1000 pg/ml). Statistwal analysts The results in the tumor and control groups were statistically evaluated using the Kruskal-Wallis test (X 2 approximation).
Results
Comparison of tsolated PBMC cultures and whole blood cell cultures In both systems there was an evident dependence of cytokine production on mononuclear cell concentrations. Fig. 1 shows a typical curve of I F N - 7 production in cultures of isolated PBMC and whole blood of the same donor at various cell concentrations. At equal cell numbers the cytokine values compared very well (except for high blood
IF N - g a m m a 70
(ng/ml)
60 50
~
40
30 20 10 0
m i
2
3
4
I
I
I
I
5
6
7
8 X i00000
PBMC/500 ~1
Fig. 1. Correlation of mononuclear cell count and mitogen-induced IFN-7 production m whole blood cell cultures and isolated PBMC cultures. In whole blood cell cultures the absolute number of monoeytes and lymphoeytes was calculated from the differential cell count and adjusted by dilution Mltogen. PWM 5 pg/ml; incubation time' 4 days.
concentrations). Ttus was not true for the T N F values, wtuch were always higher in the P B M C cultures. The effect of adding heparin, which was necessary to prevent clotting in the whole blood cultures, was also tested. In the range up to 400 U / m l heparin had no influence either on cytokine production or on the ELISA. In contrast, when using E D T A blood, only minimal stimulation was obtained. The reproducibility of the whole blood culture system was very good. Ten independent cultures of one blood sample gave very reproducible cytokine values under equal stimulation conditions, the coefficients of variation ranging between 5% and 20%. In contrast, the reproducibility of the stimulation experiments with isolated P B M C was less satisfactory.
Kinetics of cytokine productzon and the prohferatwe response Fig. 2 shows cumulative time-response curves of cytokine production in whole blood cell cultures upon stimulation with PHA. The curves for the four cytokines differed: I F N - 7 and I L - l a production steadily increased until day 4 and then reached a plateau. For T N F there was already a m a x i m u m after 24 h of incubation and often a second peak between day 4 and day 6. IL-2 secreuon reached a m a x i m u m between day 2 and day 3 and then declined. The corresponding kinetics of mitogen induced proliferation of the same cultures is also shown. The kinetics of the isolated P B M C cultures were similar. Cytokme values in whole blood cultures of healthy donors and tumor pattents In a first trial we used the whole blood system for the screening of 60 cancer patients and 60 healthy controls. We chose two of the four cytokines and measured the values of I F N - y and I L - l a in the 4 day post-induction supernatants following stimulation with P H A and PWM. In the controls I F N - 7 levels ranged from 10 to 250 n g / m l , and I L - l a values were between 50 and 500 p g / m l regardless of which mitogen was studied. Cell samples from the tumor patients had a significantly lower capacity to produce I F N - 7 and I L - l a at all mitogen concentrations used, although
194
leukocyte and lymphocyte counts were similar in both groups of individuals (Fig. 3).
IFN-gamma
(ng/=l)
250 .
MAX
Discussion We have described a test system for the measurement of four different cytokines using whole blood cell cultures and simple and reproducible enzymoimmunological tests. Comparing the whole blood method with isolated PBMC cultures we found a direct correlation for IFN-7 and IL-la concentrations if the cell numbers were adjusted in both systems. From these results we conclude that, in spite of the complexity of the cellular events taking place in these cultures, whole blood tests do indeed reflect the performance of T lymphocytes and monocytes as already postulated by other authors (Luquetti et al., 1976). In con-
200
150
~3
100
...........
ME]
50 ng/ml x6o
CPM
x
i000
14o
~=o 0 xoo controls
tumor
controls
tumor
Fig. 3. IVhtogen-mduced IFN-7 productton m whole blood cell
so
cultures of 60 tumor patients and 60 healthy controls. Incubation time: 4 days. Mltogens: 10 /~g/ml PHA (left); 5 / t g / m l PWM (right).
6o
4o
2o
• .......
Pg/~oo
p~.
laoo ~ooo
aoo
~ 2oo 6oo
x
2
3
•
(dsy)
~
7
Fig. 2. T ~ e course of cytoMne pr~uctmn m whole bkKxt ce~ cultures and the ~ i a t ~
pro~rative r ~ p o n ~ .
trast, TNF-a values were generally higher in the PBMC cultures than in the corresponding whole blood tests. We suggest that the reason for this is prestimulation of cells either through the separation procedure or cell death or both. The whole blood method is based on an optimal dilution of the blood cells in medium and has great advantages over isolated PBMC cultures. It requires only small amounts of blood and no unphysiological cell separation is involved. Another advantage is that this method is easy and rapid, making it suitable for testing large populations of patients. This was also true for the enzymoimmunological tests for the determination of the cytokines. Comparing the time course of the four cytokine values in the culture superuatants we decided to take measurements on day 4 of incubation.
195
With this test system a normal range of mitogen responsiveness could be defined for the controls and it was possible to show that the tumor patients had a demonstrable decrease in IFN--/ and IL-1 production by their mononuclear cells. We were also able to show that these reduced cytokine levels were not due to a reduced lymphocyte count in the cancer patients. This finding is in accordance with the results of Fritze et al. (1984), who showed a reduced lymphocyte activity, as measured by lymphocyte proliferation, in cultures of whole blood from cancer patients. Further investigations wdl be needed to show whether this method provides appropriate information about the state of cellular immunity and whether or not it will be useful for prognostic purposes.
Acknowledgements The authors wish to thank B. Koch and I. Pracht for their excellent technical assistance.
References Bubenik, J., Kleler, J., Tromholt, V., Herman, G and Jandlova, T. (1988) Defect m lectm-mduced mterleukin 2 production
by peripheral blood lymphocytes of pauents with lnvaslve urinary bladder carcinoma. Immunol. Lett. 18, 115 Fntze, D. and Dystant, P. (1984) Detection of impaired rmtogen responses m autologous whole blood of cancer pauents using an o p ~ d method of lymphocyte sUmulatton. Immunol. Lett. 8, 243 Gailata, H., Pracht, I., Schnudt, J., HRnng, P. and Garotta, G. (1987) A simple, rapid and large capacity ELISA for btologically active and recombinant human IFN-gamma. J. Biol. Regul. Homeost. Agents 1, 109. Ho, A.D., Moritz, T., Rensch, K., Hunstem, W and Kirchner, H. (1988) Defloency m interferon producUon of peripheral blood leukocytes from patients with non-Hodgkm lymphoma. J. Interferon Res. 8, 405 Karchner, H., Klemicke, Ch. and Ehgel, W. (1981) A whole blood techmque for testing producUon of human interferon by leukocytes. J. Immunol. Methods 48, 213. Luquetti, A. and Janossy, G. (1976) Lymphocyte acUvaUon VIII. The apphcatlon of a whole blood test to the quanUtauve analysis of PHA responsive T cells. J. Immunol. Methods 10, 7. Paganelh, R., Capoblanclu, M.R., Matncardl, P.M., Cloc, L., Sermnara, R. Dlanzam, F. and AinU, F. (1984) Defecave interferon-gamma production m Atayaa-Telang~cctasia Chn. Immunol. Immunopathol. 32, 387. Vdcec, J., Klion, A , Henrikscn-DeStefano, D., Zemtsov, A., Dawdson, D.M, Dawdson, M., Frledman-I(den, A.E. and Le, J. (1986) Defective gamma-interferon producUon m peripheral blood leukocytes of paUents with acute tuberculosls J Chn. Immunol. 6, 146.
