139
Journal of Immunological Methods, 129 (1990) 139-141 Elsevier JIM 05578
Short communication
Sodium pyruvate inhibits the spontaneous release of 51Cr from RBC in chromium release assays Arthur S. Colsky and James S. Peacock Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101, U.S.A. (Received 16 January 1990, accepted 7 February 1990)
This report describes the utility of sodium pyruvate for markedly decreasing the spontaneous release of chromium-51 from RBC in long term chromium release assays. This method can be used to increase the sensitivity and duration of cytotoxicity assays which use chromium-51-1abeled RBC as targets. Key words: Erythrocyte; Chromium release assay; Sodium pyruvate
Introduction The standard chromium release assay (CRA) is one of the most widely applied in vitro assays used to measure cell lysis (Brunner, 1968; Russel, 1981). The CRA is based on the release of preloaded 51Cr from the cell cytoplasm into the supernatant where the amount of the isotope liberated can be quantified to determine the percent cytolysis. One of the main limitations to the CRA is the high level of spontaneous release of 51Cr which precludes its application for measuring lysis over extended periods of incubation. For example, cytotoxicity by macrophage and granulocyte populations often requires incubation periods of 18 h or greater (Russel, 1981; Somerset al., 1986; Graziano et al., 1989). Such long-term killing assays commonly employ red blood cells (RBC) as susceptible targets (Imir et al., 1976; Ralph and Nakoinz, 1977; Koren and Fisher, 1981). RBC, however, are especially prone to spontaneously releasing 51Cr within relatively short incubation
Correspondence to: J.S. Peacock, Department of Microbiology and Immunology, R-138, University of Miami School of Medicine, Miami, FL 33101, U.S.A.
periods. In one method aimed at alleviating this problem, the 51Cr-labeled RBC targets are coincubated with large numbers of unlabeled autologous RBC. This use of unlabeled 'filler' cells results in a diminished spontaneous release of the isotope from the labeled targets through ill-defined mechanisms. However, since this method provides only partial resolution to this problem, and since the spontaneous release obtained by this method may not adequately reflect that which occurs in samples incubated with various effector cell populations, a method which can diminish the spontaneous release of 51Cr without the need for 'filler' cells would be of value. In this report, we show that the addition of sodium pyruvate in CRAs greatly reduces the spontaneous release of 5~Cr for extended periods. This observation has allowed us to increase both the duration and sensitivity of the CRA using RBC of diverse species origin.
Materials and methods
Chicken and mouse RBC were obtained immediately prior to each experiment by cardiac puncture of normal chickens and tail vein punc-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers.B.V. (Biomedical Division)
140 ture of B A L B / c mice, respectively. An aliquot of each RBC preparation was washed in HBSS and labeled with 100 #Ci Na51CrO4 (Amersham) by incubation for 1 h at 3 7 ° C in 5% CO2. Subsequently, the cells were washed 3 × in HBSS and adjusted to 1 x 105/ml in RPMI 1640 culture medium supplemented with 300 # g / m l L-glutamine, 20 m M Hepes buffer, 100 U / m l penicillin, 0.1 m g / m l streptomycin (all from Sigma) and 50 n g / m l gentamycin (Gibco Laboratories, Grand Island, NY), in the presence or absence of 1 mM sodium pyruvate as designated. 100 #1 of the target cell suspension was added to an equal volume of media alone or media containing appropriately concentrated effector cells in 96 well microtiter plates (Costar, Cambridge, MA) which were then centrifuged at 20 x g for 5 rain before incubation. Following incubation for designated periods at 37°C, the culture plates were gently spun down and a 100 #1 aliquot of supernatant was removed from each well and assayed for ra-
dioactivity in a LKB Clinigamma counter. The % cytotoxicity was calculated according to the formula: experimental release - spontaneous release x 100% maximum r e l e a s e - spontaneous release
In assays of antibody-dependent cellular cytotoxicity, effector cells consisted of thioglycollate (TG)-elicited peritoneal M e populations harvested by peritoneal lavage 3 - 4 days following the injection of 1 ml sterile T G broth into the peritoneal cavities of normal B A L B / c mice. The IgG fraction of rabbit anti-CRBC was purchased from Accurate Chemical Co. (Westbury, NY).
R e s u l t s and d i s c u s s i o n
A comparison of the spontaneous release of 5]Cr from RBC incubated in the presence or absence of sodium pyruvate is shown in Fig. 1. In
100 "
/
80"
lmM
•
O
"~ n-
b o-"
o-.--
6o
100
8O
Pyruvate
!
Normal
G)
fA m
_e
:6o
(/3 O
e-
O G) ¢-
/
co
o~
20
0
£:: O OZ. 09
2O
i
0
40
20
!
40
I
60
Time (hrs)
I
80
i
100
0
i
0
20
!
40
!
60
I
80
Time (hrs)
Fig. l. Sodiumpyruvate inhibits the spontaneous release of 51Cr from RBC. RBC (5000/microtiter well) were chicken(a) and mouse (b) obtained and treated as described in the materials and methods section. Spontaneous $1Cr release was assessed in a standard 5]Cr release assay for the designated incubation periods. Results are from a representative experiment and are expressed as the mean of quadruplicate samples. Maximum 51Crrelease in the presenceof 0.1~ Triton X-100 was 864 cpm for CRBC and 577 clamfor MRBC.
141
TABLE 1 SODIUM PYRUVATE DOES NOT INHIBIT SlCr RELEASE INDUCED BY EFFECTOR CELL-MEDIATED CYTOTOXICITY a E l T ratio
20 10
% lysis+ antibody 63.5 60.7
% lysis- antibody 18.9 21.0
a CRBC targets (5000/microtiter well) were incubated for 18 h with effector cells in the presence or absence of rabbit IgG anti-CRBC (25/~g/ml). Spontaneous51Cr release by CRBC in the absence of effector cells was 55.3 cpm and maximum release of the isotope by 0.1% Triton X-100 was 410.7 clam. contrast to RBC incubated in medium devoid of sodium pyruvate, which released greater than 80% of the 51Cr within 6 h of incubation, RBC cultured with 1 m M sodium pyruvate liberated only very low levels of 5tCr within the same culture period. Both mouse (Fig. l a ) and chicken (Fig. l b ) RBC exhibited very low spontaneous release of 51Cr in the presence of sodium pyruvate. Even after 2 days of incubation, mouse RBC (MRBC) and chicken RBC (CRBC) had released only 25 and 5% of the SlCr, respectively and CRBC still retained over 90% of the isotope after three days of culture. A similar effect of sodium pyruvate was also observed for human and rat RBC (data not shown). The presence of sodium pyruvate in a standard CRA did not inhibit the release of 5~Cr from the RBC targets following effector cell mediated cytotoxic attack. In a standard assay of antibody dependent cellular cytotoxicity (ADCC) (Koren and Fisher, 1981), efficient antibody-dependent RBC lysis was detected as measured by the release of large amounts of 5~Cr into the media. At an E : T ratio of 20, over 60% of CRBC targets were lysed by peritoneal exudate macrophages in the presence of CRBC-specific rabbit IgG. At the same E : T ratio, antibody-independent cytotoxicity by the same effector cells resulted in approximately 19% lysis of the erythrocyte targets. CRBC incubated in the absence of the antibody or effector cells spontaneously released only 13% of the preloaded isotope.
The mechanism by which sodium pyruvate inhibits the release of cytoplasmic 51Cr from RBC is not known. A similar effect on the spontaneous release was not observed for various tumor cell populations (data not shown). In RBC, pyruvate is reduced to lactate in a process which increases the concentration of N A D ÷ and results in an upregulation of glycolysis (Harris, 1986). Since RBC rely exclusively on glycolysis for the production of energy required in maintaining active ion gradients important in homeostasis, it can be reasoned that an increase in the availability of pyruvate for generating N A D ÷ during conversion to lactate may be a source of increased energy production for the cell. Although this is highly speculative, the passive diffusion of pyruvate across the plasma membrane would make any exogenously added sodium pyruvate readily available to the cell for use in this capacity and may serve to prevent breakdown of the cell's integrity thereby precluding the spontaneous release of various int,racellular substances including SlCr. References Brunner, K.T., Manuel, J., Cerrottini, J.-C. and Chapuis, B. (1968) Immunobiology 14, 181. Graziano, R.F., Looney, R.J., Shen, L. and Fanger, M.W. (1989) FcyR-mediated killing by eosinophils. J. lmmunol. 142, 230. Harris, R.A. (1986) Carbohydrate metabolism. I. Major metabolic pathways and their control. In: T.M. Devhn (Ed.), Textbook of Biochemistry with Clinical Correlations. John Wiley, New York, p. 261. lmir, T., Saksela, E. and Makela, O. (1976) Two types of antibody dependent cell-mediated cytotoxicity, arming and sensitization. J. Immunol. 117, 1938. Koren, H.S. and Fisher, D,G. (1981) Antibody-dependent cellular cytotoxicity (ADCC) of erythroid and tumor cells. In: D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for studying mononuclear phagocytes. Academic Press, New York, p. 813. Ralph, P. and Nakoinz, I. (1977) Antibody-dependent killing of erythrocyte and tumor targets by macrophage-related cell lines: enhancement by PPD and LPS. J. hnmunol. 119, 950. Russel, S.W. (1981) Quantification of cytolysis of neoplastic cells by release of chromium-51. In: D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for studying mononuclear phagocytes. Academic Press, New York, p. 793.
Journal of Immunological Methods, 129 (1990) 139-141 Elsevier JIM 05578
Short communication
Sodium pyruvate inhibits the spontaneous release of 51Cr from RBC in chromium release assays Arthur S. Colsky and James S. Peacock Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101, U.S.A. (Received 16 January 1990, accepted 7 February 1990)
This report describes the utility of sodium pyruvate for markedly decreasing the spontaneous release of chromium-51 from RBC in long term chromium release assays. This method can be used to increase the sensitivity and duration of cytotoxicity assays which use chromium-51-1abeled RBC as targets. Key words: Erythrocyte; Chromium release assay; Sodium pyruvate
Introduction The standard chromium release assay (CRA) is one of the most widely applied in vitro assays used to measure cell lysis (Brunner, 1968; Russel, 1981). The CRA is based on the release of preloaded 51Cr from the cell cytoplasm into the supernatant where the amount of the isotope liberated can be quantified to determine the percent cytolysis. One of the main limitations to the CRA is the high level of spontaneous release of 51Cr which precludes its application for measuring lysis over extended periods of incubation. For example, cytotoxicity by macrophage and granulocyte populations often requires incubation periods of 18 h or greater (Russel, 1981; Somerset al., 1986; Graziano et al., 1989). Such long-term killing assays commonly employ red blood cells (RBC) as susceptible targets (Imir et al., 1976; Ralph and Nakoinz, 1977; Koren and Fisher, 1981). RBC, however, are especially prone to spontaneously releasing 51Cr within relatively short incubation
Correspondence to: J.S. Peacock, Department of Microbiology and Immunology, R-138, University of Miami School of Medicine, Miami, FL 33101, U.S.A.
periods. In one method aimed at alleviating this problem, the 51Cr-labeled RBC targets are coincubated with large numbers of unlabeled autologous RBC. This use of unlabeled 'filler' cells results in a diminished spontaneous release of the isotope from the labeled targets through ill-defined mechanisms. However, since this method provides only partial resolution to this problem, and since the spontaneous release obtained by this method may not adequately reflect that which occurs in samples incubated with various effector cell populations, a method which can diminish the spontaneous release of 51Cr without the need for 'filler' cells would be of value. In this report, we show that the addition of sodium pyruvate in CRAs greatly reduces the spontaneous release of 5~Cr for extended periods. This observation has allowed us to increase both the duration and sensitivity of the CRA using RBC of diverse species origin.
Materials and methods
Chicken and mouse RBC were obtained immediately prior to each experiment by cardiac puncture of normal chickens and tail vein punc-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers.B.V. (Biomedical Division)
140 ture of B A L B / c mice, respectively. An aliquot of each RBC preparation was washed in HBSS and labeled with 100 #Ci Na51CrO4 (Amersham) by incubation for 1 h at 3 7 ° C in 5% CO2. Subsequently, the cells were washed 3 × in HBSS and adjusted to 1 x 105/ml in RPMI 1640 culture medium supplemented with 300 # g / m l L-glutamine, 20 m M Hepes buffer, 100 U / m l penicillin, 0.1 m g / m l streptomycin (all from Sigma) and 50 n g / m l gentamycin (Gibco Laboratories, Grand Island, NY), in the presence or absence of 1 mM sodium pyruvate as designated. 100 #1 of the target cell suspension was added to an equal volume of media alone or media containing appropriately concentrated effector cells in 96 well microtiter plates (Costar, Cambridge, MA) which were then centrifuged at 20 x g for 5 rain before incubation. Following incubation for designated periods at 37°C, the culture plates were gently spun down and a 100 #1 aliquot of supernatant was removed from each well and assayed for ra-
dioactivity in a LKB Clinigamma counter. The % cytotoxicity was calculated according to the formula: experimental release - spontaneous release x 100% maximum r e l e a s e - spontaneous release
In assays of antibody-dependent cellular cytotoxicity, effector cells consisted of thioglycollate (TG)-elicited peritoneal M e populations harvested by peritoneal lavage 3 - 4 days following the injection of 1 ml sterile T G broth into the peritoneal cavities of normal B A L B / c mice. The IgG fraction of rabbit anti-CRBC was purchased from Accurate Chemical Co. (Westbury, NY).
R e s u l t s and d i s c u s s i o n
A comparison of the spontaneous release of 5]Cr from RBC incubated in the presence or absence of sodium pyruvate is shown in Fig. 1. In
100 "
/
80"
lmM
•
O
"~ n-
b o-"
o-.--
6o
100
8O
Pyruvate
!
Normal
G)
fA m
_e
:6o
(/3 O
e-
O G) ¢-
/
co
o~
20
0
£:: O OZ. 09
2O
i
0
40
20
!
40
I
60
Time (hrs)
I
80
i
100
0
i
0
20
!
40
!
60
I
80
Time (hrs)
Fig. l. Sodiumpyruvate inhibits the spontaneous release of 51Cr from RBC. RBC (5000/microtiter well) were chicken(a) and mouse (b) obtained and treated as described in the materials and methods section. Spontaneous $1Cr release was assessed in a standard 5]Cr release assay for the designated incubation periods. Results are from a representative experiment and are expressed as the mean of quadruplicate samples. Maximum 51Crrelease in the presenceof 0.1~ Triton X-100 was 864 cpm for CRBC and 577 clamfor MRBC.
141
TABLE 1 SODIUM PYRUVATE DOES NOT INHIBIT SlCr RELEASE INDUCED BY EFFECTOR CELL-MEDIATED CYTOTOXICITY a E l T ratio
20 10
% lysis+ antibody 63.5 60.7
% lysis- antibody 18.9 21.0
a CRBC targets (5000/microtiter well) were incubated for 18 h with effector cells in the presence or absence of rabbit IgG anti-CRBC (25/~g/ml). Spontaneous51Cr release by CRBC in the absence of effector cells was 55.3 cpm and maximum release of the isotope by 0.1% Triton X-100 was 410.7 clam. contrast to RBC incubated in medium devoid of sodium pyruvate, which released greater than 80% of the 51Cr within 6 h of incubation, RBC cultured with 1 m M sodium pyruvate liberated only very low levels of 5tCr within the same culture period. Both mouse (Fig. l a ) and chicken (Fig. l b ) RBC exhibited very low spontaneous release of 51Cr in the presence of sodium pyruvate. Even after 2 days of incubation, mouse RBC (MRBC) and chicken RBC (CRBC) had released only 25 and 5% of the SlCr, respectively and CRBC still retained over 90% of the isotope after three days of culture. A similar effect of sodium pyruvate was also observed for human and rat RBC (data not shown). The presence of sodium pyruvate in a standard CRA did not inhibit the release of 5~Cr from the RBC targets following effector cell mediated cytotoxic attack. In a standard assay of antibody dependent cellular cytotoxicity (ADCC) (Koren and Fisher, 1981), efficient antibody-dependent RBC lysis was detected as measured by the release of large amounts of 5~Cr into the media. At an E : T ratio of 20, over 60% of CRBC targets were lysed by peritoneal exudate macrophages in the presence of CRBC-specific rabbit IgG. At the same E : T ratio, antibody-independent cytotoxicity by the same effector cells resulted in approximately 19% lysis of the erythrocyte targets. CRBC incubated in the absence of the antibody or effector cells spontaneously released only 13% of the preloaded isotope.
The mechanism by which sodium pyruvate inhibits the release of cytoplasmic 51Cr from RBC is not known. A similar effect on the spontaneous release was not observed for various tumor cell populations (data not shown). In RBC, pyruvate is reduced to lactate in a process which increases the concentration of N A D ÷ and results in an upregulation of glycolysis (Harris, 1986). Since RBC rely exclusively on glycolysis for the production of energy required in maintaining active ion gradients important in homeostasis, it can be reasoned that an increase in the availability of pyruvate for generating N A D ÷ during conversion to lactate may be a source of increased energy production for the cell. Although this is highly speculative, the passive diffusion of pyruvate across the plasma membrane would make any exogenously added sodium pyruvate readily available to the cell for use in this capacity and may serve to prevent breakdown of the cell's integrity thereby precluding the spontaneous release of various int,racellular substances including SlCr. References Brunner, K.T., Manuel, J., Cerrottini, J.-C. and Chapuis, B. (1968) Immunobiology 14, 181. Graziano, R.F., Looney, R.J., Shen, L. and Fanger, M.W. (1989) FcyR-mediated killing by eosinophils. J. lmmunol. 142, 230. Harris, R.A. (1986) Carbohydrate metabolism. I. Major metabolic pathways and their control. In: T.M. Devhn (Ed.), Textbook of Biochemistry with Clinical Correlations. John Wiley, New York, p. 261. lmir, T., Saksela, E. and Makela, O. (1976) Two types of antibody dependent cell-mediated cytotoxicity, arming and sensitization. J. Immunol. 117, 1938. Koren, H.S. and Fisher, D,G. (1981) Antibody-dependent cellular cytotoxicity (ADCC) of erythroid and tumor cells. In: D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for studying mononuclear phagocytes. Academic Press, New York, p. 813. Ralph, P. and Nakoinz, I. (1977) Antibody-dependent killing of erythrocyte and tumor targets by macrophage-related cell lines: enhancement by PPD and LPS. J. hnmunol. 119, 950. Russel, S.W. (1981) Quantification of cytolysis of neoplastic cells by release of chromium-51. In: D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for studying mononuclear phagocytes. Academic Press, New York, p. 793.
