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In vitro inhibition of the primary antibody response to sheep erythrocytes by cyclophosphamide
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
SHORT
59,617-619
(1981)
COMMUNICATIONS
In Vitro Inhibition of the Primary Antibody Response Erythrocytes by Cyclophosphamidel
The Mishell-Dutton in tvitro antibody-producing assay has been found useful for recimmunosuppressive chemicals ognizing (Archer et al., 1978). In addition to specificity, the system offers the advantage of complex cellular interactions involved to achieve the ultimate response. The disadvantage of the assay, as pointed out by Kutz et ~1. (1980), is that chemicals which require activation to reactive metabolites are not detectable. It is the purpose of this communication to report on the inclusion of a system for metabolic activation in the Mishell-Dutton assay and the inhibitory effect of cyclophosphamide when used with the described modification.
to Sheep
fraction was prepared from livers of male BALB/c mice to serve as a source of enzymes for metabolic activation. In some cases the animals were induced with phenobarbital by placing 1 mglml in the drinking water for 1 week until 24 hr prior to sacrifice by cervical dislocation. Livers were removed aseptically and homogenized in 3 ml of cold, sterile 1.15% KCVg liver. This homogenate was centrifuged at 9OOOg and the supematant (S9 fraction) removed and frozen in l-ml aliquots. When used, a tube of S9 was removed from storage at - 7O”C, thawed, and added to a solution of cofactors. This “S9 mix” contained 3 mg S9 protein, 2 mM NADP, and 35 mM isocitrate in sterile culture medium and was added directly to spleen cell suspensions at a dilution of 1 part Mix per 10 parts culture. Incubation qf‘ spleen cell suspension wjith S9 mix. Suspensions of spleen cells were adjusted to 1.5 x 10’ cells/ml in RPM1 1640 without serum and distributed into 10 x 35-mm plastic petri plates, 1 ml per nlate. To each plate was added 0.1 ml of S9 mix and cyctophosphamide (from Sigma) as indicated in the table of data. The plates were incubated at 37°C for 1 hr on a rocker platform in boxes gassed with a mixture of 10% CO,, 7% 0,. 83% N, . Following this incubation period, the contents of each plate were transferred to a sterile plastic tube and centrifuged at 2OOOg for 10 min. This pellet was resuspended in 1.1 ml RPM1 1640 with serum (1 X 10’ cells/ml) and 1 ml pkrced in a fresh plate. Following addition of antigen (5 x 106 sheep erythrocytes), the cultures were gassed with the special mixture and incubated for 5 days with gentle rocking and daily feeding.
METHODS Mi.shell-Duiton crsscry. Spleens were removed from male BALB/c mice, 6-10 weeks of age, and cultured in the presence of sheep red blood cells essentially as described by Mishell and Dutton (1967). The medium used for culture was RPM1 1640 (GIBCO) containing 10% heat-inactivated fetal calf serum. Cultures were incubated with rocking at 37°C in an atmosphere of 10% CO,, 7% O,, and 83% N, and assayed on Day 5 using the Cunningham modification of the Jeme plaque assay (Cunningham and Szenberg, 1968). All cell counts were determined with a Model ZBI Coulter Counter. Metuholic ucti~~ution system. A crude microsomal
RESULTS
AND
DISCUSSION
Preliminary experiments concerning the inclusion of liver homogenate fractions in the Mishell-Dutton assay indicated that
’ Supported by U.S. Army Research and Development Command Contract DAMD 17-78-C-8083. 617
0041-008X/81/090617-03802.00/0 Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
618
SHORT TABLE
IN VIIRO
1
RESPONSE PHOSPHAMIDE
INHIBITION OF THE PRIMARY TO SHEEP ERYTHROCYTES USING THE MISHELL-DUTTON
CP mm
S9 mix
None 1.0 None 0.1 0.3 1.0 None 0.01 0.03 0.10
None None Naive Naive Naive Naive Induced Induced Induced Induced
COMMUNICATIONS
PFCiculture 1760 ? 1707 t 2520 + 1807 2 1035 2 727 -t 2227 _f 1835 t 795 _f None
210 154 218 140 121 144 154 161 Ill
ANTIBODY BY CYCLOASSAM
106 cells 5.55 5.27 5.00 4.12 5.02 2.92 5.05 3.62 3.65 1.88
k k + T k r 2 + T 2
0.47 0.17 0.23 0.59 0.81 0.34 0.41 0.53 0.18 0.23
Note. The preparation of S9 mix from liver homogenate and method of inclusion in the assay is described in detail under Methods. Plaque-forming cells (PFC) were enumerated on Day 5 of culture and are expressed as the mean 2 SE (N = 3). Cells recovered were counted on a Coulter Counter. Background (no antigen) was 102 t 23 PFCiculture.
the S9 fraction (90001: supematant) completely inhibited the lymphocyte response to sheep erythrocytes. This effect appeared to be due to soluble factors in the homogenate since microsomal protein purified by ultracentrifugation could be added to the cultures without adverse effects. However, when the cofactors necessary for microsoma1 mixed-function oxidase activity were added to cultures (at final concentrations of 0.2 mM NADP, 3.4 mM isocitrate, and 0.1 U/ml isocitrate dehydrogenase) complete inhibition resulted, which was subsequently determined to be due to the isoneither citrate dehydrogenase. Since NADP nor isocitrate was inhibitory (nor was NADPH up to 0.05 mM), it was decided to preincubate the suspension of spleen cells with the S9 mix described under Methods, collect the cells by centrifugation, and resuspend them in fresh medium for culture with antigen. This procedure evolved from other preliminary experiments which illustrated that S9 could
be used for short periods. The use of S9 allows the elimination of isocitrate dehydrogenase from the cofactors. Cyclophosphamide (CP) was chosen as a positive control for this modification of the Mishell-Dutton assay because it is known to require metabolic activation (Connors et ul., 19741, and it is immunosuppressive in the mouse (Dean et al., 1979). The effect of CP on the primary antibody response of splenic lymphocytes to sheep erythrocytes using the modified Mishell-Dutton assay is shown in Table 1. The control value ( 1760 PFC/culture) can be compared to a mean control value from six experiments not involving the modification of 2680 ? 273. Cyclophosphamide has no effect up to 1 mM when S9 mix is not included, but produces a dose-dependent inhibition in the presence of S9 from livers of either naive or phenobarbital-induced mice. Approximate ED,, values from log dose plots are 65 p&ml when naive S9 is used and 6.3 &ml when induced S9 is used. The use of the induced preparation thus caused a IO-fold greater potency of CP. A one-way analysis of variance yields F values of 14.13 when the naive S9 is used and 54.03 when the phenobarbital-induced S9 is used. The preparation used in this experiment had been stored for 3 months at -70°C. Although, the fractions were more active when used shortly after preparation, the induced preparation was slightly inhibitory at that time. On the other hand, the naive preparation is always slightly stimulatory, an observation for which we have no explanation. The data presented here are based on a modification which needs further study and characterization. Depletion of the cultures’ adherent cell populations during the preincubation must be considered, for instance. However, the modification described for the Mishell-Dutton assay makes the assay appropriate for use with the many chemicals which require metabolic activation. As well as serving as a tool for elucidating mechanisms, the modified assay may have
SHORT
value as a predictive tool for immunotoxicity. Livers from animals treated with 3methylcholanthrene can also be used for the metabolic activation system and would be appropriate for compounds activated by aryl hydrocarbon hydroxylase.
ther improvements in the plaquing technique for detecting single antibody-forming cells. Irnrnunolo~~~ 14, 599-600. DEAN, J. H.. PADARATHSINGH, M. L., JERRELLS, T. R..
D. L.. SMITH, B. G., AND BUKOVIC-WESS, J. A. (1978). Use of an in V&W antibody-producing system for recognizing potentially immunosuppressive compounds. Int. Arch. Allergy Appl. Immun. 56, 90-93.
ARCHER,
Cox,
P. B.,
FARMER,
A.
B.,
AND
M. (1974). Some studies of the active intermediates formed in the microsomal metabolism of cyclophosphamide and isophosphamide. Biorhem. Phurmacol. 23, I IS- 129.
CUNNINGHAM,
A. J., AND
SZENBERG,
L.,
AND
NORTHING.
J. (19791.
Assess-
S. A.,
HINSDILL.
R. D.,
AND
WELTMAN,
D. J.
(1980). Evaluation of chemicals for immunomodulatory effects using in ~.ifro antibody-producing assay. Environ. ReJ. 22, 368-376.
REFERENCES
J. A.,
KEYS.
ment of immunobiological effects induced by chemicals, drugs or food additives. II. Studies with cyclophosphamide. Drlr~ Chem. Toxicol. 2, 133-153. KUTZ,
CONNORS, JARMAN,
619
COMMUNICATIONS
A. (1968).
Fur-
MISHELL,
R. 1.. AND
DUTTON,
R. W.
(1967).
Immuni-
zation of dissociated mouse spleen cell cultures from normal mice. J. &I. Med. 126, 423-442. ANNE
ALBERT Depurtment of‘ Phurmacology Medical College of Virginia Richmond. Virginia 23298 Recei\,ed September 22. 1980
N.
E.
TUCKER MUNSON
AND
APPLIED
PHARMACOLOGY
SHORT
59,617-619
(1981)
COMMUNICATIONS
In Vitro Inhibition of the Primary Antibody Response Erythrocytes by Cyclophosphamidel
The Mishell-Dutton in tvitro antibody-producing assay has been found useful for recimmunosuppressive chemicals ognizing (Archer et al., 1978). In addition to specificity, the system offers the advantage of complex cellular interactions involved to achieve the ultimate response. The disadvantage of the assay, as pointed out by Kutz et ~1. (1980), is that chemicals which require activation to reactive metabolites are not detectable. It is the purpose of this communication to report on the inclusion of a system for metabolic activation in the Mishell-Dutton assay and the inhibitory effect of cyclophosphamide when used with the described modification.
to Sheep
fraction was prepared from livers of male BALB/c mice to serve as a source of enzymes for metabolic activation. In some cases the animals were induced with phenobarbital by placing 1 mglml in the drinking water for 1 week until 24 hr prior to sacrifice by cervical dislocation. Livers were removed aseptically and homogenized in 3 ml of cold, sterile 1.15% KCVg liver. This homogenate was centrifuged at 9OOOg and the supematant (S9 fraction) removed and frozen in l-ml aliquots. When used, a tube of S9 was removed from storage at - 7O”C, thawed, and added to a solution of cofactors. This “S9 mix” contained 3 mg S9 protein, 2 mM NADP, and 35 mM isocitrate in sterile culture medium and was added directly to spleen cell suspensions at a dilution of 1 part Mix per 10 parts culture. Incubation qf‘ spleen cell suspension wjith S9 mix. Suspensions of spleen cells were adjusted to 1.5 x 10’ cells/ml in RPM1 1640 without serum and distributed into 10 x 35-mm plastic petri plates, 1 ml per nlate. To each plate was added 0.1 ml of S9 mix and cyctophosphamide (from Sigma) as indicated in the table of data. The plates were incubated at 37°C for 1 hr on a rocker platform in boxes gassed with a mixture of 10% CO,, 7% 0,. 83% N, . Following this incubation period, the contents of each plate were transferred to a sterile plastic tube and centrifuged at 2OOOg for 10 min. This pellet was resuspended in 1.1 ml RPM1 1640 with serum (1 X 10’ cells/ml) and 1 ml pkrced in a fresh plate. Following addition of antigen (5 x 106 sheep erythrocytes), the cultures were gassed with the special mixture and incubated for 5 days with gentle rocking and daily feeding.
METHODS Mi.shell-Duiton crsscry. Spleens were removed from male BALB/c mice, 6-10 weeks of age, and cultured in the presence of sheep red blood cells essentially as described by Mishell and Dutton (1967). The medium used for culture was RPM1 1640 (GIBCO) containing 10% heat-inactivated fetal calf serum. Cultures were incubated with rocking at 37°C in an atmosphere of 10% CO,, 7% O,, and 83% N, and assayed on Day 5 using the Cunningham modification of the Jeme plaque assay (Cunningham and Szenberg, 1968). All cell counts were determined with a Model ZBI Coulter Counter. Metuholic ucti~~ution system. A crude microsomal
RESULTS
AND
DISCUSSION
Preliminary experiments concerning the inclusion of liver homogenate fractions in the Mishell-Dutton assay indicated that
’ Supported by U.S. Army Research and Development Command Contract DAMD 17-78-C-8083. 617
0041-008X/81/090617-03802.00/0 Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
618
SHORT TABLE
IN VIIRO
1
RESPONSE PHOSPHAMIDE
INHIBITION OF THE PRIMARY TO SHEEP ERYTHROCYTES USING THE MISHELL-DUTTON
CP mm
S9 mix
None 1.0 None 0.1 0.3 1.0 None 0.01 0.03 0.10
None None Naive Naive Naive Naive Induced Induced Induced Induced
COMMUNICATIONS
PFCiculture 1760 ? 1707 t 2520 + 1807 2 1035 2 727 -t 2227 _f 1835 t 795 _f None
210 154 218 140 121 144 154 161 Ill
ANTIBODY BY CYCLOASSAM
106 cells 5.55 5.27 5.00 4.12 5.02 2.92 5.05 3.62 3.65 1.88
k k + T k r 2 + T 2
0.47 0.17 0.23 0.59 0.81 0.34 0.41 0.53 0.18 0.23
Note. The preparation of S9 mix from liver homogenate and method of inclusion in the assay is described in detail under Methods. Plaque-forming cells (PFC) were enumerated on Day 5 of culture and are expressed as the mean 2 SE (N = 3). Cells recovered were counted on a Coulter Counter. Background (no antigen) was 102 t 23 PFCiculture.
the S9 fraction (90001: supematant) completely inhibited the lymphocyte response to sheep erythrocytes. This effect appeared to be due to soluble factors in the homogenate since microsomal protein purified by ultracentrifugation could be added to the cultures without adverse effects. However, when the cofactors necessary for microsoma1 mixed-function oxidase activity were added to cultures (at final concentrations of 0.2 mM NADP, 3.4 mM isocitrate, and 0.1 U/ml isocitrate dehydrogenase) complete inhibition resulted, which was subsequently determined to be due to the isoneither citrate dehydrogenase. Since NADP nor isocitrate was inhibitory (nor was NADPH up to 0.05 mM), it was decided to preincubate the suspension of spleen cells with the S9 mix described under Methods, collect the cells by centrifugation, and resuspend them in fresh medium for culture with antigen. This procedure evolved from other preliminary experiments which illustrated that S9 could
be used for short periods. The use of S9 allows the elimination of isocitrate dehydrogenase from the cofactors. Cyclophosphamide (CP) was chosen as a positive control for this modification of the Mishell-Dutton assay because it is known to require metabolic activation (Connors et ul., 19741, and it is immunosuppressive in the mouse (Dean et al., 1979). The effect of CP on the primary antibody response of splenic lymphocytes to sheep erythrocytes using the modified Mishell-Dutton assay is shown in Table 1. The control value ( 1760 PFC/culture) can be compared to a mean control value from six experiments not involving the modification of 2680 ? 273. Cyclophosphamide has no effect up to 1 mM when S9 mix is not included, but produces a dose-dependent inhibition in the presence of S9 from livers of either naive or phenobarbital-induced mice. Approximate ED,, values from log dose plots are 65 p&ml when naive S9 is used and 6.3 &ml when induced S9 is used. The use of the induced preparation thus caused a IO-fold greater potency of CP. A one-way analysis of variance yields F values of 14.13 when the naive S9 is used and 54.03 when the phenobarbital-induced S9 is used. The preparation used in this experiment had been stored for 3 months at -70°C. Although, the fractions were more active when used shortly after preparation, the induced preparation was slightly inhibitory at that time. On the other hand, the naive preparation is always slightly stimulatory, an observation for which we have no explanation. The data presented here are based on a modification which needs further study and characterization. Depletion of the cultures’ adherent cell populations during the preincubation must be considered, for instance. However, the modification described for the Mishell-Dutton assay makes the assay appropriate for use with the many chemicals which require metabolic activation. As well as serving as a tool for elucidating mechanisms, the modified assay may have
SHORT
value as a predictive tool for immunotoxicity. Livers from animals treated with 3methylcholanthrene can also be used for the metabolic activation system and would be appropriate for compounds activated by aryl hydrocarbon hydroxylase.
ther improvements in the plaquing technique for detecting single antibody-forming cells. Irnrnunolo~~~ 14, 599-600. DEAN, J. H.. PADARATHSINGH, M. L., JERRELLS, T. R..
D. L.. SMITH, B. G., AND BUKOVIC-WESS, J. A. (1978). Use of an in V&W antibody-producing system for recognizing potentially immunosuppressive compounds. Int. Arch. Allergy Appl. Immun. 56, 90-93.
ARCHER,
Cox,
P. B.,
FARMER,
A.
B.,
AND
M. (1974). Some studies of the active intermediates formed in the microsomal metabolism of cyclophosphamide and isophosphamide. Biorhem. Phurmacol. 23, I IS- 129.
CUNNINGHAM,
A. J., AND
SZENBERG,
L.,
AND
NORTHING.
J. (19791.
Assess-
S. A.,
HINSDILL.
R. D.,
AND
WELTMAN,
D. J.
(1980). Evaluation of chemicals for immunomodulatory effects using in ~.ifro antibody-producing assay. Environ. ReJ. 22, 368-376.
REFERENCES
J. A.,
KEYS.
ment of immunobiological effects induced by chemicals, drugs or food additives. II. Studies with cyclophosphamide. Drlr~ Chem. Toxicol. 2, 133-153. KUTZ,
CONNORS, JARMAN,
619
COMMUNICATIONS
A. (1968).
Fur-
MISHELL,
R. 1.. AND
DUTTON,
R. W.
(1967).
Immuni-
zation of dissociated mouse spleen cell cultures from normal mice. J. &I. Med. 126, 423-442. ANNE
ALBERT Depurtment of‘ Phurmacology Medical College of Virginia Richmond. Virginia 23298 Recei\,ed September 22. 1980
N.
E.
TUCKER MUNSON