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Amphotericin B-induced immunosuppression in tumor-bearing mice
Int. J. Immunopharmac., Vol. 1, pp. 299-301 © Pergamon Press Ltd. 1979. Printed in Great Britain.
0192-0561/79/1201-0299 $02.00/0
SHORT COMMUNICATION
AMPHOTERICIN B-INDUCED IMMUNOSUPPRESSION IN TUMORBEARING MICE ANTONIO FERRANTEand Y. H. THONG Department of Paediatrics, University of Adelaide, The Adelaide Children's Hospital, North Adelaide, 5006, South Australia, Australia
(Received 1 August 1979; in final form 10 September 1979) Abstract~B16 melanoma-bearing mice had a reduced capacity to mount a delayed-type hypersensitivity response, and this response was further depressed when these mice were also treated with multiple daily doses of amphotericin B (AraB). Results from the present experiments, which were designed to simulate the clinical situation, indicate that patients receivingAmB may require frequent monitoring of immune functions during treatment.
Cell-mediated immunity (CMI) is an important defence mechanism against fungal infections (Gentry & Remington, 1971; Rogers, Balish & Manning, 1976). The immunity of the host may be greatly suppressed firstly by the tumour and, secondly by the various treatments such as corticosteroids, cytotoxic drugs and irradiation. Such changes in immunity may have a profound effect on the ability of the host to resist fungal infection and thus fungal infections are common in cancer patients. Systemic fungal infections are commonly treated with the polyene antibiotic amphotericin B (AmB) (Bennet, 1974). However, AmB may pose an additional problem for the host responding immunologically against the fungus and also against cancer because previous studies have demonstrated that AmB can suppress the immune response. In vitro AmB has been shown to suppress the mitogen-induced response of human (Tarnvik & ~ns~hn, 1974; Thong & Rowan-Kelly, 1978) and mouse lymphocytes (Ferrante, Rowan-Kelly & Thong, in press) and, when injected into mice, the drug markedly depressed the delayed-type hypersensitivity (DTH) response to sheep red blood cells (Ferrante et al., in press). In the following report, we have used the Bt6 melanoma tumour cell model to demonstrate that mice carrying a B j6 melanoma tumour display a poor DTH response which becomes further suppressed following AmB treatment.
Adelaide, and kept in an air-conditioned animal house with free access to food and water.
Amphotericin B The AmB used in the following study was Fungizone (E. R. Squibb & Sons Inc., New York). The powder was reconstituted in distilled water and further diluted to the required concentration with sterile pyrogen-free saline (0,9°70 w.v.). Although sodium deoxycholate is a diluent in the AmB preparation, previous studies showed that it did not significantly affect the DTH response at the concentrations used in these experiments and hence it was not given to control animals.
Delayed-type hypersensitivity The ability of mice to mount a DTH response to sheep red blood cells (SRBC) was used as a test for cell-mediated immunity (Liew, 1977). Mice were immunized subcutaneously (s.c.) with 1 x 10a SRBC, and then challenged with I x l0 s SRBC s.c. in the hind footpad 5 days later. Footpad thickness was measured with a dial caliper (Mercer) 24 h after challenge and the degree of DTH was expressed as the per cent increase in footpad thickness.
Anti-SRBC antibody response
Immunization was carried out by injecting mice with I x 109 SRBC intravenously (i.v.). The mice were bled 6 days later by cardiac puncture. To deterEXPERIMENTAL PROCEDURES mine the anti-SRBC antibody titre, twofold dilutions Animals (0.025 ml) of the mouse sera were made in microtitre Inbred C57B1/6J male mice, 6 - 8 weeks old, were plates in saline. Then to each well was added an equal used in the present study. They were obtained from volume of 1°7o (v/v) SRBC and the plates incubated the Institute of Medical and Veterinary Science, at 37°C for 1 h. 299
3O0
ANTONIOFERRANTEand Y. H. THONG
Maintenance and method of harvesting B16 melanoma cells The stock o f BI6 m e l a n o m a cells was obtained from D. Goh, Departments of Surgery and Microbiology and Immunology, University of Adelaide. The ceils were maintained by bi-weekly intraperitoneal (i.p.) inoculation into C 5 7 B I / 6 J mice. Harvesting of cells involved excising the tumour and pressing it through a sieve. Single cell suspensions were prepared in 0.9% saline ready for injection and checked for viability by trypan blue dye exclusion as follows: to 0.1 ml of cell suspension (2 × 106/ml) was added 0.1 ml of a 0.2% solution of trypan blue in RPMI-1640 medium, the mixture was incubated for 5 min at 37°C and the number of stained cells determined by counting in a haemocytometer. However, some reservation should be made about the accuracy of this test for cell viability (Black & Berembaum, 1964; Bhuyan, L o u g h m a n , Fracer & Day, 1976).
Statistics Statistical significance was evaluated by the Student's t-test for unpaired data. RESULTS For D T H experiments the mice were divided into 4 groups. Groups 1 and 2 received saline injections (i.p.) at day 0, while groups 3 and 4 received 5 × 105 B~6 m e l a n o m a cells. At day 7 all mice were primed with 1 × 108 SRBC s.c. Starting on the same day, groups 2 and 4 received A m B (7.5 mg/kg) as a daily i.p. injection for 4 days. At day 13, all mice were challenged with 1 × l0 s SRBC in the footpad. The D T H response was measured on day 14. The results showed that tumour-bearing mice (group 3) had a reduced capacity to mount a D T H response (Fig. 1); the per cent footpad increase was 33.7 compared to 69.2 in controls (group 1). The results also confirm previous studies that multiple doses of A m B have a suppressive effect on D T H ; the per cent footpad increase was reduced to 35.7 (group 2). O f particular interest was the finding that the combination of tumour-bearing and A m B therapy resulted in a marked suppression o f D T H , more than either tumour-bearing or A m B alone; the per cent footpad increase was only 16.8 (group 4). For antibody experiments, another lot o f mice was divided into 4 groups according to the above experimental design, except that the mice were primed with 1 × 109 SRBC i.v. on day 7 and bled by intra-cardiac puncture on day 14. The results showed that tumourbearing does not reduce the capacity to form antibody to S R B C (Fig. 2); log 2 mean titre was 7.3 (group 3) compared to 7.8 in controls (group 1). In mice
receiving A m B (group 2), the antibody titre was significantly increased to 9.8. The tumour-bearing mice receiving AraB (group 4) also showed a raised antibody titre but this difference did not reach statistical significance. CO o3 uJ Z
80-
,,, -It--
o
60--
n F-
0
co 4 0 - -
÷1 v
Z LU
co .<
_
20--
u.J t'r"
z
I 2 Control
I 3
AMB
1 4
B16
B16+AMB
Fig. 1. The effect of AmB treatment on the DTH response in normal mice and in mice bearing BI6 melanoma. Mice injected with either AraB or BI6 melanoma (groups 2 and 3 respectively) displayed a suppressed DTH response (P<0.001), whilst mice bearing BI6 melanoma and treated with AmB (group 4) showed a greater immunosuppression (0.01 < P< 0.02). Each experimental group contained 8-10 mice. Statistical significance was evaluated by the Student's t-test for unpaired data.
~ 10~ _ co ÷, co
_o tu ~ 5 i.-
xm co < I
!
I
!
I
2
3
4
Control
AMB
B16
B16+AMB
Fig. 2. The effect of AmB treatment on the haemagglutinating anti-SRBC antibody response in normal mice and in mice bearing BI6 melanoma. Neither AmB nor BI6 melanoma (groups 2 and 3 respectively) caused a depression of circulating haemagglutinating antibody level. In fact, AmB treatment (group 2) caused enhancement of the antibody response (P<0.001), although this was not observed if the mice were carrying Bt6 melanoma (group 4). Each experimental group contained 7 - 9 mice. Statistical significance was evaluated by the Student's t-test for unpaired data.
Amphotericin B-induced Immunosuppression in Tumor-bearing Mice DISCUSSION Deep fungal infections in cancer patients are notoriously difficult to treat. The elimination of these organisms generally requires the participation of host immune responses; in the cancer patient, however, immunological responses are already compromised by the presence of the tumour and cytotoxic drug therapy. In this clinical situation, further immuno-depression by AmB treatment can militate against recovery from fungal infection. The results of the present studies show that AmB given in 4 daily doses depresses even further the already depressed DTH response of tumour-bearing mice. These results confirm and extend our previous report that multiple doses of AmB have a potent sup-
301
pressive effect on DTH responses (Ferrante et al., in press). The immunopharmacological effects of single-dose AmB have been reported to be quite different. Blanke, Little, Shirly & Lynch (1977) showed that immunological responses were augmented by single injections of AmB. Medoff, Valeriote, Lynch, Schlessinger & Kobayashi (1974) also showed that AmB has immunoadjuvant properties in a mouse leukaemia system. In the clinical situation, however, treatment for systemic fungal infections with AmB requires multiple doses of this drug over many days and sometimes weeks. The present experiments were performed to simulate closely this clinical situation. The results indicate that patients receiving AmB may require frequent monitoring of immune function during treatment.
REFERENCES
BENNET, J. E. (1974). Chemotherapy of systemic mucosis. New Engl. J. Med. 290, 30-32. BHUYAN,B. K., LOUGHMAN,B. E., FRACER,T. J. & DAY, K. J. (1976). Comparison of different methods of determining cell viability after exposure to cytotoxic compounds. Expl. Cell Res. 97, 275-280. BLACK,L. & BERENBAUM,M. C. (1964). Factors affecting the dye exclusion test for cell viability. Expl. Cell Res. 35, 9--13. BLANKE,T. J., LtYrLE, J. R., SHIRLY,S. F. & LYNCH,R. G. (1977). Augmentation of murine immune responses by amphotericin B. Cell Immunol. 33, 180--190. FERRANTE,A., ROWAN-KELLY,B. & THONG,Y. H. Suppression of the immunological responses in mice by treatment with amphotericin B. Clin. exp. lmmunoL, in press. GENTRY, L. O. d~.REMINGTON,J. S. (1971). Resistance against cryptococcus conferred by intracellular bacteria and protozoa. J. Infect. Dis. 123, 22--31. LIEW, F. Y. (1977). Regulation of delayed-type hypersensitivity. I. T suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice. Eur. J. lmmunol. 7, 714-718. MEDOFF, G., VALERIOTE,F., LYNCH,R. G., SCHLESSINGER,D. & KOBAYASHI,G. S. (1974). Synergistic effect of amphotericin B and 1,3-bis(2-chloreothyl)-l-nitrosourea against a transplantable AKR leukemia. Cancer Res. 34, 974-978. ROGERS,T. J., BALISH,E. & MANNING,D. D. (1976). The role of thymus-dependent cell-mediated immunity in resistance to experimental disseminated candidiasis. J. Reticulo. Soc. 20, 291-298. o TARNVIK,A. & ANSi/HN, S. (1974). Effect of amphotericin B and clotrimazole on lymphocyte stimulation. Anticmicrob. Ag. Chemother. 6, 529-533. THONG, Y. H. & ROWAN-KELLY,B. (1978). Amphotericin B inhibits mitogen-induced human lymphocyte proliferation. IRCS J. reed. Sci. 6, 21.
0192-0561/79/1201-0299 $02.00/0
SHORT COMMUNICATION
AMPHOTERICIN B-INDUCED IMMUNOSUPPRESSION IN TUMORBEARING MICE ANTONIO FERRANTEand Y. H. THONG Department of Paediatrics, University of Adelaide, The Adelaide Children's Hospital, North Adelaide, 5006, South Australia, Australia
(Received 1 August 1979; in final form 10 September 1979) Abstract~B16 melanoma-bearing mice had a reduced capacity to mount a delayed-type hypersensitivity response, and this response was further depressed when these mice were also treated with multiple daily doses of amphotericin B (AraB). Results from the present experiments, which were designed to simulate the clinical situation, indicate that patients receivingAmB may require frequent monitoring of immune functions during treatment.
Cell-mediated immunity (CMI) is an important defence mechanism against fungal infections (Gentry & Remington, 1971; Rogers, Balish & Manning, 1976). The immunity of the host may be greatly suppressed firstly by the tumour and, secondly by the various treatments such as corticosteroids, cytotoxic drugs and irradiation. Such changes in immunity may have a profound effect on the ability of the host to resist fungal infection and thus fungal infections are common in cancer patients. Systemic fungal infections are commonly treated with the polyene antibiotic amphotericin B (AmB) (Bennet, 1974). However, AmB may pose an additional problem for the host responding immunologically against the fungus and also against cancer because previous studies have demonstrated that AmB can suppress the immune response. In vitro AmB has been shown to suppress the mitogen-induced response of human (Tarnvik & ~ns~hn, 1974; Thong & Rowan-Kelly, 1978) and mouse lymphocytes (Ferrante, Rowan-Kelly & Thong, in press) and, when injected into mice, the drug markedly depressed the delayed-type hypersensitivity (DTH) response to sheep red blood cells (Ferrante et al., in press). In the following report, we have used the Bt6 melanoma tumour cell model to demonstrate that mice carrying a B j6 melanoma tumour display a poor DTH response which becomes further suppressed following AmB treatment.
Adelaide, and kept in an air-conditioned animal house with free access to food and water.
Amphotericin B The AmB used in the following study was Fungizone (E. R. Squibb & Sons Inc., New York). The powder was reconstituted in distilled water and further diluted to the required concentration with sterile pyrogen-free saline (0,9°70 w.v.). Although sodium deoxycholate is a diluent in the AmB preparation, previous studies showed that it did not significantly affect the DTH response at the concentrations used in these experiments and hence it was not given to control animals.
Delayed-type hypersensitivity The ability of mice to mount a DTH response to sheep red blood cells (SRBC) was used as a test for cell-mediated immunity (Liew, 1977). Mice were immunized subcutaneously (s.c.) with 1 x 10a SRBC, and then challenged with I x l0 s SRBC s.c. in the hind footpad 5 days later. Footpad thickness was measured with a dial caliper (Mercer) 24 h after challenge and the degree of DTH was expressed as the per cent increase in footpad thickness.
Anti-SRBC antibody response
Immunization was carried out by injecting mice with I x 109 SRBC intravenously (i.v.). The mice were bled 6 days later by cardiac puncture. To deterEXPERIMENTAL PROCEDURES mine the anti-SRBC antibody titre, twofold dilutions Animals (0.025 ml) of the mouse sera were made in microtitre Inbred C57B1/6J male mice, 6 - 8 weeks old, were plates in saline. Then to each well was added an equal used in the present study. They were obtained from volume of 1°7o (v/v) SRBC and the plates incubated the Institute of Medical and Veterinary Science, at 37°C for 1 h. 299
3O0
ANTONIOFERRANTEand Y. H. THONG
Maintenance and method of harvesting B16 melanoma cells The stock o f BI6 m e l a n o m a cells was obtained from D. Goh, Departments of Surgery and Microbiology and Immunology, University of Adelaide. The ceils were maintained by bi-weekly intraperitoneal (i.p.) inoculation into C 5 7 B I / 6 J mice. Harvesting of cells involved excising the tumour and pressing it through a sieve. Single cell suspensions were prepared in 0.9% saline ready for injection and checked for viability by trypan blue dye exclusion as follows: to 0.1 ml of cell suspension (2 × 106/ml) was added 0.1 ml of a 0.2% solution of trypan blue in RPMI-1640 medium, the mixture was incubated for 5 min at 37°C and the number of stained cells determined by counting in a haemocytometer. However, some reservation should be made about the accuracy of this test for cell viability (Black & Berembaum, 1964; Bhuyan, L o u g h m a n , Fracer & Day, 1976).
Statistics Statistical significance was evaluated by the Student's t-test for unpaired data. RESULTS For D T H experiments the mice were divided into 4 groups. Groups 1 and 2 received saline injections (i.p.) at day 0, while groups 3 and 4 received 5 × 105 B~6 m e l a n o m a cells. At day 7 all mice were primed with 1 × 108 SRBC s.c. Starting on the same day, groups 2 and 4 received A m B (7.5 mg/kg) as a daily i.p. injection for 4 days. At day 13, all mice were challenged with 1 × l0 s SRBC in the footpad. The D T H response was measured on day 14. The results showed that tumour-bearing mice (group 3) had a reduced capacity to mount a D T H response (Fig. 1); the per cent footpad increase was 33.7 compared to 69.2 in controls (group 1). The results also confirm previous studies that multiple doses of A m B have a suppressive effect on D T H ; the per cent footpad increase was reduced to 35.7 (group 2). O f particular interest was the finding that the combination of tumour-bearing and A m B therapy resulted in a marked suppression o f D T H , more than either tumour-bearing or A m B alone; the per cent footpad increase was only 16.8 (group 4). For antibody experiments, another lot o f mice was divided into 4 groups according to the above experimental design, except that the mice were primed with 1 × 109 SRBC i.v. on day 7 and bled by intra-cardiac puncture on day 14. The results showed that tumourbearing does not reduce the capacity to form antibody to S R B C (Fig. 2); log 2 mean titre was 7.3 (group 3) compared to 7.8 in controls (group 1). In mice
receiving A m B (group 2), the antibody titre was significantly increased to 9.8. The tumour-bearing mice receiving AraB (group 4) also showed a raised antibody titre but this difference did not reach statistical significance. CO o3 uJ Z
80-
,,, -It--
o
60--
n F-
0
co 4 0 - -
÷1 v
Z LU
co .<
_
20--
u.J t'r"
z
I 2 Control
I 3
AMB
1 4
B16
B16+AMB
Fig. 1. The effect of AmB treatment on the DTH response in normal mice and in mice bearing BI6 melanoma. Mice injected with either AraB or BI6 melanoma (groups 2 and 3 respectively) displayed a suppressed DTH response (P<0.001), whilst mice bearing BI6 melanoma and treated with AmB (group 4) showed a greater immunosuppression (0.01 < P< 0.02). Each experimental group contained 8-10 mice. Statistical significance was evaluated by the Student's t-test for unpaired data.
~ 10~ _ co ÷, co
_o tu ~ 5 i.-
xm co < I
!
I
!
I
2
3
4
Control
AMB
B16
B16+AMB
Fig. 2. The effect of AmB treatment on the haemagglutinating anti-SRBC antibody response in normal mice and in mice bearing BI6 melanoma. Neither AmB nor BI6 melanoma (groups 2 and 3 respectively) caused a depression of circulating haemagglutinating antibody level. In fact, AmB treatment (group 2) caused enhancement of the antibody response (P<0.001), although this was not observed if the mice were carrying Bt6 melanoma (group 4). Each experimental group contained 7 - 9 mice. Statistical significance was evaluated by the Student's t-test for unpaired data.
Amphotericin B-induced Immunosuppression in Tumor-bearing Mice DISCUSSION Deep fungal infections in cancer patients are notoriously difficult to treat. The elimination of these organisms generally requires the participation of host immune responses; in the cancer patient, however, immunological responses are already compromised by the presence of the tumour and cytotoxic drug therapy. In this clinical situation, further immuno-depression by AmB treatment can militate against recovery from fungal infection. The results of the present studies show that AmB given in 4 daily doses depresses even further the already depressed DTH response of tumour-bearing mice. These results confirm and extend our previous report that multiple doses of AmB have a potent sup-
301
pressive effect on DTH responses (Ferrante et al., in press). The immunopharmacological effects of single-dose AmB have been reported to be quite different. Blanke, Little, Shirly & Lynch (1977) showed that immunological responses were augmented by single injections of AmB. Medoff, Valeriote, Lynch, Schlessinger & Kobayashi (1974) also showed that AmB has immunoadjuvant properties in a mouse leukaemia system. In the clinical situation, however, treatment for systemic fungal infections with AmB requires multiple doses of this drug over many days and sometimes weeks. The present experiments were performed to simulate closely this clinical situation. The results indicate that patients receiving AmB may require frequent monitoring of immune function during treatment.
REFERENCES
BENNET, J. E. (1974). Chemotherapy of systemic mucosis. New Engl. J. Med. 290, 30-32. BHUYAN,B. K., LOUGHMAN,B. E., FRACER,T. J. & DAY, K. J. (1976). Comparison of different methods of determining cell viability after exposure to cytotoxic compounds. Expl. Cell Res. 97, 275-280. BLACK,L. & BERENBAUM,M. C. (1964). Factors affecting the dye exclusion test for cell viability. Expl. Cell Res. 35, 9--13. BLANKE,T. J., LtYrLE, J. R., SHIRLY,S. F. & LYNCH,R. G. (1977). Augmentation of murine immune responses by amphotericin B. Cell Immunol. 33, 180--190. FERRANTE,A., ROWAN-KELLY,B. & THONG,Y. H. Suppression of the immunological responses in mice by treatment with amphotericin B. Clin. exp. lmmunoL, in press. GENTRY, L. O. d~.REMINGTON,J. S. (1971). Resistance against cryptococcus conferred by intracellular bacteria and protozoa. J. Infect. Dis. 123, 22--31. LIEW, F. Y. (1977). Regulation of delayed-type hypersensitivity. I. T suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice. Eur. J. lmmunol. 7, 714-718. MEDOFF, G., VALERIOTE,F., LYNCH,R. G., SCHLESSINGER,D. & KOBAYASHI,G. S. (1974). Synergistic effect of amphotericin B and 1,3-bis(2-chloreothyl)-l-nitrosourea against a transplantable AKR leukemia. Cancer Res. 34, 974-978. ROGERS,T. J., BALISH,E. & MANNING,D. D. (1976). The role of thymus-dependent cell-mediated immunity in resistance to experimental disseminated candidiasis. J. Reticulo. Soc. 20, 291-298. o TARNVIK,A. & ANSi/HN, S. (1974). Effect of amphotericin B and clotrimazole on lymphocyte stimulation. Anticmicrob. Ag. Chemother. 6, 529-533. THONG, Y. H. & ROWAN-KELLY,B. (1978). Amphotericin B inhibits mitogen-induced human lymphocyte proliferation. IRCS J. reed. Sci. 6, 21.