Int. J. Immunopharmac., Vol. 1, pp. 299-301 0192-0561/79/1201-0299 $02.00/0 © Pergamon Press Ltd. 1979. Printed in Great Britain.

S H O R T C O M M U N I C A T I O N

AMPHOTERICIN B-INDUCED IMMUNOSUPPRESSION IN TUMOR- BEARING MICE

ANTONIO FERRANTE and 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 res- ponse, 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 receiving AmB may require frequent monitoring of immune functions during treatment.

Cell-mediated immunity (CMI) is an important de- fence 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 infec- tions are commonly treated with the polyene anti- biotic amphotericin B (AmB) (Bennet, 1974). How- ever, 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 hyper- sensitivity (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.

EXPERIMENTAL PROCEDURES Animals

Inbred C57B1/6J male mice, 6-8 weeks old, were used in the present study. They were obtained from the Institute of Medical and Veterinary Science,

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 Fungi- zone (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 concen- trations 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 10 a 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 chal- lenge 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 deter- mine the anti-SRBC antibody titre, twofold dilutions (0.025 ml) of the mouse sera were made in microtitre plates in saline. Then to each well was added an equal volume of 1 °7o (v/v) SRBC and the plates incubated at 37°C for 1 h.

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Maintenance and method of harvesting B16

melanoma cells

The stock o f BI6 melanoma cells was obtained from D. Goh, Departments of Surgery and Micro- biology and Immunology, University of Adelaide. The ceils were maintained by bi-weekly intraperi- toneal (i.p.) inoculation into C57BI /6 J mice. Har- vesting 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 deter- mined by counting in a haemocytometer . However, some reservation should be made about the accuracy of this test for cell viability (Black & Berembaum, 1964; Bhuyan, Loughman, Fracer & Day, 1976).

Statistics

Statist ical s ignif icance was evaluated by the Student 's t-test for unpaired data.

RESULTS

ANTONIO FERRANTE and Y. H. THONG

receiving AmB (group 2), the antibody titre was sig- nificantly increased to 9.8. The tumour-bearing mice receiving AraB (group 4) also showed a raised anti- body titre but this difference did not reach statistical significance.

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 melanoma cells. At day 7 all mice were primed with 1 × 108 SRBC s.c. Starting on the same day, ~ 10- groups 2 and 4 received AmB (7.5 mg/kg) as a daily ~ _

co i.p. injection for 4 days. At day 13, all mice were ÷, challenged with 1 × l0 s SRBC in the footpad. The

co

DTH response was measured on day 14. _o The results showed that tumour-bearing mice tu

(group 3) had a reduced capacity to mount a D T H ~ 5 i.-

response (Fig. 1); the per cent footpad increase was 33.7 compared to 69.2 in controls (group 1). The x m results also confirm previous studies that multiple co doses of A m B have a suppressive effect on DTH; the per cent footpad increase was reduced to 35.7 (group < 2). Of particular interest was the finding that the combinat ion of tumour-bearing and A m B therapy resulted in a marked suppression o f DTH, more than either tumour-bearing or AmB alone; the per cent Fig. 2. footpad increase was only 16.8 (group 4).

For ant ibody experiments, another lot o f mice was divided into 4 groups according to the above experi- mental design, except that the mice were primed with 1 × 10 9 SRBC i.v. on day 7 and bled by intra-cardiac puncture on day 14. The results showed that tumour- bearing does not reduce the capacity to form anti- body to SRBC (Fig. 2); log 2 mean titre was 7.3 (group 3) compared to 7.8 in controls (group 1). In mice

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Control AMB 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.

I !

I 2

C o n t r o l A M B

I ! 3 4

B16 B 1 6 + A M B

The effect of AmB treatment on the haemagglu- tinating 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 com- promised by the presence of the tumour and cyto- toxic 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-

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pressive effect on DTH responses (Ferrante et al., in press). The immunopharmacological effects of single-dose AmB have been reported to be quite dif- ferent. 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.

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