Clin. exp. Immunol. (1978) 32,419-422.
The effects of thiol moiety of levamisole on both cellular and humoral immunity during the early response to a hapten-carrier complex P. J. NE VEU Laboratoire d'Immunologie et des Tumeurs, Centre Rene Gauducheau, Nantes, France
(Received 12January 1978) SUMMARY
Cell-mediated reactions to the carrier and antibody-mediated reactions to the hapten were studied in guinea-pigs treated with a single i.v. injection of the thiol moiety of levamisole (DTC), before or after immunization with a hapten-carrier complex. The results show that a heavy dose of DTC induced delayed hypersensitivity reactions to the carrier and decreased antibody synthesis to the hapten. On the other hand, with a small dose of DTC no delayed hypersensitivity could be induced, but antibody synthesis to the hapten was depressed or enhanced, depending upon whether the injection was done before or after immunization. The mechanisms whereby DTC modulated the immune response are discussed. INTRODUCTION Levamisole has been shown to enhance the immune response to erythrocytes and bacterial antigens in normal mature animals and to restore the immune defence mechanisms impaired by ageing or immaturity (see review by Renoux, 1977). In man this drug has restored delayed hypersensitivity (DH) in anergic cancer patients (Tripodi, Parks & Brugmans, 1973; Hirshaut et al., 1973; Verhaegen et al., 1973) and in elderly individuals (Renoux et al., 1973; Verhaegen et al., 1973). Levamisole possesses thiol and imidazole moieties, which behave differently in the activation of immunocompetent cells in vitro and in vivo. Sodium diethyldithiocarbamate (DTC) has previously been chosen to exemplify the role of the thiol moiety of the levamisole molecule (Renoux, 1977). To achieve a better understanding of the modulating effects of DTC on both DH and antibody synthesis, we have studied its effects on the responses of guinea-pigs to a hapten-carrier conjugate which normally induces antibody synthesis to the hapten and DH to the carrier (Benacerraf & Gell, 1959). The hapten-carrier model has previously been used to dissociate T- and B-cell functions (Playfair, 1971).The T cells are directly involved in DH to the carrier (Benacerraf & Paul, 1971), while cooperation between T and B cells is needed for antibody synthesis to the hapten (Wigzell, 1970; Segal, Globerson & Feldman, 1971). In the experiments reported here, two different doses of DTC were injected intravenously before or after the i.v. immunization with a hapten-carrier complex. The results show that the heavy dose of DTC injected before immunization made possible the induction of DH to the carrier, while it depressed antibody synthesis to the hapten. On the other hand, with the small dose of DTC no DH could be induced, but antibody synthesis to the hapten was depressed or enhanced, depending upon whether the injection was done before or after immunization. The mechanisms whereby DTC modulated the immune response are discussed. Correspondence: Dr P. J. Neveu, Laboratoire d'Immunologie et des Tumeurs, Centre Rene Gauducheau, Quai Moncousu, 44035 Nantes, France. 0099-9104/78/0600-0419$02.00 (©1978 Blackwell Scientific Publications
419
420
P. 9. Neveu MATERIALS AND METHODS
Animals. Adult Hartley guinea-pigs (Laboratoire Roger Bellon, Neuilly-sur-Seine) were used. They were housed in the same room and allocated into cages at random, irrespective of the treatment they received, and were fed laboratory chow and water ad libitum. Antigens. Bovine gamma globulins (BGG) (Miles Laboratories, Kankakee, Illinois) and human serum albumin (HSA) (Miles Laboratories, Kankakee, Illinois) were dinitrophenylated according to Eisen's method (1964). Filtration through a Sephadex G-25 column was used to remove unconjugated dinitrophenyl groups (DNP). The average number of DNP per molecule was calculated from the absorbance value of a known concentration of the conjugate solution at 360 nm. DNP48 BGG was used for immunization and DNP20 HSA for skin testing. Immunization procedures. Animals were injected intravenously with 500 ig or 10 mg of sodium diethyldithiocarbamate (Laboratoires Merck, France) in 1-0 ml of saline, 9 days before, 3 days after or on the same day they were immunized i.v. with 4 0 mg of DNP48 BGG. Skin testing. Skin tests were performed on five animals in each group at days 9, 12 and 16 after immunization. In order to test the different types of hypersensitivity, i.d. injections of antigens were done according to the schedule described by Voisin & Toullet (1966). For testing delayed hypersensitivity 0-1, 1-0 and 10 tg of BGG in 0-1 ml of saline were injected 24 hr before killing the animals. Arthus and anaphylactic types of hypersensitivity were tested by injecting 1-0 and 10 ,ug of DNP20 HSA and of BGG 140 min and 10 min before killing the animals. Evans blue (0-24 ml of a 0 5% solution per 100 g of body weight) was injected i.v. 2 hr before killing the animals (20 min after the i.d. injections for Arthus hypersensitivity).
RESULTS In the first experiment, the anaphylactic reactions to the hapten exhibited by the animals injected with 500 gig of DTC on day 0 were similar to those of the controls (Table 1). The animals injected with DTC on day 3 showed weaker reactions on day 9 and stronger reactions on day 16 than those of the TABLE 1. An anaphylactic reaction to the hapten in groups of five guinea-pigs immunized i.v. with 4 mg of DNP48 BGG
Anaphylactic reactions to the hapten on: Treatment with 500 jpg of DTC on day:
Day 9
Day 12
Day 16
-9 0 3 None
13-6±2-0 13-4±2-0 10-2±3-0* 14-2±3-6
13-4+2-8
11-0+1-6* 15-4+2-4 17-0+1-0* 14-0±2-0
12-6±1-8 15-0±2-1 14 5±3-1
The results are expressed as Evans blue extravasation diameters in mm (±s.e.) after i.d. injection of 10 jig of DNP48 HSA. * P< 0 05 as compared to untreated animals. TABLE 2. Cutaneous reactions in groups of five guinea-pigs immunized i.v. with 4 mg of DNP20 BGG
DH reactions to the carrier Treatment with 10 mg of DTC on day: -9
3 None
Anaphylactic reactions to the hapten
Day 12
Day 16
Day 12
Day 16
10-241-3 0 0
8-4* 0 0
11-0+4-0t
11-8±1*9t 15-6±0-9 16-0±1-4
14-5±1-3 16-0+2-5
The results are expressed as Evans blue extravasation diameters in mm (± s.e.) after i.d. injection of 10 ,pg of antigen. * One animal was negative. t P
The effects of thiol moiety of levamisole on both cellular and humoral immunity during the early response to a hapten-carrier complex P. J. NE VEU Laboratoire d'Immunologie et des Tumeurs, Centre Rene Gauducheau, Nantes, France
(Received 12January 1978) SUMMARY
Cell-mediated reactions to the carrier and antibody-mediated reactions to the hapten were studied in guinea-pigs treated with a single i.v. injection of the thiol moiety of levamisole (DTC), before or after immunization with a hapten-carrier complex. The results show that a heavy dose of DTC induced delayed hypersensitivity reactions to the carrier and decreased antibody synthesis to the hapten. On the other hand, with a small dose of DTC no delayed hypersensitivity could be induced, but antibody synthesis to the hapten was depressed or enhanced, depending upon whether the injection was done before or after immunization. The mechanisms whereby DTC modulated the immune response are discussed. INTRODUCTION Levamisole has been shown to enhance the immune response to erythrocytes and bacterial antigens in normal mature animals and to restore the immune defence mechanisms impaired by ageing or immaturity (see review by Renoux, 1977). In man this drug has restored delayed hypersensitivity (DH) in anergic cancer patients (Tripodi, Parks & Brugmans, 1973; Hirshaut et al., 1973; Verhaegen et al., 1973) and in elderly individuals (Renoux et al., 1973; Verhaegen et al., 1973). Levamisole possesses thiol and imidazole moieties, which behave differently in the activation of immunocompetent cells in vitro and in vivo. Sodium diethyldithiocarbamate (DTC) has previously been chosen to exemplify the role of the thiol moiety of the levamisole molecule (Renoux, 1977). To achieve a better understanding of the modulating effects of DTC on both DH and antibody synthesis, we have studied its effects on the responses of guinea-pigs to a hapten-carrier conjugate which normally induces antibody synthesis to the hapten and DH to the carrier (Benacerraf & Gell, 1959). The hapten-carrier model has previously been used to dissociate T- and B-cell functions (Playfair, 1971).The T cells are directly involved in DH to the carrier (Benacerraf & Paul, 1971), while cooperation between T and B cells is needed for antibody synthesis to the hapten (Wigzell, 1970; Segal, Globerson & Feldman, 1971). In the experiments reported here, two different doses of DTC were injected intravenously before or after the i.v. immunization with a hapten-carrier complex. The results show that the heavy dose of DTC injected before immunization made possible the induction of DH to the carrier, while it depressed antibody synthesis to the hapten. On the other hand, with the small dose of DTC no DH could be induced, but antibody synthesis to the hapten was depressed or enhanced, depending upon whether the injection was done before or after immunization. The mechanisms whereby DTC modulated the immune response are discussed. Correspondence: Dr P. J. Neveu, Laboratoire d'Immunologie et des Tumeurs, Centre Rene Gauducheau, Quai Moncousu, 44035 Nantes, France. 0099-9104/78/0600-0419$02.00 (©1978 Blackwell Scientific Publications
419
420
P. 9. Neveu MATERIALS AND METHODS
Animals. Adult Hartley guinea-pigs (Laboratoire Roger Bellon, Neuilly-sur-Seine) were used. They were housed in the same room and allocated into cages at random, irrespective of the treatment they received, and were fed laboratory chow and water ad libitum. Antigens. Bovine gamma globulins (BGG) (Miles Laboratories, Kankakee, Illinois) and human serum albumin (HSA) (Miles Laboratories, Kankakee, Illinois) were dinitrophenylated according to Eisen's method (1964). Filtration through a Sephadex G-25 column was used to remove unconjugated dinitrophenyl groups (DNP). The average number of DNP per molecule was calculated from the absorbance value of a known concentration of the conjugate solution at 360 nm. DNP48 BGG was used for immunization and DNP20 HSA for skin testing. Immunization procedures. Animals were injected intravenously with 500 ig or 10 mg of sodium diethyldithiocarbamate (Laboratoires Merck, France) in 1-0 ml of saline, 9 days before, 3 days after or on the same day they were immunized i.v. with 4 0 mg of DNP48 BGG. Skin testing. Skin tests were performed on five animals in each group at days 9, 12 and 16 after immunization. In order to test the different types of hypersensitivity, i.d. injections of antigens were done according to the schedule described by Voisin & Toullet (1966). For testing delayed hypersensitivity 0-1, 1-0 and 10 tg of BGG in 0-1 ml of saline were injected 24 hr before killing the animals. Arthus and anaphylactic types of hypersensitivity were tested by injecting 1-0 and 10 ,ug of DNP20 HSA and of BGG 140 min and 10 min before killing the animals. Evans blue (0-24 ml of a 0 5% solution per 100 g of body weight) was injected i.v. 2 hr before killing the animals (20 min after the i.d. injections for Arthus hypersensitivity).
RESULTS In the first experiment, the anaphylactic reactions to the hapten exhibited by the animals injected with 500 gig of DTC on day 0 were similar to those of the controls (Table 1). The animals injected with DTC on day 3 showed weaker reactions on day 9 and stronger reactions on day 16 than those of the TABLE 1. An anaphylactic reaction to the hapten in groups of five guinea-pigs immunized i.v. with 4 mg of DNP48 BGG
Anaphylactic reactions to the hapten on: Treatment with 500 jpg of DTC on day:
Day 9
Day 12
Day 16
-9 0 3 None
13-6±2-0 13-4±2-0 10-2±3-0* 14-2±3-6
13-4+2-8
11-0+1-6* 15-4+2-4 17-0+1-0* 14-0±2-0
12-6±1-8 15-0±2-1 14 5±3-1
The results are expressed as Evans blue extravasation diameters in mm (±s.e.) after i.d. injection of 10 jig of DNP48 HSA. * P< 0 05 as compared to untreated animals. TABLE 2. Cutaneous reactions in groups of five guinea-pigs immunized i.v. with 4 mg of DNP20 BGG
DH reactions to the carrier Treatment with 10 mg of DTC on day: -9
3 None
Anaphylactic reactions to the hapten
Day 12
Day 16
Day 12
Day 16
10-241-3 0 0
8-4* 0 0
11-0+4-0t
11-8±1*9t 15-6±0-9 16-0±1-4
14-5±1-3 16-0+2-5
The results are expressed as Evans blue extravasation diameters in mm (± s.e.) after i.d. injection of 10 ,pg of antigen. * One animal was negative. t P
