Clin. exp. Immunol. (1979) 38, 609-614.
In vivo effect of levamisole on cellular and humoral immunity in normal chickens E. SOPPI, 0. LASSILA, M. K. VILJANEN, O.-P. LEHTONEN & J. ESKOLA Departments of Anatomy and Medical Microbiology, Turku University, SF-20520 Turku 52, Finland
(Acceptedfor publication 4 May 1979)
SUMMARY
The effect of levamisole in vivo was studied on the PHA and Con A responses of chicken peripheral blood lymphocytes and on the in vivo antibody response to a thymus dependent antigen (BSA) and to a thymus independent antigen (Brucella abortus). Levamisole (0-25 mg/kg) increased significantly both the PHA and Con A responses of chicken blood lymphocytes. The antigens were given at the time of enhanced mitogenic responses and a significant increase was observed in both IgM and IgG antibodies to BSA. In contrast, no effect was obtained on antibody responses to Brucella abortus organisms. The results show that levamisole is able to enhance both humoral and cellular immune responses in normal chickens. The effect is probably mediated by the activation of the T cell function and effects only antibody responses to thymus dependent antigen. These findings confirm and extend the observations regarding the ability of levamisole to modulate immune responses.
INTRODUCTION Levamisole is a promising agent for use in the immunotherapy of patients with deficient host defense mechanisms. Both in anergic patients and in experimental animals levamisole has been shown to stimulate cell mediated immunity probably through the enhanced maturation of cells (van Eygen et al., 1976; Huskisson et al., 1976; Oettgen, Pinsky & Delmonte, 1976; Gordon & Yanagihara, 1977; Symoens & Rosenthal, 1977; Amery, 1978; Soppi et al., 1978) or through the enhanced function of macrophages (Symoens & Rosenthal, 1977; Kelly, 1978). The results concerning the effect of levamisole on humoral immunity are controversial. Both in animal and human studies levamisole had generally little or no effect on existing serum immunoglobulin levels or on specific antibody production (Symoens & Rosenthal, 1977). In anergic patients levamisole has been shown to increase the number of circulating B cells and to enhance the antibody response to typhoid, but not to influenza vaccine or to diphtheria toxoid (Lods, Dujardin & Halpern, 1975; Hirshaut et al., 1976; Delespesse et al., 1977). Recently Renoux et al. (1974, 1976, 1977) showed that the activation of murine T cells by levamisole was accompanied by a switch of anti-sheep erythrocyte (SRBC) antibodies from IgM to IgG. However, Lord & Stites (1977) demonstrated a failure of levamisole to increase delayed hypersensitivity or antibodies to SRBC in normal or T cell deprived mice. Quite recently Neveu (1978) has shown that the thiol moiety of levamisole can induce in guinea-pigs enhanced delayed hypersensitivity to carrier antigens, while depression of antibody production to a hapten was observed. In the chicken, levamisole has been shown to increase the antibody response to Newcastle disease virus vaccine (Kulkarni et al., 1973). The objective of the present study was to determine the ability of in vivo levamisole to modify cell-mediated and humoral immune response in normal chickens. Antibody responses to thymus dependent antigen, bovine serum albumin (BSA), and to thymus independent antigen Brucella abortus organisms (Brucella), were quantiCorrespondence: Dr Esa Soppi, Department of Anatomy, Turku University, SF-20520 Turku 52, Finland. 0099-9104/79/1200-0609$02-00 © Blackwell Scientific Publications
609
E. Soppi et al.
610
fied by enzyme linked immunosorbent assay (ELISA). The method permits the separate measurement of IgM and IgG antibodies to the antigens. MATERIALS AND METHODS Animals. White Leghorn line V chickens of both sexes and homozygous for the major histocompatibility locus (genotype
B'-5B15) were used at the age of 4-6 weeks. Antigens. Bovine serum albumin (BSA, Fraction V, Armour Pharmaceutical Company, Eastbourne, England) (2.5 mg) and formalin-killed Brucella abortus organisms (strain M586) (10') were injected intraperitoneally into the chickens. Levamisole. Levamisole (2,3,5,6-tetrahydro-6-phenyl imidazo (2,lb) thiazole hydrochloride, kindly provided by Janssen Pharmaceutica, Beerse, Belgium) was dissolved in saline immediately before use and sterilized by Millipore (0-22 pm) filtration. Lymphocyte cultures. Peripheral blood was collected into heparinized syringes from the wing vein of the chickens. Cultures for lymphocyte transformation were carried out as described earlier (Lassila, Eskola & Toivanen, 1976). Autopsies. At the end of the study the chickens were killed and their thymus, bursa and body weights were measured. Immunoassay for antibodies. Quantification of serum concentrations of chicken IgM and IgG antibodies to BSA and to Brucella were done by an enzyme linked immunosorbent assay (ELISA) modification of corresponding radioimmunoassay earlier developed in our laboratory (Viljanen, Granfors & Toivanen, 1975). The tests were carried out in disposable polystyrene nine-cuvette blocks (Finnpipette-Labsystems, Helsinki, Finland). The antigens were diluted in 0 05 M PBS pH 7.5 (PBS) for coupling to the cuvettes. 200 ul of BSA (1-0 mg/ml) or Brucella (106 bact/ml) were added to the cuvettes. The cuvette blocks were incubated at 37°C for 3 hr and washed three times with 0 4 ml of PBS. In order to decrease nonspecific absorption of proteins to the solid phase it was saturated by incubating the cuvettes for 16 hr at room temperature with 200 ,ul of PBS with 1% of normal sheep serum (NSS-PBS). The cuvettes were washed once with 0 4 ml of saline supplemented with 0.05% of Tween 20 (NaCl-Tw) and stored dry at 4°C until use. The serum specimens were diluted 1:16, 1:64 and 1:256 with NSS-PBS. 150 pl of these dilutions were added in triplicate to the cuvettes and incubated at 37°C for 3 hr. After washing three times with 0 4 ml of NaCl-Tw, 150 Jul of alkaline phosphatase conjugated sheep anti-chicken-p or antichicken-y diluted 1:200 with NSS-PBS were added to the cuvettes. The anti-heavy-chain antibodies were produced and purified as described earlier (Viljanen et al., 1975). The conjugation of antibodies with alkaline phosphatase was carried out according to Engvall & Perlmann (1972). After incubation for 16 hr at room temperature the cuvettes were washed as previously with NaCl-Tw. The amount of alkaline phosphatase bound to the cuvettes was quantified using p-nitrophanylphosphate (2 mg/ml, Sigma Chemical Company, St. Louis, Missouri, USA) as a substrate in glycine-MgCI2 buffer pH 10-0 (Orion Diagnostica, Helsinki, Finland). After an incubation of 30 min at 37°C the optical absorbance of the coloured products was measured at a wavelength of 405 nm in a nine-channel photometer with a vertical beam specially adapted for the cuvette blocks (Finnpipette Analyzer System FP-9, Finnpipette-Labsystems). A standard curve was prepared for each daily assay using highly positive reference sera as standards. The concentration of antibodies in the sample is expressed as a percentage of the concentration of the corresponding antibody in the reference serum. Statistics. The Student's t-test and paired T-test were used for statistical analysis of lymphocyte transformation results. The Student's t-test was used for log-transformed data about antibody concentrations.
RESULTS The experimental design is presented in Fig. 1. In pilot studies several doses of levamisole were tested (0.25-10 mg/kg). Based on these results the doses of 0-25 and 2-5 mg/kg were chosen for further studies. In the first set experiments the effect of levamisole on the mitogenic responses was studied and the animals were not immunized by the antigens. Levamisole (0.25 mg/kg) increased significantly both PHA and Con A responses on day 0 when compared to day -3 or to control chickens on day 0 (P< 0O01 and 0 05 respectively). The higher dose of levamisole (2-5 mg/kg) had a weaker effect and only the PHA response was increased significantly. Later during the experiment the mitogenic responses decreased and reached the starting level of day -3 I
.
-3 -2 Lev Lev
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0
Ag
.
.
*
*
S
S
S
S
S
*
*
.
5 Lev
6 Lev
7
*
.
9
Day
FIG. 1. Scheme for experimental design. S = blood sample for lymphocyte transformation tests (on days -3, 0, +5, +7, +9) and for antibody titrations (on days +5 and +9), Ag = injection of antigens: BSA and Brucella, Lev = injection of levamisole.
611
Levamisok and immune responses Ul) 00
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In vivo effect of levamisole on cellular and humoral immunity in normal chickens E. SOPPI, 0. LASSILA, M. K. VILJANEN, O.-P. LEHTONEN & J. ESKOLA Departments of Anatomy and Medical Microbiology, Turku University, SF-20520 Turku 52, Finland
(Acceptedfor publication 4 May 1979)
SUMMARY
The effect of levamisole in vivo was studied on the PHA and Con A responses of chicken peripheral blood lymphocytes and on the in vivo antibody response to a thymus dependent antigen (BSA) and to a thymus independent antigen (Brucella abortus). Levamisole (0-25 mg/kg) increased significantly both the PHA and Con A responses of chicken blood lymphocytes. The antigens were given at the time of enhanced mitogenic responses and a significant increase was observed in both IgM and IgG antibodies to BSA. In contrast, no effect was obtained on antibody responses to Brucella abortus organisms. The results show that levamisole is able to enhance both humoral and cellular immune responses in normal chickens. The effect is probably mediated by the activation of the T cell function and effects only antibody responses to thymus dependent antigen. These findings confirm and extend the observations regarding the ability of levamisole to modulate immune responses.
INTRODUCTION Levamisole is a promising agent for use in the immunotherapy of patients with deficient host defense mechanisms. Both in anergic patients and in experimental animals levamisole has been shown to stimulate cell mediated immunity probably through the enhanced maturation of cells (van Eygen et al., 1976; Huskisson et al., 1976; Oettgen, Pinsky & Delmonte, 1976; Gordon & Yanagihara, 1977; Symoens & Rosenthal, 1977; Amery, 1978; Soppi et al., 1978) or through the enhanced function of macrophages (Symoens & Rosenthal, 1977; Kelly, 1978). The results concerning the effect of levamisole on humoral immunity are controversial. Both in animal and human studies levamisole had generally little or no effect on existing serum immunoglobulin levels or on specific antibody production (Symoens & Rosenthal, 1977). In anergic patients levamisole has been shown to increase the number of circulating B cells and to enhance the antibody response to typhoid, but not to influenza vaccine or to diphtheria toxoid (Lods, Dujardin & Halpern, 1975; Hirshaut et al., 1976; Delespesse et al., 1977). Recently Renoux et al. (1974, 1976, 1977) showed that the activation of murine T cells by levamisole was accompanied by a switch of anti-sheep erythrocyte (SRBC) antibodies from IgM to IgG. However, Lord & Stites (1977) demonstrated a failure of levamisole to increase delayed hypersensitivity or antibodies to SRBC in normal or T cell deprived mice. Quite recently Neveu (1978) has shown that the thiol moiety of levamisole can induce in guinea-pigs enhanced delayed hypersensitivity to carrier antigens, while depression of antibody production to a hapten was observed. In the chicken, levamisole has been shown to increase the antibody response to Newcastle disease virus vaccine (Kulkarni et al., 1973). The objective of the present study was to determine the ability of in vivo levamisole to modify cell-mediated and humoral immune response in normal chickens. Antibody responses to thymus dependent antigen, bovine serum albumin (BSA), and to thymus independent antigen Brucella abortus organisms (Brucella), were quantiCorrespondence: Dr Esa Soppi, Department of Anatomy, Turku University, SF-20520 Turku 52, Finland. 0099-9104/79/1200-0609$02-00 © Blackwell Scientific Publications
609
E. Soppi et al.
610
fied by enzyme linked immunosorbent assay (ELISA). The method permits the separate measurement of IgM and IgG antibodies to the antigens. MATERIALS AND METHODS Animals. White Leghorn line V chickens of both sexes and homozygous for the major histocompatibility locus (genotype
B'-5B15) were used at the age of 4-6 weeks. Antigens. Bovine serum albumin (BSA, Fraction V, Armour Pharmaceutical Company, Eastbourne, England) (2.5 mg) and formalin-killed Brucella abortus organisms (strain M586) (10') were injected intraperitoneally into the chickens. Levamisole. Levamisole (2,3,5,6-tetrahydro-6-phenyl imidazo (2,lb) thiazole hydrochloride, kindly provided by Janssen Pharmaceutica, Beerse, Belgium) was dissolved in saline immediately before use and sterilized by Millipore (0-22 pm) filtration. Lymphocyte cultures. Peripheral blood was collected into heparinized syringes from the wing vein of the chickens. Cultures for lymphocyte transformation were carried out as described earlier (Lassila, Eskola & Toivanen, 1976). Autopsies. At the end of the study the chickens were killed and their thymus, bursa and body weights were measured. Immunoassay for antibodies. Quantification of serum concentrations of chicken IgM and IgG antibodies to BSA and to Brucella were done by an enzyme linked immunosorbent assay (ELISA) modification of corresponding radioimmunoassay earlier developed in our laboratory (Viljanen, Granfors & Toivanen, 1975). The tests were carried out in disposable polystyrene nine-cuvette blocks (Finnpipette-Labsystems, Helsinki, Finland). The antigens were diluted in 0 05 M PBS pH 7.5 (PBS) for coupling to the cuvettes. 200 ul of BSA (1-0 mg/ml) or Brucella (106 bact/ml) were added to the cuvettes. The cuvette blocks were incubated at 37°C for 3 hr and washed three times with 0 4 ml of PBS. In order to decrease nonspecific absorption of proteins to the solid phase it was saturated by incubating the cuvettes for 16 hr at room temperature with 200 ,ul of PBS with 1% of normal sheep serum (NSS-PBS). The cuvettes were washed once with 0 4 ml of saline supplemented with 0.05% of Tween 20 (NaCl-Tw) and stored dry at 4°C until use. The serum specimens were diluted 1:16, 1:64 and 1:256 with NSS-PBS. 150 pl of these dilutions were added in triplicate to the cuvettes and incubated at 37°C for 3 hr. After washing three times with 0 4 ml of NaCl-Tw, 150 Jul of alkaline phosphatase conjugated sheep anti-chicken-p or antichicken-y diluted 1:200 with NSS-PBS were added to the cuvettes. The anti-heavy-chain antibodies were produced and purified as described earlier (Viljanen et al., 1975). The conjugation of antibodies with alkaline phosphatase was carried out according to Engvall & Perlmann (1972). After incubation for 16 hr at room temperature the cuvettes were washed as previously with NaCl-Tw. The amount of alkaline phosphatase bound to the cuvettes was quantified using p-nitrophanylphosphate (2 mg/ml, Sigma Chemical Company, St. Louis, Missouri, USA) as a substrate in glycine-MgCI2 buffer pH 10-0 (Orion Diagnostica, Helsinki, Finland). After an incubation of 30 min at 37°C the optical absorbance of the coloured products was measured at a wavelength of 405 nm in a nine-channel photometer with a vertical beam specially adapted for the cuvette blocks (Finnpipette Analyzer System FP-9, Finnpipette-Labsystems). A standard curve was prepared for each daily assay using highly positive reference sera as standards. The concentration of antibodies in the sample is expressed as a percentage of the concentration of the corresponding antibody in the reference serum. Statistics. The Student's t-test and paired T-test were used for statistical analysis of lymphocyte transformation results. The Student's t-test was used for log-transformed data about antibody concentrations.
RESULTS The experimental design is presented in Fig. 1. In pilot studies several doses of levamisole were tested (0.25-10 mg/kg). Based on these results the doses of 0-25 and 2-5 mg/kg were chosen for further studies. In the first set experiments the effect of levamisole on the mitogenic responses was studied and the animals were not immunized by the antigens. Levamisole (0.25 mg/kg) increased significantly both PHA and Con A responses on day 0 when compared to day -3 or to control chickens on day 0 (P< 0O01 and 0 05 respectively). The higher dose of levamisole (2-5 mg/kg) had a weaker effect and only the PHA response was increased significantly. Later during the experiment the mitogenic responses decreased and reached the starting level of day -3 I
.
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*
I
0
Ag
.
.
*
*
S
S
S
S
S
*
*
.
5 Lev
6 Lev
7
*
.
9
Day
FIG. 1. Scheme for experimental design. S = blood sample for lymphocyte transformation tests (on days -3, 0, +5, +7, +9) and for antibody titrations (on days +5 and +9), Ag = injection of antigens: BSA and Brucella, Lev = injection of levamisole.
611
Levamisok and immune responses Ul) 00
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