Europ. J. Cancer Vol. 13, pp. 469--471. Pergamon Press 1977. Printed in Great Britain
Levamisole and Human Lymphocyte Cultures* JOSEPH WYBRAN and ANDR~ GOVAERTS Service d'Immunologie, H6pital Universitaire Saint-Pierre, Brussels Abstract--Blood lymphocytes fiom normal subjects were cultured with levamisole
in the absence or presence of phytohemagglutinin (PHA). Levamisole, by itself, did not stimulate the lymphocytes. Levamisole, at 10-* mg/lO0 ml, significantly increased the response to the mitogen. Other concentrations (from 1 mg to 10-s mg/lOO ml) did not alter the response to PHA. The same effect, at the same concentration, was observedfor isolated blood T cell rich populations in presence of PHA. Levamisole did not modify the PHA response of T cell poor populations. These experiments suggest that levamisole has an action on human T lymphocytes.
INTRODUCTION
processed in order to obtain a T cell rich population and a T cell poor population according a rosetting technique previously described [4]. This technique is based on the property of human T lymphocytes to bind to sheep red blood cells (SRBC) in a rosette formation [5]. The SRBC present in the pellet of rosettes (T cells) were lysed with an isotonic solution of NH,C1. The same lysis procedure was applied, for control purposes, to the unseparated population and the T cell poor population before culture. A pure preparation oflevamisole (Janssen Pharmaceufica, Belgium) was dissolved in medium R P M I 1640 with HEPES and passed through a Millipore filter to sterilize it. The final concentrations of levamisole had a range from 1 to 1 0 - , mg/ 100 ml. The cultures were done in plastic tubes (16 × 75 mm) containing 200,000 cells in one ml of R P M I 1640 with HEPES, L-glutamine, penicillin, streptomycin and 10% heat inactivated fetal calf serum. In some cultures, one microgramme of a PHA-P was contained in this ml of culture. To each culture was added 0.1 ml of various concentrations of levamisole. The cells were cultured, at 37°C for 3 days. Six hours before harvesting, 0"5/~Ci of 3Hthymidine (specific activity: 20 mCi/mmole) were added to each culture. T h e Cells were collected on glass fiber filters (mash filters: Dynatech Lab.), washed in saline and trichloreactic acid. The filter was ethanol dried and transferred in a vial to which scintillation fluid was added. Counting was performed using a beta counter and the results were expressed in counts/min.
LEVAMISOLE, an antihelmenthic drug, has recently been much investigated in immunology. This interest lies in the reports that it can stimulate, immune mechanisms in mice and in m a n [1, 2]. In mice, levamisole has thus been shown t o possess stimulatory activity on both humoral and cellular i m m u n e responses, when injected into animals at the same time as the antigenic stimulus [1]. Both lymphocytes and macrophages appear to be involved in the mechanisms of levamisole. In man, the drug restores the delayed hypersensitivity responses in the anergic old normal subjects or cancer patients [2]. Little is known regarding the, in vitro, effect of levamisole on human lymphocytes. Therefore, we decided to investigate if levamisole can directly stimulate human lymphocytes or influence their response to phytohemagglutinin (PHA), a rather specific T cell mitogen. M A T E R I A L AND M E T H O D S H u m a n lymphocytes were isolated from heparinized blood of six normal volunteers, on a Ficoll-Hypaque gradient. They were washed three times with a Hank's buffered salt solution and resuspended at a final concentration of 20 x 106 cells per ml of medium K P M I 1640 with HEPES (Gibco-Biocult). A fraction of these cells, further referred as unseparated population was used for these cultures [3]. The other fraction was further *This work was supported by the "Fonds National de la Recherche Scientifique M~dicale Belge". 469
470
Joseph Wybran and Andrt Govaerts Table 1. Effectof levamisole on PHA response* Levamisole concentration (rag/100 ml) 0
10 = 2
Unseparated population
64.657 +904
66.241 + 1.178
T cell rich population T cell poor population
48.324 +-730 7.174 + 206
46.920 +_659 4.324 +- 479
10- s
10-4
76.424+* 72.341 +_773 +- 1.383 57.032++ +-510 6.834 +- 226
51.910 +-552 5.921 +- 208
No PHA and
10- s
10- e
10- ~
10- e
no levamisolel"
70.421 +- 1.506
68.320 +-821
62.051 + 1.172
61.342 + 714
1.625 +-67
52.542 +-681 6.834 +- 231
49"034 +-608 6.901 + 182
46.491 +-495 8-362 +- 159
47.031 +-612 7.930 + 202
341 _+ 14 1-906 +- 252
*The results are expressed in counts/rain _+S.E.M. (6 experiments).
J'The cells were cultured without PHA and levamisole. ~These results are significantly different (P < 0.05) from the cultures done in the absence oflevamisole (first column). ]RESULTS
The results are reported as the mean of the six experiments +_ standard error of the mean for each investigated population (Table 1). It can be seen that a significant increase of PHA response was noted only for 10 -3 mg/100 ml in the unseparated population and in the T cell rich population. The statistical analyses has been done using the Student's t-test for paired data. The level of significance is P less than 0.05. Statistical analysis was also done to compare the triplicate control cultures (no levamisole, PHA one microgram) to each other culture. In the unseparated population, levamisole at 10- 4, 10- s, 10- 6, 10- 7 and 10- s mg/ 100ml increased significantly the response to P H A in only one instance out of six (not the same experiment), in 2 experiments out of 6 at 1 0 - 2 m g / 1 0 0 m l , in 4 experiments out of 6 at 10- 3 mg]100 ml. Similarly, for the T cell rich population, levamisole significantly increased the PHA response 4 times out of 6 for 1 0 - 3 m g / 1 0 0 m l , 2 times out of 6 for 10 -4 and 10 -7 rag/100 ml in one experiment for 1 0 - S m g / 1 0 0 m l . In the T cell poor population, levamisole increased the PHA response in only one instance at 10-3 rag/ 100 ml. Levamisole, by itself, in the absence of PHA, never stimulated any of the lymphocyte populations. DISCUSSION
These experiments, done to investigate the possible, in vitro, action of levamisole on human lymphocytes from normal individuals, clearly indicate that, in rather strict conditions, the drug influences the thymidine uptake in presence of PHA. Levamisole, by itself, was not mitogenic for h u m a n lymphocytes. This observation is thus different from the situation in mouse where levamisole can be slightly m i t o -
genie [6, 7]. Similar results have recently been published by Hadden et al. [7]. On the other hand, levamisole will significantly increase the PHA response of human lymphocytes. This effect is however not very dramatic since the increase is only of 15--20 %. Furthermore, it was obtained only at a concentration of 10-a mg/ 100ml (roughly 10 -7 M). This small and narrow ranged action of levamisole on PHA response probably explains the difficulties of many investigators to demonstrate this phenomenon although recently, similar data have been reported [7]. This action is also detected on isolated blood T cell rich populations. The increase is also in the range of 20%. In contrast, levamisole did not increase the PHA responsiveness of T cell poor populations. These results are thus similar to the mice system where pure T cell populations increased by 40% (at 10 -7 M) the response to PHA [7]. The fact that, in the mouse system, the T cells were isolated from the thymus rather than the blood, the purity of various preparations and T cell heterogeneity may explain these slight discrepancies. These studies have thus shown that levamisole can act on proliferative human T ceils. It remains, however, unclear whether the, in vitro, experiments can explain the, in vivo, observed effects. Indeed the, in vitro, range of activity is very small and restricted to higher concentrations than in the, in vivo, situation. It is possible that the activity oflevamisole, in low concentrations, is party due to its metabolites. Finally, these data suggest that either levamisole acts preferentially on a stimulated (proliferative) T cell, suggesting some type of antigenic stimulation as a prerequisite for levamisole activity or Ievamisole enhances the immune response as reflected in the response to mitogens.
Levamisole and Human Lymphocyte Cultures
REFERENCES 1. •G. R~ozrx and M. Re~oux, Effet ;m~unostlmulant d'un imido~azole dans l'imruunisation des souris contre l'infection par Brucella Abortus. C. R. Acad. Sci. Pads D 272, 349 (1970). 2. R. TmPODI, L. C. PARKS and J. B R u G ~ s , Drug-induced restoration of cutaneous delayed hypersensitivity in anergic patients with cancer. New Engl. ,I. Med. 289, 354 (1973). 3. J. W,e'sa.~, S. CaxrrrLER and H. H. FtrDr~rCS~RG,Isolation of normal T ceils in chronic lyrfiphatic leukemia. Lancet i~ 126 (1973). 4. J. W,/'B~N, I. HELLSTR6M, K. E. HELLSTROMand H. H. FUDENBERG,Cytotoxicity of human rosette-forming blood lymphocytes on cultivated human tumor cells. Int. J. Cancer 13, 515 (1974). 5. J. W,e'saAN and H. H. Ftn~E~Br~RG, Rosette formation, a test for cellular immunity. Trans. Ass. Amer. Physcns. 114, 239 (1971). 6. W.A. WOODS, M. J. SIEOELand M. A. Crtt~Gos, In vitro stimulation of spleen cell cultures by Poly I: Poly C and levamisole. Cell Immunol. 14, 327 (1974). 7. J. W. HA.DDEN, R. 1~. COFFEY, E. M. HADDEN, E. LOPEz-CoRRALES and G. H. Stmsmrce, Effects of levamisole and imidazole on lymphocyte proliferation and cyclic nueleotide levels. Cell. Immunol. 20, 98 (1975).
471
Levamisole and Human Lymphocyte Cultures* JOSEPH WYBRAN and ANDR~ GOVAERTS Service d'Immunologie, H6pital Universitaire Saint-Pierre, Brussels Abstract--Blood lymphocytes fiom normal subjects were cultured with levamisole
in the absence or presence of phytohemagglutinin (PHA). Levamisole, by itself, did not stimulate the lymphocytes. Levamisole, at 10-* mg/lO0 ml, significantly increased the response to the mitogen. Other concentrations (from 1 mg to 10-s mg/lOO ml) did not alter the response to PHA. The same effect, at the same concentration, was observedfor isolated blood T cell rich populations in presence of PHA. Levamisole did not modify the PHA response of T cell poor populations. These experiments suggest that levamisole has an action on human T lymphocytes.
INTRODUCTION
processed in order to obtain a T cell rich population and a T cell poor population according a rosetting technique previously described [4]. This technique is based on the property of human T lymphocytes to bind to sheep red blood cells (SRBC) in a rosette formation [5]. The SRBC present in the pellet of rosettes (T cells) were lysed with an isotonic solution of NH,C1. The same lysis procedure was applied, for control purposes, to the unseparated population and the T cell poor population before culture. A pure preparation oflevamisole (Janssen Pharmaceufica, Belgium) was dissolved in medium R P M I 1640 with HEPES and passed through a Millipore filter to sterilize it. The final concentrations of levamisole had a range from 1 to 1 0 - , mg/ 100 ml. The cultures were done in plastic tubes (16 × 75 mm) containing 200,000 cells in one ml of R P M I 1640 with HEPES, L-glutamine, penicillin, streptomycin and 10% heat inactivated fetal calf serum. In some cultures, one microgramme of a PHA-P was contained in this ml of culture. To each culture was added 0.1 ml of various concentrations of levamisole. The cells were cultured, at 37°C for 3 days. Six hours before harvesting, 0"5/~Ci of 3Hthymidine (specific activity: 20 mCi/mmole) were added to each culture. T h e Cells were collected on glass fiber filters (mash filters: Dynatech Lab.), washed in saline and trichloreactic acid. The filter was ethanol dried and transferred in a vial to which scintillation fluid was added. Counting was performed using a beta counter and the results were expressed in counts/min.
LEVAMISOLE, an antihelmenthic drug, has recently been much investigated in immunology. This interest lies in the reports that it can stimulate, immune mechanisms in mice and in m a n [1, 2]. In mice, levamisole has thus been shown t o possess stimulatory activity on both humoral and cellular i m m u n e responses, when injected into animals at the same time as the antigenic stimulus [1]. Both lymphocytes and macrophages appear to be involved in the mechanisms of levamisole. In man, the drug restores the delayed hypersensitivity responses in the anergic old normal subjects or cancer patients [2]. Little is known regarding the, in vitro, effect of levamisole on human lymphocytes. Therefore, we decided to investigate if levamisole can directly stimulate human lymphocytes or influence their response to phytohemagglutinin (PHA), a rather specific T cell mitogen. M A T E R I A L AND M E T H O D S H u m a n lymphocytes were isolated from heparinized blood of six normal volunteers, on a Ficoll-Hypaque gradient. They were washed three times with a Hank's buffered salt solution and resuspended at a final concentration of 20 x 106 cells per ml of medium K P M I 1640 with HEPES (Gibco-Biocult). A fraction of these cells, further referred as unseparated population was used for these cultures [3]. The other fraction was further *This work was supported by the "Fonds National de la Recherche Scientifique M~dicale Belge". 469
470
Joseph Wybran and Andrt Govaerts Table 1. Effectof levamisole on PHA response* Levamisole concentration (rag/100 ml) 0
10 = 2
Unseparated population
64.657 +904
66.241 + 1.178
T cell rich population T cell poor population
48.324 +-730 7.174 + 206
46.920 +_659 4.324 +- 479
10- s
10-4
76.424+* 72.341 +_773 +- 1.383 57.032++ +-510 6.834 +- 226
51.910 +-552 5.921 +- 208
No PHA and
10- s
10- e
10- ~
10- e
no levamisolel"
70.421 +- 1.506
68.320 +-821
62.051 + 1.172
61.342 + 714
1.625 +-67
52.542 +-681 6.834 +- 231
49"034 +-608 6.901 + 182
46.491 +-495 8-362 +- 159
47.031 +-612 7.930 + 202
341 _+ 14 1-906 +- 252
*The results are expressed in counts/rain _+S.E.M. (6 experiments).
J'The cells were cultured without PHA and levamisole. ~These results are significantly different (P < 0.05) from the cultures done in the absence oflevamisole (first column). ]RESULTS
The results are reported as the mean of the six experiments +_ standard error of the mean for each investigated population (Table 1). It can be seen that a significant increase of PHA response was noted only for 10 -3 mg/100 ml in the unseparated population and in the T cell rich population. The statistical analyses has been done using the Student's t-test for paired data. The level of significance is P less than 0.05. Statistical analysis was also done to compare the triplicate control cultures (no levamisole, PHA one microgram) to each other culture. In the unseparated population, levamisole at 10- 4, 10- s, 10- 6, 10- 7 and 10- s mg/ 100ml increased significantly the response to P H A in only one instance out of six (not the same experiment), in 2 experiments out of 6 at 1 0 - 2 m g / 1 0 0 m l , in 4 experiments out of 6 at 10- 3 mg]100 ml. Similarly, for the T cell rich population, levamisole significantly increased the PHA response 4 times out of 6 for 1 0 - 3 m g / 1 0 0 m l , 2 times out of 6 for 10 -4 and 10 -7 rag/100 ml in one experiment for 1 0 - S m g / 1 0 0 m l . In the T cell poor population, levamisole increased the PHA response in only one instance at 10-3 rag/ 100 ml. Levamisole, by itself, in the absence of PHA, never stimulated any of the lymphocyte populations. DISCUSSION
These experiments, done to investigate the possible, in vitro, action of levamisole on human lymphocytes from normal individuals, clearly indicate that, in rather strict conditions, the drug influences the thymidine uptake in presence of PHA. Levamisole, by itself, was not mitogenic for h u m a n lymphocytes. This observation is thus different from the situation in mouse where levamisole can be slightly m i t o -
genie [6, 7]. Similar results have recently been published by Hadden et al. [7]. On the other hand, levamisole will significantly increase the PHA response of human lymphocytes. This effect is however not very dramatic since the increase is only of 15--20 %. Furthermore, it was obtained only at a concentration of 10-a mg/ 100ml (roughly 10 -7 M). This small and narrow ranged action of levamisole on PHA response probably explains the difficulties of many investigators to demonstrate this phenomenon although recently, similar data have been reported [7]. This action is also detected on isolated blood T cell rich populations. The increase is also in the range of 20%. In contrast, levamisole did not increase the PHA responsiveness of T cell poor populations. These results are thus similar to the mice system where pure T cell populations increased by 40% (at 10 -7 M) the response to PHA [7]. The fact that, in the mouse system, the T cells were isolated from the thymus rather than the blood, the purity of various preparations and T cell heterogeneity may explain these slight discrepancies. These studies have thus shown that levamisole can act on proliferative human T ceils. It remains, however, unclear whether the, in vitro, experiments can explain the, in vivo, observed effects. Indeed the, in vitro, range of activity is very small and restricted to higher concentrations than in the, in vivo, situation. It is possible that the activity oflevamisole, in low concentrations, is party due to its metabolites. Finally, these data suggest that either levamisole acts preferentially on a stimulated (proliferative) T cell, suggesting some type of antigenic stimulation as a prerequisite for levamisole activity or Ievamisole enhances the immune response as reflected in the response to mitogens.
Levamisole and Human Lymphocyte Cultures
REFERENCES 1. •G. R~ozrx and M. Re~oux, Effet ;m~unostlmulant d'un imido~azole dans l'imruunisation des souris contre l'infection par Brucella Abortus. C. R. Acad. Sci. Pads D 272, 349 (1970). 2. R. TmPODI, L. C. PARKS and J. B R u G ~ s , Drug-induced restoration of cutaneous delayed hypersensitivity in anergic patients with cancer. New Engl. ,I. Med. 289, 354 (1973). 3. J. W,e'sa.~, S. CaxrrrLER and H. H. FtrDr~rCS~RG,Isolation of normal T ceils in chronic lyrfiphatic leukemia. Lancet i~ 126 (1973). 4. J. W,/'B~N, I. HELLSTR6M, K. E. HELLSTROMand H. H. FUDENBERG,Cytotoxicity of human rosette-forming blood lymphocytes on cultivated human tumor cells. Int. J. Cancer 13, 515 (1974). 5. J. W,e'saAN and H. H. Ftn~E~Br~RG, Rosette formation, a test for cellular immunity. Trans. Ass. Amer. Physcns. 114, 239 (1971). 6. W.A. WOODS, M. J. SIEOELand M. A. Crtt~Gos, In vitro stimulation of spleen cell cultures by Poly I: Poly C and levamisole. Cell Immunol. 14, 327 (1974). 7. J. W. HA.DDEN, R. 1~. COFFEY, E. M. HADDEN, E. LOPEz-CoRRALES and G. H. Stmsmrce, Effects of levamisole and imidazole on lymphocyte proliferation and cyclic nueleotide levels. Cell. Immunol. 20, 98 (1975).
471
