CELLULAR
IMMUNOLOGY
The Effect
21, 272-277 (1976)
of Levamisole on Human Lymphocyte Production in Vitro
MICHAEL
E. WHITCOMB, AND
Thorndike
SIDNEY
Mediator
VINCENT J. MERLUZZI, R. COOPERBAND
Memorial Laboratory, Department of Medicine, Boston City Hospital, School of Medicine, Department of Medicine, Boston University Boston, Massachusetts 02118 Received
and
Sepptcmber 16, 1975
Levamisole has previously been demonstrated to increase delayed hypersensitivity reactions in anergic patients. In order to elucidate the mechanism by which levamisole stimulates the immune response in vivo, we have studied the effect of this substance on both human lymphocyte proliferation and lymphocyte-mediator production in vitro. Our results indicate that in vitro levamisole augments the production of soluble mediators by mitogen-stimulated lymphocytes, while having no effect on lymphocyte proliferation.
INTRODUCTION In 1973, Tripodi et al. (1) reported that levamisole administration increased delayed hypersensitivity reactions in 14 of 20 anergic patients with solid tumors. Hirshaut et al. (22) and Verhaegen et al. (3) also demonstrated an increase in delayed hypersensitivity reactions in anergic cancer patients treated with levamisole, and, in addition, Verhaegen reported a similar response in 5 of 12 anergic, elderly individuals. Elucidation of the mechanism by which nonspecific immune stimulants such as levamisole augment the immune response in wivo is not only important for further understanding the nature of the immune response but may also have practical application in the mnagement of the immunosuppression associated with a variety of clinical states. Churchill and David (4) have hypothesized that levamisole might stimulate the immune response by increasing the responsiveness of sensitized lymphocytes or by augmenting defective mediator production by lymphocytes. Several investigators have studied the effect of levamisole on the proliferative response of human lymphocytes in vitro (3, 5-7). In all but one of these studies, levamisole had no effect on in vitro lymphocyte proliferation. In order to investigate further the mechanism by which levamisole may stimulate the human immune response in vivo, we have studied the effect of levamisole on both lymphocyte proliferation and lymphocyte-mediator production. Our results indicate that in vitro levamisole augments the production of soluble mediators by mitogen-stimulated human lymphocytes while having no effect on lymphocyte proliferation. 272 1976 by Academic Press, Inc. Copyright All rights o4 reproduction in any form reserved.
LEVAMISOLE
AND
LYMPHOKINE
PRODUCTION
273
METHODS Levamisole. A stock solution of levamisole (2,3,5,6-tetrahydro-6-phenyl imidazo (2, l-b) thiazole) at a concentration of 50 pg/O.l ml was prepared by dissolving levamisole in minimum essential medium containing penicillin (100 units/ml) and streptomycin (100 &ml) (MEM). The stock solution was kept frozen until use. Preparation of lymphokines. Heparinized blood was obtained from normal human donors by routine venipuncture. After sedimentation in inverted syringes, the cellrich plasma was removed and centrifuged at 15Og for 10 min. The cell pellet was resuspended in MEM and the mononuclear cells separated by Ficoll-Hypaque density centrifugation. The mononuclear cell layer was washed three times in MEM and resuspended at a concentration of 1.0 X lo6 lymphocytes/ml. The cell suspension was then divided into four equal portions, and concanavalin-A (CON-A) (10 pguglml) was added to each. This concentration of CON-A was selected because previous studies in our laboratory had demonstrated that this was a suboptimal concentration for stimuluating mediator production. Levamisole was added to three of the cultures in a concentration of 25, 50 or 100 &ml, respectively. These concentrations of levamisole were selected because they were in the range that had been demonstrated in our laboratory to cause optimal augmentation of the proliferative response of CON-A-stimulated murine spleen cells. The fourth culture served as a control. In experiments in which the direct effect of levamisole on lymphocyte-mediator production was being investigated, no CON-A was added to the cultures. All cultures were incubated for 48 hr at 37°C in a 570 CO* in air environment. After 48 hr of incubation, the cultures were centrifuged at 15Og for 10 min and the supernatant fluids removed. Residual CON-A was removed by passing the supernatant solutions through an inverted syringe containing 15 ml of G-50 Sephadex which had been swollen in MEM. The total volume of the supernatant solution was collected after passage through Sephadex, dialyzed against 20 volumes of fresh MEM for 48 hr to remove levamisole, and sterilized by passage through a Millipore filter (0.45 pm). Fetal calf serum was added to a volume of 10% and the supernatant fluid was frozen until use. Measurement of lymphocyte proliferation. Human mononuclear cell suspensions were prepared as described above. The cells were cultured in microtiter plates at a concentration of 2.5 x lo5 cells in 0.2 ml of MEM containing 10% fetal calf serum (MEM-FCS). In different experiments, phytohemagglutinin (PHA) or CON-A was employed as the stimulating mitogen. A dose-response curve was constructed with each mitogen. Levamisole was added to all cultures at a concentration of 50 pg/ml. The cultures were incubated for 48 hr in a 5% CO2 in air environment at 37°C. They were then pulsed with 0.5 &I of tritiated thymidine (Schwarz/Mann ; sp. act., 1.9) for 24 hr and sacrificed. The cells were collected on a glass-fiber by use of a multiple automated sample harvester (MASH II, Microbiologic Assoc.) and the incorporation of [ 3H] thymidine into trichloroacetic acid precipitable material was measured as an index of DNA synthesis. All cultures were performed in triplicate. Harvesting macrophages. Five-hundred-gram male Hartley strain guinea pigs were used in all experiments. Pulmonary alveolar macrophages (PAM) were harvested after the animal had been exsanguinated by cardiac puncture under pento-
274
WHITCOMB,
MERLUZZI
AND
COOPERBAND
barbital anesthesia. Benadryl was administered intraperitoneally 30 min before the lung lavage to prevent bronchospasm. Following death, the trachea was cannulated and the ventral half of the chest resected. A total lavage of 60 ml was performed by inflating the lung with 5-6-ml aliquots of physiologic saline prewarmed to 37°C. The lavage effluent was collected in prechilled glass tubes and placed in an ice bath. The effluent was then centrifuged at 200g for 10 min. The cell pellet was washed three times and resuspended at a concentration of 2.0-2.5 x JO6 cells/ml in MEM-FCS. Peritoneal exudate macrophages (PEC) were harvested 72 hr after intraperitoneal injection of 30 ml of light paraffin oil. The animal was sacrificed by intracardiac air injection, and the peritoneal cavity was lavaged with 100 ml of physiologic saline. The lavage effluent was centrifuged at 200s for 10 min. The cell pellet was washed three times and the red blood cells lysed by exposure to hypotonic saline for 5 min at 37°C. A 10% suspension (v/v) was then prepared by using MEM-FCS. Ly~phokine assays. (A) Migration inhibitory factor (MIF) : Macrophage migration inhibition was measured by the technique of Rocklin et al. (8). PEC in capillary tubes were placed in incubation. chambers and cultured for 24 hr. The area of migration was traced by means of projection microscopy and measured planimetrically. The percentage of inhibition was calculated in the following way: Percentage of migration inhibition = [ 1 - ( (area of migration in levamisole supernatant)/(area of migration in control supernatant) ) ] X 100. (B) Macrophage activating factor (MAF) : Lymphokine-induced macrophage adherence was measured using a modification of the technique of Nathan et al. (9). One milliliter of a PAM suspension (2.0-2.5 x 10F cells/ml) was plated in 10 x 35-mm plastic petri dishes and incubated for 1 hr. The dishes were then vigorously rinsed in physiologic saline. Control (1.5 ml) or levamisole (1.5 ml) supernatant solution was then added to duplicate dishes. The macrophage monolayer cultures were incubated for 48 hr at 37°C in a 5% COz in air environment. The cultures were supplemented with 0.3 ml of MEM-FCS at 24 hr. At 48 hr, the culture dishes were rinsed vigorously in physiologic saline and dried. One milliliter of 1 N NaOH was then added to each dish overnight. A 0.2-ml aliquot of this solution was then assayed for protein content by using the method of Lowry et al. (10). Baseline data were obtained by sacrificing monolayers at 1 hr and measuring the protein content as above. The percentage of adherence was calculated in the following way: Percentage of adherence = [(micrograms of protein levamisole supernatant dishes)/micrograms of protein-baseline supernatant dishes) ] X 100. RESULTS Lymphocyte proliferation. In six experiments, levamisole had no direct mitogenic effect on human lymphocytes. In addition, levamisole, at a concentration (50 @/ml) which had been demonstrated to augment CON-A-induced lymphokine production had no augmenting effect on lymphocyte proliferation over the entire doscresponse range of CON-A or PHA. Lymphokine production. In two experiments, no MIF or MAF activity could be detected in supernatant solutions from lymphocyte cultures containing only levamisole. However, in each of five experiments in which levamisole was added
LEVAMISOLE
AND
100
0
LYMPHORINE
PRODUCTION
27.5
r
I 0
25
LEVAMISOLE
50
100
(peg/ml)
FIG. 1. The effect of increasing concentrations of levamisole on the production of macrophage-activating factor in h vitro human lymphocyte cultures. The closed-circle symbols represent the means * 1 SD of five experiments in which CON-A (10 pg/ml) was present in the cultures. The results of a single representative experiment in which no CON-A was present in the cultures are depicted by the open triangles for comparative purposes.
to lymphocyte cultures containing a suboptimal stimulating concentration of CON-A, MIF and MAF activity were increased in the supernatant solutions from the cultures stimulated in the presence of levamisole. There was a progressive increase in MIF and MAF activity with increasing concentration of levamisole. Mean MAF activity as measured by macrophage adherence at 48 hr was increased in the levamisole supernatant solutions by 17, 32 and 56% when compared to the control supernatant solution containing only CON-A (Fig. 1). Mean MIF activity was 46, 64 and 80 percent greater in the three levamisole supernatants than that present in the supernatant from the cultures stimulated with CON-A alone (Table 1). Preliminary studies were performed to rule out the possibility that these results could be due to the effect of residual levamisole in the dialyzed supernatant fluids. The stock solution of levamisole directly inhibited macrophage migration and augmented lymphokine-induced macrophage adherence. Both of these effects were lost after dialysis of the stock solution, suggesting that the small molecular weight drug was entirely removed by dialysis or remained in such small concentrations that it could not be responsible for the results of these experiments. DISCUSSION Elucidation of the mechanism by which levamisole stimulates the immune response is not only of importance for understanding the nature of the immune response but also may have practical applications in the management of a variety of immunodeficient states. Among the possible modes of action by which levamisole might affect the immune response, stimulation of either macrophage or lymphocyte
276
WHITCOMB,
MERLUZZI
TABLE Effect of Levamisole Fractions
Supernatants
tested
1 2
COOPERBAND
1 on MIF
Production
Percentage of migration inhibition Levamisole (pg/ml) 25
50
100
2 75 42 69 15
36 90 67 90 1.5
53 100 87 100 59
0 0
0 0
15 0
with CON-A
1 2 3 4 5 Supernatants
AND
without
CON-A
function seems most logical. Although several studies have demonstrated that there is an increase in macrophage phagocyte activity both in viva and in vitro in the presence of levamisole, the significance of these observations in terms of immune responsiveness is unclear (11, 12). Levamisole could stimulate the immune responsiveness of lymphocytes either by increasing the proliferative response of stimulated lymphocytes or by augmenting the production of lymphocyte mediators. Several investigators have studied the effect of levamisole on the proliferative response of stimulated human lymphocytes. Verhaegen et al. (3) and Glogau et al. (5) were unable to demonstrate increased lymphocyte proliferation to either mitogen or antigen stimulation after levamisole therapy. Although Lichtenfield (6) reported an increase in the proliferative response to antigen, mitogen, or allogenic cells when levamisole was added to lymphocyte cultures, Copeland et al. (7) were unable to demonstrate such a response. In this study, levamisole had no effect on the proliferative response of human lymphocytes to either PHA or CON-A when a wide range of concentrations of stimulating mitogens was used. Although multiple concentrations of levamisole were not employed, the concentration that was used in these experiments had been demonstrated to augment human lymphocyte-mediator production and had previously been demonstrated in our laboratory to stimulate murine splenic T-cells and to augment their response to T-cell mitogens (13). Thus, while murine splenic T-cells seem to undergo a proliferative response in the presence of levamisole, this study and the majority of previously published studies have failed to demonstrate that levamisole affects the proliferative response of human lymphocytes in vitro. These observations make it somewhat unlikely that levamisole stimulates the immune response by increasing the proliferative response of human sensitized lymphocytes in vivo. Our results demonstrated that levamisole augments mediator production of mitogen-stimulated lymphocytes in vitro. A dose-dependent increase in MIF and MAF activity was present in the supernatant solutions of mitogen-stimulated lymphocyte cultures containing levamisole. Since levamisole had no direct effect on lymphokine production in the absence of CON-A, the increase in mediator
LEVAMISOLE
AND
LYMPFIOKIi%E
PRODUCTION
277
production observed in the presence of this mitogen would appear to be due to either an increase in the number of lymphocytes responding to the concentration of CON-A used in these experiments or increased production of mediators per cell by the same population of responding cells. Our current data do not allow us to distinguish these two alternatives. To the best of our knowledge, these experiments have demonstrated for the first time direct augmentation of in vitro lymphocyte-mediator production by an immune stimulant. Although transfer factor has been demonstrated to increase lymphokine production in vitro after in tivo administration, a direct effect on lymphocyte-mediator production in vitro has not been demonstrated. Similarly, there is no reported evidence of an in vitro effect on lymphocyte-mediator production by other nonspecific adjuvants. The discrepancy between lymphocyte proliferation and MIF production in levamisole-stimulated lymphocyte cultures demonstrated in this study is not inconsistent with what is now known about lymphocyte physiology. Results of recent studies have clearly demonstrated that CON-A induced lymphocyte proliferation and MIF production are separate events and may involve distinct populations of cells (14). Thus, an increase in mediator production clearly may occur in the absence of an increase in lymphocyte proliferation. The augmentation of mediator production by mitogen-stimulated lymphocytes demonstrated in this study may well explain the increased delayed hypersensitivity reactions previously reported in patients treated with levamisole. Rocklin et al. (15) have reported that in vitro MIF production could be correlated with skin test reactivity in patients with sarcoidosis and Hodgkin’s disease, while in zGtro lymphocyte proliferative responses could not. Studies of the effect of levamisole on in z&o mediator production by lymphocytes obtained from anergic patients may help to substantiate the hypothesis that augmentation of defective mediator production is the mechanism by which levamisole stimulates the immune response in Z&JO.
REFERENCES 1. Tripodi, D., Parks, I,. C., and Brugmans, J., N. E~yl. I. Med. 289, 354, 1973. 2. Hirshaut, Y., Pinsky, C., and Marquardt, H., Proc. Amer. Ass. Cancer Rcs. 14, 109, 1973. 3. Verhaegen, H., Decree, J., DeCock, W., and Verbruggen, F., N. Eugl. J. Med. 289, 1148, 1973. 4. Churchill, W. H., and David, J. R., N. E~gl. J. Med. 289, 375, 1973. 5. Glogau, R., Spitler, L., O’Connor, R., Olson, J., Ostler, P., Silverman, S., and Smolin, G., Clin. Res. 23, 291A, 1975. 6. Lichtenfield, L. J., Desner, M., Mardiney, M. R., and Wiernik, P. H., Fed. Pvor. 33, 790, 1974. 7. Copeland, D., Stewart, T., and Harris, J., Cancer Chemother. Rcs. 58, 167, 1974. 8. Rocklin, R. E., Meyers, 0. L., ant David, J. R., J. Zmmnn.ol. 104, 95, 1970. 9. Nathan, C. F., Karnovsky, M. L., and David, J. R., J. Exp. Med. 133, 1356, 1971. 10. Lowry, 0. H., Rosebrough, N. J, Farr, A I,., and Randall, R J., J. Biol. Chum. 193, 265, 1951. 11. VanGinckel, R. F., and Hoebeke, J., J. Reticuloendothel. Sac. 17, 65, 1975. 12. Al-Ibrahim, M. S., Holman, R. S., and Lawrence, H. S., Clin. Res. 23, 286A, 1973. 13. Merluzzi, V. J. Badger, A. M., Kaiser, C. W. and Cooperband, S. R., J. Clbc. ExP. Immunol., in press. 14. Rocklin, R. E., MacDermott, R. P., Chess, L., Scholssman, S. F., and David, J. R., I. Exp. Med. 140, 1303, 1974. 15. Rocklin, R. E., Sheffer, A., and David, J. R., In “Proceedings of the Sixth Leucocytc Culture Conference” (M. R. Sc h warz, Ed.), p. 743. Academic Press, New York, 1972.
IMMUNOLOGY
The Effect
21, 272-277 (1976)
of Levamisole on Human Lymphocyte Production in Vitro
MICHAEL
E. WHITCOMB, AND
Thorndike
SIDNEY
Mediator
VINCENT J. MERLUZZI, R. COOPERBAND
Memorial Laboratory, Department of Medicine, Boston City Hospital, School of Medicine, Department of Medicine, Boston University Boston, Massachusetts 02118 Received
and
Sepptcmber 16, 1975
Levamisole has previously been demonstrated to increase delayed hypersensitivity reactions in anergic patients. In order to elucidate the mechanism by which levamisole stimulates the immune response in vivo, we have studied the effect of this substance on both human lymphocyte proliferation and lymphocyte-mediator production in vitro. Our results indicate that in vitro levamisole augments the production of soluble mediators by mitogen-stimulated lymphocytes, while having no effect on lymphocyte proliferation.
INTRODUCTION In 1973, Tripodi et al. (1) reported that levamisole administration increased delayed hypersensitivity reactions in 14 of 20 anergic patients with solid tumors. Hirshaut et al. (22) and Verhaegen et al. (3) also demonstrated an increase in delayed hypersensitivity reactions in anergic cancer patients treated with levamisole, and, in addition, Verhaegen reported a similar response in 5 of 12 anergic, elderly individuals. Elucidation of the mechanism by which nonspecific immune stimulants such as levamisole augment the immune response in wivo is not only important for further understanding the nature of the immune response but may also have practical application in the mnagement of the immunosuppression associated with a variety of clinical states. Churchill and David (4) have hypothesized that levamisole might stimulate the immune response by increasing the responsiveness of sensitized lymphocytes or by augmenting defective mediator production by lymphocytes. Several investigators have studied the effect of levamisole on the proliferative response of human lymphocytes in vitro (3, 5-7). In all but one of these studies, levamisole had no effect on in vitro lymphocyte proliferation. In order to investigate further the mechanism by which levamisole may stimulate the human immune response in vivo, we have studied the effect of levamisole on both lymphocyte proliferation and lymphocyte-mediator production. Our results indicate that in vitro levamisole augments the production of soluble mediators by mitogen-stimulated human lymphocytes while having no effect on lymphocyte proliferation. 272 1976 by Academic Press, Inc. Copyright All rights o4 reproduction in any form reserved.
LEVAMISOLE
AND
LYMPHOKINE
PRODUCTION
273
METHODS Levamisole. A stock solution of levamisole (2,3,5,6-tetrahydro-6-phenyl imidazo (2, l-b) thiazole) at a concentration of 50 pg/O.l ml was prepared by dissolving levamisole in minimum essential medium containing penicillin (100 units/ml) and streptomycin (100 &ml) (MEM). The stock solution was kept frozen until use. Preparation of lymphokines. Heparinized blood was obtained from normal human donors by routine venipuncture. After sedimentation in inverted syringes, the cellrich plasma was removed and centrifuged at 15Og for 10 min. The cell pellet was resuspended in MEM and the mononuclear cells separated by Ficoll-Hypaque density centrifugation. The mononuclear cell layer was washed three times in MEM and resuspended at a concentration of 1.0 X lo6 lymphocytes/ml. The cell suspension was then divided into four equal portions, and concanavalin-A (CON-A) (10 pguglml) was added to each. This concentration of CON-A was selected because previous studies in our laboratory had demonstrated that this was a suboptimal concentration for stimuluating mediator production. Levamisole was added to three of the cultures in a concentration of 25, 50 or 100 &ml, respectively. These concentrations of levamisole were selected because they were in the range that had been demonstrated in our laboratory to cause optimal augmentation of the proliferative response of CON-A-stimulated murine spleen cells. The fourth culture served as a control. In experiments in which the direct effect of levamisole on lymphocyte-mediator production was being investigated, no CON-A was added to the cultures. All cultures were incubated for 48 hr at 37°C in a 570 CO* in air environment. After 48 hr of incubation, the cultures were centrifuged at 15Og for 10 min and the supernatant fluids removed. Residual CON-A was removed by passing the supernatant solutions through an inverted syringe containing 15 ml of G-50 Sephadex which had been swollen in MEM. The total volume of the supernatant solution was collected after passage through Sephadex, dialyzed against 20 volumes of fresh MEM for 48 hr to remove levamisole, and sterilized by passage through a Millipore filter (0.45 pm). Fetal calf serum was added to a volume of 10% and the supernatant fluid was frozen until use. Measurement of lymphocyte proliferation. Human mononuclear cell suspensions were prepared as described above. The cells were cultured in microtiter plates at a concentration of 2.5 x lo5 cells in 0.2 ml of MEM containing 10% fetal calf serum (MEM-FCS). In different experiments, phytohemagglutinin (PHA) or CON-A was employed as the stimulating mitogen. A dose-response curve was constructed with each mitogen. Levamisole was added to all cultures at a concentration of 50 pg/ml. The cultures were incubated for 48 hr in a 5% CO2 in air environment at 37°C. They were then pulsed with 0.5 &I of tritiated thymidine (Schwarz/Mann ; sp. act., 1.9) for 24 hr and sacrificed. The cells were collected on a glass-fiber by use of a multiple automated sample harvester (MASH II, Microbiologic Assoc.) and the incorporation of [ 3H] thymidine into trichloroacetic acid precipitable material was measured as an index of DNA synthesis. All cultures were performed in triplicate. Harvesting macrophages. Five-hundred-gram male Hartley strain guinea pigs were used in all experiments. Pulmonary alveolar macrophages (PAM) were harvested after the animal had been exsanguinated by cardiac puncture under pento-
274
WHITCOMB,
MERLUZZI
AND
COOPERBAND
barbital anesthesia. Benadryl was administered intraperitoneally 30 min before the lung lavage to prevent bronchospasm. Following death, the trachea was cannulated and the ventral half of the chest resected. A total lavage of 60 ml was performed by inflating the lung with 5-6-ml aliquots of physiologic saline prewarmed to 37°C. The lavage effluent was collected in prechilled glass tubes and placed in an ice bath. The effluent was then centrifuged at 200g for 10 min. The cell pellet was washed three times and resuspended at a concentration of 2.0-2.5 x JO6 cells/ml in MEM-FCS. Peritoneal exudate macrophages (PEC) were harvested 72 hr after intraperitoneal injection of 30 ml of light paraffin oil. The animal was sacrificed by intracardiac air injection, and the peritoneal cavity was lavaged with 100 ml of physiologic saline. The lavage effluent was centrifuged at 200s for 10 min. The cell pellet was washed three times and the red blood cells lysed by exposure to hypotonic saline for 5 min at 37°C. A 10% suspension (v/v) was then prepared by using MEM-FCS. Ly~phokine assays. (A) Migration inhibitory factor (MIF) : Macrophage migration inhibition was measured by the technique of Rocklin et al. (8). PEC in capillary tubes were placed in incubation. chambers and cultured for 24 hr. The area of migration was traced by means of projection microscopy and measured planimetrically. The percentage of inhibition was calculated in the following way: Percentage of migration inhibition = [ 1 - ( (area of migration in levamisole supernatant)/(area of migration in control supernatant) ) ] X 100. (B) Macrophage activating factor (MAF) : Lymphokine-induced macrophage adherence was measured using a modification of the technique of Nathan et al. (9). One milliliter of a PAM suspension (2.0-2.5 x 10F cells/ml) was plated in 10 x 35-mm plastic petri dishes and incubated for 1 hr. The dishes were then vigorously rinsed in physiologic saline. Control (1.5 ml) or levamisole (1.5 ml) supernatant solution was then added to duplicate dishes. The macrophage monolayer cultures were incubated for 48 hr at 37°C in a 5% COz in air environment. The cultures were supplemented with 0.3 ml of MEM-FCS at 24 hr. At 48 hr, the culture dishes were rinsed vigorously in physiologic saline and dried. One milliliter of 1 N NaOH was then added to each dish overnight. A 0.2-ml aliquot of this solution was then assayed for protein content by using the method of Lowry et al. (10). Baseline data were obtained by sacrificing monolayers at 1 hr and measuring the protein content as above. The percentage of adherence was calculated in the following way: Percentage of adherence = [(micrograms of protein levamisole supernatant dishes)/micrograms of protein-baseline supernatant dishes) ] X 100. RESULTS Lymphocyte proliferation. In six experiments, levamisole had no direct mitogenic effect on human lymphocytes. In addition, levamisole, at a concentration (50 @/ml) which had been demonstrated to augment CON-A-induced lymphokine production had no augmenting effect on lymphocyte proliferation over the entire doscresponse range of CON-A or PHA. Lymphokine production. In two experiments, no MIF or MAF activity could be detected in supernatant solutions from lymphocyte cultures containing only levamisole. However, in each of five experiments in which levamisole was added
LEVAMISOLE
AND
100
0
LYMPHORINE
PRODUCTION
27.5
r
I 0
25
LEVAMISOLE
50
100
(peg/ml)
FIG. 1. The effect of increasing concentrations of levamisole on the production of macrophage-activating factor in h vitro human lymphocyte cultures. The closed-circle symbols represent the means * 1 SD of five experiments in which CON-A (10 pg/ml) was present in the cultures. The results of a single representative experiment in which no CON-A was present in the cultures are depicted by the open triangles for comparative purposes.
to lymphocyte cultures containing a suboptimal stimulating concentration of CON-A, MIF and MAF activity were increased in the supernatant solutions from the cultures stimulated in the presence of levamisole. There was a progressive increase in MIF and MAF activity with increasing concentration of levamisole. Mean MAF activity as measured by macrophage adherence at 48 hr was increased in the levamisole supernatant solutions by 17, 32 and 56% when compared to the control supernatant solution containing only CON-A (Fig. 1). Mean MIF activity was 46, 64 and 80 percent greater in the three levamisole supernatants than that present in the supernatant from the cultures stimulated with CON-A alone (Table 1). Preliminary studies were performed to rule out the possibility that these results could be due to the effect of residual levamisole in the dialyzed supernatant fluids. The stock solution of levamisole directly inhibited macrophage migration and augmented lymphokine-induced macrophage adherence. Both of these effects were lost after dialysis of the stock solution, suggesting that the small molecular weight drug was entirely removed by dialysis or remained in such small concentrations that it could not be responsible for the results of these experiments. DISCUSSION Elucidation of the mechanism by which levamisole stimulates the immune response is not only of importance for understanding the nature of the immune response but also may have practical applications in the management of a variety of immunodeficient states. Among the possible modes of action by which levamisole might affect the immune response, stimulation of either macrophage or lymphocyte
276
WHITCOMB,
MERLUZZI
TABLE Effect of Levamisole Fractions
Supernatants
tested
1 2
COOPERBAND
1 on MIF
Production
Percentage of migration inhibition Levamisole (pg/ml) 25
50
100
2 75 42 69 15
36 90 67 90 1.5
53 100 87 100 59
0 0
0 0
15 0
with CON-A
1 2 3 4 5 Supernatants
AND
without
CON-A
function seems most logical. Although several studies have demonstrated that there is an increase in macrophage phagocyte activity both in viva and in vitro in the presence of levamisole, the significance of these observations in terms of immune responsiveness is unclear (11, 12). Levamisole could stimulate the immune responsiveness of lymphocytes either by increasing the proliferative response of stimulated lymphocytes or by augmenting the production of lymphocyte mediators. Several investigators have studied the effect of levamisole on the proliferative response of stimulated human lymphocytes. Verhaegen et al. (3) and Glogau et al. (5) were unable to demonstrate increased lymphocyte proliferation to either mitogen or antigen stimulation after levamisole therapy. Although Lichtenfield (6) reported an increase in the proliferative response to antigen, mitogen, or allogenic cells when levamisole was added to lymphocyte cultures, Copeland et al. (7) were unable to demonstrate such a response. In this study, levamisole had no effect on the proliferative response of human lymphocytes to either PHA or CON-A when a wide range of concentrations of stimulating mitogens was used. Although multiple concentrations of levamisole were not employed, the concentration that was used in these experiments had been demonstrated to augment human lymphocyte-mediator production and had previously been demonstrated in our laboratory to stimulate murine splenic T-cells and to augment their response to T-cell mitogens (13). Thus, while murine splenic T-cells seem to undergo a proliferative response in the presence of levamisole, this study and the majority of previously published studies have failed to demonstrate that levamisole affects the proliferative response of human lymphocytes in vitro. These observations make it somewhat unlikely that levamisole stimulates the immune response by increasing the proliferative response of human sensitized lymphocytes in vivo. Our results demonstrated that levamisole augments mediator production of mitogen-stimulated lymphocytes in vitro. A dose-dependent increase in MIF and MAF activity was present in the supernatant solutions of mitogen-stimulated lymphocyte cultures containing levamisole. Since levamisole had no direct effect on lymphokine production in the absence of CON-A, the increase in mediator
LEVAMISOLE
AND
LYMPFIOKIi%E
PRODUCTION
277
production observed in the presence of this mitogen would appear to be due to either an increase in the number of lymphocytes responding to the concentration of CON-A used in these experiments or increased production of mediators per cell by the same population of responding cells. Our current data do not allow us to distinguish these two alternatives. To the best of our knowledge, these experiments have demonstrated for the first time direct augmentation of in vitro lymphocyte-mediator production by an immune stimulant. Although transfer factor has been demonstrated to increase lymphokine production in vitro after in tivo administration, a direct effect on lymphocyte-mediator production in vitro has not been demonstrated. Similarly, there is no reported evidence of an in vitro effect on lymphocyte-mediator production by other nonspecific adjuvants. The discrepancy between lymphocyte proliferation and MIF production in levamisole-stimulated lymphocyte cultures demonstrated in this study is not inconsistent with what is now known about lymphocyte physiology. Results of recent studies have clearly demonstrated that CON-A induced lymphocyte proliferation and MIF production are separate events and may involve distinct populations of cells (14). Thus, an increase in mediator production clearly may occur in the absence of an increase in lymphocyte proliferation. The augmentation of mediator production by mitogen-stimulated lymphocytes demonstrated in this study may well explain the increased delayed hypersensitivity reactions previously reported in patients treated with levamisole. Rocklin et al. (15) have reported that in vitro MIF production could be correlated with skin test reactivity in patients with sarcoidosis and Hodgkin’s disease, while in zGtro lymphocyte proliferative responses could not. Studies of the effect of levamisole on in z&o mediator production by lymphocytes obtained from anergic patients may help to substantiate the hypothesis that augmentation of defective mediator production is the mechanism by which levamisole stimulates the immune response in Z&JO.
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