CELLULAR

IMMUNOLOGY

Interaction

IVAN

43, 62-69 (1979)

of Levamisole and Mercaptans during Thymocyte Proliferation Induced by Concanavalin A

G. OTTERNESS, ANTHONY Central Research Division,

J. TORCHIA, AND MARCIA L. BLIVEN Pfizer Inc., Groton,

Connecticut

06340

Received July 7, 1978

Levamisole enhances[sH]thymidine uptake of murine thymocytes stimulated by concanavalin A (Con A). The proliferative response of thymocytes to Con A can also be enhanced by addition of mercaptans. Six different mercaptans were examined for this effect; three of them, 2-mercaptoethanol, cysteamine, and L-cysteine, stimulated the Con A response. Addition of levamisole to an optimal stimulatory dose of 2-mercaptoethanol or cysteamine resulted in complete inhibition of cell proliferation. Three other mercaptans, penicillamine, o-cysteine, and glutathione, failed to enhance the Con A response and, in fact, were mildly inhibitory. Levamisole gave only slightly less than normal stimulation in the presence of these mercaptans. In the absence of Con A neither levamisole nor the mercaptans stimulated cell proliferation. Oxidized 2-mercaptoethanol reacted analogously to reduced 2-mercaptoethanol both in the presence and absence of levamisole. We have interpreted these results as suggesting that the effect of levamisole is dependent upon the state of activation of the lymphocyte.

INTRODUCTION The anthelmintic drug levamisole has been the subject of studies in a wide variety of human disorders. Breast cancer (l), rheumatoid arthritis (2,3), recurrent herpes simplex (4), and respiratory infections (5) are but a few of the reported favorable applications in man. The recurrent theme in each of these studies is that levamisole potentiates the immune response and this, in some way, results in clinical benefit. An immunopotentiating role for levamisole is supported by reports that levamisole also stimulates proliferation of human (6-8) and murine (9- 11) lymphoid cells in culture. However, a few reports (12,13) have suggested that levamisole may, in fact, act through an immunosuppressive mechanism. We wish to report that by manipulating lymphocyte culture conditions, levamisole can be made to either stimulate or to suppress concanvalin A-stimulated lymphocyte proliferation. MATERIALS

AND METHODS

Media. The standard medium was minimum essential medium (Eagle) with Earle’s salts (MEM) with 1% penicillin (10,000 units/ml), 1% streptomycin (10,000 &ml), 1% L-glutamine (200 mM), 1% sodium pyruvate (100 mk!), and 6% heat-inactivated fetal calf serum. In later experiments, 1% of 100x nonessential amino acids and an appropriate mercaptan were added to the standard medium. 62 0008-8749/79/030062-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Dulbecco’s modified Eagle medium was used in some experiments. It was supplemented as above and additionally with 5 x lop5 M 2-mercaptoethanol(14). Culture condition. Thymuses from 7-week-old male C57B116 mice (Jackson Laboratories, Bar Harbor, Me) were aseptically removed, the cells teased apart in MEM, filtered through sterile gauze (Johnson and Johnson, New Brunswick, N.J.), and washed twice. The viable cell numbers were determined by trypan blue exclusion. Cell proliferation was determined by the uptake of [3H]thymidine after 72 hr of culture. To 16 x 125mm tissue culture tubes (Falcon Plastics, Calif.), 5 x lo6 thymocytes in 0.25 ml, 0.25 ml of concanavalin A, 0.25 ml levamisole, and other reagents and media were added to make the final volume to 1 ml. The order of addition of reagents influences the final degree of cell proliferation. Triplicate samples were incubated at 37°C in 5% CO, for 54 hr, then pulsed with 0.1 ml of a 10 pCi/ml solution of tritiated thymidine (Schwarz/Mann, specific activity 1.9 Ci/mM) for 18 hr. The cells were harvested and the DNA precipitated with 3 ml of ice cold 10% trichloroacetic acid. The precipitates were collected on glass fiber filters (2.4 cm, GFK, Fisher Scientific), and washed three times. After drying, the filters were counted in a toluene-Omnifluor mixture on a Nuclear Chicago Scintillation Counter. Chemicals. Concanavalin A (Con A), D- and L-cysteine, D,L-penicillamine, cysteamine, and glutathione were obtained from Sigma Chemical Company, St. Louis, MO. Hepes buffer and 2-mercaptoethanol were obtained from Calbiochem, La Jolla, Calif.; oxidized 2-mercaptoethanol from Aldrich Chemical, Madison, Wis.; levamisole was obtained from American Cyanamide, Princeton, N.J. Media and supplements were purchased from GIBCO, Grand Island, N.Y. RESULTS Levamisole -Concentration

Dependence

of Lymphocyte

Stimulation

At an optimal Con A concentration of 2.5 &ml, levamisole enhances Con A stimulation in the 25-100 @ml range (Fig. 1). The dose response curve is bellshaped and the stimulation decreases progressively above 100pg/ml. No effect was seen in the lower dose ranges, 0.01-l pg/ml. The levamisole effect was maximal at optimal Con A concentration (Fig. 2). At concentrations of Con A that were not mitogenic, i.e., less than 0.625 or more than 5 pg/ml, no effect of levamisole was observed. We examined the effect of further enhancement of the basal Con A response by using the medium of Cerottini et al. (14) (Dulbecco’s modified Eagle medium with added L-glutamine, non-essential amino acids, 5 x lop5 M 2-mercaptoethanol, penicillin, streptomycin, and fetal calf serum). As expected Con A gave much greater stimulation (-6x) in the enriched medium (Figs. 3A versus 3B). However, in the enriched medium levamisole not only failed to augment the Con A response, its addition led to complete inhibition of proliferation (Fig. 3B). Further experiments showed that 2-mercaptoethanol was responsible for this phenomenon. 2-Mercaptoethanol

and Lymphocyte

Proliferation.

The effect of 2-mercaptoethanol and Con A concentration on thymocyte proliferation are shown in Fig. 4. At submitogenic or supramitogenic concentra-

OTTERNESS, TORCHIA, AND BLIVEN

lO-

3025

,025

25

2.5

25

50150

wo

Levamirola (pg/mll

FIG. 1. The effect of levamisole concentration on incorporation of [3H]thymidine by thymocytes stimulated by an optimal dose of Con A (2.5 pg/ml).

tions of Con A, 2-mercaptoethanol did not augment thymocyte incorporation of [3H]thymidine. At mitogenic concentrations of Con A, 2-mercaptoethanol enhanced [3H]thymidine incorporation, and at 10 pM and above, the optimal stimulation concentration of Con A was shifted from 2.5 to 5 &ml.

16

14

12 %c i 10 0 h L .-B e $6 z 2 4

2

0

FIG. 2. The effect of variation in levamisole concentration on the Con A-induced incorporation of [3H]thymidine by thymocytes; (0) no levamisole, (a) 25 &ml levamisole, (0) 50&ml levamisole, and (0) 75 &ml levamisole.

INTERACTION

OF LEVAMISOLE

r

65

AND MERCAPTANS

40

38

3A 0 P x 30.

f

E 0 5 s g 20 .E e p E e ,I 10

(al

Ibt

-

T

(4

iii-

(cl

FIG. 3. The effect of the culture media on the response of thymocytes to Con A and levamisole. In both panels, (a) control unstimulated cells, (b) cells stimulated with Con A (2.5 pg/ml), and(c) cells stimulated with Con A (2.5 &ml) and 50pg/ml levamisole. In panel 3A the culture medium is the standard medium based upon Eagle’s minimum essential medium. In panel 3B the culture medium is based upon Dulbecco’s minimum essential medium according to Cerottini et al. (14). The detailed ingredients and preparation of each medium is given under Materials and Methods.

The interaction between 2-mercaptoethanol and levamisole was examined by varying the 2-mercaptoethanol concentration in the presence and absence of 50 &ml levamisole (Fig. 5). Enhancement of Con A-stimulated proliferation is seen from at least 2 x lop6 M to 5 x 1O-4 M 2-mercaptoethanol, with an optimum

Gmcanavalin

A (uglml)

FIG. 4. The effect ofvarying concentrations of 2-mercaptoethanol on the Con A-induced incorporation of [3H]thymidine. The concentrations of 2-mercaptoethanol were 0 fl (a), 2 +f (O), 5 w(O), 10PM (O), and 20 fl (0).

OTTERNESS, TORCHIA, AND BLIVEN

FIG. 5. The effect of varying concentrations of 2-mercaptoethanol on the incorporation of [3H]thymidine by optimally (2.5 &ml) Con A-stimulated thymocytes in the presence (0) and absence (0) of 50 pg/rnl levamisole.

between lop5 and 10m4M. At low 2-mercaptoethanol concentrations, levamisole gave a near constant stimulation (ca. 30,000 cpm) above the control level. At higher 2-mercaptoethanol concentrations, levamisole stimulation was progressively lost until at 5 x lop5 M and above nearly complete inhibition of [3H]thymidine incorporation was obtained. Comparison

of Different

Mercaptans

The ability of each of six mercaptans to stimulate Con A-induced thymidine incorporation and to interact with levamisole was compared. 2-Mercaptoethanol, L-cysteine, and cysteamine each enhanced thymidine incorporation when added to Con A-stimulated thymocytes. Both cysteamine and 2-mercaptoethanol were approximately equivalent, whereas at the highest concentration tested, 5 m&f, L-cysteine was not able to stimulate more than half the maximum of cysteamine and 2-mercaptoethanol. When levamisole (50 pg/ml) was added to the mercaptancontaining medium, it caused marked inhibition of thymidine incorporation in both cysteamine and 2-mercaptoethanol-containing cultures, but still augmented the L-cysteine-containing cultures. Penicillamine, glutathione, and D-cysteine were without major effect on the culture. They did not stimulate the response to Con A, but rather appeared slightly inhibitory. When levamisole was added to the mercaptan-supplemented media, it enhanced the [3H]thymidine incorporation above the basal level with Con A alone (Table l), although incorporation was less than in cultures stimulated with only levamisole and Con A. The effect of oxidized 2-mercaptoethanol on the thymocyte cultures was also examined. No significant differences were found between oxidized and reduced

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AND MERCAPTANS

TABLE 1 Levamisole and Mercaptan Effects on Thymocyte Proliferation [3H]Thymidine incorporation (cpm)

Mercaptan” Control 2-Mercaptoethanol Oxidized 2-mercaptoethanol L-Cysteine D-CySteke

Cysteamine Glutathione Penicillamine

Media + levamisole (50 dml)

Media 12,353 2 106,476 2 122,926 k 24,380 ? 7,773 k 42,277 k 7,660 2 5,747 +

593 6353 5923 1300 394 5705 419 868

54,713 + 1,681 + 3,118 f 78,189 r 31,137 k 7,221 2 22,137 t 18,373 5

3357 46 85 3411 2163 1184 1368 490

B All mercaptans at 2 x lo4 M in the reduced state or 1 x lO+ M in the oxidized state. All cultures stimulated by 2.5 pg/ml concanavalin A.

2-mercaptoethanol; stimulation alone and suppression in the presence of levamisole was comparable (Table 1). On a molar basis, dose response curves were nearly identical. DISCUSSION The impetus for this study was the divergent reports of the concentration of levamisole required for activity as a stimulant in mitogenic assays (6- 11, 15, 16). When we examined the response of murine thymus cells and spleen cells (unpublished) in the presence of levamisole, only a single stimulation peak was found at concentrations of about 50 &ml. These results confirm those of Merluzzi et al. (11) and fail to show any evidence for enhancement at levamisoie concentrations of 1&ml or less (6,7,9,10). We have not examined the question of whether levamisole stimulates human lymphocytes (15, 16). Our findings indicate that the effects of levamisole may be highly dependent upon the culture system since with murine thymus cells, levamisole may stimulate, may have no effect, or may suppress the response to Con A using different culture media or mouse strains (unpublished). In our examination of the relationship between the primary mitogenic stimulus by Con A and its enhancement by levamisole two major points emerged. First, and as reported by Merluzzi et al. (ll), levamisole does not induce significant cell proliferation at nonmitogenic doses of Con A. Thus, levamisole does not itself act as a primary stimulant or mitogen. Second, the effect of levamisole is profoundly altered by the concentration of 2-mercaptoethanol in the media. Since neither levamisole (Fig. 2) nor 2-mercaptoethanol (Fig. 4) can do more than enhance the already initiated Con A response, it must be argued that both levamisole and 2-mercaptoethanol serve to amplify or decontrol a rate-limiting process. Examination of the six mercaptans in the absence of levamisole showed two types of dose response curves. Mercaptans of the first type, 2-mercaptoethanol, cysteamine, and L-cysteine, each gave rise to an enhancement of the Con A response. The further addition of levamisole in parallel cultures led to a suppression

68

OTTERNESS,

TORCHIA,

AND BLIVEN

of the response with 2-mercaptoethanol and cysteamine, but not L-cysteine. The second type of mercaptan showed no stimulatory effects on the Con A response. The further addition of levamisole to these mercaptans enhanced thymidine incorporation, albeit not to the same extent as in the absence of mercaptan. Broome and Jeng (17) divided mercaptans into those promoting lymphoid replication in culture and those which do not. The observations of Ryzewski et al. (18) that L-cysteine but not glutathione increases the number of titratable sulfhydryl groups in the lymphocyte suggests a possible metabolic basis for this difference, but the mechanism whereby mercaptans stimulate lymphocyte proliferation is undefined. It has been suggested that they act to prevent oxidation of important factors in the culture (19, 20), by changing the concentration of intracellular sulfhydryl groups (glutathione cycle, microtubules, and mitosis) (21, 22), by substituting for adherent cells (23, 24), or by lymphocyte rescue (25). The observations of Oliver, Albertini, and Berlin (26) on the importance of the sulfhydryl groups in the association of microtubules seems of particular relevance to us since we have noted that 2-mercaptoethanol promotes escape from the mitotic block caused by colcemid in this system (unpublished). However, these results should not be interpreted to mean that simply providing enough reducing units of mercaptan to the cell interior is all that is required for enhanced mitosis as is suggested by the data of Toohey (27). We found, in agreement with Engers et al. (28), that oxidized 2-mercaptoethanol is as effective as reduced 2-mercaptoethanol in promoting lymphoid replication. The mechanism underlying the phenomenon in which combination of 2-mercaptoethanol and levamisole suppresses proliferation is similarly unclear. This phenomenon does not appear to be attributable to direct cell toxicity, with viability determined by trypan blue exclusion. We have excluded the possibility that the primary effect of levamisole is on thymidine pool size (29) by showing that levamisole directly enhances cell mitosis (30). Also, Dr. J. Lombardino of these laboratories has found no evidence for a new chemical product after mixing 2-mercaptoethanol and levamisole in either aqueous or organic (chloroform) solvent. Supraoptimal stimulation with agents like Con A leads to suppression presumably by activating T-suppressor cells (3 1). When two agents which enhance proliferation are used together, one might visualize a similar suppression due to supraoptimal stimulation. Effects of levamisole might then depend upon the state of lymphocyte activation, e.g., the position on the bell-shaped response curve (Fig. 5). Thus levamisole might augment suboptimal responses, but inhibit optimal responses. Evidence has been presentedin vivo for such a dichotomy of effects (32). Precedence for the concerted effects of two positive stimuli leading to suppression has been summarized by Coutinho and Moller (33), albeit only for the case of B-cell activation. Our data might be interpreted as evidence for similar effects with T cells. ACKNOWLEDGMENT We thank Drs. J. F. Niblack

and J. G. Lombardino

for helpful discussions.

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