Clin. exp. Immunol. (1975) 22, 486-492.

In vitro stimulation of murine lymphoid cell cultures by levamisole V. J. MERLUZZI, ALISON M. BADGER, C. W. KAISER & S. R. COOPERBAND Departments of Medicine, Microbiology and Surgery, Boston University School of Medicine, Boston, Massachusetts, U.S.A.

(Received 18 February 1975) SUMMARY

Levamisole has been reported to act as an immunological adjuvant. Experiments reported here on the effect of this agent on a variety of murine lymphoid culture systems were designed to gain an insight into its mechanism of action. We have found levamisole to be a weak mitogen for mouse spleen cells producing a dose related response which peaks at 48 hr in culture. The drug acted to augment the response of spleen cells to sub-optimal concentrations of concanavalin A, but had no unusual effect on the lipopolysaccharide stimulation of B-cell DNA synthesis in vitro. Levamisole was directly stimulatory on enriched T-cell populations and was found to have two actions: (1) to stimulate a subpopulation of T cells and (2) to augment the response of suboptimal mitogen concentrations of concanavalin A. In addition, we have found that murine thymocytes stimulated by concanavalin A were greatly potentiated in the presence of levamisole, but this population of cells could not be stimulated directly by the drug. INTRODUCTION It has been shown that levamisole 2,3,5,6-tetrahydro-6-phenyl imidazo (2,1-b) thiazole, an anti-helminthic drug (Thienpont et al., 1966) may possess immunostimulatory properties in vivo. This drug has been reported to enhance the formation of antibody-producing cells in mice immunized with sheep red blood cells (Renoux & Renoux, 1972a); to restore the impaired immunological system in aged mice (Renoux & Renoux, 1972b); to enhance antibacterial immunization (Renoux & Renoux, 1971); and to increase the intensity of the graft versus host reaction in Fl hybrid recipients following inoculation of parental donor cells previously treated with levamisole (Renoux & Renoux, 1972c). In addition, these authors have reported that levamisole reduces the incidence of primary tumours and metastases in mice (Renoux & Renoux, 1972d). In contrast, there has been a report that this drug was inactive against four immunogenic transplantable tumours (Potter et al., 1974). Other laboratories have reported that levamisole augmented a chemotherapeutically induced remission of a murine leukemia (Chirigos, Pearson & Pryor, 1973), and increased the blood clearance of colloidal carbon in mice (Hoebeke & Franchi, 1973). In man, levamisole has been reported to reduce the frequency of lesions in patients with recurrent aphthous stomatitis (Verhaegen, De Cree & Brugmans, 1973), and also has been reported to restore delayed skin test responses in anergic cancer patients (Tripodi, Parks & Brugmans, 1973). Recently, levamisole has been shown to potentiate the response of tritiated thymidine incorporation into DNA of human peripheral lymphocytes treated with PHA, allogeneic Correspondence: Dr V. J. Merluzzi, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118, U.S.A. 486

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cells, and specific antigen, while having no effect on control cultures (Lichtenfeld et al., 1974). This drug also has been reported to stimulate spleen cell cultures of BALB/c mice (Woods, Siegal & Chirigos, 1974). MATERIALS AND METHODS Mice. CBA/J male and DBA/J female mice were purchased from the Jackson laboratory, Bar Harbor, Maine, and were maintained on acidified drinking water and routine mouse chow. Mice were 2-6 months old at the time of use. Within any given experiment only mice of the same age, strain and sex were used. Culture media. Culture media for measuring the DNA synthetic response of spleen cells and thymocytes consisted of Eagle's minimal essential media (MEM) with Earle's salts obtained from Flow Laboratories, Rockville, Maryland. This medium was supplemented with L-glutamine, sodium pyruvate, non-essential amino acids, 100 units of penicillin and 100 ,pg of streptomycin per millilitre and 5% (v/v) of heat inactivated (560C for 30 min) foetal calf serum (FCS) (Grand Island Biological Company, Grand Island, New York). Mitogens. Concanavalin A (con A) was obtained from Miles Laboratories, Kankakee, Illinois. Escherichia coli lipopolysaccharide (055 :B5) was obtained from Difco Laboratories, Detroit, Michigan. Levamisole. A crystalline preparation of levamisole (lot number SM15807) was kindly supplied by Dr F. J. Persico of the Ortho Research Foundation Raritan, New Jersey. On the day of use the levamisole preparation was dissolved in the appropriate culture media and immediately sterilized by filtration through Millipore filters (HA 0 45 ,pm). Culture conditions. Lymphoid cells were prepared by a proceedure similar to that described by Mishell and Dutton (Mishell & Dutton, 1967). Following cervical dislocation, the spleens and thymuses were removed asceptically and the cells were gently extruded with a small curved forceps into Hanks's balanced salt solution (HBSS) in a small Petri dish. The cells were then dispersed by repeated aspiration with a Pasteur pipette and transferred to a 15 ml conical tube on ice. The larger tissue particles were allowed to settle out for 5 min. Those cells remaining in the supernatant were then removed and washed in HBSS by centrifugation (10 min, 100 g) and resuspended to a final concentration of 20 x 106/ml in culture media. For the measurement of DNA synthesis, cultures were established in microtest plates (IS-FB-96-TC; Linbro Chemical Company, New Haven, Connecticut). Spleen cells or thymocytes (20 x 1061/ml), culture media, levamisole, and other reagents were added separately to each culture well with 25 or 50-pl droppers (Linbro). The final concentration of nucleated cells per culture well was 1 x 106 in a final volume of 0-2 ml culture media. The cultures were incubated in a humidified 5% CO2 atmosphere at 370C. They were then pulsed with tritiated thymidine (3HT) (Schwartz Mann, specific activity 1 9 C/M) after 48 hr in culture and were terminated at 64-66 hr. The incorporation of ['4C]leucine (New England Nuclear, Boston, Massachusetts) was used to measure protein synthesis. ['4C]leucine (0-125 pCi per culture well) was added at the initiation of the experiments with and without various doses of levamisole, and the cultures were terminated after 5 days of incubation. Harvesting was accomplished on a multiple automated sample harvester (Mash II) obtained from Microbiological Associates, Bethesda, Maryland). For DNA synthetic responses the procedure consisted of several saline washes followed by precipitation of the DNA onto glass fibre filters (Reeve Angel 934AH) with 5% trichloroacetic acid. The filters were then counted in a dioxane napthalene, 2,5-diphenyl oxazole (PPO) mixture in a Beckman Scintilation Counter. Harvesting cultures radiolabelled with ['4C]leucine consisted of several saline washes, followed by one wash with 0-3 M perchloric acid with 1% casamino acids, and then several washes with 0 3 M perchloric acid alone. Enriched T-cell populations. Enrichment of spleen cells to contain primarily T cells was accomplished by the nylon wool method (Trizio & Cudkowicz, 1974). Briefly, 1-5 x 108 CBA/J spleen cells in a volume of 2 ml were added to a nylon wool column that had been preincubated with medium at 37°C. The column was then incubated for 45 min and the non-adherent spleen cells were eluted with 25 ml of warm culture medium. This effluent fraction contained the T cell-rich population of spleen cells. Success of enrichment was determined by DNA synthetic responses to the T-cell mitogen, con A and the B-cell mitogen LPS.

RESULTS The mitogenic effect of levamisole on mouse spleen cells is shown in Fig. 1. CBA/J spleen cells at 1 x 106/culture well were incubated for 3 days with various doses of levamisole. The cultures were pulsed with 0 5 uCi of 3HT for the last 16 hr of incubation. The data indicates that within a dose range of 1-30 pg of levamisole the maximum rate of incorporation of 3HT into DNA occurred between 5 and 15 pg/culture well. We have at times found that

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significant stimulation with levamisole can occur with a dose as low as 0 1 pug while at doses approaching 100 pg the drug appears to be toxic; stimulatory doses of levamisole were never toxic. Similar dose curves have been found using the spleen cells from DBA/J female mice. Fig. 2 demonstrates an experiment designed to determine the optimum time for this stimulatory effect. In this experiment, untreated spleen cells and spleen cells containing 10 pg of levamisole per culture well were established at time 0 and were pulsed with 0-5 pCi 3HT for 16 hr at either 24, 48, 72 or 96 hr. As can be seen an optimal stimulatory effect occurs when the cultures were pulsed at 48 hr and terminated at 64 hr after culture initiation. 100

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In vitro stimulation of murine lymphoid cell cultures by levamisole.

Levamisole has been reported to act as an immunological adjuvant. Experiments reported here on the effect of this agent on a variety of murine lymphoi...
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