Abstract
Stimulation of the immune system by chemical agents such as poly I:poly C (1-3), polyanions (4), tilorone (5), pyran copolymer (6), and levamisole (7-9) have been demonstrated by in vivo methods. The chemicals poly I:poly C (10) and levamisole (11) are active in vitro stimulators of spleen cell cultures, and levamisole has been shown to be a stimulator of peripheral blood lymphocytes (12). We have used the in vitro mixed lymphocyte reaction to demonstrate a direct stimulatory action of levamisole on cells involved in allogeneic recognition reactions and report here that low concentrations of levamisole induce blastogenic stimulation of spleen cell cultures as well as enhance allogeneic stimulation in mixed lymphocyte cultures.
Methods and Materials. Inbred mouse strains. C57B1/6 and DBA/2 male mice, 6-8 wk of age, were used throughout the study.
Levamisole. L-2, 3, 5, 6-tetrahydro-6-phenylimidazole [2, 1-b] thiazole hydro-chloride (LMS, obtained from Janssen Pharmaceuticals, Beerse, Belgium) was dissolved in distilled water at a concentration of 1 mg/ml. Appropriate dilutions were made in RPMI-1640 medium containing 20% fetal bovine serum, 100 units/ml penicillin and 1 μg/ml streptomycin (RPMI-FBS). The drug was added to cultures in 100 /xl volume at the initiation of the cultures.
Mixed lymphocyte culture. The one way mixed lymphocyte reaction (MLR) was employed, using mitomycin C (75 Mg/ml final concentration) as target cell blocking agent (13). Spleens were removed and minced finely in RPMI-1640; single cell suspensions were prepared by forcing the pieces through a 16-gauge needle and passing the suspension through gauze pads. The cells were washed twice in 50 ml RPMI-1640 with centrifugation at 980 g for 5 min in a PR2 refrigerated centrifuge. Target cells were inactivated by treating an aliquot of the cell suspension with mitomycin C (Cal. Biochem., San Diego, CA) for 30 min at 37° followed by two 50 ml washes of RPMI-1640.
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