Alteration of the Cytotoxic Action of Sensitized Lymphocytes by Cholinergic Agents and Activators of Adenylate Cyclase

Abstract

The cytotoxic action of lymphocytes upon cells bearing alloantigens to which they are sensitized is inhibited by agents that elevate intracellular amounts of 3': 5'-cyclic AMP: prostaglandin El, cholera toxin, and theophylline. Cholinergic agents, added in the range of 1 to 100 pM, enhance cytotoxicity, an effect that is blocked by atropine. Because 2995After exsanguination of rats by aortic puncture, sensitized recipient spleen tissue and normal thymus tissue from the donor strain were excised and placed in RPMI-1640 medium containing 0.5 volume percent of 1 M HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.55) and 4% (v/v) of heat-inactivated fetal-calf serum. The organs were gently expressed through a fine steel mesh, and the cell suspensions were passed through a tube containing cotton; medium was added liberally. Subsequently, the cell suspensions were purified by Ficoll-Hypaque gradient separation (7), resulting in suspensions consisting almost exclusively of viable lymphocytes. After it was washed, 1.5 ml of the thymic cell suspension containing 1-3 X 107 cells was incubated with 300 ,uCi 5"Cr for 1 hr before it was washed twice with medium and centrifuged at 200 X g to remove free "Cr. Before the spleen and thymus cells were counted in a Coulter counter, they were suspended in RPMI-1640 medium to which 10% (v/v) heat-inactivated fetal-calf serum and 0.5 volume of 1 li HEPES buffer were added. The concentration of the suspension of spleen cells was adjusted to 5 X 10' cells per ml.Lymphocyte-mediated cytotoxicity was quantitated by a modification of Brunner's method (1). Attacking cell populations consisted of sensitized (by LBN skin grafting) or unsensitized Lewis spleen cells, and target cells were "Cr-labeled thymocytes. All media and cell mixtures were suspended in disposable 10 X 75-mm glass test tubes in triplicate. Incubations containing 5 X 104 target cells suspended in 2 ml of RPMII-1640 media with 10% (v/v) heat-inactivated fetalcalf serum and 0.5 volume percent 1 M HEPES buffer were used as controls to determine spontaneous "Cr release. 5 X 106 sensitized or unsensitized Lewis cells were added to 5 X 104 target cells in a final medium volume of 2 ml. After incubation at 370 for 30 min, all mixtures were centrifuged at 50 X g for 10 min in order to promote close cell contact. The mixtures were further incubated for 3.5 hr at 370, then centrifuged at 400 X g for 10 min. The supernatants were promptly decanted into disposable 13 X 100-mm glass test tubes and counted for "Cr release. In studies with pharmacological agents, 0.1 ml of the agent was added to triplicate control samples containing target cells alone and to triplicate samples of mixtures of attacking and target cells. 2-ml Suspensions of 5 X 104 target cells were frozen and thawed in a dry ice-ethanol bath three times to determine maximum releasable cell-bound chromium. None of the pharmacological

Effects of cholinergic agents and sodium ions on the levels of guanosine and adenosine 3′:5′‐cyclic monophosphates in neuroblastoma and neuroblastoma x glioma hybrid cells

“…Ligand binding studies using membrane fragments from the electric organ of Torpedo [33] or from intestinal smooth muscle of guinea pig [34] support the view of the existence of the receptor in 2 different states. Also in other systems biological responses (including the elevation of the level of cyclic GMP) that occur at low concentrations of cholinergic agonists are not observed at high concentrations [28,35].…”

Induction of T-Cell Differentiation In Vitro by Thymin, a Purified Polypeptide Hormone of the Thymus

“…If this be so, it is remarkable that thymin was isolated not by this effect, but by a presumably secondary neuromuscular effect detected in myasthenia gravis. Acetylcholine receptors are widely distributed in the body, being present not only in the nervous system but also in hematopoietic and lymphoid cells (27,28). Thus, it is possible that the physiological receptors for thymin are related to such acetylcholine receptors, and that the binding site of thymin is acetylcholine-mimetic.…”

The regulation of lymphotoxin release from stimulated human lymphocyte cultures: The requirement for continual mitogen stimulation

“…Numerous reports have recently determined that in order for immune cytolysis to occur, cellular events following contact are required. The addition of agents known to affect secretory processes appears to have a pronounced effect upon the lymphocytes' cytodestructive ability (28)(29)(30)(31). Increasing the intracellular levels of cyclic AMP (28,29), as well as the addition of cytochalasin B (31), greatly reduces the cytolytic activity of immune lymphocytes.…”