Human lymphocyte metabolism. Effects of cyclic and noncyclic nucleotides on stimulation by phytohemagglutinin

Abstract

ABSTRA CT The effects of extracellular nucleotides and agents which elevate intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP) concentrations on human lymphocyte metabolism have been studied. Aminophylline, isoproterenol, and prostaglandins, all of which elevate lymphocyte cyclic AMP levels, inhibited incorporation of 'H-labeled thymidine, uridine, and leucine into the DNA, RNA, and protein of phytohemagglutinin (PHA)-stimulated lymphocytes. Aminophylline inhibition was maximal only when the inhibitor was added within 1 hr after exposure of cells to PHA, suggesting that a relatively early step in the lymphocyte transformation process may be affected.The addition of various nucleotides to the culture medium also inhibited incorporation of labeled precursors. The best inhibitor, dibutyryl cyclic AMP (DU cyclic AMP), produced maximal inhibition only if present during the 1st hr after initial exposure to PHA. Among the various cyclic nucleotides derivatives of guanosine and adenine were the most effective inhibitors (substantial inhibition at 0.1 mm concentrations). However, the inhibition was not specific for nucleotides containing the cyclic phosphodiester moiety since the tri-, di-, and monophosphates of adenosine and guanosine were equally effective in diminishing thymidine uptake. The above inhibitions were not due to secondary effects of the inhibitors on the interaction of PHA with lymphocytes as judged by "25I-labeled PHA binding studies.Low concentrations (1-10 imoles/liter) of cyclic AMP produced slight stimulation of thymidine-BH uptake in resting lymphocytes (lymphocytes not stimulated with PHA). However, the effects were quite smallThese studies have been presented in part to the 53rd

Hemolytic Plaque Formation by Leukocytes in Vitro CONTROL BY VASOACTIVE HORMONES

“…produced a slight rise in cyclic AMP in BALB/BL spleen cells (comparable to the effects of histamine or isoproterenol) and significantly inhibited plaque formation. The pattern of prostaglandin effects on spleen cells from these three mouse strains parallels effects documented in mixed human leukocytes, purified human lymphocytes, human platelets, and other tissues (2,20,21): The E prostaglandins are generally potent stimulators of adenyl cyclase, while the F prostaglandins are generally inactive.…”

“…The same hormones in vitro inhibit expression of two other immunologic responses, IgE-mediated immediate hypersensitivity (1)(2)(3)(4)(5)(6) and cell-mediated (delayed) hypersensitivity (6,9), as well as the transformation of human lymphocytes by phytohemagglutinin (20), and the release of lysosomal hydrolases from phagocytic neutrophils (17). Since most of these inhibitory effects appear to be mediated by intracellular cyclic AMP, the experiments reported here were aimed at as critical an evaluation of the role of the nucleotide in inhibiting antibody release as possible.…”

Stimulation by Alcohols of Cyclic AMP Metabolism in Human Leukocytes

“…Reagent grade chemicals were used throughout. The source and method of preparation of solutions of theophylline, beef heart phosphodiesterase (PDE), prostaglandin E, (PGE,), epinephrine, L-propranolol, [3H ]cAMP, concanavalin A (Con A), and erythroagglutinating phytohemagglutinin (E-PHA) have been described (3,(17)(18)(19)(20) (21,22). Purified human neutrophils (>98% of the total nucleated cells) were harvested from the Ficoll-Hypaque cell pellet as a by-product of the lymphocyte purification procedure (23) and subsequently handled like the lymphocyte preparations.…”

“…Lymphocyte blastogenesis was examined uising a minor modification of methods previously described from ouir laboratory (18,20,23 5 min and washed with an additional 2 ml of AIB-PBS. The cells were then diluted in 0.5 ml of distilled water, sonicated, and quantitatively transferred to counting vials with 10 ml scintillation fluid and assayed.…”

Guanosine 3′:5′-Cyclic Monophosphate: A Possible Intracellular Mediator of Mitogenic Influences in Lymphocytes

“…Mitogens, such as phytohemagglutinin (PHA) and concanavalin A (Con A), have been used to induce clonal proliferation in lymphocytes, particularly those derived from the thymus. Several experiments (1)(2)(3)(4)(5)(6) have been performed in which the relationship between cyclic AMP concentration and mitogen-induced lymphocyte division has been investigated. The results indicate (a) that cyclic AMP itself does not significantly stimulate division of lymphocytes in the resting phase ("Go" or "G1" phase) of the cell cycle (1), (b) that increases in cyclic AMP concentration resulting from stimulation by hormones and polynucleotides do not induce lymphocytes to divide (2,3), and (c) that agents that promote increases in Iymphocyte cyclic AMP concentration inhibit mitogen-induced clonal proliferation (4-6).…”