Gerald Wolberg

Inhibition of lymphocyte-mediated cytolysis by adenosine analogs: Biochemical studies concerning mechanism of action

Abstract A number of adenosine (Ado) analogs have been found to inhibit lymphocyte-mediated cytolysis (LMC) in vitro at μM concentrations. Those analogs which are substrates for adenosine deaminase were more inhibitory to LMC in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of the deaminase. The inhibitory activity of most of these analogs (the exceptions being 2′-deoxyadenosine, 9-β- d -arabinofuranosyladenine and 7-deazaadenosine) was markedly enchanced by an inhibitor (Ro 20-1724) of cyclic AMP phosphodiesterase. With the exceptions of 2-fluoroadenosine, 7-deazaadenosine and formycin A, the inhibition of LMC caused by the Ado analogs was fully reversible upon removal of the analogs from the medium. In general, the Ado analogs did not cause a reduction in the pool sizes of endogenous ribonucleoside 5′-triphosphates in the lymphocytes. Some inhibitory and non-inhibitory analogs were metabolized to their corresponding 5′-triphosphates. Lymphocytes pretreated with a reversible inhibitor of LMC, 2-aminoadenosine, retained most of the resultant 2-amino-ATP during subsequent incubation in analog-free medium. Most of the Ado analogs which were inhibitory to LMC caused a substantial elevation of lymphocyte cyclic AMP; the magnitude of this elevation was enhanced by Ro 20-1724. Collectively, these results suggest that Ado and many of its structural analogs inhibit LMC by reason of their ability to stimulate the formation and consequent build-up of cyclic AMP in the cytotoxic lymphocytes. This stimulation of adenylate cyclase appears to result from the binding of an appropriate nucleoside to an adenosine receptor located on the membrane of the lymphocytes.

Metabolic formation of nucleoside-modified analogues of S-adenosylmethionine.

Abstract Evidence is presented for the metabolism of six different adenosine analogues (formycin, 7-deazaadenosine, 8-azaadenosine, 2-fluoroadenosine, purine ribonucleoside and 3′-deoxyadenosine) to their corresponding S-nucleosidylmethionine derivatives in human erythrocytes and mouse lymphocytes. The identification of these novel metabolites was based upon chromatographic and enzymatic characterization of the unique radioactive substance found in cells incubated with each of these adenosine analogues plus L-[methyl-3H]methionine. The metabolic formation of analogues of S-adenosylmethionine may contribute to the inhibition of cellular methylation reactions caused by various purine analogues.

Immunosuppressive Effects of the S-Adenosylhomocysteine Hydrolase Inhibitor, 3-Deazaadenosine

Immunosuppressive effects of 3-deazaadenosine (3-DAA), an inhibitor of S-adenosylhomocysteine hydrolase, were tested in vivo in immune assays against sheep red blood cells (SRBC), involving serum titrations for hemagglutinins and hemolysins, cellular cytotoxicity tests and the direct plaque-forming cell assay. At daily doses up to 100 mg/kg, the compound was suppressive when injected before antigen and the effect appeared to be dose-dependent (ED50 = 52.6 +/- 4.9 mg/kg). When doses of 25 mg/kg of 3-DAA were given before antigen, co-injections of 250 mg/kg of L-homocysteine (L-HC) potentiated the suppressive effect, although L-HC alone was inactive. Daily administration of 100 mg/kg of 3-DAA or 250 mg/kg of L-HC alone was not suppressive when given after the antigen; however, in combination they were able to induce suppression. The possible biochemical mechanisms of the suppression, particularly those involving the inhibition of S-adenosylmethionine-dependent methylation reactions, are discussed.

Potentiation by homocysteine of adenosine-stimulated elevation of cellular adenosine 3′,5′-monophosphate

Abstract The novel ability of l -homocysteine (Hcy) to potentiate the cellular elevation of adenosine 3′,5′-monophosphate (cAMP) caused by adenosine (Ado) is described. This effect of Hcy is highly selective in that it is not mimicked by l -cysteine, and Hcy does not potentiate the elevation of cellular cAMP caused by either 2-chloroadenosine or prostaglandin E1. Hcy also augments the Ado-stimulated increase in 2-fluoroadenosine 3′,5′-monophosphate in cells preloaded with nucleotides of 2-fluoroadenosine. Addition of Hcy to cells during their incubation with radioactive Ado results in a decrease in the cellular content of radioactive Ado and a concomitant buildup of S-adenosylhomocysteine. The enhancive effect of Hcy on the Ado-stimulated elevation of cAMP may be due to this associated reduction in the intracellular pool of Ado (due to condensation of Ado with Hcy via S-adenosylhomocysteinase) and to a resultant reduction in inhibition of adenylate cyclase by intracellular Ado, thereby allowing greater net stimulation of the cyclase by extracellular Ado.

Inhibition of neutrophil adherence to endothelial cells by 3-deazaadenosine.

Treatment of cultured human umbilical vein endothelial cells (HUVE) with tumor necrosis factor alpha (TNF-α) increases their capacity to adhere human neutrophils. We have found that 3-deazaadenosine (c3Ado), when added in conjunction with TNF-α, inhibited this increase in neutrophil adherence. This activity of c3Ado was potentiated by the addition ofl-homocysteine thiolactone (Hcy). The ability of c3Ado to inhibit neutrophil adherence to HUVE may contribute to the anti-inflammatory activity of this nucleoside analogue.

Effect of acyclovir on various murine in vivo and in vitro immunologic assay systems

In two in vitro tests, lymphocyte-mediated cytotoxicity and neutrophil chemotaxis, acyclovir showed no inhibitory effects at concentrations as high as 600 microM. The compound inhibited rosette formation with nonimmune mouse lymphocytes in vitro by approximately 50 percent at 15.8 microM. The significance of this inhibition is unclear. In four in vivo tests in mice which measured humoral and cell-mediated immunity (complement-dependent cellular cytotoxicity, complement-independent cellular cytotoxicity, delayed hypersensitivity and graft versus host reaction) acyclovir showed no inhibitory effects at single doses up to 200 mg/kg given on day 2 after antigenic stimulation. Four daily doses of acyclovir at 50 mg/kg per day had no effect on the numbers of hemolytic IgM antibody-forming cells in the spleen when assayed on day 4. At the higher dosage of 100 mg/kg per day for four days, there was a slight reduction in the numbers of these cells. There was no significant decrease in hemagglutinin or hemolysin antibody titers after four daily doses of acyclovir up to 200 mg/kg.

Inhibition of lymphocyte-mediated cytolysis by 2-fluoroadenosine--evidence for two discrete mechanisms of drug action.

Abstract 2-Fluoroadenosine (F-Ado) is a potent, irreversible inhibitor of lymphocyte-mediated cytolysis (LMC) in vitro: the irreversibility of this inhibition has been attributed to the metabolism of F-Ado to 2-fluoroadenosine t′-triphosphate (F-ATP) and 2-fluoroadenosine 3′, 5′-monophosphate (F-cAMP) within the cytotoxic lymphocytes [T. P. Zimmerman, J. L. Rideout, G. Wolberg, G.S. Duncan and G. B. Elion, J. biol. Chem.251, 6757 (1976)]. The present study was undertaken to define better the biochemical events intrinsic to the inhibition of LMC by F-Ado. Several purine ribonucleosides, which are themselves non-inhibitory towar LMC, have been found to inhibit the metabolism of F-Ado to F-ATP and F-cAMP by the cytotoxic lymphocytes. The reduction in F-cAMP formation caused by these ribonucleosides was counterbalanced by their augmentation of the elevation of lymphocytic cyclic AMP (cAMP) caused by F-Ado. While interference with the metabolism of F-Ado had little or no effect on the immediate inhibitory activity of F-Ado toward LMC, prevention of the cellular formation of F-ATP and F-cAMP did allow most of the inhibitory activity of F-Ado to be reversed after washing the lymphocytes free of exogenous F-Ado. The relative efficacy of these ribonucleosides in allowing reversibility of the inhibitory activity of F-Ado toward LMC followed the same order as did their efficacy in preventing the metabolism of F-Ado by the cytotoxic lymphocytes: 8-aza-adenosine > inosine > guanosine. Cytotoxic lymphocytes which had been preloaded with nucleotides of F-Ado (via prior incubation with F-Ado and subsequent washout of residual extracellular drug) exhibited increased inhibition of their cytolytic activity upon subsequent incubation with an inhibitor (Ro 20-1724) of cAMP phosphodiesterase. Under these latter experimental conditions, Ro 20-1724 caused a 2- to 3-fold elevation of F-cAMP in the cytotoxic lymphocytes but did not raise cAMP above control levels. These results suggest that F-Ado can inhibit LMC by either of two distinct mechanisms: (1) an extracellular mechanism, wherein F-Ado binds reversibly to an adenosine receptor present on the plasma membrane of the cytotoxic lymphocytes and reversibly activates a functionally associated adenylate cyclase, thereby causing an elevation of cellular cAMP; and (2) an intracellular mechanism, wherein F-Ado is metabolized irreversibly (during the 1- to 2-hr experimental period) by the cytotoxic lymphocytes to F-cAMP which, by reason of its ability to activate cAMP-dependent protein kinase, mimics the effect of elevated cellular levels of cAMP.

Studies Concerning the Mechanism of Action of 3-Deazaadenosine in Leukocytes

3-Deazaadenosine (c3Ado) is a fascinating compound that exhibits interesting biological activities but whose mechanism of action remains an enigma. Although the synthesis of c3Ado was first reported in 1966 (Rousseau et al., 1966), during the subsequent eleven years little was reported concerning biological activities of this adenosine analogue. During this interval, c3Ado was shown to be resistant to deamination by adenosine deaminase (Ikehara & Fukui, 1974) and to be nontoxic to tumor cells (Kitano et al., 1975; May & Townsend, 1975). In 1977, however, widespread interest in c3Ado was awakened by the discovery that c3Ado is both a substrate and a potent inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (Chiang et al., 1977); in this same report it was shown that c3Ado caused a buildup of AdoHcy and was metabolized to S-3-deazaadenosylhomocysteine (c3AdoHcy) in rat hepatocytes. Following this landmark publication, numerous reports began to appear describing diverse biological effects of c3Ado, particularly with respect to leukocyte functions (reviewed by Ueland, 1982, and by Chiang, 1985). In many of these experimental Systems, exogenously supplied L-homocysteine (Hcy) has been found to enhance the biological activity of c3Ado.

Taxol inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced human neutrophil polarization and H2O2 production while decreasing [3H]FMLP binding

We have studied the effect of taxol on two N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil functions and the possible mechanism by which it inhibits these functions. Taxol inhibited FMLP-induced human neutrophil polarization (a characteristic change in neutrophil shape in response to a chemotactic stimulus) and H2O2 generation. Taxol also decreased the specific binding of [3H]FMLP to human neutrophils at 4°C. The decreased binding of FMLP to its receptor may be responsible for the inhibition by taxol of FMLP-induced polarization and H2O2 generation.

Effects of adenosine on neutrophil polarization induced by N-formyl-methionyl-leucyl-phenylalanine, sodium propionate and colchicine

Polarization of human neutrophils (a characteristic bipolar shape change) can be induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP); sodium propionate, which causes a rapid acidification of the cytosol; or colchicine, which disrupts microtubules. We have previously reported that adenosine, endogenously produced in human neutrophil suspensions, inhibits FMLP-induced polarization. We report here that endogenously produced adenosine also inhibits sodium propionate-induced polarization but has no effect on colchicine-induced polariaation. These results suggest that neutrophil polarization may be a multistep process inducible by compounds that trigger different biochemical events.