Potentiation by thiopurines and sulfhydryl-reactive agents of the inhibition by 3-deazaadenosine of mononuclear phagocytes.

Combination effects of 3-deazaadenosine (c3Ado) on antibody-dependent phagocytosis in mouse resident peritoneal cells and human peripheral blood monocytes precultured with cytotoxic thiols, azathioprine (AZA) or 6-mercaptopurine (6-MP), and thiol-reactive agents, 2-cyclohexene-1-one (2-CH) or ethacrynic acid (ETA), are described. In the mouse cell preparations, a non-inhibitory concentration of 10 microM AZA or 6-MP potentiated the inhibition by 5 and 10 microM c3Ado of phagocytosis. Higher concentrations of AZA or 6-MP (50, 100 microM) and c3Ado (40, 50 microM) were needed to achieve similar effects in human monocytes. Both 2-CH (50 microM) and ETA (25 microM) inhibited mouse cell phagocytosis and acted synergistically with c3Ado. Precultivation of mouse cells with an inhibitor of glutathione synthesis, buthionine sulfoximine (BSO, 50 microM) caused no inhibition of phagocytosis and no potentiation of the inhibition by c3Ado, although BSO potentiated the inhibition by 2-CH (50 microM). In human monocytes, non-inhibitory concentrations (10 and 25 microM) of gold sodium thiomalate (GST), AZA, and c3Ado, but not 6-MP, potentiated the inhibition by 2-CH (25-37.5 microM) of phagocytosis. Results are discussed in connection with the possible modulation by endogenous sulfhydryl-reactive metabolites of phospholipid turnover of the effects of c3Ado.

Synergism of 3-deazaadenosine with some Fc receptor ligands in the inhibition of antibody-dependent phagocytosis.

Inhibition of phagocytosis by 3-deazaadenosine, present in vitro during the assay, is unique in that it is dependent on the concentration of IgG on Ig-coated erythrocyte target cells. This regulatory interaction is now further examined utilizing the capacity of other Fc receptor ligands, like soluble immune complexes and Ig preparations, to modulate the effect of 3-deazaadenosine on antibody-dependent phagocytosis (AD phi) in mouse resident peritoneal macrophages. Non-inhibitory concentrations of soluble immune complexes formed in mixtures of ovalbumin (OVA) antigen and rabbit anti-OVA (RAOVA) antibodies enhanced the inhibition by 3-deazaadenosine of AD phi. The effect was reversed by using F(ab')2 antibodies, that lack the Fc portion of IgG, in OVA/RAOVA mixtures. Similar synergistic effects were achieved with human gammaglobulin and IgG preparations, and were further enhanced by aggregated IgG. Another source of Fc receptor ligands, autoimmune MRL/Mp-lpr/lpr mouse sera, unlike MRL/Mp-+/+ sera, also showed synergism with 3-deazaadenosine in the inhibition of AD phi. This effect was reversed by treatment of MRL sera with F(ab')2 fragments of an anti-mouse Fc antibody. It has been pointed out that the regulatory interaction between Fc receptor ligands, like Ig-coated cells, soluble immune complexes, Ig aggregates, on one hand, and the effects of 3-deazaadenosine, on the other hand, may represent a novel mechanism by which an agent inhibits phagocytosis indirectly, by manipulating the competition among these various ligands for the macrophage Fc receptor.

Regulatory role for the immune complex in modulation of phagocytosis by 3-deazaadenosine.

The S-adenosyl-L-homocysteine hydrolase inhibitor 3-deazaadenosine (3-DAA) modulates antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (AD phi) by mouse spleen effector cells and antibody-coated erythrocyte target cells. Concentrations of this compound inhibiting ADCC caused augmentation of phagocytosis. In a modified version of these assays referred to as complement-independent cellular cytotoxicity (CICC) and complement-independent phagocytosis (CI phi), specifically immune spleen cells were the source of effector cells and antitarget antibodies. CICC and CI phi were assayed with antiserum-untreated erythrocyte target cells. Although CICC was inhibited, 3-DAA failed to induce augmentation of phagocytosis in CI phi assays. Augmentation was restored by the presence of antibody-coated targets. If 3-DAA was present before the initiation of the assay by the addition of antibody-coated targets, it also failed to augment conventional AD phi. Varying dilutions of the antiserum, used for the preparation of antibody-coated target cells, induced differential effects of 3-DAA on phagocytosis. A regulatory interaction between the target cell antigen-antibody complex and the action of 3-DAA on phagocytosis has been suggested.

Effects of 3-deazaadenosine on antibody-dependent cellular cytoxicity and phagocytosis: reciprocal inhibition and augmentation of these functions in various effector cell populations.

3-Deazaadenosine (3-DAA), an inhibitor of S-adenosyl-L-homocysteine hydrolase, caused a dose-dependent inhibition of antibody-dependent cellular cytotoxicity (ADCC) against erythrocyte target cells of mouse spleen effector cells and complete Freund's adjuvant-induced peritoneal exudate cells. Inhibition of ADCC was accompanied by a proportional augmentation of antibody-dependent phagocytosis (ADø). When resident peritoneal cells were used as the source of effectors, ADø was inhibited by 3-DAA without augmentation of ADCC. Long-term (overnight) preincubation studies showed that the effect of 3-DAA on peritoneal ADø increased with time and was irreversible. Peritoneal exudate cells elicited by sodium caseinate mediated both antibody-dependent and -independent (spontaneous) phagocytosis. 3-DAA was shown as a potent and irreversible inhibitor of spontaneous phagocytosis. Inhibition of phagocytosis in these cell preparations resulted in augmentation of ADCC. In most of these effector cell populations, effects of 3-DAA were potentiated by the presence of L-homocysteine thiolactone.

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.

Differential interference by azathioprine and 6-mercaptopurine with antibody-mediated immunoregulation: synergism of azathioprine and antibody in the control of an immune response.

Comparative studies on the effects of immunosuppressive agents azathioprine (AZ) and 6-mercaptopurine (6-MP) in antibody-mediated immunoregulation are reported. To determine the degree of interference of these agents with the generation of antibody molecules controlling an anti-SRBC response, the donors of mouse anti-SRBC sera were given daily doses of the drugs. Primary antisera from AZ-treated donors were not significantly inhibited by daily doses of 180, 270, and 360 mumol/kg in their ability to suppress the direct PFC response; on the other hand 6-MP was inhibitory at the two higher equimolar dose levels. When the antisera were used in the antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (AD phi) assays, similar differential effects of the two agents were found. Multiple doses of AZ and 6-MP given to the donors of ADCC effector cells enhanced the capacity of these cells to lyse antibody-sensitized SRBC target cells. The enhancement of ADCC with 6-MP was less manifest than that with AZ. When injected before the antigen, AZ acted synergistically with the antibody in the control of the direct PFC response. Possible mechanisms by which AZ can interact with antibody-mediated immune regulation are discussed.

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.

Differential inhibition by azathioprine and 6-mercaptopurine of specific suppressor T cell generation in mice.

Doses of 100 mg/kg/day and 55 mg/kg/day of azathioprine (AZ) and 6-mercaptopurine (6-MP), respectively, significantly suppressed anti sheep red blood cell (SRBC) responses in CBA mice, assayed by the complement-dependent cellular cytotoxicity (CDCC) test and by serum titration. Four injections of similar doses of the agents were given to donors of carrier-specific suppressor T cells, generated by two immunizations with SRBC, and transferred to syngeneic recipients sensitized with the TNP hapten on SRBC carrier. Anti-TNP response of the recipients was assayed by the CDCC, using TNP-coated EL4 target cells. Whereas 6-MP, given after or before the second immunization of the donors with carrier SRBC, caused abrogation of suppressor cell activity, equimolar doses of AZ were less inhibitory to suppressor cell generation.