concanavalin-a and bucillamine

2-Mercaptomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-758), which is the active metabolite of 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), is a novel sulphydryl anti-rheumatic drug. In this study we analyzed the effect of KE-758 on the proliferation of murine lymphocytes, and on the production of nitric oxide (NO) by RAW264.7 murine macrophage cells. We compared its effect with other sulphydryl drugs such as D-penicillamine, bucillamine and auranofin. The proliferation of lymphocytes was measured by 3H-thymidine incorporation assay. Nitrite was measured using Griess Reagent. In the absence of copper ions, KE-758, D-penicillamine and bucillamine rarely affected the proliferation of concanavarin A (ConA) activated murine splenocytes. However, in the presence of copper, pharmacological concentrations of KE-758 but not D-penicillamine and bucillamine suppressed the proliferation of murine splenocytes through a hydrogen peroxide-dependent mechanism. Auranofin markedly suppressed the proliferation regardless of the presence of copper ions by reducing the cellular viability. Furthermore, only KE-758 markedly suppressed the proliferation of phorbol myristate acetate (PMA) plus ionomycin activated murine whole blood lymphocytes (WBL) even in the absence of exogenous copper ions by a hydrogen peroxide-independent mechanism. Meanwhile, lipopolysaccharide (LPS) or LPS plus interferon-gamma (IFN-gamma) induced NO production by RAW264.7 cells were suppressed by KE-758 and auranofin but not by D-penicillamine and bucillamine. In conclusion, KE-758 is a novel immunosuppressive drug, which inhibits both lymphocyte and macrophage functions and its unique anti-rheumatic profile is distinct from that of D-penicillamine, bucillamine and auranofin.

Topics: Animals; Antirheumatic Agents; Auranofin; Cell Division; Cell Line; Concanavalin A; Cysteine; Female; Hydrogen Peroxide; In Vitro Techniques; Interferon-gamma; Ionomycin; Lipopolysaccharides; Lymphocyte Activation; Lymphocytes; Macrophages; Mice; Mice, Inbred DBA; Nitric Oxide; Penicillamine; Phenylpropionates; Recombinant Proteins; Tetradecanoylphorbol Acetate

The in vitro and in vivo effects of a new immunomodulating agent, bucillamine, on experimental autoimmune uveitis (EAU) was studied in the rat. The capacity of S-antigen-sensitized lymphocytes to proliferate in response to the antigen or to produce antigen-specific antibodies was significantly suppressed by bucillamine in culture in a dose-dependent manner. The inhibitory effect of bucillamine was significantly enhanced by adding cyclosporin A (CYA) in the culture. The in vivo effects of bucillamine alone or in combination with CYA were further examined in Lewis rats immunized with S-antigen. All untreated rats developed severe EAU 17 days after S-antigen immunization, while rats treated with either bucillamine (200 mg kg-1 day-1) or CYA (2 mg kg-1 day-1) demonstrated milder symptoms of EAU. A combination therapy with bucillamine (20 or 200 mg kg-1 day-1) and CYA (2 mg kg-1 day-1) exhibited much more significant suppression of EAU induction. Although the in vivo treatment with bucillamine or CYA had no effects on the T-cell populations of spleen cells, the combination therapy significantly decreased the CD4+ T-cell population. As for the immune responses to S-antigen in drug-treated rats, bucillamine suppressed the lymphocyte proliferation to S-antigen, which was further suppressed by combination therapy with CYA. The serum antibody levels specific to S-antigen were not affected by tested dose of bucillamine.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibody Formation; Antigens; Arrestin; Autoimmune Diseases; Cell Division; Cells, Cultured; Concanavalin A; Cyclophosphamide; Cysteine; Drug Synergism; Eye Proteins; Immune Tolerance; Male; Rats; Rats, Inbred Lew; Spleen; Uveitis

The effect of bucillamine [N-(2-mercapto-2-methylpropionyl)-L-cysteine], a new antirheumatic drug, on the concanavalin A (Con A)-induced proliferation of mouse spleen cells was compared with the effect of D-penicillamine (D-pen). Bucillamine inhibited the Con A-induced incorporation of thymidine (TdR) into mouse spleen cells in a dose-dependent fashion. At the concentration of 10(-4) M, bucillamine inhibited the incorporation by approximately 80%. The inhibitory effect of bucillamine was not enhanced by the addition of copper. In contrast, D-pen showed the same degree of inhibition only in the presence of copper. (4R)-7,7-Dimethyl-6-oxo-tetrahydro-3H-1,2,5-dithiazepine-4-carboxy lic acid (SA981), an intramolecular disulfide derivative of bucillamine, also showed the same degree of inhibition as bucillamine in the absence and presence of copper, whereas D-penicillamine disulfide did not show the inhibitory effect even in the presence of copper. The inhibitory effects of bucillamine and SA981 were not abolished significantly by the addition of catalase which restored the inhibition by D-pen plus copper. The mechanism of inhibition by D-pen plus copper is believed to involve the production of hydrogen peroxide resulting from the oxidation of D-pen. The results in this study, however, indicated that the inhibitory effect of bucillamine is not due to the production of hydrogen peroxide.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Catalase; Cell Division; Cells, Cultured; Concanavalin A; Copper; Cysteine; Male; Mice; Mice, Inbred C3H; Penicillamine; Spleen; Structure-Activity Relationship

We have screened from various thiol compounds SA96 (2-mercapto-2-methylpropanolyl-L-cysteine) as a stimulant of LPS-induced proliferative response of murine lymphocytes, and the immunopharmacological profile of this compound was studied in vitro. SA96, only at 10(-3) M concentration, caused 3H-thymidine (3H-TdR)uptake in the presence of foetal calf serum (FCS). The effects of SA96 on the proliferative response to mitogens were biphasic. The uptake 3H-TdR induced by Con A or LPS was suppressed in a dose-dependent manner below 10(-4) M, but was enhanced at 10(-3) M in the case of LPS. The response to Con A was recovered from the suppression at 10(-3) M, but did not exceed the control level. Some experiments suggested that cysteine moiety might importantly contribute to the proliferative response caused by SA96. SA96 enhanced the PFC response to SRBC in vitro at 10(-5) approximately 10(-4) M (nonmitogenic doses), but markedly inhibited the elevated response in the presence of 2-mercaptoethanol (2-ME). On the other hand, PFC response to DNP-Ficoll )a T-independent antigen) was augmented by SA96 irrespective of the presence of 2-ME. These findings suggest that SA96, a thiol compound, may be useful as an immunomodulator like levamisole.

Topics: Animals; Antibody Formation; Antibody-Producing Cells; Cattle; Concanavalin A; Cysteine; Dose-Response Relationship, Drug; Female; Fetal Blood; Lipopolysaccharides; Lymphocyte Activation; Lymphocytes; Mercaptoethanol; Mice; Mice, Inbred BALB C; Penicillamine; Spleen