Immunosuppression by D-penicillamine in vitro. Inhibition of human T lymphocyte proliferation by copper- or ceruloplasmin-dependent generation of hydrogen peroxide and protection by monocytes.

Abstract

It has been suggested that D-penicillamine is active in rheumatoid arthritis because of its capacity to function as a selective inhibitor of T lymphocyte function. The basis for the immunosuppressive action of this drug as well as mechanisms whereby the effect of D-penicillamine could be modified by elements of rheumatoid synovial tissue were examined. As previously reported, D-penicillamine, in the presence of copper ions markedly inhibited mitogen-induced human T lymphocyte DNA synthesis. Since the vast majority of copper in the body exists as an integral part of the ceruloplasmin molecule, the capacity of this cuproprotein to augment D-penicillamine-mediated inhibition of T cell function was examined. The requirement for copper ions could be entirely replaced by purified ceruloplasmin, which had been depleted of nonspecifically bound copper by passage over Chelex-100 columns. The mechanism by which D-penicillamine in the presence of either copper ions or ceruloplasmin caused inhibition of T lymphocyte responsiveness was examined. Partial protection from this inhibitory effect was accomplished by sodium borohydride. While superoxide dismutase had no protective effect, catalase was found to protect lymphocyte responsiveness totally from the inhibitory action of D-penicillamine and either copper ions or ceruloplasmin. Similarly, horseradish peroxidase and myeloperoxidase also protected responsiveness from these inhibitors while boiled catalase was without effect. These results indicate that inhibition of T lymphocyte responsiveness resulted from the generation of hydrogen peroxide. Since a number of cells likely to be present at chronic inflammatory sites, such as mononuclear phagocytes, contain enzymatic mechanisms to degrade hydrogen peroxide, the modulatory influence of these cells on the inhibition of T cell function caused by D-penicillamine and copper was examined. Monocytes, whose function was not suppressed by D-penicillamine and copper, were found to protect T lymphocyte responsiveness from the inhibitory effects of either the mixture of D-penicillamine and CuSO4 or of hydrogen peroxide. By contrast, endothelial cells, fibroblasts, or cells obtained from enzyme-digested noninflamed synovium could not protect T cells from the inhibitory effects of D-penicillamine and copper. Protection of T cells was afforded by means of a heat labile, azide-sensitive soluble factor present in lysates of human monocytes. These results indicate that the mechanism whereby D-penicillamine in the presence of copper or ceruloplasmin inhibits T lymphocyte responsiveness involves the generation of hydrogen peroxide and that other neighboring cells likely to be found w