gramicidin-a and Uveitis--Posterior

Using an in vitro lymphocyte proliferation assay we screened several cyclic peptide antibiotics (bacitracin, oleandomycin, capreomycin, colistin, virginiamycin, and gramicidin S) for their immunosuppressive activity. Gramicidin S (GrS) was found to inhibit [3H]-thymidine incorporation into concanavalin A-stimulated and E coli lipopolysaccharide-stimulated lymphocytes. In vivo studies, experimental autoimmune uveoretinitis (EAU) and pinealitis were induced in female Lewis rats by immunization with bovine S-antigen and experimental autoimmune encephalomyelitis (EAE) were induced by immunization of rats with rat brain homogenates. GrS suppressed the onset of these inflammatory diseases at nontoxic concentrations. Evidence was obtained that GrS inhibits [3H]-thymidine incorporation into lymphocytes by preventing transport of the compound across the membrane. Since GrS binds to various cell membranes, GrS would suppress the proliferation of not only lymphocytes but also of other immune cells by modifying cell membrane properties. The present study indicates that a search for compounds which cause proper cell membrane modification should be a worthwhile strategy for development of immunosuppressive drugs.

Topics: Amino Acid Sequence; Animals; Antigens; Arrestin; Autoimmune Diseases; Concanavalin A; Encephalomyelitis, Autoimmune, Experimental; Eye Proteins; Female; Gramicidin; Immunization; Immunosuppressive Agents; Lipopolysaccharides; Lymphocyte Activation; Lymphocytes; Molecular Sequence Data; Phosphoric Monoester Hydrolases; Pineal Gland; Rats; Rats, Inbred Lew; Retinitis; Spinal Cord; Spleen; Thymidine; Uveitis, Posterior

Gramicidin S (GrS) suppressed the onset of experimental autoimmune uveoretinitis (EAU) induced with S-antigen in rats, and the proliferation of mitogen-stimulated lymphocytes in culture. As an immunosuppressive mechanism of the cyclic peptide antibiotic we first postulated that the drug exerts its effect as an ionophore. Although all ionophore compounds tested suppressed lymphocyte proliferation, no correlation was observed between changes in intracellular concentrations of Na+ and K+ and the degree of immunosuppression. For example, monensin inhibited lymphocyte proliferation without affecting intracellular Na+ and K+ levels. Thus it was likely that the immunosuppressive effects of the ionophore compounds including GrS were due to their ability to modify cell membrane properties rather than their ionophore activity. We then tested the hypothesis that GrS inhibits transport of metabolic intermediates or metabolites (thymidine and methionine) into lymphocytes. The idea was experimentally supported. Further, inhibition of metabolite transport by GrS was found to be reversible. To investigate whether inhibition of metabolite uptake can be a mechanism of immunosuppression of EAU, endogenously stimulated lymphocytes were isolated from S-antigen-immunized rats at different stages of EAU and the effect of GrS on metabolite uptake by the cells was determined. The activity of lymphocytes to transport metabolites was enhanced at pre-EAU stages and the enhanced metabolite uptake was markedly inhibited by GrS. We interpreted the result to support that inhibition of metabolite uptake by GrS is probably a mechanism of immunosuppression in vivo by this drug.

Topics: Animals; Antigens; Arrestin; Autoimmune Diseases; Biological Transport; Cells, Cultured; Disease Models, Animal; Eye Proteins; Female; Gramicidin; Immunosuppressive Agents; Ionophores; Lymphocyte Activation; Lymphocytes; Methionine; Potassium; Rats; Rats, Inbred Lew; Sodium; Thymidine; Uveitis, Posterior