Flow cytometric analysis of altered mononuclear cell transmembrane potential induced by cyclosporin

Immunosuppression by the fungal metabolite cyclosporin A (CsA) is characterized by functional inhibition, rather than destruction of cells. Because activation of immune cells involves intracellular signalling events associated with modulations of cell transmembrane potential (TMP), we tested the ability of cyclosporin A (CsA) to modulate immune mononuclear cell TMP in vitro using a TMP sensitive cationic dye, dihexyloxacarbocyanine (DIOC6(3)). All analyses were performed by flow cytometry. CsA increased TMP in monocytes and lymphocytes isolated from the blood of healthy human volunteers. CsA-induced hyperpolarization was time and concentration dependent in monocytes while the lymphocyte hyperpolarization, although time dependent, was evident over the entire range of CsA concentrations tested. CsA-induced hyperpolarization of lymphocytes was dependent on potassium ion (K+) efflux as indicated by the absence of hyperpolarization in 154 mM KCl or with pretreatment with 100 microM quinine (an inhibitor of K+ channels). Monocyte hyperpolarization by CsA was not inhibited in either system. Dihydrocyclosporin C (DH-CsC), an immunosuppressive analog of CsA, also hyperpolarized mononuclear cells. The anionic TMP sensitive dye bis oxonal (diBA-C4) indicated that CsA treatment depolarized mononuclear cell plasma membranes. The mitochondrial poison carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazine (FCCP) eliminated CsA induced hyperpolarization and also indicated that CsA caused plasma membrane depolarization. We conclude that brief in vitro exposure to cyclosporin alters the transmembrane electrical potential of human lymphocytes and monocytes.