Cyclosporin A (CsA) is a potent immunosuppressive agent, which acts primarily by inhibiting T cell function, likely by preventing IL2 formation. Its wide and successful use in clinical transplantation requires a more accurate definition of its mode of action. The presumption that CsA selectively blocks IL2 production ignores the inhibition readily demonstrated on earlier metabolic events and its suppressive activity in B lymphocytes as well. We have demonstrated that the immunosuppressive cyclosporins, but not the non-immunosuppressive analog, affect ion mobilization and the resting membrane potential of cells, resulting in decreased intracellular Ca2+ concentrations. These findings, which are the earliest recorded effect of the drugs, likely contribute to the overall immunosuppressive activities of this group of drugs, especially on IL2 production and T-cell proliferation. The goals of the present proposal are to further define the ionic, biochemical, and genetic basis for cyclosporin-dependent effects on membrane potential, Ca2+ uptake and IL2 formation, to determine the interrelationship between these phenomena, and to delineate the requirements for their immunosuppressive action. Specifically in human T and B cells we will examine early activation events following receptor-ligand interaction, subsequent events which occur after a signal has reached the nucleus, and late events associated with cell division, lymphokine production or Ig secretion. Using intracellularly trapped fluorescent indicators we will monitor changes in cytosolic Ca2+ concentrations, cytosolic pH, and membrane potential. We will determine if the major effects of the cyclosporins are mediated through alteration of K+ permeability. We will assess the consequences of altering early biochemical events including phosphoinositide metabolism on the expression of c-fos, c-myc, IL2, IL2 receptor and transferrin receptor gene mRNA and ultimately on receptor expression, IL2 formation and cell proliferation or Ig secretion. In an attempt to define activities of specific T-cell subsets we will obtain T- cell subsets by sorting and will couple the drugs to liposomes and target them to specific lymphocyte subsets with monoclonal antibodies. More precise definition of the mechanism of action of the cyclosporins would provide further understanding for its use as an immunosuppressive agent, offering the potential for combining it with other forms of therapy and possibly reducing dosage, duration and hence toxicity.
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