Characterization of the inhibitory effect of lysolipids on perforin-mediated hemolysis

The ability of lysolipids to inhibit the lytic activity of perforin from cytotoxic T lymphocytes was investigated. Sublytic concentrations of various lysolipids were incorporated into the membranes of sheep red blood cells (RBC) and the cells were then lysed with purified perforin. Lysophosphatidylcholines (lysoPC) can effectively block perforin-mediated lysis at micromolar concentrations. This is in marked contrast to phosphorylcholine, the putative calcium-dependent receptor for perforin, which inhibits lysis only at greater than or equal to millimolar concentrations. Unlike the inhibitory action of lipids, the lysolipids do not show a strict dependence on headgroup composition, as lysophosphatidylserine (lysoPS) is just as effective as lysoPC. All the lysoPC tested, ranging from lysolauroyl PC to lysostearoyl PC, are good inhibitors, with lysomyristoyl PC being the most effective. Binding of lysoPC to RBC is reversible; the inhibition by lysoPC can be removed with bovine serum albumin (BSA), and washing RBC that had been pretreated with lysoPC leads to a loss of inhibition. Binding of perforin to membranes is temperature-independent and precedes a temperature-dependent, insertion/pore-formation stage; hemolysis experiments that take advantage of this fact indicate that lysoPC acts mostly by blocking perforin binding to the RBC membranes.