Free fatty acid perturbation of transmembrane signaling in cytotoxic T lymphocytes

Transmembrane signaling in CTL is found to be extremely sensitive to short term exposure to long chain free fatty acids (FFA). Both alloantigen specific target cells and the lectin Con A were used to stimulate cloned murine CTL. This stimulation was monitored by changes in intracellular calcium concentrations (Ca2+]i) using the fluorescence indicator fura-2. Treatment of the CTL cells with oleic acid (18:1) at concentrations corresponding to less than 10% (mol/mol) bound to the cell, completely inhibits target cell or Con A-mediated rise in Ca2+]i. The inhibitory effect of oleic acid is observed within seconds of addition and the inhibition is completely reversed by treating cells with fatty acid free BSA. In addition, using the fluorescence indicator 2',7'-bis(carboxyethyl)carboxyfluorescein to monitor intracellular pH, it was found that oleic acid itself acidifies the cytosol by about 0.3 to 0.4 pH units. Acidification is probably necessary, but is not sufficient to inhibit the calcium rise. Stearic acid (18:0), even at concentrations that correspond to a factor of two to three more bound to the cell than for oleic acid, had no effect on either the Ca2+]i or intracellular pH responses. Oleic acid was found to bind to cells with single site kinetics and with a number of sites and affinity corresponding to membrane lipid binding sites. Esterification of added oleic acid was negligible in the time (seconds to minutes) required to induce inhibition of the Ca2+]i response. Inasmuch as added FFA primarily binds to membrane lipid, is not appreciably esterified, and the inhibition is reversed by treatment with fatty acid free BSA, it is likely that the oleic acid effects are due to a physical perturbation of membrane lipid. Furthermore, oleic acid does not affect Con A binding or the production of inositol phosphate metabolites, suggesting that the inhibition of the response is distal to surface recognition events or receptor-phospholipase C coupling. Given the relatively low levels of FFA at which these effects occur it is possible, under conditions in which FFA levels are elevated, that FFA perturbation may modulate CTL activity.