Macrophages deficient in CTP:phosphocholine cytidylyltransferase-α are viable under normal culture conditions but are highly susceptible to free cholesterol-induced death -: Molecular genetic evidence that the induction of phosphatidylcholine

Macrophages in atherosclerotic lesions accumulate excess free cholesterol (FC) and phospholipid. Because excess FC is toxic to macrophages, these observations may have relevance to macrophage death and necrosis in atheromata. Previous work by us showed that at early stages of FC loading, when macrophages are still healthy,there is activation of the phosphatidylcholine (PC) biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase (CT), and accumulation of PC mass, We hypothesized that this is an adaptive response, albeit transient, that prevents the FC:PC ratio from reaching a toxic level; To test this hypothesis directly, we created mice With macrophage-targeted disruption of the major CT gene, CT alpha, using the Cre-lox system. Surprisingly, the number of peritoneal macrophages harvested from CT alpha ;deficient mice and their overall health under normal culture conditions appeared normal, Moreover, CT activity-and PC biosynthesis and in vitro CT activity were decreased by 70-90% but were not absent. As a likely explanation of this residual activity, we showed that CT beta2, a form of CT that arises from another gene, is induced in CT alpha -deficient macrophages. To test our hypothesis that increased PC biosynthesis is an adaptive response to FC loading, the viability of wild-type versus CT alpha -deficient macrophages under control and FC-loading conditions was compared. After 5 h of FC loading, death increased from 0.7% to only 2.0% in wild-type macrophages but from 0.9% to 29.5% in CT alpha -deficient macrophages, These data offer the first molecular genetic evidence that activation of CT alpha and induction of PC biosynthesis in FC-loaded macrophages is an adaptive response. Furthermore, the data reveal that CT beta2 in macrophages is induced in the absence of CT alpha and that a low: level of residual CT activity, presumably due to CT beta2, is enough to keep the cells viable in the peritoneum in vivo and under normal culture conditions.