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

Macrophages in atherosclerotic lesions accumulate excess free cholesterol ( FC) and phospholipid. Because excess FC is toxic to macrophages, these obse rvations may have relevance to macrophage death and necrosis in atheromata. Previous work by us showed that at early stages of FC loading, when macrop hages are still healthy,there is activation of the phosphatidylcholine (PC) biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase (CT), and acc umulation of PC mass, We hypothesized that this is an adaptive response, al beit 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. Surpri singly, the number of peritoneal macrophages harvested from CT alpha ;defic ient 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 t his residual activity, we showed that CT beta2, a form of CT that arises fr om another gene, is induced in CT alpha -deficient macrophages. To test our hypothesis that increased PC biosynthesis is an adaptive response to FC lo ading, the viability of wild-type versus CT alpha -deficient macrophages un der 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 firs t molecular genetic evidence that activation of CT alpha and induction of P C biosynthesis in FC-loaded macrophages is an adaptive response. Furthermor e, the data reveal that CT beta2 in macrophages is induced in the absence o f 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.