Abstract
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, CTalpha, using the Cre-lox system. Surprisingly, the number of peritoneal macrophages harvested from CTalpha-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 CTbeta2, a form of CT that arises from another gene, is induced in CTalpha-deficient macrophages. To test our hypothesis that increased PC biosynthesis is an adaptive response to FC loading, the viability of wild-type versus CTalpha-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 CTalpha-deficient macrophages. These data offer the first molecular genetic evidence that activation of CTalpha and induction of PC biosynthesis in FC-loaded macrophages is an adaptive response. Furthermore, the data reveal that CTbeta2 in macrophages is induced in the absence of CTalpha and that a low level of residual CT activity, presumably due to CTbeta2, is enough to keep the cells viable in the peritoneum in vivo and under normal culture conditions.
Highlights
Macrophages in atherosclerotic lesions accumulate excess free cholesterol (FC) and phospholipid
Our laboratory has studied cellular events that occur during FC loading of macrophages, and we discovered that the cells respond initially by an increase in PC biosynthesis and cellular PC mass, which appears to occur in lesional macrophages in vivo [15, 16]
Creation of Mice Whose Macrophages Are Depleted in CT␣—To obtain mouse peritoneal macrophages with decreased PC biosynthesis, the overall strategy was to create a mouse with the endogenous CT␣ locus replaced by a CT␣ gene with loxP sites flanking one or more exons critical for CT␣ function
Summary
Materials—The restriction endonucleases and other enzymes were purchased from either New England Biolabs (Beverly, MA) or Life Technologies, Inc. [␣-32P]dCTP, [methyl-3H]choline, and phospho [methyl-14C]choline were from PerkinElmer Life Sciences. Generation and Identification of Gene-targeted ES Clones—The replacement vector was transfected into 129/Sv ES cells, and G-418resistant clones were selected and screened by Southern blot analysis using a 1-kb NheI/EcoRI exon 8-containing probe (Fig. 1A) as follows. Heterozygous CT␣flox mice were identified and sibmated, and homozygous CT␣flox mice resulting from this mating (ϳ25% of the offspring) were identified by PCR using the CT382 primer (above) and a primer called CT3UL (5Ј-GAAGTAGGCACTGAACTTAGG-3Ј) just upstream of the 3Ј loxP site (see Fig. 2A and “Results”) These homozygous CT␣flox mice were bred with homozygous LysMCre mice (obtained from Dr Irmgard Forster, Technical University of Munich), in which the Cre recombinase is driven by the lysozyme promoter via gene targeting into the lysozyme locus [26]. Statistics—Results are given as means Ϯ S.E. (n ϭ 3 for all experiments except the cell death experiment in Fig. 6, where n ϭ 5 fields of cells at ϳ200 cells per field); absent error bars in the figures signify S.E. values smaller than the graphic symbols
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