DISRUPTION OF 12 15-LIPOXYGENASE EXPRESSION IN PERITONEAL-MACROPHAGES- ENHANCED UTILIZATION OF THE 5-LIPOXYGENASE PATHWAY AND DIMINISHED OXIDATION OF LOW-DENSITY-LIPOPROTEIN/

Previously, we isolated the murine ''leukocyte-type'' 12-lipoxygenase (L-12LO) cDNA from RNA of peritoneal-elicited cells that consisted pre dominantly of leukocytes (Chen, X.-S., Kurre, U., Jenkins, N. A., Cope land, N. G., and Funk, C. D. (1994) J. Biol. Chem. 269, 13979-13987). By in situ hybridization we show that the L-12LO gene is expressed abu ndantly in a subset of peritoneal macrophages but not in elicited leuk ocytes, alveolar macrophages, or bone marrow-derived macrophages. L-12 LO is highly related to human and rabbit 15-lipoxygenases, enzymes tha t have been implicated in the maturation process of red blood cells, a nd the oxidative modification of low density lipoproteins that is impl icated in atherogenesis. Accordingly, these enzymes have been referred to as 12/15-lipoxygenases. We have inactivated the L-12LO gene in mic e using homologous recombination in embryonic stem cells. Macrophage e xpression of L-12LO was abolished in homozygous deficient mice as was formation of 12-hydroxyeicosatetraenoic acid (12-HETE). In zymosan-sti mulated cells, there was significant diversion of metabolism to the 5- lipoxygenase products leukotriene C-4 and 5-HETE and in A23187-treated cells to 5-HETE only. The enhanced formation of 5-lipoxygenase metabo lites was not due to compensatory changes of 5-lipoxygenase or 5-lipox ygenase activating protein but rather an apparent substrate diversion. L-12LO-deficient mice have no obvious abnormalities in reticulocyte o r mature red blood cells, which suggest that in mice this pathway is n ot functionally important for erythrocytic development. Indices for ox idation of low density lipoprotein (measured as either thiobarbituric acid-reactive substances or the oxidant stress marker isoprostane 8-ep i-prostaglandin F-2 alpha) were identical in incubations with unstimul ated wild type and L-12LO deficient macrophages, but the zymosan-induc ed increase observed with wild-type macrophages was abolished in L-12L O-deficient cells, Thus, 12/15-lipoxygenase-deficient mice will be use ful for the study of interaction between lipoxygenase pathways and det ermination of the in vivo role of 12/15-lipoxygenase-catalyzed oxidati on of LDL in atherogenesis.