No abstract

Eicosanoid biosynthesis in animal cells either results from agonist-stimulated phospholipase activation (endogenous pathway) or from lipoprotein receptor-mediated uptake and lysosomal lipid hydrolase-dependent release of AA (exogenous pathway) (see Fig. 1 for schematic representation). LDL stimulates eicosanoid formation through delivery of substrate AA to enzymes of oxidative AA metabolism. The classical LDL receptor is a control point of the effects of LDL AA on eicosanoid formation in different tissues: LDL AA metabolism occurs in several cell types of mesenchymal and epithelial origin and generates the formation of distinct eicosanoid patterns in each case. The LDL AA pathway does appear to couple directly to the PGH synthase reaction, whereas it does not couple directly to the 5-lipoxygenase reaction. We expect that a more complete characterization of the LDL unsaturated fatty acid pathway in different tissue will yield additional information on the biochemistry of lipoproteins, AA, and eicosanoids.

We used chicken myelomonocytic cells transformed by a temperature-sensitive mutant of the myb/ets oncogene-containing avian leukemia virus E26 to study the regulation of leukotriene (LT) synthesis during macrophage differentiation. Cells exposed to arachidonic acid and the Ca2+ ionophore 23187 produced up to 180 times more LTs at the nonpermissive temperature (420C) than at the permissive temperature (3rC). Induction of LT synthesis was detectable within 2 hr after temperature shift, whereas conventional macrophage markers became evident after 2-3 days. NFormylmethionylleucylphenylalanine, opsonized zymosan, and complement factor C5a induced LT synthesis in temperature-sensitive mutant-transformed cells only when the cells were maintained at 42C, and this effect was blocked by pertussis toxin. When cells were kept at 42°C for 48 hr and then shifted back to 37C to induce retrodifferentiation, LT synthesis rates declined within 8 hr and reached near control values within 36 hr. Retrodifferentiation also led to decreased LT synthesis in response to N-formylmethionylleucylphenylalanine, opsonized zymosan, and C5a. These results indicate that activation of the 5-lipoxygenase pathway is a very early event in the macrophage differentiation pathway that is directly or indirectly controlled by the temperature-sensitive v-myb protein.Leukocytes have been shown to synthesize leukotrienes (LTs)(1) in response either to unspecific stimuli, such as Ca2l ionophores, or to specific agonists, such as the chemotactic peptide N-formylmethionylleucylphenylalanine (fMLP), opsonized zymosan, and complement factor C5a (2-5). The current view of the regulation of LT synthesis holds that these agonists stimulate the release ofarachidonic acid (A4Ach) from endogenous phospholipid stores and also activate the 5-lipoxygenase that initiates the conversion of A4Ach into LTs (2, 5). Whereas the mechanisms of A4Ach release have received considerable attention, very little is known about the mechanisms that regulate other components of the LT response system including the 5-lipoxygenase and the other enzymes of LT synthesis. One way to identify these mechanisms is to study the up-regulation of LT synthesis during leukocyte differentiation (6)(7)(8).A particularly promising approach for the study of otherwise scarce hematopoietic precursors is the use of avian leukemia retroviruses to produce homogeneous populations of transformed bone marrow cells (9 (10). Importantly, time-lapse photography has demonstrated that the differentiation of these cells into macrophages is a reversible process (12). Thus, ts-mutant-transformed cells provide a reversible macrophage differentiation system that circumvents many of the problems encountered with other differentiation systems.In the present study we used ts-mutant-transformed cells to investigate the relation between LT synthesis and macrophage differentiation. To compare the results obtained with ts-mutant-transformed cells with those obtained with differentiated macrophages, parallel experiments...

We studied the ability of low density lipoproteins (LDLs) to provide arachidonic acid (AA) for eicosanoid biosynthesis in human blood-derived monocytes. When incubated in the presence of reconstituted LDL that contained cholesteryl [1_-4C] These results demonstrate that the LDL receptor pathway preferentially promotes the synthesis of PGH synthase products in resting human blood-derived monocytes and that an additional mechanism is required to promote effective synthesis of 5-lipoxygenase pathway products from AA that originates in LDL cholesteryl esters.We and others have observed stimulatory effects of native plasma lipoproteins on the formation of prostacyclin and prostaglandin (PG) E2 in cultured endothelial cells, smooth muscle cells, and fibroblasts (1-3). Furthermore, we have shown (4) that the stimulatory effect of low density lipoprotein (LDL) on prostacyclin and PGE2 production by plateletderived growth factor-stimulated fibroblasts was a consequence of the LDL receptor-dependent delivery of arachidonic acid (AA) to the PGH synthase (EC 1.14.99.1) reaction and that it was followed by a profound inhibition of PGH synthase. This raised the possibility that the LDL pathway might have a role in the regulation of eicosanoid synthesis in fibroblasts in addition to its known role in the maintenance of cellular cholesterol homeostasis. Furthermore, since all animal cells can express the LDL receptor and also can convert AA into one or more eicosanoids (of which more than 40 with very different biological activities have been identified to date; for review, see refs. 5 and 6), it seemed possible that the LDL pathway might play a similar role in other cell types. To investigate the latter possibility, we turned our attention to human blood-derived monocytes. Unlike most mammalian cells, monocytes express both the PGH synthase pathway that leads to the formation of prostacyclin, thromboxane (TX) A2, and PGE2 (7) and the 5-lipoxygenase (EC 1.13.11.12) pathway that leads to the formation of leukotriene (LT) B4 and LTC4 (8). Furthermore, the products ofthe two pathways play distinct, and in some instances even opposing, biological roles (5). Our results demonstrate that human monocytes use the classical LDL receptor pathway (for review, see refs. 9 and 10) to deliver AA for the production of prostacyclin, TXA2, and PGE2, and that N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) induces formation of LTB4 and LTC4 from AA that originates in LDL cholesteryl esters (CEs). MATERIALS AND METHODSMaterials. fMet-Leu-Phe, AA, Ca2+ ionophore A 23187, and all other materials were from Sigma; [14C]AA and [14C]AA-CE (specific activity, 54.9 mCi/mmol; 1 Ci = 37 GBq) were from NEN.Cell Preparation and Cell Culture. Human blood-derived mononuclear cells were isolated by leukapheresis from fasting volunteers, and monocytes were purified as described (11,12). Cells were maintained in minimum essential medium supplemented with undialyzed 20%