Early reversible induction of leukotriene synthesis in chicken myelomonocytic cells transformed by a temperature-sensitive mutant of avian leukemia virus E26.

Abstract

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...

Characterization of the human 5-lipoxygenase gene promoter.

“…A study using chicken myelomonocytic cells and myb-containing oncovirus indicated a down-regulatory effect of the myb protein on 5-lipoxygenase gene expression with concomitant inhibition of macrophage lineage differentiation (19). Another observation in line with this reasoning is that c-myb protooncogene expression declines in HL-60 cells during maturation (27).…”

“…Substantial production of 5-lipoxygenase metabolites is well documented for cells of myeloid lineage such as granulocytes, basophils, and macrophages. Differentiation of immature cells is associated with enhanced 5-lipoxygenase activity, as shown in studies using HL-60 cells (16)(17)(18), chicken myelomonocytic cells (19), and mouse mastocytoma cells (20). Some of our findings may reflect the cell-specific expression of 5-lipoxygenase.…”

Differential low density lipoprotein receptor-dependent formation of eicosanoids in human blood-derived monocytes.

“…Moreover, the use of radiolabeled AA present as CE does not allow precise mass estimates of eicosanoid synthesis, because the intracellular concentration of unesterified AA derived from endogenous lipids is unknown. Although constitutive eicosanoid production of resting monocytes has been reported to be low (5,7,19,22), these considerations may become more relevant when agonist-dependent eicosanoid responses that are associated with cellular phospholipase activation and AA release are considered (Figs. 3 and 4 and Table 2).…”

“…Radiolabeled tracer eicosanoids were added to parallel samples to determine recovery of the biological samples as described (17)(18)(19). PGH synthase or 5-lipoxygenase products, respectively, were extracted and separated by reversed-phase high pressure liquid chromatography systems as reported (7,17 (Fig.…”

Cloning and Characterization of a Bifunctional Leukotriene A4 Hydrolase from Saccharomyces cerevisiae

“…On the other hand, the epoxide hydrolase activity, i.e. the production of LTB 4 , has only been detected in vertebrates, including birds, fish, and frogs (12)(13)(14)(15), and a nonmammalian form of LTA 4 hydrolase was recently purified from the African claw toad, Xenopus laevis. The toad enzyme contained zinc and exhibited both epoxide hydrolase and peptidase activity (16).…”