Altered expression and localization of 5-lipoxygenase accompany macrophage differentiation in the lung

Abstract

The alveolar macrophage (AM) exhibits a greater capacity to synthesize bioactive leukotrienes from arachidonic acid than does its circulating precursor the peripheral blood monocyte. Macrophage differentiation in the lung entails cellular residence within both the pulmonary interstitial and alveolar compartments. In the present study, we sought to determine 1) whether this enhanced metabolic activity was acquired during maturation within the alveolar space and 2) the underlying mechanisms responsible for this upregulation. Rat AMs were separated by Percoll gradient centrifugation into four density-defined subpopulations thought to reflect their degree of maturation. On stimulation with a calcium ionophore, synthesis of leukotriene B4 increased with the degree of maturation, although it was diminished in the oldest subpopulation. This maturation-dependent upregulation was not explained by increases in arachidonic acid release but was associated with increased expression of 5-lipoxygenase (5-LO) protein as determined by immunoblot analysis. Whereas 5-LO is primarily cytosolic in monocytes, it is known to be primarily intranuclear in unfractionated AMs. Here, the localization of 5-LO was investigated by immunofluorescence microscopy and was found to be predominantly nuclear in all AM subpopulations; by contrast, the protein was cytosolic in interstitial macrophages isolated by mechanical and enzymatic lung digestion. These divergent localization patterns in AMs and interstitial macrophages were verified in situ by immunohistochemical staining of sections of normal rat lung. When unfractionated AMs were isolated and maintained in culture for 3 days, a shift in 5-LO distribution from nucleus to cytosol was observed. We conclude that 1) nuclear import of 5-LO occurs within the alveolar space and is reversible on removal from the alveolar milieu and 2) leukotriene synthetic capacity increases further during AM residence within the alveolar space as a result of a progressive increase in the amount of 5-LO protein.

Inhibition of ovulation by a lipoxygenase inhibitor involves reduced cyclooxygenase-2 expression and prostaglandin E2 production in gonadotropin-primed immature rats

“…Preliminary experiments checked the cross-reactivity of the used antibodies against respective LOX antigens with lung (ALOX5 and -12) and spleen (ALOX12) positive controls. As was consistent with the previously reported results (Maruyama et al 1989, Covin et al 1998, Kawajiri et al 2000, alveolar epithelial cells in the lung and white pulp in the spleen of adult rats showed positive immunoreaction for respective antigen (data not shown).…”

“…This experiment was also carried out for three different samples at each time point. Sections of lung and spleen of adult rats served as positive controls for ALOX5 (lung) and ALOX12 (lung and spleen) immunohistochemistry (Maruyama et al 1989, Covin et al 1998, Kawajiri et al 2000.…”

Phosphorylation of Serine 271 on 5-Lipoxygenase and Its Role in Nuclear Export

“…This has been demonstrated in primary cells, such as neutrophils (33) and macrophages (53), as well as in molecular model systems, such as transfected NIH 3T3 cells (34). In addition, we have previously demonstrated that impairment of nuclear import of 5-LO, achieved by selectively inactivating a nuclear import sequence, reduces LTB 4 synthesis by more than 60% (34).…”

Leukotriene B4mediates macrophage influx and pulmonary hypertension in bleomycin-induced chronic neonatal lung injury

“…1). 5-LPO activity requires perinuclear translocation from the cytosol (9) and association with FLAP homodimer (7,27,39) and is increased by phosphorylation at serine 271 (55). LTB 4 and cysLTs exert their actions by binding to distinct G proteincoupled receptors: BLT1 and BLT2 for LTB 4 , and cysLT 1 and 2 receptors (cysLT 1 R and cysLT 2 R) for cysLTs ( Fig.…”