Interleukin-4 (IL-4) induces expression of reticulocyte-type 15-lipoxygenase-1 (15-LOX-1) in various mammalian cells via the Janus kinase/signal transducer and activator of transcription 6 (STAT6) signaling system. We studied the mechanism of 15-LOX-1 induction in A549 lung epithelial cells and found that genistein, a potent tyrosine kinase inhibitor, prevented phopsphorylation of STAT6, its binding to the 15-LOX-1 promoter, and the expression of catalytically active enzyme. In contrast, cycloheximide did not prevent 15-LOX-1 induction. Surprisingly, we found that IL-4 up-regulated the histone acetyltransferase activity of CREB-binding protein (CBP)/p300, which is responsible for acetylation of nuclear histones and STAT6. The acetylation of both proteins appears to be essential for the IL-4-induced signal transduction cascade, because inhibition of CBP/ p300 by the viral wild-type E1A oncoprotein abrogated acetylation of both histones and STAT6 and strongly suppressed transcriptional activation of the 15-LOX-1 gene. Moreover, we found that the inhibition by sodium butyrate of histone deacetylases, which apparently suppress 15-LOX-1 gene transcription, synergistically enhanced the IL-4-stimulated 15-LOX-1 expression. These data suggest that both phosphorylation and acetylation of STAT6 as well as acetylation of nuclear histones are involved in transcriptional activation of the 15-LOX-1 gene, although these reactions follow differential kinetics. STAT6 phosphorylation proceeds within the first hour of IL-4 stimulation. In contrast, CBP/p300-mediated acetylation requires 9 -11 h, and similar kinetics were observed for the expression of the active enzyme. Thus, our results suggest that in the absence of IL-4, nuclear histones may be bound to regulatory elements of the 15-LOX-1 gene, preventing its transcription. IL-4 stimulation causes rapid phosphorylation of STAT6, but its binding to the promoter appears to be prevented by nonacetylated histones. After 9 -11 h, when histones become acetylated, STAT6 binding sites may be demasked so that the phosphorylated and acetylated transcription factor can bind to activate gene transcription.

The peritoneal macrophage (Mφ) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11bhigh cells, with the remaining 5% being 12/15-LOX−. 12/15-LOX+ cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX− cells are a dendritic cell population. Resident peritoneal Mφ numbers were significantly increased in 12/15-LOX−/− mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mφ from 12/15-LOX−/− mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX−/− mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX−/− adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX+ cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX−/− mice showed elevated TGF-β1, along with increased immigration of monocytes/Mφ, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-α. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MΦ population and regulates both the recruitment of monocytes/Mφ and cytokine response to bacterial products in vivo.

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