Leukotriene D(4) induces gene expression in human monocytes through cysteinyl leukotriene type I receptor

Abstract

Background: Cysteinyl leukotrienes (CysLTs) are important mediators of innate immune responsiveness and chronic inflammatory diseases. CysLTs acting through CysLT receptors can influence the migration and activity of cells, such as eosinophils, monocytes, and dendritic cells.

Objective: We sought to determine the gene expression signature of human monocytes in response to CysLTs and to elucidate the signaling pathways involved in monocyte activation.

Methods: Gene expression was analyzed by using oligonucleotide microarrays. Responsiveness to CysLTs was assessed by using real-time PCR, calcium flux, kinase activation, and chemotaxis assays.

Results: CysLT type 1 receptor (CysLTR(1)) transcript 1 is predominantly expressed in human monocytes, and CysLTs signal through CysLTR(1) in these cells. Several immediate-early genes, including early growth response 2 and 3, FBJ murine osteosarcoma viral oncogene homolog B, activating transcription factor 3, and nuclear receptor subfamily 4 were significantly induced by leukotriene (LT) D(4). This effect was mediated by CysLTR(1) coupled to the G protein alpha inhibitory subunit, activation of phospholipase C, and inositol-1,4,5-triphosphate and store-operated calcium channels. LTD(4) induced p38 mitogen-activated protein kinase phosphorylation, a pathway also involved in the regulation of immediate-early gene expression in monocytes. LTD(4) stimulated monocyte chemotactic activity that was fully blocked by a selective CysLTR(1) inhibitor, MK571, and pertussis toxin, suggesting that CysLTR(1) coupled to the G protein alpha inhibitory subunit is a dominant functional pathway in human monocytes.

Conclusion: Our data show that CysLTs acting through CysLTR(1) can significantly influence the activation and migration of human monocytes and that these effects can be fully inhibited by CysLTR(1) antagonists.

FIG 1

LTD₄ induces EGR2 and FOSB genes in a time-dependent fashion. Monocytes were stimulated with LTD₄ (100 nmol/L) for up to 4 hours, and mRNA expression was measured by means of TaqMan analysis. Data are presented as a fold change in comparison with vehicle (ethanol)–treated cells. Mean ± SD; n = 3.

FIG 2

Calcium mobilization responses to CysLTs. Monocytes (A) and CysLTR₁-transfected HEK293 cells (B) were prepared and calcium release was measured as indicated in the Methods section. Data are presented as means from 2 separate experiments performed in triplicate. Empty pcDNA-transfected HEK293 cells treated with different concentrations of LTD₄ were used as controls.

FIG 3

Characterization of calcium mobilization responses to LTD₄ in monocytes. Elutriated monocytes were prepared and calcium release was measured as indicated in the Methods section. Cells were preincubated with inhibitors or vehicle control as indicated in the Results section and stimulated with LTD₄ (100 nmol/L). Data from one of 3 experiments from 3 donors, each with similar results, are shown.

FIG 4

The effect of LTD₄ on MAPK activation. Monocytes were stimulated with LTD₄ (100 nmol/L) for different times, and phosphorylation of p44/42 and p38 was measured (A). Cells were preincubated with MK571 (100 nmol/L) for 10 minutes and stimulated with LTD₄ (100 nmol/L) for 10 minutes (B). Data are representative of 3 independent experiments.

FIG 5

Characterization of the pathway leading to LTD₄-induced EGR2 and FOSB mRNA upregulation. Monocytes were preincubated with MK571 (MK; 100 nmol/L for 10 minutes), pertussis toxin (PTX; 100 ng/mL overnight), 2APB (50 μmol/L for 10 minutes), SB203580 (SB35; 5 μmol/L for 30 minutes), PD98059 (PD; 10 μmol/L for 30 minutes), BAPTA/AM (30 μmol/L for 30 minutes) (A and B) or with different concentrations of SB202190 (in micromoles per liter; 30 minutes) and U0126 (in micromoles per liter; 30 minutes) stimulated with LTD₄ (100 nmol/L) for 30 minutes (C and D) and subjected to TaqMan analysis. Data are shown as a fold change in comparison with control vehicle-treated cells or expressed as the percentage of fold change induced by LTD₄. Mean ± SD; n = 6 to 9. All inhibitors significantly inhibited LTD₄-induced gene expression (P < .001 in comparison with LTD₄ only–treated cells by using the Student t test).

FIG 6

Chemotactic responses of monocytes to LTD₄. A, Chemotactic activity of monocytes to different concentrations of LTD₄ or CCL2 (5 nmol/L) as a positive control (Con) was measured as described in the Methods section. LTD₄ induces chemotaxis of monocytes in a dose-dependent manner (*P < .001, ANOVA). B, Cells were preincubated with MK571 (MK; 100 nmol/L for 10 minutes), pertussis toxin (PTX; 100 ng/mL overnight), SB203580 (SB35; 5 μmol/L for 30 minutes), SB202190 (SB21; 1 μmol/L for 30 minutes), PD98059 (PD; 10 μmol/L for 30 minutes), and U0126 (10 μmol/L for 30 minutes) and stimulated with LTD₄ (100 nmol/L). Data from 3 different donors are presented as the migration index in comparison with vehicle control or percentage of migration index induced by LTD₄ (mean ± SD).