Interleukin-13-induced MUC5AC is regulated by 15-lipoxygenase 1 pathway in human bronchial epithelial cells

Abstract

Rationale: 15-Lipoxygenase-1 (15LO1) and MUC5AC are highly expressed in asthmatic epithelial cells. IL-13 is known to induce 15LO1 and MUC5AC in human airway epithelial cells in vitro. Whether 15LO1 and/or its product 15-HETE modulate MUC5AC expression is unknown.

Objectives: To determine the expression of 15LO1 in freshly harvested epithelial cells from subjects with asthma and normal control subjects and to determine whether IL-13-induced 15LO1 expression and activation regulate MUC5AC expression in human bronchial epithelial cells in vitro.

Methods: Human airway epithelial cells from subjects with asthma and normal subjects were evaluated ex vivo for 15LO1 and MUC5AC expression. The impact of 15LO1 on MUC5AC expression in vitro was analyzed by inhibiting 15LO1 through pharmacologic (PD146176) and siRNA approaches in human bronchial epithelial cells cultured under air-liquid interface. We analyzed 15 hydroxyeicosatetraenoic acid (15-HETE) by liquid chromatography/UV/mass spectrometry. MUC5AC and 15LO1 were analyzed by real-time RT-PCR, immunofluoresence, and Western blot.

Measurements and main results: Epithelial 15LO1 expression increased with asthma severity (P < 0.0001). 15LO1 significantly correlated with MUC5AC ex vivo and in vitro. IL-13 increased 15LO1 expression and stimulated formation of two molecular species of 15-HETE esterified to phosphotidylethanolamine (15-HETE-PE). Inhibition of 15LO1 suppressed 15-HETE-PE and decreased MUC5AC expression in the presence of IL-13 stimulation. The addition of exogenous 15-HETE partially restored MUC5AC expression.

Conclusions: Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma.

Figure 1.

15LO1 expression in fresh bronchial epithelial cells increases with asthma severity ex vivo. (A) Representative Western blots of 15LO1 protein expression in fresh bronchial epithelial cells from normal subjects (n = 2) and subjects with mild (n = 3) and severe (n = 3) asthma. (B) Quantitative analysis of 15LO1 protein expression in fresh bronchial epithelial cells using densitometry, with β-actin as the loading control. All values are means ± SE.

Figure 2.

Representative immunofluorescence staining of 15LO1 and MUC5AC protein in tissues demonstrating the increases and colocalization of 15LO1 with MUC5AC in a subject with severe asthma compared with normal control subject. The results of staining were viewed using confocal microscopy. 15LO1 staining is green, MUC5AC is red, and nucleus staining is blue (Draq5 stain). Colocalization of 15LO1 and MUC5AC is displayed as yellow.

Figure 3.

IL-13 induces 15LO1 expression in human bronchial epithelial cells under air–liquid interface (ALI) culture. Primary human bronchial epithelial cells were cultured with or without IL-13 (10 ng/ml) stimulation for 10 days. (A) Representative Western blots of 15LO1 protein expression induced by IL-13 (10 ng/ml for 10 d) in vitro. (B) Densitometric analysis of 15LO1 protein expression induced by IL-13, with GAPDH as the loading control. Solid line: normal control subjects. dashed line: patients with asthma. (C) Quantitative real-time RT-PCR analysis of 15LO1 mRNA induced by IL-13 in vitro, with GAPDH as the internal control. Solid line: normal control subjects. Dashed line: patients with asthma. (D) Western blot showing no change in 15LO2 expression with IL-13 stimulation in vitro.

Figure 4.

Precursor scanning identifies esterified 15-HETEs generated by activated epithelial cells. Lipid extracts from ionophore-activated cells were separated using liquid chromatography and scanned using precursor scanning as described in Methods. (A) Ions eluting at 23 minutes. *Characteristic HETE daughter fragments on MS/MS. (B) Spectrum of ions eluting in region marked by an asterisk in A, showing prominent ions at m/z 738.7 and 764.8. (C) Scheme structures of 15-HETE-PE lipids generated by human airway epithelial cells.

Figure 5.

IL-13 induces intracellular 15-HETE-PE conjugates in vitro, with no free 15-HETE detectable. Primary human bronchial epithelial cells from two patients with severe asthma (SA) and two normal control subjects (NC) were cultured with or without IL-13 (10 ng/ml) stimulation for 10 days. A23187 (10 μM) was added to medium 30 minutes before harvest, and 1 μM unconjugated 15(S)-HETE was added into culture 24 hours before harvest. Cell lysate and culture medium were harvested in Krebs buffer for LC/ESI/MS/MS analysis. All values were normalized to the corresponding basal level of each subject and expressed as the means of fold change ± SE. Two specific esterified 15-HETE-PE (738/219 and 764/219 ions) were increased in response to IL-13. (A) IL-13–induced 15-HETE-PE conjugations in vitro, with modest additional increase in 15-HETE-PE after ionophore stimulation. (B) Exogenous 15(S)-HETE (1 μM, unconjugated) increased the levels of 15-HETE-PE in the presence of IL-13 stimulation.

Figure 6.

IL-13 induces MUC5AC expression in human bronchial epithelial cells in vitro. Primary human bronchial epithelial cells from 27 subjects (10 severe asthma, 6 mild-moderate asthma, and 11 normal control) were cultured with or without IL-13 (10 ng/ml) stimulation for 10 days. Relative mRNA expression levels were calculated using the delta Ct method.

Figure 7.

Exogenous 15-HETE enhances MUC5AC expression induced by IL-13 in vitro. Primary human bronchial epithelial cells (1 severe asthma, 1 mild-moderate asthma, and 2 normal control) were cultured with or without IL-13 (10 ng/ml) stimulation for 10 days. Additional 15-HETE (1 μM, unconjugated) was added to the medium for 24 or 72 hours before harvest. Ethanol (1%) was added to the medium as the vehicle control.

Figure 8.

Pharmacologic inhibition of 15LO1 decreases IL-13–induced 15-HETE-PE conjugation and MUC5AC mRNA expression in vitro. Primary human bronchial epithelial cells were stimulated with IL-13 (10 ng/ml) for 10 days with or without the 15LO1 inhibitor (PD146176, 1 μM). (A) The pharmacologic 15 LO1 inhibitor (PD146176, 1 μM) decreased the intracellular levels of 15-HETE-PE induced by IL-13 stimulation (n = 3, 2 severe asthma, and 1 normal control). (B) PD146176 also suppressed IL-13–induced MUC5AC mRNA expression (n = 8, 2 severe asthma, 2 mild-moderate asthma, and 4 normal control).

Figure 9.

15LO1 siRNA transfection suppressed IL-13–induced MUC5AC mRNA expression in vitro, and exogenous 15-HETE neutralized this suppression. Primary human bronchial epithelial cells were transiently transfected with 15LO1 siRNA (ALOX15 siRNA) and stimulated with IL-13 (10 ng/ml) for 2 days under acute lung injury. Unconjugated exogenous 15-HETE (1 μM) was added to medium 24 hours before harvest. All values expressed as fold changes induced by IL-13 stimulation. ALOX15 siRNA transfection significantly suppressed IL-13–induced 15LO1 (A) mRNA and (B) protein expression in vitro (n = 5, 3 severe asthma, and 2 normal control). (C) ALOX15 siRNA transfection suppressed IL-13–induced MUC5AC mRNA expression and exogenous 15-HETE neutralized this suppression (n = 9, 5 severe asthma, 1 mild-moderate asthma, and 3 normal control).