Allergy Asthma Immunol Res.  2014 Jan;6(1):66-74. 10.4168/aair.2014.6.1.66.

Stimulated Bronchial Epithelial Cells Release Bioactive Lysophosphatidylcholine 16:0, 18:0, and 18:1

• Zhuge Y
• Yuan Y
• van Breemen R
• DeGrand M
• Holian O
• Yoder M
• Lum H

• KMID: 2166940
• DOI: http://doi.org/10.4168/aair.2014.6.1.66

Abstract

PURPOSE
In human subjects and animal models with acute and chronic lung injury, the bioactive lysophosphatidylcholine (LPC) is elevated in lung lining fluids. The increased LPC can promote an inflammatory microenvironment resulting in lung injury. Furthermore, pathological lung conditions are associated with upregulated phospholipase A2 (PLA2), the predominant enzyme producing LPC in tissues by hydrolysis of phosphatidylcholine. However, the lung cell populations responsible for increases of LPC have yet to be systematically characterized. The goal was to investigate the LPC generation by bronchial epithelial cells in response to pathological mediators and determine the major LPC species produced.
METHODS
Primary human bronchial epithelial cells (NHBE) were challenged by vascular endothelial growth factor (VEGF) for 1 or 6 h, and condition medium and cells collected for quantification of predominant LPC species by high performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). The cells were analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for PLA2. The direct effects of LPC in inducing inflammatory activities on NHBE were assessed by transepithelial resistance as well as expression of interleukin-8 (IL-8) and matrix metalloproteinase-1 (MMP-1).
RESULTS
VEGF stimulation of NHBE for 1 or 6 h, significantly increased concentrations of LPC16:0, LPC18:0, and LPC18:1 in condition medium compared to control. The sPLA2-selective inhibitor (oleyloxyethyl phosphorylcholine) inhibited the VEGF-induced release of LPC16:0 and LPC18:1 and PLA2 activity. In contrast, NHBE stimulated with TNF did not induce LPC release. VEGF did not increase mRNA of PLA2 subtypes sPLA2-X, sPLA2-XIIa, cPLA2-IVa, and iPLA2-VI. Exogenous LPC treatment increased expression of IL-8 and MMP-1, and reduced the transepithelial resistance in NHBE.
CONCLUSIONS
Our findings indicate that VEGF-stimulated bronchial epithelial cells are a key source of extracellular LPCs, which can function as an autocrine mediator with potential to induce airway epithelial inflammatory injury.

Keyword

Bronchial epithelium; phospholipase A2; lysophosphatidylcholine

MeSH Terms

Epithelial Cells*
Group X Phospholipases A2
Humans
Hydrolysis
Interleukin-8
Lung
Lung Injury
Lysophosphatidylcholines*
Mass Spectrometry
Matrix Metalloproteinase 1
Models, Animal
Phosphatidylcholines
Phospholipases A2
Reverse Transcriptase Polymerase Chain Reaction
RNA, Messenger
Vascular Endothelial Growth Factor A
Group X Phospholipases A2
Interleukin-8
Lysophosphatidylcholines
Matrix Metalloproteinase 1
Phosphatidylcholines
Phospholipases A2
RNA, Messenger
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Representative positive ion electrospray LC-MS-MS SRM chromatograms showing the detection of LPC species from condition medium collected from NHBE stimulated with (A) 10 nM VEGF for 1 h or (B) 10 nM VEGF for 6 h. SRM transitions: LPC 16:0 m/z 496→184 (retention time 5.6 min); LPC 18:0 m/z 524→184 (retention time 6.6 min); LPC 18:1 m/z 522→184 (retention time 5.8 min); LPC 19:0 m/z 538→184 (retention times 7.0 min). The solid line represents cells treated with VEGF, and the dashed line represents the control without VEGF. VEGF, vascular endothelial growth factor.

• 

  Fig. 2 Summary graph showing the effects of VEGF stimulation on generation of LPC16:0, LPC18:0, and LPC18:1. NHBE were treated with 10 nM VEGF for 1 (VEGF-1) or 6 h (VEGF-6) or with control buffer (C-1 and C-6, respectively); (A) the condition medium and (B) cells were collected for analysis by LC-MS-MS (Materials and Methods). *P<0.05 and **P<0.001 compared to control; †P<0.01 and ‡P<0.001, compared to 1 h VEGF; n=3-4.

• 

  Fig. 3 Increased PLA2 activity in condition medium by VEGF. The PLA2 activity in the condition medium from VEGF-stimulated or untreated (control) NHBE was determined using a phosphatidylcholine substrate analog (Red/Green BODIPY PC-A2) (Materials and Methods). Activity was measured at 10 min intervals for 120 min in the presence or absence of a selective inhibitor of sPLA2, oleyloxyethyl phosphorylcholine (oleyloxyethyl phosphorylcholine; 20 µM); n=2.

• 

  Fig. 4 PLA2 expression by NHBE. (A) Shown is a representative gel of regular RT-PCR analysis of constitutive expression of major PLA2 subtypes in NHBE and GAPDH used as an internal control. Major sPLA2 subtypes are: sPLA2-IIa, sPLA2-V, sPLA2-X and sPLA2-XIIa; other subtypes are: cPLA2-IVa and iPLA2-VI; n=3. (B) qRT-PCR analysis of PLA2 mRNA in response to VEGF treatment. NHBE were treated with VEGF (10 nM for 1 or 6 h), and collected for quantification of sPLA2-X, sPLA2-XIIa, cPLA2-IVa, and iPLA2-VI. Results reported as relative copy number normalized to GAPDH and shown as average±SE; n=2.

• 

  Fig. 5 Effects of sPLA2 inhibitor on LPC generation. The effect of the sPLA2-selective inhibitor, oleyloxyethyl phosphorylcholine (Op), on the VEGF-induced release of LPC16:0, LPC18:0 and LPC18:1 into the condition medium was determined. OP (20 µM) was added to the medium as a pretreatment of NHBE for 30 min, followed by treatment with VEGF (10 nM for 1 h) or buffer control, and the condition medium was collected for LPC determination by LC-MS-MS as described. *P<0.05, compared to control; n=3-7.

• 

  Fig. 6 Upregulation of IL-8 by exogenous LPC. (A) NHBE were treated with LPC16:0 (25 µM) for 1 or 5 h, and cells collected for qRT-PCR analysis of IL-8 mRNA; the graph shows relative copy number of IL-8 mRNA normalized to GAPDH as average±SE. (B) NHBE were treated with LPC16:0 (25 µM) for 4 h, and the condition medium collected for secreted IL-8 protein; results reported as average relative absorbance (450 nm); LPS treatment (10 µg/mL) for 4 h was used as positive control. N=4. *P<0.05 compared to control.

• 

  Fig. 7 Upregulation of MMP-1 by exogenous LPC. NHBE cells were treated with LPC16:0 (25 µM) for 1 or 5 h, and cells collected for qRT-PCR analysis for MMP-1 mRNA; graph shows the relative copy number of MMP-1 normalized to GAPDH as average±SE. *P<0.05 compared to control; n=4-8.

• 

  Fig. 8 Decreased transepithelial electrical resistance by LPC. Confluent NHBE were grown on ECIS electrodes, and real-time resistance change in response to LPC16:0 stimulation was measured for up to 5 h; shown is representative resistance change relative to baseline (from 6 separate determinations); arrow indicates time of LPC addition.

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