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Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies
Published on: March 2, 2018
Role of NF-kappaB and PPAR-gamma in lung inflammation induced by monocyte-derived microparticles
1Dipartimento Cardiotoracico e Vascolare, Laboratory of Respiratory Cell Biology, University of Pisa, Via Paradisa, 2 56124 Pisa, Italy.
Abstract:
Microparticles (MP) are phospholipid vesicles shed by cells upon activation or apoptosis. Monocyte-derived MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells; the molecular bases of such activity are unknown. Peroxisome proliferator-activated receptors (PPAR) have been demonstrated to be involved in the modulation of nuclear factor (NF)-κB transcriptional activity and inflammation. We investigated whether the upregulation of the synthesis of proinflammatory cytokines by human lung epithelial cells induced by monocyte/macrophage-derived MP involves NF-κB activation and is modulated by PPAR-γ. MP were generated by stimulation of human monocytes/macrophages with the calcium ionophore, A23187. MP were incubated with human lung epithelial cells. NF-κB translocation was assessed by electrophoretic mobility shift assay. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 synthesis was assessed by ELISA and RT-PCR. Stimulation of A549 alveolar cells with monocyte/macrophage-derived MP caused an increase in NF-κB activation and IL-8 and MCP-1 synthesis that was inhibited by pre-incubation with the PPAR-γ agonists, rosiglitazone and 15-deoxy-Δ12,14-prostaglandin-J2. Parallel experiments with normal human bronchial epithelial cells largely confirmed the results. The effects of PPAR-γ agonists were reversed by the specific antagonist, GW9662. Upregulation of the synthesis of proinflammatory mediators by human lung epithelial cells induced by monocyte/macrophage-derived MP is mediated by NF-κB activation through a PPAR-γ dependent pathway.
Insights
Monocyte-derived microparticles (MP) induce lung inflammation via NF-κB activation. Peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists inhibit this inflammatory response, revealing a PPAR-γ dependent pathway.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Microparticles (MP) are vesicles shed by activated or apoptotic cells.
- Monocyte-derived MP are known to increase pro-inflammatory mediators in lung epithelial cells.
- The underlying molecular mechanisms for this effect are not fully understood.
Purpose of the Study:
- To investigate if NF-κB activation mediates the upregulation of pro-inflammatory cytokines by monocyte-derived MP in lung epithelial cells.
- To determine if Peroxisome proliferator-activated receptors gamma (PPAR-γ) modulate this inflammatory response.
Main Methods:
- Human monocytes/macrophages were stimulated to generate MP.
- MP were incubated with human lung epithelial cells (A549 alveolar and bronchial cells).
- NF-κB translocation, Interleukin-8 (IL-8), and Monocyte Chemotactic Protein-1 (MCP-1) synthesis were assessed using electrophoretic mobility shift assay, ELISA, and RT-PCR.
Main Results:
- Monocyte/macrophage-derived MP increased NF-κB activation and IL-8/MCP-1 synthesis in lung epithelial cells.
- Pre-incubation with PPAR-γ agonists (rosiglitazone, 15-deoxy-Δ12,14-prostaglandin-J2) inhibited MP-induced inflammation.
- The inhibitory effects of PPAR-γ agonists were reversed by the antagonist GW9662.
Conclusions:
- Upregulation of pro-inflammatory mediators by monocyte/macrophage-derived MP in lung epithelial cells is mediated by NF-κB activation.
- This process is dependent on a Peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway.
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