Diesel particulate matter induces receptor for advanced glycation end-products (RAGE) expression in pulmonary epithelial cells, and RAGE signaling influences NF-κB-mediated inflammation

Abstract

Background: Receptors for advanced glycation end-products (RAGE) are cell-surface receptors expressed by alveolar type I (ATI) epithelial cells and are implicated in mechanisms of alveolar development and sustained pulmonary inflammation.

Objectives: In the present study, we tested the hypothesis that diesel particulate matter (DPM) up-regulates RAGE in rat ATI-like R3/1 cells and human primary small airway epithelial cells (SAECs), leading to an inflammatory response.

Methods and results: Using real-time reverse transcriptase polymerase chain reaction and immunoblotting, we found that RAGE mRNA and protein are up-regulated in cells exposed to DPM for 2 hr. Use of a luciferase reporter containing nuclear factor-κB (NF-κB) response elements revealed decreased NF-κB activation in cells transfected with small interfering RNA (siRNA) for RAGE (siRAGE) before DPM exposure compared with cells transfected with scrambled control siRNA (siControl). In addition, immunostaining revealed diminished nuclear translocation of NF-κB in DPM-exposed cells transfected with siRAGE compared with cells transfected with siControl before DPM stimulation. Enzyme-linked immunosorbent assay demonstrated that in R3/1 cells DPM induced secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), two cytokines induced by NF-κB and associated with leukocyte chemotaxis during an inflammatory response. Incorporating siRAGE was sufficient to significantly decrease DPM-induced MCP-1 and IL-8 secretion compared with cells transfected with siControl.

Conclusions: These data offer novel insights into potential mechanisms whereby RAGE influences pulmonary inflammation exacerbated by DPM exposure. Further research may demonstrate that molecules involved in RAGE signaling are potential targets in lessening the degree of particulate matter-induced exacerbations of inflammatory lung disease.

Figure 1

RAGE mRNA was induced by DPM in R3/1 cells and SAECs. Quantitative real-time RT-PCR revealed a significant increase in RAGE mRNA expression in R3/1 cells and SAECs exposed to 3 μg/mL DPM for 2 hr compared with unstimulated control cells. Experiments were performed in triplicate. Abbreviations: +, with; −, without. *p ≤ 0.05.

Figure 2

RAGE protein was induced by DPM in R3/1 cells and SAECs. Immunoblotting for RAGE revealed a marked increase in RAGE protein synthesis in cells transfected with siControl 24 hr before a 2 hr exposure to 3 μg/mL DPM. Densitometry of bands revealed clear reductions in DPM-induced RAGE expression when siRAGE was transfected 24 hr before DPM exposure. Abbreviations: +, with; −, without; kDa, kilodaltons.

Figure 3

RAGE inhibition by siRNA blocked activation of NF-κB by DPM in R3/1 cells. R3/1 cells were transfected with an NF-κB–Luc vector and siRAGE or siControl 24 hr before DPM exposure or fresh medium replacement. Exposure to DPM for 2 hr significantly induced nuclear NF-κB activity in siControl-transfected R3/1 cells. When siRAGE was incorporated, DPM-induced NF-κB activity was markedly decreased to below unstimulated basal levels. Abbreviations: +, with; −, without. *p ≤ 0.05.

Figure 4

DPM-induced secretion of MCP-1 and IL-8 was mediated by RAGE. (A) ELISAs demonstrate that R3/1 cells exposed to DPM for 2 hr significantly increased MCP-1 secretion. Compared with control cells exposed to DPM, siRAGE incorporation significantly decreased DPM-induced MCP-1 secretion. (B) IL-8 was significantly up-regulated by R3/1 cells after DPM exposure, and secretion of IL-8 was significantly diminished in cells previously transfected with siRAGE. Abbreviations: +, with; −, without. *p ≤ 0.05.