Receptors for advanced glycation end-products (RAGE) are members of the immunoglobulin superfamily of cell-surface receptors implicated in mechanisms of pulmonary inflammation. In the current study, we test the hypothesis that RAGE mediates inflammation in primary alveolar macrophages (AMs) exposed to diesel particulate matter (DPM). Quantitative RT-PCR and immunoblotting revealed that RAGE was up-regulated in Raw264.7 cells, an immortalized murine macrophage cell line and primary AMs exposed to DPM for 2 h. Because DPM increased RAGE expression, we exposed Raw264.7 cells and primary AMs isolated from RAGE null and wild-type (WT) mice to DPM prior to the assessment of inflammatory signaling intermediates. DPM led to the activation of Rat sarcoma GTPase (Ras), p38 MAPK and NF-κB in WT AMs and, when compared to WT AMs, these intermediates were diminished in DPM-exposed AMs isolated from RAGE null mice. Furthermore, cytokines implicated in inflammation, including IL-4, IL-12, IL-13 and TNFα, were all significantly decreased in DPM-exposed RAGE null AMs compared to similarly exposed WT AMs. These results demonstrate that diesel-induced inflammatory responses by primary AMs are mediated, at least in part, via RAGE signaling mechanisms. Further work may show that RAGE signaling in both alveolar epithelial cells and resident macrophages is a potential target in the treatment of inflammatory lung diseases exacerbated by environmental pollution.

BackgroundReceptors for advanced glycation end-products (RAGE) are immunoglobulin-like pattern recognition receptors abundantly localized to lung epithelium. Our research demonstrated that primary tobacco smoke exposure increases RAGE expression and that RAGE partly mediates pro-inflammatory signaling during exposure. However, the degree to which RAGE influences developing lungs when gestating mice are exposed to secondhand smoke (SHS) has not been determined to date.MethodsTimed pregnant RAGE null and wild type control mice were exposed to 4 consecutive days of SHS from embryonic day (E) 14.5 through E18.5 using a state of the art nose-only smoke exposure system (Scireq, Montreal, Canada). RAGE expression was assessed using immunofluorescence, immunoblotting, and quantitative RT-PCR. TUNEL immunostaining and blotting for caspase-3 were performed to evaluate effects on cell turnover. Matrix abnormalities were discerned by quantifying collagen IV and MMP-9, a matrix metalloprotease capable of degrading basement membranes. Lastly, TNF-α and IL-1β levels were assessed in order to determine inflammatory status in the developing lung.ResultsPulmonary RAGE expression was elevated in both dams exposed to SHS and in fetuses gestating within mothers exposed to SHS. Fetal weight, a measure of organismal health, was decreased in SHS-exposed pups, but unchanged in SHS-exposed RAGE null mice. TUNEL assessments suggested a shift toward pulmonary cell apoptosis and matrix in SHS-exposed pups was diminished as revealed by decreased collagen IV and increased MMP-9 expression. Furthermore, SHS-exposed RAGE null mice expressed less TNF-α and IL-1β when compared to SHS-exposed controls.ConclusionsRAGE augmentation in developing pups exposed to maternal SHS weakens matrix deposition and influences lung inflammation.

Receptors for advanced glycation end‐products (RAGE) are members of the immunoglobulin superfamily of cell‐surface receptors implicated in pulmonary inflammation. In the present study, we tested the hypothesis that RAGE mediates inflammation in primary alveolar macrophages (AMs) triggered by diesel particulate matter (DPM). Using real time RT‐PCR, we discovered that RAGE was up‐regulated in primary AMs exposed to DPM for four hours. Because DPM increased RAGE expression, we exposed primary AMs isolated from RAGE null and wild type Balb/C mice to DPM. DPM led to the activation p38 and NF‐kB and that these two signaling intermediates were diminished in similarly exposed AMs isolated from RAGE null mice. Furthermore, TNF‐a, IL‐12, and IL‐13 were all significantly decreased in culture media from DPM‐exposed RAGE null AMs compared to similarly exposed WT AMs. These results demonstrate that primary AMs orchestrate diesel‐induced inflammation, at least in part, via RAGE‐mediated mechanisms. Further work may show that RAGE signaling is a potential target in treatment of inflammatory lung diseases exacerbated by environmental pollution. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).

The receptor for advanced glycation end‐products (RAGE) has increasingly been demonstrated to be an important modulator of inflammation in cases of pulmonary disease. Published reports involving tobacco smoke exposure have demonstrated increased expression of RAGE, its participation in pro‐inflammatory signaling and its role in irreversible pulmonary remodeling. The current research evaluated the in vivo effects of short‐term tobacco smoke exposure in RAGE null and control mice compared to identical animals groups exposed to room air only. Q‐PCR and immunoblotting revealed elevated RAGE expression in controls after four weeks of exposure and an anticipated absence of RAGE expression in RAGE null mice regardless of smoke exposure. Inflammatory cell behaviors were assessed by determining the activation of ras, intracellular signaling kinases, and cytokine synthesis and secretion. Furthermore, bronchoalveolar lavage fluid (BALF) was procured from RAGE null and control animals prior to assessment for inflammatory cells and molecules. As a general theme, inflammation induced by tobacco smoke exposure was influenced by the availability of RAGE. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to tobacco smoke and further research is necessary in order to fully explain roles for receptors such as RAGE in cells coping with exposure. Grant Funding Source: Supported by the Flight Attendant’s Medical Research Institute (FAMRI) and a BYU MEG Award (PRR).