Receptors for advanced glycation end-products (RAGE) are multiligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages, and expression increases following exposure to cigarette smoke extract (CSE). The present study sought to characterize the proinflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE knockout (KO) mice compared with controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and analyzed. CSE significantly increased RAGE expression by wild-type AMs. Employing ELISAs, wild-type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates proinflammatory signaling. Conversely, RAGE KO AMs had less Ras activation compared with wild-type AMs after exposure to CSE. In RAGE KO AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed that CSE-induced inflammation may occur in part via RAGE signaling. Lastly, quantitative RT-PCR revealed that the expression of proinflammatory cytokines including TNF-α and IL-1β were detectably decreased in RAGE KO AMs exposed to CSE compared with CSE-exposed wild-type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.

Our research has implicated RAGE, a pattern recognition receptor, in inflammation induced by pulmonary epithelial cells exposed to cigarette smoke (CS). However, precise pro‐inflammatory contributions by alveolar macrophages (AMs) in the respiratory compartment remained unknown. Primary AMs were isolated from bronchoalveolar lavage fluid (BALF) and immunohistochemistry was employed using Mac‐3, a macrophage‐specific marker, to ensure the purity of the cell population. Employing quantitative PCR, we discovered that RAGE mRNA was significantly increased by primary AMs from wild type C57Bl/6 mice following CS exposure. Because CS exposure increased RAGE, we exposed freshly isolated primary AMs from RAGE null and wild type mice to CS and assessed pro‐inflammatory molecule secretion. Using a PCR based cDNA amplification approach, we discovered that Eotaxin, IL‐12, and IL‐ 3 were all up‐regulated in wild type primary AMs exposed to CS compared to CS‐exposed AMs from RAGE null mice. Furthermore, ELISAs revealed that RAGE might also regulate the secretion of these pro‐inflammatory cytokines by primary AMs that encounter CS. The data reveal that acute secretion of pro‐inflammatory cytokines by primary AMs exposed to CS occurs, at least in part, via RAGE signaling. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).

Receptors for advanced glycation end‐products (RAGE) are multi‐ligand cell surface receptors expressed by lung cells and expression increases following exposure to cigarette smoke (CS). The current study sought to characterize the pro‐inflammatory effects of RAGE in pulmonary macrophages (PMs) following CS exposure. Acute exposure to CS via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer macrophages in RAGE null mice compared to controls. Primary PMs were obtained from BAL, exposed to CS in vitro, and analyzed. CS significantly increased RAGE expression in wild type PMs. By ELISA, wild type PMs increased active Ras, a small GTPase that perpetuates pro‐inflammatory signaling, following CS exposure. Conversely, RAGE null PMs had less Ras activation compared to wild type PMs after exposure to CS. Assessment of signaling intermediates including p38 and NF‐kB revealed CS‐induced inflammation may occur in part via RAGE signaling. Lastly, qRT‐PCR and ELISA revealed that secretion of cytokines such as TNF‐α and IL‐1β were decreased in RAGE null PMs exposed to CS compared to CS‐exposed wild type PMs. These results show for the first time that primary PMs orchestrate CS‐induced inflammation, at least in part, via RAGE‐mediated mechanisms. This work was supported by the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).