Internalization of adenovirus by alveolar macrophages initiates early proinflammatory signaling during acute respiratory tract infection

Adenovirus is a common respiratory pathogen which causes a broad range of d istinct clinical syndromes and has recently received attention for its pote ntial for in vivo gene delivery. Although adenovirus respiratory tract infe ction (ARTI) results in dose-dependent, local inflammation, the pathogenesi s of this remains unclear. We hypothesized that alveolar macrophages (AM ph i) rapidly internalize adenovirus following in vivo pulmonary administratio n and then initiate inflammatory signaling within the lung. To evaluate the role of AM phi in the induction of lung inflammation during ARTI in vivo, we directly assessed adenovirus uptake by murine AM phi and correlated upta ke with the initiation of proinflammatory gene expression. Stimulation of c ytokine (tumor necrosis factor alpha [TNF-alpha], interleukin-6 [IL-6], mac rophage inflammatory protein-2 [MIP-2], and MIP-1 alpha) expression in the lung was evaluated at the level of mRNA (by reverse transcription-PCR [RT-P CR]) and protein (by enzyme-linked immunosorbent assay) and by identificati on of cells expressing TNF-alpha and IL-6 mRNA in lung tissues (by in situ hybridization) and isolated lung lavage cells (by RT-PCR). Adenovirus, labe led with the fluorescent dye (Cy3), was rapidly and widely distributed on e pithelial surfaces of airways and alveoli and was very rapidly (similar to 1 min) localized within AM phi. At 30 min after infection AM phi but not ai rway epithelial or vascular endothelial cells expressed mRNA for TNF-alpha and IL-6, thus identifying AM phi as the cell source of initial cytokine si gnaling. IL-6, TNF-alpha, MIP-2, and MIP-1 alpha levels progressively incre ased in bronchoalveolar lavage fluid after pulmonary adenovirus infection, and all were significantly elevated at 6 h (P < 0.05), To begin to define t he molecular mechanism(s) by which adenovirus initiates the inflammatory si gnaling in macrophages, TNF-alpha expression from adenovirus-infected RAW26 4.7 macrophages was evaluated in vitro. TNF-alpha expression was readily de tected in adenovirus-infected RAW cell supernatant with kinetics similar to AM phi during in vivo infection. Blockage of virus uptake at specific cell ular sites, including internalization (by wortmannin), endosome acidificati on and/or lysis (by chloroquine) or by Ca2+ chelation (by BAPTA) completely blocked TNF-alpha expression, In conclusion, results showed that during AR TI, (i) AM phi rapidly internalized adenovirus, (ii) expression of inflamma tory mediators was initiated within AM phi and not airway epithelial or oth er cells, and (iii) the initiation of inflammatory signaling was linked to virion uptake by macrophages occurring at a point after vesicle acidificati on. These results have implications for our understanding of the role of th e AM phi in the initiation of inflammation following adenovirus infection a nd adenovirus-mediated gene transfer to the lung.