Internalization of Adenovirus by Alveolar Macrophages Initiates Early Proinflammatory Signaling during Acute Respiratory Tract Infection

Abstract

ABSTRACT Adenovirus is a common respiratory pathogen which causes a broad range of distinct clinical syndromes and has recently received attention for its potential for in vivo gene delivery. Although adenovirus respiratory tract infection (ARTI) results in dose-dependent, local inflammation, the pathogenesis of this remains unclear. We hypothesized that alveolar macrophages (AMφ) rapidly internalize adenovirus following in vivo pulmonary administration and then initiate inflammatory signaling within the lung. To evaluate the role of AMφ in the induction of lung inflammation during ARTI in vivo, we directly assessed adenovirus uptake by murine AMφ and correlated uptake with the initiation of proinflammatory gene expression. Stimulation of cytokine (tumor necrosis factor alpha [TNF- α ], interleukin-6 [IL-6], macrophage inflammatory protein-2 [MIP-2], and MIP-1α) expression in the lung was evaluated at the level of mRNA (by reverse transcription-PCR [RT-PCR]) and protein (by enzyme-linked immunosorbent assay) and by identification of cells expressing TNF-α and IL-6 mRNA in lung tissues (by in situ hybridization) and isolated lung lavage cells (by RT-PCR). Adenovirus, labeled with the fluorescent dye (Cy3), was rapidly and widely distributed on epithelial surfaces of airways and alveoli and was very rapidly (∼1 min) localized within AMφ. At 30 min after infection AMφ but not airway epithelial or vascular endothelial cells expressed mRNA for TNF-α and IL-6, thus identifying AMφ as the cell source of initial cytokine signaling. IL-6, TNF-α, MIP-2, and MIP-1α levels progressively increased in bronchoalveolar lavage fluid after pulmonary adenovirus infection, and all were significantly elevated at 6 h ( P < 0.05). To begin to define the molecular mechanism(s) by which adenovirus initiates the inflammatory signaling in macrophages, TNF-α expression from adenovirus-infected RAW264.7 macrophages was evaluated in vitro. TNF-α expression was readily detected in adenovirus-infected RAW cell supernatant with kinetics similar to AMφ during in vivo infection. Blockage of virus uptake at specific cellular sites, including internalization (by wortmannin), endosome acidification and/or lysis (by chloroquine) or by Ca 2+ chelation (by BAPTA) completely blocked TNF-α expression. In conclusion, results showed that during ARTI, (i) AMφ rapidly internalized adenovirus, (ii) expression of inflammatory mediators was initiated within AMφ and not airway epithelial or other cells, and (iii) the initiation of inflammatory signaling was linked to virion uptake by macrophages occurring at a point after vesicle acidification. These results have implications for our understanding of the role of the AMφ in the initiation of inflammation following adenovirus infection and adenovirus-mediated gene transfer to the lung.