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Journal of Virology, October 2000, p. 9655-9667, Vol. 74, No. 20
Division of Pulmonary
Biology1 and Division of Pulmonary
Medicine,2 Children's Hospital Medical
Center, Cincinnati, Ohio 45229
Received 30 March 2000/Accepted 13 July 2000
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 (AM
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Internalization of Adenovirus by Alveolar
Macrophages Initiates Early Proinflammatory Signaling during Acute
Respiratory Tract Infection


) rapidly
internalize adenovirus following in vivo pulmonary administration and
then initiate inflammatory signaling within the lung. To evaluate the role of AM
in the induction of lung inflammation during ARTI in
vivo, we directly assessed adenovirus uptake by murine AM
and
correlated uptake with the initiation of proinflammatory gene expression. Stimulation of cytokine (tumor necrosis factor alpha [TNF-
], interleukin-6 [IL-6], macrophage
inflammatory protein-2 [MIP-2], and MIP-1
) 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-
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 AM
. At 30 min after infection AM
but not airway epithelial or vascular
endothelial cells expressed mRNA for TNF-
and IL-6, thus identifying
AM
as the cell source of initial cytokine signaling. IL-6, TNF-
, MIP-2, and MIP-1
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-
expression
from adenovirus-infected RAW264.7 macrophages was evaluated in vitro.
TNF-
expression was readily detected in adenovirus-infected RAW cell
supernatant with kinetics similar to AM
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 Ca2+ chelation (by BAPTA) completely
blocked TNF-
expression. In conclusion, results showed that during
ARTI, (i) AM
rapidly internalized adenovirus, (ii) expression of
inflammatory mediators was initiated within AM
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 AM
in the initiation of
inflammation following adenovirus infection and adenovirus-mediated
gene transfer to the lung.
*
Corresponding author. Mailing address: Children's
Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet
Ave., Cincinnati, OH 45229. Phone: (513) 636-6361. Fax: (513) 636-3723. E-mail: Bruce.Trapnell{at}chmcc.org.
Present address: Department of Pediatrics, Harvard Medical School,
Boston, MA 02115.
Present address: Department of Laboratory Medicine, Yamagata
University School of Medicine, Yamagata 990-23, Japan.
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