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) 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. Adenovirus, a nonenveloped DNA virus with at least 45 serotypes, is an important respiratory pathogen affecting individuals of all ages and accounting for 7 to 10% of all respiratory illnesses in infants and children, with an incidence of between 5 to 10 million infections annually in the United States alone (13, 21). Adenovirus respiratory tract infection (ARTI) occurs sporadically, epidemically, and nosocomially, presents in a wide spectrum of distinct clinical syndromes ranging from self-limited acute pharyngitis to fatal pneumonia, and has been identified as an etiological factor associated with exacerbations in individuals with chronic obstructive lung diseases (13). Despite the frequency and broad range of clinical presentations, the pathogenesis of inflammation in ARTI is poorly understood. Early information regarding host responses to adenovirus was derived from efforts to develop human adenovirus vaccines (42) or to understand the pathology of fatal adenoviral pneumonia (4). Recently, ARTI has been studied in a variety of animal models, including mice (14, 37, 58), Cotton rats (Sigmodon hispidus) (15, 39, 59, 60), and primates (8, 44, 55, 61) as part of preclinical toxicology studies for human gene therapy clinical trials for cystic fibrosis (reviewed in references 7 and 48). Studies in humans showed that administration of replication-deficient adenovirus vectors to the respiratory tract can cause dose-dependent local inflammation (12,26,33).Inflammatory responses to ARTI have been best studied in rodent models. In the Cotton rat, a permissive host for replication of human adenovirus, pulmonary histopathology consists of early and late phases similar to that seen in human ARTI (15,39). The early phase consists primarily of accumulation of neutrophils, macrophages, and monocytes and develops within the first 24 h (60). The late phase, consisting mainly of lymphocytes, is apparent by day 5 (39, 60). In mice, a nonpermissive host for human adenovirus, adenovirus early gene

Replication-deficient adenovirus vectors (Avs) have shown high-efficiency gene transfer in a variety of animal models, but demonstrated lower than expected efficiency in the intensely inflammatory milieu of the respiratory tract of individuals with cystic fibrosis (CF). Specific acquired immune responses directed at adenovirus capsid proteins are known to limit the duration of transgene expression and the effectiveness of vector readministration. In these models, however, nonspecific inflammation is also frequently noted to accompany specific immune responses. Because inflammation can occur early after Av administration, we hypothesized that inflammation may block Av-mediated gene transfer in the lung independent of specific immune responses. To evaluate this hypothesis, we measured pulmonary gene transfer and expression in the absence or presence of the potent antiinflammatory agent dexamethasone. To address and eliminate concerns over the potentially confounding effects of systemic, vector-specific acquired immune responses, evaluations were confined to a 3-day period following Av administration and were carried out, in parallel, in normal and immunodeficient (athymic) mice. Dexamethasone significantly reduced Av-associated inflammation in all animals as measured by a significant reduction of blinded, quantitative lung histopathology scores and by reduced proinflammatory cytokine release. Concomitant with reduced inflammation, gene transfer efficiency was significantly increased in both normal and immunodeficient animals as measured by transgene product activity (beta-galactosidase) in total lung homogenates 3 days after vector administration. This finding could not be explained by a direct effect of dexamethasone on transgene specific activity. To begin to understand the molecular mechanisms of Av-induced inflammatory responses, lung levels of the chemoattractive chemokines MIP-2, MIP-1alpha, and MCP-1 were quantified. All were elevated significantly in Av-exposed animals. Dexamethasone reduced levels of MCP-1 and MIP-1alpha, but not MIP-2, consistent with the observed pattern of inflammatory cell changes. Expression of several proinflammatory cytokines including TNF-alpha, IL-6, IL-1beta, and IFN-gamma were also elevated in Av-exposed animals and modulated by dexamethasone. These observations demonstrate that nonspecific inflammation is an important determinant of the efficiency of in vivo pulmonary gene transfer and expression independent of specific immune responses and may have important implications for human gene therapy for diseases of the lung.

GM-CSF gene-targeted (GM−/−) mice have impaired pulmonary clearance of bacterial and fungal pathogens by alveolar macrophages (AMs). Because AMs also clear adenovirus from the lung, the role of GM-CSF in endocytic internalization of adenovirus by AMs was evaluated. Pulmonary clearance of adenovirus was severely impaired in GM−/− mice compared to wild-type (GM+/+) mice as determined by Southern analysis of viral DNA. Internalization of adenovirus by AMs was deficient in GM−/− mice in vivo and in vitro as determined by uptake of fluorescently labeled adenovirus or by PCR quantification of adenoviral DNA internalized within AMs. An AM cell line previously established from GM−/− mice (mAM) had impaired internalization of adenovirus and transferrin-coated 100-nm latex beads compared to MH-S, a GM+/+ AM cell line. Phagocytosis of 4-μm latex beads was also impaired in mAM cells as determined by confocal and fluorescence microscopy. Retroviral vector-mediated reconstitution of PU.1 expression in cultured GM−/− AMs restored phagocytosis of 4-μm beads, endocytosis of adenovirus, and transferrin-coated 100-nm beads (independent of integrin αV and transferrin receptors, respectively), and restored normal cytoskeletal organization, filamentous actin distribution, and stimulated formation of filopodia. Interestingly, mRNA for the phosphoinositide 3 kinase p110γ isoform, important in macrophage phagocytic function, was absent in GM−/− AMs and was restored by PU.1 expression. These data show that GM-CSF, via PU.1, regulates endocytosis of small (∼100 nm) pathogens/inert particles and phagocytosis of very large inert particles and suggests regulation of cytoskeletal organization by GM-CSF/PU.1 as the molecular basis of this control.

IntroductionAlveolar macrophages (AMs) are pulmonary residents of the bone marrow-derived mononuclear phagocyte system that play a critical role in several diverse lung functions and in lung host defense. 1 AMs arise from circulating blood monocytes that enter the lungs and other tissues 2,3 and undergo terminal differentiation into tissue macrophage populations that are heterogeneous in their level of accumulation, 4,5 functional activity, 4 and expression of cell-surface molecules. 6,7 The mechanisms regulating the abundance and heterogeneity of AMs and other tissue macrophage populations are not well defined. However, exposure of macrophages to different tissue microenvironments varying in the local amount or form of hematologic growth factors capable of regulating macrophage proliferation has been proposed. 5 Macrophage colony-stimulating factor (M-CSF, also known as CSF-1) is one of several factors known to regulate the survival, proliferation, and differentiation of mononuclear phagocytic lineage cells. 8 M-CSF is abundant in blood, present in most tissues (though in lower and various levels depending on the type of tissue), and expressed as 3 biologically active forms: a circulating secreted glycoprotein isoform, a proteoglycan isoform, and a cell-surface isoform produced locally in tissues. 8,9 Recombinant M-CSF was shown to promote accumulation of murine AMs in vivo 10 and survival of human AMs in vitro, 11 and M-CSF levels are increased in the lungs of smokers in association with significant increases in numbers of AMs. 12 Granulocyte-macrophage colonystimulating factor (GM-CSF) and interleukin 3 (IL-3) are also capable of regulating macrophage proliferation. 5,13 AM numbers are increased markedly in transgenic mice expressing GM-CSF in the lung from a lung-specific transgene. 14 However, AM numbers are not reduced in GM-CSF knockout mice lacking GM-CSF, 15,16 suggesting that GM-CSF is not essential for constitutive accumulation of AMs.Osteopetrotic mice are homozygous for a naturally occurring recessive frameshift mutation (Csf-1 op or Op) in the M-CSF gene 17 and consequently have no M-CSF. 18 They thus provide a good model for studying the role of M-CSF in vivo. In Op/Op mice, various M-CSF-dependent macrophage populations are decreased in number and show developmental anomalies in a tissue-specific manner. 5,19,20 For example, bone marrow monocytes/macrophages, which usually increase progressively with age in normal mice, are reduced in number in young Op/Op mice. 5 However, bone marrow macrophages still increase in number with age in these mice in parallel with spontaneous correction of osteopetrosis. 21 Macrophage populations that increase transiently in number at 2 weeks of age in normal mice are reduced in number, with or without preservation of the transient increase (eg, in the intestine or The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C....

Surfactant protein A (SP-A) is a member of the collectin family of host defense molecules expressed primarily in the epithelial cells of the lung. To determine the role of SP-A in pulmonary adenoviral infection, SP-A-deficient (SP-A −/−) mice were intratracheally infected with a replication-deficient recombinant adenovirus, Av1Luc1. Lung inflammation was markedly increased in SP-A −/− compared with SP-A +/+ mice and was associated with increased hemorrhage and epithelial cell injury. Polymorphonuclear cells in bronchoalveolar lavage fluid (BALF) were increased in SP-A −/− mice after administration of adenovirus. Coadministration of adenovirus and purified human SP-A ameliorated adenoviral-induced lung inflammation in SP-A −/− mice. Concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β were increased in BALF of SP-A −/− mice. Likewise, TNF-α, IL-6, macrophage inflammatory protein (MIP)-1α, monocyte chemotactic protein-1, and MIP-2 mRNAs were increased in lung homogenates from SP-A −/− mice 6 and 24 h after viral administration. Clearance of adenoviral DNA from the lung and uptake of fluorescent-labeled adenovirus by alveolar macrophages were decreased in SP-A −/− mice. SP-A enhances viral clearance and inhibits lung inflammation during pulmonary adenoviral infection, providing support for the importance of SP-A in antiviral host defense.