Adenovirus infection increases iNOS and peroxynitrite production in the lung.

Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 x 10(9) plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.

Keratinocyte growth factor stimulates transduction of the respiratory epithelium by retroviral vectors.

Cell proliferation is required for transduction by standard retrovirus vectors derived from viruses in the murine leukemia virus (MuLV) group. Since proliferation rates are low in the mature pulmonary epithelium, we tested the hypothesis that the efficiency of retrovirus-mediated transduction of respiratory epithelial cells can be enhanced by stimulation of cell proliferation with recombinant human keratinocyte growth factor (rhKGF). A marked increase in proliferation of bronchiolar and alveolar epithelial cells was observed after intratracheal administration of rhKGF (30 mg/kg) to adult FVB/N mice. Two days after rhKGF or saline treatment, 10(7) AP+ FFU of LAPSN, a recombinant amphotropic retrovirus that expresses human placental alkaline phosphatase (AP), was instilled intratracheally into the mice. Transduction efficiency, measured 2 days after infection, was increased approximately 70-fold by rhKGF pretreatment. However, even after KGF treatment the total numbers of AP-expressing cells were few. Transduction efficiency was similar using either LAPSN packaged by amphotropic host range packaging cells or LAPSN pseudotyped with 10A1 MuLV envelope protein (0.091 +/- 0.006 versus 0.094 +/- 0.028 transduction events/mm2, respectively). Amphotropic vectors use Pit-2 for cell entry, while 10A1 MuLV vectors can use Pit-1 or Pit-2 for cell entry. By in situ hybridization the retroviral receptor Pit-2 (Ram-1) mRNA was expressed only in the pulmonary vasculature, and Pit-1 (Glvr-1) mRNA was expressed at low levels throughout the lung. In vitro studies demonstrated that retrovirus was inactivated by pulmonary surfactant. Stimulating proliferation of the respiratory epithelium increased retroviral transduction in vivo, but the paucity of retroviral receptors and inactivation by surfactant are additional barriers to high-level retroviral gene transfer in the lung.

Adenovirus-mediated granulocyte-macrophage colony-stimulating factor improves lung pathology of pulmonary alveolar proteinosis in granulocyte-macrophage colony-stimulating factor-deficient mice.

Mutation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene by homologous recombination causes progressive pulmonary alveolar proteinosis (PAP) in GM-CSF-deficient mice (GM-/-). The present study tested whether adenovirus-mediated expression of GM-CSF alters the progression of PAP in GM-/- mice. Adult mice were pretreated with an anti-T cell receptor (TCR) antibody to block T cell-mediated immune response, followed by intratracheal instillation of deltaE1-E3 replication-deficient adenovirus expressing mouse GM-CSF (Av1mGM). Mice were killed 1, 3, and 5 weeks after treatment to assess lungs for GM-CSF, surfactant protein B (SP-B), alveolar macrophage maturation, and type II cell proliferation. GM-CSF was detected in BAL fluid from GM-/- mice 1 week after Av1mGM treatment, and GM-CSF mRNA was detected by RT-PCR through 5 weeks. Five weeks after Av1mGM treatment, PAP was improved and SP-B decreased as assessed by ELISA and immunostaining. Increased numbers of alveolar macrophages stained with alpha-naphthyl acetate esterase (alpha-NAE) following treatment with Av1mGM. Local expression of GM-CSF with a recombinant adenovirus ameliorated PAP in the GM-/- mice in association with enhanced maturation of alveolar macrophages.

GM-CSF enhances lung growth and causes alveolar type II epithelial cell hyperplasia in transgenic mice.

The human surfactant protein (SP)-C gene promoter was used to direct expression of mouse granulocyte macrophage colony-stimulating factor (GM-CSF; SP-C-GM mice) in lung epithelial cells in GM-CSF-replete (GM+/+) or GM-CSF null mutant (GM-/-) mice. Lung weight and volume were significantly increased in SP-C-GM mice compared with GM+/+ or GM-/- control mice. Immunohistochemical staining demonstrated marked type II cell hyperplasia, and immunofluorescent labeling for proliferating cell nuclear antigen was increased in type II cells of SP-C-GM mice. Abundance of type II cells per mouse lung was increased three- to fourfold in SP-C-GM mice compared with GM+/+ and GM-/- mice. GM-CSF increased bromodeoxyuridine labeling of isolated type II cells in vitro. Type II cells, alveolar macrophages, and endothelial and bronchiolar epithelial cells were stained by antibodies to the GM-CSF receptor alpha-subunit in both GM+/+ mice and GM-CSF gene-targeted mice that are also homozygous for the SP-C-GM transgene. High levels of GM-CSF expression in type II cells of transgenic mice increased lung size and caused type II cell hyperplasia, demonstrating an unexpected role for the molecule in the regulation of type II cell proliferation and differentiation.

Recombinant adenoviral vector disrupts surfactant homeostasis in mouse lung.

Although replication-deficient adenoviruses efficiently transfer genes into epithelial cells of the lung, host immune responses limit the extent and duration of gene expression. To define further the role of inflammatory responses to first-generation, recombinant, deltaE1, deltaE3 adenovirus in lung pathology and surfactant protein homeostasis, expression of the surfactant proteins SP-A, SP-B, and proSP-C was determined by immunohistochemistry 2, 7, and 14 days following intratracheal administration of 2 x 10(9) pfu of a recombinant adenovirus, Av1Luc1, to BALB/c nu/nu and BALB/c wild-type mice. Two to 7 days after virus administration, an acute inflammatory response was observed in both mouse strains. Respiratory epithelial cells were sloughed, and extracellular accumulation of SP-A and SP-B was detected in the airways. Diminished immunostaining for SP-A and SP-B was noted in type II cells, and SP-A and SP-B mRNA expression was decreased in focal regions of the lungs from both mouse strains. One week after virus administration, immunostaining for proSP-C was markedly increased in cells lining the regenerating alveolar epithelial surfaces. Two weeks after Av1Luc1 treatment of nu/nu mice, immunostaining for SP-A, SP-B, and proSP-C was similar to those patterns observed prior to adenoviral administration. In immunocompetent wild-type mice, however, immunostaining for surfactant proteins was absent in areas associated with chronic lymphocytic infiltration. The recombinant adenoviral vector, Av1Luc1, caused acute inflammatory responses in the respiratory epithelium with disruption of surfactant protein homeostasis in both wild-type and nu/nu mice. Alterations in surfactant homeostasis persisted in wild-type mice. Thus, both acute and thymic-dependent immune responses limit transgene expression and disrupt surfactant protein gene expression and homeostasis. Because surfactant proteins are critical to host defense and to the maintenance of alveolar stability following injury, these findings raise concerns regarding both acute and chronic toxicity of first-generation recombinant adenoviral vectors for gene transfer.

Anti-T cell receptor antibody prolongs transgene expression and reduces lung inflammation after adenovirus-mediated gene transfer.

Replication-deficient delta E1a-E3 adenovirus mediates efficient gene transfer to the mouse lung; however it induces a host immune response mediated, in part, by T cells. This immune response is associated with loss of transgene expression. Monoclonal antibodies (mAb) against the T cell receptor (TCR) complex can inhibit both CD4+ and CD8+ T cell responses in vivo and are the most potent anti-T cell agents in clinical use. To determine whether such mAbs can be used to prolong adenovirus-mediated transgene expression, the vector Av1Luc1 (delta E1a-E3 recombinant adenovirus encoding the firefly luciferase gene) was administered intratracheally to C57BL/6 mice on day 0. Three days prior to adenovirus administration (day -3), mice were treated with a single i.p. injection of the anti-TCR mAb H57. Controls received phosphate-buffered saline. Animals were sacrificed on days 3, 14, 28, and 56 and lungs were assessed for transgene expression and histopathology. Luciferase activity decreased markedly in the controls by day 14, but was maintained at high levels in animals receiving anti-TCR mAb. A mild, focal, predominantly neutrophilic inflammation was observed in the alveoli of all mice 3 days after virus administration. In PBS-treated controls, this inflammation progressed to a moderate to severe multifocal, perivascular and peribronchiolar lymphoid infiltration at 14 days. B cells and T cells were present in approximately equal numbers, with CD4+ T cells predominating over CD8+ T cells by 3- to 28-fold. Treatment with H57 resulted in near-complete prevention of the lymphocytic inflammatory infiltrate and increased luciferase activity throughout the 56-day study period, in association with TCR modulation and T cell depletion. Thus, anti-TCR mAb decreases inflammation and prolongs gene expression following adenovirus-mediated gene transfer.

Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu/nu) mice.

To evaluate the role of cell-mediated immunity during gene transfer to the respiratory epithelium, the time course of luciferase activity was assessed after intratracheal administration of Av1Luc1, an E1a-E3-deleted adenoviral (Ad5) vector expressing firefly luciferase, to FVB/N, BALB/c and BALB/c-nu/nu adult mice. Adenovirus-mediated luciferase activity was rapidly lost from the respiratory tract between 2 and 14 days after treatment of both FVB/N and BALB/c wild-type mice. In the wild-type mice, loss of luciferase activity was associated with an early inflammatory response consisting of infiltration with macrophages and polymorphonuclear leukocytes and a more prolonged response characterized by lymphocytic infiltration. In the immune-deficient nu/nu mice, luciferase activity was maintained at higher levels than in immune-competent mice after exposure to virus and was associated with a distinct pattern of inflammation, consisting primarily of macrophages and polymorphonuclear cells but lacking the lymphocytic infiltrates typical of the inflammation in wild-type mice. Adenoviral DNA was rapidly cleared from the lungs of both nu/nu and wild-type mice. Markedly increased expression of proliferating cell nuclear antigen (PCNA) was observed in bronchiolar and alveolar epithelial cells and in inflammatory cells after exposure to Av1LUc1. The proliferative response of the respiratory epithelium was more extensive and persistent in wild-type than in nu/nu mice. To assess further the impact of the immune system on adenovirus-mediated gene expression, cotton rats treated with cyclosporin A or dexamethasone were exposed to Av1Luc1. Both agents decreased lung inflammation and significantly increased lung luciferase activity. The loss of lung luciferase activity is dependent, in part, on the immune-mediated clearance of respiratory epithelial cells, which may limit the extent and duration of gene expression with recombinant adenoviral vectors.