Augmentation of pulmonary host defense against Pseudomonas by FcγRIIA cDNA transfer to the respiratory epithelium
Stefan Worgall, Petr Bezdicek, Moo-Kyung Kim, Jong-Gu Park, Ravi Singh, Melpo Christofidou-Solomidou, Alice Prince, Imre Kovesdi, Alan D. Schreiber, Ronald G. Crystal
Stefan Worgall, Petr Bezdicek, Moo-Kyung Kim, Jong-Gu Park, Ravi Singh, Melpo Christofidou-Solomidou, Alice Prince, Imre Kovesdi, Alan D. Schreiber, Ronald G. Crystal
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Augmentation of pulmonary host defense against Pseudomonas by FcγRIIA cDNA transfer to the respiratory epithelium

Abstract

Fcγ receptors on the surface of phagocytic cells bind the Fc region of IgG and mediate binding, phagocytosis, and destruction of particulate antigens opsonized by the antigen-specific IgG molecule. The present study evaluates the feasibility of converting lung epithelial cells into phagocytic cells using adenovirus (Ad) vector–mediated gene transfer of FcγRIIA cDNA to induce expression of the human FcγRIIA receptor. Binding and phagocytosis of opsonized sheep red blood cells (SRBCs) by the A549 human lung epithelial cell line after Ad-mediated FcγRIIA gene transfer was demonstrated using light and fluorescence microscopy and phagocytic assays with 51Cr-labeled SRBCs. When A549 cells were infected with an Ad vector expressing a FcγRIIA mutant in which 2 of 3 cytoplasmic tyrosines have been replaced with phenylalanine, only binding, but not phagocytosis, of opsonized SRBCs was observed. In vivo expression of FcγRIIA in the lung after intratracheal administration of the AdFcγRIIA enhanced clearance of opsonized Pseudomonas aeruginosa from the lung in normal rats and in mice deficient in Fcγ receptor expression. Similar results were observed with a chimeric FcγRIIA construct containing the extracellular domain of FcγRIIIA. Together, these data demonstrate that Ad-mediated FcγRIIA receptor cDNA expression can mediate the binding and phagocytosis of opsonized particulate antigens by normally nonphagocytic cells, suggesting that gene-transfer strategies might be used to utilize nonphagocytic cells to clear bacteria or other opsonized particulate antigens from the respiratory tract.

Authors

Stefan Worgall, Petr Bezdicek, Moo-Kyung Kim, Jong-Gu Park, Ravi Singh, Melpo Christofidou-Solomidou, Alice Prince, Imre Kovesdi, Alan D. Schreiber, Ronald G. Crystal

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Quantitative assessment of binding and phagocytosis of SRBCs by A549 cel...
Quantitative assessment of binding and phagocytosis of SRBCs by A549 cells after Ad-mediated FcγRIIA cDNA gene transfer. A549 cells were infected with AdFcγRIIA, AdFcγRIIA mutant, or AdNull (all at 10 moi). After 48 hours, cells were incubated with IgG-coated 51Cr-labeled SRBCs for 60 minutes, or as a control, with nonopsonized 51Cr-labeled SRBCs for 60 minutes. To evaluate binding of IgG-coated SRBCs, the cells were washed and radioactivity of the lysate was quantified. To evaluate phagocytosis, the cells were washed and the SRBCs bound to the cell surface were removed by lysis. The A549 cells were then lysed, and radioactivity of the lysate was quantified. (a) Binding of nonopsonized SRBCs. (b) Binding of opsonized SRBCs. (c) Phagocytosis of nonopsonized SRBCs. (d) Phagocytosis of opsonized SRBCs. Shown are means ± SEM of activity (disintegrations per minute) per well of triplicate assessments.