Augmentation of pulmonary host defense against Pseudomonas by FcγRIIA cDNA transfer to the respiratory epithelium
Stefan Worgall, … , Alan D. Schreiber, Ronald G. Crystal
Stefan Worgall, … , Alan D. Schreiber, Ronald G. Crystal
Published August 15, 1999
Citation Information: J Clin Invest. 1999;104(4):409-418. https://doi.org/10.1172/JCI5432.
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Article

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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Expression of FcγRIIA by A549 cells after Ad-mediated gene transfer. Cel...
Expression of FcγRIIA by A549 cells after Ad-mediated gene transfer. Cells were infected with AdFcγRIIA and/or AdNull for 48 hours at 1 or 10 moi. Noninfected cells were used as negative controls. (a) Expression of FcγRIIA mRNA assessed by Northern analysis. Hybridized with human FcγRIIA cDNA probe (top); hybridized with a human GAPDH cDNA probe (bottom). Lane 1: naive control; lane 2: infected with AdFcγRIIA moi 1; lane 3: infected with AdFcγRIIA moi 10. The sizes of transcripts are indicated. (b–e) Expression of FcγRIIA protein on the cell surface measured by immunohistochemistry. The cells were stained with anti-FcγRIIA mAb, followed by alkaline-phosphatase–conjugated goat-anti mouse IgG and alkaline-phosphatase substrate and counterstained with hematoxylin. (b) Naive cells. (c) AdNull-infected cells (moi 10). (d) AdFcγRIIA-infected cells (moi 10). (e) AdFcγRIIA-infected cells (moi 10) incubated with isotype-matched control antibody. All panels are original magnification ×400. (f) Expression of FcγRIIA protein on the cell surface assessed by immunostaining and flow cytometry. Cells were incubated with anti-FcγRIIA mAb, followed with FITC-conjugated goat anti-mouse F(ab)2 IgG and assessed by flow cytometry in comparison to isotype-matched controls. Shown is analysis of naive controls, AdNull-infected cells (moi 10), and AdFcγRIIA-infected cells (moi 10). The horizontal line indicates the region considered positive.