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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AdFcγRIIA induced binding and phagocytosis of opsonized SRBCs in vitro. ...
AdFcγRIIA induced binding and phagocytosis of opsonized SRBCs in vitro. A549 cells grown on coverslip dishes were infected with AdFcγRIIA, AdFcγRIIA mutant, and AdNull at 10 moi. After 48 hours, the cells were incubated with IgG-coated SRBCs for 60 minutes. To evaluate binding, cells were washed, fixed, and stained by Giemsa stain. To evaluate phagocytosis, cells were washed, SRBCs bound to the cell surface were lysed, and the A549 cells were fixed and stained by Giemsa stain or eosin as indicated. (a) Binding of opsonized SRBCs by naive cells. (b) Binding of opsonized SRBCs by AdNull-infected cells. (c) Binding of opsonized SRBCs by AdFcγRIIA mutant–infected cells. (d) Binding of opsonized SRBCs by AdFcγRIIA-infected cells. (e) Phagocytosis of opsonized SRBCs by naive noninfected cells. (f) Phagocytosis of opsonized SRBCs by AdNull-infected cells. (g) Phagocytosis of opsonized SRBCs by AdFcγRIIA mutant–infected cells. (h) Phagocytosis of opsonized SRBCs by AdFcγRIIA-infected cells. Large panels are original magnification ×400 (Giemsa stain). Shown in the insets are light microscopy of original magnification ×1,000 for a–d (Giemsa stain) and fluorescent microscopy of original magnification ×1,000 (eosin) for e–h.