Jong-Gu Park

Molecular dissection of Fcγ receptor-mediated phagocytosis

Abstract Using an experimental model in COS-1 cells, we have examined the structural requirements for phagocytosis of IgG-sensitized cells by Fcγ receptors. We have established that isoforms of each of the 3 classes of the Fcγ receptors, FcγRI, FcγRII and FcγRIII, are able to transmit a phagocytic signal in the absence of the other receptor class. FcγI and RcγRIIIA require a γ-subunit for this signaling event, but FcγRIIA does not. FcγRIIA and the γ-subunit associated with FcγRI and FcγRIIIA contain 2 copies of a conserved tyrosine-containing cytoplasmic sequence, YXXL. This sequence is important for phagocytosis and is phosphorylated on tyrosine after receptor ligation. The Fcγ receptors FcγRIIB1 and FcγRIIB2 which contain only 1 copy of the YXXL cytoplasmic sequence do not include the phagocytosis of IgG-coated cells. Thus, the Fcγ receptor isoforms differ in their ability to transmit a phagocytic signal. Structure/function studies also indicate that the Fcγ receptors which induce phagocytosis differ in their requirements for phagocytic signaling.

Structure/function relationships of Fcγ receptors in phagocytosis

Abstract An important function of Fc γ receptors is the ingestion or phagocytosis of IgG sensitized cells. It has been difficult to clearly define the individual function of each receptor in phagocytosis because hematopoietic cells express multiple Fc γ receptor isoforms. To examine this issue, an in-vitro model system in COS-1 cells has been developed. When transfected with an appropriate Fc γ receptor, COS-1 cells which lack endogeneous Fc receptors, ingest IgG-sensitized cells. Using this model, a single class of human Fc γ receptor in the absence of other Fc receptors was observed to mediate phagocytosis. Furthermore, isoforms from each Fc γ receptor class can mediate phagocytosis although the requirements for phagocytosis differ. Investigation of the relationship between structure and function for Fc receptor-mediated phagocytosis established the importance of the cytoplasmic tyrosines of the receptor or its associated γ chain. For example, two cytoplasmic YXXL sequences, in a configuration similar to the conserved tyrosine containing motif found in immunoglobulin gene family receptors, are important for phagocytosis by the human Fc γ receptor, Fc γ RIIA. Fc γ RI and Fc γ RIIIA do not possess cytoplasmic tyrosines, but transmit a phagocytic signal through interaction with an associated γ -subunit which contains two YXXL sequences in a conserved motif required for phagocytosis. The human Fc γ RII isoforms, Fc γ RIIB1 and Fc γ RIIB2, do not induce phagocytosis and have only a single YXXL sequence. Crosslinking of the phagocytic Fc γ receptors induces tyrosine phosphorylation of either Fc γ RIIA or the γ chain and treatment with tyrosine kinase inhibitors reduces both phagocytosis and phosphorylation of the receptor tyrosine residues. The protein tyrosine kinase Syk, which is associated with the γ chain in monocytes/macrophages, dramatically enhances phagocytosis mediated by Fc γ RI and Fc γ RIIIA and also induces non-phagocytic Fc γ RI or Fc γ RIIIA expressing cells to acquire phagocytic capability.

Structural requirements of Syk kinase for Fcγ receptor– phagocytosis

Abstract The tyrosine kinase Syk plays a critical role in the phagocytic pathway mediated by Fcγ receptors (FcγR). In transfected COS1 cells co-expression of Syk enhances FcγR mediated phagocytosis. The other member of the Syk kinase family, the highly homologous tyrosine kinase Zap70, also plays a role in signaling by immunoglobulin gene family receptors, but does not increase the phagocytic efficiency of FcγRs. The homologous tandem SH2 and kinase domains of Syk and Zap70 are separated by a nonhomologous region referred to as the unique domain. Zap70s inability to enhance phagocytosis was not due to unique domain tyrosine 292, previously implicated in negative regulation of Zap70 function. We determined the regions of Syk important for its interaction with the phagocytic pathway. An intact kinase domain was required for Syks effect on phagocytosis. Furthermore, the Syk variant SykB, lacking 23 amino acids in the unique region, signaled for phagocytosis as efficiently as did Syk. We then constructed exchange chimeras between Syk and Zap70 and determined the contributions of the SH2, unique and kinase domains to phagocytic signaling. Our data suggest that the Syk kinase domain, which has high intrinsic kinase activity, is important for facilitating phagocytic signaling by FcγRI and FcγRIIIA.

Augmentation of pulmonary host defense against Pseudomonas by FcγRIIA cDNA transfer to the respiratory epithelium

Fcgamma 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 FcgammaRIIA cDNA to induce expression of the human FcgammaRIIA receptor. Binding and phagocytosis of opsonized sheep red blood cells (SRBCs) by the A549 human lung epithelial cell line after Ad-mediated FcgammaRIIA gene transfer was demonstrated using light and fluorescence microscopy and phagocytic assays with (51)Cr-labeled SRBCs. When A549 cells were infected with an Ad vector expressing a FcgammaRIIA 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 FcgammaRIIA in the lung after intratracheal administration of the AdFcgammaRIIA enhanced clearance of opsonized Pseudomonas aeruginosa from the lung in normal rats and in mice deficient in Fcgamma receptor expression. Similar results were observed with a chimeric FcgammaRIIA construct containing the extracellular domain of FcgammaRIIIA. Together, these data demonstrate that Ad-mediated FcgammaRIIA 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.