Sharon Hunter

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.

The effect of phosphatases SHP-1 and SHIP-1 on signaling by the ITIM- and ITAM-containing Fcγ receptors FcγRIIB and FcγRIIA

Inositol and tyrosine phosphatases have been implicated in inhibitory signaling by an Fc receptor for immunoglobulin G, FcγRIIB, in B cells, mast cells, and monocytes. Here, we propose a role for the Src homology 2 (SH2)‐containing tyrosine phosphatase‐1 (SHP‐1) in FcγRIIB‐mediated inhibition of FcγR signaling. Coexpression of SHP‐1 enhances FcγRIIB‐mediated inhibition of FcγRIIA phagocytosis in COS‐1 cells. SHP‐1 also enhances the reduction in FcγRIIA tyrosine phosphorylation that accompanies this inhibition. Significantly, tyrosine phosphorylation of Syk kinase is substantially inhibited by SHP‐1. Furthermore, the activation of SHP‐1 tyrosine phosphorylation is observed following stimulation of FcγRII in COS‐1 cells and in human monocytes. The SH2 domain containing inositol phosphatase (SHIP), SHIP‐1 also enhances FcγRIIB‐mediated inhibition of FcγRIIA, indicating that FcγRIIB can use more than one pathway for its inhibitory action. In addition, SHP‐1 and SHIP‐1 can inhibit FcγRIIA phagocytosis and signal transduction in the absence of FcγRIIB. The data support emerging evidence that SH2‐containing phosphatases, such as SHP‐1 and SHIP‐1, can modulate signaling by “activating” receptors.

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.

The monocyte Fcγ receptors FcγRI/γ and FcγRIIA differ in their interaction with Syk and with Src‐related tyrosine kinases

There are important differences in signaling between the Fc receptor for immunoglobulin G (IgG) FcγRIIA, which uses the Ig tyrosine‐activating motif (ITAM) within its own cytoplasmic domain, and FcγRI, which transmits signals by means of an ITAM located within the cytoplasmic domain of its associated γ‐chain. For example, in transfected epithelial cells and COS‐1 cells, FcγRIIA mediates phagocytosis of IgG‐coated red blood cells more efficiently than does FcγRI/γ, and enhancement of phagocytosis by Syk kinase is more pronounced for FcγRI/γ than for FcγRIIA. In addition, structure/function studies indicate that the γ‐chain ITAM and the FcγRIIA ITAM have different requirements for mediating the phagocytic signal. To study the differences between FcγRIIA and FcγRI/γ, we examined the interaction of FcγRIIA and the FcγRI/γ chimera FcγRI‐γ‐γ (extracellular domain–transmembrane domain–cytoplasmic domain) with Syk kinase and with the Src‐related tyrosine kinases (SRTKs) Hck and Lyn in transfected COS‐1 cells. Our data indicate that FcγRIIA interacts more readily with Syk than does FcγRI‐γ‐γ and suggest that one consequence may be the greater phagocytic efficiency of FcγRIIA compared with FcγRI/γ. Furthermore, individual SRTKs affect the efficiency of phagocytosis differently for FcγRI‐γ‐γ and FcγRIIA and also influence the ability of these receptors to interact with Syk kinase. Taken together, the data suggest that differences in signaling by FcγRIIA and FcγRI‐γ‐γ are related in part to interaction with Syk and Src kinases and that individual SRTKs play different roles in FcγR‐mediated phagocytosis.

Interaction of Two Phagocytic Host Defense Systems Fcγ RECEPTORS AND COMPLEMENT RECEPTOR 3

Phagocytosis of foreign pathogens by cells of the immune system is a vitally important function of innate immunity. The phagocytic response is initiated when ligands on the surface of invading microorganisms come in contact with receptors on the surface of phagocytic cells such as neutrophils, monocytes/macrophages, and dendritic cells. The complement receptor CR3 (CD11b/CD18, Mac-1) mediates the phagocytosis of complement protein (C3bi)-coated particles. Fcγ receptors (FcγRs) bind IgG-opsonized particles and provide a mechanism for immune clearance and phagocytosis of IgG-coated particles. We have observed that stimulation of FcγRs modulates CR3-mediated phagocytosis and that FcγRIIA and FcγRI exert opposite (stimulatory and inhibitory) effects. We have also determined that an intact FcγR immunoreceptor tyrosine-based activation motif is required for these effects, and we have investigated the involvement of downstream effectors. The ability to up-regulate or down-regulate CR3 signaling has important implications for therapeutics in disorders involving the host defense system.

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.