Moo-Kyung Kim

Inhibition of Allergic Inflammation in the Airways Using Aerosolized Antisense to Syk Kinase

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of FcγR and FcεR, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of β2 integrins, α4 integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.

Contrasting requirements for ubiquitylation during Fc receptor‐mediated endocytosis and phagocytosis

Fc receptors on leukocytes mediate internalization of antibody‐containing complexes. Soluble immune complexes are taken up by endocytosis, while large antibody‐opsonized particles are internalized by phagocytosis. We investigated the role of ubiquitylation in internalization of the human FcγRIIA receptor by endocytosis and phagocytosis. A fusion of FcγRIIA to green fluorescent protein (GFP) was expressed in ts20 cells, which bear a temperature‐sensitive mutation in the E1 ubiquitin‐activating enzyme. Uptake of soluble IgG complexes mediated by FcγRIIA–GFP was blocked by incubation at the restrictive temperature, indicating that endocytosis requires ubiquitylation. In contrast, phagocytosis and phagosomal maturation were largely unaffected when ubiquitylation was impaired. FcγRIIA–GFP was ubiquitylated in response to receptor cross‐linking. Elimination of the lysine residues present in the cytoplasmic domain of FcγRIIA impaired endocytosis, but not phagocytosis. The proteasomal inhibitor clasto‐lactacystin β‐lactone strongly inhibited endocytosis, but did not affect phagocytosis. These studies demonstrate a role for ubiquitylation in the endocytosis of immune receptors, and reveal fundamental differences in the mechanisms underlying internalization of a single receptor depending on the size or multiplicity of the ligand complex.

Differential kinase requirements in human and mouse Fc‐gamma receptor phagocytosis and endocytosis

Fc gamma receptors (FcγRs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (FcγR) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src‐related tyrosine kinases (SRTKs) and phosphatidyl inositol 3‐kinases (PI3K) play important roles in FcγR phagocytosis. Herein, we demonstrate that these kinases are not required for FcγR endocytosis. Endocytosis of heat‐aggregated IgG (HA‐IgG) by COS‐1 cells stably transfected with FcγRIIA or chimeric FcγRI‐γ‐γ (EC‐TM‐CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG‐sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow‐derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when FcγRIIA was cross‐linked using HA‐IgG or EA. However, inhibition of FcγR phosphorylation prevented only FcγR phagocytosis. Finally, biochemical analyses of PI3K(p85)‐Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during FcγR phagocytosis and endocytosis. Overall, our results indicate that FcγR endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition‐based model in which PI3K and c‐Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.

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.

Signal sequence within FcγRIIA controls calcium wave propagation patterns: Apparent role in phagolysosome fusion

Calcium oscillations and traveling calcium waves have been observed in living cells, although amino acid sequences regulating wave directionality and downstream cell functions have not been reported. In this study we identify an amino acid sequence within the cytoplasmic domain of the leukocyte IgG receptor FcγRIIA that affects the amplitude of calcium spikes and the spatiotemporal dynamics of calcium waves in the vicinity of phagosomes. By using high-speed microscopy to map calcium-signaling routes within cells, we have discovered that bound IgG-coated targets trigger two calcium waves traveling in opposite directions about the perimeter of cells expressing FcγRIIA. After phagocytosis, one calcium wave propagates around the plasma membrane to the site of phagocytosis where it splits into two calcium signals: one traveling to and encircling the phagosome once, and the second continuing around the plasma membrane to the point of origin. However, in a genetically engineered form of FcγRIIA containing a mutation in the cytoplasmic L-T-L motif, the calcium signal travels around the plasma membrane, but is not properly routed to the phagosome. Furthermore, these calcium pattern-deficient mutants were unable to support phagolysosome fusion, although recruitment of phagolysosome-associated proteins lysosome-associated protein 1, Rab5, and Rab7 were normal. Our findings suggest that: (i) calcium signaling is a late step in phagolysosome fusion, (ii) a line of communication exists between the plasma membrane and phagosome, and (iii) the L-T-L motif is a signal sequence for calcium signal routing to the phagosome.

Phosphorylation-independent Ubiquitylation and Endocytosis of FcγRIIA

Endocytosis of the Fc receptor FcγRIIA depends on a functional ubiquitin conjugation system, and the receptor becomes ubiquitylated upon ligand binding. Phosphorylation of tyrosines in FcγRIIA by Src family kinases is thought to be the initiating event in its signaling. However, although the Src family kinase inhibitor PP1 inhibited both ligand-induced phosphorylation of FcγRIIA and phagocytosis in ts20 cells expressing FcγRIIA, it did not inhibit receptor ubiquitylation or endocytosis of soluble ligands. Conversely, genistein and the proteasomal inhibitor MG132 did not inhibit receptor phosphorylation but strongly inhibited both receptor ubiquitylation and endocytosis. A region of the receptor lying within the immunoreceptor tyrosine-based activation motif was found to be necessary for both ubiquitylation and endocytosis. Ubiquitylation occurs at the plasma membrane before internalization. Endocytosis of FcγRIIA is dependent on clathrin but independent of the adaptor protein AP-2. These findings point to a novel mechanism for ubiquitylation and endocytosis of this immunoreceptor.

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.

Effect of locally administered Syk siRNA on allergen-induced arthritis and asthma.

New approaches for the treatment of inflammatory disorders such as rheumatic arthritis (RA) and inflammatory lung disease (asthma) are needed because a significant population of patients do not experience sustained relief with currently available therapies. The tyrosine kinase Syk plays a crucial role in inflammatory signaling pathways and has gained much attention as a potential target for treatment of inflammatory disorders. We have shown that our Syk siRNA injected directly into limb joints of arthritic mice, diminishes joint swelling and reduces levels of Syk kinase and inflammatory cytokines in joint tissue. Further, our Syk siRNA, administered via nasal instillation, inhibits recruitment of inflammatory cells to the bronchoalveolar fluid of allergen-sensitized mice. We propose that targeting Syk via localized application of Syk siRNA provides an opportunity for specific knockdown of Syk kinase with minimal potential for systemic effects.

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.