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.     

Fcγ receptors exhibit different phagocytosis potential in human neutrophils

In neutrophils, two receptors for IgG antibodies, namely FcγRIIA and FcγRIIIB are constitutively expressed, and a third one, FcγRI, can be upregulated by interferon-γ. Whether FcγRIIIB is capable of triggering phagocytosis by itself is still controversial. The main role of FcγRI has not been clearly established in these cells. To address this problem, neutrophils were treated with interferon-γ, and then phagocytosis mediated by each type of Fcγ receptor was evaluated by flow cytometry. FcγRIIA was the most efficient receptor for phagocytosis. FcγRIIIB could mediate phagocytosis but much less efficiently than FcγRIIA. Both FcγRIIA- and FcγRIIIB-mediated phagocytosis were blocked by inhibitors of Src family kinases, Syk, PI 3-K, and ERK. In contrast, interferon-γ-induced FcγRI was not able to mediate phagocytosis. Also, FcγRI did not activate ERK in the nucleus, but was however able to stimulate an efficient calcium rise. These data show that different neutrophil Fcγ receptors possess different phagocytosis capabilities: FcγRIIA and FcγRIIIB, but not FcγRI, promote phagocytosis.     

Complement receptor 3 (CD11b/CD18) mediates type I and type II phagocytosis during nonopsonic and opsonic phagocytosis,respectively

Two types of opsonic phagocytosis have been defined depending on the receptor engaged: FcgammaRs mediate type I phagocytosis of IgG-coated particles; complement receptor 3 (CR3) mediates type II phagocytosis of complement-coated particles. In addition to opsonic phagocytosis, CR3 also mediates nonopsonic phagocytosis of zymosan (Z) and Mycobacterium kansasii through engagement of distinct sites. Using Chinese hamster ovary cells stably expressing human CR3, we studied CR3-mediated ingestion of nonopsonized particles, Z or M. kansasii, compared with opsonized zymosan (OZ). We show that 1) while OZ sinks into cells, Z is engulfed by pseudopodia as visualized by electron microscopy; 2) in contrast to OZ, nonopsonic phagocytosis of Z and M. kansasii depends on Rac and Cdc42 but not on Rho activity; and 3) CR3-mediated phagocytosis of Z depends on the kinase activity of the Src family tyrosine kinase Hck, while OZ internalization does not. Therefore, CR3 mediates type I phagocytosis under nonopsonic conditions and type II under opsonic conditions. This is the first evidence that a single receptor can mediate both types of phagocytosis depending on the ligand used.     

Binding of IgG-opsonized particles to Fc gamma R is an active stage of phagocytosis that involves receptor clustering and phosphorylation

Fc gammaR mediate the phagocytosis of IgG-coated particles and the clearance of IgG immune complexes. By dissecting binding from internalization of the particles, we found that the binding stage, rather than particle internalization, triggered tyrosine phosphorylation of Fc gammaR and accompanying proteins. High amounts of Lyn kinase were found to associate with particles isolated at the binding stage from J774 cells. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an Src kinase inhibitor, but not piceatannol, an inhibitor of Syk kinase, reduced the amount of Lyn associated with the bound particles and simultaneously diminished the binding of IgG-coated particles. Studies of baby hamster kidney cells transfected with wild-type and mutant Fc gammaRIIA revealed that the ability of the receptor to bind particles was significantly reduced when phosphorylation of the receptor was abrogated by Y298F substitution in the receptor signaling motif. Under these conditions, binding of immune complexes of aggregated IgG was depressed to a lesser extent. A similar effect was exerted on the binding ability of wild-type Fc gammaRIIA by PP2. Moreover, expression of mutant kinase-inactive Lyn K275R inhibited both Fc gammaRIIA phosphorylation and IgG-opsonized particle binding. To gain insight into the mechanism by which protein tyrosine phosphorylation can control Fc gammaR-mediated binding, we investigated the efficiency of clustering of wild-type and Y298F-substituted Fc gammaRIIA upon binding of immune complexes. We found that a lack of Fc gammaRIIA phosphorylation led to an impairment of receptor clustering. The results indicate that phosphorylation of Fc gammaR and accompanying proteins, dependent on Src kinase activity, facilitates the clustering of activated receptors that is required for efficient particle binding.     

Myosin II-dependent exclusion of CD45 from the site of Fcγ receptor activation during phagocytosis

Fcγ receptor (FcγR)-mediated phagocytosis requires myosin II activity. Here we show that myosin II contributes to FcγR activation and subsequent F-actin assembly at the nascent phagocytic cup. Inhibition of myosin II attenuates phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of FcγR and binding of Syk to the ITAM. Furthermore, FcγR clusters independently of myosin II activity at the phagocytic cup, from which the receptor-like protein tyrosine phosphatase CD45 is excluded depending on myosin II activity. These findings suggest that myosin II-dependent segregation of CD45 from FcγR facilitates phosphorylation of the ITAM and triggers phagocytosis.     

Ligation of FcγR Alters Phagosomal Processing of Protein via Augmentation of NADPH Oxidase Activity

Proteolysis and the reduction of disulfides, both major components of protein degradation, are profoundly influenced by phagosomal redox conditions in macrophages. We evaluated the activation of phagocytic receptors that are known to influence activation of the phagocyte NADPH oxidase (NOX2), and its effect on phagosomal protein degradation. Population‐based and single phagosome analyses of phagosomal chemistries in murine macrophages revealed that activation of NOX2 via the Fcγ receptor (FcγR) during phagocytosis decreased rates of proteolysis and disulfide reduction. Immunoglobulin G (IgG)‐stimulated reactive oxygen species (ROS) production and the inhibition of phagosomal proteolysis and disulfide reduction were dependent on NOX2, FcγR and protein kinase C (PKC)/spleen tyrosine kinase (Syk) signaling. In contrast, low levels of ROS production were observed following the phagocytosis of unopsonized beads, which resulted in higher rates of phagosomal proteolysis and disulfide reduction. Phagosomes displayed autonomy with respect to FcγR‐mediated differences in NOX2 activation and proteolysis, as phagosomes containing unopsonized cargo retained low NOX2 activation and high proteolysis even in the presence of phagosomes containing IgG‐opsonized cargo in the same macrophage. These results show that opsonization of phagocytic cargo results in vastly different phagosomal processing of proteins through the FcγR‐triggered, PKC/Syk‐dependent local assembly and activation of NOX2.      

Interferon gamma induces actin polymerization, Rac1 activation and down regulates phagocytosis in human monocytic cells

IFNγ is a potent activator and IL-10 a powerful inhibitor of macrophage functions. However, neither all cellular functions are enhanced by IFNγ nor IL-10 inhibits all cellular responses. Thus, FcγRs-mediated phagocytosis in monocyte-derived macrophages (MDM) increases after IL-10 treatment, and decreases after treatment with IFNγ, although both IL-10 and IFNγ up regulate FcγRI expression. In this work we investigated the effect of IFNγ and IL-10 on phagocytic signaling by FcγRs in MDM. Treatment with IFNγ diminished phagocytosis of IgG-opsonized SRBC (IgG-SRBC) while treatment with IL-10 increased it. These opposite effects cannot be attributed to changes in FcγR expression induced by each cytokine. Early biochemical responses mediated by FcγRs were distinctly affected by cytokine treatment. Syk phosphorylation and the rise in [Ca²⁺]_i were higher after IL-10 treatment, whereas IFNγ treatment also increased Syk phosphorylation but had no effect on the rise in [Ca²⁺]_i. IFNγ treatment led to increased basal levels of F-actin and this effect correlated with the decrease in phagocytosis of both IgG-SRBC and non-opsonized Escherichia coli. IL-10 did not alter F-actin basal levels, and enhanced the phagocytosis of E. coli and IgG-SRBC. The level of F-actin reached after IFNγ treatment was not further increased after stimulation with IgG-SRBC or CCL5, whereas MDM treated with IL-10 showed a slightly higher response than control cells to CCL5. IFNγ increased Rac1-GTP levels. Inhibition of PI3K with LY294002 prevented IFNγ-mediated actin polymerization. Our data suggest that IFNγ induces a higher basal level of F-actin and activation of Rac1, affecting the response to stimuli that induce cytoskeleton rearrangement such as phagocytic or chemotactic stimuli.     

Leukotriene B4 augments and restores Fc gammaRs-dependent phagocytosis in macrophages

Phagocytosis by macrophages is essential for host defense, i.e. preventing invasion of pathogens and foreign materials. Macrophages engulf immunoglobulin G (IgG)-opsonized particles through the action of the receptors for the Fc of IgG (FcγRs). Leukotriene B(4) (LTB(4)) is a classical lipid chemoattractant derived from arachidonic acid. Leukotriene B(4) receptor 1 (BLT1), a high affinity LTB(4) receptor, is expressed in a variety of immune cells such as neutrophils, macrophages, and dendritic cells. Although LTB(4) has been shown to enhance macrophage phagocytosis, few studies have investigated the intracellular mechanisms involved in this in detail. Furthermore, there have been no reports of the direct cross-talk between LTB(4)-BLT1 and IgG-FcγRs signaling. Here, we show that FcγRs-dependent phagocytosis was attenuated in BLT1-deficient macrophages as compared with wild-type (WT) cells. Moreover, cross-talk between LTB(4)-BLT1 and IgG-FcγRs signaling was identified at the level of phosphatidylinositol 3-OH kinase (PI3K) and Rac, downstream of Syk. In addition, the trimeric G(i) protein (G(i)) was found to be essential for BLT1-dependent phagocytosis. Surprisingly, we found that LTB(4)-BLT1 signaling restores phagocytosis in the absence of FcγRs signaling. These data indicate that LTB(4)-BLT1 signaling plays a pivotal role in macrophage phagocytosis and innate immunity.     

Reactive oxygen intermediates enhance Fc gamma receptor signaling and amplify phagocytic capacity.

Receptors for the Fc region of IgG (Fc gamma R) mediate internalization of opsonized particles by human neutrophils (PMN) and mononuclear phagocytes. Cross-linking of Fc gamma R leads to activation of protein tyrosine kinases and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within Fc gamma R subunits, both obligatory early signals for phagocytosis. Human PMN constitutively express two structurally distinct Fc gamma R, Fc gamma RIIa and Fc gamma RIIIb, and can be induced to express Fc gamma RI by IFN-gamma. We have previously shown that stimulation of PMN through Fc gamma RIIIb results in enhanced Fc gamma RIIa-mediated phagocytic activity that is inhibited by catalase. In the present study, we have tested the hypothesis that reactive oxygen intermediates (ROI) have the capacity to regulate Fc gamma R responses and defined a mechanism for this effect. We show that H2O2 augmented phagocytosis mediated by Fc gamma RIIa and Fc gamma RI in PMN and amplified receptor-triggered tyrosine phosphorylation of Fc gamma R-associated ITAMs and signaling elements. Generation of endogenous oxidants in PMN by cross-linking Fc gamma RIIIb similarly enhanced phosphorylation of Fc gamma RIIa and Syk, a tyrosine kinase required for phagocytic function, in a catalase-sensitive manner. Our results provide a mechanism for priming phagocytes for enhanced responses to receptor-driven effects. ROI generated in an inflammatory milieu may stimulate quiescent cells to rapidly increase the magnitude of their effector function. Indeed, human monocytes incubated in the presence of stimulated PMN showed oxidant-induced increases in Fc gamma RIIa-mediated phagocytosis. Definition of the role of oxidants as amplifiers of Fc gamma R signaling identifies a target for therapeutic intervention in immune complex-mediated tissue injury.     

Immobile ligands enhance FcγR-TLR2/1 crosstalk by promoting interface overlap of receptor clusters

Innate immune cells detect pathogens through simultaneous stimulation of multiple receptors, but how cells use the receptor crosstalk to elicit context-appropriate responses is unclear. Here, we reveal that the inflammatory response of macrophages from FcγR-TLR2/1 crosstalk inversely depends on the ligand mobility within a model pathogen membrane. The mechanism is that FcγR and TLR2/1 form separate nanoclusters that interact at their interfaces during crosstalk. Less mobile ligands induce stronger interactions and more overlap between the receptor nanoclusters, leading to enhanced signaling. Different from the prevailing view that immune receptors colocalize to synergize their signaling, our results show that FcγR-TLR2/1 crosstalk occurs through interface interactions between non-colocalizing receptor nanoclusters, which are modulated by ligand mobility. This suggests a mechanism by which innate immune cells could use physical properties of ligands to fine-tune host responses.     

Differential involvement of Src family kinases in Fc gamma receptor-mediated phagocytosis

The tyrosine phosphorylation cascade originated from Fc gamma receptors (Fc gamma Rs) is essential for macrophage functions including phagocytosis. Although the initial step is ascribed to Src family tyrosine kinases, the role of individual kinases in phagocytosis signaling is still to be determined. In reconstitution experiments, we first showed that expression in the RAW 264.7 cell line of C-terminal Src kinase (Csk) inhibited and that of a membrane-anchored, gain-of-function Csk abolished the Fc gamma R-mediated signaling that leads to phagocytosis in a kinase-dependent manner. We next tested reconstruction of the signaling in the membrane-anchored, gain-of-function Csk-expressing cells by introducing Src family kinases the C-terminal negative regulatory sequence of which was replaced with a c-myc epitope. Those constructs derived from Lyn and Hck (a-Lyn and a-Hck) that associated with detergent-resistant membranes successfully reconstructed Fc gamma R-mediated Syk activation, filamentous actin rearrangement, and phagocytosis. In contrast, c-Src-derived construct (a-Src), that was excluded from detergent-resistant membranes, could not restore the series of phagocytosis signaling. Tyrosine phosphorylation of Vav and c-Cbl was restored in common by a-Lyn, a-Hck, and a-Src, but Fc gamma RIIB tyrosine phosphorylation, which is implicated in negative signaling, was reconstituted solely by a-Lyn and a-Hck. These findings suggest that Src family kinases are differentially involved in Fc gamma R-signaling and that selective kinases including Lyn and Hck are able to fully transduce phagocytotic signaling.     

Differential requirement of lipid rafts for FcγRIIA mediated effector activities

Immunoglobulin G (IgG) dependent activities are important in host defense and autoimmune diseases. Various cell types including macrophages and neutrophils contribute to pathogen destruction and tissue damage through binding of IgG to Fcγ receptors (FcγR). One member of this family, FcγRIIA, is a transmembrane glycoprotein known to mediate binding and internalization of IgG-containing targets. FcγRIIA has been observed to translocate into lipids rafts upon binding IgG-containing targets. We hypothesize that lipid rafts participate to different extents in binding and internalizing targets of different sizes. We demonstrate that disruption of lipid rafts with 8 mM methyl-β-cyclodextrin (MβCD) nearly abolishes binding (91% reduction) and phagocytosis (60% reduction) of large IgG-coated targets. Conversely, binding and internalization of small IgG-complexes is less dependent on lipid rafts (49% and 17% inhibition at 8 mM MβCD, respectively). These observations suggest that differences between phagocytosis and endocytosis may arise as early as the initial stages of ligand recognition.     

Signal transduction by human-restricted Fc gamma RIIa involves three distinct cytoplasmic kinase families leading to phagocytosis.

Recent experiments indicate an important role for Src family and Syk protein tyrosine kinases and phosphatidylinositol 3-kinase in the signal transduction process initiated by mouse receptors for IgG and leading to phagocytosis. Considerably less is known regarding signal transduction by the human-restricted IgG receptor, FcgammaRIIa. Furthermore, the relationship among the Src family, Syk, and phosphatidylinositol 3-kinase in phagocytosis is not understood. Here, we show that FcgammaRIIa is phosphorylated by an Src family member, which results in recruitment and concomitant activation of the distal enzymes Syk and phosphatidylinositol 3-kinase. Using a FcgammaRI-p85 receptor chimera cotransfected with kinase-inactive mutants of Syk or application of a pharmacological inhibitor of Syk, we show that Syk acts in parallel with phosphatidylinositol 3-kinase. Our results indicate that FcgammaRIIa-initiated monocyte or neutrophil phagocytosis proceeds from the clustered IgG receptor to Src to phosphatidylinositol 3-kinase and Syk.     

Two distinct regions of FC gamma RI initiate separate signalling pathways involved in endocytosis and phagocytosis.

Cross-linking of the high affinity receptor for IgG, Fc gamma RI, can result in both endocytosis of immune complexes and phagocytosis of opsonized particles in myeloid cells, although the cytoplasmic domain of the receptor lacks the tyrosine activation motif which has been implicated in signal transduction triggered by cross-linking of other Fc receptors. To identify the structural determinants of Fc gamma RI-mediated ligand internalization, we have expressed Fc gamma RI or truncated versions of Fc gamma RI in COS cells, either alone or in the presence of the Fc epsilon RI gamma subunit (which contains a classical tyrosine activation motif and associates with Fc gamma RI in myeloid cells), and assessed their ability to mediate endocytosis and phagocytosis. We have found that Fc gamma RI alone (in the absence of the gamma subunit) is capable of mediating endocytosis in COS cells and that the process occurs via a novel, tyrosine kinase-independent signalling pathway. Activation of this pathway following cross-linking appears to require only the receptor extracellular domain. In contrast, Fc gamma RI phagocytic function in COS cells is dependent on an interaction between the receptor transmembrane domain and the gamma subunit and is mediated by recruitment of tyrosine kinase activity. Our data therefore indicate that distinct domains of the receptor regulate ligand internalization following receptor cross-linking by either immune complexes (endocytosis) or opsonized particles (phagocytosis) and that these functions are mediated by different intracellular signalling pathways.     

Artificial Anti-Tumor Opsonizing Proteins with Fibronectin Scaffolds Engineered for Specificity to Each of the Murine FcγR Types

We have engineered a panel of novel Fn3 scaffold-based proteins that bind with high specificity and affinity to each of the individual mouse Fcγ receptors (mFcγR). These binders were expressed as fusions to anti-tumor antigen single-chain antibodies and mouse serum albumin, creating opsonizing agents that invoke only a single mFcγR response rather than the broader activity of natural Fc isotypes, as well as all previously reported Fc mutants. This panel isolated the capability of each of the four mFcγRs to contribute to macrophage phagocytosis of opsonized tumor cells and in vivo tumor growth control with these monospecific opsonizing fusion proteins. All activating receptors (mFcγRI, mFcγRIII, and mFcγRIV) were capable of driving specific tumor cell phagocytosis to an equivalent extent, while mFcγRII, the inhibitory receptor, did not drive phagocytosis. Monospecific opsonizing fusion proteins that bound mFcγRI alone controlled tumor growth to an extent similar to the most active IgG2a murine isotype. As expected, binding to the inhibitory mFcγRII did not delay tumor growth, but unexpectedly, mFcγRIII also failed to control tumor growth. mFcγRIV exhibited detectable but lesser tumor-growth control leading to less overall survival compared to mFcγRI. Interestingly, in vivo macrophage depletion demonstrates their importance in tumor control with mFcγRIV engagement, but not with mFcγRI. This panel of monospecific mFcγR-binding proteins provides a toolkit for isolating the functional effects of each mFcγR in the context of an intact immune system.     

Kinesin 5B is necessary for delivery of membrane and receptors during FcγR-mediated phagocytosis

FcγR-mediated phagocytosis is a cellular event that is evolutionary conserved to digest IgG-opsonized pathogens. Pseudopod formation during phagocytosis is a limiting step in managing the uptake of particles, and in this paper, we show that the conventional kinesin is involved in both receptor and membrane delivery to the phagocytic cup. Expression of a mutant kinesin isoform (GFP dominant negative mutant of kinesin H chain [EGFP-Kif5B-DN]) in RAW264.7 cells significantly reduced binding of IgG-sheep RBCs when macrophages were faced with multiple encounters with opsonized particles. Scanning electron microscopy analysis of EGFP-Kif5B-DN-expressing cells challenged with two rounds of IgG-sheep RBCs showed sparse, extremely thin pseudopods. We saw disrupted Rab11 trafficking to the phagocytic cup in EGFP-Kif5B-DN-transfected cells. Our particle overload assays also implicated phagosome membrane recycling in pseudopod formation. We observed reduced phagosome fission and trafficking in mutant kinesin-expressing cells, as well as reduced cell surface expression of FcγRs and Mac-1 receptors. In conclusion, anterograde trafficking via kinesin is essential for both receptor recycling from the phagosome and delivery of Rab11-containing membrane stores to effect broad and functional pseudopods during FcγR-mediated phagocytosis.     

Rho-kinase and myosin-II control phagocytic cup formation during CR,but not FcgammaR,phagocytosis

Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at CR phagosomes. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho --> ROK --> myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.     

Biological Activities of Murine Low-Affinity Fc Receptors for IgG

Murine low-affinity Fc receptors for IgG (FcγRIIbl, FcγRIIb2, and FcγRIII) bind the same IgG subclasses and are not distinguished by available anti-FcγRII/III mAbs (2.4G2). They trigger various biological activities, among which are the internalization of soluble and particulate immune complexes, cell activation, and its regulation. To determine the biological properties of the three murine receptors, each was expressed by stable transfection of corresponding cDNAs in two model cells: the murine lymphoma B cell IIA1.6 and the rat basophilic leukemia cell RBL-2H3. Biological activities of recombinant receptors were triggered with soluble immune complexes or 2.4G2 IgG in IIA1.6 cells, which express no FcγR, and with 2.4G2 Fab or F(ab′)₂, cross-linked with mouse anti-rat F(ab′)₂ in RBL, which express rat FcγR. Conditions for studying cell activation and endocytosis in both cell models are described, as are conditions for studying phagocytosis in RBL cells and antigen presentation or regulation of cell activation in IIA1.6 cells. Internalization of immune complexes was triggered by FcγRIIb2 and FcγRIII, but not by FcγRIIb1. Intracytoplasmic sequences required for phagocytosis and endocytosis could be distinguished in FcγRIIb2, but not in FcγRIII. Cell activation was restricted to FcγRIII. FcγRIII-mediated endocytosis, phagocytosis, and cell activation involved the consensus tyrosine-containing activation motif found in the intracytoplasmic domain of the γ subunit. Regulation of cell activation was induced by both FcγRII isoforms and depended on the same sequence as endocytosis. As a consequence, a single motif can determine more than one biological response of the cell, and a given response may be triggered by several motifs, borne by different FcγR.     

Lyn and Syk kinases are sequentially engaged in phagocytosis mediated by Fc gamma R

Recent data indicate that phagocytosis mediated by FcgammaRs is controlled by the Src and Syk families of protein tyrosine kinases. In this study, we demonstrate a sequential involvement of Lyn and Syk in the phagocytosis of IgG-coated particles. The particles isolated at the stage of their binding to FcgammaRs (4 degrees C) were accompanied by high amounts of Lyn, in addition to the signaling gamma-chain of FcgammaRs. Simultaneously, the particle binding induced rapid tyrosine phosphorylation of numerous proteins. During synchronized internalization of the particles induced by shifting the cell to 37 degrees C, Syk kinase and Src homology 2-containing tyrosine phosphatase-1 (SHP-1) were associated with the formed phagosomes. At this step, most of the proteins were dephosphorylated, although some underwent further tyrosine phosphorylation. Quantitative immunoelectron microscopy studies confirmed that Lyn accumulated under the plasma membrane beneath the bound particles. High amounts of the gamma-chain and tyrosine-phosphorylated proteins were also observed under the bound particles. When the particles were internalized, the gamma-chain was still detected in the region of the phagosomes, while amounts of Lyn were markedly reduced. In contrast, the vicinity of the phagosomes was heavily decorated with anti-Syk and anti-SHP-1 Abs. The local level of protein tyrosine phosphorylation was reduced. The data indicate that the accumulation of Lyn during the binding of IgG-coated particles to FcgammaRs correlated with strong tyrosine phosphorylation of numerous proteins, suggesting an initiating role for Lyn in protein phosphorylation at the onset of the phagocytosis. Syk kinase and SHP-1 phosphatase are mainly engaged at the stage of particle internalization.     

Abrogation of the Fc gamma receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides.

The role of Syk kinase in Fc gamma receptor (Fc gamma R) IIA-mediated phagocytosis was examined with two forms of antisense oligodeoxynucleotides (ODNs) designed to hybridize to human Syk mRNA. Monocytes were incubated with linear and stem-loop antisense ODNs targeted to Syk mRNA. When complexed with cationic liposomes, stem-loop Syk antisense ODN with phosphorothioate modification exhibited stability in fetal bovine and human serum. The stem-loop Syk antisense ODN at a concentration of 0.2 microM inhibited Fc gamma RIIA-mediated phagocytosis by 90% and completely eliminated Syk mRNA and protein in monocytes, whereas scrambled-control ODNs had no effect. The Syk antisense ODNs did not change beta-actin mRNA levels and Fc gamma RII cell-surface expression. In addition, stem-loop Syk antisense ODN inhibited Fc gamma RI and Fc gamma RIIIA-mediated phagocytosis. These data indicate the efficacy of stem-loop Syk antisense ODN for targeting and degrading Syk mRNA and protein and the importance of Syk kinase in Fc gamma receptor-mediated phagocytosis. Immunoblotting assay demonstrated that Fc gamma RII tyrosine phosphorylation after Fc gamma RII cross-linking did not change in the absence of Syk protein. These results indicate that Syk kinase is required for Fc gamma RIIA-mediated phagocytic signaling and that Fc gamma RII cross-linking leads to tyrosine phosphorylation of Fc gamma RII independent of Syk kinase.