Gamma RI Research Articles

Induction of Phagocytosis by a Protein Tyrosine Kinase

The transmission of extracellular signals to cellular targets by many noncatalytic surface receptors is dependent on interaction between cytoplasmic protein tyrosine kinases (PTKs) and tyrosine-containing sequences in the cytoplasmic domain of the receptor or an associated subunit. Isoforms of each of the three classes of the noncatalytic Fc gamma receptors, Fc gamma RI, Fc gamma RII, and Fc gamma RIII, are able to transmit a phagocytic signal in transfected COS-1 cells. Both Fc gamma RI and Fc gamma RIIIA require the gamma subunit for this signaling event. The protein tyrosine kinase Syk dramatically enhances phagocytosis mediated by both these receptors and increases the number of cells able to mediate phagocytosis. Two gamma chain cytoplasmic YXXL sequences are required for this effect. The action of Syk is less pronounced on the phagocytic Fc gamma RII receptor, Fc gamma RIIA, which does not require the gamma chain for phagocytosis. However, Syk allows phagocytosis by the nonphagocytic Fc gamma RII receptor Fc gamma RIIB2, which contains only a single YXXL sequence, when an additional tyrosine-containing sequence, YMTL, is introduced. These studies indicate that the efficiency of phagocytosis is markedly enhanced by the presence of a specific protein tyrosine kinase.

Activation of monocyte effector genes and STAT family transcription factors by inflammatory synovial fluid is independent of interferon gamma.

Activated monocytes play an important role in the pathogenesis of inflammatory arthritis. Blood monocytes which enter the inflamed joint become activated upon adherence to extracellular matrix and exposure to a complex inflammatory environment. We have analyzed the mechanism of monocyte activation by soluble factors present in inflammatory synovial fluid (SF). Greater than 75% of inflammatory SFs tested (a total of 22 fluids to date) increased cell surface expression and dramatically increased mRNA levels of monocyte activation markers Fc gamma RI, Fc gamma RIII, and HLA-DRA. This induction was not triggered by adherence, a known activating stimulus, and several lines of evidence showed that induction was not dependent upon interferon gamma (IFN-gamma). Induction was not prevented by neutralizing anti-IFN-gamma antibodies and IFN-gamma was not detected in the SFs using a sensitive enzyme-linked immunosorbent assay. The SFs also were not able to activate the IFN-gamma-activated transcription factor Stat1, thus providing further support for the absence of IFN-gamma. SFs did activate a related signal transducer and activator of transcription (STAT) family factor, termed Stat-SF, which bound specifically to the IFN-gamma response region (GRR), a well-characterized transcription element in the Fc gamma RI promoter. Based upon DNA-binding specificity and mobilities in gel shift assays, and reactivity with specific antisera, Stat-SF likely contains Stat3, or a closely related STAT family member. Neutralization of interleukin 6, a cytokine present in SFs which is known to activate Stat3, abolished the activation of Stat-SF and inhibited the induction of Fc gamma RI expression by SFs. These results demonstrate the activation of monocytes by inflammatory SF and suggest that monocyte activation at an inflammatory site may occur in the absence of IFN-gamma through the triggering of signal transduction pathways that activate STAT transcription factors.

Fc gamma receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent.

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. Fc gamma R recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for Fc gamma R-mediated phagocytosis, specifically Fc gamma RI(p135) and Fc gamma RIIa isoforms. The low-affinity receptor, Fc gamma RIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, Fc gamma RI has no known signaling motifs, though a functional interaction has recently been demonstrated with the gamma chain of the high-affinity receptor for IgE, Fc epsilon RI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although Fc gamma RI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either gamma chain or Fc gamma RIIa replaces that of Fc gamma RI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either Fc gamma RI or Fc gamma RIIa is replaced with that of CD2, a T cell antigen, indicate that Fc gamma R-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.

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.

Tyrosine phosphorylation and association of Syk with Fc gamma RII in monocytic THP-1 cells.

Although the cytoplasmic portion of the low-affinity receptor for immunoglobulin G, Fc gamma RII, does not contain a kinase domain, rapid tyrosine phosphorylation of intracellular substrates occurs in response to aggregation of the receptor. The use of specific tyrosine kinase inhibitors has suggested that these phosphorylations are required for subsequent cellular responses. We previously demonstrated the coprecipitation of a tyrosine kinase activity with Fc gamma RII, suggesting that non-receptor tyrosine kinases might associate with the cytoplasmic domain of Fc gamma RII. Anti-receptor immune complex kinase assays revealed the coprecipitation of several phosphoproteins, most notably p56/53lyn, an Src-family protein tyrosine kinase (PTK), and a 72 kDa phosphoprotein. Here we identify the 72 kDa Fc gamma RII-associated protein as p72syk (Syk), a member of a newly described family of non-receptor PTKs. A rapid and transient tyrosine phosphorylation of Syk was observed following Fc gamma RII activation. Syk was also tyrosyl-phosphorylated following aggregation of the high-affinity Fc gamma receptor, Fc gamma RI. The Fc gamma RI activation did not result in association of Syk with Fc gamma RII, implying that distinct pools of Syk are activated upon aggregation of each receptor in a localized manner. These results demonstrate a physical association between Syk and Fc gamma RII and suggest that the molecules involved in Fc gamma RII signalling are very similar to the ones utilized by multichain immune recognition receptors such as the B-cell antigen receptor and the high-affinity IgE receptor.

Composition of TCR-CD3 complex in human intestinal intraepithelial lymphocytes: lack of FcεRlγ chain

Human intestinal intraepithelial lymphocytes (iIEL) are a unique population of predominantly CD8 alpha beta+, TCR alpha beta+ lymphocytes and, to a lesser extent, TCP gamma delta+ lymphocytes that proliferate poorly to anti-CD3 mitogenic signals but display significant cytolytic activity. Studies in mouse model systems have shown that the gamma chain of the high-affinity receptor for IgE (Fc epsilon RI gamma) may substitute for the zeta chain in the TCR-CD3 complex of iIEL. This has suggested that the functional properties of these cells may be associated with an altered composition of the TCR-CD3 complex. We therefore analyzed the TCR-CD3 complex of normal human iIEL. One- and two-dimensional non-reducing/reducing SDS-PAGE analysis of CD3 gamma, CD3 delta, CD3 epsilon, zeta and Fc epsilon RI gamma chain immunoprecipitates of cell surface radiolabeled proteins with subunit-specific antibodies revealed a TCR-CD3 complex without associated Fc epsilon RI gamma chains. Thus, normal human iIEL contain a TCR-CD3 complex that consists predominantly of zeta homodimers in association with the alpha beta TCR and CD3 gamma, delta and epsilon, similar to the majority of peripheral lymphocytes. This indicates that the distinct properties of human iIEL are not associated with substitutions of the Fc epsilon RI gamma chain in the TCR-CD3 complex.

Oligosaccharide‐protein interactions in IgG can modulate recognition by Fcγ receptors

X-ray crystal structures of IgG-Fc provide evidence of extensive noncovalent interactions between the protein and carbohydrate moieties, and glycosylation, at Asn-297 within the Fc, has been shown to be important for effector functions mediated through Fc gamma receptors expressed on leukocytes. We have applied protein engineering in an attempt to define protein/carbohydrate interactions essential to wild-type biological activity. We demonstrate that replacement of Lys-246, Asp-249, and Glu-258, which make contacts with GlcNac and Gal on the outer alpha[1-->6] arm, do not affect recognition of human chimeric IgG3 by human Fc gamma RI and Fc gamma RII. However, replacement of Asp-265, which make contacts with the primary GlcNac sugar residue and is covalently attached to Asn-297, resulted in loss of recognition of both Fc gamma RI and Fc gamma RII. Similarly, replacement of Asp-265 in mouse IgG2b resulted in loss of recognition by mouse Fc gamma RII. These results suggest that noncovalent contacts of Asp-265 with the primary GlcNac residue are important for maintenance of recognition of IgG by Fc gamma receptors whereas contacts with GlcNac and Gal on the alpha [1-->6] arm do not have a measurable effect. This conclusion was supported by experiments in which galactose-deficient and fully galactosylated forms of a human IgG4-Fc fragment were shown to be equivalent in their ability to inhibit superoxide generation by IgG4 stimulated U937 cells.

IgG isotype-specific auto-antibodies bind preferentially to cross-linked membrane Ig.

Under equilibrium conditions, the affinities of five anti-IgG2a mAb isolated from virus-infected mice were comparable to other high-affinity auto-antibodies. Similar to rheumatoid factors, these anti-IgG2a auto-antibodies bound to aggregated or complexed IgG2a with 50 to 1500-fold higher avidity than their monomeric counterparts. Despite their high functional affinity to IgG2a, flow cytometric analysis revealed no binding or marginal mAb binding to four distinct lines of B cells expressing different densities of membrane-anchored IgG2a. If, however, surface IgG2a was cross-linked by polyclonal light chain-specific antibodies, IgM and IgA mAb binding resulted, and was detected as an increase in mean fluorescence intensity compared with isotype-matched control antibodies. The binding of one IgM mAb to cross-linked IgG2a patches of the cell surface was also visualized by confocal microscopy. Pretreatment of cells with aggregated IgG2a caused increased fluorescence intensity, demonstrating that the IgM and IgA mAb were also able to interact with IgG2a aggregates bound on the B cell surface via Fc gamma RIIB. It also permitted efficient co-ligation of the aggregated B cell receptors (BCR) with Fc gamma RIIB-fixed immune complexes known to deliver a negative signal in B cell activation. Cross-linking of IgG2a complexes bound to Fc gamma RI on macrophages or dendritic cells with antigen-specific BCR and/or T cells via their Fc gamma RIIB may accelerate the physical contact of cells involved in the antigen-specific response.(ABSTRACT TRUNCATED AT 250 WORDS)

IgG immune complexes inhibit IFN-gamma-induced transcription of the Fc gamma RI gene in human monocytes by preventing the tyrosine phosphorylation of the p91 (Stat1) transcription factor.

Immune complexes (IC) modulate Ag-driven immune responses in part by their ability to inhibit IFN-gamma-dependent MHC class II expression. Because many genes, including MHC class II Ags, transcriptionally activated by IFN-gamma require the tyrosine phosphorylation of the transcription factor p91 (Stat1), we examined whether IC could suppress IFN-gamma-induced expression of the Fc gamma receptor I gene (Fc gamma RI) in human monocytes and whether this occurred through inhibition of p91 phosphorylation. Preincubation of monocytes on gamma-globulin-coated dishes resulted in a 80% reduction in steady state levels of RNA for the Fc gamma RI gene. Nuclear run-on analysis confirmed that the inhibition was at the level of transcription. Treatment with IC resulted in no change in the IFN-gamma receptor number. In monocytes pretreated with IC, there was a 79% reduction in the formation of FcRF gamma, a p91-containing DNA binding protein complex that is rapidly activated by IFN-gamma, and which recognizes the gamma response region enhancer within the promoter of the Fc gamma RI gene. Furthermore, there was a marked reduction in the tyrosine phosphorylation of p91. Pretreatment with IC resulted in the inhibition of the tyrosine phosphorylation of the tyrosine kinases, Jak1 and Jak2, both of which are involved in IFN-gamma signal transduction. Therefore, culture of monocytes on IC inhibits IFN-gamma-induced expression of the Fc gamma RI gene by preventing tyrosine phosphorylation of p91, probably by the associated inhibition of the tyrosine kinases Jak1 and Jak2.

Fcγ‐receptor activity in the developing human placenta

The expression of Fc gamma receptors (Fc gamma R) and annexin II in 20 placentae (range 8-27 weeks' gestation) and in 3 full-term placentae was studied using monoclonal antibodies (mAbs) and soluble immune complexes. Functionally active Fc gamma R was detected on the trophoblast, endothelial cells, and stromal cells (Hofbauer cells) from the 8th week of gestation. Fc gamma RI (mAb 32.2) and Fc gamma RIII (mAb 3G8) were detected only on Hofbauer cells, whereas Fc gamma RII (IV3 and C1KM5) were detected both on Hofbauer cells and endothelial cells. Fc gamma RIII (anti-Leu-11b) and the IgG-binding molecule annexin II (mAb B1D6) were expressed by Hofbauer cells, endothelial cells, and the trophoblast. There was some variation in staining among the different specimens, but the number of positive cells as well as the staining intensity increased from the first to the second trimester. In first trimester placenta, staining was localized both to the syncytio- and cytotrophoblast, with the strongest intensity in the cytotrophoblast and at the boundary between the two cell layers. In second and third trimester placenta, staining was localized to the syncytiotrophoblast. The localization and distribution of Fc gamma R on the trophoblast during ontogeny is of interest with regard to its presumed role in the transport of IgG from mother to fetus.

The interaction between human Fc gamma RI and the gamma-chain is mediated solely via the 21 amino acid transmembrane domain of Fc gamma RI.

We have established a biological assay to investigate the nature of the non-covalent interaction between two integral type I membrane proteins, Fc gamma RI and gamma-chain. Fc gamma RI, the human high affinity receptor for immunoglobulin G (IgG), is expressed on the surface of macrophages and monocytes and mediates a broad range of important immunological functions. Fc gamma RI relies on a functional interaction with a second integral type I membrane protein, gamma-chain, to mediate many of these functions. For example, Fc gamma RI can only mediate phagocytosis of IgG-coated particles in COS cells when co-expressed with gamma-chain. We have previously shown that the cytoplasmic domain of Fc gamma RI is not necessary for this functional interaction. In this study we have used the phagocytosis assay to investigate the role of the transmembrane region of Fc gamma RI in mediating this functional interaction with gamma-chain by using mutant and chimeric forms of the receptor. Three mutants, which introduce or remove charged residues from a conserved 10 amino acid stretch of amino acids in the proximal transmembrane region of Fc gamma RI, were able to mediate phagocytosis of IgG-coated particles. In contrast, two chimeric receptors, In which 21 of the amino acids in the distal transmembrane region of Fc gamma RI were replaced with the transmembrane region of the related receptors CD2 or LFA3, were expressed but failed to interact functionally with gamma-chain to mediate phagocytosis. Thus, these mutants demonstrate that the interaction between human Fc gamma RI and gamma-chain is mediated solely via these 21 amino acids in the transmembrane domain of Fc gamma RI.

Lipopolysaccharide-dependent induction of IL-10 receptor expression on murine fibroblasts.

Although various biologic activities of IL-10 have been identified, little is known about IL-10's molecular mechanism of action. Herein we report the characterization of IL-10R on different murine cell lines and demonstrate the LPS inducibility of this protein. With the use of purified recombinant murine IL-10 and labeled IL-10-specific mAb, IL-10Rs were detected by flow cytometry. Radioligand binding analyses showed that RAW264.7 cells expressed 238 +/- 87 receptors per cell and bound ligand with a single affinity of 8.3 +/- 2.4 x 10(9) M-1. Similar studies documented IL-10R expression on murine B cells (CH27) and CD4+ Th1 cells but not murine Th2 cells, L929 or WA-17 fibroblasts, or fibrosarcoma cells. Exposure of fibroblasts to LPS-induced cellular IL-10 binding activity in a dose- and time-dependent manner. Radioligand binding analyses performed on LPS-treated fibroblasts showed cellular expression of 325 +/- 59 IL-10 binding sites and a binding affinity of Ka = 7.5 +/- 2.5 x 10(8) M-1. RT-PCR analysis confirmed the induction of the IL-10R. Functional analyses of IL-10R-expressing cells revealed that IL-10 activated a cellular transcription factor in RAW264.7 cells that bound a DNA probe containing the IFN-gamma response region from the Fc gamma RI gene. In contrast, IL-10 did not induce gamma response region binding activity in L929 fibroblasts either treated with LPS or transfected with the murine IL-10R cDNA. These results thus indicate that cellular responses to IL-10 may be influenced by both the external environment and the presence of additional signaling components within the cell.

Protein-tyrosine kinase p72syk in Fc gamma RI receptor signaling

In this report we show that gamma-interferon (IFN) induces the expression of the nonreceptor protein tyrosine kinase, p72syk, and that cross-linking the Fc gamma RI receptor in IFN-differentiated U937 cells (U937IF cells) results in the activation of syk kinase. We show that syk is tyrosine phosphorylated (12-fold increase) after Fc gamma RI cross-linking. In vitro kinase assays demonstrate that the specific kinase activity of syk increased eightfold after Fc gamma RI cross- linking. The activation of signal transduction through the Fc gamma RI receptor, as measured by the respiratory burst, is associated with the tyrosine phosphorylation and catalytic activation of the syk kinase. We show that syk coprecipitates with the gamma subunit of the Fc gamma RI, Fc gamma RI gamma. The data suggest that p72syk is involved in signal transduction through the Fc gamma RI receptor, involving the Fc gamma RI gamma subunit.

Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes

Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes.

Association of IgA-Fc receptors (Fc alpha R) with Fc epsilon RI gamma 2 subunits in U937 cells. Aggregation induces the tyrosine phosphorylation of gamma 2.

We investigated the possibility that IgA-binding chains of Fc alpha R on monocytic cells are physically associated with gamma 2 subunits of Fc epsilon RI (Fc epsilon RI gamma 2 or gamma 2). Fc alpha R was precipitated from lysates of IFN gamma-treated U937 cells, subclone 10.6, and probed by immunoblotting with Ab against human gamma 2. Fc alpha R was precipitated through anti-Fc alpha R mAbs A59 or A62, through A62 from lysates that had been exhaustively precleared of high affinity IgG-Fc receptors (Fc gamma RI) and of low affinity Fc gamma RII, and through anti-Fc alpha R mAb A77 from Fc gamma RI-precleared lysates of untreated 10.6 cells. precipitation was also performed through F(ab')2 A77 and through the native ligand of the receptor, hlgA. In all cases, Fc alpha R precipitates contained co-isolated 22-kDa gamma 2 (unreduced). The Fc alpha R alpha-chain/gamma 2 complex did not readily dissociate in 1% Nonidet P-40 as did Fc gamma RI alpha-chain/gamma 2, suggesting a novel aspect to the Fc alpha R subunit interaction. Specific Fc alpha R aggregation on cells triggered a robust respiratory burst and the tyrosine phosphorylation of several proteins. Among them was phospho-gamma 2, which migrated as a 24- to 28-kDa gamma 2 phosphoprotein on gels and was detected as a 28-kDa phosphoprotein by anti-phosphotyrosine immunoblot. Aggregated Fc alpha Rs that were precipitated from Fc alpha R-triggered cells also contained a phosphoprotein of the same mobility and immunoreactivity, as did aggregated Fc gamma RI from which the 28-kDa phosphoprotein could be more readily eluted and identified (as phospho-gamma 2). We concluded that myelocytic Fc alpha Rs are multichain complexes containing gamma 2 subunits that are tyrosine phosphorylated upon Fc alpha R aggregation. As IgA is the predominant Ig on mucosal surfaces, gamma-subunits may play an important role in mucosal immunity involving leukocytic Fc alpha R.

Protein-Tyrosine Kinase p72syk in FcγRI Receptor Signaling

In this report we show that gamma-interferon (IFN) induces the expression of the nonreceptor protein tyrosine kinase, p72syk, and that cross-linking the Fc gamma RI receptor in IFN-differentiated U937 cells (U937IF cells) results in the activation of syk kinase. We show that syk is tyrosine phosphorylated (12-fold increase) after Fc gamma RI cross-linking. In vitro kinase assays demonstrate that the specific kinase activity of syk increased eightfold after Fc gamma RI cross-linking. The activation of signal transduction through the Fc gamma RI receptor, as measured by the respiratory burst, is associated with the tyrosine phosphorylation and catalytic activation of the syk kinase. We show that syk coprecipitates with the gamma subunit of the Fc gamma RI, Fc gamma RI gamma. The data suggest that p72syk is involved in signal transduction through the Fc gamma RI receptor, involving the Fc gamma RI gamma subunit.

Physical and functional association of the high affinity immunoglobulin G receptor (Fc gamma RI) with the kinases Hck and Lyn.

The high affinity immunoglobulin G (IgG) receptor Fc gamma RI (CD64) is expressed constitutively on monocytes and macrophages, and is inducible on neutrophils. Fc gamma RI has recently been shown to be associated with the signal transducing gamma subunit of the high-affinity IgE receptor (Fc epsilon RI gamma). Induction of cytoplasmic protein tyrosine phosphorylation by Fc gamma RI cross-linking is known to be important in mediating Fc gamma RI-coupled effector functions. Recently, syk has been implicated in this role. We now report that the src-type kinases hck and lyn are physically and functionally associated with Fc gamma RI. Hck and lyn coimmunoprecipitated with Fc gamma RI from detergent lysates of normal human monocytes and of the monocytic line THP-1. Hck and lyn showed rapidly increased phosphorylation and increased exogenous substrate kinase activity after cross-linking of Fc gamma RI. These results demonstrate both physical and functional association of the Fc gamma RI/Fc epsilon RI gamma receptor complex with hck and lyn, and suggest a potential signal transducing role for these kinases in monocyte/macrophage activation.

Binding of monomeric immunoglobulin G triggers Fc gamma RI-mediated endocytosis.

Cross-linking of leukocyte Fc receptors specific for IgG (Fc gamma Rs) by multivalent IgG complexes triggers a wide range of immune functions. Many of these responses can also be stimulated in vitro using anti-Fc gamma R monoclonal antibody-containing complexes. This observation has suggested that cross-linking is the key event and that binding of IgG, which in itself does not elicit a response, is functionally passive. However, in this study we show that binding of monomeric IgG to the human high affinity receptor, Fc gamma RI, is itself sufficient to permit the receptor to enter an internalization-recycling pathway, which has a small intracellular pool. Unoccupied Fc gamma RI is not internalized and recycled in this manner. This finding may be explained by the previous observation that there is a physical association between Fc gamma RI and the cytoskeletal component, actin-binding protein (non-muscle filamin; ABP-280), which is disrupted upon IgG binding. Thus, in the absence of IgG, Fc gamma RI may be physically excluded from the endocytic pathway by tethering to the cytoskeleton. The role of cross-linking is to divert Fc gamma RI-IgG complexes from the recycling pathway, causing their retention and subsequent degradation within the cell. In contrast to Fc gamma RII-mediated endocytosis, intracellular accumulation of cross-linked Fc gamma RI-IgG complexes is not sensitive to inhibition by genistein, suggesting that the process is independent of tyrosine kinase activity.

Involvement of phosphatidylinositol 3-kinase in Fc gamma receptor signaling.

Wortmannin, a potent and selective inhibitor of phosphatidylinositol (PI) 3-kinase (Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3563-3567), prevented Fc receptor for IgG (Fc gamma R)-dependent phagocytosis of the human monocytic cell line U937 or guinea pig neutrophils. Cross-linking of Fc gamma R on the surface of U937 cells increased PI 3-kinase activity that was immunoprecipitated with antibody against phosphotyrosine or antibody against the 85-kDa regulatory subunit of PI 3-kinase. Specific cross-linking of Fc gamma R subclass Fc gamma RI or Fc gamma RII, using monoclonal antibodies against each receptor subclass and the F(ab')2 fragment of goat antibody against mouse IgG, increased anti-phosphotyrosine-precipitable PI 3-kinase activity. Treatment of cells with anti-Fc gamma RIII antibody plus the same F(ab')2 did not affect the activity, reflecting the lack of Fc gamma RIII in U937 cells. Fcy gamma R stimulation triggered prominent tyrosine phosphorylation of several proteins, among which the 115-kDa peptide showed strong association with PI 3-kinase. Thus, Fc gamma R appears to be coupled functionally, via a tyrosine kinase, to PI 3-kinase, which may regulate the phagocytotic activity of the cells.

Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1.

Immunoglobulin G (IgG) molecules are glycosylated in CH2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions. The carbohydrate attached to the IgG constant region is a complex biantennary structure. Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis. To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps. We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1. The Pro-5-produced Ab (IgG1-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complement-mediated cytolysis, and affinity for Fc gamma RI. Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, C1q binding, and C1 activation. IgG1-Lec 1 also showed reduced but significant affinity for Fc gamma R1 receptors. The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma- or Pro-5-produced counterpart, with more being cleared during the alpha-phase and with more rapid clearance during the beta-phase. Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan. Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide. Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure. Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function.