Immunoglobulin G1 Fc Domain Motions: Implications for Fc Engineering

The fragment crystallizable (Fc) region links the key pathogen identification and destruction properties of immunoglobulin G (IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fcγ receptors (FcγRs) on immune cells. The cellular response and committal to a damaging, though protective, immune response are tightly controlled at multiple levels. Control mechanisms are diverse and in many cases unclear, but one frequently suggested contribution originates in FcγR affinity being modulated through shifts in Fc conformational sampling. Here, we report a previously unseen IgG1 Fc conformation. This observation motivated an extensive molecular dynamics investigation of polypeptide and glycan motions that revealed greater amplitude of motion for the N-terminal Cγ2 domains and N-glycan than previously observed. Residues in the Cγ2/Cγ3 interface and disulfide-bonded hinge were identified as influencing the Cγ2 motion. Our results are consistent with a model of Fc that is structurally dynamic. Conformational states that are competent to bind immune-stimulating FcγRs interconverted with Fc conformations distinct from those observed in FcγR complexes, which may represent a transient, nonbinding population.     

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.     

C5a-regulated CCAAT/enhancer-binding proteins β and δ are essential in Fcγ receptor-mediated inflammatory cytokine and chemokine production in macrophages

CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPδ are known to participate in the regulation of many genes associated with inflammation. However, little is known about the activation and function of C/EBPβ and -δ in inflammatory responses elicited by Fcγ receptor (FcγR) activation. Here we show that C/EBPβ and -δ activation are induced in IgG immune complex (IC)-treated macrophages. The increased expression of C/EBPβ and -δ occurred at both mRNA and protein levels. Furthermore, induction of C/EBPβ and -δ was mediated, to a large extent, by activating FcγRs. Using siRNA-mediated knockdown as well as macrophages deficient for C/EBPβ and/or -δ, we demonstrate that C/EBPβ and -δ play a critical role in the production of TNF-α, MIP-2, and MIP-1α in IgG IC-stimulated macrophages. Moreover, both ERK1/2 and p38 MAPK are involved in C/EBP induction and TNF-α, MIP-2, and MIP-1α production induced by IgG IC. We provide the evidence that C5a regulates IgG IC-induced inflammatory responses by enhancing ERK1/2 and p38 MAPK activities as well as C/EBPβ and -δ activities. Collectively, these data suggest that C/EBPβ and -δ are key regulators for FcγR-mediated induction of cytokines and chemokines in macrophages. Furthermore, C/EBPs may play an important regulatory role in IC-associated inflammatory responses.     

Important roles of CD32 in promoting suppression of IL-4 induced immune responses by a novel anti-IL-4Rα therapeutic antibody

ABSTRACT

Asthma is characterized by airway hyperresponsiveness and inflammation, as well as underlying structural changes to the airways. Interleukin-4 (IL-4) is a key T-helper type 2 (Th2) cytokine that plays important roles in the pathogenesis of atopic and eosinophilic asthma. We developed a novel humanized anti-IL-4Rα antibody that can potently inhibit IL-4/IL-13-mediated TF-1 cell proliferation. Using monocytes isolated from human peripheral blood mononuclear cells (PBMCs), we revealed a critical role of CD32 in modulating the immune responses of monocytes in response to blockade of IL-4Rα signaling pathway. We, therefore, devised a new strategy to increase the efficacy of the anti-IL-4Rα monoclonal antibody for the treatment of asthma and other atopic diseases by co-engaging CD32 and IL-4Rα on monocytic cells by choosing IgG classes or Fc mutations with higher affinities for CD32. The antibody with selectively enhanced affinity for CD32A displayed superior suppression of IL-4-induced monocytes’ activities, including the down-regulation of CD23 expression. Intriguingly, further analysis demonstrated that both CD32A and CD32B contributed to the enhancement of antibody-mediated suppression of CD23 expression from monocytes in response to blockade of IL-4Rα signaling. Furthermore, inhibition of IgE secretion from human PBMC by the antibody variants further suggests that the complex allergic inflammation mediated by IL-4/IL-4Rα signaling might result from a global network where multiple cell types that express multiple FcγRs are all involved, of which CD32, especially CD32A, is a key mediator. In this respect, our study provides new insights into designing therapeutic antibodies for targeting Th2 cytokine-mediated allergic pathogenesis.     

Dimers and multimers of monoclonal IgG1 exhibit higher in vitro binding affinities to Fcγ receptors

The in vitro binding of monomeric, dimeric and multimeric forms of monoclonal IgG1 molecules, designated mAb1 and mAb2, to the extracellular domains of Fcγ receptors RI, RIIA and RIIIB were investigated using a surface plasmon resonance (SPR) based biosensor technique. Stable noncovalent and covalent dimers of mAb1 and mAb2, respectively, were isolated from CHO cell expressed materials. The dissociation constants of monomeric mAb1 and mAb2 were determined to be 1 nM for the FcγRI-binding and 6–12 μM for the FcγRIIA- and FcγRIIIB-binding. Dimeric mAb1 and mAb2 exhibited increased affinities, by 2-3 fold for FcγRI and 200-800 fold for FcγRIIA and FcγRIIIB. Further increases in binding were observed when the antibodies formed large immune complexes with multivalent antigens, but not in a linear relation with size. The binding properties of monomeric mAb2 were identical with and without a bound monovalent antigen, indicating that antigen-binding alone does not induce measurable change in binding of antibodies to Fcγ receptors. Dimerization is sufficient to show enhancement in the receptor binding. Given the wide distribution of the low-affinity Fcγ receptors on immune effector cells, the increased affinities to aggregated IgG may lead to some biological consequences, depending on the subsequent signal transduction events. The SPR-based in vitro binding assay is useful in evaluating Fcγ receptor binding of various species in antibody-based biotherapeutics.     

IgG4 can induce an M2-like phenotype in human monocyte-derived macrophages through FcγRI

Antibodies evoke cellular responses through the binding of their Fc region to Fc receptors, most of which contain immunoreceptor tyrosine-based activation motif domains and are thus considered “activating.” However, there is a growing appreciation of these receptors for their ability to deliver an inhibitory signal as well. We previously described one such phenomenon whereby interferon (IFN)γ signaling is inhibited by immune complex signaling through FcγRI. To understand the implications of this in the context of therapeutic antibodies, we assessed individual IgG subclasses to determine their ability to deliver this anti-inflammatory signal in monocyte-derived macrophages. Like IgG1, we found that IgG4 is fully capable of inhibiting IFNγ-mediated events. In addition, F(ab’)2 fragments that interfere with FcγRI signaling reversed this effect. For mAbs developed with either an IgG1 or an IgG4 constant region for indications where inflammation is undesirable, further examination of a potential Fc-dependent contribution to their mechanism of action is warranted.     

Fc receptors on monocytes cause OKT3-treated lymphocytes to internalize T3 and to secrete IL-2

Monocytes cause OKT3-treated T cells to secrete IL-2 and to lose cell surface T3. We have studied the fate of the "lost" T3. Immunofluorescence microscopy on permeabilized cells showed that monocytes induce T cells to internalize T3. Furthermore, experiments with radioiodinated T cells showed that the internalized T3 was not degraded and exhibited an unaltered polypeptide composition for at least 16 hr. The role of Fc receptors in inducing internalization was also investigated. Fc receptors were depleted from monocytes by allowing the phagocytes to spread on immune complexes. Such depleted monocytes exhibit a fourfold reduction in their ability to promote both internalization of T3 and production of IL-2. A comparable reduction is seen if F(ab')2 fragments of OKT3 were employed in place of intact IgG. Furthermore, monocyte Fc receptors that have been blocked by heat-aggregated human IgG also show much reduced capability for induction of OKT3-mediated T-cell proliferation. We therefore conclude that Fc receptors bind to the Fc domain of OKT3 and thereby cause OKT3-treated T cells to internalize T3 and become activated.     

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.     

From Rhesus macaque to human: structural evolutionary pathways for immunoglobulin G subclasses

The Old World monkey, Rhesus macaque (Macaca mulatta, Mm), is frequently used as a primate model organism in the study of human disease and to test new vaccines/antibody treatments despite diverging before chimpanzees and orangutans. Mm and humans share 93% genome identity with substantial differences in the genes of the adaptive immune system that lead to different functional IgG subclass characteristics, Fcγ receptors expressed on innate immune cells, and biological interactions. These differences put limitations on Mm use as a primary animal model in the study of human disease and to test new vaccines/antibody treatments. Here, we comprehensively analyzed molecular properties of the Fc domain of the four IgG subclasses of Rhesus macaque to describe potential mechanisms for their interactions with effector cell Fc receptors. Our studies revealed less diversity in the overall structure among the Mm IgG Fc, with MmIgG1 Fc being the most structurally like human IgG3, although its C_H2 loops and N²⁹⁷ glycan mobility are comparable to human IgG1. Furthermore, the Fcs of Mm IgG3 and 4 lack the structural properties typical for their human orthologues that determine IgG3’s reduced interaction with the neonatal receptor and IgG4’s ability for Fab-arm exchange and its weaker Fcγ receptor interactions. Taken together, our data indicate that MmIgG1-4 are less structurally divergent than the human IgGs, with only MmIgG1 matching the molecular properties of human IgG1 and 3, the most active IgGs in terms of Fcγ receptor binding and Fc-mediated functions. PDB accession numbers for deposited structures are 6D4E, 6D4I, 6D4M, and 6D4N for MmIgG1 Fc, MmIgG2 Fc, MmIgG3 Fc, and MmIgG4 Fc, respectively.     

Resveratrol enhances TNF-α production in human monocytes upon bacterial stimulation

Background

Resveratrol is a key component of red wine that has been reported to have anti-carcinogenic and anti-aging properties. Additional studies conducted in vitro and in animal models suggested anti-inflammatory properties. However, data from primary human immune cells and in vivo studies are limited.

Methods

A pilot study was performed including 10 healthy volunteers. Plasma cytokine levels were measured over 48 h after oral application of 5 g resveratrol.To verify the in vivo findings, cytokine release and gene expression in human peripheral blood mononuclear cells (PBMC) and/or monocytes was assessed after treatment with resveratrol or its metabolites and stimulation with several toll-like receptor (TLR)-agonists. Additionally, the impact on intracellular signaling pathways was analyzed using a reporter cell line and Western blotting.

Results

Resveratrol treated individuals showed a significant increase in tumor necrosis factor-α (TNF-α) levels 24 h after treatment compared to baseline. Studies using human PBMC or isolated monocytes confirmed potentiation of TNF-α production with different TLR agonists, while interleukin (IL)-10 was inhibited. Moreover, we observed significantly enhanced nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB) activation using a reporter cell line and found increased phosphorylation of p105, which is indicative of alternative NF-κB pathway activation.

General significance

By administering resveratrol to healthy humans and utilizing primary immune cells we were able to detect TNF-α enhancing properties of the agent. In parallel, we found enhanced alternative NF-κB activation. We report on a novel pro-inflammatory property of resveratrol which has to be considered in concepts of its biologic activity.     

Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes

We have studied the Fc receptor-mediated pinocytosis of immunoglobulin G (IgG)-containing immune complexes by mouse macrophages. IgG complexes were formed from affinity-purified rabbit dinitrophenyl IgG and dinitrophenyl modified BSA at molar ratios of 2.5-10:1. Both the specificity of binding and the fate of internalized receptors were analyzed using monoclonal and polyclonal anti-Fc receptor antibodies. Based on the susceptibility of surface-bound ligand to release by proteolysis, we have found that at 37 degrees C, 125I-labeled IgG complexes were rapidly internalized (t1/2 less than 2 min) and delivered to lysosomes; acid-soluble 125I was detectable in the growth medium within 5-10 min of uptake. However, kinetic evidence indicated that Fc receptors were not efficiently re-used for multiple rounds of ligand uptake. Instead, macrophages that were exposed continuously to saturating concentrations of IgG complexes exhibited a selective and largely irreversible removal of Fc receptors from the plasma membrane. This loss of surface receptors correlated with an increased rate of receptor turnover, determined by immune precipitation of Fc receptors from 125I-labeled macrophages. Thus, in contrast to the results obtained in the accompanying paper (I. Mellman, H. Plutner, and P. Ukkonen, 1984, J. Cell Biol. 98:1163-1169) using a monovalent ligand, these data indicate that the interaction of Fc receptors with polyvalent complexes leads to the degradation of both ligand and receptor following their delivery to lysosomes.     

Licorice-derived dehydroglyasperin C increases MKP-1 expression and suppresses inflammation-mediated neurodegeneration

Recent studies have demonstrated that microglial hyperactivation-mediated neuroinflammation is involved in the pathogenesis of several neurodegenerative diseases. Thus, inhibiting microglial production of the neurotoxic mediator tumor necrosis factor-α (TNF-α) is considered a promising strategy to protect against neurodegeneration. Here, we investigated the inhibitory effect of licorice-derived dehydroglyasperin C (DGC) on lipopolysaccharide (LPS)-induced TNF-α production and inflammation-mediated neurodegeneration. We found that DGC pre-treatment attenuated TNF-α production in response to LPS stimulation of BV-2 microglia. DGC pre-treatment attenuated LPS-induced inhibitor of κB-α (IκB-α) and p65 phosphorylation and decreased the DNA binding activity of nuclear factor-κB (NF-κB). DGC pre-treatment also inhibited LPS-mediated phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinase (ERK). Interestingly, DGC treatment of BV-2 microglia significantly increased MAPK phosphatase 1 (MKP-1) mRNA and protein expression, which is a phosphatase of p38 MAPK and ERK, suggesting that the DGC-mediated increase in MKP-1 expression might inhibit LPS-induced MAPKs and NF-κB activation and further TNF-α production. We also found that LPS-mediated microglial neurotoxicity can be attenuated by DGC. The addition of conditioned media (CM) from DGC- and LPS-treated microglia to neurons helped maintain healthy cell body and neurite morphology and increased the number of microtubule-associated protein 2-positive cells and the level of synaptophysin compared to treatment with CM from LPS-treated microglia. Taken together, these data suggest that DGC isolated from licorice may inhibit microglia hyperactivation by increasing MKP-1 expression and acting as a potent anti-neurodegenerative agent.     

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.     

p38-Mediated phosphorylation of Eps15 endocytic adaptor protein

Epidermal growth factor receptor pathway substrate 15 (Eps15) has been suggested to be involved in the endocytosis of cell surface receptors, including epidermal growth factor receptor (EGFR). Eps15 is phosphorylated at Tyr-849 upon stimulation with EGF during endocytic processes. In the present study, we found that stimulation of HeLa cells with EGF or TNF-α induced transient phosphorylation of Eps15 at Ser-796. Inhibition of p38 completely blocked phosphorylation and recombinant p38α directly phosphorylated the residue. These results demonstrate a novel stress kinase-mediated signaling pathway to Eps15 endocytic adapter protein.     

TNF receptor II fusion protein with tandemly repeated Fc domains

The extracellular domain of tumour necrosis factor (TNF) receptor II fused with the human IgG1 Fc region (TNFRII-Fc), as well as antibodies against TNF, has been used to treat rheumatoid arthritis. However, TNFRII-Fc is less effective than these antibodies in terms of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against cells bearing TNF on the cell surface. We hypothesized that these activities could be increased by fusing TNFRII with tandemly repeated Fc (TNFRII-Fc-Fc). The affinities of TNFRII-Fc-Fc for soluble TNF-α and transmembrane TNF-α and the TNF-α cytotoxicity-inhibitory activity were as potent as those of TNFRII-Fc. TNFRII-Fc-Fc showed much higher binding avidity for Fcγ receptors than TNFRII-Fc and was more potent in terms of both ADCC and CDC against cells expressing transmembrane TNF-α. TNFRII-Fc-Fc of 80 kDa, as well as TNFRII-Fc-Fc of 200 kDa, was detected. TNFRII-Fc-Fc (80 kDa) was as potent as TNFRII-Fc in terms of both ADCC and CDC. These results suggest that Fc multimerization of receptor-Fc fusion proteins can augment effector functions such as ADCC and CDC, and thereby have the potential to provide a superior therapeutic effect. This may be the case not only for TNFRII-Fc but also for other receptor-Fc fusion proteins.     

Efficient expression and purification of human aglycosylated Fcγ receptors in Escherichia coli

Effector Fc gamma receptors (FcγRs) are expressed on the surface of a variety of cells of hematopoietic lineage and serve as a bridge between adaptive and innate immune responses. The interaction between immune complexes, formed by IgG class antibodies that are crosslinked with antigen, and FcγRs triggers signaling cascades that result in numerous cellular responses including the activation or donwregulation of cytotoxic responses, cytokine release, and antibody synthesis. Here, the extracellular domains of the human type I transmembrane FcγRs were expressed in Escherichia coli and their interactions to subclass IgGs (IgG1, IgG2, IgG3, and IgG4) antibodies were analyzed. Expression using fully synthetic E. coli codon optimized FcγR genes and optimization of sequences for N‐terminal translation initiation region through mRNA secondary structure prediction enabled us to achieve high yield of purified, bacterially expressed receptors, including FcγRI and FcγRIIIa which have not been successfully expressed in bacteria until now. The aglycosylated FcγRs showed similar IgG subclass binding selectivity compared to the respective glycosylated FcγRs expressed in mammalian cells. Biotechnol. Bioeng. 2010;107: 21–30. © 2010 Wiley Periodicals, Inc.     

Ligation of Fc receptor of macrophages stimulates protein kinase C and antileishmanial activity

Fc receptors are known to express on the surface of mature monocytes macrophages and lymphocytes. In this study a ligand e.g. liposomal IgG (human IgG coupled to PE-liposome via carbodimide reaction) was developed to ligate the Fc receptor of macrophages. When liposomal IgG was incubated with macrophages at 37°C for 5 min, it induced the macrophage activation which suppress the parasite burden approximatley to an extent of 60%, 50% and 45%, when macrophages were infected with UR6, AG83 and GE1 strains of L-donovani respectively. Superior efficacy of liposomal IgG were achieved compared to the treatment with free IgG and free liposomes. The activity of protein kinase C (PKC) has been found to be higher in the Fc receptor targeted macrophage membrane fraction, suggesting its translocation from the cytosol. Staurosporine, a potent inhibitor of the enzyme protein kinase C (PKC) has been found to protect the parasite inside the macrophage indicating the role of PKC in the signaling process. The liposomal IgG treatment has been found to induce the generation of significant amount of superoxide and hydrogen peroxide which helped to suppress the parasite burden. Further when liposomal IgG were incubated with IFN- primed, LPS activated macrophages, a significant amount of NO release was also noticed, indicating its role in parasite killing. The above results suggest that Fc receptor mediated activation by liposomal IgG may be used as an alternative approach to kill parasites intracellularly.     

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.