Functional characteristics of the high affinity IgG receptor, FcγRI

IgG FcRs are important mediators of immunity and play a key role during Ab-based immunotherapy. Within the leukocyte IgG receptor family, only FcγRI is capable of IgG binding with high affinity. FcγRI exists as a complex of a ligand binding α-chain and an FcR γ-chain. The receptors' α-chain can, furthermore, elicit several functions independent of the ITAM-bearing FcR γ-chain. Functional implications of high-affinity IgG binding and mechanisms underlying FcR γ-chain-independent signaling remain unclear to this day. In this paper, we provide an overview of past literature on FcγRI and address the implications of recently described interactions between cytosolic proteins and the FcγRI α-chain, as well as cytokine-enhanced FcγRI immune complex binding. Furthermore, an analysis of potential polymorphisms within the FCGR1A gene is provided.     

Automated hydrodynamic modelling of a complex between a human IgE fragment (FcÂ3–4) and the IgE high affinity receptor FcÂRI ⋅-chain

The binding of IgE to its high affinity receptor FcεRI plays an important role in the allergic response. The interaction between soluble FcεRIα-chain (sFcεRIα) and Fcε3–4, a fragment of IgE consisting of the Cε3 and Cε4 heavy chain constant domains, has been studied using analytical ultracentrifugation (Keown et al. this volume). Here we describe the development of a simple automated hydrodynamic modelling technique and its application to this interaction. This procedure utilises sphere models of the two molecules and performs an automated systematic translational search of sFcεRIα relative to Fcε3–4. The result of this is the generation of 40,359 individual models of how the receptor can be placed relative to Fcε3–4. These are then assessed for consistency by comparing the sedimentation coefficients generated for the models to the experimentally determined sedimentation coefficients, and are displayed graphically to show allowed and disallowed complexes. From this analysis, it is clear that the complex between sFcεRIα and Fcε3–4 is compact, with the most elongated models being excluded. In addition, sFcεRIα appears not to interact with the C-terminal end of Fcε3–4, and probably binds either to the sides or face, observations which are consistent with other experimental data on the FcεRIα/IgE interaction. Automated hydrodynamic modelling also has the potential to be used for other interactions, providing a simple way of looking at a large number of models, and making rigorous studies of interacting components more feasible. Received: 31 July 1996 / Accepted: 1 December 1996     

Basis of the 1:1 stoichiometry of the high affinity receptor FcεRI-IgE complex

A soluble fragment of the high-affinity IgE receptor FcεRI α-chain (sFcεRIα) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the Cε2, Cε3 and Cε4 domains of the ε-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG₁, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the Cε2 and Cε3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging Cε2 domains. To test this hypothesis we have expressed a recombinant ε-chain fragment containing Cε3 and Cε4. This product, Fcε3–4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface FcεRI. Titration experiments, together with molecular mass measurements of the Fcε3–4/sFcεRIα complex, reveal that Fcε3–4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by Cε2 accounts for the unexpected stoichiometry. Received: 31 July 1996 / Accepted: 1 December 1996     

Efficient expression of recombinant soluble human FcγRI in mammalian cells and its characterization

The extracellular domain of human FcγRI which interacts with a human IgG was expressed as recombinant soluble human FcγRI (rshFcγRI) by Chinese hamster ovary (CHO) cell. Stable CHO cell clones with efficient expression of rshFcγRI were established based on a dihydrofolate reductase (DHFR)/methotrexate (MTX) gene-amplification system. The CHO clones efficiently produced rshFcγRI under high-density continuous culture in a bioreactor. After 53 days of culture, the number of cells had reached approximately 4 × 10? cells/mL in the bioreactor and the average production of rshFcγRI had reached 7.4 mg L-medium?1 day?1. Secreted rshFcγRI was purified to a homogeneous state using cation exchange and affinity chromatographies. The binding affinities of rshFcγRI to human IgG subclasses were determined using surface plasmon resonance analysis. The binding affinities of rshFcγRI to human IgG1/κ and IgG3/κ were high (1.59 × 10?1? and 2.81 × 10?1? M, respectively), whereas that of rshFcγRI to human IgG4/κ was lower binding affinity (1.41 × 10?? M). Binding to IgG2/κ was not detectable. Examination of circular dichroism spectra indicated that rshFcγRI was rich in β-structures and loop or turn structures, but there were few α-helices. These results may be valuable for further studies of the structure and function of human FcγRI.     

The synthesis of peptide fragments of high affinity receptor Fc?RI and their binding to allergen-specific immunoglobulins E

Using computer analysis, a minimal sequence of Arg136-Asn137-Trp138-Asp139 that can bind to IgE C3 and C4 fragments was determined for the high-affinity receptor Fc?RI, a key protein of IgE-dependent allergic reactions of the immediate type. A number of peptides containing the Arg-Asn-Trp-Asp sequence were proposed as model analogues of the Fc?RI receptor. It was shown that these peptides manifested immunobiological effects and bound to IgE. The peptides were demonstrated to bind to IgE serum antibodies specific to the Dermatophagoides pteronyssinus allergen isolated from plasma of patients with allergic bronchial asthma.     

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains ¹. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout ^{2, 3}. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml^-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses ^{4, 5}. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease ^{6, 2, 3}.     

Molecular cloning and expression of the porcine high-affinity immunoglobulin G Fc receptor (FcγRI)

Receptors for the Fc region (FcγRs) of immunoglobulin G (IgG) play a crucial role in the immune system and host protection against infection. In this study, we describe the cloning, sequencing, and expression of the high-affinity IgG receptor from pig. By screening a translated Expressed Sequence Tags database with the human FcγRI (CD64) protein sequence, we identified a putative porcine homologue. Subsequent polymerase chain reaction amplification confirmed that the identified full-length cDNA was expressed in porcine cells. Rosetting analysis shows that COS-7 cells transfected with a plasmid containing the cloned cDNA were able to bind chicken erythrocytes sensitized with porcine IgG. Scatchard analysis indicated that monomeric IgG bound to transiently transfected cells with an affinity of approximately 4×10⁷ M⁻¹. The porcine FcγRI cDNA is 1,038 nucleotides long and is predicted to encode a 346-amino-acid transmembrane glycoprotein composed of three Ig-like domains, a transmembrane region, and a short cytoplasmic tail. The overall identity of the porcine FcγRI to its human and mouse counterparts at the level of the amino acid sequence was 75% and 57%, respectively. Identification of porcine FcγRI will aid in the understanding of the molecular basis of the porcine immune system and further studies of the receptor function.Gaiping Zhang and Songlin Qiao contributed equally to this study.The GenBank accession number of the nucleotide sequence reported here is DQ026063.     

Helicobacter pylori pediatric infection changes FcεRI expression in dendritic cells and Treg profile in vivo and in vitro

H. pylori infection shows an inverse relationship with allergies. Dendritic cells regulate mucosal immune responses including the induction of T regulatory cells which are fundamental in Helicobacter pylori-induced dampening of allergies. In this respect expression of high-affinity IgE receptor (FcεRI) has been associated with a regulatory dendritic cell profile. Therefore we aimed to evaluate possible mechanisms by which H. pylori infection might modify atopy in pediatric patients. Here we show that H. pylori-infected children exhibited both increased expression of FcεRI on peripheral myeloid and plasmacytoid dendritic cells and higher levels of Foxp3 and Latency Associated Peptide on T regulatory cells. Moreover, exposure to H. pylori drove increased FcεRI expression and IL-10 secretion by both pediatric H. pylori-exposed monocyte derived dendritic cells and T cells. Finally, we show a positive correlation between expression of FcεRI in circulating myeloid DCs and total Treg cells, suggesting that in children, H. pylori infection may have a modulating role in atopy, mediated by both altered surface expression of FcεRI on children's DC and an increased T regulatory cell profile.     

Production of macrophage-activated killer cells for targeting of glioblastoma cells with bispecific antibody to FcγRI and the epidermal growth factor receptor

Objective: The aim was to determine the ability of macrophage-activated killer cells (MAK cells) obtained from peripheral blood of normal volunteers to kill glioblastoma multiforme (GBM) cell lines. Another goal was to investigate whether a bispecific antibody (bsAb) MDX-447, recognizing the high-affinity Fc receptor for IgG (FcγRI) and epidermal growth factor receptor (EGFR), would enhance MAK cell tumoricidal activity. Methods: Monocytes, from leukapheresis product, were isolated by countercurrent elutriation and differentiated into MAK cells by culture with granulocyte/macrophage-colony-stimulating factor, vitamin D₃ and interferon γ. Cells were checked for sterility, endotoxin and phenotypic markers. MAK cell functional activity was measured by a flow-cytometric phagocytosis assay. Target cells, a carcinoma cell line and two glioma cell lines expressing EGFR, were stained with PKH-26. MAK cells were labeled with fluorescein-conjugated anti-CD14. Combined effectors, targets and bsAb were incubated and the percentage of MAK cells with phagocytosed targets was determined by flow cytometry. Conclusion: We demonstrate that a large number of highly purified monocytes, isolated from peripheral blood, can be differentiated into MAK cells for use as an adjuvant for cancer treatment. After culture these cells are sterile, endotoxin-free and comprise more than 95% MAK cells. Increased amounts of CD14, CD64 and HLA-DR, which are characteristics of macrophage activation, were expressed. MAK cells were extremely phagocytic in comparison to monocytes, even in the absence of bsAb. Moreover, bsAb enhanced the tumoricidal activity of elutriated MAK cells targeted against GBM cell lines. Therefore, intracavity MAK cells armed with MDX-447 could be an effective adoptive immunotherapy for EGFR-positive GBM. Received: 5 April 2000 / Accepted: 12 July 2000     

Crystal structure of Fcγ receptor I and its implication in high affinity γ-immunoglobulin binding

Fcγ receptors (FcγRs) play critical roles in humoral and cellular immune responses through interactions with the Fc region of immunoglobulin G (IgG). Among them, FcγRI is the only high affinity receptor for IgG and thus is a potential target for immunotherapy. Here we report the first crystal structure of an FcγRI with all three extracellular Ig-like domains (designated as D1, D2, and D3). The structure shows that, first, FcγRI has an acute D1-D2 hinge angle similar to that of FcεRI but much smaller than those observed in the low affinity Fcγ receptors. Second, the D3 domain of FcγRI is positioned away from the putative IgG binding site on the receptor and is thus unlikely to make direct contacts with Fc. Third, the replacement of FcγRIII FG-loop ((171)LVGSKNV(177)) with that of FcγRI ((171)MGKHRY(176)) resulted in a 15-fold increase in IgG(1) binding affinity, whereas a valine insertion in the FcγRI FG-loop ((171)MVGKHRY(177)) abolished the affinity enhancement. Thus, the FcγRI FG-loop with its conserved one-residue deletion is critical to the high affinity IgG binding. The structural results support FcγRI binding to IgG in a similar mode as its low affinity counterparts. Taken together, our study suggests a molecular mechanism for the high affinity IgG recognition by FcγRI and provides a structural basis for understanding its physiological function and its therapeutic implication in treating autoimmune diseases.     

The FcεRIβ homologue,MS4A4A,promotes FcεRI signal transduction and store-operated Ca2+ entry in human mast cells

Members of the membrane spanning 4A (MS4A) gene family are clustered around 11q12-13, a region linked to allergy and asthma susceptibility. Other than the known functions of FcεRIβ (MS4A2) and CD20 (MS4A1) in mast cell and B cell signaling, respectively, functional studies for the remaining MS4A proteins are lacking. We thus explored whether MS4A4A, a mast cell expressed homologue of FcεRIβ, has related functions to FcεRIβ in FcεRI signaling. We establish in this study that MS4A4A promotes phosphorylation of PLCγ1, calcium flux and degranulation in response to IgE-mediated crosslinking of FcεRI. We previously demonstrated that MS4A4A promotes recruitment of KIT into caveolin-1-enriched microdomains and signaling through PLCγ1. Caveolin-1 itself is an important regulator of IgE-dependent store-operated Ca²⁺ entry (SOCE) and promotes expression of the store-operated Ca²⁺ channel pore-forming unit, Orai1. We thus further report that MS4A4A functions through interaction with caveolin-1 and recruitment of FcεRI and KIT into lipid rafts. In addition to proximal FcεRI signaling, we similarly show that MS4A4A regulates Orai1-mediated calcium entry downstream of calcium release from stores. Both MS4A4A and Orai1 had limited effects with compound 48/80 stimulation, demonstrating some degree of selectivity of both proteins to FcεRI receptor signaling over Mas-related G Protein coupled receptor X2 signaling. Overall, our data are consistent with the conclusion that MS4A4A performs a related function to the homologous FcεRIβ to promote PLCγ1 signaling, SOCE, and degranulation through FcεRI in human mast cells and thus represents a new target in the regulation of IgE-mediated mast cell activation.     

Single-particle tracking of immunoglobulin E receptors (FcεRI) in micron-sized clusters and receptor patches

When mast cells contact a monovalent antigen-bearing fluid lipid bilayer, IgE-loaded FcεRI receptors aggregate at contact points and trigger degranulation and the release of immune activators. We used two-color total internal reflection fluorescence microscopy and single-particle tracking to show that most fluorescently labeled receptor complexes diffuse freely within these micron-size clusters, with a diffusion coefficient comparable to free receptors in resting cells. At later times, when the small clusters coalesce to form larger patches, receptors diffuse even more rapidly. In all cases, Monte Carlo diffusion simulations ensured that the tracking results were free of bias, and distinguished biological from statistical variation. These results show the diversity in receptor mobility in mast cells, demonstrating at least three distinct states of receptor diffusivity.     

Genome-wide gene expression profiling of human mast cells stimulated by IgE or FcεRI-aggregation reveals a complex network of genes involved in inflammatory responses

Background

High growth (hg) modifier and background independent quantitative trait loci (QTL) affecting growth, adiposity and carcass composition were previously identified on mouse chromosomes (MMU) 1, 2, 5, 8, 9, 11 and 17. To confirm and further characterize each QTL, two panels of speed congenic strains were developed by introgressing CAST/EiJ (CAST) QTL alleles onto either mutant C57Bl/6J-hg/hg (HG) or wild type C57Bl/6J (B6) genetic backgrounds.

Results

The first speed congenic panel was developed by introgressing four overlapping donor regions spanning MMU2 in its entirety onto both HG and B6 backgrounds, for a total of eight strains. Phenotypic characterization of the MMU2 panel confirmed the segregation of multiple growth and obesity QTL and strongly suggested that a subset of these loci modify the effects of the hg deletion. The second panel consisted of individual donor regions on an HG background for each QTL on MMU1, 5, 8, 9, 11 and 17. Of the six developed strains, five were successfully characterized and displayed significant differences in growth and/or obesity as compared to controls. All five displayed phenotypes similar to those originally attributed to each QTL, however, novel phenotypes were unmasked in several of the strains including sex-specific effects.

Conclusion

The speed congenic strains developed herein constitute an invaluable genomic resource and provide the foundation to identify the specific nature of genetic variation influencing growth and obesity.     

Long-term cultured human skin mast cells are suitable for pharmacological studies of anti-allergic drugs due to high responsiveness to FcεRI cross-linking

Human skin mast cells proliferated in the presence of interleukin (IL)-4+SCF (expanding 18-fold in 8 weeks) and acquired profound responsiveness towards high affinity IgE receptor (FcεRI) cross-linking, liberating about 75% of their histamine. In a proof-of-concept, we found that these cells are useful for pharmacological testing. Even a subtle inhibition of degranulation can be visualized. This model might prove valuable in tests of novel anti-allergic drugs.     

Regulation of the High Affinity IgE Receptor (FcεRI) in Human Neutrophils: Role of Seasonal Allergen Exposure and Th-2 Cytokines

The high affinity IgE receptor, FcεRI, plays a key role in the immunological pathways involved in allergic asthma. Previously we have demonstrated that human neutrophils isolated from allergic asthmatics express a functional FcεRI, and therefore it was of importance to examine the factors regulating its expression. In this study, we found that neutrophils from allergic asthmatics showed increased expression of FcεRI-α chain surface protein, total protein and mRNA compared with those from allergic non asthmatics and healthy donors (p<0.001). Interestingly, in neutrophils isolated from allergic asthmatics, FcεRI-α chain surface protein and mRNA expression were significantly greater during the pollen season than outside the pollen season (n = 9, P = 0.001), an effect which was not observed either in the allergic non asthmatic group or the healthy donors (p>0.05). Allergen exposure did not affect other surface markers of neutrophils such as CD16/FcγRIII or IL-17R. In contrast to stimulation with IgE, neutrophils incubated with TH2 cytokines IL-9, GM-CSF, and IL-4, showed enhanced FcεRI-α chain surface expression. In conclusion, these results suggest that enhanced FcεRI expression in human neutrophils from allergic asthmatics during the pollen season can make them more susceptible to the biological effects of IgE, providing a possible new mechanism by which neutrophils contribute to allergic asthma.     

The enhanced expression of estrogen-related receptor α in human bladder cancer tissues and the effects of estrogen-related receptor α knockdown on bladder cancer cells

Estrogen-related receptor α (ERRα) belongs to the superfamily of nuclear orphan receptors. However, the role of ERRα in bladder cancer remains unknown. This study examined the expression of ERRα in bladder cancer tissues and explored the molecular mechanisms of ERRα in bladder cancer progression. The expression of ERRα in bladder cancer tissues from 61 patients was determined by immunohistochemistry. We performed quantitative real-time polymerase chain reaction assay to detect the gene expression levels and carried out Western blot assay to measure protein levels. In vitro functional assays, including colony formation, Cell Counting Kit-8, Transwell invasion, and migration assays, were performed to detect bladder cancer cell growth, proliferation, invasion, and migration, respectively. Flow cytometry was used to determine the cell apoptotic rate of bladder cancer cells. Among the 61 detected bladder cancer tissues, 39 bladder cancer tissues showed positive ERRα immunoreactivity. Higher ERRα immunoreactivity score was significantly associated with TNM stage, tumor grade, distant metastasis, and poor survival in patients with bladder cancer. Univariate and multivariate analyses showed that ERRα immunoreactivity was an independent prognostic factor for overall survival in patients with bladder cancer. ERRα was found to be upregulated in bladder cancer cell lines, and knockdown of ERRα suppressed bladder cancer cell growth, proliferation, invasion, and migration; promoted bladder cancer cell apoptosis; and inhibited the epithelial-mesenchymal transition of bladder cancer cells. On the other hand, bladder cancer cell proliferation, invasion, and migration were significantly enhanced after cells were transfected with an ERRα-overexpressing vector. In vivo tumor growth and metastasis assays showed that ERRα knockdown resulted in remarkable inhibition of tumor growth and tumor metastasis in nude mice. Collectively, our results suggest that the enhanced expression of ERRα may play a key role in the development and progression of bladder cancer and ERRα may serve as an important prognostic factor for bladder cancer.     

Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells

We have isolated the bovine and human genes for Müllerian inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Müllerian duct during development of the male embryo. The mRNA sequence of bovine MIS, determined from an analysis of cDNA and genomic clones, codes for a protein of 575 amino acids containing a 24 amino acid leader peptide. The human gene has five exons that code for a protein of 560 amino acids. A comparison of the bovine and human MIS proteins reveals a highly conserved C-terminal domain that shows marked homology with human transforming growth factor-beta and the beta chain of porcine inhibin. Animal cells transfected with the human gene secrete biologically active MIS, which causes regression of the rat Müllerian duct in vitro.