Human FcγRI Research Articles

Molecular cloning and identification of full-length cDNA encoding high affinity Fc receptor for bovine IgG (FcγRI)

The receptor I for the Fc region of immunoglobulin G (FcγRI) is a member of the Ig superfamily with a high affinity, and it mediates antibody-dependent cellular cytotoxicity and immune complex clearance. In this study, a cDNA encoding the bovine FcγRI was cloned. The full-length cDNA sequence is 1050 bp long with a short 5′- and long 3′-untranslated end regions, which codes for 349 amino acids and contains a signal peptide, an extracellular region with three Ig-like domains, and transmembrane and intracytoplasmic domains. Five potential N-linked glycosylation sites are recognized in this sequence. Compared with the sequences of human and mouse FcγRI, the homologies of nucleotide sequences are 80 and 69% and homologies of deduced amino acid sequences are 66 and 55%, respectively. It is shown that the sequences of the monomeric IgG binding domain in these three species of FcγRI are highly conserved.

The influence of the hinge region length in binding of human IgG to human Fcγ receptors

Interactions between human IgG with human FcγRI and FcγRIIa (R131) were studied to investigate the role of the hinge region of IgG3 and IgG1 in the binding of the antibodies to FcγR. It was found that a hinge deletion mutant of IgG3 (IgG3 m15) was reduced in its ability to bind to FcγRI and FcγRIIa but was more potent at activating ADCC by activated lymphocytes (FcγRIIIa-mediated), compared to the wild-type version of IgG3. The human IgG1 allotype G1m(a,z) was more efficient at binding to FcγRI than the two IgG3 antibodies tested. The IgG1 and IgG3 wild type antibodies were better able to bind to FcγRII than the hinge deletion mutant version of IgG3. The data suggest a role for the hinge region in influencing FcγR mediated effector functions in IgG3.

Lysis of murine B lymphoma cells by transgenic phagocytes via a human Fc gamma RI x murine MHC class II bispecific antibody.

The class I IgG receptor (Fc gamma RI) on cytotoxic effector cells has been reported to initiate destruction of tumour cells by effector cells in vitro. We are aiming at developing an immunocompetent model to evaluate the cytotoxic capacity of human Fc gamma RI for the rejection of tumour cells in vivo. Therefore, we recently generated a transgenic mouse strain expressing human Fc gamma RI on monocytes, macrophages, and neutrophils. In these mice, the human receptor is up-regulated by granulocyte-colony-stimulating factor (G-CSF) and is able to trigger cellular responses. Subsequently, in the present study the B cell lymphoma IIA1.6 cell line is selected as a tumour target, and a human Fc gamma RI-directed antitumour bispecific antibody (bsAb) is constructed and characterized. Fab' fragments of mAb 22, which bind hFc gamma RI at an epitope that is distinct from the ligand binding site, were chemically linked to Fab' fragments of rat anti-(mMHC class II antigens) mAb M5/114, yielding bsAb 22 x M5/114. This bsAb was able to bind simultaneously to hFc gamma RI and mMHC class II antigens in a dose-dependent fashion. Binding of 22 x M5/114 to Fc gamma RI was not inhibited in the presence of human IgG. It is important to note that, MHC-class-II-expressing IIA1.6 lymphoma cells were lysed by whole blood from G-CSF-treated transgenic mice in the presence of bsAb 22 x M5/114. No lysis by whole blood from non-transgenic mice or from transgenic animals that had not received G-CSF was observed. These results indicate that human Fc gamma RI is able to mediate lysis of murine IIA1.6 lymphoma cells by transgenic effector cells via bsAb 22 x M5/114. A trial with transgenic mice, evaluating the efficacy of these hFc gamma RI-directed bsAb in combination with G-CSF for treatment of IIA1.6 B cell lymphoma, is currently in progress.

FcR γ-Chain Is Essential for Both Surface Expression and Function of Human FcγRI (CD64) In Vivo

Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface-expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma-chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA-IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo.

Information technology law: What does the future hold?

IgG responses are crucial in antiviral defence and instrumental for the serodiagnosis of infections. Fcγ receptors (FcγRs), which recognize the Fc-part of IgG, differ regarding their IgG binding affinity, IgG subclass preference, cellular expression profile and pathogen elimination mechanisms elicited upon activation. Assessing their activation in vitro is of fundamental importance, but technically difficult. Therefore, a novel assay for measuring antiviral IgG antibodies triggering activation of individual host Fcγ receptors was established. The assay comprises the co-cultivation of virus-infected target cells with immune IgG antibodies and mouse BW5147 hybridoma cells stably expressing chimeric FcγR–CD3ζ chain molecules consisting of the extracellular domain of human FcγRIIIA, FcγRIIA or FcγRI, respectively, fused to the transmembrane and intracellular domains of the mouse CD3ζ chain. Triggering of the chimeric FcγR receptors by immune complexes formed on the surface of IgG-opsonized virus-infected target cells resulted in FcγR activation leading to IL-2 secretion by BW5147 cells, which was quantified as a surrogate marker in an ELISA. Target cells infected with various human pathogenic viruses including herpes simplex virus type 1 (HSV-1), Epstein–Barr virus (EBV), human cytomegalovirus (HCMV), measles virus (MV), and respiratory syncytial virus (RSV) or displaying human immunodeficiency virus-1 (HIV-1) gp120 evoke dose-dependent IgG responses demonstrating the universal applicability of the assay. Taken together, a new reliable and simple tool for measuring antibodies triggering activation of Fcγ receptors was established. This assay will be instrumental for defining novel correlates of IgG immunity and the design of new therapeutic IgGs.

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.

Use of the 5′ -flanking region of the mouse perforin gene to express human Fcγ receptor I in cytotoxic T lymphocytes

Expression of the gene encoding the cytolytic granule protein perforin is restricted to cytotoxic lymphocytes. To undertake a functional analysis of the immediate 5'-promoter region of the mouse perforin gene, we transiently transfected mouse perforin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs into cytotoxic T, T lymphoid, B-lymphoid, and nonlymphoid cell lines. The transcriptional activity of the perforin promoter was restricted to cytotoxic lymphocytes. The perforin promoter was controlled by several positive (in perforin-positive cells) and negative (in perforin-negative cells) cis-acting regions, spread over at least 1.1 kilobases. The most specific expression of the CAT reporter gene in the interleukin-2-dependent cytotoxic T cell line CTLL-R8 was obtained with the mouse perforin promoter encompassing positions -1104 to +1 in relation to the RNA cap site. This construct expressed 65- to 70-fold higher CAT activity than the promoterless CAT construct in perforin-expressing cells but only 1- to 5-fold higher CAT activity than the promoterless construct in nonlymphoid cells. On the basis of these data, we used this most specifically active mouse perforin promoter, -1104 to +1, to express in CTLL-R8, a chimeric human receptor comprising the extracellular domains of human Fc gamma RI and the transmembrane and intracellular domains of TCR zeta. Selection in G418-containing medium produced CTLL-R8 transfectant clones that (1) expressed high levels of human Fc gamma RI mRNA; (2) expressed cell surface Fc gamma RI as demonstrated by immunoprecipitation and their ability to bind the Fc portion of human and mouse monoclonal antibodies (mAbs) in an isotype-specific manner, and (3) bound RBC expressing mucin-1 (Muc-1) peptide in the presence of a chimeric mouse-human anti-Muc-1 mAb. Activation of CTLL-R8 transfectants upon engagement of the human Fc gamma RI was evidenced by their ability to lyse tumor target cells in an mAb isotype-dependent manner. The successful expression of a functional chimeric gene in CTLL-R8 suggests that the mouse perforin promoter represents a novel reagent for expressing exogenous genes in cytotoxic T lymphocytes.

Human Fcγ RI triggering of the mononuclear phagocyte respiratory burst

This study investigates the role played by Fcγ RI and Fcγ RII in triggering the respiratory burst induced in human monocyte-like U937 cells by monoclonal chimaeric anti-NIP antibodies expressing the human IgG subclass and mouse IgG2b heavy chains. Respiratory burst activity was measured as Superoxide generation. Four separate lines of evidence indicate a predominant role for Fcγ RI in triggering Superoxide generation induced by erythrocytes sensitized with up to the maximum of 100,000 IgG molecules per cell. Firstly, erythrocytes sensitized with mouse IgG2b anti-NIP antibodies which are not recognized by human Fcγ RI, did not induce a response but when residue Glu-235 was replaced by Leu to give the lower hinge sequence of mouse IgG2a which is recognized by Fcγ RI, the mutant bound to Fcγ RI and induced a response equal to 80% of that given by chimaeric human IgG3. Chimaeric human IgG3 antibodies with amino acid substitutions in the lower hinge showed reduced activity and the greatest reductions (< 32% of wild type antibody activity) were associated with changes at Leu-235 which is critical for recognition by Fcγ RI. Secondly, chimaeric human IgG4 antibodies which are not recognized by FcγRII, were able to induce Superoxide generation. The rank order of abilities of chimaeric human IgG subclass antibodies to induce responses was IgG3 > IgG1 > IgG4 ⪢ IgG2. Thirdly, responses induced by chimaeric human IgG were inhibited by concns of monomeric human IgG3 in the nM range. Finally, chimaeric human IgG3 induced responses were inhibited by anti-Fcγ RI, but not anti-Fcγ RII monoclonal antibodies. Consistent with a major role for FcγRI in triggering the responses of U937 cells, erythrocytes sensitized with chimaeric human IgG3 did not induce Superoxide generation by neutrophils which express FcγRII and FcγRIII, or eosinophils which express Fcγ RII, but neither of which expresses Fcγ RI.

Canine monocytes express a receptor specific for murine Fcγ2a and Fcγ3 immunoglobulins

Murine monoclonal antibodies (mAb) of IgM and IgG subclasses that do not bind to canine cells have been used to detect the expression of Fc receptors on canine peripheral blood mononuclear cells and the cell line MAXEY-DH82. Murine Ig of IgG2a and IgG3 isotypes bound specifically to canine peripheral blood monocytes and to MAXEY-DH82, whereas Ig of IgG1, IgG2b, and IgM did not. No other cell types, including resting and activated peripheral blood lymphocytes, expressed this canine Fc receptor (cFcR) specific for murine IgG2a and IgG3. MAXEY-DH82 was used to characterize this Fcγ2a/γ3 receptor. Binding of murine IgG2a and IgG3 is trypsin sensitive and partially suppressed by phosphatidyl inositol-phospholipase C (PI-PLC) treatment, indicating that this cFcγ2a/γ3 receptor is a lipid-anchored protein. Preincubation of MAXEY-DH82 with canine sera or canine IgG prevented the binding of murine γ2a/γ3 Ig, demonstrating that this receptor is a cFc receptor for canine IgG. The cFcγR expressed on canine monocytes and on MAXEY-DH82 is probably the analog of the murine and human FcγRI. The specific expression of this analog by canine cells could be used in identifying and purifying canine monocytes.

Chimeric Fc receptors identify immunoglobulin-binding regions in human Fc gamma RII and Fc epsilon RI.

Fc gamma RII and Fc epsilon RI are functionally distinct cell surface receptors for immunoglobulin (Ig); Fc gamma RII binds IgG with low affinity, whereas Fc epsilon RI binds IgE with high affinity, yet they are homologous in structure and sequence having extracellular regions containing two Ig-like domains with 38% amino acid identity. Chimeric receptors derived from human Fc gamma RII and Fc epsilon RI were produced by exchanging homologous regions of the two receptors to define binding region(s) for IgG in Fc gamma RII and IgE in Fc epsilon RI. Firstly, a chimeric form of the Fc epsilon RI alpha chain was produced by replacing the transmembrane region and cytoplasmic tail with that of Fc gamma RII. This mutant alpha chain could be expressed on the cell surface independently of associated beta and gamma subunits, and retained high-affinity IgE binding, indicating that the extracellular region of the Fc epsilon RI alpha chain is sufficient for high-affinity IgE binding. Secondly, to identify the role of the individual domains in Fc binding of both Fc gamma RII and Fc epsilon RI, chimeric receptors were generated by exchanging the first extracellular domains between Fc gamma RII and the alpha chain mutant and used to demonstrate that the second extracellular domain of both receptors contains region(s) directly involved in Ig binding. Additional chimeric receptors were constructed to localize the Ig interactive regions in domain two of Fc gamma RII and Fc epsilon RI; these identified a single region of IgG binding in Fc gamma RII located between residues Ser136 to Val169, and at least three independent IgE binding regions in the Fc epsilon RI alpha chain, between residues Trp87 to Lys128, Tyr129 to Asp145, and Ser146 to Val169.

Genomic organisation and sequence of the extracellular domain exons of the bovine Fc γRI receptor, and evidence for restricted binding of ruminant IgG to U937 cells

Southern blotting of bovine genomic DNA and hybridization with a human Feγ RI cDNA probe, p 135, has identified a single copy of the bovine FcγRI gene. A bovine genomic lymphocyte library in λEMBL3 was screened with probe p135. A positive λ clone, 15.5.4, containing the three extracellular domain exons of Feγ RI, has been cloned, mapped and sequenced. Each extracellular domain is encoded within a single exon. All three domains are assigned to the C-2 set of the Ig superfamily with 58% identity between amino acid residues of bovine, human and mouse Fc γRI. Pairs of cysteine residues are conserved in each domain as potential sites for intra-chain disulphide bonding. Human monocytoid U937 cells were used as a model to test binding homology within the Fc γRI family. The binding of IgG isotypes to IFN-γ stimulated U937 cells was determined by FACScan flow cytometry. U937 cell Fc γRI receptor does not bind bovine or ovine IgG isotypes. On the basis of these studies and by comparison of the Fc determinant region sequences of IgG, the introduction of species specificity in Fc γRI/IcG interaction by evolutionary drift is proposed.

Characterization of the 5′-flanking transcriptional regulatory region of the human Fcγ receptor gene, Fcγ RIIA

The human Fcγ receptor gene Fcγ RIIA is expressed in platelets, neutrophils, monocytes and macrophages. Understanding the regulation of expression of Fcγ RIIA will enhance our knowledge of regulated gene expression and immune function m these cells. We cloned a 3.65 kb region of the 5′ end of the Fcγ RIIA gene and characterized 3.4 kb of previously unreported sequence of the 5′-flanking region. Primer extension studies and RNase protection analyses of mRNA from HEL, K562 and U937 cells revealed multiple transcription start sites. One transcription start site mapped to a 5′-untranslated (5′UT) exon approximately 1 kb 5′ to the ATG translation initiation codon, while a second start site mapped near the ATG codon. Reverse transcription combined with PCR (RT-PCR) employing an oligonucleotide m the putative 5′UT exon and an antisense oligonucleotide in the translated region yielded products which confirm that transcription starts in this 5′UT exon 881 bp upstream of the ATG codon. Sequence analysis of the RT-PCR products showed two related RNA splice products which use alternative 3'-consensus AG splice acceptor sites. Fcγ RIIA mRNA thus has three distinct potential 5′UT regions, two alternatively spliced forms from the start site in the 5′UT exon and the third from the start site near the ATG codon. Comparisons of the human Fcγ RIIA 5′-flanking region whith human Fcγ RI and miouse Fcγ RIIβ genes as well as with other genes expressed in megakaryocytes, neutrophils and monocytes reveal structural similarities and shared promoter elements.

Interaction of aglycosyl immunoglobulins with the IgG Fc transport receptor from neonatal rat gut: Comparison of deglycosylation by tunicamycin treatment and genetic engineering

The role of carbohydrate in the structure and function of immunoglobulin Fc regions has been studied using the interaction of a monoclonal mouse IgG2b anti-NIP antibody with the IgG Fc transport receptor from neonatal rat gut. An aglycosyl variant of this immunoglobulin, in which site-directed mutagenesis had been used to eliminate the carbohydrate attachment site in the C H2 domain by changing Asn297 to Ala, was compared in this system to aglycosyl immunoglobulin prepared from immunoglobulin-secreting cells treated with tunicamycin to inhibit N-linked glycosylation. Loss of carbohydrate from the heavy chain was confirmed for both methods by Western blotting of the separated chains with Concanavalin A, and no significant differences in circular dichroism spectra were found between glycosylated and non-glycosylated mutants. Removal of carbohydrate by site-directed mutagenesis had no effect on binding of the immunoglobulin to the Fc transport receptor (FcTR) in vitro or transport from the gut to blood in vivo. Short-term clearance from circulation and degradation by gut contents in vitro were similarly unaffected. Mutation of Glu235 to Leu, an alteration that allows binding to human monocyte Fcγ RI, did not alter the interaction with FcTR. However, treatment of wild-type or aglycosyl mutant cells with tunicamycin resulted in immunoglobulin which was less stable, cleared more rapidly and was transported slightly less efficiently. These findings indicate that the binding site for the FcTR may be unique among Fc-binding ligands, and that tunicamycin treatment may cause alterations in the immunoglobulin molecule in addition to loss of N-linked carbohydrate.

Molecular definition of interaction sites on human IgG for Fc receptors (huFcγR)

Evidence from several experimental approaches allows us to conclude that the primary amino acid sequence of the lower hinge region (residues 234–237) of human IgG molecules determines recognition by human FcγRI, FcγRII and FcγRIII. Glycosylation of the CH2 domain is also essential, although the carbohydrate is not accessible for direct interaction with ligands. The role of the carbohydrate moiety may be to maintain a protein conformation that allows accessibility to amino acid side chains essential for ligand recognition and binding. It appears logical that the evolutionarily-related Fcγ R molecules should interact with overlapping non-identical sites on the IgG molecule.

Monoclonal antibodies identify three IgG Fc receptors in normal human central nervous system

Functional Fc receptors have been described in the central nervous system (CNS) in the subependymal periventricular regions, leptomeninges, including brain perivascular tissues, and choroid plexus. The distribution of this receptor activity suggests a role in protection of adjacent nervous tissue from IgG-opsonized antigens, including microorganisms. In this report, we have utilized monoclonal antibodies to human FcγRI, II, and III; 32, IV.3, and 3G8, respectively, to immunohistochemically examine the distribution of these receptors in the CNS. FcγRI was only occasionally present in the CNS where it was identified most often in the choroid plexus. FcγRII was the predominant receptor in brain. It was consistently present in leptomeninges, including brain perivascular regions, arachnoid granulations, and choroid plexus stroma. Some samples of subependymal periventricular tissue also displayed FcγRII. FcγRIII was only identified in subependymal periventricular tissue but not in choroid plexus and arachnoid. These results demonstrate that regions of normal adult brain which produce cerebral spinal fluid (CSF) and border on CSF and vascular compartments display FcγR heterogeneity consistent with that of blood monocytes and systemic macrophages.