Molecular cloning and expression of the mouse high affinity Fc receptor for IgG

Full length cDNA clones encoding the mouse Fc gamma RI were isolated by using redundant oligonucleotide probes based on previously determined amino acid sequence of protein bound to an IgG2a antibody column. Sequence analysis of cDNA clones indicates that mouse Fc gamma RI is a transmembrane glycoprotein that is composed of three disulfide bonded extracellular Ig binding domains unlike Fc gamma RII of man and mouse. These extracellular domains contain five potential sites of N-linked glycosylation; three sites in the first domain and one in each of the second and third domains. In addition a transmembrane region is present followed by a cytoplasmic tail of 84 amino acids. Analysis of the amino acid sequence of the first two extracellular domains of Fc gamma RI indicate that these are highly homologous to the extracellular domains of Fc gamma RII; the third domain is different and shows a lower level of homology to other FcR domains but is clearly related to the Ig super-family. Transfected cells expressing Fc gamma RI were shown to bind immune complexes of rabbit IgG; and monomeric IgG2a bound to transiently transfected cells with an affinity of approximately 5 x 10(7) M-1, i.e. the receptor was of high affinity and therefore was by definition Fc gamma RI. Northern analysis demonstrated that Fc gamma RI mRNA could be detected in the Fc gamma RI+ myeloid cell lines WEH1 3B and J774. Finally, Southern analysis indicated that Fc gamma RI is likely to be encoded by a single copy gene of approximately 9 kb.     

Chimeric Fc receptors identify functional domains of the murine high affinity receptor for IgG.

Chimeric Fc gamma R have been generated between the mouse high affinity receptor for IgG (Fc gamma RI) and the low affinity receptor for IgG (Fc gamma RII) by exchanging the first two domains of the three-domain extracellular structure of Fc gamma RI with the homologous two-domain extracellular structure of Fc gamma RII. Studies of the affinity and specificity of binding of mouse Ig classes to these receptors defined functional regions of Fc gamma RI and showed some surprising results. After removal of the third extracellular domain of Fc gamma RI, the remaining two domains (domains 1 and 2) retained the capacity to bind Ig in the form of immune complexes, however, they bound monomeric IgG2a with a reduced affinity. Surprisingly, these two domains in the absence of the third domain bound not only IgG2a but also IgG1 and IgG2b, i.e., the third domain of Fc gamma RI suppresses the intrinsic capacity of the first two domains to act as a low affinity Fc gamma RII-like molecule. Linking the third extracellular domain of Fc gamma RI to the two extracellular domains of Fc gamma RII resulted in a receptor that retained the specificity and affinity of Fc gamma RII. Thus, the removal of domain 3 from Fc gamma RI resulted in the conversion of Fc gamma RI to an "Fc gamma RII-like" receptor. These findings indicate that domains 1 and 2 of Fc gamma RI form an Ig-binding motif, and although domain 3 is not essential for Fc binding by Fc gamma RI, it plays a crucial role in determining the specific high affinity interaction of Fc gamma RI with IgG2a.     

Soluble monomeric IgG1 Fc

Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.     

Human leukocyte Fc (IgG) receptors: quantitation and affinity with radiolabeled affinity cross-linked rabbit IgG.

Quantitative studies have been made of Fc receptors on human leukocytes derived from peripheral blood, thymus, tonsil, and spleen. The relative affinities and average numbers of receptors per cell were determined by measuring the binding of 125I-labeled, affinity cross-linked trimers of rabbit IgG to various populations of cells. In parallel, the sizes of receptor-bearing populations were determined by fluorescence microscopy. Fc receptors could be detected on leukocytes from peripheral blood and spleen, but not from tonsil or thymus. In the peripheral blood, the highest density of receptors was found on polymorphonuclear leukocytes; a subpopulation of lymphocytes had somewhat fewer receptors per cell, and circulating monocytes had the lowest receptor density. Among splenocytes, most of the receptors were found on myeloid cells and monocytes. In all populations, the affinity of Fc receptors for the trimer was about the same. At 0 degrees C the average value for the association constant was 5 x 10(7) M-1.     

Interferon gamma induces in human neutrophils and macrophages expression of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I or CD64)

Immune interferon (IFN-gamma) induces in human neutrophils accumulation of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I, CD64) with a mechanism that is independent from de novo protein synthesis and from activation of the Na+/H+ antiporter. Monocyte-derived macrophages can also be induced to express high levels of Fc gamma R-I mRNA by IFN-gamma, without requirement of protein synthesis. Unlike what is observed in neutrophils, induction by IFN-gamma of macrophage Fc gamma R-I mRNA was significantly depressed by the Na+/H+ antiporter inhibitor amiloride. These results indicate that phagocytes' Fc gamma R-I mRNA induction by IFN-gamma is regulated by different mechanisms depending on the target cells.     

Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences

Many biological functions, including control of the homeostasis and maternofetal transfer of serum gamma-globulins, are mediated by the MHC class I-related neonatal FcR (FcRn). A correlation exists in mice between the binding affinity of IgG1/Fc fragments to FcRn at pH 6.0 and their serum t(1/2). To expand this observation, phage display of mutagenized Fc fragments derived from a human IgG1 was used to increase their affinity to both murine and human FcRn. Ten variants were identified that have a higher affinity toward murine and human FcRn at pH 6.0, with DeltaDeltaG (DeltaG(wild type) - DeltaG(mutant)) from 1.0 to 2.0 kcal/mol and from 0.6 to 2.4 kcal/mol, respectively. Those variants exhibit a parallel increase in binding at pH 7.4 to murine, but not human, FcRn. Although not degraded in blood in vitro, accumulated in tissues, nor excreted in urine, their serum concentration in mice is decreased. We propose that higher affinity to FcRn at pH 7.4 adversely affects release into the serum and offsets the benefit of the enhanced binding at pH 6.0.     

Functional ion channel formation by mouse macrophage IgG Fc receptor triggered by specific ligands

The mouse macrophage Fc gamma 2b/gamma 1R has previously been purified with the aid of the monoclonal antibody 2.4G2. That this Fc gamma R functions as a ligand-dependent ion channel is supported by the following evidence. Employing [3H]tetraphenylphosphonium ([3H]Ph4P+) as a probe for membrane potential changes in intact cells, we found a biphasic change in membrane potential following treatment with immune complexes, monoclonal antibody 2.4G2 IgG and 2.4G2 Fab-Sephadex particles. We observed an immediate depolarization followed by prolonged hyperpolarization. [3H]Ph4P+ uptake experiments with plasma membrane vesicles prepared from J774 macrophages showed that binding of ligands to the FcR led to transmembrane monovalent cation flow. Similar [3H]Ph4+ uptake experiments were done with phospholipid vesicles containing purified and reconstituted Fc gamma 2b/gamma. Following challenge with specific ligands, transmembrane monovalent cation flow was observed. Purified FcR was reconstituted into planar lipid bilayers; exposure to ligands led to transient bilayer conductance increase. THe conductance change was resolved into single channel events. Quin-2 measurements showed an increase of free cytosolic calcium levels in macrophages following exposure of cells to different ligands of the FcR. An optimal range of calcium was found to be required for phagocytosis, below and above which inhibition of ingestion occurred.     

Polymorphonuclear leukocyte migration through human amnion membrane

A new in vitro model has been developed for studying migration of human polymorphonuclear leukocytes (PMN) through living native cellular and matrix barriers. Human amnion membrane consists of a single layer of epithelium bound to a continuous basement membrane interfacing an avascular collagenous stroma. Living amnion was placed in plastic chambers with separate compartments on each side of the membrane. PMN were introduced on the epithelial side of the amnion, and a Millipore filter (Millipore Corp., Bedford, Mass.) was placed against the stromal side. In response to N-formylmethionyl-leucyl- phenylanlanine (FMLP) chemoattractant, PMN penetrated the full thickness of the amnion and were collected and counted on the filter. The rate of PMN traversal of the amnion was dependent on the concentration of FMLP (optimal at 10(-8)M) as well as the slope of the FMLP gradient across the amnion. The route of PMN migration was studied by transmission electron microscopy. PMN first attached to the epithelial surface, then infiltrated between intercellular junctions. PMN migrated around or through tight junction and hemidesmosome attachments. The PMN then penetrated the basement membrane and migrated through the dense collagenous stroma. The present amnion migration system has characteristics of the in vivo inflammatory state not described in any previous method for monitoring PMN migration in vitro. Prior methods have not used native epithelium, whole basement membrane, or collagenous stroma. PMN penetration of these barriers occurs in the normal inflammatory response and probably involves biochemical mechanisms not required for simple migration through the pores of an artificial filter. The amnion system can be useful for future biochemical and morphological studies of PMN penetration of these barriers and possible repair processes that may follow.     

The low affinity Fc receptor for IgG functions as an effective cytolytic receptor for self-specific CD8 T cells

We have recently described a population of self-Ag-specific murine CD8(+) T cells with a memory phenotype that use receptors of both the adaptive and innate immune systems in the detection of transformed and infected cells. In this study we show that upon activation with IL-2 with or without Ag, between 10 and 20% of the activated self-specific CD8(+) T cells express the low affinity FcR for IgG. By contrast, all IL-2-activated NK cells express high levels of this FcR. The FcR comprises the FcgammaRIIIalpha and FcRgamma subunits. However, the FcRgamma subunit also associates with the CD3 complex, and this association probably contributes to the low expression of FcR in activated cells. Although the FcR is expressed at a low level on activated self-specific CD8(+) T cells, it functions very efficiently as a cytolytic receptor in ADCC. FcR-dependent killing occurred in the absence of TCR stimulation, but could be augmented by concurrent stimulation of the TCR. In addition to mediating ADCC, engagement of the FcR on self-specific CD8(+) T cells results in the production of both IFN-gamma and TNF-alpha. This is the first report of an activating FcR on self-specific murine CD8(+)alphabeta TCR(+) T cells and establishes the importance of innate immune system receptors in the function of these self-specific CD8(+) T cells.     

Crystal structure of the human leukocyte Fc receptor, Fc gammaRIIa.

Fc gamma receptors bind IgG to initiate cellular responses against pathogens and soluble antigens. We have determined the three-dimensional structure of the extracellular portion of human Fc gammaRIIa to 2.0 A resolution providing a structural basis for the unique functions of the leukocyte FcR family. The receptor is composed of two immunoglobulin domains and arranged to expose the ligand-binding site at one end of domain 2. Using alanine mutants we find that the binding sites for IgG1 and 2 are similar but the relative importance of specific regions on the receptor varies. In crystals, Fc gammaRIIa molecules associate to resemble V(L)V(H) dimers, suggesting that two Fc gammaRIIa molecules could cooperate to bind IgG in an asymmetric manner.     

Human monocyte chemiluminescence triggered by IgG aggregates. Requirement of phospholipase activation and modulation by Fc receptor ligands

Human mononuclear cells stimulated with soluble IgG aggregates generated chemiluminescence, a response attributable to monocytes. Some requirements of this reaction were examined by preincubation of the cells with a variety of inhibitors. The protease antagonists TPCK and TLCK, the phospholipase inhibitors quinacrine and BPB, and the calcium channel blocker verapamil were all inhibitory at micromolar concentrations. The oxygen metabolite scavengers SOD and catalase were less inhibitory. These findings are consistent with a major role for arachidonic acid metabolites in the generation of light. Modulation of monocyte Fc-mediated chemiluminescence also occurred by preincubation of the cells with Fc ligands. While IgG aggregates and monomeric IgG blocked Fc-dependent chemiluminescence, IgG Fc fragments were stimulatory of this response.     

Cynomolgus and pigtail macaque IgG subclasses: characterization of IGHG genes and computational analysis of IgG/Fc receptor binding affinity

Macaques are the most widely used experimental nonhuman primate (NHP) species. Rhesus (Macaca mulatta, Macmul), cynomolgus (Macaca fascicularis, Macfas), and pigtail (Macaca nemestrina, Macnem) macaques continue to be popular models for vaccine and infectious diseases research, especially HIV infection and AIDS, and for the development of antibody-based therapeutic strategies. Increased understanding of the immune system of these species is necessary for their optimal use as models of human infections and intervention. In the past few years, the antibody/Fc receptor system has been characterized in a stepwise manner in these species. We have continued this characterization by identifying the four IG heavy gamma (IGHG) genes of Macfas and Macnem in this study. Our results show that these genes share a high degree of similarity with those from other NHP species, while presenting consistent differences when compared to human IGHG genes. Furthermore, comparison of Macfas IGHG genes with those described in other studies suggests the existence of polymorphism. Using sequence- and structure-based computational tools, we performed in silico analysis on multiple polymorphic Macfas IgG and their interactions with human IgG Fc receptors (FcγR), thus predicting that Macfas IGHG polymorphisms influence IgG protein stability and/or binding affinity towards FcγR. The presence of macaque IGHG polymorphisms and macaque/human amino acid changes at locations potentially involved in antibody functional properties indicate the need for cautious design and data interpretation of studies in these models, possibly requiring the characterization of antibody/Fc receptor interactions at the individual level.     

Subclass specificity of the Fc receptor for human IgG on K562

The erythroleukemic cell line K562 bears a 40-kDa Fc receptor (Fc gamma RII) serologically related to and with a similar molecular weight as the Fc gamma R present on a broad range of leukocytes. The human IgG subclass specificity of the Fc gamma R on K562 was investigated using IgG aggregates of defined size, obtained from purified human myeloma proteins. The monoclonal antibody IV.3, which reacts with the Fc gamma RII present on various cell types, totally prevented binding of 125I-IgG2 trimers to K562. Experiments with radiolabeled IgG2 trimers showed that K562 cells bound a mean of 156,764 +/- 9895 molecules per cell with an association constant (Ka) of 1.8 +/- 0.7 X 10(8) M-1. Similar results were obtained with IgG3 oligomers. IgG3 and IgG2 trimers were about two- to threefold more effective in inhibiting binding of 125I-IgG2 trimers to K562 than IgG1 and IgG4 trimers. These results were confirmed by inhibition experiments using IgG monomers. The subclass specificity of the Fc gamma RII on K562 (i.e., IgG2 = IgG3 greater than IgG1 = IgG4) is quite distinct from the one reported for the Fc gamma RI and III of human cells (i.e., IgG1 = IgG3 greater than IgG4 and IgG2).     

Solid-phase synthesis and cyclization of a large branched peptide from IgG Fc with affinity for Fc gammaRI.

A solid phase approach has been used to synthesize a large branched disulphide peptide from IgG Fc, Ac-F-C*-A-K-V-N-N-K-D-L-P-A-P-I-E-K(Ac-E-L-L-G-G-P-S-V-F)-C*-I-NH2. This peptide combines the lower hinge region of IgG and a proximal beta-hairpin loop, both implicated in binding to Fc gammaRI. Solid phase Tl(tfa)3 cyclization of the linear branched peptide resulted in a poor yield of cyclic hinge-loop peptide (11%) most likely due to steric hindrance caused by the branch. However, if addition of the branch was preceded by solid phase Tl(tfa)3 cyclization of the loop, the yield was excellent at 75%. Cyclic hinge-loop peptide was active in displacing IgG2a from Fc gammaRI expressed on monocyte cell lines with an IC50 of 40 microM, whereas the linear form of this peptide was inactive. The Fc hinge-loop peptide demonstrates the potential for a non-mAb high affinity, immunomodulatory ligand for Fc gammaRI.     

Structural polymorphism of the human monocyte 40 kilodalton Fc receptor for IgG

The 40 kD monocyte Fc receptor for IgG is capable of binding murine IgG1 and of supporting an IgG1 anti-T3 T lymphocyte proliferative response among approximately 80% of Caucasian individuals (responders), whereas the 40 kD Fc receptor on monocytes of the remaining individuals (nonresponders) is incapable of interacting with murine IgG1. By using a monoclonal antibody (mab IV3) that reacts with the 40 kD receptor, we found that the monocyte 40 kD receptors from responder and nonresponder individuals cannot be distinguished by either electrophoretic mobility on SDS-polyacrylamide gels, or by the number of receptors per cell as determined by indirect immunofluorescence. However, isoelectric focussing of the purified radioiodinated 40 kD receptor revealed that the monocyte receptor from all of four nonresponder individuals evaluated has a single distinctive pattern of multiple, regularly spaced bands, whereas the pattern of the 40 kD monocyte receptor from 11 responder individuals is of two sorts. One (seen in four of 11 responders) consists of multiple, regularly spaced bands that are asynchronous with the nonresponder pattern, and the other (seen in seven of 11 responders) consists of multiple bands that correspond in mobility to all of the bands of both of the other two patterns. The incidence of these three patterns suggests that the 40 kD Fc receptor is encoded by a single structural gene with two alleles, both of which are expressed.     

Identification of the rat IgA Fc receptor encoded in the leukocyte receptor complex

Human FcRI (CD89) is a myeloid-specific IgA Fc receptor encoded in the leukocyte receptor complex. Thus far, no gene coding for FcRI has been identified in mice. Here, we show that, unlike mice, rats have the gene (Fcar) coding for FcRI. The rat Fcar gene has an exon-intron structure essentially identical to that of the human counterpart and is encoded in the leukocyte receptor complex on Chromosome 1. Southern blot analysis using the rat Fcar as a probe revealed hybridizing bands in Chinese and Syrian hamsters and gerbils, but not in mice, indicating that Fcar was lost in the lineage leading to mice after the divergence of rats and mice. Identification of FcRI in rats should facilitate the elucidation of the in vivo role of this receptor.The sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank databases under accession numbers: AB109766, AB109767, and AB109768