Dimeric FcγR ectodomains detect pathogenic anti-platelet factor 4-heparin antibodies in heparin-induced thromobocytopenia.

Essentials FcγRIIa mediates life-threatening heparin-induced thrombocytopenia (HIT). Most anti-platelet factor (PF)4-heparin IgGs are not pathogenic so diagnosis of HIT is challenging. Dimeric rsFcγRIIa was used to quantify receptor-binding activity of anti-PF4-heparin antibodies. Dimeric rsFcγRIIa binding specifically correlated with occurrence of HIT. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is a major and potentially fatal consequence of antibodies produced against platelet factor 4 (PF4)-heparin complexes following heparin exposure. Not all anti-PF4-heparin antibodies are pathogenic, so overdiagnosis can occur, with resulting inappropriate use of alternative anticoagulation therapies that have associated risks of bleeding. However, definitive platelet functional assays are not widely available for routine analysis. Objectives To assess the utility of dimeric recombinant soluble FcγRIIa (rsFcγRIIa) ectodomains for detecting HIT antibodies. Patients/Methods Plasma from 27 suspected HIT patients were tested for pathogenic anti-PF4-heparin antibodies by binding of a novel dimeric FcγRIIa ectodomain probe. Plasmas were also tested by the use of PF4-heparin IgG ELISA, the HemosIL AcuStar HIT IgG-specific assay, and a serotonin release assay (SRA). Results The dimeric rsFcγRIIa test produced no false positives and excluded four samples that were positive by IgG ELISA. In this small patient cohort, the novel assay correctly assigned 93% of the suspected HIT patients, with two of the HIT patients being scored as false negatives. The improved discrimination of the novel assay over the IgG ELISA, which scored four false positives, supports the mechanistic interpretation that binding of dimeric rsFcγRIIa detects pairs of closely spaced IgG antibodies in PF4-heparin immune complexes. Conclusions This study found the cell-free, function-based dimeric rsFcγRIIa assay to be convenient, simple, and potentially predictive of HIT. The assay had improved specificity over the IgG ELISA, and correlated strongly with the AcuStar HIT IgG-specific assay, warranting further evaluation of its potential to identify HIT in larger patient cohorts.

Raft localisation of FcgammaRIIa and efficient signaling are dependent on palmitoylation of cysteine 208.

Ligand-dependent aggregation of FcgammaRIIa initiates multiple biochemical processes including the translocation to detergent resistant membrane domains (DRMs) and receptor tyrosine phosphorylation. Palmitoylation of cysteine residues is considered to be one process that assists in the localisation of proteins to DRMs. Within the juxtamembrane region of FcgammaRIIa there is cysteine residue (C208) that we show to be palmitoylated. Mutation of this cysteine residue results in the disruption of FcgammaRIIa translocation to DRMs as empirically defined by insolubility at high Triton X-100 concentrations. This study also demonstrates that the lack of lipid raft association diminishes FcgammaRIIa signaling as measured by receptor phosphorylation and calcium mobilisation functions suggesting that FcgammaRIIa signaling is partially dependent on lipid rafts.

Isolation, tissue distribution, and chromosomal localization of a novel testis-specific human four-transmembrane gene related to CD20 and FcepsilonRI-beta.

CD20 and the beta subunit of the high affinity receptor for IgE (FcepsilonRIbeta) are related four-transmembrane molecules that are expressed on the surface of hematopoietic cells and play crucial roles in signal transduction. Herein, we report the identification and characterization of a human gene, TETM4, that encodes a novel four-transmembrane protein related to CD20 and FcepsilonRIbeta. The predicted TETM4 protein is 200 amino acids and contains four putative transmembrane regions, N- and C-terminal cytoplasmic domains, and three inter-transmembrane loop regions. TETM4 shows 31.0 and 23.2% overall identity with CD20 and FcepsilonRIbeta respectively, with the highest identity in the transmembrane regions, whereas the N- and C-termini and inter-transmembrane loops are more divergent. Northern blot and RT-PCR analysis suggest that TETM4 mRNA has a highly restricted tissue distribution, being expressed selectively in the testis. Using fluorescence in situ hybridization and radiation hybrid analysis, the TETM4 gene has been localized to chromosome 11q12. The genes for CD20 and FcepsilonRIbeta have also been mapped to the same region of chromosome 11 (11q12-13.1), suggesting that these genes have evolved by duplication to form a family of four-transmembrane genes. TETM4 is the first nonhematopoietic member of the CD20/FcepsilonRIbeta family, and like its hematopoietic-specific relatives, it may be involved in signal transduction as a component of a multimeric receptor complex.

The interaction of Fc alpha RI with IgA and its implications for ligand binding by immunoreceptors of the leukocyte receptor cluster.

This study defines the molecular basis of the FcalphaRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcalphaRI. Biosensor analysis of mutant FcalphaRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcalphaRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcalphaRI was generated based on the structures of killer Ig-like receptors, which share 30-34% identity with FcalphaRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcalphaRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcalphaRI interaction that were observed depending on whether FcalphaRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.

Monoclonal antibodies and synthetic peptides define the active site of FcepsilonRI and a potential receptor antagonist.

Defining the structure of the human high-affinity receptor for IgE, Fc,RI, is crucial to understand the receptor:ligand interaction, and to develop drugs to prevent IgE-dependent allergic diseases. To this end, a series of four anti-FcepsilonRI monoclonal antibodies (mAbs), including three new mAbs, 47, 54, and 3B4, were used in conjunction with synthetic FcepsilonRI peptides to define functional regions of the Fc IgE-binding site and identify an antagonist of IgE binding. The spatial orientation of the epitopes detected by these antibodies and their relationship to the IgE-binding region of FcepsilonRI was defined by a homology model based on the closely related FcepsilonRIIa. Using recombinant soluble FcRI-alpha as well as FcepsilonRI-alpha expressed on the cell surface, a series of direct and competitive binding experiments indicated that the mAbs detected nonoverlapping epitopes. One antibody (15-1), previously thought to be located close to the IgE-binding site, was precisely mapped to a single loop within the IgE-binding site by both mutagenesis and overlapping synthetic peptides encompassing the entire extracellular domain. A synthetic peptide epsilonRI-11, containing the amino acids 101-120 and the mAb 15-1 epitope, inhibited IgE binding and may form the basis for the development of a useful receptor-based therapy.

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A.

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.

Immunoglobulin G3 antibodies specific for the 19-kilodalton carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein 1 transfer protection to mice deficient in Fc-gammaRI receptors.

Merozoite surface protein 1 (MSP-1(19)) is a leading malaria vaccine candidate. Specific antibodies contribute to immunity; binding to macrophages is believed to represent the main action of malaria antibodies. We show that an MSP-1(19)-specific immunoglobulin G3 (IgG3) monoclonal antibody can passively transfer protection to mice deficient in the alpha chain of Fc-gammaRI whose macrophages cannot bind IgG3.

Domain one of the high affinity IgE receptor, FcepsilonRI, regulates binding to IgE through its interface with domain two.

The high affinity receptor for IgE, FcepsilonRI, binds IgE through the second Ig-like domain of the alpha subunit. The role of the first Ig-like domain is not well understood, but it is required for optimal binding of IgE to FcepsilonRI, either through a minor contact interaction or in a supporting structural capacity. The results reported here demonstrate that domain one of FcepsilonRI plays a major structural role supporting the presentation of the ligand-binding site, by interactions generated within the interdomain interface. Analysis of a series of chimeric receptors and point mutants indicated that specific residues within the A' strand of domain one are crucial to the maintenance of the interdomain interface, and IgE binding. Mutation of the Arg(15) and Phe(17) residues caused loss in ligand binding, and utilizing a homology model of FcepsilonRI-alpha based on the solved structure of FcgammaRIIa, it appears likely that this decrease is brought about by collapse of the interface and consequently the IgE-binding site. In addition discrepancies in results of previous studies using chimeric IgE receptors comprising FcepsilonRIalpha with either FcgammaRIIa or FcgammaRIIIA can be explained by the presence or absence of Arg(15) and its influence on the IgE-binding site. The data presented here suggest that the second domain of FcepsilonRI-alpha is the only domain involved in direct contact with the IgE ligand and that domain one has a structural function of great importance in maintaining the integrity of the interdomain interface and, through it, the ligand-binding site.

Mouse FcgammaRI: identification and functional characterization of five new alleles.

The mouse Fcgr1 gene encoding the high-affinity IgG receptor (FcgammaRI) exists as two known alleles, FcgammaRI-BALB and FcgammaRI-NOD, and these alleles exhibit functional differences. To determine whether other alleles exist in mouse strains, Fcgr1 coding regions from 35 strains of mice were sequenced and a further five alleles were identified. The FcgammaRI-BALB and NOD alleles are now designated the "a" and "d" alleles, respectively. Analysis of the five new alleles revealed that although no polymorphisms were observed in the two leader exons, nucleotide and subsequent amino acid changes were observed in the exons encoding the extracellular domains, and transmembrane and cytoplasmic tail. The cDNA of the seven alleles (a-g) were isolated and transiently transfected into COS cells, and IgG-binding studies were performed. Receptors encoded by four of the five new alleles (b, c, f, g) bound IgG2a with high affinity, displaying IgG binding characteristics similar to the a allele (previously FcgammaRI-BALB). The d allele (previously FcgammaRI-NOD) and the e allele [derived from Mus spretus (SPRET/Ei)] encoded receptors which showed broader specificity by binding monomeric IgG2a, IgG2b, and IgG3.

Biochemical analysis and crystallisation of Fc gamma RIIa, the low affinity receptor for IgG.

Fc gamma RIIa is one of a family of specific cell surface receptors for immunoglobulin. Fc gamma RIIa, which binds immune complexes of certain IgG isotypes, plays important roles in immune homeostasis. However, the precise characteristics of IgG binding and three-dimensional structure of Fc gamma RIIa have not been reported. This study describes the affinity of the Fc gamma RIIa:IgG interaction as well as biochemical characterisation of recombinant Fc gamma RIIa that has been used to generate high quality crystals. Equilibrium binding analysis of the Fc gamma RII:IgG interaction found, IgG3 binds with an affinity of K(D) = 0.6 microM, as expected. Unlike other Fc gamma R, IgG4 also bound to Fc gamma RIIa, K(D) = 3 microM, clearly establishing Fc gamma RIIa as an IgG4 receptor. Biochemical analysis of mammalian and insect cell derived Fc gamma RIIa established the genuine N-terminus with Q being the first amino acid in the sequence Q, A, A, A, P... extending the N-terminus further than previously thought. Furthermore, both potential N-linked glycosylation sites are occupied. Electrospray ionisation mass spectrometry (ESMS) indicate that the N-glycans of baculovirus derived Fc gamma RIIa are core mannose oligosaccharide side chains. Finally, we describe the first crystallisation of diffraction quality crystals of soluble Fc gamma RIIa. Orthorhombic crystals diffract X-rays beyond 2.1 A resolution in the space group P2(1)2(1)2 with cell dimensions a = 78.8 A, b = 100.5 A, c = 27.8 A. This marks a significant advance towards understanding the three-dimensional structure of Fc gamma RIIa and related FcR proteins that share high amino acid identity with Fc gamma RIIa.

Differential dissociation kinetics explain the binding preference of insulin-like growth factor binding protein-6 for insulin-like growth factor-II over insulin-like growth factor-I.

Insulin-like growth factor binding protein-6 binds insulin-like growth factor-II with a marked preferential affinity over insulin-like growth factor-I. The kinetic basis of this binding preference was studied using surface plasmon resonance. Binding of insulin-like growth factor-I and insulin-like growth factor-II to immobilized insulin-like growth factor binding protein-6 fitted a two-site binding kinetic model. Insulin-like growth factor-I and insulin-like growth factor-II association rates were similar whereas the dissociation rate was approximately 60-fold lower for insulin-like growth factor-II, resulting in a higher equilibrium binding affinity for insulin-like growth factor-II. The equilibrium binding affinities of a series of insulin-like growth factor-II mutants were also explained by differential dissociation kinetics. O-glycosylation had a small effect on the association kinetics of insulin-like growth factor binding protein-6. The insulin-like growth factor binding properties of insulin-like growth factor binding protein-6 are explained by differential dissociation kinetics.

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.

Fine structure analysis of interaction of FcepsilonRI with IgE.

The high affinity receptor for IgE (FcepsilonRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcepsilonRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the alpha-subunit (FcepsilonRIalpha). In this study, the IgE binding site of human FcepsilonRIalpha has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcepsilonRIalpha and the functionally distinct but structurally homologous low affinity receptor for IgG (FcgammaRIIa) have been used to localize two IgE binding regions of FcepsilonRIalpha to amino acid segments Tyr129-His134 and Lys154-Glu161. Both regions were capable of independently binding IgE upon placement into FcgammaRIIa. Molecular modeling of the three-dimensional structure of FcepsilonRIalpha-D2 has suggested that these binding regions correspond to the "exposed" C'-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129-His134 and Lys154-Glu161 regions of FcepsilonRIalpha was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159 decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116 in the B-C loop region, which lies adjacent to the C'-E and F-G loops, has suggested Trp113 also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcepsilonRIalpha-IgE interaction for the possible treatment of IgE-mediated allergic disease.

Identification of residues in the first domain of human Fc alpha receptor essential for interaction with IgA.

The FcR family contains multiple receptors for Igs, of which the most distantly related ( approximately 20%) is the IgA receptor (human Fc alpha R), being more homologous ( approximately 35%) to another family of killer-inhibitory receptor-related immunoreceptors with a 19q13.4 chromosomal location in humans. This study of the Fc alpha R demonstrated that, like several IgG receptors, Fc alpha R is a low affinity receptor for Ab (Ka approximately 106 M-1). Rapid dissociation of the rsFc alpha R:IgA complex (t1/2 approximately 25 s) suggests that monomer IgA would bind transiently to cellular Fc alpha Rs, while IgA immune complexes could bind avidly. Mutagenesis of histidyl 85 and arginyl 82, in the FG loop of domain 1, demonstrated that these residues were essential for the IgA-binding activity of Fc alpha R, while arginyl 87 makes a minor contribution to the binding activity of the receptor. This site is unusual among the Fc receptors (Fc gamma RII, Fc gamma RIII, and Fc epsilon RI), in which the ligand binding site is in domain 2 rather than domain 1, but like Fc alpha R, the FG loop comprises part of the ligand binding site. The putative F and G strands flanking the Fc alpha R ligand binding site are highly homologous in the other killer-inhibitory receptor-related immunoreceptors, suggesting they comprise a conserved structural element on which divergent FG loops are presented and participate in the specific ligand interactions of each of these receptors.

Gain-of-function mutations in FcgammaRI of NOD mice: implications for the evolution of the Ig superfamily.

It has been postulated that, during evolution of the Ig superfamily, modifications of the function of individual receptors might occur by acquisition of exons and their subsequent modification, though evidence of this is lacking. Here we have analysed the interaction of mouse IgG subclasses with high-affinity FcgammaRI (CD64) which contains three Ig-like domains and is important in innate and adaptive immunity. This analysis has identified a mechanism by which the postulated modification of newly acquired exons provides gains in function. Thus, the most widely distributed FcgammaRI allele in mice (e.g. BALB/c), bound only a single IgG subclass, IgG2a, with high affinity. However, non-obese diabetic (NOD) mice expressed a unique allele that exhibits broader specificity and, in addition to binding IgG2a, FcgammaRI-NOD bound monomeric IgG3 and bound IgG2b with high affinity, an IgG subclass not bound by FcgammaRI of other mouse strains, either as monomer or multivalent immune complexes. Analysis of mutants of FcgammaRI wherein segments of the interdomain junctions were exchanged between FcgammaRI-BALB and FcgammaRI-NOD identified these regions as having major influence in 'gain-of-function' by the NOD form of FcgammaRI. Nucleotide sequence analysis of intron/exon boundaries encoding the interdomain junctions of the FcgammaRI alleles showed these to have arisen by mutation to alter existing or create new mRNA splice donor/acceptor sites, resulting in generation of modified junctions.

Identification of the mouse IgG3 receptor: implications for antibody effector function at the interface between innate and adaptive immunity.

Mouse IgG3 appears early in immune responses independently of T cell help and, as such, is an early effector molecule of the immune system. Yet, a specific IgG3 cellular receptor remains undefined. In transfection experiments, mouse Fc gammaRI was clearly able to bind immune complexes of IgG3, whereas mouse Fc gammaRII could not. Furthermore, macrophages from mice expressing Fc gammaRII and Fc gammaRIII but lacking Fc gammaRI were unable to phagocytose IgG3 immune complexes, thus identifying mouse Fc gammaRI as the sole receptor for IgG3 immune complexes. Competition studies demonstrated that monomeric mouse IgG3 could inhibit IgG2a binding to mouse Fc gammaRI with an ID50 approximately 10(-7) M (fivefold lower than IgG2a). The identification of mouse Fc gammaRI as the IgG3 receptor establishes Fc gammaRI as a participant in events at the interface between innate and adaptive immunity, implying a greater role for this receptor in the development of normal and pathologic immune responses than previously recognized.

The second and third extracellular domains of FcgammaRI (CD64) confer the unique high affinity binding of IgG2a.

FcgammaRI (CD64) is functionally unique as it is the only FcgammaR able to bind monomeric IgG with high affinity. FcgammaRI is also structurally distinct, containing an extracellular Ig-interactive region of three Ig-like domains in contrast to the two domains of the low affinity receptors FcgammaRII and FcgammaRIII. Previous studies have demonstrated that the third domain of FcgammaRI plays a crucial role in high affinity IgG binding of the receptor, with the first and second domains together forming a low affinity IgG binding motif. In this study the individual functional contributions of the first and second domains of FcgammaRI to IgG binding have been investigated. Chimeric FcgammaR were generated by exchanging extracellular domains between mouse FcgammaRI and the structurally related yet distinct low affinity receptor for IgG, mouse FcgammaRII. The replacement of both domains 1 and 2 of FcgammaRI with domains 1 and 2 of FcgammaRII results in a dramatic change in IgG binding characteristics, as this receptor loses the capacity to bind monomeric IgG with high affinity and also demonstrates a broader specificity (binding not only IgG2a but also IgG1 and 2b. IgG3 was not tested). However, the substitution of FcgammaRII domain 2 of this chimeric receptor with domain 2 of FcgammaRI (generating a chimeric receptor with domain 1 of FcgammaRII linked to domains 2 and 3 of FcgammaRI) was found to reconstitute the specific high affinity monomeric IgG2a binding of wild-type FcgammaRI, albeit with a slightly reduced affinity (1.8-fold lower than wild-type FcgammaRI). These findings suggest that it is the specific interaction between domains 2 and 3 of FcgammaRI, with domain 1 playing a supporting role in maintaining the conformational stability of the receptor, that is the major structural requirement to confer the unique Ig binding characteristics of FcgammaRI.

Redirected cytotoxic effector function. Requirements for expression of chimeric single chain high affinity immunoglobulin e receptors.

The aim of this study was to construct a single chain chimeric FcepsilonRIalpha receptor capable of effector function in leukocytes, including cytotoxic lymphocytes. To determine the most effective single chain FcepsilonRIalpha receptor with respect to IgE binding and signaling function, a variety of chimeric gene constructs were transiently transfected into COS-7 cells. The most effective chimera consisted of four parts including: wild-type or mutated extracellular domains (Trp130 --> Ala130, W130A) of FcepsilonRIalpha, membrane proximal and transmembrane regions of FcgammaRIIa, and intracellular CD3zeta (epsilonIIaIIazeta). Scatchard analysis indicated that these FcepsilonRIalpha chimeric receptor bound ligand with an affinity of 0.9 to 2.2 x 10(9) -1. Ligand binding capacity was dramatically reduced with the deletion of 11 membrane proximal amino acids of FcepsilonRIalpha; however, function was restored by substitution with the equivalent region of FcgammaRIIa, suggesting a crucial requirement for a "spacer" segment between the transmembrane and extracellular ligand binding domain. Chimeras that bound IgE effectively also mediated phagocytosis. Chimeric receptors that contained transmembrane zeta were expressed as multimers and consequently did not bind IgE effectively; however, cotransfection of these chimeras with gamma-chain largely reconstituted IgE-mediated phagocytosis. The mouse cytotoxic T lymphocyte cell line, CTLLR8 was stably transfected with epsilonIIaIIazeta, and cloned transfectants were demonstrated to lyse target cells in an anti-FcepsilonRIalpha or IgE antibody-dependent manner. Therefore, functional single chain chimeric FcepsilonRIalpha receptors were expressed in the absence or presence of associated zeta or gamma molecules and were used to redirect killer lymphocytes to target cells.

Extracellular mutations of non-obese diabetic mouse FcgammaRI modify surface expression and ligand binding.

The non-obese diabetic mouse (NOD) expresses a unique form of the high affinity receptor for IgG (FcgammaRI), containing multiple mutations that result in substitutions and insertions of amino acids and a truncated cytoplasmic tail. As a result of these major changes, receptor affinity for IgG increases 10-fold over that of wild-type FcgammaRI from BALB/c mice, while the specificity for ligand is retained. Kinetic analysis revealed that while the association rate of IgG with FcgammaRI from NOD mice (FcgammaRI-NOD) and FcgammaRI from BALB/c mice (FcgammaRI-BALB) is similar, IgG bound much more tightly to FcgammaRI-NOD as revealed by a profoundly diminished dissociation rate. Transfection studies demonstrated that FcgammaRI-NOD was expressed at one-tenth of the level of FcgammaRI-BALB. Although mouse FcgammaRI was previously not known to associate with the FcepsilonRI gamma-subunit, transfection of COS-7 cells demonstrates that like human FcgammaRI, mouse FcgammaRI is also able to associate with this signaling subunit. Furthermore, expression levels of FcgammaRI-NOD were not restored by the presence of the FcepsilonRI gamma-subunit. The difference in the levels of expression was mapped to mutations in the extracellular region of FcgammaRI-NOD as replacement of the extracellular domains with those of human FcgammaRI or FcgammaRI-BALB restored expression to that of human FcgammaRI or FcgammaRI-BALB.

Recombinant soluble Fc gamma RII inhibits immune complex precipitation.

Control of IgG immune complex formation and deposition is important in determining the nature and extent of subsequent immune effector responses, and appears to be aberrant in some autoimmune diseases. In this study we demonstrate that recombinant soluble Fc gamma RII (rsFc gamma RII) is an effective modulator of immune complex formation, delaying immune precipitation in a manner which is dose-dependent, and can be specifically inhibited by anti-Fc gamma RII MoAb Fab' fragments. This inhibitory role in immune precipitation also provides a possible mechanistic explanation for our previous demonstration of the efficacy of rsFc gamma RII as an inhibitor of immune complex-induced inflammation in the Arthus reaction in vivo. RsFc gamma RII inhibited immune complex precipitation in two different experimental systems. First, rsFc gamma RII inhibited the precipitation of 125I-bovine serum albumin (BSA)-anti-BSA complexes in a dose-dependent manner, while an irrelevant protein (soybean trypsin inhibitor) had no effect on the precipitation of the immune complexes. Moreover, rsFc gamma RII inhibited the precipitation of ovalbumin (OVA)-anti-OVA complexes as determined by turbidimetric analysis, where the inhibition of immune complex precipitation by rsFc gamma RII was dose-dependent and was specifically blocked by prior incubation with Fab' fragments of a blocking MoAb to Fc gamma RII. RsFc gamma RII could inhibit the precipitation of BSA-anti-BSA complexes in the presence of excess bystander IgG and did not inhibit complement-mediated prevention of immune precipitation, demonstrating that rsFc gamma RII did not block C1 binding to the BSA-anti-BSA complex. Unlike complement, rsFc gamma RII could not cause re-solubilization of pre-formed precipitated BSA-anti-BSA complexes. Soluble Fc gamma Rs have been detected in biological fluids of normal and inflammatory disease patients, yet the role of sFc gamma R is still unclear. However, they now play a potential role in the modulation of immune complex solubility.