Human Fc Receptor Research Articles

Engineering Human IgG1 Affinity to Human Neonatal Fc Receptor: Impact of Affinity Improvement on Pharmacokinetics in Primates

The pH-dependent binding of Igs to the neonatal FcR (FcRn) plays a critical role in the in vivo homeostasis of IgGs. Modulating the interaction between Fc and FcRn through protein engineering is one method for improving the pharmacokinetics of therapeutic Abs. Recent studies disputed the direct relationship between increasing FcRn affinity and improved pharmacokinetic properties. In this work, we studied the pharmacokinetics of two human IgG1 Fc variants in cynomolgus monkey to further clarify the affinity-pharmacokinetic relationship. First, we report a number of novel Fc point mutations and combination variants, including some with primate-specific FcRn-binding improvements. By studying these variants along with some previously described variants across a wide range of affinities, we discovered a direct correlation of pH 6 affinity improvements with neutral pH improvements, suggesting that all of the tested variants exhibit similar pH dependency in FcRn binding. We then evaluated the pharmacokinetics of variants N434A and N434W, which, respectively, gave approximately 4- and 80-fold improvements in pH 6-binding affinity to both human and nonhuman primate FcRn. Surprisingly, clearance of N434W was similar to that of wild type. N434W is the first variant studied in primates that exhibits significant binding to FcRn at pH 7.4, and its clearance substantiates the principle that too much affinity improvement, i.e., beyond that of N434W, does not yield improved pharmacokinetics. In contrast, N434A exhibited a approximately 2-fold decrease in clearance in cynomolgus monkey, supporting the notion that modest increases in pH 6 FcRn affinity can result in improved pharmacokinetics in primates.

Functional and clinical consequences of Fc receptor polymorphic and copy number variants

Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leucocytes, thereby linking the humoral and cellular components of immunity. Indeed, engagement of Fc receptors by immunoglobulins initiates a range of immunoregulatory processes that might also play a role in disease pathogenesis. In the circulation, five main types of immunoglobulins (Ig) exist - namely IgG, IgA, IgE, IgM and IgD and receptors with the ability to recognize and bind to IgG (Fc gamma receptor family), IgE (Fc epsilon RI and CD23), IgA (CD89; Fc alpha/microR) and IgM (Fc alpha/microR) have been identified and characterized. However, it is astonishing that nearly all the known human Fc receptors display extensive genetic variation with clear implications for their function, thus representing a substantial genetic risk factor for the pathogenesis of a range of chronic inflammatory disorders.

Functional Expression of the Human Neonatal Fc-receptor, hFcRn, in Isolated Cultured Human Syncytiotrophoblasts

Materno-fetal IgG transfer in the mature human placenta involves transport across the syncytiotrophoblast (STB) and fetal endothelial cell layer. The MHC class I-related Fcγ-receptor (hFcRn) localized in STB as well as in endothelial cells is involved in overall IgG transfer from the maternal into the fetal circulation. Functional hFcRn is a heterodimer of a transmembrane α-chain and β2-microglobulin. To establish the basis for future studies to unravel the mechanism of IgG transport in STB, we investigated hFcRn α-chain and β2-microglobulin expression in cytotrophoblasts (CTB) isolated from human term placentae and cultured in vitro under conditions where differentiation into multinuclear STB takes place (≥48h). Northern blot analysis demonstrated up-regulation of α-chain mRNA after 48h of in vitro cultivation. Likewise, hFcRn α-chain and β2-microglobulin were at the limit of detection by immunofluorescence microscopy in CTB immediately after isolation, but their expression increased upon STB formation. hFcRn α-chain co-localized with β2-microglobulin in multinuclear STB and formed a functional, i.e. low pH IgG binding, receptor as shown by affinity isolation. The in vitro differentiated STB exhibited specific, low pH-dependent IgG binding to the plasma membrane. In conclusion, these cultures can now be applied to study the role of hFcRn in IgG transport and trafficking in STB cultures in vitro.

Structural characterization of a human Fc fragment engineered for extended serum half-life

The first three-dimensional structure of a human Fc fragment genetically engineered for improved pharmacokinetics properties is reported. When introduced into the C H2 domain of human immunoglobulin G (IgG) molecules, the triple mutation M252Y/S254T/T256E (‘YTE’) causes an about 10-fold increase in their binding to the human neonatal Fc receptor (FcRn). This translates into an almost 4-fold increase in the serum half-life of YTE-containing human IgGs in cynomolgus monkeys. A recombinantly produced human Fc/YTE fragment was crystallized and its structure solved at a resolution of 2.5 Å using molecular replacement. This revealed that Fc/YTE three-dimensional structure is very similar to that of other human Fc fragments in the experimentally visible region spanning residues 236–444. We propose that the enhanced interaction between Fc/YTE and human FcRn is likely mediated by local effects at the substitutions sites. Molecular modeling suggested that potential favorable hydrogen bonds along with an increase in the surface of contact between the two partners may account in part for the corresponding increase in affinity.

Inhibition of destructive autoimmune arthritis in FcγRIIa transgenic mice by small chemical entities

The interaction of immune complexes with the human Fc receptor, FcgammaRIIa, initiates the release of inflammatory mediators and is implicated in the pathogenesis of human autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, so this FcR is a potential target for therapy. We have used the three-dimensional structure of an FcgammaRIIa dimer to design small molecule inhibitors, modeled on a distinct groove and pocket created by receptor dimerization, adjacent to the ligand-binding sites. These small chemical entities (SCEs) blocked immune complex-induced platelet activation and aggregation and tumor necrosis factor secretion from macrophages in a human cell line and transgenic mouse macrophages. The SCE appeared specific for FcgammaRIIa, as they inhibited only immune complex-induced responses and had no effect on responses to stimuli unrelated to FcR, for example platelet stimulation with arachidonic acid. In vivo testing of the SCE in FcgammaRIIa transgenic mice showed that they inhibited the development and stopped the progression of collagen-induced arthritis (CIA). The SCEs were more potent than methotrexate and anti-CD3 in sustained suppression of CIA. Thus, in vitro and in vivo activity of these SCE FcgammaRIIa receptor antagonists demonstrated their potential as anti-inflammatory agents for autoimmune diseases involving immune complexes.

Ligand binding and antigenic properties of a human neonatal Fc receptor with mutation of two unpaired cysteine residues

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule that regulates the half-life of IgG and albumin. In addition, FcRn directs the transport of IgG across both mucosal epithelium and placenta and also enhances phagocytosis in neutrophils. This new knowledge gives incentives for the design of IgG and albumin-based diagnostics and therapeutics. To study FcRn in vitro and to select and characterize FcRn binders, large quantities of soluble human FcRn are needed. In this report, we explored the impact of two free cysteine residues (C48 and C251) of the FcRn heavy chain on the overall structure and function of soluble human FcRn and described an improved bacterial production strategy based on removal of these residues, yielding approximately 70 mg.L(-1) of fermentation of refolded soluble human FcRn. The structural and functional integrity was proved by CD, surface plasmon resonance and MALDI-TOF peptide mapping analyses. The strategy may generally be translated to the large-scale production of other major histocompatibility complex class I-related molecules with nonfunctional unpaired cysteine residues. Furthermore, the anti-FcRn response in goats immunized with the FcRn heavy chain alone was analyzed following affinity purification on heavy chain-coupled Sepharose. Importantly, purified antibodies blocked the binding of both ligands to soluble human FcRn and were thus directed to both binding sites. This implies that the FcRn heavy chain, without prior assembly with human beta2-microglobulin, contains the relevant epitopes found in soluble human FcRn, and is therefore sufficient to obtain binders to either ligand-binding site. This finding will greatly facilitate the selection and characterization of such binders.

FCRL2 expression predicts IGHV mutation status and clinical progression in chronic lymphocytic leukemia

AbstractCD38 and ZAP-70 are both useful prognostic markers for B-cell chronic lymphocytic leukemia (CLL), but are variably discordant with IGHV mutation status. A total of 5 human Fc receptor–like molecules (FCRL1-5) have tyrosine-based immunoregulatory potential and are expressed by B-lineage subpopulations. To determine their prognostic potential in CLL, FCRL expression was compared with IGHV mutation status, CD38 and ZAP-70 expression, and clinical features from 107 patients. FCRL1, FCRL2, FCRL3, and FCRL5 were found at markedly higher levels on CLL cells bearing mutated IGHV genes than on unmutated CLL cells or CD19+ polyclonal B lymphocytes. Univariate comparisons found that similar to CD38 and ZAP-70, FCRL expression was strongly associated with IGHV mutation status; however, only FCRL2 maintained independent predictive value by multivariate logistic analysis. Strikingly, FCRL2 demonstrated 94.4% concordance with IGHV mutation compared with 76.6% for CD38 and 80.4% for ZAP-70. Compared with other indicators, FCRL2 was also superior at predicting the time to first therapy; the median treatment-free interval was 15.5 years for patients with high FCRL2 expression compared with 3.75 years for FCRL2-low patients. Our studies indicate that FCRL2 has robust predictive value for determining IGHV gene mutation status and clinical progression and thus may further improve prognostic definition in CLL.

Structure–activity relationships of a peptide inhibitor of the human FcRn:human IgG interaction

A family of five peptides was previously discovered by phage display techniques that binds to the human neonatal Fc receptor (FcRn) and inhibits the human IgG:human FcRn protein–protein interaction [ Proc. Nat. Acad. Sci. U.S.A. 2008, 105, 2337–2342]. The consensus peptide motif consists of the sequence GHFGGXY where X is preferably a hydrophobic amino acid, and also includes a disulfide bridge enclosing 11-amino acids in varying positions about the consensus sequence. We describe herein the structure–activity relationships of one of the five peptides in binding to FcRn using surface plasmon resonance and IgG:FcRn competition ELISA assays. Modifications of the peptide length, cyclization, and the incorporation of amino acid substitutions and dipeptide mimetics were studied. The most potent analogs exhibited a 50- to 100-fold improvement of in vitro activity over that of the phage-identified peptide sequence.

Ex vivo simulation of IgG Fc and T-cell receptor functions: An application to inflammatory bowel disease

Human leukocyte IgG Fc receptors (FcgammaR) and T-cell receptors (TCR) are primary molecules involved in inflammatory and immune pathways. These 2 receptors were stimulated in whole blood for 2-4 hours with immune complex and specific agonistic antibody, respectively, and various mRNAs were quantified by a method we developed previously. FcgammaR stimulation induced tumor necrosis factor superfamily 2 (TNFSF2), TNFSF8, TNFSF15, interleukin 1B (IL1B), IL8, CCL chemokine 2 (CCL2), CCL3, CCL4, CCL11, CCL20, CXCL chemokine 1 (CXCL1), CXCL2, and CXCL3 mRNA, whereas TCR stimulation induced different subsets of mRNA such as TNFSF1, TNFSF2, TNFSF5, TNFSF6, TNFSF9, TNFSF14, IL6, CCL2, CCL8, CCL20, and CXCL10. Interestingly, respondents and nonrespondents were identified for each mRNA. When we applied this method to inflammatory bowel disease, the respondent populations of TCR-induced TNFSF2 (= TNFalpha), TNFSF5, TNFSF14, CCL2, CCL8, and CCL20 mRNA were significantly higher in Crohn's disease (CD) patients than in healthy controls or those with ulcerative colitis (UC). No difference was found for FcgammaR-mediated responses. The respondent population of TCR-induced TNFSF2 showed significantly (P = 0.05) higher incidence of multiple surgeries than did nonrespondents. These data demonstrate an underlining hyperfunction of TCR in peripheral-blood leukocytes in CD patients. The ex vivo simulation demonstrates an underlining hyperfunction of TCR in peripheral-blood leukocytes in CD patients and may form the basis of a relatively noninvasive test for distinguishing these IBDs.

Primary roles for human neutrophil Fc receptors in the initiation of nephrotoxic glomerulonephritis

Antibody‐antigen complexes promote inflammatory and autoimmune diseases. Mice deficient in γ‐chain of Fc‐receptors are protected in antibody mediated glomerulonephritis and lupus nephritis and FcR‐bearing macrophages have been implicated as the primary initiators of disease. However, how well studies of diseases mediated by murine receptors accurately reflect human inflammation remains unclear. Here we show that neutrophil selective transgenic expression of the two uniquely human FcγRIIA and FcγRIIIB in γ‐chain deficient mice (IIA+IIIBtg/γ−/−) was sufficient to promote progressive glomerulonephritis associated with glomerular neutrophil accumulation, glomerulosclerosis, macrophage and T cell accumulation, renal failure and mortality. These responses far exceeded those in wild‐type mice despite similar expression levels of activating receptors suggesting that the human and mouse FcγRs are not functionally equivalent. Both FcγRIIIB and FcγRIIA promoted robust neutrophil recruitment while FcγRIIA alone mediated tissue injury. The latter was consistent with the robust adhesion dependent H2O2 production in IIAtg/γ−/− and the lack there of in IIIBtg/γ−/ −. Thus, human neutrophil FcγRs serve as molecular links between immune complexes and renal injury suggesting a new paradigm for FcγRs on neutrophils in the initiation of antibody mediated‐glomerulonephritis.Funded by HL065095, AR050800 and Arthritis Foundation Postdoctoral Fellowship.

The yin and yang of Fcγ receptors in ITP

In this issue of Blood , Breunis and colleagues examine the role of genetic variation in the human Fc receptors for IgG in the susceptibility to idiopathic thrombocytopenic purpura (ITP). The Fcγ receptors contribute to the susceptibility to autoimmune diseases such as ITP in several ways: (1)

A strategy for bacterial production of a soluble functional human neonatal Fc receptor

The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus (human) or newborn (rodents), and may translocate IgG over mucosal surfaces. FcRn interacts with the Fc-region of IgG and domain III of albumin with binding at pH 6.0 and release at pH 7.4. Knowledge of these interactions has facilitated design of recombinant proteins with altered serum half-lives and/or altered biodistribution. To generate further research in this field, there is a great need for large amounts of soluble human FcRn (shFcRn) for in vitro interaction studies. In this report, we describe a novel laboratory scale production of functional shFcRn in Escherichia coli ( E. coli) at milligram level. Truncated wild type hFcRn heavy chains were expressed, extracted, purified from inclusion bodies under denaturing non-reducing conditions, and subsequently refolded in the presence of human β 2-microglobulin (hβ 2m). The secondary structural elements of refolded heterodimeric shFcRn were correctly formed as demonstrated by circular dichroism (CD). Furthermore, functional and stringent pH dependent binding to IgG and human serum albumin were demonstrated by ELISA and surface plasmon resonance (SPR). This method may be easily adapted for the expression of large amounts of other FcRn species and MHC class I related molecules.

High affinity Fc receptor binding and potent induction of antibody-dependent cellular cytotoxicity (ADCC) in vitro by anti-epidermal growth factor receptor antibody cetuximab

3041 Background: Cetuximab is an IgG1 monoclonal antibody specific for human epidermal growth factor receptor (EGFR). Cetuximab acts as a functional antagonist by blocking ligand (EGF and TGFa) binding to EGFR and, therefore, inhibits EGFR activation and downstream signaling in tumor cells. Methods: In the present study, we analyzed the binding activity of cetuximab to human Fc receptors and tested whether cetuximab can elicit antibody-dependent cellular cytotoxicity (ADCC) in vitro using tumor cell lines expressing various levels of EGFR. Results: Cetuximab bound to human FcRI and FcRIII with high affinity (EC50 of 0.13 nM and 6 nM, respectively), whereas an aglycosylated form of cetuximab or an IgG2 antibody to EGFR (panitumumab) did not bind to FcRI and FcRIII. A panel of head and neck, colon, and breast tumor cell lines were tested for cetuximab-mediated ADCC using human peripheral blood mononuclear cells (PBMC) from normal donors as effector cells. We found that cetuximab-induced ADCC to these tumor cell lines ranging from 20–90% specific killing. Interestingly, the extent of ADCC induced by cetuximab correlated with the level of EGFR expression on cell surface. The aglycosylated form of cetuximab or panitumumab, did not elicit ADCC of EGFR expressing tumor cells, even though cetuximab and panitumumab bind to EGFR with similar affinity (Kd=87 pM and 83 pM, respectively). To identify the immune cell population responsible for ADCC, NK cells were isolated from PBMC and tested in an ADCC assay. Purified NK cells elicited high levels of specific killing of cetuximab bound tumor cells, suggesting that NK cells are one of effector cell populations in human PBMCs responsible for the observed ADCC activity. Conclusions: Thus, cetuximab can effectively link effector cells to EGFR expressing tumor cells and mediate potent ADCC against EGFR-expressing human tumors. [Table: see text]

The Importance of Human FcγRI in Mediating Protection to Malaria

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcγRI. This important finding documents the capacity of FcγRI to mediate potent antimalaria immunity and supports the development of FcγRI-directed therapy for human malaria.

Unique tissue distribution and molecular heterogeneity of human Fc receptor for IgA and IgM (Fcα/μR) (53.17)

Abstract Fcα/μR consists of an extracellular region with an N-terminal single Ig-like domain and a membrane proximal stalk region, a transmembrane (TM) segment and a cytoplasmic tail. Fcα/μR binds the polymeric IgM and IgA and is expressed by follicular dendritic cells in germinal centers, small intestinal Paneth cells, renal tubular epithelial cells and serous small epithelial cells of salivary glands in humans. Analyses of its transcripts and genomic DNA revealed the existence of an additional translation start site upstream of the previously isolated Fcα/μR cDNA and a splice variant in kidney tissues. The latter was generated from the direct splicing of exon 5 (Ig-like domain) to exon 7 (TM) and resulted in a reading frameshift in exon 7, generating a novel 48-aa hydrophilic C-terminal tail. To determine the roles of the upstream start site and the alternative splice variant, those cDNA were subcloned into an expression vector prior to establishment of the mouse cell lines stably expressing human Fcα/μR transgene. Transductants with the full-length Fcα/μR cDNA irrespective of the start sites expressed Fcα/μR on their cell surface, whereas those with the Fcα/μR splice variant expressed Fcα/μR associated with intracellular, not plasma, membranes. Protein blot analysis of these cell lysates revealed that the Fcα/μR proteins correspond to their predicted molecular masses. (Supported by NIH/NIAID AI052243)

The conserved histidine 166 residue of the human neonatal Fc receptor heavy chain is critical for the pH‐dependent binding to albumin

The MHC class I-related neonatal Fc receptor (FcRn) serves in the homeostatic regulation of IgG and albumin by increasing their half-lives. FcRn may bind IgG and albumin simultaneously, and in a pH-dependent manner, with ligand binding at pH 6.0-6.5 and release at pH 7.0-7.4. The FcRn-IgG interaction has been extensively characterized at the amino acid level and shown to depend on conserved histidine residues in the IgG-Fc part that interact with negatively charged residues in the alpha-2 domain of FcRn. The recently discovered FcRn-albumin interaction remains to be elucidated. Guided by the pH dependence of the FcRn-albumin interaction, we compared the sequence of the FcRn alpha-2 domain from eleven different species, and identified histidine residues that were conserved in all (H166) or seven (H161) of these. Both residues are located directly opposite to the IgG interaction site in the folded molecule. We did in vitro mutagenesis (H161A or H166A) in combination with interaction studies (ELISA and surface plasmon resonance) with recombinant, soluble, purified receptors and IgG and albumin to investigate the role of the two histidine residues. Our results show clear evidence that the conserved H166 is a key player in the FcRn-albumin interaction.

Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.

Generation and characterization of a novel tetravalent anti-CD22 antibody with improved antitumor activity and pharmacokinetics

The purpose of this study was to prepare a tetravalent anti-human CD22 recombinant antibody with improved antitumor activity and a half life longer than that of its divalent counterpart. We compared the ability of tetravalent vs. divalent antibody to associate/dissociate to/from CD22-positive Daudi cells, to interact with murine and human Fcγ receptors (FcγR), to bind human complement component C1q, to inhibit the growth of tumor cells, to diffuse into various tissues, to be internalized by Daudi cells, to react with human neonatal Fc receptors (FcRn), and to persist in the circulation of normal mice. As compared to the murine or chimeric divalent antibodies, the chimeric tetravalent counterpart has a longer half life in mice. It also has an affinity for FcRns that is identical to that of human IgG. The tetravalent antibody has increased antitumor activity in vitro and completely conserved effector functions (binding to FcγR-positive cells and to C1q) in vitro. Despite its 33% higher molecular weight, it penetrates mouse tissues as well as its divalent antibody counterpart. Based on the improved in vitro performance and pharmacokinetics of the tetravalent antibody it will now be tested for its antitumor activity in vivo.

C/EBPα functionally and physically interacts with GABP to activate the human myeloid IgA Fc receptor (FcαR, CD89) gene promoter

AbstractHuman Fcα receptor (FcαR; CD89), the receptor for the crystallizable fragment (Fc) of immunoglobulin A (IgA), is expressed exclusively in myeloid cells, including granulocytes and monocytes/macrophages, and is considered to define a crucial role of these cells in immune and inflammatory responses. A 259-base pair fragment of the FCAR promoter is sufficient to direct myeloid expression of a reporter gene and contains functionally important binding sites for CCAAT/enhancer-binding protein α (C/EBPα) (CE1, CE2, and CE3) and an unidentified Ets-like nuclear protein. Here, we show that the Ets-binding site is bound by a heterodimer composed of GA-binding protein α (GABPα), an Ets-related factor, and GABPβ, a Notch-related protein. Cotransfection of GABP increased FCAR promoter activity 3.7-fold through the Ets-binding site. GABP and C/EBPα synergistically activated the FCAR promoter 280-fold. Consistent with these observations, in vitro binding analyses revealed a physical interaction between the GABPα subunit and C/EBPα. This is the first report demonstrating both physical and functional interactions between GABP and C/EBPα and will provide new insights into the molecular basis of myeloid gene expression. (Blood. 2005;106:2534-2542)

Development of spontaneous multisystem autoimmune disease and hypersensitivity to antibody‐induced inflammation in Fcγ receptor IIa–transgenic mice

The major human Fc receptor, FcgammaRIIa, is the most widespread activating FcR. Our aim was to determine the role of FcgammaRIIa in a transgenic mouse model of immune complex-mediated autoimmunity and to characterize the development of spontaneous autoimmune disease. Arthritis was induced in normal and FcgammaRIIa-transgenic mice by immunization with type II collagen (CII) or by transfer of arthritogenic anti-CII antibodies. Also, mice that spontaneously developed autoimmune disease were assessed by clinical scoring of affected limbs, histology and serology, and measurement of autoantibody titers and cytokine production. FcgammaRIIa-transgenic mice developed collagen-induced arthritis (CIA) more rapidly than did archetypal CIA-sensitive DBA/1 (H-2q) mice, while nontransgenic C57BL/6 (H-2b) mice did not develop CIA when similarly immunized. Passive transfer of a single dose of anti-CII antibody induced a more rapid, severe arthritis in FcgammaRIIa-transgenic mice than in nontransgenic animals. In addition, most immune complex-induced production of tumor necrosis factor alpha by activated macrophages occurred via FcgammaRIIa, not the endogenous mouse FcR. A spontaneous, multisystem autoimmune disease developed in aging (>20 weeks) transgenic mice (n = 25), with a 32% incidence of arthritis, and by 45 weeks, all mice had developed glomerulonephritis and pneumonitis, and most had antihistone antibodies. Elevated IgG2a levels were seen in mice with CIA and in those with spontaneous disease. The presence of enhanced passive and induced autoimmunity, as well as the emergence of spontaneous autoimmune disease at 20-45 weeks of age, suggest that FcgammaRIIa is a very important factor in the pathogenesis of autoimmune inflammation and a possible target for therapeutic intervention.